The Mouse in Biomedical Research, 2nd Edition Volume II Diseases
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THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION Volume II Diseases EDITED
BY
James G. Fox
Muriel T. Davisson
Fred W. Quimby
Division of Comparative Medicine, MIT Cambridge, MA
The Jackson Laboratory Bar Harbor, ME
Laboratory Animal Research Center The Rockefeller University New York, NY
Stephen W. Barthold
Christian E. Newcomer
Abigail L. Smith
Center for Comparative Medicine Schools of Medicine and Veterinary Medicine University of California Davis, CA
Research Animal Resources and Department of Molecular and Comparative Pathobiology Johns Hopkins University Baltimore, MD
School of Veterinary Medicine University of Pennsylvania Philadelphia, PA
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Table of Contents Volume I History, Wild Mice, and Genetics List of Reviewers List of Contributors Foreword Preface
1.
Building a Better Mouse: One Hundred Years of Genetics and Biology
x xi xiii xv
10. 1
Herbert C. Morse III
Mouse Embryology: Research Techniques and a Comparison of Embryonic Development between Mouse and Man
165
Matthew H. Kaufman 2.
Systematics of the genus Mus
13
Priscilla K. Tucker
11.
Gamete and Embryo Manipulation
211
K.C. Kent Lloyd 3.
The Secret World of Wild Mice
25
Grant R. Singleton and Charles J. Krebs 12. 4.
Breeding Systems: Considerations, Genetic Fundamentals, Genetic Background, and Strain Types
Mouse Strain and Genetic Nomenclature: an Abbreviated Guide
225
Martin Hrabé de Angelis, Dian Michel, Sibylle Wagner, Sonja Becker, and Johannes Beckers 53
Melissa L. Berry and Carol Cutler Linder 5.
Chemical Mutagenesis in Mice
13.
Gene-Specific Mutagenesis
261
K.C. Kent Lloyd 79
Janan T. Eppig
14.
Gene Transfer Studies Using Mouse Models
267
Robert G. Pergolizzi and Ronald G. Crystal 6.
The Mouse Genome
99
Mark D. Adams 7.
Gene Mapping
15.
Mouse and Human Pluripotent Stem Cells
281
Leslie F. Lock
115
Muriel T. Davisson 16. 8.
Genetic Monitoring
135
Cytogenetics Muriel T. Davisson and Mary Ann Handel
289
Lucia F. Jorge-Nebert, Sandrine Derkenne, and Daniel W. Nebert
Richard R. Fox, Michael V. Wiles, and Petko M. Petkov 9.
Drugs and the Mouse: Pharmacology, Pharmacogenetics, and Pharmacogenomics
145 Index
321
v
vi
TA B L E O F C O N T E N T S
Volume II Diseases
10.
Retroelements in the Mouse
269
Herbert C. Morse III List of Reviewers List of Contributors Foreword Preface
x xi xiii xv
Viral Diseases
11.
Sendai Virus and Pneumonia Virus of Mice (PVM)
281
David G. Brownstein
12.
DNA Viruses
Cardioviruses: Encephalomyocarditis Virus and Theiler’s Mouse Encephalomyelitis Virus
311
Howard L. Lipton, A.S. Manoj Kumar, and Shannon Hertzler 1.
Murine Cytomegalovirus and other Herpesviruses
1 Bacterial Diseases
Geoffrey R. Shellam, Alec J. Redwood, Lee M. Smith, and Shelley Gorman 13. 2.
Mouse Adenoviruses
325
Roger G. Rank
49
Katherine R. Spindler, Martin L. Moore, and Angela N. Cauthen
Chlamydial Diseases
14.
Clostridial Species
349
Kimberly S. Waggie 3.
Mousepox
67
R. Mark L. Buller and Frank Fenner
4.
Parvoviruses
15.
Enterobacteriaceae, Pseudomonas aeruginosa, and Streptobacillus moniliformis
365
Hilda Holcombe and David B. Schauer
93
Robert O. Jacoby and Lisa Ball-Goodrich 16. 5.
Polyoma Viruses
Aerobic Gram-positive Organisms
389
Cynthia Besch-Williford and Craig L. Franklin
105
Thomas L. Benjamin 17.
RNA Viruses
Helicobacter Infections in Mice
407
James G. Fox and Mark T. Whary 6.
Mouse Hepatitis Virus
141 18.
Stephen W. Barthold and Abigail L. Smith
Mycoplasma pulmonis, other Murine Mycoplasmas, and Cilia-Associated Respiratory Bacillus
437
Trenton R. Schoeb 7.
Lymphocytic Choriomeningitis Virus
179
Stephen W. Barthold and Abigail L. Smith
19.
Pasteurellaceae
469
Werner Nicklas 8.
Lactate Dehydrogenase-Elevating Virus
215 Mycotic and Parasitic Diseases
Jean-Paul Coutelier and Margo A. Brinton
9.
Reoviridae Richard L. Ward, Monica M. McNeal, Mary B. Farone, and Anthony L. Farone
235
20.
Fungal Diseases in Laboratory Mice Virginia L. Godfrey
507
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TA B L E O F C O N T E N T S
21.
Protozoa
517
3.
Katherine Wasson
22.
Helminth Parasites of Laboratory Mice
Reproductive Biology of the Laboratory Mouse
91
Kathleen R. Pritchett and Robert Taft
551
4.
Kathleen R. Pritchett
Endocrinology: Bone as a Target Tissue for Hormonal Regulation
123
Krista M. Delahunty and Wesley G. Beamer 23.
Arthropods
565 5.
David G. Baker
Hematology of the Laboratory Mouse
133
Nancy E. Everds Miscellaneous Diseases 6. 24.
The Tumor Pathology of Genetically Engineered Mice: A New Approach to Molecular Pathology 581
Spontaneous Diseases in Commonly Used Mouse Strains
171
Fred W. Quimby and Richard H. Luong
Robert D. Cardiff, Robert J. Munn, and Jose J. Galvez
25.
Clinical Chemistry of the Laboratory Mouse
Management, Techniques, and Husbandry
7. 623
Gnotobiotics
217
Richard J. Rahija
Cory Brayton 8. 26.
Zoonoses and other Human Health Hazards
Management and Design: Breeding Facilities
235
William J. White
719
Christian E. Newcomer and James G. Fox 9. Index
747
Design and Management of Research Facilities for Mice
271
Neil S. Lipman
Volume III Normative Biology, Husbandry, and Models List of Reviewers List of Contributors Foreword Preface
10.
Nutrition
321
Graham Tobin, Karla A. Stevens and Robert J. Russell x xi xiii xv
Normative Biology
11.
Health Delivery and Quality Assurance Programs for Mice
385
Diane J. Gaertner, Glen Otto and Margaret Batchelder
12.
Environmental and Equipment Monitoring
409
J. David Small and Rick Deitrich 1.
Gross Anatomy
1
Vladimír Komárek
2.
Mouse Physiology Robert F. Hoyt, Jr., James V. Hawkins, Mark B. St. Claire, and Mary B. Kennett
13.
23
Biomethodology and Surgical Techniques
437
Alison M. Hayward, Laura B. Lemke, Erin C. Bridgeford, Elizabeth J. Theve, Courtnye N. Jackson, Terrie L. Cunliffe-Beamer, and Robert P. Marini
viii 14.
TA B L E O F C O N T E N T S
In Vivo Whole-Body Imaging of the Laboratory Mouse 489 Simon R. Cherry Use of Mice in Biomedical Research
Foreword Preface
xiii xv
Overview
1
Fred W. Quimby and David D. Chaplin 15.
Behavioral Testing
513
Douglas Wahlsten and John C. Crabbe
16.
Cardiovascular Disease: Mouse Models of Atherosclerosis
1.
The Molecular Basis of Lymphoid Architecture in the Mouse
57
Carola G. Vinuesa and Matthew C. Cook 535
Nobuyo Maeda, Raymond C. Givens, and Robert L. Reddick
2.
The Biology of Toll-like Receptors in Mice
109
Osamu Takeuchi and Shizuo Akira 17.
Convulsive Disorders
565 3.
Mariana T. Todorova and Thomas N. Seyfried
Genomic Organization of the Mouse Major Histocompatibility Complex
119
Attila Kumánovics 18.
Eye Research
595
Richard S. Smith, Patsy M. Nishina, John P. Sundberg, Johann Zwaan, and Simon W.M. John
4.
Some Biological Features of Dendritic Cells in the Mouse
135
Kang Liu, Anna Charalambous, and Ralph M. Steinman 19.
Genetic Analysis of Rodent Obesity and Diabetes
617
Sally Chiu, Janis S. Fisler, and Craig H. Warden 5. 20.
Mouse Models in Aging Research
637
Kevin Flurkey, Joanne M. Currer, and D.E. Harrison
21.
Mouse Models of Inherited Human Neurodegenerative Disease 673
Mouse Skin Ectodermal Organs
155
Maria D. Iglesias-Ussel, Ziqiang Li, and Matthew D. Scharff
6.
Karl Herrup
22.
Mouse Models Revealed the Mechanisms for Somatic Hypermutation and Class Switch Recombination of Immunoglobulin Genes
Mouse Natural Killer Cells: Function and Activation
169
Francesco Colucci 691 7.
Maksim V. Plikus, John P. Sundberg, and Cheng-Ming Chuong
Cytokine-activated JAK-STAT Signaling in the Mouse Immune System 179 Bin Liu and Ke Shuai
23.
Quality Control Testing of Biologics
731 8.
William R. Shek
Signal Transduction Events Regulating Integrin Function and T Cell Migration in the Mouse
195
Lakshmi R. Nagarajan and Yoji Shimizu Index
759 9.
Volume IV Immunology List of Reviewers List of Contributors
x xi
Mouse Models of Negative Selection Troy A. Baldwin, Timothy K. Starr, and Kristin A. Hogquist
207
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TA B L E O F C O N T E N T S
10.
Peripheral Tolerance of T Cells in the Mouse
223
14.
Vigo Heissmeyer, Bogdan Tanasa, and Anjana Rao
Mouse Models to Study the Pathogenesis of Allergic Asthma
291
Chad E. Green, Nicholas J. Kenyon, Scott I. Simon, and Fu-Tong Liu 11.
The Genetics of Mouse Models of Systemic Lupus
243
Srividya Subramanian and Edward K. Wakeland 15. 12.
Inhibitory Receptors and Autoimmunity in the Mouse 261
The Mouse Trap: How Well Do Mice Model Human Immunology?
303
Javier Mestas and Christopher C.W. Hughes
Menna R. Clatworthy and Kenneth G.C. Smith Index 13.
Mouse Models of Immunodeficiency B. Anne Croy, James P. Di Santo, Marcus Manz, and Richard B. Bankert
275
313
List of Reviewers for Chapters in this Volume Baker, David G. Besselsen, David G. Brayton, Cory Brownstein, David Buchmeier, Michael Bunte, Ralph Castleman, William Clark, H. Fred Clifford, Charles B. Compton, Susan R. Conner, Margaret E. Eckhardt, Laurel Fister, Richard Foil, Lane Franklin, Craig Gardner, Murray Jacoby, Robert O. Kaltenboeck, Bernhard Kuo, Cho-chou “Ted” Lindsay, J. Russell Maggio-Price, Lillian Maronpot, Robert Murphy, Frederick A. Ramsey, Kyle Rand, Michael S. Schauer, David B. Shek, William R. Taylor, Nancy Weisbroth, Steven H.
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Louisiana State University, Baton Rouge, LA University of Arizona, Tucson, AZ Johns Hopkins University School of Medicine, Baltimore, MD University of Edinburgh, Edinburgh, UK Scripps Research Institute, La Jolla, CA University of Pennsylvania, Philadelphia, PA College of Veterinary Medicine, University of Florida, Gainesville, FL Children’s Hospital of Philadelphia, PA Charles River Laboratories, Inc., Wilmington, MA Yale University, New Haven, CT Baylor College of Medicine, Houston, TX Hunter College of CUNY, New York City, NY Charles River Laboratories, Inc., Wilmington, MA Louisiana State University, Baton Rouge, LA University of Missouri, Columbia, MO University of California, Davis, CA Yale University, New Haven, CT Auburn University, Auburn, AL University of Washington, Seattle, WA University of Alabama, Birmingham, AL University of Washington, Seattle, WA National Institute of Environmental Health Sciences, Research Triangle Park, NC University of Texas Medical Branch, Galveston, TX Midwestern University, Downer’s Grove, IL University of Arizona, Tucson, AZ Massachusetts Institute of Technology, Cambridge, MA Charles River Laboratories, Inc., Wilmington, MA Massachusetts Institute of Technology, Cambridge, MA McLean, VA
Contributors David G. Baker Division of Laboratory Animal Medicine School of Veterinary Medicine Louisiana State University Baton Rouge, LA 70803 Lisa Ball-Goodrich Section of Comparative Medicine Yale University School of Medicine New Haven, CT 06520-8016 Stephen W. Barthold Center for Comparative Medicine Schools of Medicine and Veterinary Medicine University of California Davis, CA 95616 Thomas L. Benjamin Department of Pathology Harvard Medical School Boston, MA 02115 Cynthia Besch-Williford Research Animal Diagnostic Laboratory University of Missouri, COVM Columbia, MO 65211 Cory Brayton Department of Comparative Medicine Johns Hopkins University School of Medicine Baltimore, MD 21205 Margo A. Brinton Department of Biology Georgia State University Atlanta, GA 30302 David G. Brownstein Research Animal Pathology Core Institute for Medical Cell Biology University of Edinburgh Edinburgh, Scotland EH16 4TJ
R. Mark L. Buller Department of Molecular Microbiology and Immunology St. Louis University Health Sciences Center St. Louis, MO 63104 Robert D. Cardiff Department of Pathology and Laboratory Medicine Center for Comparative Medicine University of California at Davis Davis, CA 95616 Angela N. Cauthen Department of Natural Sciences Clayton College and State University Morrow, GA 30260 Jean-Paul Coutelier Unité de Médecine Expérimentale Université Catholique de Louvain Bruxelles, Belgium Mary B. Farone Biology Department Middle Tennessee State University Murfreesboro, TN 37132 Anthony L. Farone Biology Department Middle Tennessee State University Murfreesboro, TN 37132 Frank Fenner John Curtin School of Medical Research Australian National University Canberra, Australian Capital Territory, Australia James G. Fox Division of Comparative Medicine Massachusetts Institute of Technology Cambridge, MA 02139
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CONTRIBUTORS
Craig L. Franklin Research Animal Diagnostic Laboratory University of Missouri, COVM Columbia, MO 65211
Monica M. McNeal Infectious Diseases Children’s Hospital Medical Center Cincinnati, OH 45229-3039
Jose J. Galvez Department of Pathology and Laboratory Medicine Center for Comparative Medicine University of California at Davis Davis, CA 95616
Martin L. Moore Department of Medicine Vanderbilt University Medical Center Nashville, TN 37232-2650
Virginia L. Godfrey Division of Laboratory Animal Medicine Department of Pathology and Laboratory Medicine University of North Carolina Chapel Hill, NC 27599-7115 Shelley Gorman Telethon Institute for Child Health Research Centre for Child Health Research University of Western Australia Nedlands 6907 Western Australia Shannon Hertzler Department of Neurology Evanston Hospital Northwestern University Evanston, IL 60201 Hilda Holcombe Division of Comparative Medicine Massachusetts Institute of Technology Cambridge, MA 02139 Robert O. Jacoby Section of Comparative Medicine Yale University School of Medicine New Haven, CT 06520-8016 A.S. Manoj Kumar Department of Neurology Microbiology-Immunology, and Biochemistry Molecular and Cell Biology Evanston Hospital Northwestern University, Evanston, IL 60201 Howard L. Lipton Department of Neurology Microbiology-Immunology, and Biochemistry Molecular and Cell Biology Evanston Hospital Northwestern University, Evanston, IL 60201
Herbert C. Morse, III Laboratory of Immunopathy National Institute of Allergy and Infectious Diseases NIH Rockville, MD 20852 Robert J. Munn Center for Comparative Medicine University of California at Davis Davis, CA 95616 Christian E. Newcomer Research Animal Resources and Department of Molecular and Comparative Pathobiology Johns Hopkins University Baltimore, MD 21205 Werner Nicklas Central Animal Laboratories German Cancer Research Centre D-69120 Heidelberg Germany Kathleen R. Pritchett Charles River Laboratories Research Models and Services Domaine des Oncins l′Arbresle, Lyon 69592 France Roger G. Rank Department of Microbiology and Immunology University of Arkansas for Medical Sciences Little Rock, AR 72205 Alec J. Redwood Department of Microbiology University of Western Australia Nedlands 16907 Western Australia David B. Schauer Division of Comparative Medicine Massachusetts Institute of Technology Cambridge, MA 02139
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CONTRIBUTORS
Trenton R. Schoeb Department of Genetics Division of Genomics University of Alabama at Birmingham Birmingham, AL 35294-0024 Geoffrey R. Shellam Department of Microbiology University of Western Australia Nedlands 6907 Western Australia Lee M. Smith Department of Microbiology University of Western Australia Nedlands 6907 Western Australia Abigail L. Smith School of Veterinary Medicine University of Pennsylvania Philadelphia, PA 19104 Katherine R. Spindler Microbiology and Immunology University of Michigan School of Medicine Ann Arbor, MI 49109-0620
Kimberly S. Waggie Preclinical Development ZymoGenetics Inc. Seattle, WA 98102 Richard L. Ward Infectious Diseases Children’s Hospital Medical Center Cincinnati, OH 45229-3039 Katherine Wasson Center for Comparative Medicine University of California at Davis Davis, CA 95616 Mark T. Whary Division of Comparative Medicine Massachusetts Institute of Technology Cambridge, MA 02139
Foreword for Volume II The second edition of The Mouse in Biomedical Research reflects the revolution in mouse biology inaugurated during the past quarter century. It is exemplified by the heavy flavoring of genetics in Volume I and the focus of Volume IV on immunology. These trends re-emphasize the indispensability of mice for biomedical research and the influence of genetic engineering in driving it. Setting mouse genes in motion through molecular wizardry has accelerated understanding of mammalian biology and disease at a stunning pace. It also has provoked domination of mouse populations by novel strains which are scientifically and financially among biomedicine’s most precious assets. Therefore, the health and, implicitly, the diseases of laboratory mice are transcendent issues for investigators and laboratory animal medicine specialists. The roster of health risks for mice also has evolved significantly since the original edition of this text. Improvements in housing, husbandry and health care have reduced the impact of traditional infections, such as mycoplasmosis, viral pneumonias and enteridites, acariasis and endoparasitism. However, they remain, although weakened, incompletely dispatched and, therefore, worthy subjects for this text. Additionally, specific pathogen-free mice, which constitute the lion’s share of contemporary mousedom, are, by definition, highly susceptible to these adventitious infections. Of equal or greater importance, historically notorious agents have been superceded epidemiologically by helicobacters and recently recognized parvoviruses, both of which carry the means for serious disruption of research. Awareness of “new” agents is illustrated further by contemporary investigations of norovirus infection in mice, which are gathering interest as this edition goes to press, and thus too late for formal inclusion. (See Wobus, C.E. et al. 2006, J. Virol., 80, 5104–5112). Lastly, current understanding of agent-host interactions and their
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effects on health and research is based largely on responses of standard stocks and strains. Serendipitous phenotypes resulting from genetic engineering are likely to change these interactions in ways that are currently hard to predict. Fundamental information on pathogenesis and related factors, provided here, are essential to detect and assess divergent outcomes. It is no surprise, in these contexts, that infectious agents continue dominate contemporary murine health concerns and this volume. The potential effects of genetic engineering on the expression and prevalence of non-infectious diseases are illustrated by chapters on tumor pathology and spontaneous diseases. They also highlight the value of phenotyping for murine health. While researchers often seek categorical effects (ie predicted or desired mode of gene expression on a given cell type, tissue or organ), erudite phenotyping is the sentinel for unexpected or undesirable lesions which may compromise potentially novel models. The text also shows that mouse medicine has matured into a highly sophisticated discipline during the past 25 years. Reagents and methods for the early and accurate detection and diagnosis of disease are arguably the most visible vanguards of this advance. Nevertheless, the second edition also illustrates that collaborations with geneticists, embryologists, immunologists, epidemiologists and other scientific colleagues is developing a new and improved generation of health care paradigms and the laboratory animal experts to deliver them.
ROBERT O. JACOBY, DVM, PhD NEW HAVEN, CONNECTICUT
Preface The American College of Laboratory Animal Medicine (ACLAM) was formed in 1957 in response to the need for specialists in laboratory animal medicine. The college has promoted high standards for laboratory animal medicine by providing a structured framework to achieve certification for professional competency and by stressing the need for scientific inquiry and exchange via progressive continuing education programs. The first edition of “The Mouse in Biomedical Research” consisting of four volumes, and published in 19811983 was a part of the College’s effort to fulfill those goals. It is one of a series of comprehensive texts on laboratory animals developed by ACLAM over the past three decades: “The Biology of the Laboratory Rabbit” was published in 1974, “The Biology of The Guinea Pig” in 1976 and a twovolume work “Biology of The Laboratory Rat” in 1979 and 1980. Also, in 1979 the College published a two-volume text on “Spontaneous Animal Models of Human Disease”. In 1984 the first edition of “Laboratory Animal Medicine” appeared in print followed by “Laboratory Hamsters” in 1987. The second edition of The Biology of the Laboratory Rabbit was published in 1994. A two-volume treatise on “Nonhuman Primates in Biomedical Research” was published in 1995 and 1998. A text “Anesthesia and Analgesia in Laboratory Animals” was published in 1997 followed by the second edition of “Laboratory Animal Medicine” in 2002. Most recently, the second edition of “The Laboratory Rat” was published in 2005. The estimated annual use of 100 million-plus mice worldwide attests to the importance of the mouse in experimental research. The introduction of genetically engineered mice has only increased the usefulness of the mouse model in biomedical research. In no other species of animal has such a wealth of experimental data been utilized for scientific pursuits. Knowledge of the mouse that has been accumulated is, for the most part, scattered throughout a multitude of journals, monographs and symposia. It has been 25 years since the publication of the first edition of the “Mouse in Biomedical Research”. The intent of this second edition is to build upon the framework of the first edition, rather than simply to update and duplicate the earlier effort. The intended purpose of this text is to assemble established scientific data emphasizing recent information on the biology and use of the laboratory mouse. Separation of the material into multiple volumes was essential because of the number of
subject areas covered. The four volumes consist of 80 chapters coauthored by 167 scientists. The information in Volume 1 serves as a primer for scientists new to the field of mouse research. It provides information about the history, basic biology and genomics of the laboratory mouse (Mus musculus), as well as basic information on maintenance and use of mouse stocks. Mouse origins and relationships are covered in chapters on history, evolutionary taxonomy and wild mice. Genetics and genomics of the mouse are covered in chapters on genetic nomenclature, gene mapping, cytogenetics and the molecular organization of the mouse genome. Maintenance of laboratory mice is described in chapters on breeding systems for various types of strains and stocks and genetic monitoring. Use of the mouse as a model system for basic biomedical research is described in chapters on chemical mutagenesis, gene trapping, gene therapy, pharmacogenetics and embryo manipulation. Volume 2 entitled Diseases departs from the first edition of the same title by discussing specific disease-causing microorganisms, whereas the first edition discussed infectious diseases affecting specific organs and tissues. This volume consists of 26 chapters subdivided into RNA viruses and DNA viruses, as well as bacterial, mycotic and parasitic infections. These chapters not only provide updates on pathogenesis, epidemiology and prevention of previously recognized murine pathogens, but also include chapters on newly recognized disease-causing organisms: mouse parvovirus, cilia-associated respiratory bacilli and Helicobacter spp. A separate category, consisting of 3 chapters, discusses zoonoses, tumor pathology of genetically engineered mice and spontaneous diseases in commonly used mouse strains. Volume 3 encompasses 23 chapters whose contents provide a broad overview on the laboratory mouse’s normative biology, husbandry and its use as a model in biomedical research. This consists of chapters on behavior, physiology, reproductive physiology, anatomy, endocrinology, hematology and clinical chemistry. Other chapters cover management, as well as nutrition, gnotobiotics and disease surveillance. Individual chapters describe the mouse as a model for the study of aging, eye research, neurodegenerative diseases, convulsive disorders, diabetes and cardiovascular and skin diseases. Chapters on imaging, surgical and other research techniques and the use of the mouse in assays of biological products also are included.
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xvi Volume 4 is a completely new addition to this series, dedicated to mouse immunology. It is based on the vast body of knowledge which has made the mouse the model of choice when studying immunity in human beings. Arguably more is known about the immune system in mice than any other species except human. In large part this is due to the power of genetic engineering to delineate molecular mechanisms. This volume includes an overview of mouse immunology, including both the innate and adaptive immune systems, followed by 15 chapters (mini-reviews), each dealing with a specific area of immunology. The overview addresses broad concepts concerning molecular and cellular immunology and cites both current references and the appropriate chapter, for more detailed information, from the mini-reviews which follow. The 15 chapters illustrate the power of genetic engineering in dissecting each component of the immune response from the development of lymphoid tissues to signal transduction pathways in activated cells. Individual chapters address: The Genomic Organization of the MHC, Toll-like Receptors, The Molecular Basis of Lymphoid Architecture, The Biology of Dendritic Cells, Somatic Hypermutation and Class Switching, Natural Killer Cell Function and Activation, Cytokine Mediated Signaling, Signal Transduction Events Regulating Integrin Function and T-Cell Migration, Central Tolerance in T-Cells, Peripheral Tolerance in T-cells, Inhibitory Receptors and Autoimmunity. The volume also includes the use of mice in studies of Systemic Autoimmunity, Immunodeficiency, Allergic Airway Inflammation and the Differences Between Mouse and Human Immunology. This treatise was conceived with the intent to offer information suitable to a wide cross section of the scientific community. It is hoped that the four volumes will serve as a standard reference source for scientists using mice in biomedical research. Students embarking on scientific careers also will benefit from the broad coverage of material presented in compendium format. Certainly, specialists in laboratory animal
P R E FA C E
science will benefit from these volumes; technicians in both animal care and research will find topics on surgical techniques, management and environmental monitoring of particular value. The editors wish to extend special appreciation to the contributors to these volumes. Authors were selected because of knowledge and expertise in their respective fields. Each individual contributed his or her time, expertise and considerable effort to compile this resource treatise. In addition, the contributors and editors of this book, as with all volumes of the ACLAM series texts, have donated publication royalties to the American College of Laboratory Animal Medicine for the purpose of continuing education in laboratory animal science and comparative medicine. This book could not have been completed without the full support and resources of the editors’ parent institutions which allowed us the time and freedom to assemble this text. A special thanks is also extended to the numerous reviewers of the edited work whose suggestions helped the authors and editors present the material in a meaningful and concise manner. We also thank the editorial staff of Elsevier for their assistance. Finally, we especially acknowledge with deep appreciation the editorial assistance of Lucille Wilhelm, whose dedication and tireless commitment, as well as good humor, throughout this project were of immeasurable benefit to the editors in the completion of this text.
JAMES G. FOX STEPHEN W. BARTHOLD MURIEL T. DAVISSON CHRISTIAN E. NEWCOMER FRED W. QUIMBY ABIGAIL L. SMITH
Chapter 1 Murine Cytomegalovirus and Other Herpesviruses Geoffrey R. Shellam, Alec J. Redwood, Lee M. Smith, and Shelley Gorman
I. Introduction and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Properties of the Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Virion Structure and Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Virion Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Replication of MCMV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Virus Strains: Antigenic and Genetic Relationships . . . . . . . . . . . . . . . III. Growth In Vitro and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. In Vitro Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Propagation in Permissive Murine Cells . . . . . . . . . . . . . . . . . . . . . 2. Nonpermissive Murine Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Non-Murine Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Features of MCMV Replication In Vitro . . . . . . . . . . . . . . . . . . . . . B. Infection of Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Natural History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Natural Infection of Laboratory Mice . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mode of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Host Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Effect of Age, Dose, and Route of Inoculation . . . . . . . . . . . . . . . . . . . 1. Age and Dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Route of Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Genetic Control of Host Resistance or Susceptibility to MCMV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Resistance to Acute Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mechanisms of Host Resistance to Acute Infection In Vivo . . . . . . 3. Mouse Strain Variation in the Resolution of Chronic MCMV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. In Vitro Studies of Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Latency and Reactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
D. Suitability of MCMV as a Model of HCMV Infection and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Intrauterine Infection and Congenital Disease . . . . . . . . . . . . . . . . . 2. Interstitial Pneumonitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Cardiovascular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Adrenalitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Infection of the Central Nervous System . . . . . . . . . . . . . . . . . . . . . 7. Retinitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Effect on the Developing Ear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Effects on Hemopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. The Immune Response to MCMV Infection . . . . . . . . . . . . . . . . . . . . . . . . 1. Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Macrophages and Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Production of Interferon and Other Cytokines . . . . . . . . . . . . . . . . . 4. Natural Killer (NK) Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. B Cell–Mediated Immune Responses . . . . . . . . . . . . . . . . . . . . . . . 6. T Cell–Mediated Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . 7. Immune Evasion by MCMV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Molecular Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Development of Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Mouse Thymic Virus: Mouse T Lymphotrophic Virus (MTLV) or Murid Herpesvirus 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties of the Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Pathogenesis and Cell Tropism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION AND HISTORY
Murine cytomegalovirus (MCMV) is a very well studied virus of laboratory mice. Together with the cytomegaloviruses of the rat, guinea pig, human, and other species, MCMV belongs to the b herpesvirinae subfamily of the Herpesviridae. The CMVs exhibit distinctive features that include specificity for their natural host species and the ability to establish persistent and latent infections, which are generally asymptomatic in the immunocompetent host. The discovery of the cytomegaloviruses had its origins in early studies of the etiology of a distinctive cytopathology associated with intranuclear inclusions and cellular enlargement. These cellular changes, which were termed cytomegalia (Goodpasture and Talbot 1921), were observed in the tissues of humans (Jesionek and Kiolemenoglou 1904), guinea pigs (Jackson 1920), rats (Thompson 1932), mice (Findlay 1932; Thompson 1934), and other species. The viral nature of the causative agent was first suggested by experiments in guinea pigs (Cole and Kuttner 1926), in which the inoculation of filtered homogenates of submaxillary glands induced cellular inclusions in the recipients. Murine cytomegalovirus was first isolated in tissue culture in 1954 by Margaret Smith from the salivary gland tissue of
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infected laboratory mice (Smith 1954). This followed earlier demonstrations that the etiological agent of intranuclear inclusion bodies in murine tissues could be transmitted by the inoculation of tissue homogenates into healthy mice (Kuttner and Wang 1934; McCordick and Smith 1936). Human cytomegalovirus (HCMV) was confirmed as the viral agent responsible for human salivary gland disease following its isolation in human cells in vitro (Rowe et al. 1956; Smith 1956; Weller et al. 1957). Subsequently, guinea pig cytomegalovirus (Hartley et al. 1957) and rat cytomegalovirus (RCMV) (Bruggeman et al. 1982) were isolated. The term cytomegalovirus was introduced by Weller and colleagues (Weller et al. 1960). Over the last 20 years, murine cytomegalovirus has become one of the best-studied viral infections of laboratory mice and is the subject of a very extensive literature. There are several reasons for this. First, MCMV research has benefited from the similarities between the diseases caused by human CMV (HCMV) and MCMV in their respective host species. There has been an increased awareness of the importance of HCMV-associated diseases in recipients of solid organ or bone marrow transplants and in HIV/AIDS, where HCMV is a very important cause
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of morbidity and mortality. The role of HCMV in inducing immunopathological diseases such as pneumonitis, retinitis, adrenalitis, and atherosclerosis has received much attention. Because of the strict species-specificity of the cytomegaloviruses, HCMV cannot be studied experimentally in animal models, and increasingly MCMV infection of mice has been used as a model of HCMV-associated diseases in humans. Second, there has been strong interest in how MCMV engages with the immune system. While T cell responses have been shown to be responsible for recovery from MCMV infection, the innate immune system, especially natural killer (NK) cells, has been found to be very important in controlling the extent of MCMV infection in mice. Research in this area has provided fascinating insights into how NK cells interact with viral pathogens. A host gene that controls resistance to MCMV in mice, Cmv1, has been identified and shown to encode a receptor molecule on NK cells. Interestingly, despite the presence of strong innate and adaptive immune responses, MCMV infection persists. This is due in part to virus-encoded proteins that assist the virus to evade the host response, particularly cell-mediated responses. MCMV has become one of the best-studied viruses for understanding the function of these immune evasion proteins. Finally, the strong growth of molecular virology over the last 20 years has significantly influenced the direction of research on MCMV. The genomes of MCMV and several other CMVs have been sequenced, and this information has been enormously valuable for understanding gene function, the relatedness between CMVs, and their evolution. The ability to clone the MCMV genome into a bacterial artificial chromosome has greatly facilitated the production of mutant viruses for the study of gene function, and the ease with which this can be studied in vivo in mice has been of great benefit. In all these areas of progress there has been one common feature that has contributed to the popularity of this virus model. This is that MCMV is a natural pathogen of its host species, the house mouse, infecting both laboratory mice and free-living wild Mus musculus domesticus. As with herpesviruses in general, which are believed to have co-evolved with their particular host species (Karlin et al. 1994; McGeoch and Cook 1994; McGeoch et al. 1995), the infection of mice by MCMV is considered to be a highly evolved host-parasite relationship that is free of the bias introduced by using a virus in its unnatural host. New studies, which are exploring the mutual adaptations made by MCMV and its natural host, will provide an important direction for research over the next few years. Given the size of the literature concerning MCMV, the reader may wish to consult other reviews that deal with the history, immunobiology, or pathogenesis of this virus in more detail than is possible here (Lussier 1975b; Hudson 1979; Osborn 1982; Staczek 1990; Hudson 1994a; Price and Olver 1996; Sweet 1999; Reddehase et al. 2002; Vink et al. 2001;
Gutermann et al. 2002; Scalzo 2002; Lee et al. 2002; Fairweather et al. 2001).
II.
PROPERTIES OF THE VIRUS A.
Classification
The cytomegaloviruses are large, enveloped, doublestranded DNA viruses with an icosahedral capsid that belong to the Betaherpesvirinae subfamily of the family Herpesviridae (van Regenmortel et al. 2000). There are three genera. The genus Cytomegalovirus contains human cytomegalovirus (Human herpesvirus 5). Muromegalovirus contains mouse cytomegalovirus (Murid herpesvirus 1, MuHV-1), which is also the type species, and rat cytomegalovirus (Murid herpesvirus 2, MuHV-2). The genus Roseolovirus contains Human herpesvirus 6 (HHV-6). The general characteristics of the cytomegaloviruses include strict species specificity, an ability to induce cytomegalia in infected cells, cell-associated replication in cell culture, a slow replicative cycle, and the establishment of persistent and latent infection in the natural host. Infection is generally asymptomatic unless the host is immunosuppressed or has an immature immune system. In these situations, infection may result in morbidity or even mortality. The house mouse, Mus domesticus, is considered to be the natural host for MCMV. Because of its common usage, the term murine cytomegalovirus (MCMV) rather than Murid herpesvirus 1 will be used throughout this chapter. Apart from mouse thymic virus (Murid herpesvirus 3), which is described elsewhere in this chapter, other herpesviruses of rodents have been discovered. One of them, mouse herpesvirus strain 68 (MHV-68), was isolated from bank voles in Slovakia (Blaskovic et al. 1980). This virus, which is also known as Murid herpesvirus 4, has been assigned to the Rhadinovirus genus of the Gammaherpesvirinae subfamily of the Herpesviridae (van Regenmortel et al. 2000). However, it appears that MHV-68 is not a natural pathogen of house mice. Although MHV-68 exhibits the features of a herpesvirus infection when it is deliberately inoculated into laboratory mice, including a productive infection in the lungs (Sunil-Chandra et al. 1992) and the establishment of persistence and latency (Stewart et al. 1998; Flano et al. 2000), there have been no reports of natural infection of colonies of laboratory mice with MHV-68. Recent serological evidence also indicates that free-living wild house mice are not naturally infected with MHV-68 (J. Stewart, personal communication). In contrast, MHV-68 appears to be endemic in free-living wood mice (Apodemus sylvaticus) in the United Kingdom although, interestingly, evidence of infection was only rarely found in voles in this study (Blasdell et al. 2003). Other studies from Slovakia
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GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
have reported a sero-prevalence of 1%–12% in wild rodents depending on the locality (Kozuch et al. 1993; Mistrikova and Blaskovic 1985), but the prevalence in particular rodent species was not recorded. As a naturally occurring rodent gammaherpesvirus that induces persistent and latent infection when inoculated into inbred laboratory mice, MHV-68 has received considerable attention in recent years for its potential as a model of human gammaherpesvirus infection. The immunobiology of MHV-68 has been widely studied in inbred mice, and there is a large body of literature. The viral genome has been sequenced (Virgin et al. 1997). Nonetheless, MHV-68 will not be discussed further in this chapter because the house mouse does not appear to be the natural host species. MHV-68 has been the subject of a number of comprehensive reviews to which the reader is referred (Nash et al. 2001; Doherty et al. 2001; Blackman et al. 2000; Stevenson et al. 2002; Stewart 1999; Virgin and Speck 1999). The Tupaia herpesvirus (THV) of tree shrews (Mirkovic et al. 1970; McCombs et al. 1971) has been sequenced (Baker and Darai 2001) and has been found to resemble MCMV and other β herpesviruses. However, it is not a virus of wild or laboratory mice and will not be considered further here. Finally, a cytomegalovirus has been isolated from deer mice (Peromyscus maniculatas) in North America (Rizvanov et al. 2003). The virus, which has been designated Peromyscus cytomegalovirus (PCMV), has been characterized as a cytomegalovirus based on physical and biological properties and genetic homology with several genes of other CMVs (Rizvanov et al. 2006). It does not replicate in mouse 3T3 cells, and it is not known whether it infects laboratory mice or free living Mus species.
B. 1.
Virion Structure and Replication
Virion Structure
Herpesvirus virions are spherical and comprise four morphologically distinct elements: the core, capsid, tegument, and envelope. The core encompasses the dsDNA viral genome, which is packaged as a single linear molecule into the protein capsid that exhibits icosahedral symmetry. The tegument is a poorly defined layer of proteinaceous material between the capsid and envelope which may be equivalent to the matrix of other viruses. It contains a number of proteins. The envelope is a lipid bilayer that contains a number of different integral viral glycoproteins (van Regenmortel et al. 2000). The virions of the cytomegaloviruses, including MCMV, share these distinctive features. However, while the diameter of single virions of MCMV is approximately 230 nM, the virions are quite pleiomorphic and may include a high proportion of multicapsid virions containing a number of capsids enclosed within a common membrane (Hudson et al. 1976b). The production of multicapsid virions appears to be an unusual property of MCMV.
The genomes of cytomegaloviruses are significantly larger than those of other herpesviruses, being over 200 kbp in size (Mocarski and Courcelle 2001a). The genome of the Smith strain of MCMV (ATCC VR-194) is 230, 278bp (Rawlinson et al. 1996), which is very similar in size to the genomes of HCMV and RCMV (Chee et al. 1990; Vink et al. 2000). The MCMV genome was originally described as encoding 170 open reading frames (Rawlinson et al. 1996). However, since this time, several new spliced genes have been identified within the genome (Ciocco-Schmitt et al. 2002; Loewendorf et al. 2004; Scalzo et al. 2004). Using comparative genomics techniques and new gene predicting algorithmns, the coding potential of human CMV has been reinterpreted recently (Davison et al. 2003; Murphy, Rigoutsos, et al. 2003 Murphy, Yu, et al. 2003), and using similar approaches, a further 34 open reading frames have been predicted to exist within the MCMV genome (Brocchieri et al. 2005). The MCMV genome comprises a single unique sequence with short terminal direct repeats and several short internal repeats (Ebeling et al. 1983; Mercer et al. 1983; Rawlinson et al. 1996). Unlike HCMV, the linear genome of MCMV does not have an isomeric structure because it lacks the larger terminal or internal repeat sequences (Ebeling et al. 1983; Mercer et al. 1983). The nomenclature devised for naming MCMV genes numbers the genes from the 5′ to the 3′ end of the genome. MCMV genes with homologs in HCMV are assigned the uppercase prefix M, while genes with no sequence identity with HCMV genes are identified by the lowercase prefix m (Rawlinson et al. 1996). The MCMV and HCMV genomes are co-linear over the central 180 kb, and there is significant similarity to the HCMV genome, especially for 78 open reading frames located in the central region of the genome (Rawlinson et al. 1996). MCMV shares with other members of the herpesvirus family a number of evolutionarily conserved proteins that are involved in processes such as DNA replication and virion maturation and structure. However, there are two regions of the genome that contain genes unique to MCMV. These are the m02 gene family at the left-hand end and the m145 gene family at the right-hand end of the genome. Many of the genes in these unique regions encode immune evasion proteins (Rawlinson et al. 1996). The capsid that encloses the viral genome is composed of 162 capsomers that exhibit icosahedral symmetry. Much of the information available on MCMV capsid proteins is inferred from their homology with the proteins of HCMV. The HCMV capsid proteins are also homologous to those found in the alphaherpesvirus, herpes simplex virus (HSV). The capsids of CMVs are larger and incorporate a larger genome than other herpesviruses. The capsid of MCMV is composed of seven proteins. The major capsid protein (MCP), the minor capsid protein, the minor capsid binding protein, and the smallest capsid protein are encoded by the MCMV genes M86, M85, M46, and m48.5, respectively. There are also three distinct assemblin-related proteins, which are encoded by M80, M80a,
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
and M80.5 and associate with capsids (Baldick and Shenk 1996; Gibson 1996; Gibson, Baxter, et al. 1996; Gibson, Clopper et al. 1996; Chen et al. 1999). The MCP is the most strongly conserved protein among herpesviruses. The tegument of HCMV is composed of up to of 25 proteins, many of which are phosphorylated and have the prefix pp. By electron microscopy, the tegument is seen to have an ordered structure, particularly proximal to the capsid. The genes encoding the tegument proteins of HCMV have homologues in MCMV. These include M32 (pp150), M48 (large tegument protein) M83 (pp65), M99, and M82 (Rawlinson et al. 1996). Several transcriptional transactivators such as M82 (pp71) have also been localized to the tegument. However, the function of most tegument proteins remains undefined. Individual tegument proteins are conserved in the b herpesvirinae but are not shared with members of the α or γ herpesvirinae. The envelope of MCMV is composed predominantly of lipids obtained from the intracellular membranes of the host cell. The CMV envelope also contains a considerable number of viral encoded glycoproteins, which exceed the number of envelope glycoproteins found in other viruses. It is more pleiomorphic than the envelope of other herpesviruses, and is a distinguishing feature of the virus when observed by electron microscopy. The major glycoprotein is the highly conserved glycoprotein B (gB), which is a dominant B-cell antigen in CMV-infected animals and humans. It has been found in every herpesvirus and is one of the most highly conserved herpesvirus proteins. The amino acid homology between MCMV and HCMV gB is 45% (Rapp et al. 1992). The herpes virus gB is essential for viral entry into the cell (Little et al. 1981). Additional MCMV glycoproteins gL, gH, and gO (encoded by M115, M75, and m74 respectively) closely resemble their HCMV counterparts, which form the heterotrimeric complex gC111 (Huber and Compton 1998). This complex is essential for the entry of HCMV. 2.
Replication of MCMV
The life cycle of MCMV is initiated when virions bind to receptors on susceptible cells and enter the cytoplasm, where the viral envelope is removed and the capsid is transported to the nucleus. VIRAL ENTRY The cellular receptors used by MCMV have not been demonstrated conclusively. However, more than one receptor is likely to be involved. A role for MHC class I molecules (Wykes et al. 1993) and beta-2-microglobulin (Wykes et al. 1992) in facilitating the entry of MCMV has been demonstrated in vitro, although a significant role for MHC class I molecules was not confirmed by others in vitro or in vivo (Polic et al. 1996; Tay et al. 1995). In addition, heparan sulfate proteoglycans contribute to MCMV binding and entry (Price et al. 1995). These molecules also contribute to the binding of HCMV to target cells (Compton et al. 1993). HCMV uses the epidermal growth factor receptor (EGFR) (Wang et al. 2003) and integrin αvβ3 (Wang et al. 2005) as
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co-receptors for viral entry, using HCMV gB and gH, respectively. Integrin αvβ1 is also a co-receptor for HCMV (Feire et al. 2004). The binding of HCMV to these molecules initiates translocation of integrin αvβ3 into lipid rafts in the cell membrane. This results in interaction with EGFR and coordinated signaling, which triggers potent cooperative effects in target cells that appear to be necessary for successful viral infection (Wang et al. 2005). The role of these molecules as possible co-receptors in MCMV infection is yet to be determined. Following viral attachment to cell surface molecules, the HCMV envelope fuses with the cell surface in a pH-independent manner (Compton et al. 1992), involving the HCMV gCIII heterotrimeric complex (Keay and Baldwin et al. 1991; Huber and Compton 1998). It is assumed that MCMV enters cells in a similar manner. In support of this, the deletion of MCMV gL prevents initiation of MCMV infection (J. Allan, personal communication). Viral nucleocapsids then enter the cytoplasm and are transported through nuclear pores to the nucleus. The transcription of MCMV genes, the replication of viral DNA, and the subsequent assembly of capsids occurs in the nucleus of infected cells. REPLICATION OF VIRAL DNA MCMV DNA replication appears to follow the same sequence of events that occurs in the replication of other herpesviruses, via circular and/or concatermeric intermediates (Marks and Spector 1988). The sequences at the termini of the genome fuse via a 3′ nucleotide extension to form the intermediates for MCMV replication, by a rollingcircle model (Marks and Spector 1988). MCMV genomes circularize as soon as 2 hr after infection (Marks and Spector 1988). Host DNA synthesis is inhibited by more than 95% by 10–12 hr post infection (p.i.) (Moon et al. 1976), and the onset of viral DNA synthesis in mouse embryo fibroblasts (MEF) occurs during 8–16 hr p.i. depending on the mitotic phase of the host cells (Misra et al. 1978; Moon et al. 1976; Muller and Hudson 1977; Muller et al. 1978). By 22 hr p.i. the equivalent of 900 viral genomes per cell have been synthesized (Misra et al. 1977). The origin of replication of MCMV is known as the oriLyt replicator gene region, which extends over 1.7 kb (Masse et al. 1997). The oriLyt region in MCMV is extremely rich in repeat sequences that act as binding elements for various transcription factors. MCMV-encoded proteins that are required for origindependent replication include the DNA polymerase (M54), a polymerase accessory protein (M44), the single-stranded DNA binding protein (M57), and a helicase-primase complex of the viral proteins M70, M102, and M105 (Elliott et al. 1991, Rawlinson et al. 1996). The maturation of the MCMV genome involves the processing and cleavage of newly synthesized concatermeric viral DNA into genome-length monomers, prior to packaging into preformed nucleocapsids in the cell nucleus. Herpesvirus-conserved pac1 and pac2 DNA sequence motifs are required for cleavage and packaging of the MCMV genome (McVoy et al. 1998).
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GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
REGULATION OF MCMV GENE EXPRESSION Gene expression is temporally controlled in a cascade manner. All betaherpesviruses (Honess and Roizman 1974) including MCMV (Keil et al. 1984; Misra et al. 1978) have three gene families, α, β, and γ, that are temporally regulated. These genes are expressed in the immediate early (IE), early (E), and late (L) phases of viral replication, respectively. The IE phase occurs immediately after viral DNA enters the nucleus and is controlled by the major IE promoter (MIEP) in MCMV (Dorsch-Hasler et al. 1985) or HCMV (Boshart et al. 1985). The IE genes do not require de novo protein synthesis, and their transcription is not inhibited by protein inhibitors such as cyclohexamide (Chantler and Hudson 1978). In MCMV, the MIEP controls expression of the ie1 (m123) and ie3 (M122) genes (Keil et al. 1987a,b). MCMV ie1 (pp89) and ie3 are transcriptional activators (Koszinowski et al. 1986; Messerle et al. 1992). The ie3 gene is essential for MCMV replication (Angulo et al. 2000). A third IE gene, ie2, not present in HCMV (Chee et al. 1990), is transcribed from a different promoter and in the opposite direction (Keil et al. 1987a). The ie2 gene is not essential for MCMV replication either in vitro or in vivo in the situations that have been examined so far (Manning and Mocarski 1988; Cardin et al. 1995). While IE gene expression is confined to discrete regions of the genome, early gene transcripts come from various regions of the genome (Marks et al. 1983; Keil et al. 1984). Entry into the E phase is dependent on de novo protein synthesis and is therefore inhibited by cyclohexamide. There is an absolute requirement for IE gene expression prior to E gene expression (Honess and Roizman 1974), while E gene expression can be regulated by proteins derived from IE genes or from other E genes (Buhler et al. 1990). Genes transcribed during the E phase of viral replication include those required for entry into the L phase of viral replication, as well as other genes, such as the immune evasion genes (Ziegler et al. 1997). Entry into the L phase of viral replication requires DNA synthesis and is consequently inhibited by phosphonoacetic acid (Misra et al. 1977; Chantler and Hudson 1978). MCMV DNA replication requires proteins synthesized during the E phase of replication and occurs in the nucleus. The L phase of viral replication occurs approximately 16 hours after infection (Moon et al. 1976; Keil et al. 1984). However, both viral DNA replication and the kinetics of virus production are likely to be dependent on the cell culture system or cell type studied (Misra and Hudson 1977; Andrews et al. 2001). L phase genes mostly encode structural proteins. MORPHOGENESIS OF MCMV Along with viral gene transcription and DNA replication, the formation of capsids and the packaging of viral DNA occurs in the nucleus of infected cells (reviewed by Gibson 1996). Capsid proteins are produced in the cytoplasm and are transported back to the nucleus across the nuclear membrane. The transport of capsid proteins is due either to their small size, which allows passage across the nuclear pore
complex, or to the presence of a nuclear localization signal. Large capsid proteins such as MCP, that do not contain nuclear localization signals are transported in association with those that do (Wood et al. 1997; Plafker and Gibson 1998). Viral DNA is packaged into complete capsids and transported to the cytoplasm via the nuclear membrane, where the capsids acquire their primary envelope as they bud through the membranes. The two conserved herpesvirus genes UL31 (Chang and Roizman 1993; Reynolds et al. 2001; Fuchs et al. 2002) and UL34 (Purves et al. 1992; Klupp et al. 2000; Reynolds et al. 2001) are involved in this process, and loss of these genes impairs primary envelopment of HSV-1 and pseudorabies virus (PrV) (reviewed in Mettenleiter 2002). In MCMV infection, the virus penetrates the nuclear membrane with the aid of M50/p35 (a homologue of UL34) and a partner MCMV gene M53/p38 (a homologue of UL31). M50/M53 recruit cellular kinases to the inner nuclear membrane, specifically to the nuclear lamina. The nuclear lamina is a filamentous network that prevents budding. Recruitment of cellular kinases results in phosphorylation and degradation of the lamina (Muranyi et al. 2002), and facilitates egress of the virus from the nucleus. The mechanism of egress of herpesviruses from infected cells remains incompletely understood. The envelope and tegument of perinuclear virions differ from those of mature virions (Gershon et al. 1994; Granzow et al. 1997). In addition, the phospholipid content of mature HSV-1 virions differs from that of the nuclear membrane (van Genderen et al. 1994). Finally, the glycoproteins found in the mature virion that are responsible for viral entry into new host cells are not required for primary envelopment (Granzow et al. 2001). The synthesis and processing pathways of the major envelope glycoprotein complex (gp52/105/150) of MCMV have been characterized (Loh 1991). Ultrastructural differences are also apparent between primary enveloped virions and mature virions. These data suggest a secondary envelopment; however, the exact mechanisms by which the virus loses the primary envelope and tegument and acquires mature tegument and envelope has not been fully elucidated. Several models of herpesvirus egress have been proposed (reviewed by Mettenleiter 2002). However, it is suggested that secondary envelopment of virions is preceded by de-envelopment in the cytoplasm. Recent studies with α- and β- herpesviruses suggest that the primary enveloped virus migrates to the rough endoplasmic reticulum that is contiguous with the nuclear membrane, and exits into the cytoplasm, losing its primary envelope (reviewed by Mettenleiter 2002). The virus then migrates to an intracellular compartment, possibly the trans Golgi network, where the virions gain their tegument and final envelope, complete with glycoproteins (Gershon et al. 1994; Mettenleiter 2002; Mettenleiter 2004). The mature enveloped virions are now present in secretory vesicles that are transported to the plasma membrane, where they are released into the extracellular space by exocytosis. Little is known of this process, although UL20 and gK from HSV-1 and PrV have been implicated (reviewed in Mettenleiter 2004).
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
The formation of multicapsid virions, which appears to be unique to MCMV infection (see IIIA, 4), is also not well understood. It has been postulated (Weiland et al. 1986) that they are formed when capsids exit the nucleus via nuclear pores and enter cytoplasmic aggregates of capsids. These capsids then receive an envelope via budding into extended cytoplasmic vacuoles derived from the Golgi apparatus. The multicapsid virions are also believed to be released from the cell by exocytosis (Weiland et al. 1986).
C.
Virus Strains: Antigenic and Genetic Relationships
A number of strains of MCMV have been described, but only the Smith and K181 strains have been widely used. The Smith strain was derived from the original isolation of MCMV by Margaret Smith (Smith 1954) and has been the most commonly used strain. The Smith strain has been held by the American Type Culture Collection for a number of years as ATCC number VR-194, recently reissued as VR-1399. However, the strain has been passaged in many different laboratories, resulting in the emergence of variants that exhibit different biological properties (reviewed by Osborn 1982). A more recent report characterizes the variant Vancouver strain, which emerged following the serial passage of the Smith strain in cell culture in Hudson’s laboratory over a number of years (Boname and Chantler 1992). This strain was found to have a 9.4 kb deletion spanning the XbaI I/L junction and a 0.9 kb insertion in the EcoR1 K fragment. It exhibits altered tissue tropism, failing to grow in the salivary gland, while exhibiting enhanced in vitro growth compared to the parental Smith strain. The K181 strain arose in Osborn’s laboratory from the salivary gland passage of the Smith strain in mice, following selection for virulent variants (cited in Misra and Hudson 1980). K181 reached 100-fold higher titers in the salivary glands than Smith (Misra and Hudson 1980), but exhibited different plaque morphology and lower yield in cell culture (Hudson et al. 1988). Differences in restriction endonuclease profiles between Smith and K181 were found for BamH I, Bgl II, EcoRI, HindIII, and XbaI restriction enzymes (Hudson et al. 1988). Where K181 genes such as m133 (Lagenaur et al. 1994), M55 (Elliott et al. 1991; Xu et al. 1996), M75 (Xu et al. 1992), and the termini of the short direct repeats (Marks and Spector 1984) have been sequenced, only minor differences between the DNA and predicted amino acid sequences of Smith and K181 have been found (Rawlinson et al. 1996). However, the exact origin of K181 is unknown. There are three possibilities: it could be a variant of Smith, an endogenous MCMV present in the laboratory mice, or a recombinant between Smith and an endogenous virus. As discussed by Hudson, the use of variants of the Smith strain may explain some of the discrepant results obtained by different investigators (Hudson 1994a). Clearly, there remains a need for reliable reporting of the strain of MCMV used, together
7
with a description of the mode of passage, passage history, and its biological properties. A comparison of the restriction endonuclease profiles of the strain and the ATCC stock of the Smith strain would be valuable. Indeed, restriction enzyme digest analysis of the viral DNA was important in determining that a virulent MCMV strain that was thought to be Smith (Chalmer et al. 1977) had a restriction a pattern identical to that of the K181 strain (Xu et al. 1992) described previously (Hudson et al. 1988). This strain has now been designated K181 (Perth) to identify its laboratory history (Scalzo et al. 1992). Finally, the use of a low-passage master stock would minimize the emergence of variants. Nonetheless, as discussed elsewhere, even a few salivary gland passages of K181 (Perth) in genetically resistant mice were sufficient to mutate the MCMV m157 gene, which is a ligand for the natural killer cell receptor Ly49H controlling MCMV resistance in this mouse strain (Voigt et al. 2003). Following the complete sequencing of the Smith strain (ATCC: VR-194) (Rawlinson et al. 1996), the genetic basis for biological differences between variants of the Smith strain as well as differences between other strains and isolates of MCMV will be readily determined. The construction of bacterial artificial chromosomes of the Smith (Messerle et al. 1997) and K181 (Redwood et al. 2005) strains will facilitate the study of genes associated with viral virulence. Because of its greater virulence in mice (Misra and Hudson 1980), K181 has proved to be useful for in vivo studies of host innate resistance (Chalmer et al. 1977; Grundy (Chalmer) et al. 1981; Allan and Shellam 1984; Scalzo et al. 1992) and the induction of immunopathological diseases (Lenzo et al. 2002; Lawson et al. 1990; Olver et al. 1994; Price et al. 1990), without the need for either immunosuppression or large viral doses. Several viruses that resemble MCMV have been isolated from wild rodents. Raynaud and colleagues isolated an MCMV-like agent from Apodemus sylvaticus (Raynaud and Barreau 1965), and Diosi and colleagues isolated and briefly characterized a similar viral agent from Microtus arvalis (Diosi et al. 1972). These viruses induce characteristic cytopathic effects in mouse embryo fibroblasts, and multicapsid virions are produced (Hudson et al. 1976b) suggesting that they may be related to MCMV. However, as these viruses have not been characterized further, it is not possible to consider them as isolates of MCMV at this stage. Finally, however, it is important to recognize that research on MCMV over the last 50 years has been based on the use of a single strain of MCMV, the Smith strain, and laboratoryderived variants of this strain. These viruses have been maintained by either salivary gland passage in laboratory (usually inbred) mice or by passage in cultured embryo fibroblasts or fibroblastic cell lines, a cell type that is not a primary target of infection in vivo. It is highly likely therefore that the strains or variants of MCMV that are in common use have acquired significant genetic and biological differences from early passages of the Smith isolate.
8
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
This issue has also been recognized in HCMV research (Mocarski and Courcelle 2001b), where the widely used AD169 strain was isolated only 2 years after the isolation of the Smith strain of MCMV (Rowe et al. 1956). AD169 has been maintained by in vitro culture, mainly in fibroblastic cell lines, and multiple variants have arisen in different laboratories. In addition to the use of other strains of HCMV such as Toledo, which was more recently derived from a clinical specimen, and Towne, a strong emphasis in recent HCMV research has been placed on the use of freshly derived clinical isolates. These are now widely used in the in vitro testing of antiviral agents as well as in studies identifying the viral genes associated with particular cell or tissue tropism, disease causation, or immune evasion. A single strain of MCMV that has been strongly adapted by laboratory passage for over 50 years cannot be expected to replicate the range or extent of clinical conditions that are associated with natural HCMV infections in humans. Therefore, if MCMV is to continue to be relevant as a model system for understanding HCMV infections, it is important that more emphasis is placed on the use of recent isolates of MCMV, and care is taken to avoid laboratory adaptation. Free-living wild mice are naturally infected with MCMV (Mannini and Medearis 1961; Rowe et al. 1962; Gardner et al. 1974; Booth et al. 1993; Smith et al. 1993; Singleton et al. 1993, 2000, Gorman et al., 2006) and have been the source of new isolates of MCMV (Booth et al. 1993). Twenty-six isolates of MCMV were obtained from the salivary glands of seropositive wild Mus domesticus that were trapped on subantarctic Kerguelen Island and on the Australian mainland at Geraldton, Nannup, and Walpeup (Booth et al. 1993). The viruses were originally characterized by their induction of cytomegaly in cultured mouse embryo fibroblasts, the reactivity of viral proteins with antibodies to MCMV by Western blotting, and the neutralization of these strains by sera from mice immunized with the K181 strain (Booth et al. 1993). Subsequently, viral genes such as gB and m157 and the H2LLdrestricted cytotoxic T cell epitope within ie1 of many of these strains have been sequenced and compared with the Smith sequence (Xu et al. 1996; Voigt et al. 2003; Lyons et al. 1996). Restriction enzyme analysis of these isolates using EcoRI, XbaI, and HindIII has revealed unique restriction profiles that differ from K181, and demonstrates that natural variation occurs throughout the MCMV genome (Booth et al. 1993). Western blot analysis of viral proteins from these isolates revealed considerable antigenic cross-reactivity among the new isolates when a polyclonal antiserum to K181 was used. However, significant variations in the electrophoretic mobility and intensity of staining of certain proteins were also observed. Interestingly, when 15 of these strains were compared for their ability to replicate in the salivary gland of weanling BALB/c mice, marked differences were observed. Nine of the strains did not replicate to significant titers, while replication was observed with the remaining 6 strains. The failure of 9 strains
that were originally isolated from the salivary gland to replicate there following their isolation and plaque purification is intriguing, especially since all isolates replicated equally well in vitro (Booth et al. 1993; and unpublished observation). This study was also the first report of mixed infection with different MCMV strains in a single mouse. Three mice were found to harbor genetically different strains in their salivary glands. In one mouse, identified as G3, 4 genetically different strains, G3A, G3B, G3C, and G3E, were detected by restriction enzyme analysis. Mixed infection is presumed to have occurred in apparently normal adult mice. This phenomenon appears to be widespread in wild mice (Gorman et al. 2006). For HCMV, mixed infections with different strains have also been described, although most reports relate to immunosuppressed patients. Mixed infections may arise by simultaneous infection with different strains or by sequential infection with individual strains. However, in the latter case, infection would have to be established in the presence of an existing immune response. This eventuality may be favored by the failure of cytomegaloviruses to induce sterilizing immunity. A recent experimental study has established that sequential infection with the G4 and then the K181 strains occurs despite the presence of MCMV-specific antibody and cytotoxic T cells (Gorman et al. 2006). The benefit to the virus of mixed infection is the possibility of complementation between genetically different strains (Cicin-Sain et al. 2005), resulting in persistent infection and enhanced transmission. Studies in laboratory mice offer an excellent means of elucidating this phenomenon.
III.
GROWTH IN VITRO AND IN VIVO A.
In Vitro Propagation
The cytomegaloviruses exhibit species specificity, and infection in vivo is restricted to their natural host species (van Regenmortel et al. 2000). The same is generally true in vitro, although there have been reports of MCMV infecting cell cultures derived from species other than the mouse. 1.
Propagation in Permissive Murine Cells
In permissive cells MCMV replicates to high titer, achieving yields of 10–100 plaque-forming units (pfu) per cell, resulting in the death of infected cells (Hudson 1994a). A feature of MCMV infection is the induction of a characteristic cytoplasmic effect (CPE) in which nuclear and cytoplasmic swelling and chromatin margination occurs (Lussier 1975b) and the formation of both intranuclear and intracytoplasmic inclusions is observed (Ruebner et al. 1966). MCMV replicates in the cell nucleus and buds into the perinuclear cisternae to associate with secondary lysosomes and the Golgi apparatus to form
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
intracytoplasmic inclusions (Joseph et al. 1978). These intracellular changes are known as cytomegalia. MCMV can be propagated in a variety of cells or cell lines in vitro (reviewed by Hudson 1994a), and generally replicates most productively in murine embryo fibroblasts (MEF) and in murine fibroblastic cell lines (Osborn 1982). Cell lines that support productive MCMV infection include two fibroblastic lines, NIH 3T3 cells, and the mouse embryo line SC-1, as well as the mouse mammary tumor cell line C127I (Smee et al. 1989). However, for the detection of MCMV by plaque assay, MEF were found to be 10 times more sensitive than these cell lines (Smee et al. 1989). Nonetheless, the use of MEF has some disadvantages. Their preparation is labor intensive, they become resistant to the growth of MCMV after serial passage in culture (Misra and Hudson 1977), and they have a finite life span due to programmed senescence. More recently, the continous murine bone marrow stromal cell line M2-10B4 was found to be as permissive for MCMV replication as MEF (Lutarewych et al. 1997). Other murine cells that are permissive for MCMV include central nervous system stem cells (Kosugi et al. 2000) and microglial cells (Schut et al. 1994). Mast cells can be infected by MCMV, but less than 12% release infectious virus (Gibbons et al. 1990). Similarly, resident peritoneal macrophages can support MCMV infection in culture (Shanley and Pesanti 1983), although reports differ on the extent of infection (Brautigam et al. 1979; Selgrade and Osborn 1974; van Bruggen et al. 1989). As a result of infection, various macrophage functions are down-regulated (Price et al. 1987). Immature dendritic cells also support MCMV replication, although mature dendritic cells are much less permissive (Andrews, Andoniou, et al. 2001; Mathys et al. 2003). In general, the replication of MCMV in these various cells or cell lines results in lower yields than are achieved in MEF (Kosugi et al. 2000; Shanley and Pesanti 1983). 2.
Nonpermissive Murine Cells
In these cells, a low-level infection may occur, resulting in yields of virus of less than 1 pfu per cell (Hudson 1994a). Such cells include the L929 fibroblast line, the Y-1 adrenal cell line, and J774A.1 and other macrophage lines (Hudson 1994a). In other cell types or lines, infection is abortive, and only the transcription of viral genes occurs without the production of viral DNA or infectious virus. Examples include murine T and B cells (Hudson 1994a). 3.
Non-Murine Cells
Several studies have shown that MCMV can replicate in certain primary and continous cell lines derived from other species. These include primary rat embryo fibroblasts (Smith et al. 2005), the rat NRK cell line (Hudson 1994a), African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells (Kim and Carp 1971).
9
However, a number of heterologous cells and cell lines were not found to support MCMV replication, including human cell lines W1-38, HeLa, Hep-2 cells (Kim and Carp 1971), and MRC-5, KB, and the African green monkey cell line Vero (Hudson 1994a). 4.
Features of MCMV Replication In Vitro
The replication of MCMV has been studied extensively in mouse embryo fibroblasts and has been reviewed previously (Osborn 1982; Hudson 1994a). KINETICS OF MCMV REPLICATION In brief, following adsorption of the virus to the fibroblasts, there is a lag period of 16–18 hours before infectious progeny are detected. The lag period is followed by the release of extracellular virus which continues over 18–36 hours p.i. with the maximum yield reached at 30–36 hours p.i.. Infected cells remain viable for 36–48 hours p.i.. As discussed by Hudson (Hudson 1994a), infected cells contain an average of 3000 viral genomes but only release about 100 pfu, which is in part due to the packing of MCMV genomes into multicapsid virions, which contain a number of nucleocapsids but may each produce only a single pfu. These figures may need revision in view of the more sensitive assays that are now available. Two additional factors, centrifugal enhancement of infection and the cell growth cycle, have been shown to influence the replication of MCMV in permissive cells. CENTGRIFUGAL ENHANCEMENT Low-speed centrifugation of the viral inoculum against the cell monolayer at 1000g for 30 minutes results in a 10 to 100-fold increase in the number of pfu that are detected (Osborn and Walker 1968; Hudson et al. 1976a; Hudson 1988, 1994a). The conditions required are precise, and the virus and cells must be centrifuged simultaneously within 60 minutes of inoculation of MCMV. The phenomenon is not dependent on the presence of multicapsid virions and is also applicable to HCMV (Woods et al. 1987) and a variety of other viruses (Pietroboni et al. 1989), although the effect seems to be most pronounced with MCMV. The mechanism is not completely understood, but may involve stabilization of the initial weak interactions between the virus and cellular receptors (Hodgkin et al. 1988). EFFECT OF THE CELL GROWTH CYCLE In permissive cells, the replication of MCMV depends upon the cell cycle (Keil et al. 1984). Production of viral progeny was found to be most effective in cells synchronized at the S phase (Muller and Hudson 1977) and least effective at the G0 phase. This cell cycle dependency is probably due to the occurrence of maximal activity of ribonucleotide reductase (RNR) during the S phase. The IE1 protein of MCMV increases the activity of R2, an enzymatic subunit of RNR, and the M45 protein is a viral homologue of the R1 enzyme subunit of RNR (Brune et al. 2001;
10
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
Lembo et al. 2000). These viral products act to up regulate the catalysis of RNR to convert ribonucleotide diphosphate into deoxyribonucleoside and increase the dNTP pool for viral DNA synthesis and replication. Other cellular proteins utilized by MCMV to regulate the cell cycle and enhance viral DNA synthesis include dihydrofolate reductase, thymidylate synthase (Cavallo et al. 2001), and interferon (IFN)-inducible 204 (Rolle et al. 2001). MULTICAPSID VIRIONS An unusual feature of the replication of MCMV is the production of multicapsid virions, as well as virions containing a single capsid. Multicapsid virions are particles with from 2 to 20 or more capsids enclosed in a common envelope (Hudson et al. 1976b). All size classes of virion appear to be infectious (Hudson et al. 1976b; Chong and Mims 1981), although a multicapsid virion is a single infectious unit. Large multicapsid virions are readily disrupted during purification; free nucleocapsids are noninfectious (Chong and Mims 1981). Mixtures of multicapsid and single virions are produced during in vitro infection of murine embryo cells and fibroblastic 3T3 cells (Hudson et al. 1976b; Chong and Mims 1981; Weiland et al. 1986). Multicapsid virions are also produced during the replication of MCMV in vivo, and have been reported in the liver (Papadimitriou et al. 1984), lung (Reddehase et al. 1985), and spleen (cited in Hudson et al. 1976b). However, multicapsid virions have not been detected in the salivary glands (Hudson et al. 1976b; Chong and Mims 1981), perhaps reflecting the different morphogenesis of MCMV in this organ. The morphogenesis of multicapsid and single virions of MCMV is discussed in Section II, B, 2. The ratio between multicapsid and single virions produced in vivo is not known, but in vitro the multicapsid form accounts for 90%–95% of all virions produced (Hudson et al. 1976b; Chong and Mims 1981). The production of multicapsid virions is not a feature of other herpesvirus infections. Occasional virions encompassing two nucleocapsids have been reported in infections with guinea pig CMV (Fong et al. 1979), rat CMV (Bruggeman et al. 1982), and herpes simplex virus (Nii et al. 1968; Watson 1973), perhaps reflecting the occasional budding of two nucleocapsids into the same vesicle. The production of multicapsid virions has not been detected with HCMV (Hudson et al. 1976b).
B.
Infection of Mice
The replication of MCMV in mice, including the kinetics of the infection, the organs and tissues that support viral growth, the establishment of persistent and latent infection, and host parameters that influence the outcome of the infection (age, mouse strain, route of inoculation) is discussed in Section V. Mice have also been used for virus titration and for virus isolation. Virus can be quantified in vivo by the LD50 assay, in
which adult mice are inoculated intraperitoneally with serial dilutions of viral stock. Interestingly, the effect of varying the dose of virus is pronounced, with mortality changing from 0% to 100% over a 4-fold increase in dose (Selgrade and Osborn 1974; Chalmer et al. 1977; Trgovcich et al. 2000). Hence, serial 1:2 dilutions of virus stock are employed, and ideally 10 mice are used for each virus dilution. Given the well-established age-dependent variation in susceptibility to MCMV (Osborn 1982 and Section V, A, 1), mice of a specified age are used, and the age should be stated. At high virus doses, mice begin to show signs of illness by the second or third day, exhibiting hunching, ruffled fur, reduced activity, and weight loss. Deaths usually occur around day 5. The cause of death is unknown, but as will be discussed in Section V, this assay was very useful in studies of host genetic control of resistance to MCMV in adult inbred mice. The difference in LD50 dose between the most susceptible (BALB/c) and the most resistant mouse strain (B10.BR) was about 30-fold (Grundy (Chalmer) et al. 1981), but even differences of 2- to 4-fold could be measured accurately to distinguish BALB/c from the more resistant C57BL/6 strain (Grundy (Chalmer) et al. 1981). However, this assay raises animal welfare issues, and it is usually not permissible now to use mortality as an endpoint. As an alternative, assessing morbidity by the daily or twice-daily recording of body weight may be useful, since large doses of MCMV cause marked body weight loss, with mice losing 20%–30% of their body weight as the infection progresses (Pomeroy et al. 1998; Bolger et al. 1999). Again, the group size should be sufficiently large to allow firm conclusions to be drawn. ID50 assays involving the inoculation of adult mice with serial dilutions of MCMV, followed by assays for seroconversion, have not been widely used. This may be because MCMV infection does not generally induce high antibody titers in a primary infection. Also, because MCMV is very successful at establishing persistent infection, even rather small viral inocula are often sufficient to allow MCMV to reach the salivary gland, where high viral titers may result. Antibody titers may not therefore adequately reflect the original viral dose. Where the kinetics of infection and the organs involved are known, measuring the viral titer by the plaque assay in a particular organ such as the spleen or liver has been used to discriminate between mouse strains on the basis of innate resistance to MCMV (Allan and Shellam 1984; Mercer and Spector 1986; Scalzo et al. 1992). Mice are commonly used for the preparation of virulent stocks of MCMV by salivary gland passage. Virus harvested from other organs such as the spleen, liver, or kidneys exhibits reduced virulence (Eizuru and Minamishima 1979). Passage of salivary gland–derived MCMV in cell culture results in its attenuation, with effects being observed following even one passage in vitro. However, virulence is restored following salivary gland passage (Osborn and Walker 1971). The mechanism of loss of virulence in cell culture remains to be determined.
11
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
Interestingly, even the virulence of salivary gland stocks of MCMV differs according to the time of harvest (Selgrade et al. 1981). Salivary glands also contain factors that inhibit the virulence of MCMV; this property increases after weaning (Mims and Gould 1979). Concern about the presence of cytokines and hormones in salivary gland tissues has convinced Podlech and colleagues (2002) to use tissue culture–prepared virus, despite its reduced virulence. The method of salivary gland passage has been described previously (Osborn and Walker 1970; Osborn 1982; Chalmer et al. 1977). Briefly, inbred female 21-day-old (weanling) mice of a susceptible strain such as BALB/c are inoculated i.p. with a sublethal dose of MCMV. The dose will depend on the strain of MCMV in use. Using the virulent K181 (Perth) strain, we inoculate approximately 5 × 103 pfu per mouse, and harvest salivary glands 17 days later. With this dose, a low level of mortality is observed and high titer stocks are achieved. A 10% w/v stock is prepared from homogenized salivary gland tissue, and after clarification by low-speed centrifugation, the stock is stored in small vials in either liquid nitrogen or at −80°C.
IV. A.
EPIZOOTIOLOGY Natural History
The natural host for MCMV in the wild is considered to be the house Mus musculus domesticus (Mus domesticus). MCMV infection in free-living Mus domesticus is widespread in different parts of the world. In North America, Rowe and colleagues sampled wild mice from a large number of locations and found that most mice were infected with MCMV (Rowe et al. 1962). Similarly, wild mice trapped in 3 separate locations in California showed evidence of infection (Gardner et al. 1974). In Australia, the seroprevalence of MCMV was found to be high in wild Mus domesticus. In mice trapped in wheat fields in 7 separate locations spread across eastern and southeastern Australia, almost 100% had antibodies to MCMV (Smith et al. 1993). Similarly, wild house mice on the Western Australian islands of Thevenard (Moro et al. 1999) and Boullanger (Moro et al. 2003) and the oceanic islands of Kerguelen (Booth et al. 1993), Macquarie (Moro et al. 2003), and Gough (G.R. Singleton, personal communication) exhibited very high rates of MCMV infection. Mus domesticus is a member of the Mus musculus complex of species, which consists of domesticus, musculus, molossinus, castaneus, and bactrianus (Guenet and Bonhomme 2003). A discussion of the taxonomy of the musculus-domesticus complex of species lies outside the scope of this review and is considered in the companion volume of this series. There do not appear to have been studies of the ability of strains of MCMV
obtained from Mus domesticus to infect other members of the Mus musculus complex. Similarly, there are no reports of isolations of cytomegaloviruses from members of this complex other than from Mus domesticus. Thus the question of whether members of this complex other than Mus domesticus can serve as a reservoir of MCMV in the wild remains unanswered. This worldwide distribution of MCMV in wild Mus domesticus reflects not only the very close association between the virus and its host, which is a feature of herpesviruses, but also the successful establishment of populations of house mice in environmentally diverse parts of the world. It is presumed that Mus domesticus reached the Americas, Australasia, and the subantarctic islands during human exploration and colonization (Guenet and Bonhomme 2003). The habitats in which MCMV has been detected in Mus domesticus range from temperate regions in California (Gardner et al. 1973) to arid islands (Moro et al. 1999, 2003) and dry wheatlands with sandy soils (Smith et al. 1993; Singleton et al. 1993, 2000) in Australia to cold subantarctic environments (Booth et al. 1993; Moro et al. 2003). In all environments, MCMV is ubiquitous, with high seroprevalence rates. Interestingly, there was a strong relationship between population size and the extent of MCMV infection. In the mallee wheatlands of southeastern Australia where mouse plagues erupt periodically, the seroprevalence of MCMV was 20%–30% during periods of low mouse abundance but reached approximately 75%–95% at times of high mouse density (Singleton et al. 2000). These trends were supported in other studies (Singleton et al. 1993; Smith et al. 1993).
B.
Natural Infection of Laboratory Mice
Early studies documented natural MCMV infection in laboratory mice. The first isolation of MCMV was made from salivary gland stock that was derived from naturally infected laboratory mice (Smith 1954). Subsequently, studies that employed virus isolation in cell culture or suckling mice found that the rate of infection was 2%–3% (Rowe et al. 1962) or less (Mannini and Medearis 1961), although these are likely to be underestimates. In the 1980s, naturally occurring MCMV infections in laboratory colonies were detected by serological techniques. Using sera provided by different commercial suppliers, 140 of 256 sera contained antibodies to MCMV, and mice from 8 of the 9 suppliers were found to be antibody positive (Anderson et al. 1986). These findings have not been reproduced. Indeed, in a similar study, no anti-MCMV antibodies were detected in sera from mice provided by 4 commercial suppliers (Classen et al. 1987). Naturally occurring MCMV infections in laboratory mice have not been reported in recent years. Nonetheless, MCMV infection may be a problem in laboratory colonies in some parts of the world. A serological survey of 556 mouse sera from 40 laboratory animal facilities in China found a high seroprevalence
12
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
to MCMV, with up to 60% of mice in some institutions being antibody positive (Tang et al. 1992).
C.
Mode of Transmission
A distinctive feature of the cytomegaloviruses is their ability to establish long-term persistence of infectious virus in the salivary gland, as demonstrated in mice (Brodsky and Rowe 1958; Medearis 1964), rats (Bruggeman et al. 1983, 1985), and humans (Lucht et al. 1998). It is presumed that the transfer of infectious virus in saliva to susceptible individuals is the major mode of transmission. In addition, in humans and guinea pigs but not in mice or rats, CMV infection of pregnant mothers may result in transplacental infection of the fetus. The salivary glands in mice and man consist of three paired major glands, which in rank order of salivary output are the submandibular, parotid, and sublinguinal glands. Each gland is connected to the oral cavity by a single excretory duct. In the mouse each gland is limited to one type of secretory cell: serous in the submandibular and parotid glands and mucous in the sublinguinal gland. In addition to these major glands, there are a number of minor glands that in man contribute <10% of the salivary output, such as the anterior and posterior linguinal glands and the labial, buccal, molar, and palatine glands. All three major glands in mice and humans support CMV infection, but in the mouse, CMV infection appears to be more pronounced in the submandibular gland, followed by the sublinguinal and parotid glands (Mims and Gould 1979; Ruebner et al. 1966). Infection of the minor glands has been reported in mice on the basis of histopathological evidence (Ruebner et al.1966), but the level of infection appears to be low. The acinar epithelial cells are the major site of MCMV replication in the salivary gland (Henson and Strano 1972), from which the virus is shed into ducts leading to the mouth. Infectious virus is present in both saliva and throat swabs of infected mice (Henson and Neopolitan 1970; Mannini and Medearis 1961; Mims and Gould 1979). The salivary gland tropism of MCMV is controlled by at least two viral genes, m133 (sgg1) and m131/129 (MCK-2). The gene sgg1 appears to specifically control the growth of MCMV in acinar cells (Manning et al. 1992; Lagenaur et al. 1994). In contrast, MCK-2 encodes a CC chemokine homolog (Fleming et al. 1999; Saederup et al. 1999) that enhances dissemination of MCMV to the salivary gland by leucocyte-associated viremia (Saederup et al. 2001). Other secretions such as tears and urine also contain infectious virus (Medearis 1964). However, while HCMV is present in breast milk in humans (Stagno et al. 1980), this route of transmission has not been adequately studied in mice. Nonetheless, protective antibody can be passed to suckling mice via colostral fluids (Medearis and Prokay 1978). Infectious virus has been detected in epididymal sperm and seminal vesicles of infected male mice, as well as in uterine
sperm from mated females (Neigbour and Fraser 1978), demonstrating the likelihood of sexual transmission of MCMV. This is supported by a study of artificial insemination with MCMV that resulted in infection (Young et al. 1977). Other artificial means of transmission have been reported and include skin grafts (Shelby et al. 1988), cardiac transplants (Rubin et al. 1984), and blood transfusions (Hamilton and Seaworth 1985). Whether salivary or sexual contact or biting is involved, close proximity is required for transmission, since MCMV is not readily transmitted from cage to cage in the laboratory (Mannini and Medearis 1961), unlike certain other murine viral pathogens. In free-living wild mice, seroprevalence rates of over 90% are common, and MCMV can be readily isolated from the salivary gland (Booth et al. 1993). Therefore, the opportunities for transmission are high in wild mice, but, again, population density is important (Section IV, A). Transmission of MCMV among wild-derived mice has been studied in large secure outdoor enclosures in a rural setting (Farroway et al. 2002, 2005). Infection of the population from infected founder mice was readily established and, using a second distinct MCMV strain, it was found that prior infection with one strain did not prevent the spread of a second strain within the population (Farroway et al. 2005). This observation demonstrates that mixed MCMV infections can be acquired naturally in free-living mice despite prior immunity, thus supporting the notion that MCMV does not induce sterilizing immunity.
D.
Host Range
The cytomegaloviruses are considered to be host-specific, and this is an important characteristic that distinguishes members of the b herpesvirinae (van Regenmortel et al. 2000). Early studies showed that homogenates containing the agent of salivary gland inclusion bodies in guinea pigs could not infect rabbits, rats, or kittens, while readily transmitting the infection to guinea pigs (Cole and Kuttner 1926). An infectious agent was found to be the cause of the characteristic inclusion bodies in the salivary glands of the guinea pig, hamster, and mouse, and in each case the infection proved to be entirely species-specific in its effect (Kuttner and Wang 1934; Kuttner and T’ung 1935). More recent studies showed that the so-called Osborn strain of MCMV did not cause significant mortality when inoculated intracerebally (i.c.) into newborn rats in a dose of 103 pfu, while the same dose of RCMV caused 75% mortality (Priscott and Tyrrell 1982). Conversely, RCMV was more lethal for newborn rats than newborn mice when given i.c. (Priscott and Tyrrell 1982), and the Maastricht strain of RCMV was reported not to replicate in mouse tissues (Bruggeman et al. 1982). However, another study showed that much larger i.p. doses of 3–8 × 106 pfu of salivary gland stock of the Smith strain of MCMV killed 70%–100% of suckling rats, and virus could be isolated from the organs of moribund animals. The cause of
13
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
death or titers of MCMV were not determined, and a comparison with RCMV was not performed (Smith et al. 1986). Similarly, when MCMV expressing green fluorescent protein was inoculated into adult rats, the virus could be detected in rat tissues (van Den Pol et al. 1999), but no evidence of a productive infection was reported. Few studies have examined the issue of the host range of MCMV in species other than laboratory mice and rats. The possibility that a small Australian native mouse could be infected by MCMV was examined in an experiment of nature. House mice were inadvertently introduced to Thevenard Island off the coast of Western Australia, where there is a relic population of the small native mouse Leggadina lakedownensis. The house mice subsequently became established and occupied the same habitat as the native mice, frequently being captured together in the same pit traps. Cohabitation is unusual because on mainland Australia Mus domesticus normally dominate their preferred habitats, displacing native murids (Chapman 1981). Despite this coexistence, no evidence was obtained for the transmission of MCMV from the highly MCMV-seropositive house mice to the native mice (Moro et al. 1999). Deliberate inoculation with 2 × 104 pfu of salivary gland stock of MCMV strain K181 in the laboratory also failed to induce MCMV infection in L. lakedownensis (Moro et al. 1999) or in the other Australian native rodents Pseudomys australis or Rattus tunneyi (Singleton, personal communication). However, MCMV inoculation did induce an antibody response in L. lakedownensis, establishing that this native rodent could respond immunologically to MCMV and that the ELISA used could detect antibodies to MCMV in free-living L. lakedownensis on Thevenard Island if present (Moro et al. 1999). Overall, these various types of studies generally support the concept of species-specificity. Given the likelihood that CMVs use ubiquitous molecules such as the integrins as cellular receptors, the need to distinguish between viral entry with immediate early gene expression and full productive infection is obvious when distinguishing between infection of natural or other hosts.
V. A.
PATHOGENESIS
Effect of Age, Dose, and Route of Inoculation
immature mice than in adults. In newborn BALB/c mice, the LD50 dose for the K181 (Perth) strain inoculated i.p. is approximately 2 pfu, using salivary gland virus. However, in adult BALB/c, the LD50 is approximately 5 × 104 pfu (Fitzgerald et al. 1990). The resistance of mice to lethal MCMV infection matures after weaning (Booss and Wheelock 1975; Selgrade and Osborn 1974) and continues to increase until around 8 weeks of age (G. R. Shellam, unpublished observation), after which it changes little until old age (Grundy (Chalmer) et al. 1981). For this reason it is important that the age of mice used in experimental work is accurately reported, and it is suggested that 8-week-old mice are used for studies investigating mouse strain variation in response to MCMV. As will be discussed below, resistance to MCMV is genetically controlled, with inbred mouse strains varying in their resistance to infection and lethal disease. Of course, age also influences this response, with adult mice exhibiting the highest levels of resistance. Even so, genetically controlled host resistance is evident in newborn mice and in mice inoculated in utero (Shellam and Flexman 1986; Fitzgerald et al. 1990; Fitzgerald and Shellam 1991). 2.
Route of Inoculation
The pathogenesis of infection in adult mice has been studied using a variety of routes of inoculation of MCMV (Osborn 1982). Most studies have employed the intraperitoneal (i.p.) route, which results in the infection of visceral organs such as the spleen and liver by the second day of infection (Allan and Shellam 1984) and the dissemination of MCMV to other tissues and organs. Footpad inoculation has also been used because it allows the host response in the draining popliteal lymph node to be studied (Reddehase et al. 1984a; Sinickas et al. 1985). The intranasal (i.n.) route is considered to be the best approximation to natural infection where salivary contact is involved (Jordan 1978). Intramuscular or subcutaneous inoculation mimics natural infection by biting. Interestingly, there do not appear to have been any studies of the kinetics of infection, organ and tissue tropism, or the host response following naturally acquired infection. B.
Genetic Control of Host Resistance or Susceptibility to MCMV
These parameters all influence the outcome of MCMV infection significantly. As they have been comprehensively reviewed previously (Osborn 1982), they will be covered only briefly here. 1.
Age and Dose
The greater susceptibility to virus infection of newborn compared with adult animals has been well documented (Sigel 1952). MCMV is also more virulent in young, immunologically
Inbred mouse strains vary markedly in their susceptibility to MCMV infection. These differences have enabled detailed genetic studies to be undertaken, resulting in the mapping of susceptibility and resistance traits and the identification of the genes that control them. In this regard, MCMV is probably the best-studied murine virus. In 1936, the first detailed study of mouse salivary gland virus, which at that time was known only for its ability to induce
14
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
intranuclear inclusions in cells, demonstrated that C57BL mice were more resistant to the development of visceral lesions than were Buffalo or Swiss strain mice (McCordick and Smith 1936). Many years later it was shown that inbred CBA mice were more resistant to the virus than C57BL (Selgrade and Osborn 1974). Subsequently, BALB/c mice were shown to develop high viral titers and extensive splenic necrosis, whereas C57BL/6, C57BL/10, and CBA mice did not (Mims and Gould 1978b).
than mortality showed that when fetuses of the susceptible BALB/c strain were inoculated in utero with 11 pfu of MCMV on day 15 of pregnancy, they developed significantly higher titers in the brain than fetuses of the resistant C3H, CBA, C57BL/10, and B10.BR strains (Fitzgerald and Shellam 1991). Similarly, following inoculation on the day of birth, BALB/c mice developed higher virus titers in the spleen and liver than CBA or B10.BR mice (Shellam and Flexman 1986).
1.
2.
Resistance to Acute Infection
Comprehensive analyses of resistance to lethal infection among a variety of inbred mouse strains and congenic strains were undertaken by Chalmer (Grundy) and colleagues (Chalmer et al. 1977; Grundy (Chalmer) et al. 1981). These studies have provided the basis for most of the investigations of this phenomenon over the last 25 years. Using salivary gland–derived stocks of MCMV that were inoculated i.p. into adult mice of a variety of inbred strains, it was found that CBA and C3H mice were more resistant to lethal MCMV infection than BALB/c or C57BL mice. Furthermore, using the BALB/c congenic strains carrying the H-2b, H-2g, or H-2k haplotype rather than the H-2d haplotype of BALB/c mice, resistance was found to be associated with H-2k (Chalmer et al. 1977). In these studies the Smith strain of MCMV was used (Chalmer et al. 1977), but later RFLP analysis indicated that the strain was K181 (see Section II, C), which is now described as K181 (Perth). In a subsequent, more detailed study using LD50 analysis, it was shown that the possession of the H-2k haplotype rendered BALB/c mice 10.2 times more resistant to MCMV than BALB/c, and C3H (H-2k) were also more resistant than BALB/c (Grundy (Chalmer) et al. 1981). The C57BL/10 strain was moderately resistant compared to BALB/c, while the C57BL/10 congenic bearing the H-2k haplotype (B10.BR) was very resistant. In addition, comparison between two mouse strains bearing the H2b haplotype (BALB.B and C57BL/10) showed that non-H-2 associated genes confer resistance in C57BL/10 (and C57BL/6) mice (Grundy (Chalmer) et al. 1981). Thus two sets of genes, H-2 associated and non-H-2 associated, were found to confer resistance to the lethal effects of MCMV. A further study that measured virus titers rather than mortality in inoculated mice revealed that susceptible BALB/c mice had higher titers of MCMV in the spleen, liver, and other organs than resistant C3H and CBA mice (Allan and Shellam 1984). Subsequently, in a study that used plaque and infectious center assays and in situ hybridization, it was shown that the course of the acute infection in susceptible BALB/c and resistant C3H mice was different from the second day, with an early block in the dissemination of the splenic infection in C3H mice (Mercer and Spector 1986). Grundy reported that newborn mice of all strains were equally susceptible to the lethal effects of MCMV (Grundy (Chalmer) et al. 1981). However, later studies that measured viral titer rather
Mechanisms of Host Resistance to Acute Infection In Vivo
Genetically controlled host resistance to MCMV is mediated by innate mechanisms. This conclusion is based on the observation that resistance acts early in infection, regulating viral titers in major visceral organs by 1–2 days p.i. (Allan and Shellam 1984; Mercer and Spector 1986), before the induction of T cell and antibody responses. The existence of protective responses in immunologically immature fetal or newborn mice, albeit at a lower level, also supports this contention. An important role for NK cells in genetically controlled resistance was suggested by the activation of NK cell responses within 24 hours of MCMV infection in genetically resistant but not susceptible strains (Bancroft et al. 1981), and by the increase in MCMV titers in beige mutant C57BL/6 and other strains in which the beige mutation abrogated NK cell activity (Shellam et al. 1981; Shellam et al. 1985). NK cells were clearly implicated in controlling MCMV infection in a study that used antiasialo GM-1 antibodies to deplete NK cells in C57BL/6 mice; viral titers, viral dissemination, and MCMV-induced hepatitis were significantly enhanced soon after infection in antibody-treated mice (Bukowski et al. 1983, 1984). A role for NK cells in controlling MCMV, MHV, and vaccinia virus but not LCMV infection was established (Bukowski et al. 1983). The adoptive transfer of NK cells protected suckling mice from MCMV but not LCMV infection (Bukowski et al. 1985). An important step in establishing that NK cells control MCMV infection was the demonstration that NK cells with the distinctive large granular lymphocyte morphology accumulated in the liver by day 3 p.i. before the influx of cytotoxic T cells (McIntyre and Welsh 1986). A recent study has demonstrated the accumulation of NK1.1+ cells around foci of infection in situ (Andrews, Farrell, et al. 2001). The clear difference in MCMV titers in the spleens of resistant C57BL/6 and susceptible BALB/c mice (Allan and Shellam 1984) enabled a genetic analysis of this characteristic to be undertaken (Scalzo et al. 1990). Evidence was obtained for a single autosomal dominant gene, designated Cmv1 with 2 alleles, Cmv1r (C57BL/6) and Cmv1s (BALB/c). The use of the recombinant inbred (RI) strains CXB, constructed between BALB/c and C57BL/6, provided a provisional location for Cmv1 on chromosome 6, indicating it was a non-H-2 gene (Scalzo et al. 1990). Cmv1 was not responsible for restricting the spread of HSV-1 in C57BL/6 mice. The use of a larger set of RI mice constructed between resistant C57BL/6 and susceptible DBA/2 strains (BXD) enabled
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
Cmv1 to be located on distal chromosome 6, closely linked to NK1.1 and other loci within a region defined as the natural killer cell complex (Scalzo et al. 1992). The central role of NK cells in the genetically controlled restriction of MCMV in the spleen of resistant C57BL/6 mice was established by showing that the in vivo depletion of CD4+ and CD8+ T cells by monoclonal antibodies had no effect, while an anti-NK1.1 monoclonal antibody removed the restriction of MCMV replication in the spleens of Cmv1r strains (Scalzo et al. 1992). The location of Cmv1 within the natural killer complex was analyzed by high-resolution mapping, which localized Cmv1 to a region in close proximity to the Ly49 cluster of genes that encode a family of C-type lectin receptors expressed by NK cells (Depatie et al. 1997, 2000; Forbes et al. 1997; Brown et al. 1999). The Ly49H molecule, which is known to be an NK cell activation receptor, was found to mediate the Cmv1 effect (Brown et al. 2001; Daniels et al. 2001; Lee et al. 2001). It is now clear that this is achieved by the binding of Ly49H on the surface of NK cells to an MCMV-encoded glycoprotein, m157, which is a member of the m145 gene family of glycoproteins (Arase et al. 2002; Smith et al. 2002), several of which function in immune evasion (Farrell et al. 1997; Krmpotic et al. 2002). Interestingly, m157 mutates rapidly under selective pressure from Ly49H+ NK cells; passage of MCMV through Ly49H+ BALB.B6-CT3 mice gave rise to MCMVs with mutations in m157 that rendered the virus resistant to Ly49H+ NK cell responses (Voigt et al. 2003). Furthermore, isolates of MCMV obtained from wild mice showed natural mutations in m157 that resulted in a failure to activate NK cells by Ly49H (Voigt et al. 2003). Two very recent reports have described another mechanism by which NK cells interact with MCMV that is separate from the Ly49H-m157 interaction. Ly49P, which is an activating receptor on NK cells, was shown to recognize MCMV-infected cells expressing the H-2k haplotype (Desrosiers et al. 2005; Dighe et al. 2005). 3. Mouse Strain Variation in the Resolution of Chronic MCMV Infection
Knowledge of the genetic control of host resistance to MCMV has been gained through studies on acute infection in the spleens and livers of infected mice. Little attention has been given to how host genes might regulate the response to persistent infection, which occurs principally in the salivary glands. Following MCMV inoculation i.p., the virus does not reach the salivary glands until 6–8 days p.i. reaching peak titers between about days 14 to 20, after which titers decline over weeks or months. From scattered reports there appears to be considerable variation among mouse strains in the persistence of MCMV in these glands. However, persistence has not been compared in a number of mouse strains using the same preparation of MCMV in a single study.
15
Although peak titers may be higher in BALB/c than in some other strains (Mercer and Spector 1986), BALB/c clear the virus by about 30 days (Mercer and Spector 1986), while the virus persists longer in moderate titers in C57BL/6 (Jonjic et al. 1994), ICR/HA (Henson and Neopolitan 1970), and C3H mice (Mercer and Spector 1986). In outbred Swiss or Swiss Webster mice, MCMV persists in the salivary gland for at least a year (Brodsky and Rowe 1958; Ruebner et al. 1966; Medearis 1964). These mouse strain patterns of viral persistence are not related to the control of acute infection in the spleen or liver, where C3H and C57BL/6 are more resistant than BALB/c (Allan and Shellam 1984; Mercer and Spector 1986). 4.
In Vitro Studies of Resistance
Innate resistance to MCMV has also been demonstrated in cell culture. Mouse embryo fibroblasts (MEF) from the same mouse strains that were resistant or susceptible to acute infection in vivo exhibited the same pattern of response in vitro (Harnett and Shellam 1982). Thus, regardless of whether in vitro growth of MCMV was measured by cytopathic effect score, virus yield, plaque count, plaque size, or the time of onset of cytopathic effect, MEF from C3H or CBA mice were more resistant to MCMV than were MEF from BALB/c or C57BL mice. Similarly, the titer of MCMV produced in tracheal organ cultures from different mouse strains reflected the in vivo resistance status of these strains (Nedrud et al. 1979; Shellam et al. 1981; Harnett and Shellam 1982). Resistance status also influenced the effect of MCMV on mitogen responses in vitro (Allan et al. 1982). The study of resistance to MCMV at the cellular level was extended by Price and colleagues, who compared the replication of MCMV in normal peritoneal macrophages derived from a number of resistant and susceptible mouse strains (Price et al. 1987; Price et al. 1990). Using a variety of assessments to enumerate infected cells, this model system enabled small differences in resistance between mouse strains to be identified. These studies showed that H-2-associated genes were effective in controlling MCMV infection in vitro, and that the MHC class I genotype of cells determined the outcome of the infection. The presence of Kd, Kb, Dd, Ks and/or Ds, Kq and/or Dq alleles was associated with susceptibility, and the presence of Kk, Kf, Dk, Df, or Db with resistance to infection. No role for non-H-2-associated mechanisms was identified in these in vitro studies. This type of resistance has been shown to be largely attributable to NK cells in vivo. The possibility that MHC class I molecules were involved in early events in viral entry and replication, perhaps acting as a cellular receptor, was considered (Price et al. 1990). The MHC class I light chain, β2 microglobulin (β2m), which is required for maintaining the correct tertiary conformation of class I molecules on the cell surface, was found to influence the susceptibility of cells to MCMV in vitro. Using viral stocks and MEF that were grown in serum-free medium lacking β2m, the
16
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
infectivity of MCMV was reduced, and it was restored by the addition of β2m (Wykes et al. 1992). These relatively modest effects of β2m were restricted to genetically susceptibile (H-2d) cells; the infectivity of MCMV for resistant (H-2k) cells was not enhanced by β2m. The role of MHC class I molecules in MCMV infection in vitro was explored further using cells lacking MHC class I molecules, which were resistant to infection. Following transfection with class I genes, the expression of class I molecules restored infection, with H-2Dd and H-2Kb molecules being the most effective (Wykes et al. 1993). The alpha-1-domain of Dd appeared to be critically involved in potentiating infection. Overall, these various studies established a role for MHC class I molecules in facilitating MCMV infection in vitro and established a hierarchy of class I alleles that potentiated infection. Nevertheless, it is possible that the role of MHC class I molecules as viral receptors is relatively minor, as in vivo studies employing β2m knockout mice that lack MHC class I molecules found that MCMV infection was not affected (Polic et al. 1996; Tay et al. 1995). Other cell surface molecules such as the heparan sulfate proteoglycans are also involved in enhancing the infection of cells with MCMV (Price et al. 1995) and other herpesviruses. The important role played by integrin molecules in the entry of HCMV (see Section II, B, 2) suggests that such molecules may also be involved in MCMV infection. Regardless of any role for class I molecules as viral receptors, the effect of H-2 genes on susceptibility or resistance to acute MCMV infection in vivo is distinctive. Titers of MCMV in the spleen are 10- to 100-fold higher in congenic mice expressing H-2d rather than H-2k (Scalzo et al. 1992). As already discussed, recent research may partly explain the role of H-2k in resistance; H-2k molecules were shown to interact with activated NK cells in the MA/My strain to induce the clearance of MCMV. The critical interaction appears to involve Ly49P on NK cells and H-2Dk on MCMV infected cells (Desrosiers et al. 2005; Dighe et al. 2005). The identification of genes providing protection against MCMV has only just begun. Using information from random germline mutagenesis, Beutler and colleagues (2005) have postulated that approximately 290 genes comprise the MCMV resistome.
C. 1.
Latency and Reactivation
Pathogenesis
Persistent infection is a common feature of the β herpesviruses. The infection may be either chronic, in which infectious virus is produced at very low levels for long periods in particular organs or tissues such as the salivary gland, or latent, in which the viral genome is present in certain cells in the body but infectious virus cannot be detected. Reactivation from latency occurs under appropriate conditions that usually
involve immunosuppression to yield infectious virus that may induce disease and be transmitted to susceptible hosts. The disease that occurs following reactivation is usually mild because of the modifying effects of prior immunity (Jonjic et al. 1994; Reddehase et al. 1994). Following initial infection, infectious MCMV is cleared quickly from most tissues (Allan and Shellam 1984; Mercer and Spector 1986) except the salivary gland. The main mediators are the innate immune system, especially NK cells (Bukowski et al. 1983; Scalzo et al. 1992), as well as adaptive responses, where CD8+ T cells are critically involved at most sites (Reddehase et al. 1985; Podlech et al. 1998, 2000). However, viral genomes remain in latently infected cells and reactivate to produce infectious virus following immunosuppression. Immunosuppression may involve the use of anti-lymphocyte serum (Gardner et al. 1974), a combination of anti-lymphocyte serum and cortisone (Jordan et al. 1977), cyclophosphamide (Mayo et al. 1977), γ-irradiation (Balthesen et al. 1993), pregnancy (Baskar et al. 1985), or surgery with intra-abdominal bacterial infection (Cook et al. 2002). Latent infection has also been detected in vitro in tissue explant cultures of salivary and prostate glands (Cheung and Lang 1977), spleen (Jordan and Mar 1982), and cardiac tissue (Wilson et al. 1985) from latently infected mice. The transplantation of the organ containing latent MCMV into naive recipients results in the reactivation of the virus in the new host (Hamilton and Seaworth 1985; Schmader et al. 1995). The nature of the latent state of MCMV has been a key question for many years. Because of the relative insensitivity of assays for infectious virus, it has not been possible until recently to distinguish been the two main concepts—the maintenance of the latent viral genome in the absence of virus production (molecular latency) and the presence of low-level chronic infection that is below the detection limit of infectivity assays (Reddehase et al. 2002; Hudson 1994a). The use of very sensitive assays for detecting infectivity has recently indicated that molecular latency exists in the spleen and kidney (Pollock and Virgin 1995) and lungs (Kurz et al. 1997). The presence of latent MCMV in various organs, which was suggested by tissue explant culture, has been confirmed using PCR to detect latent viral DNA in various organs including salivary glands, lung, spleen, liver, heart, kidney, adrenal glands, and myeloid cells (Klotman et al. 1990; Balthesen et al. 1993; Balthesen, Dreher, et al. 1994; Collins et al. 1993; Brautigam et al. 1979; Mitchell et al. 1996; Pollock et al. 1997). Using a bone marrow transplantation model, Reddehase and colleagues demonstrated that while infectious MCMV was cleared sequentially from tissues and organs in a primary infection, viral DNA was cleared only from bone marrow and intravascular leukocytes, remaining latent in other organs for the life span of the host (Balthesen, Susa, et al. 1994; Reddehase et al. 1994; Kurz et al. 1997, 1999). The clearance kinetics also excluded the bone marrow as a source of latent MCMV in other tissues. In contrast, myeloid progenitors in
17
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
human bone marrow are thought to be a major site of latency for HCMV and the source of latent virus in tissues outside the bone marrow (Kondo et al. 1994; Slobedman and Mocarski 1999). Analysis of the kinetics of clearance of latent viral DNA in human tissues is needed to resolve this discrepancy. A comparative study has revealed that the load of latent viral DNA in tissues or organs is directly related to the level of virus replication that occurred during the primary infection. Furthermore, the risk of reactivation is related to the overall load of latent MCMV DNA present (Reddehase et al. 1994). If this is also true for HCMV in humans, then, as stated by Reddehase and colleagues (2002), the risk of recurrence of HCMV may depend on the age at which primary infection occurs [perinatal > childhood > adult life]. As expected, a reduction in the load of latent MCMV in mice following therapy with antiviral CD8+ T cells reduced the risk of recurrence (Steffens, Kurz, et al. 1998). These various studies also raised an interesting question. Since the production of infectious MCMV usually results in cell death, the cells that are responsible for producing most of the infectious virus may not be the same cells in which latency occurs, in line with the paradigm established for herpes simplex virus (Reddehase et al. 2002). The recognition of molecular latency in the absence of lowlevel chronic infection as the characteristic latent state in MCMV infection has allowed detailed studies of the mechanism of reactivation from latency and recurrence of infectious MCMV to be undertaken. As already discussed, it has been established that the recurrence of infectious MCMV following reactivation is controlled by the immune system, and CD8+ T cells play the leading role (Polic et al. 1998). However, the molecular reactivation of latent MCMV genomes is regulated by different means. This has been largely determined by analysis of MCMV gene expression in latently infected lungs. It is clear now that latent viral genomes are not necessarily transcriptionally silent. The highly regulated transcriptional program can be interrupted at many checkpoints before assembly and release of infectious virus. The regulation of IE gene expression is an early checkpoint on the way from latency to recurrence. The 1E1/3 transcriptional unit gives rise to 1E1 and 1E3 mRNAs by differential splicing (Keil et al. 1987b; Messerle et al. 1992). This is driven by the P1/3 promoter with a strong upstream enhancer, which serves as a molecular switch, connecting 1E1/3 transcription to the cellular environment. External stimuli, such as the pro-inflammatory cytokine TNF α, act as the first signal in the reactivation pathway, inducing transcription factors that activate the enhancer by binding to defined sequence motifs. 1E3 is believed to be the major transactivator of E gene expression (Messerle et al. 1992; Angulo et al. 2000). MCMV latency is controlled after the initiation of 1E1/3 transcription (Kurz et al. 1999), and this is the second checkpoint in the pathway of molecular reactivation. Reddehase and colleagues have proposed a multistep model of MCMV reactivation and the subsequence recurrence of infectious virus, in which
many checkpoints may be involved (Kurz and Reddehase 1999; Reddehase et al. 2002). Intriguingly, when the early part of this pathway was studied in lung pieces from the lungs of latently infected mice, it was found that latency-associated 1E1 transcription exhibited a random pattern among individual lung pieces. It was calculated that 1 transcriptional focus occurred per 25,000 latent viral genomes, indicating that the expression of ie1 is a rare event (Kurz et al. 1999). Although the reason for the random expression of ie1 in latently infected lungs is not known, it may reflect low-level expression of cytokines such as TNF-α which could activate the sporadic transcription of 1E1. Alternatively, activated CD8+ T cells secreting TNF-α may be responsible (Reddehase et al. 2002). In this concept, TNF-α activates the enhancer, resulting in 1E1 transcription, the synthesis of 1E1 protein, and the presentation of the immunodominant 1E1 nonapeptide to 1E1-specific memory CD8+ T cells. In turn, these cells are activated and release TNF-α, which activates 1E1 expression in another latently infected cell. Although there is more to discover about the latent state of MCMV, it is likely that this knowledge will be generally applicable to HCMV. The mouse model provides the means of studying molecular reactivation in vivo and should continue to provide important insights into how latent CMV infections are regulated. D.
Suitability of MCMV as a Model of HCMV Infection and Disease
Because of the strict species specificity of cytomegaloviruses, it is not possible to undertake experimental studies of HCMV pathogenesis due to the restriction of growth of HCMV in animal models. There is one exception to this: mice with the severe combined immunodeficiency syndrome with engrafted human tissues (SCID-hu mice) support the replication of HCMV in human tissue grafts (Mocarski et al. 1993). A variety of fetal human tissues have been engrafted under the kidney capsule of SCID or SCID beige mice, including thymus, liver, lung, and colon (Mocarski et al.1993; Wang et al. 2005), as well as retinal tissue (Bidanset et al. 2001). HCMV infection is restricted to the grafted human tissues and reaches high titers over 1 month, with persistence for at least 9 months (Mocarski et al. 1993). Since clinical strains of HCMV replicate well in SCID-hu mice while AD169 does not, the model has been very valuable for demonstrating that genes within a 15-kb segment present in clinical strains but not in AD169 are crucial for the replication of HCMV in vivo (Wang et al. 2005). However, the model has significant limitations. It does not replicate the multi-organ infection observed in infected humans, and infection of endothelial and smooth muscle cells of human origin cannot be readily studied. Meaningful studies of HCMV latency or vaccination are also not possible (Wang et al. 2005). Since MCMV infection of mice shares many features with HCMV infection of humans, the mouse model has been used
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GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
extensively for studying the pathogenesis of acute, latent, and recurrent infections. The use of RCMV and guinea pig CMV in their natural hosts has also been valuable for gaining understanding of the pathogenesis of HCMV because these models may reproduce several aspects of HCMV disease (atherosclerosis and congenital disease, respectively) more precisely than MCMV. The merits of all three animal models have been reviewed (Price and Olver 1996). However, the mouse model remains the best studied and has provided a wealth of information about the virus-host relationship. An overview of the usefulness of the mouse model in reproducing HCMV disease is provided in Table 1-1. 1.
Intrauterine Infection and Congenital Disease
HCMV is the leading viral cause of congenital infections in humans in the developed world. With an incidence of 0.2% to 2.2% per live birth in the United States, it is predicted that 40,000 infected infants are born annually in the United States alone, 4000–6000 of whom will experience long-term neurological damage (Britt and Alford 1996). Symptomatic infection is largely restricted to infants born to mothers who experienced a primary rather than a reactivated HCMV infection during pregnancy. Clinical abnormalities in infants with symptomatic
congenital HCMV infection include low body weight, thrombocytopenia, hepatosplenomegaly, microcephaly, and chorioretinitis, and in some, organ dysfunction and death may occur (Britt and Alford 1996). The neurological damage is not reversible and accounts for the long-term morbidity associated with this infection. Hearing loss is the most common neurologic abnormality, and congenital HCMV infection is a leading cause of nonheritable hearing loss in the United States (Hicks et al. 1993). There is thus a clear need for experimental studies to investigate all aspects of intrauterine infection. Unfortunately, there is no conclusive evidence that MCMV can establish intrauterine infection in mice, and most experimental studies use guinea pigs because guinea pig CMV crosses the placenta and initiates fetal infection (Staczek 1990). The early attempts to establish a mouse model of intrauterine infection with MCMV have been reviewed extensively (Osborn 1982; Hudson 1994a) and will not be described in detail. These studies failed to detect in utero infection of mouse embryos following MCMV infection of pregnant female mice, using a variety of inoculation routes, sampling times, and assays for viral detection (Mannini and Medearis 1961; Johnson 1969; Landsdown and Brown 1978). However, several studies using latently infected mothers reported low-level or latent infection of fetuses (Chantler et al.
TABLE 1-1
SUITABILITY OF MCMV AS A MODEL OF HCMV-INDUCED DISEASE Human disease
Features of mouse model
Congenital infection
No transplacental infection, but fetal wastage following maternal infection resembles human condition. MCMV introduced by artificial means affects fetal development of neural tube. No congenital infection. Artificial inoculation results in microphthalmia and cerebral atrophy in fetal mice. Infection of newborns i.c. results in severe necrotizing ependymitis, encephalitis, and cerebral malformation, as in human congenital infection. Infection of adult nude or SCID but not normal mice results in CNS infection and encephalitis. Induction of retinitis in adults requires direct supraciliary intra-ocular infection. Progressive focal necrotizing retinitis observed, enhanced by CD8+ depletion. Mouse not a model for hearing loss due to congenital infection. Infection at birth results in perilabyrinthitis, whereas congenitally infected humans develop endolabyrinthitis. Resembles the human disease in essential features; arises after immunosupression, but viral replication is only indirectly related to pathology. Severity of disease reduced but not prevented by antiviral drugs. Antiviral T cell response necessary for disease development, but the relative importance of CD4 or CD8+ T cells is controversial. Resembles human disease. In adults, virus replicates in hepatocytes. Inflammatory foci are composed mainly of T cells; are initiated by NK cells, IFN-γ, and MIP-α. NK cell–produced IFN-γ important in controlling murine infection. In lethal infection, direct viral cytopathic effects are dominant. High TNF-α and IFN-γ titers may have a toxic rather than beneficial effect. Resembles the human disease observed after heart transplantation. Normal mice experience acute and persistent cardiac inflammation mediated mainly by CD8+ T cells in the presence of very low levels of virus. Viral dose and host genetics influence outcome. MCMV-induced cardiac myosin-autoantibodies may contribute, although no parallels described in humans. HCMV may contribute to disease by enhancing inflammation in blood vessels, augmenting migration of smooth muscle cells, and promoting their proliferation by blocking apoptosis. Mouse model demonstrates similar features but is mainly used for mechanistic studies using gene knockout mice. Adrenal infection in immunodeficient mice resembles adrenal necrosis detected in HIV/AIDS patients. Resembles effect of HCMV. In model of irradiation and auto-reconstitution of bone marrow, infusion of antiviral CD8+ T cells prevents hemopoietic failure and provides basis for human immunocytotherapy. MCMV prevents marrow reconstitution by inhibiting production of hemopoietic cytokines by stromal cells.
Infection of CNS
Retinitis Effect on the developing ear Interstitial pneumonitis
Hepatitis
Myocarditis
Atherosclerosis
Adrenalitis Hemopoietic failure
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
1979; Baskar et al. 1985), suggesting that either transplacental infection of a reactivated virus from latently infected mothers or transmission within the germ line had occurred. It has not been established why MCMV does not readily cross the placenta. The murine placenta is hemotrichorial, with multiple cell layers separating the maternal and fetal circulations, in contrast to the hemodichorial structure of the human (and guinea pig) placenta (Pereira et al. 2005). This is considered the most likely reason for the failure of MCMV to cross the placenta. However, there is no evidence to suggest that the murine placenta is intrinsically resistant to the passage of viruses. Polyoma virus (McCance and Mims 1977), ectromelia (Mims 1969), lymphocytic choriomeningitis virus (Traub 1936; Lehmann-Grube 1964), and Coxsackie viruses (Selzer 1969) all cross the murine placenta. If the structure of the murine placenta is a barrier to viral transmission to the fetus, it must be much more effective against MCMV. It is also possible that the Smith strain, which has been used in all studies, has lost its ability to cross the placenta during its adaptation to passage in laboratory mice or cell culture. Further studies of intrauterine infection with MCMV should be undertaken using a variety of viral isolates recently derived from wild mice. Furthermore, the ability of MCMV to infect fetal mice in utero could be investigated in free-living wild mice that are naturally infected with MCMV. Despite the inability of MCMV to cross the placenta and establish productive infection of the fetuses, the infection of pregnant females results in extensive fetal wastage with increased fetal death, fetal resorption, delayed births, and reduced body weight of newborn pups (Mannini and Medearis 1961; Medearis 1964; Johnson 1969; Landsdown and Brown 1978; Huang et al. 1986; Fitzgerald and Shellam 1991; Neighbour 1976). This effect was dose-dependent and varied with the age of the embryo and the timing of the infection (Huang et al. 1986). Time-dependent effects were also observed when pregnant females were infected with MCMV at earlier times (Neighbour 1976). When the infection was timed to coincide with ovulation and pre-implantation development, implantation frequently failed to occur. However, when mice were infected 14 days before or 4 days after mating, the pregnancy rate and preimplantation loss was unaffected. The timing of infection is also important in CMV infections in other species (Griffiths and Hsiung 1980; Boppana et al. 1993; Stagno et al. 1986). A role for genetically determined host resistance in modulating the effect of MCMV infection on fetal outcome was observed in pregnant BALB/c, BALB.K, and CBA mice that were inoculated on day 8 of pregnancy (Fitzgerald and Shellam 1991). In susceptible BALB/c, the percentage of dead or resorbed fetuses was significantly higher in infected compared with control mothers, while in the two genetically resistant strains, no adverse effect of infection was observed. Maternal infection resulted in reduced fetal body weight in all strains, particularly in genetically susceptible BALB/c mice. Infectious virus was not detected in the fetuses either by plaque or co-cultivation assays. Therefore it is likely that the effect of MCMV infection
19
on the fetuses was indirect, reflecting either placental dysfunction following infection or maternal illness affecting maternal metabolism or the immune system. In women who experience a primary HCMV infection during pregnancy, increased fetal loss in the absence of infection of the fetus or the placenta has been reported (Griffiths and Baboonian 1984). Thus, while MCMV does not readily infect fetuses transplacentally, it mimics the indirect effects of maternal HCMV infection on fetal outcome. The study of Fitzgerald and Shellam (1991) also established that genetically determined innate resistance to MCMV influences the level of infection within the fetus itself following direct in utero inoculation of MCMV. Viral titers in fetuses, which were directly inoculated in utero at day 15 of gestation, reflected the resistance status of the mouse strain. These findings suggest that if genetic factors do play a role in modulating HCMV infection in humans, they could influence the outcome of primary HCMV infection during pregnancy. EFFECT OF MCMV ON EMBRYONIC DEVELOPMENT When MCMV was inoculated into the endometrial lumina of pregnant mice 4 days after coitum at the time of embryonic implantation, fetal development was affected. Litter sizes were reduced, and the incidence of abnormal fetuses was significantly increased (Baskar et al. 1987). The fetal damage included maldevelopment of the neural tube and head and ectodermal abnormalities. Infectious MCMV was recovered from the embryos by co-cultivation with MEF. To mimic sperm-mediated MCMV transfer to ova, MCMV DNA was micro-injected into uninfected fertilized murine ova that were cultured and transferred to pseudo-pregnant mice (Baskar et al. 1993). Again, reduction in litter size, fetal growth retardation, maldevelopment, or death as observed. Using PCR and in situ hybridization, MCMV was found in the brains, skin, and salivary glands of the fetuses. However, a recent study in which MCMV was inoculated intratesticularly found no evidence of virus transmission to fertilized oocytes, blastocysts, fetal tissues, or newborn animals following mating of infected males with uninfected females (Tebourbi et al. 2001). Nonetheless, despite the artificial nature of some of these studies, they show that the presence of MCMV in the genital tract at the time of embryonic implantation has the potential not only to initiate fetal infection but also to interfere with morphogenesis. INFECTION OF GONADAL TISSUES MCMV infects the gonadal tissues of both sexes. In infected males, MCMV was recovered from the epididymal sperm, seminal vesicles, and testes (Neighbour and Fraser 1978; Baskar et al. 1986) as already discussed, and was present in the testes during acute infection in athymic nude mice and in the testes of latently infected immunocompetent mice (Dutko and Oldstone 1979). MCMV was found within Leydig cells (Baskar et al. 1983) and in spermatocytes and spermatozoa (Dutko and Oldstone 1979). In females,
20
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
MCMV infects the ovaries (Mims and Gould 1979), where it has been located in stromal cells (McCordick and Smith 1936; Brautigam and Oldstone 1980). These studies establish the potential for the sexual transmission of MCMV. EFFECT ON REPRODUCTION The presence of MCMV in gonadal tissues suggests that it may affect reproductive outcomes. While this has not been well studied in laboratory mice, it has been examined in laboratory-reared, wild-derived Mus domesticus housed in secure outdoor enclosures (Farroway et al. 2002). MCMV spread rapidly through founder populations and their offspring. There was little impact on breeding performance. However, the body condition of young mice born in enclosures with MCMV was affected, and where 2 strains of MCMV were present, there was a 20% reduction in the survival of young males. Nonetheless, there was no effect of MCMV on the rate of increase of the populations overall. 2.
Interstitial Pneumonitis
Cytomegalovirus-associated interstitial pneumonitis is a frequent complication in immunosuppressed patients undergoing allogeneic transplantation, especially bone marrow transplantation (BMT). While the incidence of HCMV infection is similar after autologous and allogeneic bone marrow transplantation, HCMV-associated interstitial pneumonitis is more common after allogeneic bone marrow transplantation with concomitant graft versus host disease, where fatalities are often high (Wingard et al. 1988; Meyers et al. 1986; Miller et al. 1986). A diffuse infiltration of mononuclear cells into the lung parenchyma leading to congestion of the septa are features of this disease. The etiology of the disease is complex. Rather than simply reflecting the lack of host control of this opportunistic agent due to immunosuppression, the disease may be immunopathological in nature (Grundy et al. 1987). The replication of HCMV in the lung appears to be only indirectly related to the development of pathological effects, and a host antiviral immune response is an essential component in the development of the disease. MOUSE MODEL The use of MCMV has been very important in understanding the etiology of this disease. Although interstitial pneumonitis has been reported in the absence of immunosuppression in adult BRVS mice following i.n. MCMV infection (Jordan 1978), immunosuppression is usually required. Following the i.n. inoculation of 8 × 104 pfu of MCMV in adult BALB/c mice, followed by a single dose of cyclophosphamide, extensive interstitial pneumonitis developed 10–14 days later (Shanley et al. 1982). However, if the administration of cyclophosphamide was continued, interstitial pneumonitis was not observed, even though the titers of MCMV were increased. The pathology of the disease resembled that seen in humans; mononuclear cells infiltrated the alveolar septa, reducing the alveolar spaces, and accumulation of amorphous eosinophilic material was observed. Lung weights were markedly increased
(Shanley et al. 1982). While the severity of the disease was proportional to virus production, the use of antiviral drugs reduced but did not prevent the development of the disease (Shanley and Pesanti 1985; Shanley et al. 1985, 1988). Other means of immunosuppression such as antilymphocyte serum (Brody and Craighead 1974), graft versus host disease (Shanley et al. 1987, 1988), irradiation (Reddehase et al. 1985), or malnutrition (Price et al. 1990) have also been successful in inducing pneumonitis in infected mice. In these studies, pneumonitis was associated with an influx of T cells into the lungs (Shanley et al. 1987; Shanley and Ballas 1985). In contrast, T cell–deficient athymic nude mice infected with MCMV developed pneumonitis with a progressive focal nodular interstitial pattern, which was quite different from the diffuse pneumonitis seen in the cyclophosphamide model (Shanley et al. 1997). Coalescence of focal areas in the lungs of nude mice suggested that lung pathology was related directly to viral damage in these mice (Shanley et al. 1997). PNEUMONITIS AS AN IMMUNOPATHOLOGICAL DISEASE Using data obtained from the mouse model and from clinical reports, Grundy and colleagues proposed that CMV-pneumonitis is an immunopathological disease that arises following immunosuppression, is triggered by CMV infection in the lung, and is mediated by T cells responding to viral antigens expressed in lung tissue. Since antiviral therapy reduced but did not eliminate pneumonitis, it was suggested that after inducing the response, the continued presence of the virus was not necessary (Grundy et al. 1987, 1985; Grundy 1990). However, this hypothesis has proved to be controversial (Barry et al. 2000; Morris 1993; Podlech et al. 2000). Identifying CD4+ T cells as critical for the development of immunopathology in Grundy’s hypothesis, Barry and colleagues have contended that CD4+ T cells are only present at very low levels in the blood soon after transplantation in humans, when most cases of pneumonitis occur, and that NK cells and CD8+ T cells are the major cell populations present in the lungs (Barry et al. 2000). Podlech and colleagues have emphasized the strongly protective role of antiviral CD8+ T cells in controlling MCMV in the lungs and their major role in controlling HCMV in transplant recipients (Podlech et al. 2000). Instead, it is proposed that early after BMT, when CD8+ T cell responses are low, CMV replicates in the lung without restriction, inducing a cytokine storm involving interferon-γ from NK cells stimulating the release of TNF-α from alveolar macrophages (Barry et al. 2000). Morris takes a somewhat different view, arguing that the immunosuppression associated with transplantation increases the likelihood that CMV viremia will result in pneumonia and that protective CD8+ T cell and NK cell responses will be reduced. The pathological effects in the lung of CMV, irradiation, and cytotoxic drugs act together to enhance the development of pneumonia (Morris 1993). More research is needed to resolve these opposing concepts and to account for the relative lack of interstitial pneumonitis in HCMV-infected AIDS patients. The value of the mouse model
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
is clearly evident. It provided the data for the original concept and also the proof that the reconstitution of CD8+ T cells after BMT was responsible for controlling CMV infection (Reddehase et al. 1985). It will also be valuable for elucidating the antigenic targets of the response in the lung, the role of cytokines, and the interplay between the cell types involved. The use of strains of MCMV recently derived from wild mice may also enhance the relevance of the model. 3.
Hepatitis
MCMV-induced hepatitis in mice has been widely studied as a model of hepatitis induced by HCMV. In humans, the liver is frequently infected in cases of disseminated HCMV infection, both in immunocompetent and immunocompromised individuals (Varani and Landini 2002). In vitro, HCMV replicates in hepatocytes, which are the major target cells, producing infectious virus. In vivo, the virus also replicates in bile duct epithelial cells and stromal cells (Sinzger et al. 1999). Early and late HCMV antigens have been detected in the livers of acutely infected patients (Sinzger et al. 1999; Barkholt et al. 1994). Thus, human liver cells can be infected by HCMV in vivo and in vitro. In symptomatic congenital HCMV infection, hepatitis is common. Clinical features include hepatomegaly, which may persist for months, increased levels of serum transaminases, and hyperbilirubinemia with jaundice. Cytomegalic cells are seen most commonly in the bile duct epithelium and infrequently in hepatocytes, indicating that the bile duct epithelium is an important site of HCMV replication in infants (Kosai et al. 1991). In immunocompetent adults, HCMV occasionally induces mononucleosis (Britt and Alford 1996), and in these cases the liver is often involved, with accompanying fever and moderately elevated transaminase activity. A mild, nonspecific hepatitis with a mononuclear cell infiltrate is observed (Ten Napel et al. 1984). Cytotoxic T cells are the predominant cell type (Pape et al. 1983), and may be responsible for rapidly clearing HCMV infected cells in the liver (Pape et al. 1983; Asanuma et al. 1999). Cytomegalovirus infection of the liver is more pronounced in transplant patients. Up to 20% of liver transplant patients have CMV hepatitis (Paya et al. 1989) and, in severe cases, a high HCMV viremia, high levels of serum transaminases, and prolonged fever is common. Histopathologic studies show the presence of typical inclusion bodies in hepatocytes, but with an inflammatory response that is reduced compared to hepatitis in immunocompetent individuals. Early and late HCMV antigens have been detected, providing evidence of productive infection. The hepatic cell damage observed in transplant patients is thought to be caused directly by the virus rather than by the inflammatory response (Varani and Landini 2002). Interestingly, the liver is rarely involved in HCMV infection in HIV-positive patients. Given the morbidity associated with HCMV hepatitis, further research is needed and the study of MCMV-induced hepatitis in mice is amply justified.
21
MCMV HEPATITIS IN MICE The ability of MCMV to infect the liver and induce hepatitis was recognized in the early studies of this virus (McCordick and Smith 1936; Ruebner et al. 1964, 1966; Henson et al. 1967). The liver is infected soon after i.p. inoculation. Hepatocytes become infected by 24 hours p.i., with the extent of infection depending on the dose and route of inoculation. Kupffer cells do not appear to be infected when salivary gland virus is used (Mims and Gould 1978a). At sublethal doses of MCMV, foci of infection develop and increase in size as inflammatory cells are attracted to sites of virus replication. Genetically determined host resistance influences this response; in highly resistant CBA mice, few inflammatory cells were observed compared with the less resistant C57BL strain (Selgrade and Osborn 1974). Viral titers peak in the liver at 2–4 days p.i., with higher titers detected in the livers of susceptible BALB/c than resistant C3H or CBA mice (Allan and Shellam 1984; Mercer and Spector 1986). Infection is more quickly resolved in the livers of resistant mice. The effect of MCMV infection on the liver has been studied in various ways. Foci of infected cells and cells with intranuclear inclusions have been analyzed histologically (Papadimitriou et al. 1982; Olver et al. 1994; Orange et al. 1995). Nuclear DNA has been measured by cytophotometry on liver sections (Papadimitriou and Shellam 1981), and the extent and location of viral antigen expression has been measured by immunocytochemistry (Shanley et al. 1993; Trgovcich et al. 2000; Olver et al. 1994). The measurement of serum transaminase levels has also proved valuable (Papadimitriou et al. 1982; Shanley et al. 1993; Bolger et al. 1999). The levels of alanine transaminase (ALT) and aspartate transaminase (AST) in serum are elevated by MCMV infection, but ALT is more liver specific and appears to be a more sensitive measure of MCMV-induced liver damage (Bolger et al. 1999). The liver is a major target of MCMV infection following i.p. or i.v. inoculation. Several studies have shown that severe hepatitis, which is induced by lethal doses of MCMV in susceptible strains of mice, is likely to be the cause of the early mortality observed (Shanley et al. 1993; Trgovcich et al. 2000). At doses of 1 × 105 pfu and higher, BALB/c mice developed high titers of MCMV in the liver by day 2 that persisted until the deaths of the mice from day 5. Large areas of necrosis were evident in liver sections from day 2 p.i., becoming confluent by day 5 PI with evidence of acute hepatic dystrophy and hypoxic necrosis (Trgovcich et al. 2000). Serum levels of AST were markedly augmented. However, inflammatory infiltrates were modest, suggesting that direct cytopathic effects rather than an immunopathological response were responsible for the liver damage. The very high serum levels of interferon-γ and TNF-α, which developed just before mortalities occurred, were taken as evidence of a toxic rather than a protective role for these cytokines in lethal MCMV infection (Trgovcich et al. 2000). At sublethal doses, titers of MCMV were lower and were reduced by day 5, serum AST titers were not elevated significantly, and, although interferon γ levels were elevated by day 4, they began
22
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
to decline by day 5. Genetically resistant B10.BR mice exhibited very low MCMV titers in the liver and little or no elevation of transaminases following i.p. inoculation of a dose of MCMV that was lethal for susceptible BALB/c mice (Shanley et al. 1993). During sublethal infection, however, different mechanisms are involved. Viral titers in the liver declined by day 5 p.i., and foci of inflammatory cells accumulated over the first week p.i., clearing after day 11 with a few foci remaining for 7–8 months in susceptible strains (Olver et al. 1994). From early times p.i. Mac-1+ cells were present in cellular infiltrates, and CD4+ and CD8+ T cells were present after the second day, peaking at day 7 and declining to control levels in resistant CBA and C57BL/6 mice by day 56. However, in susceptible BALB/c and A/J mice, CD8+ T cells persisted for over 30 weeks, dispersed throughout the liver parenchyma. Interestingly, in NK cell–deficient beige mice, the levels of CD8+ T cells were higher at day 7 in both C57BL/6 and CBA mice (Olver et al. 1994). NK cells are important in controlling MCMV in the liver as well as in other tissues. In C57BL/6 beige mice, MCMV replication in the liver was increased (Shellam et al. 1981) and hepatitis was more pronounced (Papadimitriou et al. 1982). NK cells with the distinctive large granular lymphocyte morphology are attracted to the liver during MCMV infection (McIntyre and Welsh 1987). Depletion of NK cells resulted in increased viral titers and hepatitis (Orange et al. 1995; Bukowski et al. 1983). Interestingly, the production of IFN-γ by NK cells was shown to be important in the control of MCMV in the liver (Orange et al. 1995). There is a dichotomy in the effector mechanisms of NK cells, with the production of IFN-γ by NK cells being the major NK effector mechanism in the liver, while in the spleen, cytotoxicity involving perforin is the means by which NK cells control MCMV infection (Tay and Welsh 1997). In the liver, IFN-γ appears to control infection at least in part by the induction of the antiviral molecule nitric oxide (Tay and Welsh 1997). However, very high levels of IFN-γ may also have adverse effects (Pomeroy et al. 1998). A role for TNF-α in the exacerbation of MCMV- induced liver disease has been demonstrated. It caused early hepatic necrosis independent of NK cell or T cell responses, and induced pathology that was largely responsible for liver damage at early times after infection. TNF-α was required for increased levels of serum transaminases but not for the development of inflammatory foci in the liver (Orange et al. 1997). It appears that TNF-α was also responsible for the progressive MCMV-induced liver disease in immunodeficient mice, which resulted in the death of the animals (Orange et al. 1997). 4.
Cardiovascular Diseases
MYOCARDITIS Viral infection has been recognized as one of the etiological agents of myocarditis in humans (Friman et al. 1995; Feldman and McNamara 2000), which may progress to dilated cardiomyopathy. This is a serious and often terminal
condition (Friman and Fohlman 1997; Kawai 1999). Viruses commonly associated with myocarditis include the picornaviruses, particularly Coxsackie viruses, orthomyxoviruses, and herpesviruses (Friman et al. 1995; Huber 1997). HCMV infection has been associated with the development of myocarditis (Wink and Schmitz 1980; Cohen and Corey 1985); cytomegalic cells with intranuclear inclusions are found in the endothelium and myocardial cells in the hearts of infants and adults with generalized CMV disease (Ahvenainen 1952; Myerson et al. 1984). CMV infection is a common major complication in heart transplant recipients and AIDS patients, causing increased rates of allograft rejection and cardiac dysfunction (Fernando et al. 1994; Herskowitz et al. 1994). Furthermore, heart transplant recipients experiencing a primary HCMV infection following transplantation have a 46% incidence of developing myocarditis (Arbustini et al. 1992). Myocardial damage after HCMV infection may persist and lead to dilated cardiomyopathy (Ando et al. 1992; Maisch et al. 1993). However, myocarditis remains difficult to diagnose, and a viral etiology is even more difficult to establish (Feldman and McNamara 2000; Aretz 1987; Lieberman et al. 1993). Hence, an animal model of cytomegalovirus myocarditis is important for establishing the pathogenesis of disease. The induction of myocarditis in mice by MCMV has been well studied and has provided valuable insights into the mechanisms of virusinduced cardiac damage (reviewed in Fairweather et al. 2001). MCMV infection results in the appearance of inflammatory lesions in the heart of both adult (Mims and Gould 1979; Papadimitriou et al. 1982; Bartholomaeus et al. 1988; Leung et al. 1986; Gang et al. 1986; Craighead et al. 1991; Price et al. 1991) and newborn mice (Lussier 1974; Fitzgerald et al. 1990; Price et al. 1991). These lesions persist and may become calcified (Lussier 1974; Gang et al. 1986). The main features of the model are exemplified in a recent study (Lenzo et al. 2002). In adult BALB/c mice, the inoculation of 1 × 104 pfu of salivary gland–derived MCMV [K181 (Perth)] results in myocarditis. At days 3–5 p.i., a few inflammatory foci are seen, without histological evidence of myocyte necrosis. Soon afterward, a mixed cellular infiltrate comprising lymphocytes, macrophages, and polymorphonuclear leucocytes develops around areas of myocyte necrosis. This acute focal myocarditis peaks at day 7 and declines by day 21. A chronic phase of myocarditis, which is characterized by a more dispersed interstitial inflammatory infiltrate with larger areas of necrosis, develops by day 28 p.i., is still prominent at day 56 (Lenzo et al. 2002), and persists until at least day 100 (Price et al. 1991; Lawson et al. 1990). In contrast, C57BL/6 mice inoculated with the same dose develop only a minimal acute response, with minor focal inflammation at days 5–7 p.i. and complete resolution of myocarditis by about 14 days. The size of the inflammatory foci was also generally smaller in C57BL/6. By measuring the number of inflammatory foci per heart section, clear-cut differences in the kinetics of the response and in mouse strain variations in the response could be determined (Lenzo et al. 2002).
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
Other studies have also investigated mouse strain variations in the development of myocarditis (Lawson et al. 1990; Price et al. 1991). In order to separate the effects of genetically determined resistance to MCMV infection from any genetic effects that might influence the development of myocarditis per se, adult mice received doses of MCMV that were adjusted in proportion to their sensitivity to lethal infection (Price et al. 1991). Genetically resistant BALB.K and C3H mice bearing the H-2k haplotype developed myocarditis that was as severe as that in susceptible BALB/c mice, indicating that in this kind of study, H-2 genes did not affect resistance to myocarditis other than through modulation of the viral load. The level of MCMV replication in the heart of adult BALB/c mice is, however, very low (Craighead et al. 1991, 1992; Gang et al. 1986; Lenzo et al. 2002; Lawson et al. 1990). Infectious virus generally is cleared from cardiac tissue by 7–10 days p.i., during which time apoptosis of cardiac cells is observed. It was also shown that MCMV replicates in and lyses cardiac myocytes in vitro (Lenzo et al. 2002; Lawson et al. 1990). Despite the absence of infectious virus, the ie1 and gB genes of MCMV can be detected in the heart for up to 100 days in infected adult BALB/c mice (Lenzo et al. 2002). The presence of viral iel and gB RNA transcripts up to day 35 indicates active viral replication, even though this cannot be detected by plaque assay. Beyond this time during chronic myocarditis (days 35–100 PI), viral ie1 RNA transcripts are detected, while viral gB RNA transcripts are not, suggesting that a latent infection had been established in the heart. The ability of MCMV to establish latency in heart tissue has been demonstrated previously (Wilson et al. 1985; Rubin et al. 1984). The cellular infiltrates around infected or necrotic cells consist of Mac-1+ cells and CD4+ T cells, as well as CD8+ T cells, which predominate. Inflammatory T cells persist in the hearts of infected BALB/c mice for long periods (Price et al. 1991; Lenzo et al. 2002). ROLE OF T CELLS AND NK CELLS IN MYOCARDITIS A primary role for T cells in the development of myocarditis has been established. T cell–deficient BALB/c nu/nu mice did not develop myocarditis during acute infection (Lawson et al. 1989) and, similarly, immunocompetent mice in which CD4+ and CD8+ T cells were depleted failed to develop the disease by day 9 p.i., when the acute phase of the disease occurs (Fairweather et al. 2001). However, the relative importance of CD4+ and CD8+ T cells remains unclear. In BALB/c mice, monoclonal antibody depletion or reconstitution of irradiated thymectomized mice with naive CD4+ or CD8+ T cells before MCMV infection demonstrated that CD4+ T cells were responsible for myocarditis at day 21 p.i. (Craighead et al. 1992). However, a similar study using antibody depletion demonstrated a role for both subtypes, but with CD8+ cells playing the major role at day 9 p.i. (Fairweather et al. 2001). Interestingly, in the latter study, a clear protective role for NK1.1+ NK cells was established. The depletion of NK1.1+ NK cells from C57BL/6 or
23
BALB.B6–Cmv1r congenic mice resulted in high levels of myocarditis, comparable to the levels in BALB/c mice (Fairweather et al. 2001). Presumably, NK cells and T cells are protective through the reduction of the viral load that reaches the heart, while T cells also accumulate around the sites of myocardial infection and initiate disease. ROLE OF ANTIBODIES Antibodies may also play a role in myocarditis. In response to MCMV infection, both BALB/c and C57BL/6 mice produce antibodies that recognize myosin. However, only susceptible BALB/c mice produce antibodies during chronic myocarditis that interact with the cardiac isoform of myosin (O’Donoghue et al. 1990; Lawson et al. 1992). The passive transfer of affinity-isolated IgG antibodies to cardiac myosin from late immune sera of infected BALB/c mice induces cellular inflammation and myocardial necrosis in naive mice (Lawson et al. 1992). These cardiac myosin–reactive antibodies cross-react with MCMV polypeptides and the S2 region of cardiac myosin (Lawson et al. 1992; Fairweather et al. 1998; Lawson 2000). Furthermore, several monoclonal antibodies that were raised against MCMV and neutralize the virus also react with cardiac myosin (Lawson et al. 1991). Interestingly, cardiac myosin itself is an autoantigen that can induce myocarditis in mice in the absence of viral infection, inducing high titer anti-myosin antibodies (Lawson et al. 1992). Thus, the candidate autoantigen, cardiac myosin, is capable of inducing immunopathological responses in the heart. On the basis of these findings, a possible role for molecular mimicry between MCMV proteins and cardiac myosin in the chronic phase of MCMV myocarditis has been proposed (Lawson 2000). Using evidence obtained from the Coxsackievirus B3 model of myocarditis in mice, which resembles MCMV myocarditis in many ways (Fairweather et al. 2001), the effect of the immunomodulator lipopolysaccharide (LPS) on MCMV myocarditis has been studied (Lenzo, Fairweather, et al. 2001). LPS enhanced myocarditis and serum levels of TNF in BALB/c mice and in the resistant C57BL/6 strain. However, the role of cytokines in MCMV myocarditis in the absence of immunomodulators remains to be determined. RELEVANCE OF THE MOUSE MODEL As well as providing insights into HCMV myocarditis, the relevance of the laboratory model is demonstrated by two additional studies. First, six genetically different isolates of MCMV obtained from wild Mus domesticus induced typical myocarditis in inbred BALB/c mice. Furthermore, myocarditis was detected histologically in 30% of wild mice, all of which were seropositive for MCMV. This does not necessarily imply that the myocarditis was due to MCMV infection. Sera from BALB/c mice infected with wild isolates as well as sera from free-living wild mice, contained antibodies that reacted with MCMV and the S2 region of cardiac myosin (Fairweather et al. 1998). These observations suggest that myocarditis induced in the laboratory model resembles the disease that occurs in free-living wild mice.
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GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
Second, the mouse model has been useful for determining the source of primary CMV infection in cardiac transplantation. Hearts from actively or latently infected mice were a source of primary infection in immunosuppressed recipients (Rubin et al. 1984). This study provides valuable information about CMV infection in human transplantation. ATHEROSCLEROSIS A number of infectious agents have been implicated in atherosclerosis (Mattila et al. 1998), and accumulating evidence supports a role for HCMV. The association between HCMV and atherosclerosis has been recently reviewed (Degre 2002). It is based on epidemiological studies and the demonstration of HCMV antigens and DNA in atherosclerotic plaque tissue, as well as on investigations into the mechanisms by which HCMV infection of vascular endothelial and smooth muscle cells could augment the development of atherosclerosis or restenosis. Perhaps because atherosclerosis is a complex multifactorial disease in which infection is only one of several etiologies, evidence for a causal role for HCMV is not yet strong. However, HCMV infection could contribute in several ways, including augmenting inflammation through the activation of NF-κb (Speir et al. 1998) and the release of proinflammatory cytokines and chemokines, enhancing the migration of smooth muscle cells (Zhou et al. 1999) and binding and inhibiting p53, thus blocking apoptosis and promoting the proliferation of smooth muscle cells (Tanaka et al. 1999). HCMV may also act as a prothrombotic agent (Pryzdial and Wright 1994). The ability of RCMV to induce vascular disease in rats has made it the best animal model in which to study the role of cytomegaloviruses in atherosclerosis and transplantation vasculopathy (Span et al. 1992; Kloover et al. 2000; Martelius et al. 2000; Epstein et al. 1996). However, the mouse model has the advantage of the availability of a large number of gene knockout strains and viral mutants for mechanistic studies. As a result, there has been an increase in the use of this model recently. MCMV infection of BALB/c mice results in the expression of MCMV antigens in vascular smooth muscle and endothelial cells, with inflammatory lesions and an increase in low-density lipoprotein cholesterol in the serum (Berencsi et al. 1998; Dangler et al. 1995). In C57BL/6 apolipoprotein-E knockout mice that are genetically susceptible to the development of atherosclerosis, MCMV infection causes a significant increase in the size of atherosclerotic lesions (Hsich et al. 2001), in the expression of pro-atherosclerotic genes in the aorta (Burnett et al. 2004), and in the levels of monocyte chemoattractant protein-1 (MCP-1), which is known to exacerbate atherogenesis (Rott et al. 2001). MCMV infection induced IL-6 from endothelial cells, which enhanced the production of MCP-1 (Rott et al. 2003). In transgenic mice over-expressing MCP-1 in the myocardium and pulmonary arteries, MCMV infection accelerated myocarditis and pulmonary artery inflammation (Froberg et al. 2001). A role for MCMV M33, a G protein–coupled receptor homolog (Davis-Poynter et al. 1997) resembling HCMV US28, in the migration of vascular smooth
muscle cells has been established (Melnychuk et al. 2005). The contribution of MCMV to atherogenesis has been recently reviewed (Froberg 2004). 5.
Adrenalitis
Early studies reported adrenal involvement following MCMV infection (McCordick and Smith 1936; Mims and Gould 1979), but subsequent reports differ about the level of infection achieved. Using the Smith strain, Shanley found no evidence of infection in the adrenal glands of immunocompetent adult BALB/c mice (Shanley and Pesanti 1986). In contrast, the K181 (Perth) strain of MCMV established acute infection in the adrenal glands of adult BALB/c (Price et al. 1996). However, when athymic nude or irradiated BALB/c mice were used, MCMV replicated to high titers in the adrenals (Shanley and Pesanti 1986; Reddehase et al. 1988). This resembled the situation with AIDS patients, in whom extensive adrenal necrosis associated with presumed CMV infection is observed (Tapper et al. 1984), and loss of adrenal function may occur (Pulakhandam and Dincsoy 1990). In MCMV-infected nude mice, the progressive destruction of the adrenals could be arrested by the adoptive transfer of normal spleen cells (Shanley and Pesanti 1986) or CD4+ but not CD8+ T cells from immune mice (Shanley 1987). In irradiated mice, CD8+ but not CD4+ T cells from immune mice protected against adrenal infection (Reddehase et al. 1988). Adrenal infection did not appear to compromise adrenal function in immunocompetent BALB/c mice, as assessed by the levels of circulating ACTH (Price et al. 1996). The levels of corticosterone increased after infection. Also in this strain, an adrenocortical response was important for survival, as adrenalectomized mice died at doses up to 5-fold lower than those they could usually tolerate (Price et al. 1996). A discussion of the physiological role of the adrenal gland in MCMV infection is beyond the scope of this review. A recent review (Silverman et al. 2005) covers this topic comprehensively. 6.
Infection of the Central Nervous System
In humans, HCMV infection of the CNS occurs during severe intrauterine infection. At birth, the majority of these infants show evidence of microcephaly, neurologic abnormalities, periventricular calcification, mental retardation, sensorineural hearing loss, and impaired vision (Bopana et al. 1992; Boppana et al. 1997; Ramsay et al. 1991). CNS involvement also occurs as a complication of HCMV infection of AIDS patients (Cheung and Teich 1999), in whom glial and neuronal cells may be infected (Wiley et al. 1986) and ependymal and subependymal regions of the parenchyma are involved. The retina and cochlea are also often infected (Keithley et al. 1989; Rabb et al. 1992). The mouse does not provide a natural model of congenital infection of the CNS. To circumvent this, Tsutsui (1995) directly
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
infected fetuses in utero at various stages of gestation. Microphthalmia and cerebral atrophy developed in fetuses inoculated at 8.5 days of gestation, and viral-infected cells were widely distributed in the mesenchyme, suggesting that mesenchymal infection may be critical in disrupting organogenesis. Infection at day 15.5 led to massive necrosis of the brain. In another investigation, infection of CNS stem cells prepared from fetal brains suppressed growth and inhibited neuronal differentiation, suggesting that these outcomes could be the primary causes of brain disorders in congenital CMV infection (Kosugi et al. 2000). The cerebral infection of newborn mice also gives rise to changes similar to those seen in human congenital infections. Intracerebral inoculation of suckling mice resulted in severe necrotizing ependymitis and encephalitis followed by marked cerebral malformation (Lussier 1973; Lussier 1975a). Clinical signs include mild ataxia, hind leg weakness, and runting. Necrosis and mineralization of the subventricular zone, cerebral cortex, and hippocampus were detected at autopsy. In vitro studies have established that most cell types of the fetal and adult brain support MCMV infection (Schneider et al. 1972; Willson et al. 1974). The ability of MCMV to infect the CNS of adult athymic nude or SCID mice has been studied as a parallel to HCMV infection of the CNS in AIDS patients (Reuter et al. 2004). While immunocompetent mice were resistant to CNS infection following peripheral inoculation, immunodeficient mice showed evidence of CNS infection from 21 days p.i. Many different cell types became infected in the CNS, resulting in meningitis, encephalitis, and choroiditis. Using MCMV tagged with green fluorescent protein, MCMV-infected Mac3+/CD45+ leucocytes were identified in the blood stream and in the brain, suggesting that these cells were the source of infection of the CNS. Despite the ability to establish CNS infection in adult, immunodeficient mice, it appears nonetheless that the cells of the developing brain are intrinsically more susceptible to MCMV infection (van den Pol et al. 2002). 7.
Retinitis
Interest in ocular infections with MCMV has arisen because of the emergence of HCMV-associated retinitis in patients who are immunosuppressed by HIV infection. Prior to the use of highly active antiretiroviral therapy (HAART) to control HIV, HCMV retinitis was the leading cause of vision loss and blindness among patients with AIDS (Cunningham and Margolis 1998), and it remains a chronic, sight-threatening ophthalmic problem among HIV patients who do not respond to HAART (Jabs and Bartlett 1997). Ocular abnormalities including chorioretinitis also affect 20%–25% of symptomatically congenitally infected infants (Pass et al. 1980). The features of HCMV retinitis include typical inclusions and cytomegaly of retinal neurons and retinal pigment epithelium, inflammation in the retina consisting of neutrophils or mononuclear cells, and full-thickness
25
retinal necrosis and necrosis of the retinal pigment epithelium (de Venecia et al. 1971; Egbert et al. 1980; Pepose et al. 1985). In AIDS patients with HCMV retinitis, choroiditis is also frequently observed and, less commonly, HCMV infection of cells of the optic nerve (Pepose et al. 1985; Grossniklaus et al. 1987). In immunocompetent mice infected i.p. with MCMV, the virus was recovered from homogenates of eye tissue over the first week and from intraocular fluids most frequently on days 11–21 and from explant cultures of the eye and optic nerve as long as 120 days after infection. However, ocular abnormalities were not detected, and the focal retinal necrosis that is common in HCMV retinitis in humans was not observed (Bale et al. 1984). Similarly, MCMV inoculation by the intraocular route (anterior chamber or intravitreal) results in only minimal disease (Hayashi et al. 1985; Bale et al. 1990), although latent MCMV was detected in ocular tissue (Bale et al. 1990). The use of immunosuppression by cyclophosphamide following inoculation of MCMV behind the lens resulted in uveal infection and focal retinal necrosis involving the outer retinal layers, but lacked the features of HCMV retinitis (Holland et al. 1990). However, the use of the supraciliary route for intraocular inoculation was successful at inducing progressive focal necrotizing MCMV retinitis in adult, immunocomponent BALB/c mice (Atherton et al. 1991). Depletion of CD8+ and to a lesser extent CD4+ T cells induced acute retinitis that progressed to retinal necrosis (Atherton et al. 1992). The depletion of NK cells also promoted the development of retinitis in this model (Bigger et al. 1998). The adoptive transfer of CD8+ T cells from MCMV-immune mice protected against the development of retinitis induced by MCMV in T cell–depleted mice (Bigger et al. 1999). The perforin cytotoxic pathway was recently found to be more important than the Fas/FasL cytotoxoic pathway in protecting against MCMV retinitis in C57BL/6 mice (Dix et al. 2003). Mice with the retrovirus immunodeficiency syndrome MAIDS exhibited enhanced frequency and severity of MCMV retinitis. This was found to be directly related to a decrease in the perforin cytotoxic pathway in these mice. In conclusion, MCMV does not induce retinitis unless it is administered intraocularly by the supraciliary route. Nonetheless, valuable information about protective immune mechanisms has been obtained using the mouse model. 8.
Effect on the Developing Ear
Congenital HCMV infection in humans, whether symptomatic or asymptomatic, is considered to be the leading cause of sensorineural deafness (Fowler et al. 1997; Hicks et al. 1993). Hearing loss is the most common neurologic abnormality in these infants, and may range from mild to profound (Britt and Alford 1996). Of concern is the high rate of hearing loss in infants with no clinical evidence of infection. Hearing loss may progress during the first years of life in congenitally infected children, suggesting the presence of ongoing CNS infection (Britt and Alford 1996).
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GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
The mouse does not provide a model of congenital CMV infection leading to hearing loss. However, the newborn mouse ear appears to be equivalent in its embryological development to the ear of the human fetus at 15 weeks’ gestation. The intracranial inoculation of newborn mice with MCMV resulted in infection of the inner ear, inducing perilabyrinthitis in the cochlea, although this was not achieved by i.p. or i.n. inoculation (Davis and Strauss 1973; Davis and Hawrisiak 1977). Infection of cranial nerve ganglion cells and Schwann cells was observed, and in explants of trigeminal ganglia from infected newborn mice, Schwann cells, satellite cells, and neurons showed evidence of infection (Davis et al. 1979). In vitro organ culture of the inner ear from mice infected as newborns established that MCMV replicates in mesenchymal cells (Davis 1981). These observations contrast with the effect of HCMV on the inner ear following congenital infection in humans, where endolabyrinthitis involving the replication of HCMV in epithelial rather than mesenchymal cells is observed (Davis and Hawrisiak 1977). Thus, MCMV infection of newborn mice does not appear to replicate the pathological effects on the developing ear observed following congenital HCMV infection. The role of viruses including CMVs in vestibular neuritis and viral infections of the inner ear has been further reviewed (Davis and Johnsson 1983; Davis 1993). 9.
Effects on Hemopoiesis
The effects of MCMV on the bone marrow have been widely studied to find explanations for the defects in hemopoiesis that accompany HCMV infections in human BMT. In the absence of an adequately reconstituted immune system, BMT patients are prone to debilitating HCMV-induced diseases, particularly interstitial pneumonitis. Although rates of HCMV disease in BMT have declined markedly in recent years due to improved clinical management (reviewed in Pass 2001), HCMV infection remains a significant clinical concern. The mouse model has proved to be ideal for studying this problem. In normal adult mice, sublethal MCMV infection causes a marked atrophy of the bone marrow over the first week p.i., without establishing significant infection in bone marrow cells. Recovery occurs from day 7 p.i. (Gibbons et al. 1994). MCMV interferes with hemapoiesis by reducing the colony forming units–spleen (CFU-S) and CFU-granulocyte/macrophage (CFU-GM) (Gibbons et al. 1995). In persistently infected mice, no effects on the bone marrow were observed, but after 5-fluorouracil treatment, marrow recovery was delayed in infected mice. Stromal cells from infected mice showed a reduced capacity to support hemopoiesis and harbored latent infection (Mori et al. 1999). In experiments employing sublethal irradiation and autoreconstitution of bone marrow from surviving stem cells, MCMV infection prevented marrow reconstitution and interfered with the earliest step in hemopoiesis, the generation of CFU-S-1, without establishing marrow infection (Mutter et al. 1988).
However, the adoptive transfer of antiviral CD8+ T cells prevented hemopoietic failure (Mutter et al. 1988). This study reached the important conclusion that CD8+ T cells normally play a vital role in preventing the anti-hemopoietic effect of MCMV infection. In human allogeneic BMT, the high incidence of lethal HCMV disease therefore probably results from incomplete reconstitution by histoincompatible hemopoietic cells. Consequently, a delay in the generation of immune effector cells results in a failure to control HCMV disease (Mutter et al. 1988). Further studies in the mouse model of auto-reconstitution of bone marrow have established that MCMV-induced bone marrow failure is associated with a greatly reduced capacity of bone marrow stromal cells to produce the essential hemopoietic cytokines, stem cell factor, granulocyte colony stimulating factor, and IL-6 (Mayer et al. 1997). MCMV was shown to impair the engraftment of bone marrow cells in the stroma, which was associated with a lack of hemopoietic progenitor cells expressing SCF receptors and a reduced level of SCF gene expression in stromal cells (Steffens et al. 1998). A recent study suggests a useful therapeutic approach to protect against lethal MCMV infection in either congenic or allogeneic BMT. The inclusion of small numbers of common lymphoid progenitors accelerates immune reconstitution and protects against MCMV infection (Arber et al. 2003). Thus, the mouse model has provided valuable insights into the mechanisms of CMV-induced failure of bone marrow transplantation. It has also established the crucial protective role of CD8+ T cells and the possible use of these cells or common lymphoid progenitors in immunocytotherapy.
VI.
THE IMMUNE RESPONSE TO MCMV INFECTION
Immune responses in mice infected with MCMV involve both the innate and adaptive arms of the immune system. To establish persistent infection, MCMV has evolved mechanisms to modulate immune responses. MCMV also replicates in cells regulating immune responses, including macrophages and dendritic cells. This topic is the subject of a large literature, and the interested reader is directed to more comprehensive reviews. 1.
Immunosuppression
MCMV infection results in significant but temporary suppression of the immune system. A range of responses are affected, including responses to mitogens, antigens, allograft rejection, delayed type hypersensitivity responses, and the clearance of other pathogens (reviewed in Osborn 1982). While MCMV infection may cause splenic necrosis (Mims and Gould 1978b) and the loss of T and B cells (Trgovcich et al. 2000), early evidence suggested that the major effect of MCMV was on
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
regulatory cells (Kelsey et al. 1977; Osborn 1982). In support of this, recent evidence shows that MCMV infection of dendritic cells interferes with their key coordinating role in the immune system (Andrews, Andoniou, et al. 2001). Furthermore, infection of another important accessory cell, the macrophage, results in its functional impairment (see below) and loss of response to cytokines, which enhance its response and defense against pathogens (Popkin et al. 2003). Other effects of MCMV on the immune response are discussed in Section 7 below. 2.
Macrophages and Dendritic Cells
Blood monocytes and tissue macrophages play a pivotal role in the pathogenesis of MCMV infection (Hanson et al. 1999). Macrophages are major target cells in many tissues following infection with MCMV (Hudson et al. 1978; Mercer et al. 1988; Mims and Gould 1978a; Selgrade and Osborn 1974; Stoddart et al. 1994) and may harbor latent viral DNA (Brautigam et al. 1979; Koffron et al. 1998; Pollock et al. 1997). Circulating blood monocytes disseminate virus during acute infection and differentiate into mature macrophages, an event that favors MCMV replication (Cavanaugh et al. 1996; Collins et al. 1994; Heise, Connick, et al. 1998; Hesie, Pollock, et al. 1998b; Heise and Virgin 1995; Mitchell et al. 1996). Macrophages are also important mediators of inflammatory and innate responses to infection and, when activated, infiltrate sites of infection (Heise and Virgin 1995) to initiate early antiviral responses by producing tumor necrosis factor-α (TNF-α) or interferon-γ (IFN-γ) (Hanson et al. 1999). However, IFN-γ inhibits the replication of MCMV in macrophages (Presti et al. 2001). The phagocytic function of macrophages is altered following infection with MCMV (Katzenstein et al. 1983; Shanley and Pesanti 1983; van Bruggen et al. 1989). Macrophages contribute to protection by decreasing the viral load and acting as antigen-presenting cells to activate T cell responses (Hamano et al. 1998). Macrophages may protect other highly permissive cell types from MCMV infection (Hanson et al. 1999). In vitro replication of MCMV is slower in macrophages than in fibroblasts (Heise and Virgin 1995; Shanley and Pesanti 1983; van Bruggen et al. 1989). As initiators of immune responses, dendritic cells (DC) contribute to the control of MCMV infection. Immature DC process antigen and, once activated, migrate to lymphoid organs, where they initiate primary immune responses and activate naive and activated T cells. Through the release of cytokines and chemokines, activated DC also play a role in the recruitment and activation of NK cells, NKT cells, macrophages, and B cells. Various subsets of DC limit MCMV replication during infection. Plasmacytoid DC infected with MCMV in vitro produce IFN-α/β, which induces other DC to produce IL-12, in particular the CD11b+ subset (Dalod et al. 2002, 2003). IFN-α/β also promotes the accumulation of the plasmacytoid DC and
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the maturation of CD8α+ DC (Dalod et al. 2003). CD8α+ DC are also essential for the expansion of Ly49H+ NK cells via a mechanism involving the production of IL-12 and IL-18 (Andrews et al. 2003). Recent studies have identified two phases in the response of DCs to MCMV. Upon initial infection, immature DC transiently up regulate MHC and costimulatory molecules (Andrews et al. 2001; Mathys et al. 2003). However, within 4 days, when most immature DC are productively infected, the cell surface expression of MHC and costimulatory molecules is reduced, and the DC lose their ability to prime T cells or respond to maturation stimuli. Mature DC are either infected at a lower level (Andrews et al. 2001) or are not productively infected with MCMV (Mathys et al. 2003). 3.
Production of Interferon and Other Cytokines
IFN is an important inducer of nonspecific host defenses during acute infection with MCMV. The protective role of IFN was first shown by the administration of antiserum specific for IFN-α/β, which significantly reduced the resistance of mice to MCMV infection (Grundy et al. 1982). Many other studies have demonstrated the importance of IFNs for controlling MCMV infection by administering neutralizing antibodies or exogenous cytokines to infected mice (Allan and Shellam 1985; Chong et al. 1983; Cousens et al. 1997; Heise and Virgin 1995; Lucin et al. 1992; Martinotti et al. 1990, 1992, 1993; Orange and Biron 1996b; Orange et al. 1995; Quinnan and Manischewitz 1987). During MCMV infection, IFN α/β peaks were detected in plasma at 6 hr (Shellam et al. 1981) and 48 hr (Allan and Shellam 1985) and again at 10 days PI (Tarr et al. 1978). IFN-γ levels in sera peaked at 40 hr PI (Cousens et al. 1997; Orange and Biron 1996b; Ruzek et al. 1997). IFN-α/β induces an antiviral state during early times of infection, prior to the development of adaptive host immune responses. The kinetics of the IFN-α/β response mimic NK cell activation and proliferation (Bukowski et al. 1984; Shellam et al. 1983). NK cell activity is enhanced by IFN within the first few days of infection (Welsh 1986). IFN has effects on other antiviral mechanisms via NK-cell independent pathways, as shown by prophylactic treatment with IFN-β of NK-cell deficient mice (Bukowski et al. 1987). IFN-α inhibits MCMV replication by impairing viral IE gene transcription (Martinotti et al. 1993). This is achieved by down-regulating the activity of transcription factors such as NF-κB that activate the IE enhancer in the nuclei of infected cells (Gribaudo et al. 1993). IFN-α/β also has regulatory effects on the subsequent expression of endogenous cytokines including 1L-12 and IFN-γ, which induce protective immune responses during MCMV infection (Cousens et al. 1997). IFN-γ is a key regulator during all phases of MCMV infection (Presti et al. 1998). Control of acute infection by MCMV is mediated by IFN-γ secretion by NK cells (Heise and Virgin 1995; Orange et al. 1995) and T cells (Karupiah et al. 1998;
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GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
Shanley et al. 2001). Functions of IFN-γ during MCMV infection include macrophage activation (Heise and Virgin 1995), enhancement of antigen presentation by infected cells to CD8+ T cells via MHC class I–dependent pathways (Hengel et al. 1994), and inhibition of lytic MCMV replication and gene expression at the cellular level (Heise and Virgin 1995; Lucin et al. 1994; Presti et al. 1998). IFN-γ may also regulate chronic MCMV replication in the salivary glands (Jonjic et al. 1989; Koszinowski et al. 1990; Lucin et al. 1992; Presti et al. 1998). NK cells are the dominant source of IFN-γ soon after infection. MCMV has evolved immune modulation strategies to reduce the effectiveness of IFN-γ in infected cells (Popkin et al. 2003). MCMV induces the production of other cytokines that are detectable in sera during the acute stage of infection, including IL-18, IL-12, TNF-α, IL-1α, and IL-6 (Pien et al. 2000; Ruzek et al. 1997). Using mice with cytokine deficiencies or neutralized cytokine functions, IL-6 was a pivotal mediator of the glucocorticoid response, and IL-1 contributed to IL-6 production (Ruzek et al. 1997). There may be organ-specific cytokine responses to MCMV infection. There were higher concentrations of IL-10 in the lungs of BALB/c than C57BL/6 mice following infection (Geist and Hinde 2001). 4.
Natural Killer (NK) Cells
The protective role of NK cells in MCMV infection was identified over 20 years ago and remains a very active area of research (see also Section V, B, 2). The NK cell response is a preformed defense mechanism that is active before the development of acquired immunity. NK cells are large granular lymphocytes that lyse a restricted variety of target cells upon contact. NK cells control MCMV replication by direct lysis of infected cells (Bukowski et al. 1985; Shellam et al. 1981) and by producing cytokines (Orange et al. 1995). Direct killing requires NK cells to be localized in close proximity to virusinfected target cells. However, NK cells can recycle after direct lysis to kill other infected cells. NK cells also regulate downstream T cell responses (Su et al. 2001). NK cells respond rapidly to MCMV infection (Bancroft et al. 1981), and limit the severity, extent, and duration of acute infection (Shellam et al. 1981; Bukowski et al. 1983, 1984), and may also regulate viral persistence in the salivary gland (Bukowski et al. 1984). The importance of NK cells in controlling MCMV infection has been shown by depletion or adoptive transfer of NK cells. Depletion of NK cell activity in adult mice using antibodies to asialo-GM1, or NK1.1 monoclonal antibody rendered C5BL/6 mice susceptible to MCMV (Bukowski et al. 1984; Scalzo et al. 1992; Shanley 1990; Welsh et al. 1990, 1991, 1994). Infection of newborn mice with MCMV is usually lethal. However, the adoptive transfer of cloned NK cells or NK cell–enriched fractions from naive adults to neonatal mice prevented mortalities (Bukowski et al. 1985, 1988) by reducing splenic viral titers (Welsh 1986). Genetic susceptibility to infection by MCMV correlates with the inability to mount a sufficient
NK cell response (Bancroft et al. 1981; Shellam et al. 1981). The interaction between NK cell receptors and viral ligands is discussed in Section V, B. Cytokines produced during acute MCMV infection have specific NK cell activation roles. The production of IFN-α/β is required for the accumulation, blastogenesis, and cytotoxicity of NK cells (Biron 1997; Orange and Biron 1996b; SalazerMather et al. 2002). The cytotoxic activity of NK cells is greatly augmented by the presence of IFNs, although they also protect the target cells from lysis (Welsh 1986). MCMV-induced IL-12 is responsible for early NK cell IFN-γ production and viral control, as in vivo IL-12 neutralization by antibody treatment blocks these events. However, treatment with neutralizing IL-12 failed to alter these NK cell responses at 7–9 days p.i. (Orange and Biron 1996a). IFN-α stimulated IFN-γ production by NK cells, but also inhibited NK cell cytotoxicity (Orange and Biron 1996a). NK cells may utilize different mechanisms to regulate viral infection in different organs. NK cells accumulate in the spleen and liver at the sites of viral replication in an IL-12, IFN-γ, and TNF-α dependent manner (Dokun et al. 2001). In the spleen of C57BL/6 mice, NK cells control MCMV via a perforindependent (IFN-γ independent) cytotoxic mechanism (Tay and Welsh 1997). In contrast, IFN-γ produced by NK cells is a major immune regulator of infection in the liver (Orange et al. 1995; Orange and Biron 1996 a, 1996b; Tay and Welsh 1997). The formation of early inflammatory foci in the liver (Bukowski et al. 1983; Orange et al. 1997; Olver et al. 1994) is dependent upon the rapid accumulation of NK cells, IFN-γ, and macrophage inflammatory protein-1α (MIP-1α) (Salazer-Mather et al. 2002). In C57BL/6 mice, liver pathology occurred independently of NK and T cells. However, NK cells may limit the damage elicited by TNF-α (Orange et al. 1997). NK1.1+T (NKT) cells also regulate the innate immune response, especially in tumor surveillance. However, NKT cells are not directly involved in restricting MCMV infection, but when appropriately activated, produce IFN-γ, which activates NK cells to control MCMV infection (van Dommelen et al. 2003). 5.
B Cell–Mediated Immune Responses
The humoral immune response is stimulated during acute MCMV infection. Neutralizing antibody was detected by 3 days p.i. (Araullo-Cruz et al. 1978), while non-neutralizing antibody was first detected in sera at 8–10 days p.i., when titers were 10-fold higher than neutralizing titers (Manischewitz and Quinnan 1980). Antibody titers to MCMV persisted until as late as 6 months p.i. in BALB/c mice (Classen et al. 1987). Longterm persistence of CMV in the salivary glands may contribute to serum titers of antibody, as it does in the rat model (Kloover et al. 2002). Serum IgM antibodies were detected in mice as early as 3–5 days, while IgG antibody was detected between 5–7 days, reaching peak levels at 20 days p.i. (Lawson et al. 1988). The IgG isotype response to MCMV antigens is predominantly
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
IgG2a (Price et al. 1993). IgA antibodies were not detected in the sera of MCMV-infected mice (Lawson et al. 1988). Antibody is not critical for controlling primary MCMV infection, as high antibody titers occur in mice with significant viral titers in organs (Lawson et al. 1988). Antibody also is not essential for recovery from a primary MCMV infection, as mutant mice devoid of B cells recovered from infection (Jonjic et al. 1994). Nonetheless, the role of antibody is important, as mice depleted of immunoglobulin from birth were 10-fold more susceptible to MCMV than normal mice (Selgrade et al. 1976). Passive administration of neutralizing antibody to MCMV can restrict virus replication but not the establishment of viral latency (Araullo-Cruz et al. 1978; Farrell and Shellam 1991; Shanley et al. 1981). Antibodies may also limit the reactivation of latent virus and its subsequent dissemination. Factors that influence the production of antibody include the MCMV strain, viral dose, genetic resistance, and secondary infections. Antiviral antibody was detected after the administration of 10–100 but not 1 pfu of MCMV (K181) (van Dommelen and Shellam, unpublished observation). There was no correlation between antibody titers and mouse resistance status (Lawson et al. 1988; Price et al. 1993). However, the specific viral proteins recognized vary between mouse strains (Farrell and Shellam 1989). A large number of viral proteins are recognized by antiMCMV antibody. More than 50 viral antigens were recognized by rabbit sera raised against extracts of MCMV-infected mouse cells (Chantler and Hudson 1978). These proteins included both structural and nonstructural components of the virion (Selgrade et al. 1983). Several structural glycoproteins of MCMV stimulate production of neutralizing antibodies, including gp52, gp105, gp87, and the gp150 complex (Loh 1991; Loh et al. 1988; Loh and Qualtiere 1988). MCMV-induced increases in serum immunoglobulin levels are driven by cytokines such as IL-6 (Karupiah et al. 1998). IFN-γ is important for the predominance of IgG2a antibody isotype compared to the IgG1 isotype in MCMV infection (Karupiah et al. 1998). The appearance of autoantibodies in the serum of MCMV-infected mice (Bartholomaeus et al. 1988) may be due to polyclonal activation of B cells (Price et al. 1993) and may contribute to autoimmune diseases that accompany MCMV infection in mice (Bartholomeaus et al. 1988). Such antibodies have broad cross-reactivity with auto-antigens, conventional antigens, and unrelated viral antigens (Karupiah et al. 1998; Price et al. 1993). 6.
T Cell–Mediated Immune Responses
Both CD4+ and CD8+ T cells play critical roles in the immune response to MCMV. The role of T cells in protective immunity was initially observed in T cell–deficient nude mice, which were very susceptible to MCMV (Grundy and Melief 1982; Starr and Allison 1977). CD8+ T LYMPHOCYTES Adoptive transfer of the CD8+ subset of sensitized T lymphocytes limited MCMV dissemination,
29
prevented tissue destruction, and protected mice against lethal disease (Reddehase et al. 1984a, 1988; Reddehase, Mutter, et al. 1987; Reddhase, Mutter, Munch, et al. 1987). Cytotoxic T lymphocytes (CTL) are crucial for the clearance of acute MCMV infection in BALB/c mice. CTL lysed MCMV-infected fibroblasts in vitro and were found in the spleens of mice from day 3, peaking on day 7–8 (Ho 1980; Quinnan et al. 1978; Sinickas et al. 1985) and were active until 14 days p.i. (Ho 1980). Sensitized CTL recognize both structural and nonstructural viral antigens. However, almost 50% of CTL in BALB/c mice are primarily directed against the nonstructural pp89 (IE1) protein of the Smith strain (Koszinowski, Reddehase, et al. 1987; Reddehase et al. 1984b; Reddehase and Koszinowski 1984). The pp89 protein is recognized by specific CTL in conjunction with the MHC class I H-2d locus (Alexander-Miller et al. 1993; Koszinowski, Keil, et al. 1987). BALB/c (H-2d) mice were protected against lethal MCMV challenge by vaccination with a recombinant vaccinia virus expressing pp89 (Del Val et al. 1988; Koszinowski, Keil, et al. 1987; Volkmer et al. 1987), which, however, did not prevent infection and morbidity (Jonjic et al. 1988). Epitope mapping studies, using vaccinia virus recombinants expressing pp89 synthetic peptides, have shown that pp89 contains an immunodominant CD8+ T cell epitope, YPHFMPTNL (Del Val et al. 1988; Reddehase et al. 1989), of which only 5 amino acids are essential for CTL recognition (Koszinowski et al. 1991). IE1-specific (pp89) CD8+ T cells dominate during the acute phase of infection (Holtappels, PahlSeibert, et al. 2000). Most IE1-specific CD8+ T cells belong to the CD62lo subset of resensitized memory effector cells. IE1specific CTL are likely to be frequently resensitized during latent infection of the lungs and may be involved in maintaining latency (Holtappels, Pahl-Seibert, et al. 2000). Nucleotide sequencing of MCMV isolates derived from wild mice identified variations between amino acids 147 and 192 of pp89, which included the region encompassing the CTL epitope (amino acid residues 168–176) (Lyons et al. 1996). Four groups of variants at this locus were defined. Some wild isolates and the laboratory strains K181 and Vancouver had complete identity with the Smith strain (Lyons et al. 1996). Polyclonal pp89 (Smith)-specific CTL only weakly recognized target cells infected with MCMV from most variant groups (Lyons et al. 1996). Immunization of mice with YPHFMPTNL conferred significant protection against the laboratory isolate K181, but there was no protection of mice challenged with G4 or N1 isolates (Lyons et al. 1996). Other CTL epitopes identified within the MCMV genome include H-2d restricted CTL epitopes from the m04, M45, and m164 gene products (Gold et al. 2002; Holtappels, Thomas, et al. 2000; Holtappels et al. 2002). Eighty percent of memory CD8+ T cells in spleen and pulmonary infiltrates were specific for the ie1 and m164 peptides (Holtappels et al. 2002). M83 and M84 may also encode epitopes that are recognized by CD8+ T cells (Holtappels et al. 2001). M84 encodes a protein that shares significant amino-acid homology with the HCMV pp65
30
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
tegument protein, a major target of protective CTL in humans (Morello et al. 2000). CD8+ T cell responses are not essential for the early control of MCMV replication in the presence of effective NK cell responses, such as those present in genetically resistant CBA and C57BL/6 mice (Lathbury et al. 1996). Susceptible mouse strains (BALB/c, A/J) are dependent upon CD8+ T cells. However, congenic BALB/c mice containing the resistance Cmv1r allele did not require a CD8+ T cell response for viral clearance (Lathbury et al. 1996). Thus, NK cell and CD8+ T cell responses are independently responsible for viral clearance, although CD8+ T cells are required for recovery from infection. Karrer and colleagues (2003) determined that the kinetics of the acute CD8+ T cell response after MCMV infection were characterized by rapid expansion of activated T cells followed by a contraction phase. Thereafter, MCMV-specific memory CD8+ T cells steadily accumulated over time in the spleen, lymph nodes, liver, lungs, and blood. At 1 year p.i., 20% of all CD8+ T cells were specific for the pp89 epitope, and there was also a gradual restriction in the use of the variable region of the TCR β-chain (Karrer et al. 2003). Continuous or repetitive exposure to antigen during latency may enlarge these memory T cell populations over time, as observed in HCMV infection of human populations (Gillespie et al. 2000; Weekes et al. 1999). CD4+ T LYMPHOCYTES CD4+ helper T cells are not absolutely required for initiating CD8+ T cell–mediated immune responses (Ahmed et al. 1988; Jonjic et al. 1989; Reddehase et al. 1988) but are required for viral clearance in certain organs and may enhance CD8+ T cell responses. In mice depleted of the CD4+ subset, clearance of replicating virus occured in infected tissues except for the salivary glands (Jonjic et al. 1989; Lucin et al. 1992). CD4+ lymphocytes can compensate for the absence of CD8+ lymphocytes (Jonjic et al. 1990; Koszinowski et al. 1991). IFN-γ is required for CD4+ T cell activity in the salivary glands (Lucin et al. 1992). CD4+ T cells are also required for the MCMV-specific DTH and IgG antibody responses (Lathbury et al. 1996). The response to MCMV in the salivary gland is intriguing. MCMV continues to persist for long periods despite the presence of CD8α+ DC, γ/δ T cells, NK cells, CD4+ and CD8+ T cells, and the expression of 1FNγ, IL-10, and CC chemokines (Cavanaugh et al. 2003). 7.
Immune Evasion by MCMV
A number of the genes of MCMV and other herpesviruses are responsible for establishing and maintaining the virus in its host. They control a variety of processes, including moderating the immune response, or immune evasion, the inhibition of apoptosis, and specific cell tropism. These genes are not
required for viral replication and can often be deleted without in vitro consequence. A list of the known immune invasion genes is shown in Table 1-2. Detailed discussion of these genes can be found in various reviews (Alcami and Koszinowski 2000; Scalzo 2002; Yewdell and Hill 2002; Krmpotic et al. 2003; Mocarski, 2004). The products of immune evasion genes of MCMV affect various aspects of the immune system. Some examples of the genes involved are m129/131 and M33, which encode a chemokine homolog MCK-2 and a receptor respectively, M27, whose product inhibits the interferon response, and m147.5, which inhibits antigen presentation by DC (Table 1-2). Others, such as those that down-regulate MHC class II expression (Alcami and Koszinowski 2000), antigen presentation by macrophages (Popkin et al. 2003), or DC (Andrews et al. 2001), are yet to be characterized. The best-studied of the immune evasion genes of MCMV are those that subvert NK or T cell responses. Given the importance of NK cells in combating MCMV infection, it is not surprising that MCMV encodes a number of proteins that inhibit NK cell function (Table 1-2). A homolog of cellular class I molecules that inhibits the NK cell–mediated clearance of MCMV in vivo is encoded by m144 (Farrell et al. 1997). It has been suggested that the m144 protein acts as a decoy for NK cells by engaging inhibitory NK cell receptors, although the receptor has not been identified (Farrell et al. 1997; Cretney et al. 1999; Kubota et al. 1999). As will be discussed below, the viral m152 gene inhibits the clearance of MCMV by CD8+ T cells. However, the m152 product also inhibits NK cells by down-regulating the expression of the MHC class 1–like molecule RAE-1, which is a ligand for the NK cell–activating receptor NKG2D on NK cells (Cerwenka et al. 2000; Lodoen et al. 2003). NKG2D has two additional activating ligands, MULT-1 (Carayannopoulos et al. 2002) and H-60 (Malarkannan et al. 1998; Diefenbach et al. 2000). MCMV also interferes with their expression on infected cells. The products of m145 and m155 down-regulate the expression of MULT-1 and H-60, respectively, and the deletion of these genes from MCMV enhances clearance of the virus due to NK cell–specific effects (Krmpotic et al. 2002; Lodoen et al. 2003; Ogasawara et al. 2003; Hasan et al. 2005; Krmpotic et al. 2005). Finally, as already discussed, MCMV can evade Ly49H+ NK cells by mutations in the viral ligand m157 (Voigt et al. 2003). Since mutations in m157 affecting Ly49H binding occur in wild isolates of MCMV, this interaction may not occur frequently in nature. It is likely that m157 normally binds inhibitory NK cell receptors such as Ly49I (Arase et al. 2002) in wild mice, which show significant variability in the NK gene complex (Scalzo et al. 2005). The viral genes affecting NK cell function have all been found within the m145 gene family, but it is likely that additional NK cell inhibitory genes exist within the m02 gene family (Oliveira et al. 2002).
31
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
TABLE 1-2
IMMUNE EVASION BY MCMV Viral gene
Function
Mechanism
Reference*
m04 m06
Blocks CTL function Blocks CTL function
[1–4] [2,3,5]
M27 M33
Type I and type II IFN resistance Migration of cells, including smooth muscle cells Promotes inflammation and dissemination to salivary gland Unknown Inhibits NK cell function Inhibits NK cell function
Binds MHC I and remains associated on cell surface Down-regulates MHC I by targeting molecule for lysosome degradation Down-regulates STAT-2 Receptor homologue binds RANTES, functional homologue of HCMV US28 Chemokine homologue, macrophage chemoattractant
m129/131 m138 (fcr-1) m144 m145 m147.5 m152
Inhibits Ag presentation? Blocks CTL function Inhibits NK cell function
m155
Inhibits NK cell function
m157
Activates NK cells Inhibits NK cells?
Unknown
Complement resistance
Binds antibody MHC class I homology. Mechanism unknown Down-regulates MULT-1, a ligand for the NK cell activating receptor NKG2D on infected cells Specifically down-regulates CD86 on dendritic cells Retains MHC I in ERGIC Down-regulates RAE-1, a ligand for the NK cell activating receptor NKG2D on infected cells Down-regulates H-60, a ligand for the NK cell activating receptor NKG2D on infected cells Activates NK cells via Ly49H in C57BL/6 mice Presumed function is to bind NK cell inhibitory receptors such as Ly49I Up regulation of CD46 via MCMV responsive element in CD46 gene promoter
* References are given in full in the bibliography. 1. Kleijnen et al. 1997 7. Melnychuk et al. 2005 2. Wagner et al. 2002 8. Davis-Poynter et al. 1997 3. LoPiccolo et al. 2003 9. Waldhoer et al. 2002 4. Kavanagh et al. 2001 10. Fleming et al. 1999 5. Reusch et al. 1999 11. Saederup et al. 2001 6. Zimmermann et al. 2005 12. Saederup et al. 1999 13. Crnkovic-Mertens 1998
T cells, particularly CD8+ T cells, play a vital role in viral clearance and in the control of chronic infection and recurrence from latency (Krmpotic et al. 2003). The importance of CD8+ T cells is reflected in the number of mechanisms employed by MCMV to circumvent their effect. MCMV acts on both the antigen presentation and effector stages of the immune response. The virus inhibits the capacity of DC to present antigen to naive T cells (Andrews et al. 2001), as well as inhibiting the effector phase of the response by down-regulating the expression of MHC class I molecules, thus reducing the recognition and killing of infected cells by CD8+ T cells. Three MCMV genes, m04, m06, and m152, appear to account for all the inhibitory effects of MCMV on MHC class I function, at least in vitro (Wagner et al. 2002). The product of m04, glycoprotein (gp) 34, binds MHC class I in the ER but does not inhibit its expression on the cell surface. Rather, it is transported to the cell surface as a complex with class I molecules where CD8-mediated killing of infected cells is inhibited (LoPiccolo et al. 2003). The m06
14. 15. 16. 17. 18. 19. 20.
Thale et al. 1994 Farrell et al. 1997 Cretney et al. 1999 Krmpotic et al. 2005 Loewendorf et al. 2004 Ziegler et al. 1997 Krmpotic et al. 2002
21. 22. 23. 24. 25. 26. 27.
[6] [7–9] [10–12] [13,14] [15,16] [17] [18] [2–4,19,20] [20,21] [22,23] [24–26]
[27]
Lodoen et al. 2003 Lodeon et al. 2004 Hasan et al. 2005 Scalzo et al. 1992 Arase et al. 2002 Smith et al. 2002 Nomura et al. 2002
gene encodes gp48, which interferes with the MHC class I pathway of antigen presentation by binding to complexes of MHC class I molecules and antigenic peptides, targeting them for degradation in the lysosome (Reusch et al. 1999). The reduced cell surface expression of these complexes results in reduced recognition and killing of these cells by CD8+ T cells (Reusch et al. 1999; Wagner et al. 2002; LoPiccolo et al. 2003). The m152 gene product, gp40, prevents the transport of class I molecules to the cell surface, causing them to be retained in the ERGIC (Del Val et al. 1992; Ziegler et al. 1997). Since m152 also down-regulates the NK cell ligand RAE-1, this viral gene exerts an inhibitory effect on both CD8+ T cells and NK cells. While these in vitro studies have established a role for m04, m06, and m152, their role in vivo is less clear. Deletion of m152 from MCMV results in enhanced clearance of MCMV in vivo by CD8+ T cells and NK cells (Krmpotic et al. 2002). However, a virus constructed by the deletion of m04, m06, and
32
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
m152 was cleared no more effectively than wild-type virus in vivo (Gold et al. 2004). Understanding the role of immune evasion in vivo is complicated by a number of factors. For example, the m04, m06, and m152 products exhibit distinct preferences for certain MHC class I allelic forms (Wagner et al. 2002), and their downregulation of MHC class I molecules may therefore vary among inbred mouse strains. Furthermore, the m152 product gp40 exhibits dual functions; gp40 also down-regulates H-60, which is a ligand for the NKG2D receptor on NK cells (Krmpotic et al. 2002). Thus, the effect of m152 varies among inbred mouse strains according to the interactions with particular ligands. Finally, isolates of MCMV exhibit polymorphisms in a number of immune evasion genes (Smith et al. 2006). The G4 isolate encodes a variant of m157 that does not activate Ly49H (Voigt et al. 2003). Hence, Ly49H+ mouse strains are no more resistant to G4 than are Ly49H- mouse strains. Given the complexity of the effects of MCMV immune evasion proteins, understanding how they influence the dynamic interaction between MCMV and its host poses a significant challenge.
VII. A.
DIAGNOSIS Serology
Antibody assays have been the standard means of screening for evidence of MCMV infection. Serum neutralization assays were originally used (Mannini and Medearis 1961), and their sensitivity has been improved by the addition of complement (Kim and Carp 1973; Lawson et al. 1988) and the use of MCMV antigen derived from virus passaged in cell culture rather than the salivary gland (Chong et al. 1981; Lawson et al. 1988). Since the 1980s, the use of the enzyme-linked immunosorbent assay (ELISA) has been widespread (Anderson et al. 1983, 1986; Classen et al. 1987; Lawson et al. 1988). ELISA was found to be more sensitive than nuclear anticomplement immuofluorescence (NAIF) or complement fixation assays (Anderson et al. 1986), although NAIF was reported to be more sensitive for the detection of antibodies in the acute stage of the infection (Anderson et al. 1983). Another study showed that ELISA and indirect immunofluorescence were of comparable sensitivity (Classen et al. 1987). ELISA is the method of choice for routine screening of sera from mouse colonies, and it is used by most diagnostic laboratories. However, there are situations in which serological assays are not suitable. Immunodeficient, genetically modified mice such as nude, SCID, RAG gene, or immunoglobulin gene knockout mice cannot produce anti-MCMV IgG antibodies, so sensitive molecular techniques such as the polymerase chain reaction (PCR) are employed (see below). PCR may also be used to confirm MCMV infection in mice whose antibody titers are borderline positive.
B. Molecular Detection Real-time quantitative PCR has recently been developed for the detection of MCMV (Wheat et al. 2003; Farroway et al. 2005) and, as would be predicted, it is much more sensitive than the plaque assay or ELISA for detecting infection. However, given that viral genomes are likely to be restricted to particular tissues in latently infected mice (see Section V, C), it is more feasible to use the ELISA for routine screening. Real-time qPCR has recently been adapted for the detection of mixed infection with different strains of MCMV in individual mice based on detection of different ie1 genotypes (Gorman et al. 2006).
VIII. CONTROL AND PREVENTION With the introduction of regular screening programs for murine pathogens by commercial suppliers of laboratory mice and the widespread use of specific pathogen-free mice in research, naturally occurring MCMV infections in mouse colonies no longer occur. Since MCMV does not infect the fetus transplacentally, the use of cesarean derivation to maintain the disease-free status of laboratory animals ensures that mouse colonies will remain free of MCMV. However, the potential for cross-infection with MCMV derived from experimentally infected mice remains. MCMV appears not to be transmitted from cage to cage (Mannini and Medearis 1961), although a conflicting result has been reported (Anderson et al. 1983). The physical separation of cages of MCMV-infected mice from other mice and the use of filter tops would therefore be prudent. An even greater risk attends the housing of wild-caught mice in the same facility as laboratory colonies. Because wild mice are naturally infected with MCMV (see Section IV) and other viruses, as well as bacterial and parasitic pathogens, these animals should only be housed in a separate quarantine facility with stringent barrier procedures and comprehensive monitoring. There is no need to consider the use of vaccines or other measures to control MCMV in mouse colonies. However, antiviral compounds have been used widely in research, particularly when the control of CMV infections in human patients is modeled in murine studies (Bolger et al. 1999; Lenzo, Shellam et al. 2001; Shanley et al. 1985, 1988). Ganciclovir and cidofovir are very effective against MCMV, and the 50% inhibitory concentrations in vitro are about 5 and 0.24 µM respectively (Okleberry et al. 1997; Lenzo, Shellam, et al. 2001). The use of foscarnet (Smee et al. 1995; Lenzo, Shellam, et al. 2001), acyclovir (Shanley et al. 1985), and HPMPC (Bolger et al. 1999) has also been reported. A number of antiviral drugs have been tested against MCMV and show promise (Rybak et al. 2000). Natural products with anti-MCMV properties also have been described (Hudson 1994a).
33
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
1.
Development of Vaccines
While there is no necessity to develop vaccines to control MCMV infection in colonies of laboratory mice, the mouse model has been used to test strategies for vaccination against HCMV and for evaluating the usefulness of MCMV as a persistent viral vector encoding heterologous antigens. VACCINES AGAINST MCMV OR HETEROLOGOUS ANTIGENS The ability of vaccines based on a single MCMV epitope to induce protection against MCMV challenge has been established using the viral peptide in an adjuvant formulation (Scalzo et al. 1995), a recombinant vaccinia vector expressing the peptide epitope (Del Val et al. 1991), or plasmids expressing the viral epitope (Morello et al. 2000). Alternatively, a vaccine based on MCMV that has been genetically disabled through the deletion of gL shows promise (J. Allan, personal communication). A different approach has been to use the property of viral persistence and the induction of long-term memory (Karrer et al. 2003) exhibited by MCMV in developing the virus as a vector for vaccinating against heterologous antigens. A recombinant MCMV vaccine expressing epitopes from influenza A or lymphocytic chorio-meningitis viruses induced CD8+ memory T-cell populations that expanded with time and responded rapidly to challenge with either of these viruses without the need for boosting (Karrer et al. 2004). VIRALLY VECTORED IMMUNOCONTRACEPTION A novel use of MCMV as a vaccine vector is the development of an immunocontraceptive vaccine for possible use as a disseminating agent to control population explosions of house mice in rural Australia (Shellam 1994). A recombinant MCMV vaccine was constructed encoding the murine zona pellucida 3 (ZP3) protein, which coats the egg and is involved in the binding of sperm in the fertilization reaction. Complete and long-lasting sterility was induced following a single vaccination of female BALB/c mice (Lloyd et al. 2003; Redwood et al. 2005) or wild mice (Lloyd et al. 2006). Vaccination was accompanied by the production of antibodies to ZP3 and a profound reduction in ovarian follicles.
IX.
MOUSE THYMIC VIRUS: MOUSE
T LYMPHOTROPHIC VIRUS (MTLV) OR MURID HERPESVIRUS 3 A.
Introduction and History
Mouse thymic virus (MTV) was first described in 1961, due to the occurrence of thymic necrosis during serial passage of organ homogenates in newborn Swiss mice (Rowe and Capps 1961). Inoculation of newborn mice with homogenized necrotic thymus
tissue resulted in necrosis of the thymus in recipient mice. Sera from inoculated mice did not contain antibodies against a range of mouse viruses (Rowe and Capps 1961). The properties of the infectious agent were compatible with those of a virus, being filterable, not affected by antibiotics, and not culturable on bacteriological media. Based on morphology and lability, mouse thymic virus was classified as a herpesvirus (Parker et al. 1973). Despite efforts, a permissive tissue culture system has not been established for mouse thymic agent, limiting subsequent research. The genome of MTV has not been studied.
B.
Properties of the Virus
Mouse thymic virus has typical herpesvirus morphology. Within infected thymus tissues, MTV capsids are icosahedral, ranging from 95 nm to 110 nm in diameter. Enveloped capsids are spherical, ranging from 125 nm to 165 nm in diameter (Parker et al. 1973; Athanassious et al. 1990). The virus displays the same properties of heat (50°C, 30 minutes) and ether (diethyl ether, 2 hours, 20 minutes) lability of other herpesviruses. Infection of newborn mice with MTV results in thymic necrosis and acute immunosuppression over 2–3 weeks. The thymus regenerates in most animals within 1–2 months; however, mice continue to shed virus asymptomatically into the saliva (Rowe and Capps 1961; Parker et al. 1973; Cross et al. 1979; Cohen et al. 1975). All mouse strains tested were found to be susceptible to MTV, but the rate at which thymus necrosis occurred varied between strains (Cross et. al. 1979). Neonatal infection of selected strains of mice results in histologically and serologically evident autoimmune disease, similar to organspecific autoimmune diseases in humans (Morse et al. 1999), possibly due to altered control of self-reactive T cells. Initial attempts were made to culture the virus on primary mouse embryo and kidney cultures, spleen and thymus explants, and several tumor cell lines, all without success (Rowe and Capps 1961). Similarly, several other murine and non-murine cells and cell lines have been used with no apparent virus growth or cytopathic effect (Morse 1988; Hudson 1994b). One unpublished observation (cited in Morse and Valinsky 1989) states that MTV is able to grow in “several T lymphoblastoid lines,” including three CD4+ human lines. However, this has not been repeated elsewhere. When housed with mice i.p. inoculated with MTV, test animals are seropositive by IFA, CF, and ELISA 21–30 days after cohousing, and MTV was identified in the salivary glands of contact-infected mice after 90 days of cohousing (St-Pierre et al. 1987; Lussier et al. 1988a, 1988b). Transmission of MTV from mothers to offspring was observed following inoculation with MTV 24 hours after giving birth. In this instance, virus was identified in the salivary glands of the offspring 21–27 days after inoculation, although no anti-MTV antibody could be detected in these mice (St-Pierre et al. 1987). However, it is unclear if this transmission of MTV was via milk or simply due
34
GEOFFREY R. SHELLAM, ALEC J. REDWOOD, LEE M. SMITH, AND SHELLEY GORMAN
to contact transmission from the mother. Inoculation of pregnant females at various times of gestation yielded no evidence of transplacental transfer of MTV, as all fetuses were MTV-negative and no abortion was recorded (St-Pierre et al. 1987). MTV was originally isolated from a laboratory strain of mouse (Rowe and Capps 1961) and was subsequently identified in other mouse colonies (Cross et al. 1979). Little is known about naturally occurring MTV infection in wild mice, although 4 out of 15 wild-mouse serum samples were positive for MTV by IFA. MTV was identified by infectivity assay in a small number of mainly seronegative wild mice in this study (Cross et al. 1979). Interestingly, MTV and MCMV were frequently isolated from the same populations of mice, and co-infection of MTV with MCMV results in increased MCMV-related mortality, perhaps due to the immunosuppressive effect of MTV (Cross et al. 1979).
i.p. injection. Orally inoculated animals seroconverted to the virus (Morse 1989). ADULT MICE Mice inoculated with MTV as adults (or after 7 days of age) do not develop either autoimmune conditions or thymic necrosis. Within the thymus there is no change in populations of CD4+ cells. In these animals, MTV is detectable in the salivary gland from 7 days p.i. onwards, with virus shedding into the saliva. No virus is detectable in the visceral organs. From day 14 p.i., mice are seropositive for anti-MTV antibodies by IFA, with these antibodies persisting over 70 days (Cross et al. 1979). Nude mice can be infected with MTV, but there is a low level of virus shedding from infected animals (Morse 1988), indicating the requirement of T cells for viral persistence.
D. C.
Pathogenesis and Cell Tropism
NEWBORN MICE In mice infected as newborns, MTV typically induces thymic necrosis. Three days after infection, nuclear inclusions are visible in thymocytes (mostly CD4+ 8+ and CD4+ 8-), which are almost completely destroyed within 1–2 weeks. At this point, the thymus may only be 20% of normal weight. Thymocyte numbers then begin to recover, and within 1–2 weeks the thymus appears macroscopically and histologically normal, with the composition of CD4+ cell subsets returning to normal (Cross et al. 1979; Morse et al. 1999; Morse 1989). As the age of the mouse increases prior to infection, the effect of the virus on the thymus is decreased until from 6 days of age the thymus is not susceptible to MTV-related damage (Cross et al. 1979). In BALB/c and A strain mice, but not in C57BL/6, C3H, or DBA/2 mice, 30%–40% of infected newborns develop autoimmune gastritis, which is characterized by the infiltration of mononuclear cells into the gastric mucosa and destruction of parietal and chief cells (Morse et al. 1999). Smaller numbers of mice from other strains develop oophoritis or antibodies to thyroglobin. In no case was MTV found in the affected tissues. Following i.p. inoculation in newborns, virus can be detected in the thymus between 3 and 10 days after infection, with a peak titer at day 7. Viremia is seen between 3 and 7 days p.i., with MTV identifiable in the visceral organs between days 3 and 14. Virus can be detected in the salivary glands from 5 days p.i., and persists more than 200 days later. At approximately 300 days p.i., virus is still shed into the saliva (Cross et al. 1979). No serum anti-MTV antibodies can be detected by IFA in animals infected as newborns. Interestingly, in newborn animals inoculated orally with MTV, thymic necrosis also occurs, with thymic appearance at 9 days p.i. indistinguishable from that of animals inoculated by
Diagnosis
As there is no information available on the genome of MTV (although it is presumably a ds-DNA virus), there are no methods such as PCR described for the detection of viral genomes within infected animals. Newborn mice infected with MTV produce very little antibody. However, mice infected as adults produce significant amounts of serum anti-MTV antibodies, which persist for long periods. These antibodies can be detected by indirect immunofluorescence (IFA), complement fixation (CF), or ELISA. In the IFA test, cells from thymuses of MTV-infected mice are dispersed and fixed onto slides and subsequently incubated with serial dilutions of serum from test animals. Anti-MTV antibodies bound to infected cells are identified using labeled anti-mouse conjugates (Lussier et al. 1988b). Homogenates of thymuses from 7-day-old mice inoculated with MTV as newborns are used as sources of antigen for both CF and ELISA. In the CF test, serial dilutions of heat-inactivated test sera are tested against a standard antigen dilution, and in the ELISA, a standard dilution of MTV antigen is coated onto microtiter plates prior to incubation with serial dilutions of test serum. Bound anti-MTV antibodies are detected using labeled anti-mouse antibodies (Lussier et al. 1988a, 1988b). In all tests, antibody titer is determined as the reciprocal of the highest dilution giving a positive result. During experimental infection of mice with MTV (by i.p. inoculation or by contact with infected animals), all 3 tests identified seropositive animals with similar sensitivities, although ELISA testing resulted in higher serum antibody titers (Lussier et al. 1988b). Mice were seropositive for MTV up to 230 days after infection. Serum antibody may also be titrated using a neutralization assay in which serum is incubated with a known titer of virus, that is subsequently inoculated into newborn mice. Alternatively, virus can be titrated and the ID50 calculated using an infectivity assay, where thymus homogenates are inoculated into newborn mice. In both cases, thymic necrosis is scored macroscopically after 10–14 days (Cross et al. 1979; Morse 1990).
1. MURINE CYTOMEGALOVIRUS AND OTHER HERPESVIRUSES
In none of the serological tests is there any cross-reaction between MTV and MCMV (Cross et al. 1979).
ACKNOWLEDGMENTS We acknowledge the support of the National Health and Medical Research Council of Australia through Project Grant 254636, the Pest Animal Control Co-operative Research Centre, the Grains Research and Development Corporation, and the University of Western Australia. We are very grateful to Sandra Jones, Catherine Gangell, and Fiona Stanley for assistance with the preparation of this review.
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Chapter 2 Mouse Adenoviruses Katherine R. Spindler, Martin L. Moore, and Angela N. Cauthen
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. History and Isolations of Mouse Adenoviruses, Antigenic Relationships, Virus Strains, and Virus Mutants . . . . . . . . . . . . . . . . . . . . . III. Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Molecular Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. MAV-1 Genome Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. MAV-1 E1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. MAV-1 E3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. MAV-1 E4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. MAV-1 Major Late Promoter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Growth of Mouse Adenoviruses In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . A. In Vitro Infection, wt MAV-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. In Vitro Infection, MAV-1 E1A Mutants . . . . . . . . . . . . . . . . . . . . . . . C. In Vitro Infection, MAV-1 E3 Mutants . . . . . . . . . . . . . . . . . . . . . . . . . VI. Clinical Disease and Pathogenesis of Mouse Adenoviruses . . . . . . . . . . . . A. Wild-Type MAV-1 Infection In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . B. MAV-1 E1A Mutant Infection In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . C. MAV-1 E3 Mutant Infection In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . D. Immune Response to MAV-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Innate Immune Response to MAV-1 . . . . . . . . . . . . . . . . . . . . . . . . 2. Cell-Mediated Immune Response to MAV-1 . . . . . . . . . . . . . . . . . 3. Humoral Immune Response to MAV-1 . . . . . . . . . . . . . . . . . . . . . 4. Model of MAV-1 Immunopathogenesis . . . . . . . . . . . . . . . . . . . . . E. MAV-2 Infection In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Host Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Host Range and Prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Diagnosis, Control, and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
The first mouse adenovirus was isolated by Hartley and Rowe while trying to establish the Friend leukemia virus in tissue culture from mice (Hartley and Rowe 1960). Mouse adenoviruses THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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are useful for study of adenovirus pathogenesis in the natural host, in which they cause acute and persistent infections (Smith and Spindler 1999). Such studies are not possible with the species-specific human adenoviruses (hAds). The availability of immunocompetent and immunodeficient inbred mouse Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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strains, immunological reagents for mice, and tools for genetic mapping studies combine to make mouse adenovirus studies ideal for understanding virus-host interactions. Commercial mouse suppliers standardly monitor for mouse adenoviruses, and the viruses have been eliminated in commercial mouse colonies and are rare if not absent in institutional colonies (Richter 1986). Methods for propagating and titrating the virus have been described (Cauthen and Spindler 1999a). The hAds were isolated from patients with respiratory illness independently by Rowe et al. (1953) and Hilleman and Werner (1954). The molecular biology of the hAds has been extensively studied in the years since their discovery (Shenk 2001). In addition to their use as models for studying DNA replication and mRNA transcription and processing, hAds are being widely used to develop gene therapy and vaccine vectors (GomezRoman and Robert-Guroff 2003; Hart 2003; Imperiale and Kochanek 2004; Thomas et al. 2003). Far less is known about the pathogenesis of hAds, in part due to the strict species specificity of the adenoviruses. There are currently more than 51 distinct hAd serotypes, some of which cause respiratory disease; others are associated with diseases including conjunctivitis and gastroenteritis (reviewed in Horwitz 2001). Adenoviruses are associated with acute pneumonia in children in developing countries, where they are a major cause of illness and death (Kajon et al. 1996). Severe adenovirus infections occur in immunocompromised people (Kojaoghlanian et al. 2003), including AIDS patients or those undergoing bone marrow or solid organ transplantation (Blanke et al. 1995; Carrigan 1997; Flomenberg et al. 1994). Pediatric bone marrow transplant patients are particularly at risk of hAd infection and mortality (Gavin and Katz 2002; Hale et al. 1999; Walls et al. 2003). In productive infections, the mouth, nasopharynx, or ocular conjunctiva are the initial site of hAd entry, with replication in epithelial cell types (Horwitz 2001). Like the mouse adenoviruses, hAds also cause persistent infections (Lukashok and Horwitz 1999). A study using sensitive real-time PCR coupled with lymphocyte purification suggests that human mucosal T lymphocytes are the site of hAd persistence (Garnett et al. 2002).
II.
HISTORY AND ISOLATIONS OF MOUSE
ADENOVIRUSES, ANTIGENIC RELATIONSHIPS, VIRUS STRAINS, AND VIRUS MUTANTS Although mouse adenoviruses have been isolated numerous times, there are only two serotypes, murine adenovirus 1 and murine adenovirus 2, as classified by the International Committee on Taxonomy of Viruses (2000). These are currently categorized as belonging to two different species, murine adenovirus A and murine adenovirus B, respectively. This is supported by data on serum neutralization (Lussier et al. 1987), growth characteristics (Smith et al. 1986), and genome restric-
tion analysis (Hamelin et al. 1988; Hamelin and Lussier 1988; Jacques, Cousineau, et al. 1994). Because these viruses are not infectious for infant rats (Smith and Barthold 1987) and because of the host species specificity of adenoviruses, we favor nomenclature that uses “mouse” instead of “murine,” and virus abbreviations as suggested by Ishibashi and Yasue (1984). Mouse adenovirus type 1 (MAV-1) was the first mouse adenovirus isolated (Hartley and Rowe 1960) and has also been designated in the literature as FL, MAdV-1, MAdV-FL, and MAd-1. Mouse adenovirus type 2 (MAV-2), also known as strain K87, was isolated from the feces of healthy mice by Hashimoto et al. (1966). In the work describing the isolation of MAV-1 and its classification as an adenovirus, Hartley and Rowe (1960) demonstrated a serologic relationship between MAV-1 and the hAds. Guinea pig serum from hAd-inoculated animals reacted against MAV-1 antigens. Similar results were obtained by Larsen and Nathans (1977) using serum against the hAd group antigen, hexon (a capsid protein). Hashimoto et al. (1966) used serum against hAd3 raised in guinea pigs to demonstrate a positive complement fixation reaction of MAV-2 antigen. For both MAV-1 and MAV-2, anti-MAV sera raised in mice are poorly reactive against hAd antigens (Hartley and Rowe 1960; Hashimoto et al. 1966). The antigenic relationships between MAV-1 and MAV-2 have been examined in several studies. In cross-neutralization tests between the two serotypes, there is a one-sided relationship between them (Wigand et al. 1977). MAV-2 antiserum neutralizes both MAV-1 and MAV-2, whereas MAV-1 antiserum neutralizes MAV-1 but only weakly neutralizes MAV-2. A similar partial serological relationship was identified by Lussier et al. (1987). Smith et al. (1986) found that MAV-1 antiserum neutralizing antibody titer is fourfold higher with MAV-1 than with MAV-2. MAV-1 has been more extensively studied than MAV-2. MAV-1 is available from the American Type Culture Collection (cat. no. VR550), but it should be noted that genome sequence differences and slight pathogenesis differences were found between the “ATCC” strain and the strain that we and others obtained directly from Steven Larsen (referred to as “standard”) (Ball et al. 1991). This was somewhat surprising, since the ATCC strain was deposited by Dr. Larsen. Evidently the ATCC and standard viruses have different passage histories. As discussed below (Section IV) the complete MAV-1 DNA sequence has been compiled, but there have been no sequences reported for MAV-2. A restriction map of MAV-2 has been determined (Jacques, D’Amours, et al. 1994). Mutant strains of MAV-1 have been constructed by sitedirected mutagenesis techniques using the standard strain as the starting virus. Because the standard MAV-1 genome has two EcoRI sites, these were mutated singly to obtain viruses that have a single EcoRI site in either early region 1 (E1) (Smith et al. 1996) or early region 3 (E3) (Beard and Spindler 1996). The resulting viruses, pmE301 and pmE101, have been used to
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2. MOUSE ADENOVIRUSES
construct mutants in E1A and E3, respectively (Beard and Spindler 1996; Cauthen et al. 1999; Smith et al. 1996). These virus mutants were named based on the types of mutations that were introduced (pm, point mutation; dl, deletion) and the gene where the mutation lies, E1 or E3, followed by an isolation number. The in vitro and in vivo growth characteristics of these viruses are discussed below (Sections V, B and C and VI, B and C). A naturally occurring MAV-1 mutant was isolated by Winters et al. (1981). This variant exhibits a large-plaque phenotype in cell culture and causes clinical disease with a distinct pulmonary tropism in adult C3H/HeJ mice. The mutation(s) responsible for this phenotype have not been mapped.
III.
PHYSICAL PROPERTIES
MAV-1 and MAV-2 share many physical properties, including ether resistance, thermal inactivation at 56°C, and a size of ∼80–90 nm (Hartley and Rowe 1960; Hashimoto et al. 1966). Both mouse adenoviruses lack hemagglutinating activity, unlike the hAds. MAV-1 has a buoyant density in CsCl like that of the hAds, 1.34 g/ml (Larsen and Nathans 1977; Wigand et al. 1977). MAV-1 is inactivated by 50% ethanol (Larsen and Nathans 1977).
IV. A.
MOLECULAR GENETICS
mapping of E1, E3, early region 4 (E4), and identification of the major late promoter (MLP) (Ball et al. 1989, 1991, 1988; Beard et al. 1990; Cai et al. 1992; Cauthen and Spindler 1996; Kring et al. 1992; Kring and Spindler 1990; Raviprakash et al. 1989; Song et al. 1996, 1995; Weber et al. 1994). Davison et al. (2003) have analyzed the genetic content, phylogeny, and evolution of the family Adenoviridae, and they have submitted a third-party annotation for MAV-1 (AC_000012). It should be noted that their annotation includes some predicted genes for MAV-1 that are not in agreement with published experimental evidence. For example, their MAV-1 E1A annotation does not take into account E1A transcription mapping and cDNA sequencing data (Ball et al. 1989). Other differences are indicated for IVa2, the DNA polymerase, and pTP genes, which could be correct; these regions have not been transcription mapped. MAV-1 has inverted terminal repeats (ITRs) that are 93 nucleotides (nt) long (Ball et al. 1991, 1988; Temple et al. 1981). These ITRs have the first 18 nt that are highly conserved among the adenoviruses and that are essential for replication of the viral DNA (Challberg and Rawlins 1984; Lally et al. 1984; Tamanoi and Stillman 1983). Unlike hAds, MAV-1 does not encode virus-associated (VA) RNAs (Meissner et al. 1997). Although MAV-1 polypeptide III (penton base) shares high amino acid identity with the hAds, it does not have the arginineglycine-aspartic acid (RGD) motif found in many hAds and thought to be important for viral internalization via cellular integrins (Meissner et al. 1997). However, it has a leucine-aspartic acid-valine (LDV) motif recognized by other integrins, present as LDL and, like porcine adenoviruses, MAV-1 has an RGD sequence in the C-terminus of the fiber protein.
MAV-1 Genome Features
The complete sequence (30,944 base pairs) of the doublestranded DNA genome of MAV-1 has been determined (Meissner et al. 1997) (accession number NC_000942). The ordering of genes in the MAV-1 genome, shown in Fig. 2-1, has been accomplished through sequence comparison with hAds, transcription
B.
MAV-1 E1
Genes expressed prior to early Ad viral replication are designated as “early,” while those expressed at or after the time of DNA replication are “late.” The E1 region in MAV-1
Fig. 2-1 Genomic organization of MAV-1. The genome is indicated by the horizontal lines in the center, and map units are indicated below the line. Circles on the end of the genome indicate the terminal protein. Genes whose transcription has been mapped are indicated by arrows; for E3 and E4, multiple transcripts are indicated by only a single arrow. Open reading frames with similarity to proteins of hAds are indicated by the boxes. Genes transcribed in the rightward direction are indicated above the genome, and genes transcribed in the leftward direction are indicated below. The major late promoter (MLP) is indicated by the arrowhead. DNApol, DNA polymerase; pTP, terminal protein precursor; DBP, DNA binding protein. (Adapted from Smith and Spindler, 1999, with permission.)
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corresponds in location to the E1 region of hAds, at the left end of the genome (Ball et al. 1988), and the mRNAs are transcribed in a rightward direction (Ball et al. 1989). E1 encodes three mRNAs that overlap; one is designated E1A, and the other two correspond to hAd mRNAs that encode E1B 19K and E1B 55K proteins (Ball et al. 1989). The three mRNAs are 3′ coterminal, and the three proteins share a common C-terminal sequence. The MAV-1 E1A protein sequence has little overall sequence similarity to the hAd E1A 289 aa protein (encoded by the 13S E1A mRNA). However, Ball et al. (1989) showed that MAV-1 E1A has approximately 40% sequence similarity in conserved regions 1 (CR1), CR2, and CR3 compared to CR1, CR2, and CR3, respectively, of the hAd 289 aa protein (Moran and Mathews 1987). MAV-1 CR2 is the most similar to that of hAds, with approximately 50% sequence similarity (Ball et al. 1988). The predicted amino acid sequence similarity of the MAV-1 E1B coding regions compared to that of the hAd 19K and 55K proteins is 37% and 42%, respectively (Ball et al. 1988). No further studies involving the E1B proteins have been reported. The E1A protein is detected in MAV-1 infections in cell culture at both early and late times by immunoprecipitation with polyclonal antiserum raised against amino acids 27–200 of the E1A protein (Smith et al. 1996; Ying et al. 1998). The E1A protein is predicted to have a molecular weight of 22 kDa, but migrates slightly larger than 30 kDa, likely due to its phosphorylation (Smith et al. 1996). hAd E1A proteins are also phosphorylated (Gaynor et al. 1982; Yee and Branton 1985a; Yee et al. 1983). However, the significance of the phosphorylation of E1A in MAV-1 infections is not known. MAV-1 viruses with mutations in E1A are described in Table 2-1. The E1A protein expression patterns were evaluated by Western blot analysis and immunoprecipitation (Smith et al. 1996; Ying et al. 1998). The dlE102 (CR2 deletion) and dlE106 (CR3 deletion) E1A proteins migrate faster than the wild-type (wt) E1A, as expected. pmE109 and pmE112, initiator methionine mutants, do not synthesize detectable levels of E1A protein.
TABLE 2-1
EFFECTS OF MAV-1 E1A MUTANTS IN CELL CULTURE AND OUTBRED SWISS MICE E1A mutant
Effect of the mutationa
dlE105 dlE102 dlE106 pmE109 pmE112
Deletion of CR1 region (amino acids 35–78) Deletion of CR2 region (amino acids 111–129) Deletion of CR3 region (amino acids 135–154) Mutation of initiator ATG to TTG Mutation of initiator ATG to CAC
aSmith
Average LD50 (log PFU)b 103.5 100.9 102.6 103.5 103.9
et al. 1996 et al. 1998; the average LD50 for wt virus in these experiments was 10-1.5. bSmith
Although dlE105 (CR1 deletion) has a 43 amino acid deletion, it produces protein that migrates slower than the wt protein; a similar phenomenon is seen in hAd 5 E1A CR1 deletion proteins (Egan et al. 1988). hAd E1A is a transcriptional regulator (reviewed in Gallimore and Turnell 2001), and it is required for activation of early viral transcription (Berk et al. 1979; Jones and Shenk 1979). A plasmid encoding the left end of the MAV-1 genome (including the E1A coding region) transactivates the hAd5 E3 promoter in both HeLa cells and mouse L929 cells, albeit at a level lower than that of hAd E1A (Ball et al. 1988). Although MAV-1 E1A is able to transactivate the hAd5 E3 promoter, unlike hAd E1A, it does not appear to stimulate the expression of the other early mRNAs (E1A, E1B, E2, E3, and E4), at least in 3T6-infected cells at a multiplicity of infection (MOI) of 5 (Ying et al. 1998). Thus, the importance of the transactivation by MAV-1 E1A in reporter assays of transfected cells (Ball et al. 1988) is not understood. hAd E1A CR2 encodes a retinoblastoma protein (pRB) binding motif, (D)-L-X-C-X-E, that is conserved as (D)-L-R-C-Y-E in MAV-1 E1A CR2. MAV-1 E1A protein binds to mouse pRb and related proteins (Smith et al. 1996). This was shown both for in vitro transcribed and translated pRb protein (Smith et al. 1996) and for the endogenous pRb in virus-infected cells (L. Fang et al. 2004). This binding is primarily dependent on the presence of E1A CR2 (Smith et al. 1996). Similar experiments also showed that related pRb family proteins p107 and p130 bind to MAV-1 E1A (Smith et al. 1996; L. Fang et al. 2004). For the in vitro–produced p107, CR2 is necessary and sufficient for binding (Smith et al. 1996); p107 binds to hAd E1A in the CR2 region (Dyson, Guida, Münger, et al. 1992; Harlow et al. 1986; Whyte et al. 1989; Yee and Branton 1985b). In both the pRb and the p107 experiments in which infected cell lysates were mixed with in vitro translated pRb or p107, the E1A CR1 deletion mutant protein bound to pRb and p107 at greatly reduced levels compared to wt E1A (Smith et al. 1996). This suggests that CR1 may play a cooperative or stabilizing role in pRb and p107 binding to CR2 of E1A, as has been shown for hAd E1A (Dyson, Guida, McCall, et al. 1992; Ikeda and Nevins 1993). The functional relevance for the interaction between MAV-1 E1A and pRB was shown by experiments in SAOS-2 cells, which lack pRb (Smith et al. 1996). SAOS-2 cells transfected with mouse pRb alone exhibit an arrested growth phenotype that is reversed when MAV-1 E1A and pRb are introduced into the cells together.
C.
MAV-1 E3
Transcription mapping of the E3 region of MAV-1 indicated that this region produces three early mRNAs that are 5′ and 3′ coterminal (Beard et al. 1990). Each E3 mRNA has three exons; the first and second are identical among the three mRNAs, and the last intron of each is different due to differential splicing.
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Thus, the predicted E3 proteins share a 72 amino acid N-terminus with unique C-terminal domains. The three mRNAs transcribed at early times are referred to as class 1 mRNA, which gives rise to the E3 gp11K protein, class 2, and class 3 mRNAs (Beard et al. 1990). Only the E3 gp11K protein has been detected in wt virus infection of cultured cells (Beard and Spindler 1995). A fourth type of mRNA transcribed at late times (after viral DNA replication) is also detected that encodes E3 gp11K (Beard et al. 1990; Cauthen and Spindler 1999b). A polyclonal antiserum that specifically recognizes the unique portion of the E3 gp11K protein was used to demonstrate that E3 gp11K is detected in infected 3T6 cells beginning at 16 hours postinfection (h PI) and is detected until at least 48 h PI (Beard and Spindler 1995). The E3 gp11K protein can also be immunoprecipitated from radiolabeled infected cell lysates at both early and late times in infection. Additionally, the gp11K protein is recognized by polyclonal antiserum that is specific for the common portion of the E3 proteins (Beard and Spindler 1996). This antiserum fails to detect the other E3 proteins in MAV-1 infection of 3T6 cells, presumably because the proteins produced by the class 2 or class 3 mRNA are absent from infections in cell culture or are produced at levels too low for detection (Beard and Spindler 1996; Cauthen et al. 1999; Cauthen and Spindler 1999b). The E3 gp11K protein appears to be approximately 14K in infected cell lysates, but this is larger than the size of the product produced during in vitro transcription and translation of a plasmid containing the E3 gp11K gene (Beard and Spindler 1995). This size difference can be attributed to modifications made to the protein in infected cells: the cleavage of the signal sequence (predicted at amino acids 1 to 37) at the N-terminus of the E3 gp11K protein, and glycosylation at the predicted N-glycosylation consensus site (N-X-S/T) located at amino acid 56. The E3 gp11K protein localizes to the ER of the cell and is a peripheral membrane protein, as determined by alkaline extraction and phase separation experiments (Beard and Spindler 1995). E3 gp11K is transcribed and translated as an early mRNA and protein. However, a large mRNA expressed at late times can also encode E3 gp11K (Cauthen and Spindler 1999b). This expression of E3 gp11K is due to alternative splicing of a late transcript encoding a capsid protein, pVIII. pVIII is predicted to be translated from an unspliced late mRNA. However, if the primary pVIII transcript is spliced at the “E3” sites, a fusion protein consisting of sequences of pVIII and E3 gp11K is predicted to occur (Cauthen and Spindler 1999b), since they are translated in the same reading frame and their coding regions partially overlap (Raviprakash et al. 1989). It is thought that the signal sequence of the E3 gp11K coding region allows the pVIII-E3 gp11K fusion protein to enter the ER and get processed, producing a mature E3 gp11K (Cauthen and Spindler 1999b). The relevance of transcription and translation of E3 gp11K at late times is not known. The E3 mRNAs and their putative proteins have been identified or described, but the functions of these proteins have not
yet been discovered. hAd E3 proteins are involved in immune evasion (Fessler et al. 2004). For example, the hAd2/5 E3 gp19K prevents the display of class I major histocompatibility complex (MHC) antigens on the surface of infected cells, and this has been proposed as a mechanism enabling persistence of hAds (Levine 1984; Wold and Gooding 1991). However, unlike hAds, MAV-1 does not prevent the display of class I MHC antigens on the surface of infected mouse cells in culture (Kring and Spindler 1996). D. MAV-1 E4 The transcription map of MAV-1 E4 was determined and indicates that E4 encodes seven classes of mRNAs that are 3′ coterminal (Kring et al. 1992). The predicted coding regions of three of these mRNAs have some sequence similarity to hAd E4 proteins. The MAV-1 E4 orf a/b has 48% sequence similarity to the hAd2 E4 34K (orf 6) protein (Ball et al. 1991; Kring et al. 1992). MAV-1 orf a/c has 69% sequence similarity to the hAd2 E4 11K (orf 3) protein. The N-terminus of MAV-1 orf d has 55% sequence similarity to hAd2 E4 orf 2 (Kring et al. 1992), and the entire MAV-1 orf d has 60% sequence similarity to hAdE4-orf6/7 (L. Fang and K. Spindler, unpublished data). Further studies involving the E4 region of MAV-1 have not been reported. E. MAV-1 Major Late Promoter The MLP in hAds directs the synthesis of the late messages, and the MLP in MAV-1 was mapped to a similar region in the genome using ribonuclease protection assays and primer extension analysis (Song et al. 1996). The MAV-1 MLP has a TATA box, an inverted CAAT box, an SP1 binding site, and a DE1 element (Song et al. 1996). Notably, there is an absence of a USF-binding site, an initiator element (INR), and a DE2 element found in hAds and other mammalian viruses (Song and Young 1997). The lack of an INR may explain the finding of more than one start site of the MAV-1 MLP. Song and Young (1997) used a mutational analysis to demonstrate that the TATA box, SP1 site, and CAAT box elements are important for the MAV-1 MLP to function at normal levels in cells. Additionally, gel mobility shift assays were employed to show that the SP1 protein binds to the MAV-1 MLP with high affinity.
V.
GROWTH OF MOUSE ADENOVIRUSES IN VITRO A.
In Vitro Infection, wt MAV-1
Temple et al. (1981) reported that MAV-1 viral DNA synthesis is first observed at 35 h PI, even at MOIs of up to 800 PFU/cell
K AT H E R I N E R . S P I N D L E R , M A RT I N L . M O O R E , A N D A N G E L A N . C A U T H E N
when using [3H]thymidine labeling. However, using 32PO4 labeling and analysis of viral DNA prepared by the Hirt method (1967), we observed MAV-1 DNA synthesis as early as 20 h PI in L929 cells infected at a MOI of 10 PFU/cell (Fig. 2-2). Onestep growth curves in 3T6 cells and mouse brain microvascular endothelial cells (MBMEC) show that MAV-1 exhibits a typical eclipse period, with an increase in virus titer relative to input virus at 36–48 h PI (Cauthen et al. 1999; Ying et al. 1998). Similar results were seen in a two-step growth curve in mouse L929 cells (Fig. 2-3). Cytopathic effects are first detected at 36–48 h and are similar to other adenoviruses: Cells infected with wild-type MAV-1 round up, become refractile, and eventually detach from the substrate (Fig. 2-4). Analysis of [35S]-labeled infected cell proteins by electrophoresis indicates that unlike hAds, MAV-1 does not efficiently shut off host cell protein synthesis (Antoine et al. 1982; Ying et al. 1998). At least two mechanisms are proposed for the selective translation of viral mRNAs in hAd-infected cells resulting in shutoff of
107
106
Titer (PFU)
54
105
104
103 0
24
48
72
96
120
144
168
h PI
Fig. 2-3 Two-step growth curve of MAV-1 on L929 cells. Monolayers were infected with MAV-1 at a MOI of 0.1, harvested at the indicated times, and titrated by plaque assay on L929 cells.
Fig. 2-2 Time course of MAV-1 DNA replication. L929 cells were mock infected or infected at a MOI of 10. The cultures were labeled for one hour with 32PO and harvested at the indicated times PI. Hirt supernatants containing viral 4 DNA were isolated (Hirt 1967) and digested with HindIII and RNase A and electrophoresed on 0.7% agarose gels, dried and autoradiographed. The sizes of the restriction fragments are indicated on the right. In a parallel experiment performed with hAd5 in HeLa cells, DNA replication was first detected at 16 h PI (data not shown).
host protein synthesis (reviewed in Shenk 2001). One of these is via the VA RNAs; the lack of VA RNAs in MAV-1 infected cells (Meissner et al. 1997) could be responsible for defective host protein synthesis shutoff in MAV-1 infection. The other proposed mechanism of host protein synthesis shutoff in hAd-infected cells is via an inactivation of eIF-4F by dephosphorylation observed late after hAd infection; whether this occurs in MAV-1 infection has not been reported. The receptor for MAV-1 has not been described. hAds use a two-step mechanism for entry. The protruding hAd fiber protein interacts with the cellular receptor (Philipson et al. 1968), which has been shown for a majority of hAd serotypes to be an immunoglobulin superfamily member, the Coxsackie-adenovirus receptor (CAR) (Bergelson et al. 1997; Tomko et al. 1997). In the second step, the penton base interacts with host integrin molecules to promote entry (Wickham et al. 1993). The murine homolog of CAR (mCAR) has been identified, and it can serve as a receptor for hAds (Bergelson et al. 1998; Tomko et al. 1997). However, it is not known whether mCAR is a receptor for MAV-1 or whether MAV-1 uses integrins for entry. Productive MAV-1 infections occur in cell lines that do not express mCAR, such as mouse L929 cells and NIH3T3 fibroblasts (Tomko et al. 1997), suggesting that mCAR is not required for entry. It is believed that MAV-1 does not replicate productively in cultured human cells (Antoine et al. 1982; Larsen and Nathans 1977; K. Spindler, unpublished data), although there is one early conflicting report (Sharon and Pollard 1964). Infection of human cells does not yield infectious virus (Larsen and Nathans 1977); viral DNA synthesis occurs, but there appears to be a defect at
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2. MOUSE ADENOVIRUSES
mock
wt
pmE312
Fig. 2-4 Cytopathic effects of MAV-1 infection. Mouse 3T6 fibroblasts were mock-infected or infected with wt or pmE312 mutant virus at a MOI of 1 and photographed at 2.5 days PI. Wild-type virus consistently caused cells to release from the culture dish, whereas pmE312 did not, even upon prolonged incubation (data not shown). (Adapted from Cauthen 1998, with permission.)
the level of formation of virus particles due to defects in virus structural proteins, particularly hexon (Antoine et al. 1982).
B. In Vitro Infection, MAV-1 E1A Mutants Mouse 3T6 cells and 37.1 cells (a 3T6-derived cell line that expresses MAV-1 E1A protein) infected at a MOI of 5 with each of the E1A mutant viruses (Section IV, B) give yields of virus approximately like those of wt virus (Ying et al. 1998). Similarly, serum-starved 3T6 cells and transgenic mouse embryo fibroblasts (MEFs) from pRb+/+, pRb+/-, and pRb−/− embryos infected at a MOI of 5 give yields of E1A mutant viruses similar to that of wt virus, indicating that at high MOIs, E1A is not required to stimulate progression of the cell cycle and DNA replication (Ying et al. 1998). However, infection of 3T6 cells, mouse brain microvascular endothelial cells (MBMECs), and MEFs at MOIs of 0.05 resulted in a two log unit reduction in viral yield for E1A null mutant pmE109 (Fang and Spindler 2005; M. Moore and K. Spindler, unpublished data). Multiplicity-dependent growth of mutant viruses has been observed previously for hAds (Gaynor and Berk 1983; Imperiale et al. 1984; Nevins 1981), human cytomegalovirus (Bresnahan and Shenk 2000; Oliveira and Shenk 2001), and
herpes simplex virus 1 (Cai and Schaffer 1992; Chen and Silverstein 1992; Everett et al. 2004), but its significance is unknown. The hAd E1A protein protects cells from the interferon (IFN) response (IFN-α/β and -γ) by inhibiting the ISGF3 transcription factor, thereby reducing expression of IFN-stimulated genes (ISGs) (Ackrill et al. 1991; Gutch and Reich 1991; Leonard and Sen 1996). Similarly, MAV-1 E1A protects mouse 3T6 cells from IFN-α/β and IFN-γ (Kajon and Spindler 2000). E1A can provide this protection from IFN-α/β in trans to vesicular stomatitis virus (VSV). The presence of MAV-1 E1A (either in 37.1 cells or virus-infected cells) correlates with a reduction in steady-state levels of ISGs, suggesting that the mechanism of protection from IFN effects is like that of the hAds. We have preliminary evidence that MAV-1 E1A is able to bind to STAT-1 (L. Fang and K. Spindler, unpublished data), as has been suggested for hAd5 E1A (Look et al. 1998).
C.
In Vitro Infection, MAV-1 E3 Mutants
A series of mutations were made in E3 of MAV-1 to determine the individual effects of the three putative E3 proteins (Section IV, C) on virus replication in cell culture and on
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TABLE 2-2
EFFECTS OF MAV-1 E3 MUTANTS IN CELL CULTURE AND MICE E3 mutant virus
Effect of the mutation
pmE310 pmE312 pmE314 dlE303 dlE307 dlE309
No early expression of E3 protein; late expression of E3 gp11K No early expression of E3 protein; late expression of E3 gp11K No expression of E3 proteins at early or late times in infection Produces E3 gp11K mRNA only; E3 gp11K detected in cell culture Produces E3 class 2 mRNA only; no protein detected in cell culture Produces E3 class 3 mRNA only; no protein detected in cell culture
a
Average LD50 (log PFU) 101.5 102.9 a 105.2 103.7 103.6 104.7 a
Reference Beard and Spindler 1996 Cauthen and Spindler 1999b Cauthen et al. 1999 Beard and Spindler 1996 Beard and Spindler 1996 Beard and Spindler 1996
This value is from a single experiment.
growth and pathogenesis in mice. The E3 mutant viruses and the effects of their mutations on the synthesis of the putative E3 proteins are shown in Table 2-2. The E3 mutant viruses have growth patterns similar to those of wt virus in mouse 3T6 cells at a high multiplicity (MOI of 5–10). pmE310, pmE314, and dlE309 grow to titers similar to that of wt virus (Beard and Spindler 1996; Cauthen and Spindler 1999b). In addition, pmE314 virus grew as well as wt virus in MBMECs (Cauthen and Spindler 1999b), which were tested since endothelial cells are one of the target cells of MAV-1 in vivo (Charles et al. 1998; Kajon et al. 1998). dlE303 and dlE307 grow to titers approximately 10- to 50-fold lower than that of wt virus (Beard and Spindler 1996). pmE312 grows to titers approximately 10-fold lower than that of wt virus (Cauthen and Spindler 1999a). dlE303, dlE307, and pmE312 all exhibited slightly smaller plaques than wt virus (N. Cauthen and K. Spindler, unpublished data), and pmE312 showed a unique cytopathic effect in 3T6 cells (Fig. 2-4). The multiplicity dependence seen for MAV-1 E1A (Section V, B) is not seen for E3. E3 mutants has yields like wt virus, even at a MOI of 0.05 (M. Moore and K. Spindler, unpublished data).
VI.
CLINICAL DISEASE AND PATHOGENESIS OF MOUSE ADENOVIRUSES A.
Wild-Type MAV-1 Infection In Vivo
MAV-1 permits the study of a replicating Ad in vivo and provides a good model of Ad pathogenesis. hAds do not replicate in rodents, but high dose (108 to 1010 PFU per animal) intranasal (i.n.) infection of cotton rats and mice induces pulmonary disease (Ginsberg et al. 1991; Pacini et al. 1984). In contrast to hAds, MAV-1 doses as low as 1–100 PFU cause fatal disease in newborn and adult mice (Spindler et al. 2001; van der Veen and Mes 1973). The outcome of MAV-1 infection depends on the virus dose, the mouse strain and age, the inoculation route, and the strain of virus.
When mice survive an acute MAV-1 infection, they can become persistently infected (reviewed in Smith and Spindler 1999). Infectious MAV-1 was found at high titers in urine of infected mice at 11 months PI (Rowe and Hartley 1962) and 24 months PI (van der Veen and Mes 1973). Infectious virus was found in kidney 70 days PI (Ginder 1964) and in liver 52 days PI (Wigand 1980). Virus particles were detected in urine, and viral DNA was detected in brains, spleens, and kidneys of mice 55 weeks PI (Smith et al. 1998). E1A mutants of MAV-1 persist for up to 55 weeks PI (Smith et al. 1998) (Section VI, B). The mechanism by which MAV-1 persists is not understood (Smith and Spindler 1999). This long-term shedding of MAV-1 is paralleled in human adenoviruses, which have been found shed in feces up to 2 years after infection (Fox et al. 1969). Human adenoviruses have also been isolated from urine of patients with AIDS (de Jong et al. 1983). Mouse adenoviruses have not been reported to be oncogenic, and MAV-1 virions and viral DNA do not cause transformation of cloned rat embryo fibroblast (CREF) cells, unlike hAd virions and viral DNA (K. Spindler, unpublished data). The effects of mouse age on infection are as follows. In adult mice MAV-1 induces clinical signs of disease and dose-dependent acute encephalomyelitis in outbred (Kring et al. 1995), C57BL/6 (B6) (Guida et al. 1995), 129 Sv/Ev (M. Moore and K. Spindler, unpublished data), and SJL/J (Spindler et al. 2001) mice. Adult BALB/c mice are resistant to MAV-1-induced disease, but high dose infection results in ruffled fur, hyperpnea, and conjunctivitis (Charles et al. 1998; Guida et al. 1995; Moore et al. 2004). In suckling mice MAV-1 produces fatal, disseminated disease and myocarditis (Blailock et al. 1967; Hartley and Rowe 1960), and intranuclear inclusions typical of adenovirus infections are seen in endothelial cells of the brain (Heck et al. 1972). Similar widely disseminated disease is seen in suckling mice infected with both the “standard” and “ATCC” strains of MAV-1 (Ball et al. 1991) (Section II). In both adult and suckling mice, regardless of dose, infection results in viremia as early as 1 day PI and is detected in tissues at 3 days PI (Heck et al. 1972; Spindler et al. 2001); viral DNA is found in tissues as early as 2 days PI (Ball et al. 1991). The effects of mouse strain differences in the disease outcome are discussed below (Section VII).
2. MOUSE ADENOVIRUSES
Inoculation of MAV-1 by different routes does not result in major differences in pathogenesis. Outbred mice infected i.n. with MAV-1 exhibit slightly fewer disease signs and have protracted infection kinetics compared to mice infected intraperitoneally (i.p.) (Kajon et al. 1998; Wigand 1980). High dose (106 PFU) i.n. infection of newborn mice results in a robust macrophage infiltrate in the lung at 3 days PI (Gottlieb and Villarreal 2000). Intravenous (i.v.) infection of inbred mice results in levels of virus in brain and spleen, early antibody responses, and survival in B6 and B cell–deficient mice similar to what is seen in i.p. infection (Moore et al. 2004). Intracerebral (i.c.) inoculation of outbred mice results in significant infection of the adrenal gland (Margolis et al. 1974), an organ which also has significant viral DNA levels when infected i.p. (Kring et al. 1995). Similar to mice infected i.p. with MAV-1 (Spindler et al. 2001), susceptible and resistant adult inbred mice infected i.c. showed replication of the virus in brain and spleen (Fig. 2-5A). Interestingly, in the resistant BALB/c mice, virus was detected at high levels in the spleen as early as 2 days PI and was even detected in one susceptible SJL spleen at 2 days PI. The high levels of virus found earlier in BALB/c spleens were surprising, since BALB/c mice are resistant to the virus (Guida et al. 1995; Spindler et al. 2001). One interpretation of the data is that earlier high levels of virus in spleen correlate with or stimulate a stronger innate immune response in BALB/c mice that is able to control subsequent replication in resistant mice. To determine whether
57 the i.c. injections would result in a difference in viral titers at 8 days PI, mice were infected with three low doses of MAV-1 (Fig. 2-5B). Higher levels of virus were found in the brains and spleens of SJL mice than BALB/c mice at every dose (except spleens at the lowest dose), similar to results of infection of SJL mice by the i.p. route (Spindler et al. 2001; Welton et al. 2005). Taken together, the data suggest a model where MAV-1 replicates equally well in susceptible SJL and resistant BALB/c mouse brains, but as the infection proceeds, viral replication is controlled in resistant mice but not in susceptible mice, resulting in higher viral loads at later times after infection. Pregnant mice infected with MAV-1 show histological signs of infection, but the virus does not cross the placenta (Lipps and Mayor 1980; Margolis et al. 1974). Maternal antibodies to MAV-1 are protective for suckling mice (Hartley and Rowe 1960; Lipps and Mayor 1982). In most strains of MAV-1-infected adult mice, MAV-1 infects cells of the monocyte/macrophage lineage and endothelial cells of the vasculature throughout the mouse; highest levels of virus are found in the spleen and central nervous system (CNS) (Charles et al. 1998; Guida et al. 1995; Kajon et al. 1998; Kring and Spindler 1990). MAV-1 nucleic acid is also detected by in situ hybridization (ISH) in the renal tubular epithelium of adult outbred mice infected i.p. or i.n. (Kajon et al. 1998; Smith et al. 1998). Viral DNA is detected in organ homogenates of bowel, pancreas, spleen, adrenal gland, kidney, liver, lung, heart, brain,
Fig. 2-5 Intracerebral MAV-1 infections of mice. A. Anesthetized susceptible SJL/J and resistant BALB/c mice were injected i.c. with 104 PFU MAV-1 in a volume of 30 µl. Mice were euthanized at the indicated days PI (dpi), and brain and spleen titers were determined by plaque assay. Each symbol represents an individual mouse; the short horizontal lines indicate the mean values. The arrow indicates the input dose in PFU/g, assuming brains have a mass of 0.3 g. The asterisk and dotted line indicate the level of detection. B. Mice were infected i.c. with the indicated dose of virus, and brain and spleen titers were determined at 8 days PI. P < 0.03 for the 3 PFU brain and spleen titers between SJL and BALB/c mice; P = 0.002 for the 30-PFU brain and spleen titers.
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and spinal cord of i.p. infected outbred mice (Kring et al. 1995); most likely this is due to infection of endothelium throughout the mice. MAV-1 has not been observed in the brain parenchyma; it is restricted to the vascular endothelium (Charles et al. 1998; Guida et al. 1995; Kajon et al. 1998). Mice with MAV-1-induced encephalomyelitis exhibit histological evidence of perivascular edema in the CNS, and moribund infected mice also exhibit endothelial cell reactivity, vasculitis, vascular wall degeneration, and viral inclusion bodies in microvascular endothelial cells (Charles et al. 1998; Guida et al. 1995; Kajon et al. 1998; Kring et al. 1995; Spindler et al. 2001).
B.
Sublethal doses of γ-irradiation result in increased levels of MAV-1 in infected mice 42–55 weeks PI (Smith et al. 1998). The percentage of mice with viral DNA–positive organs (brain, kidney, and spleen) increases in post-irradiation mice previously infected with wt or E1A mutant viruses, peaks in the first week following irradiation, and returns to pre-irradiation percentages by 3 weeks PI. Thus MAV-1 can persist in the brains, spleens, and kidneys of infected mice for up to 55 weeks PI, the virus can be shed in the urine, and E1A is not required for the persistence of the virus. It is not known whether the virus produced is infectious virus, nor is it known whether persistence is due to a chronic or latent infection.
MAV-1 E1A Mutant Infection In Vivo
The 50% lethal dose (LD50) of each E1A mutant virus (Section IV, B) was determined and is shown in Table 2-1. The LD50 values for the E1A mutants are 1.5–5 log units higher than that of wt virus, indicating that the E1A protein is important in MAV-1 pathogenesis. pmE112, the virus that does not synthesize the E1A protein, is the least virulent (Smith et al. 1998). The disease signs observed in the mutant virus–infected mice are identical to those of wt-infected mice, and the onset of disease is dose-dependent. Studies of the E1A null mutants, pmE109 and pmE112, were carried out in mice to evaluate histopathology and the presence and quantity of viral DNA in mice (Smith et al. 1998). Neither wt nor E1A null mutant viruses produce detectable levels of virus, as measured by dot blot analysis of DNA, in the spleens at 5 d PI in outbred mice infected with 104 PFU. Brains of wt and pmE109-infected mice have similar levels of viral DNA, but the other E1A null mutant, pmE112, produces significantly less viral DNA in the mouse. At lower dose infections, near the LD50 for the wt virus, no viral DNA is detected in brains or spleens of the pmE109- or pmE112-infected mice at 5 or 14 d PI. Mutations in the E1A null mutants did not revert in vivo, because PCR amplification and sequencing of viral DNAs recovered from infected mice showed they were identical to the starting mutant viruses (K. Smith and K. Spindler, unpublished data). The E1A mutant viruses exhibit histopathology similar to that of wt virus during the acute phase of disease (Smith et al. 1998). When doses of 104 PFU are used, the tropism of pmE109 and pmE112 is similar to that of wt virus, with the exception that pmE112 is also found in thymus of infected mice. Persistence of wt and E1A mutant MAV-1 was evaluated using an immunocapture assay of urine from virus-infected mice (Smith et al. 1998). During the 12–22 week period PI, mice infected with wt and E1A mutant viruses all shed MAV-1. From 42–55 weeks PI, only wt-infected mice shed detectable levels of virus. At 42 weeks PI viral DNA is detected in brains, spleens, and kidneys of mice infected with wt and E1A mutant viruses by PCR amplification and ISH, indicating that a persistent MAV-1 infection can be established in the absence of E1A.
C.
MAV-1 E3 Mutant Infection In Vivo
Table 2-2 shows the LD50 values for infection of outbred mice by E3 mutant viruses (Section V, C), which are all less virulent than wt virus; the E3 null mutant pmE314 has the most severe defect in virulence. The elevated LD50 levels for each of the E3 mutant viruses suggest that each of the three E3 gene products plays a role in the pathogenesis of MAV-1 in vivo. Infections with E3 mutant viruses result in the same clinical signs of disease in mice as wt virus, and the onset of disease is dependent on the dose of virus (Cauthen et al. 1999; and C. Beard, N. Cauthen, and K. Spindler, unpublished data). Similar to wt virus, pmE314 is found primarily in endothelial cells of the brain and spinal cord and in endothelial cells and stationary macrophages in the spleen (Cauthen et al. 1999). Outbred mice given 105 or 106 PFU of pmE314 die 3 or 4 d PI with large numbers of viral inclusion bodies and ISH evidence of virus; it is thought that at this dose the mice die of an overwhelming infection of endothelial cells. Lower doses (103 or 104 PFU) of pmE314 given to outbred mice result in fewer and less severe histopathological changes than wt virus, particularly with respect to inflammation and endothelial damage. Viral nucleic acid is distributed in a similar pattern in the brains and spinal cords of both pmE314- and wt-infected mice, albeit at slightly lower levels in pmE314-infected brains and spinal cords. The functions of the E3 gene products have not yet been elucidated, thus the mechanism by which the lack of E3 results in reduced inflammation in pmE314 infection is unknown. D. 1.
Immune Response to MAV-1
Innate Immune Response to MAV-1
The innate immune response to MAV-1 includes early increases in the steady-state levels of the mRNAs of cytokines and chemokines (Charles et al. 1999; Charles et al. 1998). Charles and coworkers quantitated cytokine and chemokine mRNAs in mock-infected and MAV-1-infected B6 and BALB/c brains because BALB/c mice are more resistant to MAV-1-induced encephalomyelitis than B6 mice (Guida et al. 1995). MAV-1
2. MOUSE ADENOVIRUSES
increases the mRNA steady-state levels of the cytokines IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, and lymphotoxin (LT) in B6 and BALB/c brains 4 days PI (Charles et al. 1998) and transiently increases expression of IL-12 mRNA by macrophages in CBA/Ht mice (Coutelier et al. 1995). MAV-1 also increases the mRNA steady-state levels of the chemokine receptors CCR1–CCR5 in B6 and BALB/c brains 4 days PI (Charles et al. 1999). Steady-state mRNA levels of the chemokines IFN-γ-induced protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and T cell activation gene 3 (TCA-3) are increased by MAV-1 infection in B6 but not BALB/c brains 4 days PI, suggesting that innate immune responses contribute to the MAV-1-induced encephalomyelitis in B6 mice. IFN-α/β signaling plays a role in MAV-1 pathogenesis and is a determinant of MAV-1 organ tropism. In single-cycle infectious yield reduction assays in vitro, MAV-1 is more resistant than VSV to pretreatment with mouse IFN-α/β (Kajon and Spindler 2000). However, MAV-1 replication is not completely resistant to IFN-α/β, since virus yields are reduced about 10-fold in high concentrations of IFN. E1A mutants of MAV-1 are more sensitive to IFN-α/β in vitro than wt MAV-1. These data indicate that MAV-1 E1A counteracts the IFN-α/β antiviral response in vitro. The role of IFN-α/β in MAV-1-induced encephalomyelitis was examined by comparing the pathogenesis of MAV-1 in 129 Sv/Ev and IFN-α/βR−/− mice (M. Moore and K. Spindler, unpublished data), which are defective for type I IFN signaling (M¨uller et al. 1994). IFN-α/βR−/− mice had higher levels of infectious MAV-1 in spleens at 4 and 7 days PI and exhibited a more disseminated MAV-1 infection at 7 days PI than control mice (M. Moore and K. Spindler, unpublished data). However, this disseminated MAV-1 infection in IFN-α/βR−/− mice did not show clinical or histopathological differences from infection of control mice, and survival was not different between IFN-α/βR−/− mice and controls given a 700 PFU dose of virus. Virus levels in brain were high in both 129 Sv/Ev (control) and IFN-α/βR−/− mice. These results suggest that IFN-α/β signaling is correlated with reduced MAV-1 replication in the spleen and prevention of widespread infection of vascular endothelial cells. However, IFN-α/βR signaling does not prevent MAV-1-induced encephalomyelitis or limit MAV-1 replication in the brain. ISGs whose steady-state RNA levels were increased by MAV-1 infection in vitro and in vivo were identified first by cDNA arrays and confirmed by Northern analysis (M. Moore and K. Spindler, unpublished data). These ISGs included the transcription factors interferon regulatory factor 7 (IRF-7), interferon regulatory factor 1 (IRF-1), and signal transducer and activator of transcription 1 (STAT-1). 2. Cell-Mediated Immune Response to MAV-1
Outbred mice infected i.p. with a sublethal dose develop MAV-1-specific cytotoxic T cells (CTL) that are detectable 4 days PI, peak at 10 days PI, then rapidly decline (Inada and Uetake 1978a, 1978c). These kinetics are paralleled by
59 cell-mediated immunity measured by induction of macrophage migration inhibitory factor (Inada and Uetake 1978b) and are typical of acute viral infections in mice. Several studies implicate a protective role for adaptive immunity in MAV-1-induced disease. MAV-1-infected athymic nu/nu (T cell–deficient) mice on a mixed NIH Swiss and C3H/HeN background succumb to a wasting disease with characteristic duodenal hemorrhage and intranuclear adenovirus particles in endothelial cells (Winters and Brown 1980). Mice that are homozygous for the severe combined immunodeficiency (SCID) mutation (T cell– and B cell–deficient) on a CB.17 or BALB/c background succumb to MAV-1 infection with diffuse hepatic injury that resembles Reye syndrome pathology (Charles et al. 1998; Pirofski et al. 1991). RAG-1−/− (T cell– and B cell–deficient) mice are more susceptible to MAV-1 infection than B6 controls (Moore et al. 2004). These studies suggest that adaptive immunity protects adult mice from MAV-1-induced disease. Furthermore, sublethal irradiation of inbred mice that are resistant to MAV-1 infection renders them susceptible, suggesting that resistance to MAV-1 infection has an immunological basis (Spindler et al. 2001). Infection of mice deficient for T cells, T cell subsets, and T cell–related functions revealed that T cells cause acute immunopathology and are required for long-term survival in MAV-1-induced encephalomyelitis (Moore et al. 2003). Brains harvested from MAV-1-infected mice lacking α/β T cells or perforin have less histological evidence of MAV-1 encephalomyelitis and less cellular inflammation than brains harvested from control mice. Mice lacking α/β T cells, MHC class I (β2m−/−), or perforin have fewer disease signs at 8 days PI than control B6 mice, whereas mice lacking MHC class II have acute disease signs like B6 controls, such as hunched posture, ataxia, and ruffled fur. Thus, CD8+ CTL contribute to disease severity in the acute phase of MAV-1 infection. Similar to virus-induced disease in other virus infections, MAV-1-induced disease in B6 mice depends on virus dose and cell-mediated immunity (Moore et al. 2003), supporting the view that antigen quantity controls T cell–mediated immunity (Zinkernagel and Hengartner 2001). Mice lacking α/β T cells succumb to MAV-1 infection 9 to 16 weeks PI. These mice have detectable viral loads in spleens and brains at 3 weeks PI and high viral loads in spleen and brains when moribund (Moore et al. 2003). In contrast, control B6 mice clear MAV-1 to a level below the plaque assay detection limit by approximately 12 days PI, and no infectious virus is recovered 12 weeks PI from B6 mice (Moore et al. 2003; M. Moore and K. Spindler, unpublished data). Somewhat surprisingly, neither MHC class-I deficient, MHC class II-deficient, CD8−/−, CD4−/−, perforin-deficient, nor IFN-γ-deficient mice have any detectable infectious virus in spleens or brains at 12 weeks PI. Since almost all α/β T cells are either CD8+ or CD4+ (Mombaerts et al. 1992), these results suggest that having either CD8+ or CD4+ effector α/β T cells is sufficient for α/β T cell–mediated clearance of MAV-1.
60 3.
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Humoral Immune Response to MAV-1
Early studies showed that MAV-1, like hAds, induces a strong humoral immune response. Outbred mice infected i.p. with a sublethal dose of MAV-1 develop high neutralizing antibody (nAb) titers 2 weeks PI, and nAb titers increase for one year then decline (van der Veen and Mes 1973). MAV-1 infection of CBA/Ht mice with 107 50% infectious doses (ID50) results in significant splenic B cell proliferation at 10 days PI (Coutelier et al. 1990) and, like other viral infections of mice, stimulates predominantly antiviral IgG of the IgG2a subtype (Coutelier et al. 1990, 1988, 1987). MAV-1 acts as a T cell–independent (TI) Ag of the TI-2 type (Moore et al. 2004); that is, like polyomavirus, it induces TI antibodies in immunocompetent but not Xid mice (SzomolanyiTsuda and Welsh 1998). Early TI antiviral IgM plays a crucial role in protection against disseminated MAV-1 infection (Moore et al. 2004). In contrast to mice lacking T cells, mice lacking B cells are more susceptible to acute MAV-1-induced disease than B6 controls. B cell–deficient mice die early (7 to 10 days PI), and T cells are not required for survival of acute infection (Moore et al. 2003). These findings are consistent with TI B cell responses being critical for protection against MAV-1-induced encephalomyelitis. Mice lacking Bruton’s tyrosine kinase (Btk) have reductions in serum immunoglobulin, conventional B cells, and peritoneal B-1 cells (Khan et al. 1995). Btk is required for survival of acute MAV-1 infection, since Btk-deficient mice succumb to MAV-1 infection, (Moore et al. 2004). This was the first demonstration that Btk plays a role in protection from virus-induced disease in mice. Btk-deficient and µMT mice, deficient for B cells and on B6.129 and B6 strain backgrounds, respectively, succumb to acute MAV-1 infection with systemically high viral loads and histological evidence of hepatitis in addition to the histological evidence of MAV-1-induced encephalomyelitis (Moore et al. 2004). B cell–deficient mice on a BALB/c background (Jh mice) are more susceptible to acute MAV-1-induced disease than BALB/c controls, and succumb with systemically high viral loads and evidence of significant hepatitis. However, moribund Jh mice do not exhibit encephalomyelitis; MAV-1-infected Jh mice likely die of hemorrhagic enteritis. SCID (T cell– and B cell–deficient) mice on a BALB/c background succumb to acute MAV-1 infection with evidence of liver infection but no histological evidence of hepatitis (Charles et al. 1998). One explanation for this strain-specific pathology is as follows. In the absence of B cells, MAV-1 replicates to high titers throughout the mouse (Moore et al. 2004). In the presence of T cells, acute MAV-1 infection elicits dose-dependent immunopathology (Moore et al. 2003). T cell–mediated immunopathology may be directed to various organs in different mouse strains by strain-specific innate immune responses, for example, differential chemokine expression in the brains of MAV-1-infected B6 and BALB/c brains (Charles et al. 1999). Data showing that MAV-1 replicates to high levels in the brains of Jh mice without inducing encephalomyelitis (Moore et al. 2004) support this
model rather than a model in which receptor differences account for differential pathology in MAV-1-infected B6 and BALB/c mice (Charles et al. 1998). 4.
Model of MAV-1 Immunopathogenesis
T cells, B cells, and type I IFN each have a distinct protective role in MAV-1 pathogenesis, and the data suggest their roles are interrelated. B cell–deficient mice succumb to disseminated infection with high virus loads throughout the mouse (Moore et al. 2004). T cell–mediated immunopathology is implicated in exacerbating disease in B cell–deficient mice (Moore et al. 2003, 2004). Similar to B cell–deficient mice, type I IFN–deficient mice also had a more disseminated infection with higher virus loads than control mice (M. Moore and K. Spindler, unpublished data). However, unlike B cell–deficient mice, type I IFN–deficient mice did not exhibit more clinical disease or more histopathological evidence of disease than control mice, even in organs with high virus loads, such as the liver (M. Moore and K. Spindler, unpublished data). This suggests that type I IFN controls MAV-1 replication but also contributes to T cell–mediated immunopathology.
E. MAV-2 Infection In Vivo Hashimoto and colleagues studied the pathogenesis of MAV-2 in mice infected perorally (Hashimoto et al. 1970). The virus grows in the intestinal tract and is shed in feces for 3 weeks after infection, but there are no clinical signs of disease. Inbred DK1 mice given 2 × 105 TCID50 of virus show virus replication from 3–14 days PI, with a peak of virus yield from 7–14 days PI. At the peak times of infection, virus is seen by immunofluorescence and electron microscopy in epithelial (columnar, goblet, and Paneth) cells of the ileum (Takeuchi and Hashimoto 1976). Mesenchymal cells are not infected; the virus has a specific tropism for the villus epithelium. The infection results in little cytopathic effect, but infected epithelial cells are shed at a high rate into the gut lumen. Interestingly, MAV-2 peroral infection of BALB/c nude (nu/nu) mice, which lack T cells, results in prolonged viral proliferation in the gut, but viral replication is suppressed 6 weeks PI (Umehara et al. 1984). MAV-2 is not found in organs other than the gut. Antiviral resistance in BALB/c nu/nu mice 6 weeks PI does not correlate with interferon levels or NK cell activity, and the resistance is not affected by administration of anti-asialo GM1 antibody or carrageenan (Umehara et al. 1987). However, antiviral resistance is completely abolished by cyclophosphamide treatment. Cyclophosphamide is a carcinogen and mutagen that is toxic to actively cycling cells, reduces peripheral lymphocytes, and reduces serum IgG levels in nu/nu mice. Mice rechallenged with MAV-2 28 days after initial infection are resistant to virus growth (Hashimoto et al. 1970). The data suggest that antibody
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responses are responsible for the antiviral resistance of nu/nu mice to MAV-2.
VII.
HOST GENETICS
Different strains of outbred and inbred mice have been shown to differ in their susceptibility to MAV-1 (Guida et al. 1995; Kring et al. 1995; Spindler et al. 2001). Adult B6 mice infected with MAV-1 succumb to a fatal hemorrhagic encephalitis whereas BALB/c mice do not (Guida et al. 1995). B6 mice show clinical signs of acute CNS disease accompanied by histological evidence of hemorrhage and inflammation, and high levels of virus in brain and spinal cord. In contrast, infected BALB/c mice do not have detectable levels of viral mRNA in brain or spleen, and they lack histological and clinical signs of disease. Enteritis has been seen in BALB/SCID (T and B lymphocyte–deficient) mice (Charles et al. 1998), and B lymphocyte–deficient mice on a BALB background (Jh) (Moore et al. 2004) likely succumb due to a hemorrhagic enteritis. These results suggest that there are mouse strain differences in tropism and cause of death. SJL/J mice are more than four log units more susceptible to MAV-1 than most inbred strains of mice (Spindler et al. 2001). A sublethal dose of gamma irradiation renders resistant mice susceptible, and there are no differences in viral yield in ex vivo MAV-1 infection of primary cells from susceptible and resistant mice. Susceptible mice have higher virus loads in brain and spleen than resistant mice but only modest differences in histopathology. The results suggest that immune response differences may account for differences in susceptibility. A genetic mapping approach (positional cloning) is being used to identify the host gene(s) for susceptibility to MAV-1 (Welton et al. 2005).
VIII.
HOST RANGE AND PREVALENCE
The mouse strain used when MAV-1 was isolated was not specified by Hartley and Rowe (1960), but MAV-1 infects inbred and outbred strains of mice in the Mus genus. There is a report of adenovirus isolation from Peromyscus (Reeves et al. 1967) and a report of a seropositive wild rodent (Kaplan et al. 1980), but these do not provide strong support for infections of non-Mus mice. The host range of MAV-1 in cells in culture is limited to mouse cells; infection of a variety of human and monkey cells does not result in infectious virus (Antoine et al. 1982; Larsen and Nathans 1977). A survey reported in 1966 testing mice in U.S. laboratory colonies indicated that evidence of MAV-1 infection was only found in 4 of 34 colonies, much less frequently than other viruses tested (Parker et al. 1966). A survey from 1984–1988 of
laboratory colonies in 10 European countries showed no mouse adenovirus infections (Kraft and Meyer 1990). MAV-1 is virtually absent from commercial colonies, which are now monitored routinely for MAV-1 (Otten and Tennant 1982). Commercial and laboratory colonies are not usually tested for MAV-2 (A. Smith, personal communication). It has been postulated that MAV-1 occurred enzootically in infected laboratory colonies as a silent infection that was transmitted orally to cage mates (Richter 1986). Although infection can be transmitted to cage mates, there is no seroconversion for animals held in the same animal room but in separate cages (Hartley and Rowe 1960). Mice kept in close contact with MAV-1-infected mice seroconvert by day 21 PI (Lussier et al. 1987). Animals placed on bedding obtained from cages of MAV-1-infected animals do not show signs of disease, seroconversion, or shedding of virus in urine (Smith et al. 1998). The prevalence of mouse adenoviruses in the wild has not been systematically studied. However, a serological survey of wild M. domesticus in southeastern Australia indicated that of mice isolated from 14 sites, 37% were seropositive for MAV-2 and 0% were seropositive for MAV-1 (Smith et al. 1993). There was significant variation in MAV-2 seroprevalence at two study sites in southeastern Australia investigated over 13 months, and again, no evidence of MAV-1 was seen (Singleton et al. 1993). M. domesticus was introduced to an island off the northwest coast of western Australia and first identified in 1986 (Moro et al. 1999). A study of mice from this island in 1994–1996 indicated no serological evidence of MAV-1 or MAV-2 in M. domesticus or in an indigenous mammal, the short-tailed mouse Leggadina lakedownensis.
IX.
DIAGNOSIS, CONTROL, AND PREVENTION
MAV-1 infection can be diagnosed by serological testing by ELISA; test kits are available commercially (Charles River Laboratories). Complement-fixing antibodies are detected in mice inoculated i.p. with 104 TCID50 as early as 14 days PI (Lussier et al. 1987). Neutralizing Ab is detected in mice infected with 1 PFU at 12 days PI (Moore et al. 2004). Because commercial colonies are free of mouse adenoviruses (Section VIII), control is unlikely to be needed. If necessary, control in an infected colony can be achieved by embryo rederivation (Richter 1986; Trentin et al. 1966). Mice to be infected with MAV-1 should be maintained in a room where their cages and bedding can be autoclaved after use. Because the virus appears to require close mouse-mouse contact for transmission (Hartley and Rowe 1960; Lussier et al. 1987), it is straightforward to work with these mice using standard microisolator techniques. In 20 years of performing MAV-1 infections in animal facilities in four different buildings at two institutions, we have never had mock-infected experimental mice (in separate cages) or sentinel mice (even in open cages) in the same
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room as infected mice show disease signs or become seropositive for MAV-1 (K. Spindler, unpublished data). ACKNOWLEDGMENTS We thank Lei Fang for critical reading of the manuscript. This work was supported by NIH R01 AI023762.
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65 Winters, A. L., Brown, H. K., and Carlson, J. K. (1981). Interstitial pneumonia induced by a plaque-type variant of mouse adenovirus. Proc. Soc. Exp. Biol. Med. 167, 359–364. Wold, W. S. M., and Gooding, L. R. (1991). Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions. Virology 184, 1–8. Yee, S.-P., and Branton, P. E. (1985a). Analysis of multiple forms of human adenovirus type 5 E1A polypeptides using an antipeptide antiserum specific for the amino terminus. Virology 146, 315–322. –– –– ––. (1985b). Detection of cellular proteins associated with human adenovirus type 5 early region 1A polypeptides. Virology 147, 142–153. Yee, S.-P., Rowe, D. T., Tremblay, M. L., McDermott, M., and Branton, P. E. (1983). Identification of human adenovirus early region 1 products using antisera against synthetic peptides corresponding to the predicted carboxy termini. J. Virol. 46, 1033–1013. Ying, B., Smith, K., and Spindler, K. R. (1998). Mouse adenovirus type 1 early region 1A is dispensable for growth in cultured fibroblasts. J. Virol. 72, 6325–6331. Zinkernagel, R. M., and Hengartner, H. (2001). Regulation of the immune response by antigen. Science 293, 251–253.
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Chapter 3 Mousepox R. Mark, L. Buller, and Frank Fenner
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Properties of the Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Virion Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Virion Structure and Composition . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Virion Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Strains of Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hampstead Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Moscow Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. NIH-79 Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Other strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Growth In Vitro and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Growth in Tissue Culture and Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Chick Embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Factors Affecting Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Gender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Mechanism of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Intradermal Inoculation and Scarification . . . . . . . . . . . . . . . . . . . . 2. Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Arthropod Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Intraperitoneal Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Intranasal Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Lower Respiratory Tract Inoculation . . . . . . . . . . . . . . . . . . . . . . . . 7. Intracerebral Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Intrauterine Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Virus Spread in the Animal Following Footpad Infection . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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C. Gross and Microscopic Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Susceptible Mouse Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Resistant Mouse Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Innate and Adaptive Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Innate Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Adaptive Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Genetics of Resistance to Lethal Mousepox . . . . . . . . . . . . . . . . . . . . . IX. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Host Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Strains of Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Prevalence and Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. United States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Other Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Epizootic Mousepox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Enzootic Mousepox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Virus Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Molecular and Antigen Detection Techniques . . . . . . . . . . . . . . . . . . . . XI. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Depopulation and Disinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Serological Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Rederivation of Mouse Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Sentinel Surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Quarantine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Mouse Antibody Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .....................................................
I.
INTRODUCTION
First recognized in 1930 (Marchal 1930), when the use of mice as experimental animals in virology was just beginning (see Burnet 1960, Table 3-1), infectious ectromelia, or mousepox, has had a rather different history in the four continents where laboratory mice have been extensively used: Europe, North America, Australia, and Japan. In Europe and Japan it was soon found to be present, usually as an unrecognized enzootic infection, in many breeding colonies. As well as threatening potentially valuable mouse stocks, this infection complicated much virological research involving serial passages of viruses or tumors in mice, and the presence of enzootic mousepox has also rendered suspect several studies of the nature of the disease itself. By chance, most mouse colonies established in North America were free from the disease. When the virus was inadvertently imported into the United States from Europe with mouse strains or mouse tissues (cell lines or tumors), there were sometimes disastrous outbreaks in colonies (see Briody 1955; Whitney 1974; Lipman et al. 2000); hence, quarantine precautions were instituted, and research with the
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virus in the United States was carried out with great care. Enzootic mousepox does not occur in Australia. Following the recognition of the relation of the virus to vaccinia virus by Burnet and Boake (1946), mousepox has been used as a model for research in several fields, notably the pathogenesis of generalized infections, experimental epidemiology, and the cellular immune response. In this age of concern over the potential use of pathogens as weapons of mass destruction, there is a renewed interest in development of prophylactics and therapeutics against smallpox. The mousepox model is arguably the best mouse model available for this purpose, as it provides a much greater dynamic range for evaluating antivirals and vaccines and shares a number of important similarities with smallpox (Buller 2004; Schriewer et al. 2004). Also, studies with ectromelia have suggested methods for generating more virulent orthopoxviruses that possibly could be used as bioweapons. For example, by accident, Jackson et al. (2001) using ectromelia virus (ECTV) as a vector for immunocontraception of feral mouse populations discovered that ECTV expressing IL-4 was able to break through vaccine-induced immunity.
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TABLE 3-1
TAXONOMIC POSITION OF ECTROMELIA VIRUS Virus
Animals found naturally infected
Camelpox Cowpox Ectromelia Monkeypox Raccoonpox Skunkpox Taterapox Uasin Gishu pox Vaccinia Variola Volepox
Camel Cow, man, rodents, carnivores, elephant, okapi Mice, ? vole African monkeys, anteater, great apes, man, squirrels Raccoon Skunk Tatera kempi (an African gerbil) Horse Man, cow, buffalo, rabbit Man Vole
II.
histocompatibility complex (MHC) antigens in viral infections. In the 1980s and 1990s, Brownstein (1997) provided insights into the genetic basis of resistance to mousepox; most recently, Karupiah and colleagues explored the role of innate/immune host responses for recovery from infection, and Buller and coworkers investigated the genetic basis for ECTV virulence (reviewed by Esteban et al. 2005). In 2003, the annotated genomic sequence of the Moscow strain of ECTV was published (Chen et al. 2003). Mousepox is a troublesome and often devastating disease that interferes with experiments involving mice. This chapter will examine the natural history of mousepox in laboratory mice and the measures that can be used in prevention and control. In a less detailed manner, we will explore some results that have emerged from the use of mousepox as a model infection by virologists and immunologists.
HISTORY
Mousepox was first recognized by Marchal (1930) as an epizootic disease of laboratory mice in England, following investigation of unusually high mortality in mice received from commercial breeders by the National Institute of Medical Research at Hampstead. She named it infectious ectromelia because of frequent amputation of the extremities during the outbreak that she investigated. Soon after, Barnard and Elford (1931) demonstrated by ultraviolet microscopy that the virion (“elementary particle”) was similar in size and shape to that of vaccinia virus. However, no serological comparisons with other poxviruses were made until 1946, when Burnet and Boake (1946) demonstrated by hemagglutination inhibition (HI) (Nagler 1944) that ectromelia and vaccinia viruses were closely related. Fundamental studies with mousepox have been carried out in Australia, first at the Walter and Eliza Hall Institute in Melbourne and then at the John Curtin School of Medical Research in Canberra, in Spain at Madrid, and in the United States at the National Institutes of Health, Yale University, and Saint Louis University. During the 1930s, Greenwood and collaborators (1936) used ECTV as a model pathogen in experimental epidemics in mice, and in 1946 Fenner (for review, see Fenner 1949a) revived this work with the added knowledge of the taxonomic status of the virus. He soon found (Fenner 1948b) that infection produced a rash and revealed the pathogenesis of mousepox (Fenner 1948a) as a useful model for generalized viral exanthemata (Fenner 1948d). He suggested that the disease be called mousepox and the virus ectromelia virus (cf. smallpox and variola virus). During the 1950s and early 1960s, Mims and colleagues expanded Fenner’s work on pathogenesis using fluorescent antibody staining to probe events at the cellular level (for reviews, see Mims 1964, 1966). Blanden and colleagues exploited the mousepox model to explore the role of cell-mediated immunity in poxvirus infections and demonstrate the role of major
III.
PROPERTIES OF THE AGENT A.
Classification
Three distinct species of poxviruses produce natural infections of rodents: infectious ectromelia or mousepox virus (Marchal 1930), cowpox virus (Chantrey et al. 1999), and Turkmenia rodent virus (Marennikova et al. 1978), which is a close relative of cowpox virus. All three are orthopoxviruses with strong serological cross-reactivity to other orthopoxviruses (see Table 3-1). Comparative molecular studies have shown that the DNAs of orthopoxviruses are closely related, with greater than 90% nucleotide identity exhibited over the central ~100 kbp of their genomes (Bellett and Fenner 1968; Müller et al. 1978; Gubser et al. 2004). Serological comparisons of ECTV with other orthopoxviruses have been made mainly by neutralization and HI tests. Cross-neutralization by vaccinia virus–immune sera is readily demonstrable in pock-reduction tests on the chorioallantoic membrane (McCarthy and Downie 1948), and ECTV can be distinguished from vaccinia and cowpox viruses in plaque-reduction assays (McNeill 1968). Although they cross-react, the HI titers of ECTV and vaccinia virus antiserum are substantially higher with homologous antigen (Fenner 1947a).
B.
Virion Morphology
Poxviruses are the largest of all animal viruses and can be visualized by light microscopy, although details of virion structure remain obscure. With the advent of high-resolution electron microscopy, the morphology of the virion structure began to be revealed during the 1940s and 1950s. Ectromelia virus is a
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2.
Immature particles
IMV
Virion Stability
Like other orthopoxviruses, ECTV is relatively resistant to heat and to many disinfectants (see Fenner 1949a; Bhatt and Jacoby 1987c). For example, virus infectivity was still detectable following 50 days at room temperature or treatments for 24 hr with 10% ether, 0.5% chloroform, 1% phenol, or 0.01% formalin. Virus infectivity was not detectable following a 10-minute incubation with 2% phenol or 40% alcohol.
IV.
Fig. 3-1 An electron micrograph of a thin section of orthopoxvirusinfected cells. The structure of the virion reveals a condensed nucleoprotein organization of DNA. The core assumes a dumbbell shape surrounded by a core wall invaginated by large lateral bodies, which are in turn enclosed within a membrane to form intracellular mature virus (IMV). The circular and partially circular shapes are intermediate stages in virus assembly (immature particles).
typical orthopoxvirus, morphologically indistinguishable from the prototype species, vaccinia virus. The intracellular mature virus (IMV) appears to be an oval or brick-shaped structure of about 200 to 400 nm in length, with axial ratios of 1.2 to 1.7 (see Fig. 3-1) (Peters 1956).
C. 1.
Physical Properties
Virion Structure and Composition
The virion contains a noninfectious, linear, 67% A+T-rich, double-stranded DNA genome of 209,771 base-pairs in length (Chen et al. 2003). The virion has more than 100 polypeptides, arranged in four distinct structures (core, lateral body, membrane, and envelop), as determined by electron microscopy of thin sections and negatively stained preparations of purified virions. The membrane contains more than eight proteins, of which 129, 72, and 97 are targets of neutralizing antibody (Moss 2003). The extracellular enveloped virus (EEV) is an IMV virion with a second membrane (see below). The unique EEV membrane has six major proteins, with the 155 and 135 proteins implicated as important targets of protective antibody. Like most other orthopoxviruses, ECTV produces a hemagglutinin (151) that by analogy with vaccinia virus (Payne and Norrby 1976) is part of the viral envelope, the presence of which is not necessary for infectivity.
STRAINS OF VIRUS
Isolates from Manchester, England (McGaughey and Whitehead 1933), Paris (Schoen 1938), Germany (Kikuth and Gönnert 1940), Moscow (Andrewes and Elford 1947), Japan (Ichihashi and Matsumoto 1966), and the United States (New 1981; Dick et al. 1996) were found to be almost indistinguishable from the original Hampstead strain isolated by Marchal (1930) by serologic or genetic means. For example, restriction endonuclease analysis of genomic DNA from Hampstead and Moscow strains revealed an almost identical pattern of fragments following digestion with restriction endonucleases Hind III and Xho I (Mackett and Archard 1979). Similarly, DNA sequence analysis detected nucleotide identity of 99.5% over the entire length of the genomes of the Moscow strain and an isolate from an outbreak at the Naval Medical Research Institute in the United States (referred to as the NAV isolate), even though these isolates were obtained roughly 50 years apart (Chen et al. 2003). Taken together, the serologic and genomic nucleotide sequence similarity suggests that there is little genetic diversity among the strains; however, there are distinct biologic differences. Fenner (1949c) found that virulence and transmissibility varied independently and differed between strains. The Moscow strain was highly virulent and highly infectious; the Hampstead (mouse-passaged) strain had similar virulence but was less infectious. The Hampstead (egg) strain had low virulence and low infectivity. Table 3-2 illustrates differences in the LD50 of several strains of ECTV in BALB/c mice. The Hampstead, Moscow, and NIH-79 strains have been extensively characterized, as summarized below.
A.
Hampstead Strain
The original isolation was made by Marchal (1930). This strain was used in most of the early studies characterizing mousepox, and was used in Greenwood’s epidemiological experiments (Greenwood et al. 1936). A mouse-passaged Hampstead strain retained its high virulence, but egg passage led to a substantial reduction in its virulence for mice (Fenner 1949c), although it
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was then more readily adapted to growth on the rabbit cornea (Paschen 1936).
V.
GROWTH IN VITRO AND IN VIVO A.
B.
The Moscow strain of virus was isolated by Professor V. D. Soloviev and coworkers and is highly virulent and highly infectious. It was used extensively in experiments by a succession of Australian workers (Fenner, Mims, Roberts, Blanden, and Karupiah), by Andrewes and Elford, and more recently by Buller. The Moscow strain continues to be the most frequently studied ECTV strain.
C.
NIH-79 Strain
The NIH-79 strain of virus was isolated by Dr. Anthony Allen, Veterinary Resources Branch, National Institutes of Health (Allen et al. 1981). It was used by both Buller and Wallace and Bhatt and Jacoby to study the clinical response, pathogenesis, and transmissibility of ECTV in inbred strains of mice. The NIH-79 strain is less virulent than the Moscow strain, causing less clinical illness and fewer deaths among genetically resistant C57BL/6 mice and susceptible BALB/c mice (Bhatt et al. 1988; see Table 3-2).
D.
Other Strains
Japanese workers have made considerable use of strains recovered in Japan, as well as the Hampstead strain. Of these, the Ishibashi strain was the most thoroughly studied and was shown to have a larger plaque size than the Hampstead strain (Ichihashi and Matsumoto 1966). The NAV strain was isolated from commercial, pooled mouse sera, which was the source of a 1995 outbreak of mousepox in a laboratory mouse colony at the Naval Medical Research Institute in the United States (Dick et al. 1996; see Section IX, C).
TABLE 3-2
LETHAL DOSE50 OF ECTROMELIA VIRUS STRAINS AFTER FOOTPAD ROUTE INOCULATION IN BALB/CBYJ MICEa Virus strain Washington Univ. St. Louis 69 Moscow Beijing 70 NIH-79 Ishibashi I-III aR.
Replication
Moscow Strain
M. L. Buller, unpublished results
LD50 in PFU 1.0 × 101 2.8 × 101 3.9 × 101 9.3 × 101 7.9 × 102 4.3 × 104
Key features of the intracellular replication cycle of orthopoxviuses are shown in Fig. 3-2 (Moss 2003). The virion containing early RNA transcription machinery attaches to and fuses with the plasma membrane. Within 15 minutes, transcription machinery is activated (uncoating I). Early genes are expressed that code for a variety of functions that modify the host cell for optimal virus replication, attentuate the host response to infection, and mediate virus synthetic processes. After further uncoating (II), and between 1.5 to 6 hr PI, the virus genome is replicated via concatamers. Virus DNA is replicated in a region of the cytoplasm that is “cleared” of organelles and shows characteristic staining patterns by light and electron microscopy. Kato et al. (1955) called these structures B-type inclusion bodies and Cairns (1960) referred to them as viral factories. From progeny DNA templates, intermediate genes encoding late transcription factors are expressed, leading to the sequential synthesis of late gene RNA and proteins. Late genes encode the early transcription system, enzymes, and structural proteins necessary for virion assembly. By 4 hr PI, virion morphogenesis commences with the formation of membrane structures in the intermediate compartment of the cell and the packaging of resolved unit length genomic DNA. The IMV has one membrane derived from the intermediate compartment. Some IMV acquire an additional double layer of intracellular membrane derived from the trans Golgi network that contains unique virus proteins (intracellular enveloped virus, IEV). These IEVs are transported to the periphery of the cell, where fusion with the plasma membrane ultimately results in release of EEV or, if attached to the exterior surface of the plasma membrane, remain as cell-associated enveloped virus (CEV). While IMV and CEV/EEV are infectious, the external antigens on the two virion forms are different. EEV are thought to be most important in cell-to-cell spread and systemic disease. In addition to these forms, ECTV also encodes a major late protein of 130 kDa that forms characteristic spherical, acidophilic, cytoplasmic masses in infected epithelial cells, but rarely in liver cells (Kato et al. 1963). Kato et al. (1955) called these structures A-type inclusion or Marchal bodies (ATIs). Virions may be occluded within the ATI, providing a means of environmental survival. Electron microscopic examination of infected L cells showed that with the Hampstead strain, all mature viral particles that developed in the B-type inclusion were occluded within the ATIs, whereas with the Ishibashi strain, the ATIs were devoid of virions (Matsumoto 1958; Ichihashi and Matsumoto 1966). The genetic basis for this observation likely resides in the presence or absence of an intact p4c gene product on the surface of the IMV particle (McKelvey et al. 2002). The Moscow strain
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Fig. 3-2 General orthopoxvirus replication cycle. IMV, intracellular mature virus; IEV, intracellular enveloped virus; CEV, cell-associated enveloped virus; and EEV, extracellular enveloped virus.
lacks an intact p4c and does not occlude virions (R. M. L. Buller, unpublished observations).
B. 1.
Growth in Tissue Culture and Eggs
Chick Embryo
Ectromelia virus infection of chick embryos was described simultaneously by Paschen (1936) and Burnet and Lush (1936a). Both grew the virus on the chorioallantoic membrane, and Burnet and Lush showed that inoculation of dilute virus suspensions facilitated development of discrete foci (pocks), which could be counted. This titration method was exploited by Fenner (1948a) for quantitative studies of mousepox infection. Chorioallantoic membrane inoculation with large doses of virus was usually followed by death of the embryo 4 or 5 days later, and there were often scattered areas of necrosis in the livers and spleens of these embryos.
Serial passage of ECTV on the chorioallantois sometimes modified its character. Paschen (1936) found that egg-passaged virus was more suitable for infection of the rabbit and guinea pig cornea and skin than was mouse live-passaged virus. Serial chorioallantoic passage of the Hampstead strain of ECTV (50–60 passages intermittently over a period of 10 years) resulted in greatly reduced virulence for mice (Fenner 1949c). No change in the high virulence of the Moscow strain of virus occurred after 20 consecutive passages on the chorioallantois. 2.
Tissue Culture
Ectromelia virus multiplies in cells from human (HeLa and human amnion), monkey (BSC-1), mouse (L and primary embryonic fibroblasts), and chicken (primary embryo fibroblasts) sources. Ichihashi and Matsumoto (1966) found that the Ishibashi strain of ECTV produced much larger plaques on chick embryo fibroblast monolayers than the Hampstead strain. Plaque production in mouse fibroblasts can be improved by the
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inclusion of DEAE-dextran in the overlay medium. The plaque assay in mouse L cells or fibroblasts or monkey BSC-1 cells is as sensitive as the chorioalloantoic membrane assay, but more reproducible. The tissue culture plaque assay has been adopted by most investigators as the standard assay method (Blanden 1974). Comparative studies determined the following relationship between virus particles (counted by electron microscopy) and various biological titration units: mouse (strain C57BL) footpad inoculation, 1 ID50 and 1 LD50 = 1.5 and 25 particles, respectively; chorioallantoic membrane, 1 pock-forming unit = 12 particles; chick embryo fibroblasts or mouse embryo fibroblasts, 1 plaque-forming unit = 19–20 particles (Schell 1960b).
VI.
CLINICAL DISEASE A.
Clinical Disease
Clinical disease differs among mouse strains and routes of inoculation both in natural epizootics and experimental infections. Briody (1966) observed that in natural epizootics A, BC, DBA/1, DBA/2, and CBA strains usually did not recover from infection whereas C57BL/6 mice were resistant to severe disease. Schell (1960b) showed that C57BL mice were resistant to lethal experimental infection when infected by the footpad route, but not other routes. ID50/LD50 ratios for footpad, intravenous, intranasal, intracerebral, and intraperitoneal inoculation routes were 5.4, 4.0, 3.7, 1.3, and 1.0, respectively. Early workers using outbred mice (Marchal 1930; McGaughey and Whitehead 1933; Schoen 1938) described two forms of the disease: a rapidly fatal form in which apparently healthy mice died within a few hours of the first signs of illness 6–7 days post-infection (PI) and had extensive necrosis of liver and spleen, and a chronic form characterized by ulcerating lesions of the feet, tail, and snout. Fenner (for review, see 1949a) showed that in every case there was a stage in which virus multiplied to high titer in the liver and spleen. Some mice died at this stage, but survivors almost invariably developed a generalized rash. Subsequently, it was shown that clinical signs were greatly affected by mouse genotype (see Section VIII, C). Clinical disease in inbred mouse strains occurred in two patterns (Wallace and Buller 1985; Bhatt and Jacoby 1987a,b). Ruffled fur, hunched posture, and prostration were observed in all dying mice following footpad inoculation, which mimics natural infections. For strains such as A, DBA/2, C3H, and BALB/c, death usually ranged from 6 to 14 days PI, depending on the infecting dose of virus. The ID50 and LD50 were similar. With C57BL/6 and AKR mice, over a range of viral doses,
clinical signs were minimal and the LD50 dose was 4 or 5 log10 higher than the ID50 dose.
B. 1.
Factors Affecting Clinical Disease
Age
Age affects the response of genetically susceptible mice (Fenner 1949d). Both the Moscow and Hampstead (egg) strains produced higher mortalities in suckling mice and in mice about a year old than in 8-week-old mice, the differences being more pronounced with the less virulent Hampstead virus. The increased severity was evident after footpad inoculation and in long-term experimental transmission experiments. In suckling mice there was a very short delay between peripheral inoculation of the virus and its appearance in the liver and spleen. In the year-old animals this interval, and the survival time of the mice, were the same as in 8-week-old animals. However, lethal titers of virus in the liver and spleen were attained only in occasional 8-weekold mice but occurred in most older animals. The causes of these differences were not elucidated, but may relate to a less effective immune system. 2.
Gender
Sexual dimorphism to disease has been observed in BALB/cJ and A/J mouse strains, and appears to have a partial hormonal basis (Buller et al. 1985). Day of death differences were noted between males and females, and day of death was modulated in males by castration or treatment with estrogen. This sex-related difference in severity of disease, although evident in the parental strains, was much more apparent in back-crossed populations (Wallace et al. 1985; Buller et al. 1986). The female mice had a longer time to death and higher proportion of survivors than males.
VII. A. 1.
PATHOGENESIS
The Mechanism of Infection
Intradermal Inoculation and Scarification
Infection by scarification, by footpad inoculation, or by instillation of virus into the cornea was followed by a disease indistinguishable from naturally acquired mousepox except for the localization of the primary lesion. Fenner (1948a) studied the pathogenesis of Hampstead and Moscow strains following footpad inoculation of small doses of virus. The incubation period, viral dissemination, and pathology all resembled that found in natural infections. The results with both strains of
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virus and in subsequent experiments with immunized mice indicated that the infection followed a constant pattern (Fenner 1949b) (see Section VII, B for a detailed description of virus spread within the animal). Other methods of inoculation (discussed below) caused lesions that differ considerably from those found in natural disease. 2.
Feeding
Fenner (1947c) found that infection by feeding virus occurred only if very large doses of virus were used, but the ensuing disease resembled that following natural infections. However Gledhill (1962a,b) and subsequently Horzinek and Höpken (1965) found that mice occasionally could be infected orally with much smaller doses, but that the infections were usually inapparent. Gledhill showed that chronic infection of Peyer’s patches occurred, and that small amounts of virus could be excreted in feces for as long as 119 days, sometimes associated with chronic tail lesions that also released small amounts of virus. In Gledhill’s experience, such carrier mice did not spread mousepox to uninfected mice by contact, nor was he able to “activate” acute mousepox in the carriers. Nevertheless, such mice clearly constitute a reservoir from which infection could be transferred by the inoculation of contaminated tissue suspensions. More recently, Wallace and Buller (1985) found that following intragastric inoculation, virus was isolated from feces of C57BL/6J mice for as long as 46 days and for up to 29 days from feces of BALB/cByJ mice. Transmission to cage mates from intragastrically infected C57BL/6J and BALB/cByJ occurred up to 36 and 30 days, respectively, after infection. 3.
Arthropod Vectors
Although this mode of transfer is important in several other poxvirus diseases, such as fowlpox and myxomatosis, Fenner was unable to demonstrate mechanical transmission of mousepox by mosquitoes (F. Fenner, unpublished experiments). Guillon (1970) has suggested another possible mode of transfer by arthropods. He found that the rat mite, Ornithonyssus bacoti, became infectious after a blood meal and suggested that it (and perhaps also cockroaches [Guillon 1975]) might act as a passive vector. 4.
Intraperitoneal Inoculation
This has been a commonly used way of passing ECTV. The lesions differ considerably from those observed in the natural disease, and it is obvious that some accounts of mousepox pathology are based upon the results of intraperitoneal inoculation (Fenner 1947b; Schell 1960b). There is, of course, no primary skin lesion, but in acutely fatal cases the necrosis of the liver and spleen resembles that found after natural infection. In addition, there is usually peritonitis with ascites, pancreatic edema, and considerable pleural fluid. In the rare animals that
Fig. 3-3 Postmortem appearances after intraperitoneal injection of ectromelia virus. Necrosis of the liver, hyperemia of the intestine, and peritoneal and pleural effusions are seen.
survive acute infection, general peritonitis is more pronounced (Fig. 3-3). There is a great excess of peritoneal and pleural fluid, the peritoneal surfaces of the liver and spleen are covered with a white exudate, the walls of the intestine are thickened and rigid, and there is often peritoneal fat necrosis. Extensive adhesions develop later. Survivors develop a characteristic rash. Survival times among mice inoculated intraperitoneally are usually 2 or 3 days shorter than those for mice given the same dose of virus inoculated into the foot. 5.
Intranasal Inoculation
Historically, mousepox has been induced unintentionally during mouse lung passage of influenza virus, illustrating that
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inoculation of the respiratory tract can result in generalized disease. Some workers (see, e.g., Kikuth and Gönnert 1940) suggested that ECTV acquires marked pneumotropic properties after serial lung passage, because deaths from pneumonia then occur with little macroscopic evidence of lesions in liver and spleen. When small doses of virus are inoculated intranasally, there is usually little change in the lungs except for the development of patchy congestion (Fenner 1947b; Jacoby and Bhatt 1987). The survival time of fatal cases is approximately the same after footpad inoculation with the same dose of virus, and lesions in liver and spleen are characteristic of acute, naturally acquired mousepox. With larger doses of virus, congestion of the lungs becomes more pronounced and pneumonia may occur. When very large doses are given, death occurs, with patchy or complete consolidation of the lungs and little change in the liver and spleen. Histologically, there is early exudation into the alveoli and bronchi and focal necrosis of bronchial epithelium. ATIs are seen in bronchial epithelium, histiocytes, pleural cells, and eventually in alveolar epithelium. When the lungs, liver, and spleen of fatal cases were examined virologically, titers in apparently normal liver and spleen were very high, just below the threshold at which demonstrable necrosis occurred (F. Fenner, unpublished observations; Ipsen 1945). Necrosis has been found in a few mice that survived for 6 (instead of the usual 4 or 5) days after the intranasal inoculation of a large dose of ECTV. Using fluorescent antibody staining, Roberts (1962a) showed that either macrophages or alveolar mucosal cells were initially infected, but it was the macrophages that carried virus to the pulmonary lymph nodes and thus to the bloodstream, from which it was taken up by the liver and spleen, in which multiplication then proceeded. The apparent pneumotropism is due to the fact that the local reaction, which occurs after the intranasal inoculation of very large doses of virus, kills the animal before there is time for the characteristic changes in the liver and spleen to occur. Similar findings were observed with inbred A/J and BALB/c mice (Jacoby and Bhatt 1987). Low doses of virus in small volumes (under 10 µl) resulted in local upper respiratory tract infections with little involvement of the lung. Death resulted from liver necrosis. A/NCr mice are highly susceptible to this route of infection and LD50 values of 0.3 PFU have been obtained. BALB/c mice are at least 100-fold more resistant (Bhatt and Jacoby 1987a; R. M. L. Buller, unpublished observations). 6.
Lower Respiratory Tract Inoculation
Mice can also be infected with small particle aerosols (mass median diameter of ~0.5 µm). In A/NCr mice, LD50 values have been achieved with presented doses as low as 36 PFU (Buller 2004; Schrewier et al. 2004). As with the intranasal route, infection with low doses of aerosolized virus results in death attributable to liver necrosis with only modest lung involvement.
7.
Intracerebral Inoculation
Kanazawa (1937), Jahn (1939), and Schoen (1938) described intracerebral passage of ECTV, and Kikuth and Gönnert (1940) confirmed their results. Jahn (1939) could not find ATIs in the brain, but Kikuth and Gönnert (1940) described them in neural cells and also in macrophages. It is apparent that after intracerebral inoculation, ECTV rapidly enters the systemic circulation, since virus titers in liver and spleen of fatal cases are always high (F. Fenner, unpublished observations), and except after large doses of virus, characteristic lesions develop (Kanazawa 1937). Jahn (1939) found no evidence of increased neurotropism after 20 brain-to-brain passages in mice. 8.
Intrauterine Infection
Mims (1969) showed that many pregnant mice infected intradermally with the attenuated Hampstead (egg) strain of ECTV survived, but there was extensive growth of virus in the placenta and infection of the fetuses. Infected fetuses died either in utero or soon after birth, and fluorescent antibody staining showed that there was widespread growth of virus throughout their bodies. Schwanzer et al. (1975) obtained similar results. In resistant strains of mice from enzootically infected colonies, intrauterine infection might present as a drop in reproductive success due to fetal deaths; however, in a largely immune population, this problem would probably not occur. Additional problems could arise from the use of mouse embryo cell cultures derived from infected embryos (Germer et al. 1961).
B.
Virus Spread in the Animal Following Footpad Infection
Fenner (1947b, 1949c) believed that the usual portal of entry of the ECTV in natural infections was through small abrasions of the skin. Using fluorescent antibody staining, Roberts (1962b) showed that after scarification, the first cells infected were dermal macrophages. Spread in the dermis initiated “island foci” of epidermal infection in advance of the more slowly spreading main dermal focus. Virus spread from the primary lesion through successive rounds of infection, with multiplication and liberation usually accompanied by cell necrosis. Spread to the lymphatics, often via infected macrophages, caused a sequential infection of local lymph nodes and internal organs (primary viremia; see Fig. 3-4). During the incubation period of 4 to 6 days PI, the virus multiplied locally and in the spleen and liver, and a primary lesion developed at the site of entry (Fig. 3-5). The subsequent disease course depended on the degree of virus replication in the liver and spleen. From the work of Mims (1964), it appeared that the phagocytic cells of the liver and spleen were important for the spread of the virus within the tissues. Virus from spleen and liver tissue released into circulation (secondary viremia) caused
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DAY 0
1
Peripheral routes of infection: lung and skin Regional lymph node: Multiplication
INCUBATION PERIOD
2 Blood-Stream: primary viraemia 3 Spleen and Liver: multiplication necrosis
4
5
Blood-stream: secondary: viraemia
6
Skin: focal infection: multiplication
7 Swelling of Foot: (primary lesion)
DISEASE
8
9
focal infections of the skin and sometimes of the kidneys, lungs, intestines, and other organs. If virus titers escalated, death occurred within 0–4 days of the appearance of the primary lesion (Fenner 1949b), and viral titers in the spleen and liver reached levels of l09 or 1010 pock-forming units per gram. The virus content of the skin was often very high, but not enough time had elapsed for skin lesions to develop. If the virus failed to reach a lethal concentration in the internal organs, virus deposited previously in the epidermis eventually caused multifocal necrosis, producing a generalized rash (Fig. 3-6). Healing of the skin lesions usually occurred in about a week, often leaving hairless scars. The severity of the rash depended upon the degree of viremia, which appeared to be directly related to virus concentration in liver and spleen. Virus can be detected in blood between 3 to 13 days PI, with peak titers of 104 PFU/ml detected on day 8 or 9 (Fenner 1949b). Viral multiplication in each organ followed a logarithmic course; however, titers fell steeply coincident with the onset of circulating antibody and cell-mediated immunity. Rash development was also affected by virus dose, route of inoculation, and mouse strain. It is important to note that all these observations refer to mousepox in young adult mice of a genetically susceptible Hall Institute outbred mouse strain; clinical signs are very different in disease-resistant mouse strains that have non-H-2 linked resistance genes (Section VIII, C).
Early eash Papules
C.
10 1.
11 Severe rash Ulceration Fig. 3-4
Diagram illustrating the pathogenesis of mousepox (Fenner 1948d).
Fig. 3-5
Gross and Microscopic Pathology
Susceptible Mouse Strains
Fenner described the gross and microscopic pathology of mousepox in susceptible outbred Hall Institute mice (Fenner 1948a). More recently, others have reported similar findings in
The primary lesion of mousepox on the left eyebrow of a naturally infected mouse, 8 days (left) and 14 days (right) after infection.
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A
B
C Fig. 3-6 The rash of mousepox as it appears 14 days after infection. (A) Normal mouse of a susceptible strain. (B) Normal mouse after depilation to reveal the rash. (C) In a naturally infected hairless mutant mouse (not athymic). (Courtesy of the Zentralinstitut für Versuchstiere, Hanover, Federal Republic of Germany.)
susceptible inbred mouse strains (BALB/c, DBA/2, and C3H) (Allen et al. 1981; Jacoby and Bhatt 1987). The following description is based mainly on Fenner’s work with susceptible outbred mouse strains. A. SKIN The primary gross lesion is localized swelling, usually surmounted by a minute breach of the surface (Fig. 3-5).
It rapidly increases in size, with pronounced edema of the surrounding tissues. Later, a hard, adherent scab forms and drops off after a week or two, leaving the site of the primary lesion marked by a deep, hairless scar that often persists for life (Fig. 3-7). The earliest histologic lesions precede the described gross change and may reflect viral replication that then had been in
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Fig. 3-7
Mouse that had recovered from mousepox, showing amputation of the foot (“ectromelia” of Marchal) and scars on the face.
progress for a week. The dermis and subcutaneous tissues may be edematous, and there may be lymphocytic infiltration of the dermis. ATIs may be seen in the overlying epidermal cells at the summit of the lesion (Fig. 3-8). Epidermal necrosis leads to ulceration and widespread dermal necrosis. Subsequent scab formation promotes healing. The secondary rash appears 2 or 3 days after development of the primary lesion as slightly raised, pale areas 2 or 3 mm in diameter. They may increase in number and size, ulcerate, and in animals that survive, heal by scarring 3 weeks after infection. Conjunctivitis and blepharitis may occur frequently during the secondary rash, and in severe cases ulcers can be found on the tongue and buccal mucous membrane. The first histologic changes of the secondary rash occur as localized areas of epidermal hyperplasia, with dark-staining nuclei surrounded by vacuoles; epidermal cells may contain intracytoplasmic ATIs. As areas of proliferation and edema increase in size, they become macroscopically visible as pale, slightly raised macules. Numerous ATIs are then present in epidermal cells. Fresh macules develop, and earlier lesions reveal epidermal necrosis of the superficial cells. Massive necrosis, which follows quickly, is accompanied by widespread inflammatory edema and lymphocytic infiltration of the dermis, and is expressed grossly as papules that convert to ulcers with closely adherent scabs. B. LIVER The liver is invariably affected during acute infection and is a prominent site of ECTV replication. The liver may remain macroscopically normal until within 24 hr of death, when it appears enlarged and studded with white foci representing hepatocytic necrosis (Fig. 3-9). Hepatic necrosis can extend rapidly into large semiconfluent zones. In animals that
survive, the liver usually returns to its normal macroscopic appearance, but occasionally numerous white necrotic foci are noted, especially along the anterior border of the median lobe. These foci represent the progression of viral lesion into abscesses. Histologically, little change is apparent until gross lesions also appear, that is, within a day or so of death in rapidly fatal cases; however, fluorescent antibody staining has shown that infection begins in littoral cells of the hepatic ducts, from which it spreads to contiguous parenchymal cells (Mims 1959). Numerous scattered foci of necrosis then develop and, in fatal cases, spread rapidly to semiconfluence (Fig. 3-9). Parenchyma at the margins of necrotic areas may display hepatocytic regeneration with many multinucleate cells, even in fatal cases. Liver regeneration commences early and is active, especially in nonfatal cases, and fibrosis does not occur. Portal tracts may be lightly infiltrated with lymphoid cells, but ATIs are rarely found in infected hepatocytes. The necrotic foci occasionally seen after recovery from infection consist of hyaline necrotic tissue. Survivors may have small accumulations of lymphocytes, usually in portal triads and occasionally in the liver parenchyma. C. SPLEEN The spleen often develops macroscopic changes at least a day earlier than the liver, and higher titers of virus can be found in the spleen each day until death, when virus titers in the spleen and liver are approximately the same. Fluorescent antibody studies (Mims 1964) revealed that virus probably reaches the spleen in infected lymphocytes, which initiate infection in splenic follicles. Infected follicles undergo necrosis, whereas neighboring follicles may show proliferative changes indicative of antibody production in the spleen.
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3. MOUSEPOX
A
B Fig. 3-8 Section of the skin of the foot of a mouse injected with ectromelia virus in the footpad 6 days earlier. (A) Low power. (B) High power. Mann’s stain. Almost every epithelial cell contains an eosinophilic ATI (arrows).
Initial gross lesions of splenic necrosis include pale, slightly depressed zones, which can become semiconfluent. In surviving mice, residual lesions are more common in spleen than liver, and vary from small raised plaques about 1 mm in diameter consisting of hyperplastic serosa to prominent parenchymal fibrosis. These changes constitute the most frequent and reliable gross evidence that a mouse has sustained and recovered from mousepox (Fig. 3-10). Histologically, the early splenic changes consist of hyperplastic lymphoid follicles, congestion of the red pulp, and focal necrosis of lymph follicles. Necrosis can extend rapidly to efface large segments of the parenchyma. D. OTHER LYMPHOID TISSUES Regional lymph nodes draining the primary lesions can be enlarged and show prominent to
confluent necrosis where pyknotic nuclear debris is observed in a featureless background. Numerous ATIs may be present. Lymphocytosis is characteristic of nonregional lymph nodes during recovery. Allen and colleagues (1981) reported thymic necrosis as a major finding in the 1979 National Institutes of Health mousepox outbreak. The necrosis was attributed to direct virus replication using immunohistochemistry, but the disintegration of the cortical thymocytes also pointed to the possibility of a secondary contribution from the stress of severe disease. E. INTESTINE The intestine is often engorged, and the Peyer’s patches can be enlarged in fatal cases. Greenwood et al. (1936) observed necrosis and ATIs in cells of the epithelium in about 65% of acutely fatal cases. Briody (1955, 1959) has commented
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A
B
Fig. 3-9 Acute hepatic necrosis in mousepox. A mouse sacrificed when moribund, 7 days after the injection of a large dose of ectromelia in the footpad. (A) Gross photograph of internal organs. (B) Higher power. There is extensive necrosis of the liver, with little inflammatory reaction.
upon the high frequency with which a hyperemic or bloodfilled small intestine was observed during epizootics of mousepox in the United States, especially in genetically highly susceptible strains of mice. No other organs are regularly affected in natural mousepox, but occasionally, especially in very young mice, necrotic, hemorrhagic foci can occur in the kidneys and in the urinary bladder. Necrosis may be especially prominent in the renal convoluted tubules, with ATIs in tubular epithelium, and sometimes free in the lumina. Renal fibrosis and lymphocytic infiltrations may occur among survivors. 2.
Resistant Mouse Strains
The pathology in genetically resistant C57BL/6 mice is distinctive. First, Jacoby and Bhatt (1987) and then Karupiah et al. (1993a) observed that pathology in ECTV-infected C57BL/6 mice was more restrictive than in susceptible mouse strains. Necrosis in spleen and liver was mild and accompanied by mononuclear cell inflammation and hyperplasia of
lymphoid tissues. Bone marrow, intestines, and oral tissues were relatively uninvolved.
VIII.
INNATE AND ADAPTIVE IMMUNE RESPONSES
The recovery of resistant C57BL6 mice from a primary peripheral (footpad) infection with ECTV is dependent on the seamless integration of innate defense mechanisms, including type I and II interferons, natural killer cells, and monocytes, as well as immune responses characterized by CD8+ cytotoxic T lymphocytes, CD4+ T cells, and antibody.
A.
Innate Resistance
The innate response is activated in the first few hours of infection. It is characterized by the production of soluble
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3. MOUSEPOX
the development of cytotoxic T cells, when natural killer cell lytic activity would be expected to restrict virus replication and spread (Müllbacher et al. 1999).
B.
Fig. 3-10 Scarring of the spleen after recovery from mousepox.
mediators, including interferons and the inflammatory cytokines: interleukin-1, tumor necrosis factor-α, IL-6, and IL-18. In addition, neutrophils, monocytes, and natural killer cells are activated and migrate toward the focus of infection. The importance of these early responses for recovery from infection was determined using genetic or pharmacological approaches to specifically block individual pathways. Karupiah et al. (1993a) found both interferon-α/β and interferon-γ were essential for recovery of resistant C57BL/6 mice. Further studies by Karupiah et al. (1993b) suggested that part of the antiviral activity of interferon-γ was mediated through nitric oxide production. Macrophages were also shown to be crucial for recovery of C57BL/6 mice, as lethal infections followed macrophage depletion by liposome-mediated intracellular delivery of dichloromethylene bisphosphonate (Karupiah et al. 1996). Natural killer cells are also necessary for recovery, as their depletion with rabbit anti-asialo-GM1 or mouse anti-NK1.1 monoclonal antibody resulted in lethal infections (Jacoby et al. 1989; R. M. L. Buller and G. Karupiah, unpublished observations). Consistent with these findings, perforin-deficient mice had enhanced virus replication early during infection, prior to
Adaptive Immunity
The importance of the adaptive response in recovery was initially determined by showing that recovered mice were immune to reinfection (Marchal 1930; Greenwood et al. 1936). Fenner (l949c) showed that 2 weeks after infection, mice were solidly immune to reinfection by footpad inoculation of the virus. This immunity declined slowly, but even after a year, multiplication of the virus was confined to the local skin lesion, and only in occasional animals could virus be isolated from the spleen. Local virus replication in the foot, with consequent swelling, was associated with elevated HI titers. Long-term observation of recovered mice illustrated the epidemiological importance of durable immunity. Only 3 out of 168 immune mice developed signs of reinfection, and this was restricted to a local lesion in the foot (Fenner 1948c). Cell-mediated immunity is critical for recovery from mousepox (for reviews, see Blanden 1974; Müllbacher, Hla, et al. 2003; Müllbacher and Blanden 2004). Pioneering experiments by Blanden (1970) showed that mice pretreated with antithymocyte serum died from otherwise sublethal doses of virus due to uncontrolled viral growth in target organs. These mice had impaired cytotoxic T cell responses but normal neutralizing antibody responses, elevated interferon levels in the spleen, and unchanged innate resistance in target organs. Anti-thymocyte serum–treated mice were protected from lethal infection by the transfer of immune spleen cells harvested 6 days after immunization of donor mice (Blanden 1971a). The transferred cells rapidly eliminated infection from the target organs of the recipients, while no anti-ECTV antibody or interferon was detected in the recipients. The active cells were CD8+ T cells. The kinetics of their generation and the requirement for sharing of H-2K or H-2D genes between donor and recipient identified them as cytotoxic T cells. The importance of CD8+ T cells for recovery from disease was confirmed using C57BL/6 mice depleted of CD8+ T cells by monoclonal antibody therapy or C57BL/6 β2m-/- mice lacking functional MHC class I surface molecules (Karupiah et al. 1996). As expected, C57BL/6 mice lacking granzyme A and B or perforin, which are necessary for FasLindependent CD8+ T cell killing of virus-infected cells, were also highly susceptible to lethal infection (Müllbacher et al. 1996; Müllbacher, Hla, et al. 1999; Müllbacher, Waring, et al. 1999). CD4+ T cells of the helper class, which recognize antigens dependent on the I region of the H-2 complex, are also important for recovery from ECTV infection. In C57BL/6 mice, CD4+ T cells were required for the optimal generation of cytotoxic T cells, although mice survived in their absence (Buller, Holmes, et al. 1987; Karupiah et al. 1996). C57BL/6 mice lacking CD4+ T cells sustained low levels of virus replication in most
82 organs and very high levels of replication in skin (Karupiah et al. 1996). The CD4+ T cells are likely important in facilitating immunoglobulin class switching in the production of anti-ECTV antibody, and in the activation of monocytes during hepatic infection (Blanden 1971b). B cell responses are important in protection from reinfection. Serum from recovered mice contained neutralizing (Burnet and Lush 1936a), HI (Burnet and Boake 1946), and precipitating (Horzinek 1965) antibodies. Serum-neutralizing antibody was not detected until day 8 PI, and virus-specific IgG was detected ~7–10 days PI of C57BL/6 mice (Buller et al. 1983; Chaudhri et al. 2004). Very large doses of interferon or immune serum were relatively ineffective against established infection in target organs, though high serum antibody titers could be demonstrated in the recipients of immune serum (Fenner 1949b; Blanden 1971a). These findings indicate that innate and adaptive responses are necessary for recovery from ECTV infection. Interferons help activate the innate response, including natural killer cells that slow the spread of virus until virus-specific immune CD8+ T cells can further retard viral spread by lysing infected cells before the maturation and assembly of progeny virions. In resistant C57BL/6 mice, cell-mediated immune responses by CD8+ and CD4+ T cells occur soon after infection; for example, virus-specific precursor cytotoxic T cells were detectable in the draining popliteal lymph node 2–3 days after footpad inoculation (O’Neill and Brenan 1987). Cytotoxic T cell activity reached peak levels in the spleen ~5 days later and returned to background levels as early as day 10 PI (Blanden and Gardner 1976). CD8+ T cells supplemented by virus-specific CD4+ T cells attract blood monocytes, which contribute to the elimination of the infection by phagocytosis and intracellular destruction of virus. Delayed type hypersensitivity was detectable by the footpad-swelling test 5–6 days after infection (Fenner 1948a; Owen et al. 1975). Activated macrophages locally produce interferon that may increase the efficiency of virus control and elimination. Recently, Karupiah and colleagues examined ECTV-infected susceptible (BALB/c and A/J) and resistant (C57BL/6) mice strains for cytokine production by CD4+ and CD8+ T cells, cytolytic activity of natural killer and T cells, and virus titers in various tissues, and concluded that polarized type 1 cytokine responses and cell-mediated immunity determined genetic resistance to mousepox (Chaudhri et al. 2004). Two features of the immunological response are important in explaining the natural history of mousepox. First, the effectiveness of cell-mediated immunity helps to explain the absence of clinical signs in some resistant genotypes. Second, lactogenic (humoral) immunity maternal antibody is very effective in protecting highly susceptible mouse pups from lethal infection (Fenner 1948b). Suckling mice attained a serum antibody titer of up to half that present in the mother, and following weaning on the 21st day, the antibody titer fell until the 6th week, when it was no longer detectable. Limited investigations on the spread of the disease in two breeding colonies suggested that
R. MARK, L. BULLER, AND FRANK FENNER
maternal antibody might play an important role in the persistence of the virus in laboratory colonies (see Section IX, D).
C.
Genetics of Resistance to Lethal Mousepox
Fenner’s characterizations of mousepox (Sections VI and VII) were carried out using highly susceptible non-inbred mice. The clinical picture and pathological findings are different if genetically resistant mice are studied. Some studies reported by Schell (1960a,b) in genetically resistant C57BL mice are pertinent. Schell found no difference in the infectivity endpoint of a viral suspension titrated in susceptible outbred mice and C57BL mice, but the titer of virus in the footpad of C57BL mice ceased to rise 6 days after infection, and the highest titers in blood, liver, and spleen were 2 to 3 logs lower than in outbred susceptible mice. Roberts (1964) showed that the rate of growth of ECTV in the littoral (reticuloendothelial) cells of the livers of mice depended upon the virulence of the strain of virus used (virulent Hampstead [mouse] versus attenuated Hampstead [egg]), whereas the subsequent growth of virus in the parenchymal cells of the liver depended upon the mouse strain. Additional studies by O’Neill and Blanden (1983b) suggested that C57BL mice have cells or factors resistant to radiation (950 rads) that impose an early barrier to viral spread from a peripheral site of infection via lymphocytes or blood to liver and spleen. Differences in immunological responses were also noted between mouse strains. Neutralizing antibody, delayed type hypersensitivity, and active immunity were demonstrable 1 or 2 days earlier in C57BL mice compared to outbred mice (Schell 1960a). Breeding experiments by Schell (1960b) and Ermolaeva et al. (1974) demonstrated that resistance was largely determined by a single dominant gene that was not linked to coat color or H-2 genotype (O’Neill et al. 1983a). Studies by Wallace et al. (1985) using additional susceptible DBA/2J, A/J, and BALB/cByJ mice and resistant C57BL/6J and AKR/J mice confirmed that resistance was determined by one or more independently assorting autosomal loci with dominant alleles for resistance in AKR/J and C57BL/6J mice and recessive alleles in A/J, BALB/cByJ, and DBA/2J mice. In the (B6 × A)F1 × A backcross, resistance to lethal infection appeared to be controlled by a single autosomal dominant gene, provisionally termed Rmp-1 (resistance to mousepox). Subsequent experiments by Brownstein and colleagues (1992) using a (C57BL/6 × DBA/2) F1 × DBA/2 backcross identified three additional C57BL/6 genes, Rmp-2, Rmp-3, and Rmp-4 and mapped the location of all four genes. Rmp-1 maps to an interval of chromosome 6 that includes the natural killer cell gene complex including the NKR-P1 and Ly-49 families of proteins (Delano and Brownstein 1995). Rmp-2 maps to an interval of chromosome 2 that includes the structural gene for the fifth component of complement (Brownstein et al. 1992). Rmp-3 and Rmp-4 map to chromosome 17 and chromosome 1, respectively (Brownstein
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et al. 1992; Brownstein and Gras 1995). Thus the genetics of resistance to mousepox are complex and can vary depending on which strain of ECTV is used (Brownstein et al. 1989).
IX.
EPIZOOTIOLOGY A.
1.
Host Range
Species
The mouse can be infected by all routes of inoculation with ECTV. Several other species of laboratory animals apart from Mus musculus have been tested for susceptibility to ECTV, as summarized below. A. OTHER SPECIES OF MUS There are two reports suggesting that mousepox might occur as a disease of wild rodents, other than of wild house mice associated with an outbreak in laboratory mice (e.g., McGaughey and Whitehead 1933). Gröppel (1962) examined wild mice belonging to three genera (Microtus, Apodemus, and Cletrionomys) that were captured in several rural localities in Germany, distant from possible contamination from laboratory mice. His observations may suggest that several of the Apodemus animals were infected with an agent that was probably ECTV. Kaplan et al. (1980) found that sera from several voles and woodmice captured in the wild in the United Kingdom contained complement-fixing antibodies against an orthopoxvirus that he proposed was ECTV. More recent studies suggest that the orthopoxvirus that is maintained in these British rodent populations is cowpox virus (Crouch et al. 1995; Bennett et al. 1997; Hazel et al. 2000). Resistance to severe mousepox varied across four genera of rodents (Buller et al. 1986). Experimental infections of representative species from the genus Mus (caroli, cookii, spretus, and cervicolor), and the genus Nannomys (minutoides) resulted in high mortality. Coelomys pahari and Pyromys platythrix could be infected, as indicated by seroconversion, but showed no signs of disease. B.
Mooser (1943) found that when peritoneal fluid from moribund mice, which were infected with both ECTV and Rickettsia prowaseki, was inoculated intraperitoneally into rats, there was a severe rickettsial peritonitis. Characteristic ectromelia ATIs were also seen in smears of the peritoneal fluid, suggesting that ECTV had replicated.
RAT Reports that rats can be naturally infected with ECTV and may indeed be a reservoir of the virus in nature (e.g., Iftimovici et al. 1976) are unconvincing. Burnet and Lush (1936a) showed that when large doses of virus were inoculated intranasally into the rat, an inapparent infection of the olfactory mucosa occurred, accompanied by the development of neutralizing antibody. This finding was confirmed by Reames (1940). Intradermal inoculation of large doses of ECTV produced seroconversion but no lesions, or at most a tiny papule at the site of injection (F. Fenner, unpublished observations). Intraperitoneal inoculation was also without effect except for seroconversion. Repeated ECTV inoculations into rats produced no further increase in antibody titer.
C. RABBIT Early workers found the rabbit was resistant to ECTV infection, although Paschen (1936) reported that virus that had been passed several times on the chorioallantois produced infection of the rabbit’s cornea, with the production of ATIs, and could then be passed to the rabbit skin. Burnet and Boake (1946) found that two rabbits inoculated intravenously with a large dose of virus died 6 and 10 days later, and hemagglutinin was present in suspensions of liver and spleen. Intradermal inoculation of ECTV produced indurated papules. Such papules regularly appeared about 4 days after intradermal inoculation of large doses of virus and sometimes ulcerated a few days later (F. Fenner, unpublished observations). In rabbits immunized by a prior infection, the papule formation and regression were accelerated, reaching their maximum size by the second day and fading by the sixth day. Such intradermal infections also resulted in seroconversion. Christensen et al. (1966) showed that ECTV could be used to immunize rabbits against rabbitpox, although it was preferable to use vaccinia virus for this purpose because of the danger to the mouse colony. D. GUINEA PIG Paschen (1936) found that inoculation of the guinea pig foot or cornea with egg-passaged ECTV but not with mouse liver virus produced typical ATIs. Intradermal inoculation of chick embryo membrane preparations of the virus regularly produced local indurated lesions, and HI antibodies were detected in the sera 14 days later (F. Fenner, unpublished observations). Intraperitoneal inoculation of large doses of ECTV caused no signs, but was followed by seroconversion. E. HAMSTER According to Flynn and Briody (1962), Syrian hamsters are not susceptible to infection with ECTV. F. MAN The only conscious attempt at infection of humans with ECTV was scarification of two men who had been vaccinated with vaccinia virus several times previously (F. Fenner, unpublished observations). In both a small papule appeared on the second day, became slightly vesicular in one on the fourth day, and disappeared by the eighth day. No significant increase in antibody titer was observed. Packalén (1947) showed the Laigret-Durand strain of “mousepathogenic murine typhus rickettsia,” like the epidemic typhus strain studied by Mooser (1943), owed its mouse pathogenicity to its ECTV content. Thus active ECTV, either alone or mixed with rickettsiae, was inoculated subcutaneously into hundreds of thousands of humans in doses of up to 1000 “mouse units” (Laigret and Durand 1939, 1941; Packalén 1945). No local or
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TABLE 3-3
TABLE 3-4
INFLUENCE OF GENOTYPE ON MORTALITY IN AN EPIZOOTIC OF MOUSEPOXa
COMPARISON OF INFECTIVITY AND MORTALITY OF ECTROMELIA VIRUS IN INBRED STRAINS OF MICEa
Case fatality rate Genotype
Stock mice (%)
DBA/1 A C3H MA/Nd C57BL/6 BALB/c AKR MA Sy N Sy D
84 84 71 2 <1 <1 <1 <1 <1 <1
Lethality (LD50)b
Mouse strain
Breeding colony (%) 61 71 28 <1 <1 15 <1 2 – –
C57BL/6J AKR/J BALB/cByJ DBAl2J A.By/SNJ C3H/HeJ
1.0 1.0 6.9 6.2 6.3 7.1
Infectivity (ID50)b,c 7.0 – 7.1 6.2 7.0 7.2
aNIH-79 strain of ECTV, titer 106.3 plaque-forming units per mL in BS-C-1 cells. Mice were inoculated with 0.05 mL by the footpad route. Only male mice 6 to 10 weeks of age were used in this study. bExpressed as negative log base 10. cInfection defined by positive enzyme-linked immunosorbent assay. From Wallace and Buller (1985).
aThe figures given refer to the 3-week period after onset, when no effort was made to control the disease in 4000 stock mice but during which all ill mice were immediately removed from the breeding colony of 2,000 mice. From Briody et al. (1956).
general reaction of any significance was reported (Laigret and Durand 1941). 2.
Strains of Mice
As noted previously, susceptibility to ECTV and the resulting severity of disease are critically dependent on the mouse genotype (Trentin 1953; Briody 1955, 1959; Briody et al. 1956). Briody and coworkers demonstrated that mortality was greatest in DBA/1, A, and C3H mice, while MA/Nd, C56BL/6, BALB/c, AKR, and MA mice experienced little or no mortality (Table 3-3). Wallace and Buller (1985b) observed similar patterns of resistance and susceptibility following direct experimental infection (Table 3-4). C57BL/6 mice, because they typically develop asymptomatic or mild infections, can act as “silent reservoirs” of ECTV infection. Consistent with these observations, Wallace and Buller (1985) showed that C57BL/6 index mice transmitted infection by contact between 6 and 17 days following footpad inoculation of index mice that developed only swelling and necrosis at the site of virus inoculation (Fig. 3-11). Similar results were obtained by Bhatt and Jacoby (1987b) using C57BL/6 and BALB/c mice. Briody (1966) assembled information on the response of multiple mouse strains from several epizootics in the United States (Table 3-5) (Briody 1955, 1959; Briody et al. 1956; Trentin 1953). He concluded from this survey and from experiments with individual mice that, as a rule, mice of the strains most susceptible to lethal infection (A, BC, DBA/1, DBA/2, and CBA) did not survive long enough to permit the primary skin lesion or generalized rash to develop to the stage of virus release. Thus these strains had weak potential for disseminating infection.
Development of an epizootic in these mice was critically dependent on other mice (C3H, A, SWR, and CF1) that lived long enough to develop extensive skin lesions. Enzootic infection, without obvious disease, would be expected if the population consisted solely of C57BL/6 mice. C57BL/6, BDF1, BALB/c, and CAFl mice were regarded by Briody as ideal for maintaining subclinical enzootic infections, but able to initiate explosive epizootics when less-resistant strains (C3H, A, SWR, and CF1) were exposed. Briody further suggested that neither infection nor disease would result if the population groups were MA/Nd, AKR, MA, or C58 mice. Factors other than genotype, notably passive and active immunity, play a role in the epizootic behavior of mousepox (see Section VIII, A and B). For example, both in U.S. (Trentin 1953; Trentin and Ferrigno 1957) and European mouse colonies (Guillon 1975), initial outbreaks were characterized by acute deaths with predominantly visceral lesions (even among C57BL mice). Once enzootic infection was established, the incidence of acute deaths decreased, and subacute, chronic, or inapparent infections dominated.
B. 1.
Prevalence and Distribution
Europe
Mousepox was recognized as a relatively common infection of laboratory mouse colonies in Europe during the first half of the twentieth century (Marchal 1930; Fairbrother and Hoyle 1937; McGaughey and Whitehead 1933; Gledhill 1962a; Hornus and Thibault 1939; Schoen 1938; Kikuth and Gönnert 1940; Schell 1964; Andrewes and Elford 1947; Mooser 1943; Briody 1959). In parts of Europe, outbreaks were so common that few were ever reported. In the past 20 years there have been no confirmed reports of laboratory epizootics, although
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120
Infected Contact Mice (%)
100
80
60
40
20
0 1-3
3-5
5-7
7-9
9-11 11-13 Exposure period
13-15
15-17
17-19
Fig. 3-11 Transmission of ectromelia virus from index, footpad-infected C57BL/6 mice to cage mates. Mice were screened for seroconversion by ELISA at 28 days following exposure.
ECTV may still exist in mouse colonies and in archived tumor lines and virus stocks, especially in eastern Europe and in the countries of the former Soviet Union. 2.
United States
Enzootic mousepox has not been reported in the United States, but epizootics have occurred (see Section IX, C). Briody (1966) found no evidence of enzootic mousepox in serological surveys involving over 100,000 serologic assays from various mouse colonies in different parts of the United States. However, ECTV has been unwittingly imported into the United States
several times with mice, mouse tissues, and mouse sera, sometimes causing devastating outbreaks (Melnick and Gaylord 1953; Trentin 1953; Poel 1954; Dalldorf and Gifford 1955; Briody 1955; Briody et al. 1956; Whitney 1974; Dick et al. 1996; Lipman et al. 1999). 3.
Other Regions
Mousepox may be enzootic in some mouse colonies in China. At least one epizootic of mousepox in the United States was attributable to contaminated mouse sera imported from China (see below; Lipman et al. 1999).
C.
Epizootic Mousepox
TABLE 3-5
THE RESPONSE OF MICE IN EPIZOOTICS OF MOUSEPOX IN THE UNITED STATESa Mice
Skin lesions
Usual result
MA/Nd, AKR, MA, Sy N, Sy D C57BL6, BDF, BALB/c, CAF A, BC, DBA/1, DBA/2, CBA C3H, A SWR, CFI
Absent Minimal Minimal Extensive Extensive
No infection Recovery Death Death Death or recovery
a
From Briody (1966).
Outbreaks of mousepox occurred in laboratory mice when ECTV was inadvertently introduced into colonies of susceptible animals (“natural epizootics”), and deliberate, controlled epizootics have been used for the experimental study of mousepox epizootiology. The last major outbreak of mousepox in the United States occurred in the late 1970s and early 1980s at the National Institutes of Health and a number of academic institutions including Saint Louis University, Washington University in St. Louis, Yale University, University of Minnesota, and the University of
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Utah (Wallace 1981; New 1981). The likely cause of the outbreak was the exchange among scientists of mouse strains that were not commercially available. Pooled commercial mouse serum has also been a source of infection. In 1995 at the Naval Medical Research Institute in Bethesda, Maryland, such serum was used to stabilize H. Plasmodium yoelii that was inoculated into BALB/cByJ mice (Dick et al. 1996), but the source of the mouse serum was not disclosed. In 1999, an outbreak of mousepox at Weill Medical College of Cornell University was attributed to a transplantable tumor cell line (Lipman et al. 2000). The tumor line had previously been shown to be free of viral agents by standard mouse antibody production (MAP) testing, and the source of ECTV was traced to commercial pooled mouse sera used as a supplement in chemically defined media used to grow the tumor line. It was subsequently determined that 43 liters of contaminated mouse serum had been imported from China and distributed to major suppliers throughout the United States. Similarly, mouse-passage tumors derived from mice obtained from other countries may be the source of an epizootic. This was formally tested by Buller, Weinblatt, et al. (1987), who found ECTV infectivity persisted in T cell lymphoma lines for greater than 35 days, whereas replication of virus in hybridomas was more varied and to lower levels. As recently as 1974, mousepox was introduced into laboratories of the National Institutes of Health in Bethesda through the importation of a tumor from London, which had earlier (1972) been sent from a laboratory in Prague where mousepox was enzootic (Whitney et al. 1981).
D.
Enzootic Mousepox
At least three mechanisms probably contribute to maintenance of enzootic ECTV. High levels of genetic resistance and lack of clinical signs exhibited by some mouse genotypes are likely important. So is maternal-derived immunity (Fenner 1948b), which as noted earlier protected young mice from fatal disease; however, the resulting lesions on such animals were infectious. Since the protective effect of maternal antibody is lost within 4 weeks, mice infected as weanlings are highly susceptible to disease, and deaths may be common (Fenner 1948b). A third possibility, clearly involving mouse genotype, is persistence of a clinically inapparent infection. The most convincing evidence of this is the report by Gledhill (1962a,b) of infection of Peyer’s patches and lesions in tail skin, referred to in Section VII, A, 2. Some authors believe these persistent infections can be activated by various kinds of stress, including the inoculation of tissue homogenates. Kikuth and Gönnert (1940) described two persistently infected strains of mice. Strain “Do,” which contained the more unhealthy-looking mice as compared to the other four strains, never showed external signs regarded as typical for ECTV infections. Some animals
had ruffled hair, occasionally “pyodermia” occurred on different parts of the body and extremities, and a few mice had blepharitis, but no other significant signs were apparent. The other infected strain, “Vo,” showed even less. It is likely that the pyodermia and blepharitis were lesions of the secondary rash of mousepox, and these animals probably constituted the source of infection for other members of the colony. No mention is made of undue mortality in the infected strains. Fenner (1948c) was unable to demonstrate persistence in the mouse strains that he used, although he found that the lungs and spleen of two mice that had recovered from mousepox, 28 and 75 days earlier, yielded virus. Both clinically inapparent and persistent infections are likely dependent upon the host genotype. If they do occur in a mouse colony, they are important both as potential sources of virus for natural mouse-to-mouse spread and as a source of virus that might be unwittingly transferred by subinoculation.
X.
DIAGNOSIS
Following the pattern established for the diagnosis of smallpox in the Smallpox Eradication Program of the World Health Organization, a series of rapid diagnostic tests were developed for assaying poxvirus virions in the homogenized tissues (or scabs) from suspected cases, and for screening sera for orthopoxvirus-specific antibody. Historically, mousepox was diagnosed with a combination of serologic and in vivo tests. Serologic assays included screening of sera for HI antibodies or, for rapid diagnosis, scab material was tested for antigen by gel diffusion tests (Briody 1959; Carthew et al. 1977). In vivo testing involved the inoculation of genetically susceptible, nonimmune mice, with suspected specimens followed by observation for the signs of mousepox as described earlier, while similarly treated vaccinated mice proved resistant to disease. Currently, ELISA and polymerase chain reaction (PCR) are the assays of choice (Buller et al. 1983; Buller 2000).
A.
Clinical Signs
Ectromelia, cowpox, and Turkmenia rodent viruses are the most likely poxviruses that would be isolated from rodents in nature, but there are no published studies identifying cowpox or Turkmenia rodent viruses as the causative agent of an epizootic outbreak in a vivarium. As discussed previously, the clinical signs of mousepox are dependent on the age and strain of mice. Mousepox in disease-susceptible strains can present initially with ruffled face and body but active behavior, and progress to depression, hunched posture, and conjunctivitis. Prior to death, the mouse shows little or no movement, and marked respiratory distress. In disease-resistant mice, the clinical signs can include conjunctivitis and typical exanthematous rash, which can be
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observed on exposed skin, especially the tail. Alternatively, the mice may show no obvious clinical signs at all. This variability in symptomatic response emphasizes the need for methods of diagnosis other than the clinical picture.
B.
Serology
Current serology-based assays are sensitive and rapid for detecting ECTV antibodies, but only recognize genus-specific antigens common to most orthopoxviruses. For this reason the ELISA used to screen sera for anti-ECTV antibodies employs vaccinia virus as the capture antigen. Vaccinia virus is easier to grow than ECTV and posses no risk to the vivarium. Sera from mice are tested first by ELISA. Positive results are confirmed by indirect immunofluorescence (Buller et al. 1983; Dick et al. 1995). Indirect immunofluorescence and immunohistochemistry can be used to test for virus antigen in mouse tissue or cultured cells infected with specimens from suspected cases (Dick et al. 1995; Lipman et al. 2000).
C.
Pathology
The dominant pathological finding in fatal cases is focal or pan necrosis in liver. Light (Marchal 1930) and electron microscopic (Leduc and Bernhard 1962) observations are used to detect ATI (Fig. 3-8) and B-type inclusion bodies and poxvirus particles respectively. The eosinophilic cytoplasmic ATIs are pathognomonic for mousepox, but can be sometimes difficult to see in liver sections; however, they are abundant in infected keratinocytes of skin lesions.
D.
Virus Isolation
Passage of concentrated viral suspensions on the chorioallantoic membrane produced confluent lesions that when ground up exhibited characteristic hemagglutination (Burnet and Boake 1946). Suitably diluted suspensions produced characteristic small pocks on the chorioallantoic membrane and characteristic cytopathic effect in tissue culture cells derived from the epithelium of mouse (L929) or monkey (BSC-1, CV-1, or vero) (Dick et al. 1995; Lipman et al. 2000).
E.
XI.
CONTROL AND PREVENTION
With the development over the last several decades of specific pathogen-free mouse colonies, large-scale production of widely used mouse strains has been established in healthy, ECTV-free facilities. There have been periodic efforts to limit the spread of the virus between colonies of mice in different laboratories (Shope 1954; Institute 1973; Anslow et al. 1975; Wallace 1981). Quarantine and regulation of the importation and distribution of ECTV or materials infected with it are mandatory in the United States in order to protect the large, uninfected mouse colonies of that country (Shope 1954; Anslow et al. 1975). However, there is some doubt as to how well such regulations were observed (Briody 1959), and in any case, they offer no protection against unsuspected sources of infection. If mousepox is identified in a colony, four procedures may be followed: eradication by culling and disinfection, control by vaccination, or serological screening of sentinel mice or at-risk populations in combination with quarantine or rederivation. The most effective strategy, however, is to prevent the introduction of ECTV-infected mice or specimens into the mouse colony in the first place.
A. 1.
Control
Depopulation and Disinfection
The National Institutes of Health Committee of which Shope was chairman recommended that infected colonies should be destroyed, and materials (viral inocula, tumors, etc.) derived from such animals incinerated, followed by thorough sterilization of mouse rooms, cages, equipment, etc. (Shope 1954). Sterilization can be achieved with formalydehyde gas or a range of contact sterilants, including fresh solution of 10% (v/v) bleach, Spor-Klenz, or envirocide. Colonies of mice suspected of infection should be isolated and quarantined. All materials harvested from mice received from foreign sources, and mouse strains themselves, should be regarded as potentially infected with ECTV, and any suspected or proved outbreaks should be promptly reported and their sources investigated. In certain situations, the mouse strain(s) may be of sufficient value that destruction is not an appropriate course of action. In this case vaccination, quarantine, or derivation (see below) may be the option of choice.
Molecular and Antigen Detection Techniques
Orthopoxvirus species can be identified and differentiated by polymerase chain reaction amplification of genomic DNA from tissue or scabs using primer-pairs based on sequences coding for the major protein component of the cowpox virus ATI or the hemagglutinin (HA) genes (Meyer et al. 1994; Ropp et al. 1995).
2.
Vaccination
Vaccination with the IHD-T strain of vaccinia virus, by any route of inoculation, will protect mice against mousepox. The protection, however, is not absolute, especially against virulent strains of virus, and it wanes with time (Fenner 1947a, 1949b). The strain of vaccinia virus, the age and genotype of the mouse,
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the concentration of virus, and the route of inoculation all affect the immune response, which may vary from lethal infection at one extreme to failure to produce an antibody response at the other (Owen et al. 1975). The practice of vaccination was reexamined by Buller and Wallace (1985). They found that vaccination protected for at least 12 weeks against clinically apparent disease when challenged with cage mates infected with the NIH-79 strain of ECTV. Importantly, 4–8 weeks after vaccination, mice were again capable of transmitting virus to nonvaccinated cage mates. Also, it is difficult serologically to distinguish between a vaccinated mouse and one that has seroconverted following infection with ECTV. This study suggested that a more prudent method to control an outbreak is the combination of quarantine and serologic surveillance with the removal of serologic reactors, unless irreplaceable strains of mice are at risk (see below). Mice that are vaccinated should not be used in immunologic experiments, as the vaccination process can affect future immunologic responses against other antigens (R. M. L. Buller, unpublished observations). 3.
Serological Screening
With modern barrier facilities, stringent animal husbandry protocols, quarantine, and sentinel surveillance programs, risks from the unintended introduction of mouse pathogens into a mouse colony have been substantially reduced. A localized outbreak of mousepox is therefore easier to quarantine and can be managed by serologic screening of the affected mouse population with the culling of mice that are seropositive for orthopoxvirus antibodies. In C57BL/6 mice, both ECTVspecific IgM and IgG antibodies were detected from 7 to 10 days PI (Buller et al. 1983; Chaudhri et al. 2004). The anti-ECTV IgG antibodies continued to rise for 30 days. Valuable mouse strains that have been exposed to mice infected with ECTV can be quarantined in direct contact with a susceptible mouse strain (see below). The mice are then sequentially tested for anti-ECTV antibodies over a 6–8 week period. Failure of the mice to seroconvert and the contact mouse to show clinical signs of mousepox would suggest the quarantined mouse could be reintroduced into the animal colony 4.
Rederivation of Mouse Strains
Mims (1969) observed there was extensive growth of virus in the placenta and infection of fetuses of outbred mice infected intradermally with the Hampstead (egg) strain of ECTV. Bhatt and Jacoby (1987b) found that infections of BALB/c mice with the NIH-79 strain were self-limiting. Recovered mice did not transmit mousepox to their progeny or breeding partners. The disparity in the findings between these two studies may be due to the use of different mouse and virus strains; however, based on our current understanding of the immune response to ECTV, it is likely that mice recovered from disease will no longer be a
source of infectious virus and can be used to prepare embryos for rederivation of the strain.
B.
Prevention
Prevention is the most cost-effective means of preventing mousepox. The major features of a prevention program are serologic surveillance of the colony, the quarantine of mice coming from facilities that have questionable health surveillance programs, and MAP testing of all cell lines and specimens that have originated from mice or that have been passaged in mice. Recent experience suggests that the use of commercial mouse sera, with any cell line or substance destined for inoculation into mice, should be avoided. 1.
Sentinel Surveillance
One approach for monitoring the vivarium for mouse pathogens including ECTV is to provide each mouse room, cubicle, or rack with a cage containing two to four specificpathogen-free mice that are exposed to bedding or fecal material from selected cages containing mice on experiment. Every 3 months, at least two mice are bled and submitted for gross necropsy, histopathology, and standard bacteriology and parasitology tests. The serum is screened against antigens of common mouse pathogens including ECTV. Historically the HI test, using vaccinia virus to provide the hemagglutinin, was used to screen sera (Briody 1959). The HI test has been replaced by ELISA for screening of sera for orthopoxvirus-specific antibody (Buller et al. 1983). A second confirmatory test using immunofluoresence is carried out on positive sera. 2.
Quarantine
Mice imported from a noncommercial source or imported from outside of the United States should be quarantined, and sera tested for orthopoxvirus antibodies at least twice over a 6–8 week quarantine period. In addition, sentinel mice can be exposed to the soiled bedding from the cage containing the quarantined mice, as ECTV is relatively stable and infectivity was still detectable in biologic material such as blood and serum following 4 days at room temperature (Bhatt and Jacoby 1987c). Also, Fenner (1949a) showed that the Moscow strain of ECTV, when sprayed onto sawdust bedding in clean cages, was capable of efficient infection of outbred mice. Finally, healthy, genetically susceptible mice (e.g., strains A/J, DBA/2, or C3HeJ) can be cohoused as sentinel animals with the imported mice for a few weeks to reveal the possible presence of ECTV. If the imported mice are to be used in breeding, this test necessitates segregating the males and females during this period of contact or, alternatively, introducing only ovariectomized females as susceptible contact mice. The sentinel mice should
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be observed for clinical signs and their sera screened for orthopoxvirus antibodies 6–8 weeks after cohousing. 3.
Mouse Antibody Production
Transplantable tumors, cell lines, or other biological materials derived from mouse-passage and destined to be inoculated into mice must be first MAP tested in genetically susceptible mouse strains for clinical signs of mousepox or the induction of orthopoxvirus antibodies. This approach could be adapted to use PCR as the detection method; however, the assay sensitivity may be low due to the transient nature of the ECTV viremia. Valuable tumor lines suspected of contamination with ECTV may be passed for at least two transplant generations in vaccinated mice, followed by a third generation in unvaccinated mice. If sera from the third-generation passage are negative for orthopoxvirus antibodies and the mice show no signs of mousepox, then the cell line can be tentatively reported as ECTV-free, but continued vigilance will still be warranted.
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Chapter 4 Parvoviruses Robert O. Jacoby and Lisa Ball-Goodrich
I. II. III. IV. V. VI. VII.
History and Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antigenic and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology and Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. MPV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. MVM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Effects on Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. MVM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. MPV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. HISTORY AND NOMENCLATURE In 1966, Crawford reported the isolation of a passenger virus from a mouse adenovirus stock (Crawford, 1966). It had cell culture, physical, and chemical characteristics that resembled rat virus (Kilham and Olivier, 1959), the prototype virus for the family Parvoviridae, including the capacity for autonomous replication. He named the agent minute virus of mice (MVM) and the initial strain MVM-CR. Additional isolates have since been identified, including from naturally infected mice. Two strains in particular have been widely used to investigate parvovirus biology: a plaque-purified strain prepared by Tattersall and Bratton (1966) that was initially named MVM-T THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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but was then renamed MVMp (p for “prototype”) and an allotropic strain, MVMi (i for “immunosuppressive”), isolated from a transplantable lymphoma by Bonnard and coworkers (Bonnard et al. 1976). Recently, the International Committee on Taxonomy of Viruses (Van Regenmortel et al. 2000) has considered renaming MVM mice as mouse minute virus (MMV). To the best of our knowledge, this potential change has not been formally accepted. Therefore, we use the acronym MVM in this chapter. For many years, MVM was viewed as the sole parvovirus serotype affecting laboratory mice (Cross and Parker, 1972). During the mid-1980s, however, diagnostic laboratories began to detect mouse sera that reacted with MVM in generic, but not Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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serotype-specific, tests (A.L. Smith, unpublished data). These findings suggested the existence of a murine parvovirus antigenically distinct from MVM. Subsequently, McKisic and colleagues (McKisic et al. 1993) isolated a lymphocytotropic parvovirus from mice. The newly recognized agent had adverse effects on in vitro immune responses, including lytic growth in a CD8+ T cell clone and inhibition of cloned T cell proliferation after stimulation with antigen or IL-2. Similar parvoviruses were soon isolated from naturally infected mice (Smith, unpublished data; Smith et al. 1993). The new serotype was initially—and unfortunately—named “mouse orphan parvovirus” to convey vague understanding of its biological significance, despite a causative association with immune dysfunction. However, it was subsequently renamed mouse parvovirus (MPV) (BallGoodrich and Johnson, 1994) to conform with the nomenclature of parvoviruses in other mammalian species. Retrospective serology suggested that MPV strains had been present in American mouse colonies for at least 20 years (Smith, unpublished data). The discovery of this new serotype, with some properties distinct from MVM, highlights the importance of employing both generic and serotype-specific tests for the identification of murine parvoviruses. The detection of multiple MPV isolates indicated a need for standardized nomenclature. We proposed adaptation of the system introduced by Parrish to classify canine and feline parvoviruses (Parrish, 1990). Thus, the prototype strain of MPV was designated MPV-1a, and two subsequent isolates were named MPV-1b and MPV-1c, with additional isolates to be named in alphabetized sequence (Jacoby et al. 1996).
Fig. 4-1
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Isolates of additional parvovirus serotypes would be designated MPV-2a and so on. Strong sequence homology between conserved regions of the MVM and MPV genomes encoding for nonstructural proteins suggests that the name MPV could encompass all strains of both serotypes and facilitate a unified nomenclature based on the Parrish system. Because of their similarities, we describe the properties of MVM and MPV collectively, except where drawing distinctions between them is warranted.
II.
MOLECULAR ANALYSIS
Murine parvoviruses are small (15–28 nm), non-enveloped single-stranded (ss) DNA viruses (Cotmore and Tattersall, 1987; Ward and Tattersall, 1982). As with other autonomous parvoviruses, they require cellular factors expressed during cell division and differentiation for productive replication. These factors are thought to account for their predilection for mitotically active cells. Genomic analysis has shown that the 5-kb genome is bracketed by terminal palindromes involved in replication (Fig. 4-1). The plus-sense strand of the double-stranded replicative intermediate contains two large and several smaller open reading frames (ORF). The larger ORFs are driven by promoters at map positions 4 and 38 and utilize alternative splicing to generate multiple transcripts. The P4 transcripts encode two nonstructural proteins, NS1 and NS2, that are involved in viral transcription and replication and are highly
Physical and genetic map of a murine parvovirus.
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conserved among the rodent parvoviruses. The P38 transcripts encode the two major viral (capsid) proteins, VP1 and VP2, which are serotype-specific. All of VP2 is contained within VP1, but VP1 contains an additional 143 amino acids at the N terminus. VP3 is generated by cleavage of 19 N-terminal amino acids from VP2, and is present in differing amounts in virions. The virion coat is composed of 60 copies of VP1, VP2, and VP3, 54 of which are VP2 or VP3.
III.
REPLICATION
MVM replicates in monolayer cultures of mouse fibroblasts (A9 cells), C6 rat glial cells, SV40-transformed human newborn kidney (324K cells), T cell lymphomas (S49 and EL4 cells), and rat or mouse embryo cells and produces cytopathic effects that can include the development of intranuclear inclusions (Tattersall and Cotmore, 1986). MPV, by contrast, is very difficult to cultivate in vitro. L3, a line of cloned CD8+ T lymphocytes, is the only line known to support MPV with any degree of reliability (McKisic et al. 1993), although there is preliminary evidence that MPV will replicate in 324K cells after serial passage (S. Jennings, personal communication). Nevertheless, the identification of simpler and hardier in vitro cultivation methods for MPV remains a challenge for parvovirus research. The replication of murine parvoviruses has been determined primarily through in vitro studies of MVM infection (Cotmore and Tattersall, 1987; Tattersall and Bratton, 1983; Tattersall and Cotmore, 1986) and consists of four outcomes, one representing productive infection and three representing nonproductive infection. Productive infection, the most common outcome, begins with the binding of the virion to an undefined cell surface receptor(s). The virus is internalized by endocytosis, transported to the nucleus, and the positive strand is synthesized to form a duplex DNA replicative intermediate. Viral transcription and translation result in the production of NS proteins, which up-regulate the synthesis of both NS and VP proteins. A burst of viral DNA replication, through duplex DNA intermediates, occurs a short time later. VP1 and VP2 self-associate to form capsids, and predominantly minus-sense viral ssDNA is packaged. Additional functions defined for NS1 and NS2 include modulation of transcription from cellular promoters and cytostatic and cytotoxic functions presumed to down-regulate cellular processes not required for viral replication (Brandenberger et al. 1990; Legendre and Rommelaere, 1992). Replication typically leads to cell lysis and the release of newly synthesized virions. For nonproductive infection, restrictive infection has been demonstrated for two MVM strains. Thus, MVMp and MVMi undergo productive infection in fibroblasts and lymphocytes, respectively, but the growth of each strain is restricted in the reciprocal cell type. Abortive infection may occur when MVM
infects cells of a different species. Significant viral transcription and protein synthesis occur with limited or no genomic DNA replication or production of infectious virus. Cryptic infection may occur in nonreplicating cells that are normally capable of supporting productive infection. Since nonreplicating cells do not proceed through S phase, virus replication is inhibited until the cell is stimulated to divide. Because nonproductive infections have been demonstrated only for MVM and only in vitro, their influence on the health or experimental performance of mice with natural parvovirus infections is currently unknown. The unique replication strategy of parvoviruses has been utilized to characterize viral tropisms and replication in vivo. Single-stranded genomic probes of either plus-sense, which detect double-stranded (ds) DNA replicative intermediates and ssDNA, or minus-sense, which detect dsDNA replicative intermediates and RNA transcripts, can distinguish between actively infected cells in which replication and viral transcription are occurring from those in which only genomic DNA or dsDNA are present (Bloom et al. 1989; Jacoby et al. 1995).
IV.
ANTIGENIC AND PHYSICOCHEMICAL PROPERTIES
Sequence analysis of the MPV genome indicates that regions encoding NS proteins are strongly homologous to those of MVM (Ball-Goodrich and Johnson, 1994). This homology is conserved at the protein and antigenic level, since immune sera to MVM NS proteins also detect MPV NS proteins. NS1 and NS2 proteins are equivalent in size and ratio in both MPV- and MVM-infected cells. The capsid proteins, which are more divergent, provide serotype specificity. Parvoviruses are highly resistant to many classical methods of inactivation such as dessication, heating, and exposure to lipid solvents or chaotropic agents, including urea and sodium dodecyl sulfate (Harris et al. 1974; Tattersall and Cotmore, 1986). This feature is due to their small size, stable construction, and lack of a lipid envelope. A recent study showed that exposure to wet heat (90ºC) for at least 10 minutes was required to inactivate MVM; infectious virus was present in samples treated for 1 hour at temperatures up to 80ºC (Boschetti, 2003). This study also showed that NaOH concentrations 0.1M (pH 12.8) were required for efficient (1 minute) inactivation of filtered (dissociated) MVM. However, MVM aggregates were resistant even to prolonged (60 minute) NaOH treatment. Parvoviruses appear to be inactivated by formalin and oxidizing agents such as sodium hypochlorite and sodium chlorite (Saknimit et al. 1988). Although these studies assessed only rat and canine parvoviruses, there is no reason to suspect that these agents would not be equally effective against murine parvoviruses. There does not appear to be documented information addressing the virucidal capacity of chlorine dioxide, which is widely used as an
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oxidizing disinfectant in animal resource facilities, nor did we find information on the virucidal effectiveness of oxidizing agents during common application such as by spray or wipe.
V.
CLINICAL SIGNS
Natural parvovirus infections of mice, for all intents and purposes, are clinically silent, regardless of host age. Therefore, the most common evidence of infection is seroconversion, which occurs, in immunocompetent mice, 7 to 14 days after initial exposure to the virus (Jacoby et al. 1995). The absence of clinical signs also is generally characteristic for experimental infections, even in immunodeficient mice. However, experimental infection of neonatal BALB/c, SWR, SJL, CBA, and C3H mice with MVM(i) has led to morbidity and mortality due to internal hemorrhage and/or accelerated involution of hepatic hematopoiesis (Brownstein et al. 1991). An early study demonstrated granuloprival cerebellar hypoplasia after combined intracerebral and intraperitoneal inoculation of neonates with MVM, and also reported cerebellar hypoplasia in several contact-exposed control mice (Kilham and Margolis, 1970). More recently, the intranasal inoculation of MVMi into newborn BALB/c mice caused neurological signs associated with active replication in proliferative centers of the cerebrum and in young cerebellar neurons (Ramirez et al. 1996). Nevertheless, the incidence of brain lesions in naturally infected mice should be considered extremely low.
VI.
EPIZOOTIOLOGY
The mouse appears to be the primary and sole natural host of murine parvoviruses. Although a large proportion of rat sera tested by Parker contained low-titer reactivity to MVM (Parker et al. 1970), it disappeared after kaolin treatment, illustrating that the reactions were nonspecific. MVM can infect rats and hamsters inoculated parenterally or during fetal development, but such conditions are far removed from natural exposure (Kilham and Margolis, 1970, 1971). An MPV-like virus has been detected in hamsters, but its potential for cross-infection of mice is unknown (Besselsen et al. 1996, 1999). The prevalence of murine parvoviruses is attributable to their infectivity, persistence (at least for MPV) in infected mice, resistance to environmental inactivation, and contamination of biologicals used for animal inoculation (Parker et al. 1970; Nicklas et al. 1993). A U.S. survey conducted in 1997 (Jacoby and Lindsey, 1997) found that parvovirus infections were present in about 25% of barrier-maintained mouse colonies and 45% of conventional colonies among leading U.S. biomedical research institutions. No comparable surveys of commercial colonies have been reported, but there is anecdotal evidence
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that they endure occasional outbreaks (S. Compton, personal communication). Several European laboratories have reported parvoviruses as common contaminants in specimens of mouse origin (Kraft and Meyer, 1990; Nicklas, 1993). Natural murine parvovirus infections appear to result from oronasal exposure, but parenteral inoculation of contaminated biologicals, such as transplantable tumors, are potential sources of infection. Oral exposure facilitates primary replication in the small intestine followed by systemic spread. Viral DNA can be detected in kidney, intestine (and feces), and lung during acute infection (Brownstein et al. 1991; Jacoby et al. 1995; Smith et al. 1993) suggesting that excretion occurs in urine, feces, and, potentially, exhaled air. These results also imply that the alimentary tract is a portal for viral entry. Significant excretion through the respiratory tract seems doubtful, since viruspositive cells are sparse in this organ system. Therefore, the transmission of infection probably occurs primarily by ingestion of contaminated food, bedding, or feces. Further, the comparatively slow rate of cage-to-cage spread suggests that transmission is primarily by contaminated fomites. MVM appears to produce self-limiting infection in infant and adult mice. Thus, no infection or transmission was detected beyond 4 weeks after oronasal inoculation of neonatal or adult mice (Smith, 1983; Smith and Paturzo, 1988). In contrast, MPV appears to persist among infant and adult mice after the development of humoral immunity. For example, mice inoculated neonatally transmitted virus to cagemates for up to 6 weeks, and mice inoculated as young adults transmitted infection for up to 4 weeks (Smith et al. 1993), despite the onset of seroconversion 7–10 days after inoculation. There is some corollary data from a natural enzootic suggesting that juvenile animals present a greater hazard than older mice for spread of infection (Shek et al. 1998). Further, molecular hybridization studies indicate that MPV DNA can persist in lymphoid tissues of adult mice for at least 9 weeks (Jacoby et al. 1995) whereas no evidence of MVM was found after 3 weeks (Jacoby et al. unpublished data). Preliminary evidence from mouse antibody production tests using samples from naturally infected mice imply that MPV DNA-positive lymphoid tissues harbor infectious virus (Shek et al. 1998). We add the possibility that although continuous contact exposure to infected animals or soiled bedding induces seroconversion within 3 weeks, periodic exposure to low doses of virus may extend this interval (S. Compton, unpublished data). Suckling mice in enzootically infected colonies are protected from infection with homologous virus by maternally acquired immunity, which generally decays within 2–3 months. Subsequent infection of such mice as adults elicits active immunity. However, immunity to MVM may not confer cross-immunity to MPV (Hansen et al. 1999). There is no evidence that murine parvoviruses are transmitted in utero after infection of dams by a natural route. As noted above, parvoviruses resist environmental inactivation, which prolongs risks for the spread and duration of epizootic and enzootic infections.
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VII.
PATHOLOGY AND PATHOGENESIS
Despite in vitro evidence that productive parvovirus replication leads to cell death, necrosis is not an obvious feature of active infection in mice. This fact, coupled with low sensitivity of immunostaining, has led to reliance on in situ hybridization (ISH) for contemporary pathogenesis studies. Although MPV and MVM infections, especially in adult mice, are similar, they are described separately to emphasize several differences.
A.
MPV
As noted above, MPV infection appears to begin in the small intestine, since parvovirus-specific ISH signal can be detected among enterocytes in the small intestine within several days after oral inoculation of virus (Jacoby et al. 1995) (Fig. 4-2). However, virus-positive cells rapidly become more prominent among mononuclear cells and capillary (or lymphatic) endothelium in the lamina propria. Acute infection extends to Peyer’s
Fig. 4-3 Cervical lymph node from an adult BALB/c mouse 6 days after oronasal inoculation with MPV. In situ hybridization demonstrates virus-positive cells at the margin of a germinal center.
Fig. 4-2 Duodenal villus in an adult BALB/c mouse 6 days after oronasal inoculation with MPV. In situ hybridization demonstrates virus-positive cells (arrows) among enterocytes and in the lamina propria.
patches, lymph nodes, thymus, spleen, lung, kidney, and liver (Figs. 4-3 and 4-4). Thus, lymphocytotropism occurs early in infection, and may contribute to viremic dissemination. This also suggests that at least some virus-positive cells in intestinal lamina propria are intra-epithelial lymphocytes, a notion compatible with the in vitro tropism of MPV for T cells. Seroconversion during the second week of infection marks a gradual decrease in virus-positive cells in all infected tissues. However, positive cells can be detected in lymph nodes and splenic white pulp through at least 9 weeks, primarily among germinal centers (Figs. 4-5 and 4-6). It is unclear whether virus targets specific lymphoid cell subsets in vivo. The random distribution of virus-positive cells during acute infection, which includes paracortical regions of lymph nodes and periarteriolar lymphoid sheaths in spleen, followed by the localization of virus during persistent infection suggests that multiple lymphoid cell types can be infected. This possibility also is supported by the characteristics of immune dysfunctions associated with MPV infection (see below). Infection of hematopoietic tissue (splenic red pulp) is sparse and appears limited to early stages
Fig. 4-4 Distribution of virus-positive tissues during MPV infection of adult BALB/c mice as detected by in situ hybridization using a random-primed probe.
of infection. Thus, hematopoietic cells are not conspicuous targets as they can be in MVM infection of neonates or immunodeficient adults (Segovia et al. 1999). During persistent infection, lymphoid tissues contain virionpositive cells but no evidence of viral transcripts or dsDNA. This suggests that virus is quiescent, or that replication is occurring at levels undetected by ISH. Therefore, signal amplification strategies such as polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR) may be required to detect viral transcription and replication during persistent infection.
B.
Fig. 4-5 Germinal center of a mesenteric lymph node demonstrating viruspositive cells 9 weeks after oronasal inoculation with MPV. In situ hybridization.
MVM
MVM infection in adult mice reveals patterns similar to those for MPV; that is, enterotropism followed by lymphocytotropism (Jacoby et al. unpublished data). However, hybridization signal is rarely detected after 3 weeks, consistent with the concept that MVM is a self-limiting infection in immunocompetent mice (Smith, 1983). As noted above, neonatal mice of several strains are subject to hemorrhagic lesions after experimental inoculation of MVMi (Brownstein et al. 1991). These include infarction of the renal papilla in BALB/c,
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Survey of tissues from orally inoculated adult BALB/cByJ mice for MPV by in situ hybridization using a 32P probe labeled by random-priming.
Fig. 4-6
Distribution of tissues in MPV-infected mice containing cells labeled with a minus-sense hybridization probe and indicative of viral replication
SWR, SJL, CBA, and C3H mice. Further, DBA/2 neonates can develop intestinal hemorrhage and accelerated involution of hepatic hematopoiesis. In contrast, C57BL/6 neonates appear resistant to vascular disease. MVMi replicates equally well in resistant (C57BL/6) and susceptible (C3H) mice, but in situ hybridization and immunohistochemistry have shown that fewer target cells (endothelium, lymphocytes, and erythropoietic precursors in the liver) are infected in resistant mice. One explanation offered for these findings is that genetic susceptibility facilitates the accumulation of NS proteins, which are known to be cytotoxic in vitro. This possibility was supported by studies with an MVMi NS2 mutant that revealed that the pathogenicity of MVMi depended on expression of NS2 (Brownstein et al. 1992). Companion studies with intertypic recombinants between MVMi and MVMp indicated that the pathogenicity of MVMi also depended on allotropic determinant(s) encoded by the capsid genes. This region of the MVMi
genome, which determines tropism in vitro, also appears to determine lymphotropism and endotheliotropism in vivo. Kimsey and coworkers compared the effects of MVMp and MVMi (Kimsey et al. 1986) and found that neither strain affected T cell function in adult mice. However, MVMi caused disseminated infection, runting, and mild immunodeficiency in mice inoculated as neonates, effects attributed to lymphocytotropism. MVM appears to be more pathogenic for hematopoietic tissue than MPV. In vitro and in vivo studies complementary to those described above for neonates have demonstrated infection of hematopoietic stem and committed progenitor cells by MVMi (Lamana et al. 2001; Segovia et al. 1995, 2003). MVMi replicated in primary myeloid cells of long-term bone marrow cultures, resulting in high titers of infectious virus and acute myelosuppression in the cultures. In vivo infection of newborn BALB/c mice resulted in a significant decrease in
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femoral and splenic cellularity and delayed myeloid depression. In immunodeficient SCID mice, MVMi infection resulted in suppression of myeloid and erythroid progenitors in the bone marrow and lethal leukopenia (Segovia et al. 1999). Hematopoietic tropism aside, MPV and MVM share a predilection for the intestine and lymphoid tissues, but MPV infection persists significantly after serocoversion, whereas MVM infection appears to be more self-limiting. These findings increase concerns not only about the epizootiology of MPV infection, but also about long-term influences of MPV on immune function.
VIII.
DIAGNOSIS
The simple structure of rodent parvoviruses has stimulated development of valuable reagents to detect infection. These include generic and serotype-specific antigens for use in serological tests, antibodies to viral proteins for use in immunohistochemistry, PCR primers for detection of viral DNA in infected tissues and feces, and strand-specific molecular probes that help distinguish sites of active viral replication (Jacoby et al. 1995; Besselsen et al. 1995, 1998). In practical terms, however, the detection of parvovirus infection continues to rely heavily on serologic assays for anti-parvovirus antibodies. Generic serological tests capitalize on extensive cross-reactivity among conserved (NS1) antigens. Therefore, MVM NS proteins can be used to detect antibodies to both MVM and MPV. This feature is exploited in immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) using virus-infected cells (Riley et al. 1996; Smith, 1985; Smith et al. 1982). In addition, the NS1 ELISA utilizes recombinant (r) MVM NS1 protein as antigen and serves as a useful screening test for parvovirus infection (Riley et al. 1996). Although ELISAs using whole parvovirus virions or rNS1 as antigen are comparably specific, the latter appears to be more sensitive for generic detection of antibodies to murine parvovirus (Riley et al. 1996). However, the rNS1 ELISA may give false negative reactions if NS proteins are not elicited uniformly among strains and ages of infected mice (Besselsen, 2000). VP2 antigens, because they are serotype-specific, are essential for the serological differentiation of MVM infection from MPV infection. In this context, recently developed ELISAs utilizing recombinant MPV and MVM VP2 proteins or empty virions offer sensitivity and specificity that render the HAI assay outmoded. (Ball-Goodrich et al. 2002; Livingston et al. 2002). While current serological assays to detect and differentiate murine parvoviruses have great diagnostic value, we add a cautionary note: They may not be reliably applicable to newly recognized strains. This concern is exemplified by a preliminary report that a putative antigenically distinct strain of MPV, designated MPV-2, may not be detected by serological assays for MPV-1 (Dhawan et al. 2004).
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The mouse antibody production (MAP) test can be used to detect parvovirus infections in mouse tissues or tissue products, but it is time-consuming and expensive (de Souza and Smith, 1989). Virus isolation has similar drawbacks, but it is a valuable option to confirm MVM infection. Established cell lines frequently used for MVM isolation include A9 murine fibroblasts, NB324K human kidney cells, C6 rat glial cells, and BHK-21 cells (Tattersall and Bratton, 1983; Cotmore and Tattersall, 1987). MPV, as mentioned previously, grows efficiently only in selected T cell lines and clones. Tissue explantation culture, where small segments of tissue harvested from suspect hosts are cultivated for several weeks to allow amplification of virus prior to inoculation and testing of indicator monolayer cultures, is an additional option to detect MVM (Smith and Paturzo, 1988). Neither this option or co-cultivation of lymphoid cells has been tested systematically to detect MPV infection. More recently, molecular diagnostics have replaced MAP testing and virus isolation for specific and sensitive detection of parvovirus infection. A generic PCR assay, which amplifies a conserved portion of the NS1 gene, detects MVM and MPV (Riley et al. 1996). Virus-specific PCR assays amplify gene segments within the capsid protein genes (Ball-Goodrich and Johnson, 1994; Besselsen et al. 1995; Redig and Besselsen, 2001; Yagami et al. 1995). Both assays are effective in assessing feces as well as tissues from infected animals for parvoviral DNA. Testing of DNA from mesenteric lymph nodes by PCR can be especially useful because the nodes are a consistent site of acute and persistent infection. There also is recent evidence that PCR assays can be used to monitor ventilated cage racks for the presence of parvovirus-infected mice by testing filters placed strategically in the exhaust plenum for parvoviral DNA (Compton et al. 2004). While it is too soon to conclude that such monitoring will play a major role in the early detection of parvovirus infection, this approach illustrates emerging possibilities for molecular diagnosis in rodent preventive medicine. ISH with strand-specific probes is valuable for detecting genomic and replicative viral DNA in paraffinembedded tissues (Jacoby et al. 1995). Immunostaining of paraffin-embedded tissues has, thus far, proved less reliable for histologic evaluation of infection.
IX.
EFFECTS ON RESEARCH
The effects of murine parvoviruses on research are attributable to their predilection for mitotically active cells. The prevalence of clinically silent infections implies that interference will most likely be manifested as distortions of biological responses that depend on cell proliferation. Their historical association with transplantable neoplasms exemplifies this phenomenon, but the clearest demonstrations of the potential impact of parvovirus interference pertain to immune dysfunction.
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A.
MVM
Early reports showed that MVMi suppressed lymphocyte proliferation in vitro in response to mitogens, abrogated the generation of cytolytic T cells in primary mixed lymphocyte cultures, and inhibited T cell–dependent B cell responses in vitro (Bonnard et al. 1976: Engers et al. 1981; McMaster et al. 1981). These effects were attributed to virus-induced cytolysis. However, as noted previously, the immunosuppressive effects of MVMi were mild in vivo and observed only in infant mice (Kimsey et al. 1986). Further, MVMi has the specific potential to interfere with the hematopoietic system in newborn or severely immunocompromised mice. MVMp, which is not lymphocytotropic, was not immunomodulatory in vitro or in vivo. MVM contamination of transplantable neoplasms is a demonstrable risk. Therefore, infection can be introduced to a colony through inoculation of contaminated cell lines. Failure to establish long-term cell cultures from infected mice or a low incidence of tumor “takes” should alert researchers to the possibility of MVM contamination.
B.
MPV
MPV can interfere with the ability of cloned T cells to thrive and proliferate (McKisic et al. 1993). Further characterization of the immunosuppressive properties of MPV showed that infection reduced both cytokine- and antigen-induced T cell proliferation in vitro, apparently without affecting cell viability (McKisic et al. 1996). These results suggested that infection need not be patently lytic to alter effector functions. Virus replication was essential to perturb immune functions since heat inactivation abrogated these effects. These experiments also suggested that MPV infection impaired a signaling event common to both cytokine- and antigen-stimulated proliferation. MPV infection of adult mice can cause immune dysfunction that can persist after seroconversion. Both CD4+ and CD8+ T cell–mediated responses are altered during acute MPV infection. Modulation of ovalbumin-induced CD4+ T cell proliferation was observed in BALB/c mice as early as 3 days after virus inoculation, although this function was restored within 2 to 3 weeks (McKisic et al. 1996). Additionally, MPV appears to modulate immune responses differentially, depending on the anatomical source of T cells. Specifically, the proliferative responses of splenic and popliteal lymph node cells obtained from infected, ovalbumin-primed mice were suppressed, while proliferation of mesenteric lymph node cells was augmented. Inoculation of BALB/c mice with MPV resulted in abnormal CD8+ T cell rejection of tumor and skin allografts for at least 3 weeks post-infection (McKisic et al. 1996, 1998). Adult BALB/c mice infected 1, 2, or 3 weeks prior to receiving an inoculum of sarcoma I cells developed smaller tumors that
were rejected more quickly compared to uninfected controls. Similarly, MPV infection, either before or after skin transplantation, potentiated the rejection of full thickness allografts. The alteration of tumor and skin graft rejection was not due to virus infection of the tumor or the graft. Also, the specific proliferative and cytolytic capacity of alloantigen-reactive lymphocytes from tumor- and skin-graft-sensitized infected mice was diminished. Therefore, MPV appeared to accelerate graft rejection while inducing dysfunction of alloreactive T cells. Surprisingly, syngeneic grafts on MPV-infected mice were rejected at the same rate as allogeneic grafts, whereas mice inoculated with MVMi, MVMp, or heat-inactivated MPV-1 did not reject syngeneic grafts. Depletion of T lymphocytes prior to grafting or passive immunization with anti-MPV immune serum prevented the rejection of syngeneic grafts. Thus, MPV infection of skin-grafted mice appeared to disrupt normal mechanisms of peripheral tolerance. Modulation of immune responses by MPV raises concerns about the full potential for such effects on immunologic research using mice. It is worth noting that the course and effects of parvovirus infection have not been examined significantly in genetically engineered mice, many strains of which are known to be immunologically dysfunctional. Therefore, the true impact of infection is potentially much greater than current perceptions imply.
X.
CONTROL AND PREVENTION
Because MVM does not persist in immunocompetent mice, control and elimination should employ quarantine for at least 1 month, combined with thorough disinfection of the environment. Quarantine also assumes that MPV infection has been ruled out. If serotyping of virus remains problematic, i.e., if MPV infection remains a possibility, more stringent approaches may be preferable. Elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced. For mice that are not easily replaced, persistent infection coupled with low risk for transplacental transmission favor Cesarean rederivation or embryo transfer as attractive options to eliminate infection. Thorough decontamination is applicable to all parvovirus outbreaks because of the environmental stability of these viruses. Chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent. While fogging contaminated rooms with formalin may be useful, it also presents increased risk for inadvertent chemical exposure. We have been encouraged by the effectiveness of sequential washing of surfaces, followed by chlorine dioxide and several days of drying. Prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice.
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REFERENCES Ball-Goodrich, L.J., and Johnson, E.A. (1994). Molecular characterization of a newly recognized mouse parvovirus. J. Virol. 68, 6476–6486. Ball-Goodrich, L.J., Hansen, G., Dhawan, R., Paturzo, F.X., and Vivas-Gonzalez, B.E. (2002). Validation of an enzyme-linked immunosorbent assay for detection of mouse parvovirus infection in laboratory mice. Comp. Med. 52, 160–166. Besselsen, D.G. (1998). Detection of rodent parvoviruses by PCR. Meth. Mol. Biol. 92, 31–37. Besselsen, D.G., Besch-Williford, C.L., Pintel, D.J., Franklin, C.L., Hook, R.R., and Riley, L.K. (1995). Detection of newly recognized rodent parvoviruses by PCR. J. Clin. Microbiol. 33, 2859–2863. Besselsen, D.G., Gibson, S.V., Besch-Williford, S.C.L., Purdy, G.A., Knowles, R.L., Wagner, J.E., Pintel, D.J., Franklin, C.L., Hook, R.R., and Riley, L.K. (1999). Natural and experimentally induced infection of Syrian hamsters with a newly recognized parvovirus. Lab Anim. Sci. 49, 305–312. Besselsen, D.G., Pintel, D.J., Purdy, G.A., Besch-Williford, C.L., Franklin, C.L., Hook, R.R., and Riley, L.K. (1996). Molecular characterization of newly recognized rodent parvoviruses. J. Gen. Virol. 77, 899–911. Besselsen, D.G., Wagner, A.M., and Loganbill, J.K. (2000). Effect of mouse strain and age on detection of mouse parvovirus by use of serologic testing and polymerase chain reaction analysis. Comp. Med. 50, 498–502. Bloom, M.E., Alexandersen, S., Mori, S., and Wolfinbarger, J.B. (1989). Analysis of parvovirus infections using strand-specific hybridization probes. Virus Res. 14, 1–26. Bonnard, G.D., Manders, E.K., Campbell, D.A., Herberman, R.B., and Collins, M.J. (1976). Immunosuppressive activity of a subline of the mouse EL-4 lymphoma. J. Exp. Med. 143, 187–205. Boschetti, N., Wyss, K., Mischler, A., Hostettler, T., and Kempf, C. (2003). Stability of minute virus of mice against temperature and sodium hydroxide. Biologicals. 31, 181–185. Brandenberger, A., Legendre, D., Avalosse, B., and Rommelaere, J. (1990). NS-1 and NS-2 may act synergistically in the cytopathogenicity of parvovirus MVM(p). Virology 174, 576–584. Brownstein, D.G., Smith, A.L., Jacoby, R.O., Johnson, E.A., Hansen, G., and Tattersall, P. (1991). Pathogenesis of infection with a virulent allotropic variant of minute virus of mice and regulation by host genotype. Lab. Invest. 65, 357–364. Brownstein, D.G., Smith, A.L., Johnson, E.A., Pintel, D.J., Naeger, L.K., and Tattersall, P. (1992). The pathogenesis of infection with minute virus of mice depends on expression of the small nonstructural protein NS2 and on the genotype of the allotropic determinants VP1 and VP2. J. Virol. 66, 3118–3124. Compton, S.R., Homberger, F.X., Paturzo, F.X., and MacArthur Clark, J. (2004). Efficacy of three microbiological monitoring methods in a ventilated cage rack. Comp. Med. 54, 382–392. Cotmore, S.F., and Tattersall, P. (1987). The autonomously replicating parvoviruses of vertebrates. Adv.Virus Res. 33, 91–174. Crawford, L.V. (1966). A minute virus of mice. Virology 26, 602–612. Cross, S.S., and Parker, J.C. (1972). Some antigenic relationships of the murine parvoviruses: minute virus of mice, rat virus, H-1 virus. Proc. Soc. Exp. Biol. Med. 139, 105–108. de Souza, M., and Smith, A.L. (1989). Comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses. J. Clin. Microbiol. 27, 185–187. Dhawan, R., Henderson, K.S., Cowley, J.P., Wundereich, M.L., Blank, W.A., and Shek, W.R. (2004). Serological and nucleic acid sequence characterization of a newly identified mouse parvovirus strain. Cont. Topics Lab. Anim. Med. 43, 43 (Abstract). Engers, H.D., Louis, J.A., Zubler, R.H., and Hirt, B. (1981). Inhibition of T cell-mediated functions by MVM(i), a parvovirus closely related to minute virus of mice. J. Immunol. 127, 2280–2285.
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Hansen, G.M., Patruzo, F.X., and Smith, A.L. (1999). Humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus. Lab. Anim. Sci. 49, 380–384. Harris, R.E., Coleman, P.H., and Morahan, P.S. (1974). Stability of minute virus of mice to chemical and physical agents. Appl. Microbiol. 28, 351–354. Jacoby, R.O., and Ball-Goodrich, L.J. (1995). Parvovirus infections of mice and rats. Sem. Virol. 6, 329–333. Jacoby, R.O., Ball-Goodrich, L.J., Besselsen, D.G., McKisic, M.D., Riley, L.K., and Smith, A.L. (1996). Rodent parvovirus infections. Lab. Anim. Sci. 46, 370–380. Jacoby, R.O., Johnson, E.A., Ball-Goodrich, L.J., Smith, A.L., and McKisic, M.D. (1995). Characterization of mouse parvovirus infection by in situ hybridization. J. Virol. 69, 3915–3919. Jacoby, R.O., and Lindsey, J.R. (1997). Health care for research animals is essential and affordable. FASEB Journal 11, 609–614. Jacoby, R.O., Smith, A.L., and Ball-Goodrich, L.J. Unpublished data. Kilham, L., and Margolis, G. (1970). Pathogenicity of minute virus of mice (MVM) for rats, mice and hamsters. Proc. Soc. Exp. Biol. Med. 133, 1447–1452. Kilham, L., and Margolis, G. (1971). Fetal infection of hamsters, rats and mice induced with minute virus of mice (MVM). Teratology 4, 43–62. Kilham, L., and Olivier, L. (1959). A latent virus of rats isolated in tissue culture. Virology 7, 428–437. Kimsey, P.B., Engers, H.D., Hirt, B., et al. (1986). Pathogenicity of fibroblastand lymphocyte-specific variants of minute virus of mice. J. Virol. 59, 8–13. Kraft, V., and Meyer, B. (1990). Seromonitoring in small laboratory animal colonies. A five year survey: 1984–1988. Z. Versuchstierkd. 33, 29–35. Lamana, M.L., Albella, B., Bueren, J.A., and Segovia, J.C. (2001). In vitro and in vivo susceptibility of mouse megakaryocyte precursors to strain I of parvovirus minute virus of mice. Exp. Hematol. 29, 1303–1309. Legendre, D., and Rommelaere, J. (1992). Terminal regions of the NS protein of the parvovirus minute virus of mice are involved in cytotoxicity and promoter trans inhibition. J. Virol. 66, 5705–5713. Livingston, R.S., Besselsen, D.G., Steffen, E.K., Besch-Williford, C.L., Franklin, C.L., and Riley, L.K. (2002). Serodiagnosis of mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirusexpressed VP2 proteins. Clin. Diagn. Lab. Immunol. 5, 1025–1031. McKisic, M.D., Lancki, D.W., Otto, G., Padrid, R., Snook, S., Cronin, D.C., Lohmar, P.D., Wong, T., and Fitch, F.W. (1993). Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells. J. Immunol. 150, 419–428. McKisic, M.D., Macy, J.D., Jr., Delano, M.L., Jacoby, R.O., Paturzo, F.X., and Smith, A.L. (1998). Mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic graft rejection. Transplantation 65, 1436–1446. McKisic, M.D., Paturzo, F.X., and Smith, A. L. (1996). Mouse parvovirus infection potentiates rejection of tumor allografts and modulates T cell effector functions. Transplantation 61, 292–299. McMaster, G.K., Beard, P., Engers, H.D., and Hirt, B. (1981). Characterization of an immunosuppressive parvovirus related to the minute virus of mice. J. Virol. 38, 317–326. Nicklas, W., Kraft,V., and Meyer, B. (1993). Contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses. Lab. Anim. Sci. 43, 296–300. Parker, J.C., Collins, M.J., Cross, S.S., and Rowe, W.P. (1970). Minute virus of mice. II. Prevalence, epidemiology, and occurrence as a contamination of transplanted tumors. J. Natl. Cancer Inst. 45, 305–310. Parrish, C.R. (1990). Emergence, natural history, and variation of canine, mink, and feline parvoviruses. Adv. Virus Res. 38, 403–450. Ramirez, J.C., Fairen, A., and Almendral, J.M. (1996). Parvovirus minute virus of mice strain I multiplication and pathogenesis in newborn mouse brain are restricted to proliferative areas and to migratory cerebellar young neurons. J. Virol. 70, 8109–8116.
4. PARVOVIRUSES
Redig, A.J., and Besselsen, D.G. (2001). Detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays. Comp. Med. 51, 326–331. Riley, L.K., Knowles, R., Purdy, G., Salome, N., Pintel, D., Hook, R.R., Franklin, C.L., and Besch-Williford, C.L. (1996). Expression of recombinant parvovirus NS1 protein by a baculovirus and application to serologic testing of rodents. J. Clin. Microbiol. 34, 440–444. Saknimit, M., Inatsuki, I., Sugiyama, Y., and Yagami, K. (1988). Virucidal efficacy of physico-chemical treatments against coronaviruses and parvoviruses of laboratory animals. Jikken Dobutsu 37, 341–345. Segovia, J.C., Bueren, J.A., and Almendral, J.M. (1995). Myeloid depression follows infection of newborn mice with the parvovirus minute virus of mice (strain I). J. Virol. 69, 3229–3232. Segovia, J.C., Gallego, J.M., Bueren, J.A., and Almendral, J.M. (1999). Severe leukopenia and dysregulated erythropoiesis in SCID mice persistently infected with the parvovirus minute virus of mice. J. Virol. 73, 774–784. Segovia, J.C., Guenechea, G., Gallego, J.M., Almendreal, J.M., and Bueren, J.A. (2003). Parvovirus infection suppresses long-term repopulating hematopoietic cells. J. Virol. 77, 8495–8503. Shek, W.R., Paturzo, F.X., Johnson, E.A., Hansen, G.M., and Smith, A.L. (1998). Characterization of mouse parvovirus infection among BALB/c mice from an enzootically infected colony. Lab. Anim. Sci. 48, 294–297. Smith, A.L. (1983). Response of weanling random-bred mice to inoculation with minute virus of mice. Lab. Anim. Sci. 33, 37–39. Smith, A.L. (1985). An enzyme immunoassay for identification and quantification of infectious murine parvovirus in cultured cells. J. Virol. Meth. 11, 321–327. Smith, A.L. Unpublished data.
103 Smith, A.L., Ball-Goodrich, L.J., and Jacoby, R.O. Unpublished data. Smith, A.L., Davis, D.E.M., Gardener, E.P., Paturzo, F.X., and Kornblatt, A.N. (1982). Comparison of methods for detection of serum antibody to minute virus of mice. Lab. Anim. Sci. 32, 417. Smith, A.L., Jacoby, R.O., Johnson, E.A., Paturzo, F.X., and Bhatt, P.N. (1993). In vivo studies with an “orphan” parvovirus of mice. Lab. Anim. Sci. 43, 175–182. Smith, A.L., and Paturzo, F.X. (1988). Explant cultures for detection of minute virus of mice in infected mouse tissue. J. Tissue Cult. Meth. 11, 45–47. Tattersall, P., and Bratton, J. (1983). Reciprocal productive and restrictive virus-cell interactions of immunosuppressive and prototype strains of minute virus of mice. J. Virol. 46, 944–955. Tattersall, P., and Cotmore, S.F. (1986). The rodent parvoviruses. In Viral and Mycoplasmal Infections of Rodents: Effects on Biomedical Research (P.N. Bhatt, R.O. Jacoby, H.C. Morse, and A.E. New, eds.), pp. 305–348. Academic Press, Orlando, FL. Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Carstens, E., Estes, M., Lemon, S., Maniloff, J., Mayo, M.A., McGeoach, D., Pringle, C.R., and Wickner, R.B. (eds). (2000). Virus taxonomy. Seventh report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego. Ward, D.C., and Tattersall, P. (1982). Minute virus of mice. In The Mouse in Biomedical Research. (H.L. Foster, J.D. Small, and J.G. Fox, eds.), Vol. II, pp. 313–334. Academic Press, New York. Yagami, K., Goto, Y., Ishida, J., Ueno, Y., Kajiwara, N., and Sugiyama, F. (1995). Polymerase chain reaction for detection of rodent parvoviral contamination in cell lines and transplantable tumors. Lab. Anim. Sci. 45, 326–328.
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Chapter 5 Polyoma Viruses Thomas L. Benjamin
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The Polyoma Virus-Mouse System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Virus-Cell Interactions in Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Productive and Nonproductive Infections . . . . . . . . . . . . . . . . . . . . 2. Cell Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Natural and Laboratory Infections of Mice . . . . . . . . . . . . . . . . . . . . . . III. The Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Polyoma Tumor Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Tissue Interactions in Tumor Formation . . . . . . . . . . . . . . . . . . . . . . . . IV. Polyoma Tumorigenesis as a General Model of Cancer . . . . . . . . . . . . . . . A. Initiation and Early Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Genomic Instability and Further Progression . . . . . . . . . . . . . . . . . . . . C. Overcoming Host Defense Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . 1. Apoptotic Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Invasion and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Viral Determinants—Structural Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . A. VP1 and Self-Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Virus-Like Particles and Gene Delivery . . . . . . . . . . . . . . . . . . . . . . B. Determinants of Pathogenicity in VP1 . . . . . . . . . . . . . . . . . . . . . . . . . C. Pathogenicity Determinants in the Sialic Acid Binding Pocket of VP1 D. The Minor Structural Proteins VP2 and VP3 . . . . . . . . . . . . . . . . . . . . VI. Viral Determinants—Tumor Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Primary Structures and Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Molecular Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Genetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Determinants of Pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Viral Regulatory Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Host Determinants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cell Receptors and Machinery of Virus Uptake . . . . . . . . . . . . . . . . . . B. Effects of the Host Genetic Background . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
106 107 107 107 108 108 109 109 109 110 110 111 111 111 112 112 113 113 113 113 114 116 116 116 118 119 120 121 122 122 123
Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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IX. Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. SV40 and JCV T Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Polyoma T Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Use of Polyoma Virus Regulatory Sequences in Transgene Expression X. The Pneumotropic Virus of Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Perspectives and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
“Under normal conditions, the mouse polyoma virus remains perfectly harmless in its natural carrier host . . . yet has a formidable pathogenic potential . . . and may be prompted to induce the development of tumors.” Ludwik Gross in Oncogenic Viruses, 2nd ed, Pergamon Press, 1970
Important contributions to cancer research have come through studies of oncogenic viruses. Viruses play a role in 15–20% of human cancers worldwide. These include members of the papilloma, hepadna, herpes, and retrovirus groups (Blattner 1999; Eckhart 1998; Wong et al. 2002; Zur Hausen 1991). Human cancers of nonviral etiology arise through genetic alterations affecting many of the same cellular processes and pathways as those disrupted by oncogenic viruses. DNA-containing oncogenic viruses alter pathways that regulate cell growth and survival in order to promote their own replication. Mutations in elements of these pathways arise somatically in many types of cancer. Such mutations may also be present in the germline leading to inherited predisposition to the development of certain cancers. Research on oncogenic viruses has led to the discovery and elucidation of several of these important pathways. The mouse has long been a subject of basic cancer research. Extensive histories document the use of the mouse in studies of viruses along with radiation and chemicals as carcinogenic agents. These experimental cancers in the mouse derive relevance to human cancer from the similarities of many types of cancer in the two species, from the extensive homology between the mouse and human genomes, and from advantages of working with the mouse from the genetic standpoint (Balmain 2002; Balmain and Harris 2000). Mouse models have originated from selective breeding of naturally occurring cancers (e.g., the so-called “spontaneous” leukemias [Gross 1983a]), from phenotypic screens following germline mutagenesis (Moser et al. 1990; Su et al. 1992), and from the construction of transgenic and knockout mice involving various viral oncogenes, tumor suppressor genes, and protooncogenes. Among the many agents that induce cancer in mice, none acts more broadly or rapidly than the mouse polyoma virus (Py). This virus was named for its extraordinary ability to induce multiple tumors following inoculation into newborn mice
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126 126 127 128 128 129 130 130
(Eddy 1982; Gross 1983b, 1953; Stewart 1960; Stewart et al. 1958). Py is the oldest representative and prototype of the polyoma virus group. This group includes viruses of rodent and avian hosts as well as several viruses of nonhuman primates and humans. The primate polyomavirus SV40 has been found in association with several types of cancer in humans, but an etiological role remains to be established (Carter et al. 2003; Garcea and Imperiale 2003; Gazdar et al. 2002; Klein et al. 2002; Lopez-Rios et al. 2004; Poulin and DeCaprio 2006; Shah et al. 2004; Vilchez and Butel 2004; Wong et al. 2002)). In addition to inducing tumors, Py also induces other pathological conditions, depending on the particular virus strain and genetic background of the host. These include an acute and frequently fatal runting syndrome in newborn animals (Bauer et al. 1995; Bolen et al. 1985), demyelinating and paralytic syndromes (McCance et al. 1983; Sebesteny et al. 1980), polyarteritis with two histologically distinct lesions in the large vessels (Dawe et al. 1987), a myeloproliferative disorder in SCID mice (Szomolanyi-Tsuda et al. 1994)), hydrocephalus (Holtz 1964; Li and Jahnes 1959), and a role in autoimmune disease (Tonietti et al. 1970). These manifestations of Py infections have received little attention relative to the oncogenic properties of the virus. Since the last edition of this book, enormous progress has been made in understanding the molecular biology, genetics, structure, and pathogenic properties of Py. Advances have followed from the cloning and sequencing of various wild-type and mutant viral genomes and from applications of sitedirected mutagenesis to the functional analysis of viral gene products (Carmichael and Benjamin 1980; Deininger et al. 1979, 1980; Freund et al. 1992; Hattori et al. 1979; Soeda et al. 1980; Soeda and Griffin 1978). Studies with wild-type and mutant virus strains have been carried out in cell culture systems (Carmichael et al. 1984, 1982; Cook and Hassell 1990; Griffin and Maddock 1979; Mes-Masson et al. 1984; Templeton and Eckhart 1982; Templeton et al. 1986) and in the mouse (Bronson et al. 1997; Freund, Bronson, et al. 1992; Freund, Calderone, et al. 1991; Freund et al. 1998; Freund, Dawe, et al. 1992; Freund, Dubensky, et al. 1992; Freund et al. 1987; Freund, Sotnikov, et al. 1992). Results of these investigations have demonstrated the limits of in vitro cell transformation as a reliable model of tumor induction and point to the need to uncover new cellular targets of the viral T (tumor) antigens
5. P O LY O M A
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(Benjamin 2001). Structural studies of the virus have revealed surprising and important features. These bear on fundamental aspects of virus structure and assembly (Eisenberg 1982; Garcea and Liddington 1997; Garcea et al. 1987; Klug 1983; Rayment et al. 1982), on virus-receptor interactions (Stehle and Harrison 1996, 1997; Stehle et al. 1994), and on pathways of cell entry (Tsai et al. 2003). Critical determinants of pathogenicity residing in the structural proteins as well as in the T antigens have been elucidated at the molecular level. “Viruslike particles” made with structural proteins of Py are being explored for purposes of cell targeting and gene delivery (Fortstova et al. 1995; Shin and Folk 2003). Studies have also begun to define some of the host genetic factors underlying susceptibility and resistance to tumor induction by the virus (Benjamin 2001). The earlier chapter and monograph by Bernice Eddy (1982), the review by Sarah Stewart (1960), and the chapter on polyoma virus in Oncogenic Viruses by Ludwik Gross (1983) remain vital sources on the early years of Py research. More recent reviews and chapters have appeared on various aspects of the genetics, cell, and molecular biology of Py infections (Dilworth 2002; Gottlieb and Villarreal 2001; Griffin and Dilworth 1983; Norkin 1982), as well as structure (Garcea and Liddington 1997) and pathogenesis (Benjamin 2001). The chapter on Polyomaviridae in Fundamental Virology provides important details and extensive references on the molecular biology of polyoma viruses (Cole and Conzen 2001).
TABLE 5-1
SITES OF TUMOR INDUCTION BY POLYOMA VIRUS Common Sites Epithelial Mammary gland Skin (hair follicle) Thymus Salivary glands Mesenchymal Subcutaneous connective tissue Bone Renal medulla Less Common Sites Epithelial Thyroid Ovary Adrenal medulla Teeth (enamel epithelium) Sweat glands Mesenchymal Mesothelium (pleural and peritoneal) Vascular endothelium
genetic and physiological bases of host resistance and susceptibility to tumor induction by Py.
A. 1.
II.
THE POLYOMA VIRUS-MOUSE SYSTEM
Py is one of the few tumor viruses amenable to study in its natural host. Genetic determinants of both virus and host can thus be investigated as they affect tumor induction in a variety of tissues. Though the broadest range of tumor types is seen in the mouse, Py also induces tumors in rats, hamsters, guinea pigs, and other rodent species. When inoculated as newborns, mice of certain genetic backgrounds develop an array of solid tumors arising from at least a dozen distinct cell types (Table 5-1). By 3–4 months of age, individual animals typically bear tumors of multiple types and with a combined tumor mass that may approach 25% or more of total body weight. Fifty years after its discovery, Py continues to provide a useful system for experimental cancer research (Benjamin 2001). This derives principally from knowledge of its three tumor or “T” antigens. These early viral gene products perform essential replicative as well as transforming functions. Py T antigens activate protooncogenes and inactivate tumor suppressor genes, disrupting cellular pathways commonly altered in human cancers. Given this extraordinary ability to induce tumors, it is equally remarkable that mice of certain genetic backgrounds develop few or no tumors when inoculated with the virus. An important challenge lies in understanding the
Virus-Cell Interactions in Culture
Productive and Nonproductive Infections
Py can readily be grown in tissue culture and studied in its replicative or lytic cycle and in its ability to induce neoplastic transformation. The viral genome is a ~5.3 kb circular DNA comprised of a short noncoding regulatory region of ~450 bp and roughly 4.8 kb of coding sequences (Cole and Conzen 2001). Viral DNA is packaged as a “minichromosome” using histones from the cell (Frearson and Crawford 1972; Schaffhausen and Benjamin 1976). DNA extracted from the virus is infectious and can be used in both productive and nonproductive infections. Overlapping reading frames and alternative splicing are utilized to encode three early proteins corresponding to the large, middle, and small T antigens, and three late structural proteins made up of the major (VP1) and minor (VP2 and VP3) capsid proteins. Mouse cells normally undergo a lytic or “productive” infection. Early and late genes are expressed, leading to production of progeny virus particles and to the death of the host cell within roughly 48 hours. Rat and hamster cells undergo primarily a “nonproductive” infection in which the virus fails to complete its replication, thus allowing the cells to survive. Nonproductive infection leads to transformation, which can be either abortive or stable. The latter is accompanied by integration of the viral DNA into host DNA. Abortively transformed cells fail to maintain the viral genome and revert to a normal phenotype (Stoker 1968). Radiobiological evidence (Benjamin 1965) and later molecular
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and genetic evidence showed that only about half of the genome, that is, that corresponding to the early region encoding the T antigens, is required for transformation. Continued expression of the T antigens directs and maintains the transformed state of the cell. Late genes are not expressed and infectious virus is not produced in transformed cells. The genetics and molecular biology of the virus have been explored in depth using these cell culture systems (Cole and Conzen 2001). 2.
Cell Transformation
In vitro cell transformation results from nonproductive infection and has been studied most frequently using established fibroblasts from rats or hamsters. A small fraction of the cells that are exposed to virus emerge with altered properties that readily distinguish them from their normal progenitors. Changes associated with transformation arise rapidly and coordinately based on actions of the T antigens. At a molecular level, these viral oncoproteins intervene in multiple pathways through interactions with cellular protein kinases, protein phosphatases, and with factors involved in transcription and DNA replication (see Section VI below). The efficiency of transformation by the virus is several orders of magnitude lower than for plaque formation. Py-transformed cells undergo changes in growth properties and morphology (Table 5-2), providing the means for rapid and quantitative assays of cell transformation (Benjamin 1974). Quantitative assays of transformation are based on focus formation on monolayers of cells growing in a liquid medium on plastic or glass surfaces. In this assay, foci stand out as clones of cells with distinct morphology and in multilayered growth patterns that stand out above the monolayer of normal cells. When cloned and studied further, cells from such foci exhibit loss of contact inhibition of growth and increased saturation densities, measured as the maximum number of cells per unit of surface area in the culture dish. Equally useful are assays based on anchorage-independent growth, in which cells acquire the ability to grow when suspended in a semisolid medium such as soft agar or methylcellulose (Benjamin 1974; Macpherson and Montagnier 1964). Transformed cells selected by either method are essentially the same. When tested in low
TABLE 5-2
PROPERTIES OF POLYOMA VIRUS TRANSFORMED CELLS Alterations in Growth Control Loss of contact inhibition Loss of anchorage dependence Decreased serum requirement Morphological and Structural Changes Decrease substratum adhesion Cytoskeletal changes Alterations in the plasma membrane
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concentrations of serum, they grow better than their normal progenitors. This decreased dependence on serum-derived growth factors represents another aspect of altered growth control in transformed cells (Benjamin 1974). Transformation also involves morphological changes. These are reflected in a decreased adhesion to glass or plastic surfaces, often accompanied by decreased synthesis or secretion of fibronectin or other extracellular matrix component. Transformation is accompanied by changes in the cytoskeleton, principally the loss of bundles of actin microfilaments or “stress fibers” (Pollack et al. 1975), and by structural changes in the plasma membrane as measured by increased agglutinability of cells by certain plant lectins (Burger 1969). Transformed cells exhibiting these collective properties have frequently been shown to have acquired the ability to grow as tumors when inoculated into normal syngeneic or immunocompromised nonsyngeneic hosts.
B.
Natural and Laboratory Infections of Mice
In the infected newborn mouse, Py disseminates widely through successive cycles of productive infection. The natural transmission of the virus occurs most likely through a respiratory route (Rowe 1961; Rowe et al. 1961), leading first to infection in the lung followed by the kidney as the major site of amplification (Dubensky et al. 1991). Following intranasal inoculation of newborn mice, virus is seen to replicate in Clara cells lining the bronchi and bronchioles; transient hyperplasia along the bronchi is also noted (Gottlieb and Villarreal 2000). When transmitted to newborns in the wild, Py persists as a silent infection with no development of tumors or other discernible pathological condition (Gardner et al. 1974; Gross 1983b; Stewart 1960). Virus is shed from infected carriers in the urine and is also found in saliva and feces. The virus is stable and recoverable from bedding and nesting materials as well as from aerosols (Rowe 1961; Rowe et al. 1961). The virus is highly immunogenic. Infected mice develop and maintain high titers of neutralizing or hemagglutinationinhibiting antibodies for extended periods and perhaps for life. The “mouse antibody production” or MAP test is a sensitive way to detect small amounts of Py in infected tissues or environmental samples (Rowe, Hartley, Estes, et al. 1959; Rowe, Hartley, Law, et al. 1959). Antibodies to the virus are transmitted to newborns via the milk (Stewart et al. 1960). Maternal antibody appears to be a major factor explaining the difference in outcomes between infections in the wild and in the laboratory. T cell immunity is also induced and maintained (see Section VIII, B below). Tumor development in laboratory mice depends on many factors. Both the virus strain and host genetic background are critical. Administration of virus to newborns within the first hours of birth is of great importance. Delays beyond the first 12–18 hrs result in sharp declines in tumor frequency and in
5. P O LY O M A
complete resistance if the virus is given after several days (Eddy 1982; Gross 1983b). Different routes of inoculation have been used to induce tumors, most commonly subcutaneous or intraperitoneal. Litters born in the laboratory to uninfected mothers acquire no protective antibody. This lack of protection coupled with administration of virus soon after birth and prior to full maturation of the immune system allows rapid amplification and dissemination of virus (Dubensky et al. 1991), which is a necessary prelude to tumor development. Lytic infection has been documented in over 30 different cell types in 4–10-day-old mice infected as newborns (Dawe, Freund, Mandel et al. 1987). When tumors arise, the viral DNA is present either as low-copy integrated DNA or as free unintegrated DNA, which can be present in high copy numbers without signs of late viral gene expression or cell lysis (Dubensky et al. 1991; Talmage et al. 1992). Lytic lesions and virus-producing cells are found within some tumors, particularly those of epithelial origin (Talmage et al. 1992). Apart from its role as an oncogenic virus, Py is being used to develop a mouse model of nephropathy associated with the BK human polyoma virus. BK virus–associated nephropathy is becoming appreciated as a major factor in dysfunction and failure of kidney transplants. This model uses murine kidney transplants to explore this pathogenesis. Preliminary studies show that Py replicates to 1,000-fold higher levels in allogeneic than syngeneic transplants in acutely infected mice. Such mice also show accelerated allograft failure and a larger anti-donor T cell response (Han Lee et al. 2006). This model should prove valuable in probing the virologic and immunologic determinants that contribute to renal allograft rejection.
III. A.
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THE TUMORS
The Polyoma Tumor Profile
Py induces an array of solid tumors of both epithelial and mesenchymal origin. The most common epithelial tumors are intraductal mammary carcinomas, which arise in male as well as female animals, basal cell carcinomas (hair follicle tumors or trichoepitheliomas), carcinomas of all of the salivary glands, and thymic epitheliomas. Tumors of mesenchymal origin include renal sarcomas, hemangiosarcomas, osteosarcomas, and fibrosarcomas. Tumors also arise at lower frequencies in the ovary derived from the surface epithelium, male reproductive tract, lung, thyroid and adrenal glands, among other sites (Dawe, Freund, Mandel, et al. 1987). A closer analysis of the rare and common epithelial tumors suggests that these comprise a particular “polyoma tumor constellation” (Dawe 1981). Epithelial target cells in this constellation are terminally differentiated derivatives of primitive ectoderm and endoderm, deriving from an anatomically defined region of
the embryo. Interestingly, the organs and tissues affected by Py are similar to the set of tissues affected in the sex-linked recessive condition of anhidrotic ectodermal dysplasia in man (Dawe 1981; Goodman 1977; Reed et al. 1970; Tannhauser 1936).
B.
Tissue Interactions in Tumor Formation
Studies using intact and dissociated salivary gland rudiments have demonstrated the importance of epithelial-mesenchymal cell interactions in the induction of salivary gland tumors by Py (Dawe 1981; Dawe et al. 1966; Dawe et al. 1971). Intact gland rudiments from various stages of development can be infected ex vivo and transplanted back to syngeneic hosts where they develop tumors. However, when epithelial and mesenchymal components are first separated and then infected and transplanted, no tumors develop. Experiments have been carried out with infected chimeric rudiments reconstituted from chromosomally marked components. Results of these experiments demonstrate that the tumors derive from the epithelial and not the mesenchymal component. The latter, however, plays an essential role of induction, conferring on the epithelium the capacity to be transformed by Py. Interestingly, the neoplastic salivary gland epithelium, once established, can substitute for normal mesenchyme in inducing competence of normal epithelium to be transformed (Dawe 1968). Epithelial-mesenchymal interactions are also important in the development of kidney and tooth bud tumors (Eddy 1982). A subset of Py salivary gland tumors arise with a distinct lymphocytic component. Serial transplantation of these lymphoepitheliomas results in a gradual loss of the epithelial component and replacement by pure lymphoma. The blast-like lymphocytes bear markers of immature cortical thymocytes (Dawe et al. 1990; Hoot and Kettman 1989a, 1989b). When transplanted subcutaneously, the lymphomas disseminate rapidly to virtually all organs. Importantly, these lymphomas are free of viral DNA (Dawe et al. 1990). When transplanted to F1 recipients, lymphoma cells appear to be recruited from the host (Harrod and Kettman 1992). The term “epineoplasm” has been used to describe these tumors because they arise within a Pyinduced tumor and may derive some influence from the latter without being infected or indirectly acquiring viral DNA. The evolution of these epineoplasms suggests that the Py-transformed epithelial component exerts an inductive effect, mimicking normal thymic epithelium and causing immature T lymphocytes to proliferate and eventually undergo neoplastic conversion (Dawe et al. 1990). Results of these investigations demonstrate an important difference between cell transformation in vitro and tumor induction in vivo. The former is clearly a “cell autonomous” process, resulting from direct virus-cell interaction with individual clonally derived cells. In contrast, in at least some tissues, tumor development has features of a “non–cell autonomous”
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process, requiring inductive effects within tissues for cellular targets to develop, and with the emerging tumor cells acquiring properties able to induce neoplastic development of neighboring normal cells without transfer of the viral genetic material. The cellular and molecular mechanisms involved in these tissue interactions are not understood.
IV.
POLYOMA TUMORIGENESIS AS A GENERAL MODEL OF CANCER
Certain basic features characterize cancer development across species. Cancers are generally understood to evolve in a multistep process driven by genetic changes, with selection and progression occurring at each stage. The earliest steps of initiation require genetic change(s) that alter controls on cell replication. These changes confer a “transformed phenotype” to the cell. Steps beyond cell transformation per se are required for persistent tumor growth, for acquisition of invasive and metastatic properties, and for overcoming various defense mechanisms of the host. Stages in the development of different tumors have been described conceptually and operationally in different ways. Fig. 5-1 presents an outline of the steps in tumor development useful for assessing the extent to which the Py-mouse system exhibits or fails to exhibit general features commonly associated with cancer. It also serves to identify gaps in our knowledge where further investigations are needed.
Fig. 5-1
A.
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Initiation and Early Progression
The initiating events in tumorigenesis by Py can be viewed as corresponding to those of cell transformation in vitro, with the establishment of the viral genome and expression of the T antigens in the target cell. The coordinate expression of these viral proteins perturbs multiple signaling pathways, leading to neoplastic transformation. Acquisition of the viral genome is a single event defined kinetically but carries with it multiple functional consequences for the cell. The multiple effects exerted through the T antigens may well bypass the need for further hits in the form of cellular mutations. Py tumors grow rapidly to a size that threatens the life of the host typically within 3–4 months. Initiating events of transformation lead to the establishment of a microscopic tumor of not more than a few hundred cells and a size of roughly a cubic millimeter. These small tumors cease their growth, limited by the supply of oxygen. Further expansion requires neovascularization. Endothelial cells in nearby vessels are induced to migrate, divide, and form new vessels that invade and nourish the tumor. This “angiogenic switch” is an early and crucial step in tumor formation in general (Folkman and Hanahan 1991). It involves an interplay of pro- and anti-angiogenic factors secreted by tumor cells themselves and by surrounding stroma (Hahnfeldt et al. 1999). The former include VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), and osteopontin, and the latter angiostatin and endostatin. Large primary tumors induced by Py are obviously efficient in inducing angiogenesis, and
Steps in tumor development in the Polyoma virus mouse system. See Section IV.
5. P O LY O M A
cells from a variety of Py tumors have been shown to produce pro-angiogenic factors in culture (Velupillai and Benjamin, unpublished results). Histological examination reveals that mice carrying gross tumors also have numerous occult or microscopic tumors present throughout the body and particularly in tissues where large tumors commonly develop (Dawe, Freund, Mandel, et al. 1987). Why only some of the initial tumors induced by Py progress to the overt stage is not understood but may depend on the angiogenic switch. The step of angiogenic conversion may reflect some stochastic event leading to development of gross tumors that then inhibit further progression of microscopic tumors through the elaboration of anti-angiogenic factors. Further investigation is necessary to test this as well as other hypotheses.
B.
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Genomic Instability and Further Progression
Mutations in cellular genes may follow the establishment of the viral genome, although evidence that such changes are necessary for the development of Py tumors is lacking. Karyotype analyses of primary tumors indicate that tumor cells are primarily diploid or pseudodiploid, with occasional cells showing abnormalities (unpublished results). While exhibiting some heterogeneity, Py tumors do not generally become aneuploid. Neither do they show signs of specific chromosomal loss, duplication, or rearrangements. The question of genomic instability in primary and transplanted Py tumors should be investigated further using approaches of greater resolution and sensitivity such as comparative genomic hybridization and assays for microsatellite instability. Genomic instability in cancer is commonly linked with the mutational loss or inactivation of p53. Interestingly, Py tumors show no evidence of p53 inactivation. This is supported by the finding that virus-induced tumors arise significantly earlier in p53-null mice than in normal mice, implying that the virus itself has no effective means of nullifying the actions of p53 (Dey et al. 2000). The apparent failure of Py to directly inactivate or inhibit p53 is surprising in view of the fact that SV40 and other DNA tumor viruses have various mechanisms to antagonize p53. Unlike transformed cells and tumors induced by SV40 in which p53 becomes stabilized and inhibited through interactions with the large T antigen, Py tumors show no signs of either stabilization or degradation of p53 (Dey et al. 2000). Short-term cultures established from primary tumors induced by Py generally retain p53 and the ability to respond to DNA damage. Loss of p53 may occur if the tumor cells are propagated in culture for any length of time (Qian et al. 1997), but this may reflect the drive for loss of p53 and pRb pathways that accompanies the establishment of cells in culture (Dey et al. 2001; Harvey and Levine 1991; Qian et al. 1997). A somewhat different picture emerges from studies of lytic infection. This infection is accompanied by induction of a p53
response accompanied by interaction between Py large T antigen and a phosphorylated form of p53 (Dey et al. 2002). The potential effects of triggering a p53 response during virus replication are overcome by large T interaction with pRb and middle T with phosphatidylinositol 3-kinase (see Section IV, C below). Consistent with the continued expression and function of p53 in Py tumors is the fact that the tumor cells do not generally exhibit an immortal phenotype. Sequential subculturing of cells from Py tumors leads to senescence and crisis, similar to what is seen with normal cells from mouse embryos. Cultures of primary tumors may give rise to established cell lines more readily than cultures of embryonic cells but the tumor cells in general have not acquired an immortal phenotype in vivo (J. Carroll, T. Benjamin, unpublished results). Independent evidence that immortalization is not required for Py tumor development comes from studies with mutant strains of Py that have lost the ability to bind and inactivate the retinoblastoma protein pRb. These mutants have lost the ability to immortalize cells in culture, yet are fully able to induce tumors (Freund, Bronson, et al. 1992). That Py tumors develop rapidly without signs of gross genomic instability, apparently without inactivating p53 and without undergoing immortalization are surprising and atypical features of this system.
C. 1.
Overcoming Host Defense Mechanisms
Apoptotic Responses
Host defenses at the cellular and systemic levels act to prevent persistent tumor growth. Viral infections as well as certain oncogenic growth signals trigger apoptotic responses. How Py and other tumor viruses overcome apoptotic and growth arrest responses is only partially understood. Apoptotic signals generated by Py infection appear to be both p53-dependent and independent (Dahl et al. 1998). Like DNA damage and other forms of stress or injury, Py infection triggers a p53 response, potentially leading to growth arrest or apoptosis (Dey et al. 2000; Heinrichs and Deppert 2003). Various actions of the T antigens overcome these responses. Although middle T activates p53, acting through ARF and possibly MDM-2 (Lomax and Fried 2001), this pathway can be overridden by small T which blocks the signaling between middle T, and p53 (Moule et al. 2004). Binding and inactivation of the tumor-suppressor protein pRb by the large T antigen acts downstream of p53 in the cell cycle. This interaction effectively bypasses the G1arrest checkpoint brought about by p53 induction of the cyclin-cdk inhibitor p21 (Dey et al. 2002, Freund et al. 1994). Py large T also binds to p21 and may inhibit its functions directly (Cho et al. 2001). Py initiates anti-apoptotic responses through several mechanisms involving the T antigens. Activation of the
112 phosphatidylinositol 3-kinase/Akt pathway by middle T confers protection against apoptotic death (Dahl et al. 1998). Phosphorylation of BAD and caspase-9 by Akt effectively block apoptosis (Cardone et al. 1998). Induction of the proapoptotic factor BAX by p53 is effectively countered downstream by this middle T pathway. Middle T activation of the map kinase (MAPK) pathway via SHC/Grb-2 and Ras may also lead to protection against apoptosis. Small T activates the MAPK pathway through binding and inhibiting the protein phosphatase PP2A. The Akt and MAPK pathways are known to counter apoptotic signals and promote survival in different cell systems (Bonni et al. 1999; Datta et al. 1999; Eisenmann et al. 2003). Large T may also exert an anti-apoptotic effect by binding p150Sal2 (Li et al. 2001), a cellular transcription factor with pro-apoptotic activity (Li et al. 2004). 2.
Immune Responses
The larger DNA viruses of the adeno, herpes, and pox groups utilize a variety of mechanisms to evade detection by the immune systems of their hosts. These include interfering with antigen presentation by blocking maturation or causing degradation of class 1 MHC molecules, by expression of class I–like decoys to evade detection by NK cells, and by encoding cytokine mimics that interfere with the functions of normal host cytokines (Ploegh 1998). SV40 has been reported to downregulate MHC class I expression via its small T antigen (Moreno et al. 2004), but polyoma viruses in general, with their much smaller genomes, are not known to employ these other mechanisms of immune evasion. Immunocompetent mice respond to Py infection by vigorous and long-lasting humoral responses with high titers of neutralizing antibodies. T cell responses protect against tumor growth as well as productive viral infection. These responses do not totally eliminate the virus, but allow the establishment of persistent and usually silent infections. These features of infection by polyoma viruses under natural conditions are found in humans as well as in mice. How persistence is established and maintained is not fully understood. Py tumors are highly immunogenic, leading to rejection in most immunocompetent hosts. Rejection is based primarily on effective immunosurveillance mediated by virus-specific CD8+ T lymphocytes. The classically defined virus-specific transplantation antigens or TSTAs of Py and SV40 are derived from the respective viral T antigens. The latter are processed and presented as MHC-restricted peptides providing targets for cytolytic CD8+ T cells. In H-2k mice, an immunodominant peptide from the middle T antigen is an important target for rejection (Lukacher et al. 1999; Velupillai et al. 1999), and sequences from Py and SV40 large T antigens have been identified as H-2b-restricted antigenic peptides (Berke, Palmer, et al. 1996; Kemball et al. 2005; Mylin et al. 2000). In-depth studies have been carried out on H2-restricted CTL responses to SV40 large T antigen. In H-2b mice, responses develop in a
THOMAS
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hierarchical fashion to a series of epitopes (Mylin et al. 2000; Tevethia et al. 2001). Interestingly, expansion of Py-specific CD8+ T cells occurs in some susceptible hosts, but these cells lack CTL effector functions and fail to lyse tumor cells expressing the appropriate viral peptide (Moser et al. 2001; Moser and Lukacher 2001). The lack of cytolytic activity in these T cells is due to expression of the inhibitory natural killer cell receptor CD94/NKG2A (Moser et al. 2002). This inhibition of CTL effector function appears to be one of the important mechanisms underlying tumor susceptibility (see further in Section VIII, B below). Gene knockouts have been used along with other approaches to establish the importance of immune functions in controlling virus replication and persistence as well as in tumor rejection. Class I MHC molecules are clearly important for recognition and elimination of Py tumor cells (Berke and Dalianis 2000; Drake and Lukacher 1998). Absence or immunodepletion of CD4 and CD8 inhibit tumor rejection (Berke, Wen, et al. 1996; Ljunggren et al. 1994). CD8+ CTLs and memory cells function in controlling virus production and regulating persistence (Byers et al. 2003). Antibody-mediated clearance of virus occurs in T cell–deficient as well as in normal mice (Szomolanyi-Tsuda et al. 2001, 2000; Szomolanyi-Tsuda and Welsh 1996). Empty “virus-like particles” and subviral assemblies of VP1 are able to induce IgM responses, but only live virus induces an IgG response (Szomolanyi-Tsuda et al. 1998). Serial transplantation of Py transformed or tumor cells in immunocompetent mice is not routinely successful but can be achieved. The establishment of transplantable Py tumors may be accompanied by downregulation of MHC molecules and selection, but this does not result from any known action of the viral genes. Mouse strains that are susceptible to tumor induction fail for various reasons to mount effective T cell responses to the virus. Mechanisms of immune evasion in the Py-mouse system are thus primarily a function of the host genetic background and not the virus (see further in Section VIII, B below).
D.
Invasion and Metastasis
Py tumors most often remain confined to the tissue and site of origin. This is true even of very large tumors, such as those that frequently arise in the mammary glands. These intraductal carcinomas fill extensive portions of the ductal system of a single gland without crossing the basement membrane (Freund, Dawe, et al. 1992). Mammary tumors and salivary gland tumors have been noted to metastasize to the lung, however (Gross, 1983b; Stewart 1960; Stewart et al. 1958). This occurs most likely by hematogenous spread (Stewart 1960), with tumor emboli entering the bloodstream from the necrotic centers of rapidly growing tumors. Certain experimental manipulations give rise to invasion and metastasis in a regular fashion. Mammary tumors
5. P O LY O M A
may become invasive upon serial transplantation to syngeneic hosts, demonstrating that the tumors have the potential to progress if given sufficient time. In transgenic mice expressing the Py middle T antigen, mammary tumors become invasive and metastatic (Guy et al. 1992). The host genetic background can also influence the likelihood of specific primary tumor types to become invasive and to metastasize.
V.
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VIRUSES
VIRAL DETERMINANTS—STRUCTURAL PROTEINS
Three features of the virus—its structural proteins, its noncoding sequences, and its tumor antigens—contribute at different levels to the process of tumor induction. The major capsid protein VP1 binds to cell surface sialic acid as an essential component of viral receptors. Sialic acids are abundantly expressed on cell surfaces, enabling the virus to bind and potentially enter a large number of host cell types. Regulatory sequences in the viral DNA contain multiple enhancer elements with binding sites for a variety of widely expressed cellular transcription factors. The three T antigens act collectively to perturb multiple signaling pathways that regulate cell proliferation and survival.
A.
VP1 and Self-Assembly
Py is an icosahedral or roughly spherical particle roughly 45 nm in diameter. The outer shell is made up of 72 morphological subunits or capsomeres composed of assemblies of the major capsid protein VP1. The arrangement of capsomeres in the virus shell is such that 12 are “pentavalent,” that is, surrounded by five other capsomeres, and 60 are “hexavalent,” surrounded by six capsomeres. Early theories on virus structure predicted that spherical (as well as rod-shaped) viruses could self-assemble from single protein species following certain simple rules (Caspar and Klug 1962; Crick and Watson 1956, 1957). For icosahedral viruses of more than a minimum size, the notion of “quasi-equivalence” was introduced to explain how local bonding interactions between proteins in neighboring capsomeres could be conserved in nearly identical fashion to allow self-assembly (Caspar and Klug 1962). The notion of quasiequivalence implied that the hexavalent capsomeres be comprised of hexamers and the pentavalent ones of pentamers of VP1. Low-resolution X-ray diffraction analysis first revealed the surprising finding that the hexavalent as well as the pentavalent capsomeres were both comprised of pentamers of VP1 (Rayment et al. 1982). The “all pentamer” structure of Py and SV40 capsids does not allow for the anticipated quasi-equivalent bonding between VP1 molecules. Instead, as revealed by high-resolution crystallographic studies of SV40, three distinctly different interactions
involving the C-terminal tails of VP1 are found at interpentamer contacts between hexavalent and pentavalent pentamers (Liddington et al. 1991; Stehle et al. 1996). Calcium-binding sites and disulphide bonds are important in stabilizing the virus particle (Garcea and Liddington 1997; Stehle et al. 1996). The ability to form virus shells is intrinsic to VP1, as shown by studies with recombinant protein. Py VP1 expressed and purified from E. coli assembles in vitro to form various polymorphic structures, including virus shells of the native size and structure (Salunke et al. 1986, 1989). Although assembly occurs spontaneously under non-physiological conditions in vitro, cellular chaperones such as hsp 70 bind VP1 in vivo and catalyze proper assembly in vitro and in vivo (Chromy et al. 2003; Cripe et al. 1995). Virus shells assembled in vitro bind to resting cells and induce an “early response” with activation of c-myc and c-fos (Zullo et al. 1987). Post-translational modifications of VP1 may play a role in virus assembly in vivo (Garcea et al. 1985). These various studies carried out with Py VP1 have significance for mechanisms of self-assembly of protein structures and practical implications for DNA packaging and delivery. 1.
Virus-Like Particles and Gene Delivery
Polyoma virus is able to infect and deliver its DNA to a wide variety of cell types in its natural host. Pseudovirions, that is, particles that contain cellular DNA fragments rather than viral DNA, form naturally during infection, suggesting that Py particles may be useful for encapsidation and delivery of nonviral DNA (Aposhian et al. 1972; Yelton and Aoishian 1972). Efforts to package DNA in vitro using Py empty capsids have been described (Slilaty and Aposhian 1983). Wild-type and variously engineered forms of VP1 have been expressed in E. coli or insect cells and used to form virus-like particles (VLPs) in vitro (Salunke et al. 1989) and in vivo (Montross et al. 1991). Aided by knowledge of the threedimensional structure of the virus (Stehle, 1996, 1997; Stehle et al. 1994), insertions have been made into portions of VP1 that form loops on the virus surface. Sequences encoding portions of the urokinase plasminogen activator (uPA) have been used to generate VLPs that bind to the uPA receptor on cells (Shin and Folk 2003). Efforts have also been made to couple antibody fragments specific for tumor cells to VP1 (May et al. 2002; Stubenrauch et al. 2001). Mutant Py DNA and reporter genes have been incorporated into pseudocapsids in a baculovirus system and examined for transfer into rat cells (Forstova et al. 1995). Transfer to organ cultures of rabbit cornea and to mice has also been attempted with some success (Krauzewicz, Cox, et al. 2000; Krauzewicz, Stokrova, et al. 2000).
B.
Determinants of Pathogenicity in VP1
Wild-type laboratory strains of Py fall into two types, designated “large plaque” and “small plaque” in reference to the size
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and appearance of plaques formed on monolayers of mouse embryo fibroblasts or 3T3 cells. Both types replicate and transform cells well in culture. They differ profoundly, however, in their abilities to replicate and induce tumors in the mouse. Large plaque strains rapidly establish a disseminated infection and induce the expected broad range of tumors when inoculated into newborn mice. In contrast, small plaque strains are severely restricted in their replication and induce few or no tumors (Dawe, Freund, Mandel, et al. 1987). Furthermore, occasional tumors that arise following inoculation with small plaque strains do so after an extended latency, and they represent a narrow part of the tumor spectrum, being strictly of mesenchymal origin. Results from sequencing and recombination studies have shown that the key difference between large and small plaque strains resides in a single amino acid substitution at position 91 of VP1, where large plaque stains encode glutamic acid and small plaque strains glycine. The substitution affects the ability of the virus to replicate and spread in the newborn mouse (Dubensky et al. 1991) and to induce tumors (Freund, Calderone, et al. 1991). This major determinant of pathogenicity in VP1 also governs the plaque size and hemagglutination properties of the virus (Freund, Garcea, et al. 1991). A variant large plaque strain has been found to have virulent properties. Newborn mice inoculated with this variant show severe runting and succumb within the first 2–3 weeks, well before any tumors appear (Bauer et al. 1995; Bolen et al. 1985; Rowe, Hartely, Estes et al. 1959). Morbidity is due to widespread lytic infection causing kidney failure, hemorrhages in the brain, and destruction of developing tooth buds, rendering the pups unable to suckle. The virulent behavior can be attenuated by dilution of the virus inoculum. At sublethal doses, animals survive and go on to develop the typical high tumor profile of standard large plaque strains (Bauer et al. 1995). DNA sequencing and characterization of recombinants between virulent and tumorigenic strains revealed the importance of a substitution at position 296 of VP1. Standard tumorigenic strains encode valine and the virulent strain alanine. These virus strains of different pathogenicity show characteristic differences in their abilities to hemagglutinate guinea pig erythrocytes across a range of pHs, indicating that the substitutions in VP1 in some manner affect the
L.
BENJAMIN
binding of the virus to receptor(s) on erythrocytes (Bauer et al. 1995). Variants of Py have been selected for altered host range properties, and some of these have alterations in their VP1-coding sequences (Mezes and Paolo 1994; Ricci et al. 1992). Small deletions of two amino acids in VP1 confer enhanced ability to replicate in undifferentiated mouse myoblasts and also in embryonal carcinoma cells. These same mutants show altered patterns of replication in various tissues of the mouse. Some mutants also have duplications in the enhancer region and thus may owe their altered host range to events at the level of transcriptional regulation in different cells as well as to steps of receptor recognition and cell entry. C.
Pathogenicity Determinants in the Sialic Acid Binding Pocket of VP1
Sialic acid is an essential component of Py receptors, as shown by binding studies and by the fact that pretreatment of cells with neuraminidase blocks infection (Cahan and Paulson 1980; Cahan et al. 1983; Fried et al. 1981). Oligosaccharide chains ending with a terminal sialic acid in an α2,3-linkage to galactose are features of receptors for both large and small plaque strains. Small plaque strains also bind to branched carbohydrate chains, carrying a second sialic acid attached to the third sugar in an α2,6 linkage (Cahan and Paulson 1980; Cahan et al. 1983; Fried et al. 1981). Large plaque strains, both tumorigenic and virulent, bind only the straight and not the branched chain sialic acids. Table 5-3 gives the receptor-binding properties and key amino acid substitutions in VP1 for the three types of virus distinguished by their different biological behaviors in mice. A surprising aspect of these findings is that small plaque viruses, which are restricted relative to large plaque strains in their ability to replicate and induce tumors (Dawe, Freund, Mandel et al. 1987), bind to a broader class of sialic acid–containing receptors (Freund, Calderone, et al. 1991). X-ray crystallography has provided an understanding of how these single amino acid substitutions in VP1 confer important biological properties to the virus. These studies, carried out with whole virus and with VP1 pentamers in complexes with
TABLE 5-3
DETERMINANTS OF PATHOGENICITY IN THE SIALIC ACID BINDING POCKET OF VP1a 91
296
G
V
E E G
V A A a
‘Non-pathogenic’ (few or no tumors): attenuated spread due to recognition of branched chain sialic acid ‘pseudo-receptors’, dictated by 91G. Tumorigenic: efficient virus spread coupled to failure to recognize ‘pseudo-receptors’, dictated by 91E. Virulent: rapid disseminated infection causing early death, decreased avidity for receptor, dictated by 296A (coupled with 91E). Non-pathogenic: ‘pseudo-receptor’ recognition (91G) overrides virulence (296A).
Amino acids encoded at positions 91 and 296 are indicated. A – alanine, E – glutamine acid, G – glycine and V - valine
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VIRUSES
115
Fig. 5-2 Amino acid substitution in VP1 regulates virus spread. Substitution of glycine for glutamic acid at position 91 of VP1 leads to sharp reductions in the ability of the virus to spread and induce tumors in each of four susceptible mouse strains. The 91G substitution allows the virus to bind to branched sialic acids (‘pseudoreceptors’) leading to attenuated virus spread. See Sections V-B and C. [Bauer et al. 1999 reprinted with permission from Academic Press.]
sialic acid-containing oligosaccharides, show that the critical determinants map to carbohydrate binding pockets on the virus surface (Stehle and Harrison 1996, 1997; Stehle et al. 1994). The glutamic acid–glycine substitution at position 91 confers on the virus the ability to discriminate between straight and branched receptors. Steric interference and electrostatic repulsion between the glutamic acid and the inner α2,6-linked sialic acid effectively prevents large plaque strains from binding to the branched structure. The absence of a side chain in the glycine at this position readily allows small plaque strains to accommodate the second sialic acid in the branched oligosaccharide. It thus appears that the branched oligosaccharides function as “pseudoreceptors,” capturing and preventing small plaque viruses from attaching to functional straight chain receptors. Whole mouse section hybridization can be used as a semi-quantitative measure of virus spread and replication (Bauer et al. 1995; Dubensky et al. 1991, 1984). Fig. 5-2 demonstrates the attenuating effect of the glycine substitution using this approach. The same result is apparent in each of four different mouse strains, indicating that the putative
pseudoreceptors are broadly and perhaps universally expressed in mice (Bauer et al. 1999). The alanine for valine substitution that distinguishes “virulent” from “tumorigenic” large plaque strains results in a slightly weaker binding to the straight chain receptor. This conservative replacement results in the disappearance of a hydrophobic interaction between the methyl group of valine and the terminal sialic. The effect of replacement of valine-296 by alanine on the dissociation between VP1 and sialic acid, though minor, is nevertheless expected to result in a significantly weaker avidity of binding by the virus, which must engage multiple receptor molecules in a cooperative manner (Bauer et al. 1999). A lower avidity for receptors may well be a critical factor, as it would facilitate dissociation of virus from cellular debris, leading to the more rapid spread that characterizes the virulent strain. Site-directed mutagenesis and whole mouse section hybridization experiments confirm the predictions from X-ray crystallography (Bauer et al. 1999). The effects of substitutions at these two positions in VP1 are illustrated in Fig. 5-3.
Fig. 5-3 Effects of single amino acid replacements in the sialic acid binding pocket of VP1. Standard wild type strains and their VP1 types are PTA (91E, 296V), LID (91E, 296A) and RA (91G, 296V). Results with site-directed mutants demonstrate the effect of the virulence determinant 296A in increasing virus spread (PTA-V296A) and of 91G in limiting virus spread (PTA-E91G and LID-E91G). 91G enabling recognition of branched chain sialic acids (‘pseudoreceptors’) and overrides the effect of the virulence determinant (PTA-Y296A/E91G). See Sections V-B and C and Table 5-3 [Bauer et al. 1999; reprinted with permission from Academic Press.]
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The prototype nontumorigenic, tumorigenic, and virulent strains are readily distinguished by the degree of replication and spread that occurs over the first 8–10 days following injection into newborn mice. Single amino substitutions introduced into the prototype strains alter the extent of replication in the directions predicted from structural studies and known interactions with sialic acid receptors. Substituting G for E at position 91 in either tumorigenic (PTA) or virulent (LID) large plaque strains results in greatly reduced replication in the mouse, presumably by opening up the pocket on the virus surface, allowing binding to branched pseudoreceptors (Bauer et al. 1999, Stehle and Harrison 1996, 1997). The 91G substitution in these strains not only attenuates virus spread but also leads to significant reductions in tumorigenicity and virulence (Bauer et al. 1999). Introduction of 91E into the small plaque RA strain results in a significant increase in its ability to replicate and spread, presumably by preventing recognition of pseudoreceptors. The virulence determinant 296A when introduced into the large plaque PTA increases its ability to spread, matching the levels seen in the prototype virulent strain LID. When 91E and 296A are introduced together into RA, virus replication is further enhanced and the double mutant acquires virulent behavior. As might be expected, when both pseudoreceptor recognition (91G) and virulence determinants (296A) are present together, the former dominates the biological behavior. Thus, 296A, which increases replication when introduced into either PTA or RA-91E, has little or no effect when introduced into either RA or PTA-91G. The determinant allowing pseudoreceptor recognition thus overrides the determinant conferring virulence. Because prototype laboratory strains of Py have been derived and manipulated in culture in various ways over many years, the question of which type(s) of VP1 predominates in nature is unclear. Results of a recent study have shown that newly isolated strains of Py from wild-trapped mice all have the VP1 type of the PTA strain. Like PTA, these isolates are also highly tumorigenic in the laboratory. Persistance in the natural host as a “silent pathogen” appears to reflect an adaptation satisfied by the 91E and 296V determinants, in other words, by viruses that exhibit neither highly virulent behavior nor the attenuated spread that accompanies pseudoreceptor recognition (unpublished observations).
D.
The Minor Structural Proteins VP2 and VP3
Low-resolution difference analysis between full particles and empty capsid shells reveals a cavity inside the VP1 pentamers (Griffith et al. 1992). Each of these cavities is occupied by a single molecule of one of the minor capsid proteins VP2 or VP3 (Barouch and Harrison 1994; Chen et al. 1998). VP2 (319 amino acids) and VP3 (206 amino acids) are translated in frame
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with VP2 carrying unique N-terminal sequences. Both proteins appear to be essential for infectivity (Sahli et al. 1993). VP1 is needed for the efficient transport of VP2 and VP3 into the nucleus where virus assembly occurs (Forstova et al. 1993; Stamatos et al. 1987). The amino terminus of VP2 is myristoylated (Streuli and Griffin 1987). VP2 mutants with alterations at the amino terminus that prevent myristoylation have been made and partially characterized. While some substitutions of the amino terminal glycine to which the myristoyl group is conjugated lead to loss of infectivity (Krauzewicz et al. 1990), others allow incorporation of unmyristoylated VP2 into particles that retain some infectivity (Sahli et al. 1993). These virions with unmyristoylated VP2 are 15 to 20-fold less infectious than wild-type virus. They also show a significant delay in initiating viral DNA and viral protein synthesis, suggesting a block in some early step of infection involving uncoating or disassembly. The non-myristoylated VP2 mutant is defective or delayed in entry of cells both in culture and in the mouse as shown by the increased latency and decreased incidence of tumors. Following intranasal inoculation, replication is limited mainly to the lungs (Sahli et al. 1993).
VI.
VIRAL DETERMINANTS—TUMOR ANTIGENS A.
Primary Structures and Functions
Viral proteins expressed in Py tumor cells were first recognized as “tumor” or “T” antigens by their reactivity with sera from tumor-bearing animals. Three distinct proteins encoded in the early region of the viral genome have been identified as T antigens. All three have essential roles in virus replication, cell transformation, and tumorigenesis. Transcripts from the early viral promoter undergo alternative splicing to give rise to the mRNAs for the large, middle, and small T antigens (Treisman, Cowie, et al. 1981). The three species have shared sequences in their first exons and unique sequences encoded by overlapping reading frames in their second exons (see Fig. 5-4A). Large T is a ≈100 kD nuclear protein of 782 amino acids, middle T a ≈56 kD membrane-bound protein of 421 amino acids, and small T a ≈22 kD protein of 195 residues found predominantly in the cytoplasm but also in the nucleus (Ichaso and Dilworth 2001). A 79–amino acid N-terminal sequence is shared among all three T antigen species. This region contains a “Dna J” domain that is also present in the T antigens of SV40 and other polyoma viruses (Genevaux et al. 2003; Pipas 1992). This domain binds to heat shock protein(s) with co-chaperone functions that in turn mediate interactions with other cellular proteins (Nemethova et al. 2004; Sheng et al. 1997; Whalen et al. 2005). The long open reading frame in exon 2 of large T contributes sequences with multiple functions necessary for large T’s role
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Fig. 5-4
The polyoma virus T antigens. (A) Structures. (B) Interactions with cellular proteins. See Section VI.
in binding to viral DNA, initiation of viral DNA synthesis, and regulation of transcription during productive infection. Productive infection by Py is accompanied by induction of a complete round of cellular DNA synthesis (Dulbecco et al. 1965). The synthesis of viral DNA depends on the virus being able to override G1 arrest and promote entry of the host cell into S phase. These events of cell cycle progression are determined by binding of large T to the tumor suppressor pRb, leading to E2F-dependent transcription of S-phase genes (Freund et al. 1994). The binding of pRb by large T is also essential for immortalization of primary cells in culture (Freund, Bronson, et al. 1992; Larose et al. 1991). The middle and small T antigens share an extended sequence of 112 amino acids encoded by sequences in the large T intron. This region is critical for the binding of the protein phosphatase
PP2A to these two T antigens and for the binding of pp60c-src to middle T (Pallas et al. 1988). Middle T and small T utilize the same splice donor site and different splice acceptor sites and thus acquire different C-terminal extensions. Small T has a short extension of only four residues. Middle T acquires 230 amino acids with a single transmembrane sequence close to the C-terminus. This region of middle T contains multiple sites of serine and tyrosine phosphorylation critical to its functions (see Sections VI B and VI C below). Middle T is considered the major viral oncogene. It is able by itself to transform most but not all established lines of rodent fibroblasts (Lomax and Fried 2001; Mor et al. 1997; Treisman, Norak, et al. 1981). When expressed under the control of an exogenously regulated promoter, middle T can reversibly control essential aspects of the transformed phenotype (Raptis et al.
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1985). For transformation of primary or non-established cells, however, cooperation between large T and middle T is required (Rassoulzadegan et al. 1982). Small T synergizes with large T and middle T to promote cell cycle progression by several mechanisms (Marti and Ballmer-Hofer 1999; Schuchner et al. 2001; Schuchner and Wintersberger 1999). Cooperation between the Py T antigens has been studied in detail using REF52 cells, an unusual established line of rat embryo fibroblasts that retains p53 and that, like primary cells, cannot be transformed by middle T alone. Expression of a dominant negative form of p53 allows middle T to transform REF52 cells (Mor et al. 1997). Middle T induces growth arrest in these cells, and this inhibition is overcome if small T or large T is co-expressed (Marti and Ballmer-Hofer 1999). Middle T expression in REF52 cells leads to a p53-dependent cell cycle arrest due to activation of ARF, product of the alternative reading frame locus p16INK4A (Lomax and Fried 2001). This pathway is inhibited by small T which overcomes the growth arrest and allows middle T to transform REF52 cells (Moule et al. 2004).
B.
Molecular Interactions
Unlike oncogenes of retroviruses that derive from cellular protooncogenes, the Py T antigens do not derive from cellular genes and do not possess activities associated with protooncogenes such as protein kinases, transcription factors, or G proteins. Instead, they interact with cellular proteins that either possess or regulate these activites. The functions of the T antigens are understood largely in terms of the cellular proteins with which they interact. Most of the currently known interactions are depicted in Fig. 5-4B. The significance of some of these interactions is not fully understood. The large T antigen of both polyoma and SV40 binds to their respective viral origins of replication and act as ATP-dependent DNA helicases (Gaudray et al. 1980; Seki et al. 1990). In conjunction with DNA polymerase/primase α and other host DNA replication proteins, large T acts to unwind the origin and initiate viral DNA replication. X-ray diffraction and biochemical studies have revealed important structural and functional details about interactions of the SV40 large T protein with the viral replication origin (Gai et al. 2004; Li, Zhao, et al. 2003; Simmons 2000). The Dna J domain in large T is essential for binding heat shock protein hsp70 and for mediating the effects of large T bound to pRb (Sheng et al. 1997). Transactivation of S phase genes depends on an intact Dna J domain (Nemethova et al. 2004). Productive infection by Py leads to induction of a DNA damage-like p53 response with induction of p21Cip1/Waf1 and BAX (Dey et al. 2002). This is accompanied by phosphorylation of p53 and binding of large T. Interaction of large T with pRb is required for induction of the p53 response. Productive infection by Py is also accompanied by a modest induction of ATM, an
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upstream regulator of DNA damage responses, and this induction leads to increased virus yields (J. Dahl and T. Benjamin 2005). It thus appears that induction of a DNA damage response in productively infected cells is in some manner turned to the virus’ advantage. In contrast to productive infection, there is little evidence for functional interaction between Py large T and p53 in tumors. Full-length large T is not always expressed in these cells (Talmage et al. 1992). When expressed, Py large T does not stabilize p53 in the manner of SV40 large T, which binds and prevents the degradation of p53. Studies comparing p53deficient and normal mice have shown no evidence that Py inhibits p53 during the course of tumor induction, and tumorderived cell lines retain functional p53 and the ability to respond to DNA damage (Dey et al. 2000). Polyoma large T binds the p53 transcriptional co-activators p300/CBP (Cho et al. 2001; Nemethora and Wintersberger 1999). Binding of these co-activators with histone acetyltransferase activity as well as the PCAF acetyltransferase is essential along with the Dna J domain for transactivation of E2F-responsive genes (Nemethova et al. 2004). Association of large T with the PCAF and GCN5 acetyltransferases leads to acetylation of large T and stimulation of viral DNA replication (Xie et al. 2002). It is unclear whether the association of large T with p300/CBP is involved in the hyperacetylation of viral chromatin (Schaffhausen and Benjamin 1976). The TAZ protein, a transcriptional co-activator with PDZbinding domain (Kanai et al. 2000), binds to all three Py T antigens (Tian et al. 2004). Viral infection inhibits transactivation by TAZ (Tian et al. 2004). Large T interacts with the CREB protein, mediator of transactivation through cyclic AMP–responsive elements, and has a direct effect in transactivation at CREB/ATF sites (Love et al. 2005). This effect of large T is independent of pRb binding and also independent of cyclic AMP and protein kinase A (Love et al. 2005). The middle and small T antigens alter signaling pathways involving protein phosphorylation. Both rival proteins have effects on early gene expression and rival DNA replication (Chen et al. 2006). Middle T functions primarily by binding to and activating the tyrosine kinase activity of the protooncogene product pp60c-src (Courtneidge and Smith 1983). Other members of the src kinase family also interact with middle T (Dilworth 2002; Kornbluth et al. 1987; Kypta et al. 1988; Thomas et al. 1993). Middle T binds to the protein phosphatase PP2A (Walter et al. 1990). The association of middle T with pp60c-src depends on its binding to PP2A (Glover et al. 1999), consistent with observations on various middle T mutants that fail to bind both. In complexes with pp60c-src, middle T not only activates c-src kinase activity but also serves as a substrate, undergoing phosphorylation on at least three tyrosine residues. Phosphorylation of these tyrosines leads to binding of three cellular proteins with SH2 or phosphotyrosine binding domains with important functions in mitogenic signaling. Three pathways
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are activated by these interactions—via SHC to the ras-MAP kinase pathway (Campbell et al. 1994; Dilworth et al. 1994), via phosphatidylinositol 3-kinase to Akt (Kaplan et al. 1986; Talmage et al. 1989), and via phospholipase-Cγ (Su et al. 1995), which generates inositol tris phosphate and diacylglycerol as further messengers. The downstream effects of these pathways reach far into regulatory networks of the cell, affecting many aspects of cell behavior. Research on middle T and its functions have been reviewed in detail (Dilworth 2002; Gottlieb and Villarreal 2000; Ichaso and Dilworth 2001). Small T acts via binding to PP2A (Pallas et al. 1990). This interaction has the potential to alter multiple signal transduction pathways regulated by reversible serine and threonine phosphorylation. Some reports indicate that PP2A may also act on phosphotyrosine (Agostinis et al. 1996; Fellner et al. 2003; Ugi et al. 2002). Signal transduction pathways involving protein phosphorylation in general, including those altered by the middle and small T proteins, frequently converge on transcriptional machinery, leading to transactivation of specific genes. Both small T and middle T signal to elements in the rival enhancer (Chen et al. 2006). Middle T acting through the phosphatidylinositol 3-kinase and MAP kinase pathways leads to activation of AP-1 transcription factors (Oliveira et al. 1998). Small T of SV40 is able to transactivate cyclins A and D1 as well as to downregulate the cdk inhibitor p27 (Skoczylas et al. 2004; Watanabe et al. 1996). PP2A-dependent and independent actions of SV40 small T have been recognized (Moreno et al. 2004). Small T of Py transactivates cyclin A and, in concert with large T, increases cyclin E-cdk activity by elimination of the cdk inhibitor p27 (Schuchner and Wintersberger 1999). These actions of small T depend on its interaction with PP2A. Small T also acts via PP2A in some manner to block signaling to p53 by ARF (Moule et al. 2004).
C.
Genetic Studies
The roles of the T antigens in virus replication and cell transformation have been investigated by several genetic approaches. The earliest approaches were based on the isolation of conditional lethal mutants of the virus. Temperature-sensitive mutants that replicate at 31°C but not at 39°C were examined for their abilities to transform cells. From a large set of such mutants, one class emerged that defined an “initiation function” for transformation. This class of mutant, designated ts-a, could only transform cells at the permissive temperature; once the cells were transformed at the lower temperature, they retained the transformed phenotype at the nonpermissive temperature (Fried 1965). The initiation function defined by ts-a mutants most likely corresponds to the step of integration of viral DNA into host DNA. In productive infection, this function is required to initiate viral DNA replication. A host range selection based on growth in Py-transformed 3T3 cells and absence of growth on normal 3T3 cells was employed to
119 take advantage of the possibility of complementation between the integrated virus expressing the transforming genes and mutants defective in those gene(s). Such mutants proved to be defective for transformation and were designated hr-t for being “host range transformation-defective” (Benjamin 1970). Mapping of the mutations in these two classes of mutant was achieved by DNA fragment complementation (Feunteun et al. 1976). Ts-a mutants map to the large T antigen, in its unique region downstream of middle T. Hr-t mutants map to the common middle T/small T region encoded by large T intron sequences (see Fig. 5-4A). T antigen analyses (Schaffhausen et al. 1978; Silver et al. 1978) and DNA sequencing (Carmichael and Benjamin 1980; Hattori et al. 1979) confirm the assignment of the middle and small T antigens as products of the hr-t gene. The role of the hr-t gene in productive infection lies at least in part at the level of virus assembly and encapsidation (Garcea et al. 1985; Garcea and Benjamin 1983). In transformation, middle T has the properties of a “maintenance function”, that is, one continuously required to maintain the transformed phenotype (Raptis et al. 1985). These two groups of conditional lethal mutants complement each other efficiently in virus growth and cell transformation and therefore define physiologically and genetically distinct functions (Fluck and Benjamin 1979; Fluck et al. 1977). Site-directed mutagenesis and DNA restriction technologies have afforded a powerful “reverse genetic” approach to better understand structure-function relationships in the T antigens. In particular, they have allowed the functions of T antigens in regions of overlapping reading frames to be dissected and analyzed separately. Introduction of a termination codon upstream of the hydrophobic sequence presumed to be the membraneanchoring sequence in middle T results in the failure of middle T to associate with membranes and consequently in a failure to associate with pp60c-src and to transform cells (Carmichael et al. 1982; Templeton and Eckhart 1982). The truncated and soluble form of middle T can substitute for small T in productive infection (Templeton et al. 1986). Various other mutations in and around the membrane-spanning sequence of middle T also affect transformation (Dahl et al. 1992; Markland, Cheng, et al. 1986). Eliminating the splice acceptor site for the middle T antigen creates a mutant virus that encodes large and small T but not middle T. This mutant is transformation-defective in most but not all respects (Liang et al. 1984). Substitution of the tyrosine at position 315 of middle T with phenylalanine results in a greatly weakened ability to transform cells (Carmichael et al. 1984). Phosphorylation of this tyrosine serves as a binding site for the regulatory subunit of phosphatidylinositol 3-kinase, triggering one of the critical signaling pathways from middle T (Talmage et al. 1989). Substitutions in the –NPXY-motifcontaining tyrosine 250 results in transformation-defective virus mutants (Campbell et al. 1994; Dilworth et al. 1994; Druker et al. 1992; Markland, Oostra, et al. 1986), while substitution of tyrosine-322 has only a minimal effect (Su et al. 1995). Transformation-defective middle T mutants blocked in signaling via SHC and phosphatidylinositol 3-kinase are able to
120 stimulate serum-starved cells to synthesize cellular DNA, but a double mutant blocked in both of these pathways is inactive. Small T, acting via PP2A, is also able to promote cell cycle progression (Mullane et al. 1998). Several deletion mutants have been made in the region affecting both the middle and large T antigens. With their compound defects, these mutants have various effects on virus replication and transformation (Bendig et al. 1980; Griffin and Maddock 1979; Linder et al. 1990; Nilsson and Magnusson 1984; Smolar and Griffin 1981). Additional cellular targets of the viral T antigens have been identified more recently using a “tumor host range” or THR selection procedure. THR mutants are selected for being able to grow in certain mouse tumor cell lines but not in normal primary mouse cells (Li et al. 2001). Instead of using Py-transformed cells with integrated viral genes that can directly complement (Benjamin 1970), the THR selection uses spontaneous or chemical carcinogen–induced tumor cells as permissive hosts. The rationale is that such tumor cells are likely to have undergone a loss of some unknown tumor suppressor gene or other factor with which the virus must interact in order to grow efficiently. A Py mutant unable to grow in normal cells but able to replicate to some extent in the tumor cell is presumed to have lost the ability to target that factor. Two THR mutants have been isolated and used to identify cellular proteins that bind to the T antigens. The first is the Sal2 protein that binds to the C-terminus of large T. The Sal2 protein is an orthologue of the homeotic transcription factor Spalt in Drosophila. It blocks viral DNA replication, and this inhibition is overcome by binding to large T. In normal mouse tissues, the Sal2 protein is expressed at highest levels in the ovary, followed by lung, kidney, and brain. The human homologue of Sal2 is also highly expressed in ovarian surface epithelial cells. Ovarian carcinomas that derive from the surface epithelium frequently lose expression of the Sal2 protein. Restoration of Sal2 to human ovarian carcinoma cells suppresses tumor growth in SCID mice. Sal2 possesses p53-like functions in inducing apoptosis and in transactivating p21Cip1/Waf1 (Li et al. 2004). Studies of an independently isolated THR mutant have led to the identification of the transcriptional co-activator TAZ (Kanai et al. 2000) as a binding target of all three T antigens (Tian et al. 2004). Investigations of the normal functions of TAZ have revealed some aspects of its role as a co-regulator of transcription, acting in conjunction with Runt domain and other transcription factors (Mahoney et al. 2005; Park et al. 2004). A TAZ knockout mouse shows phenotypes unrelated to cancer but develops polycystic kidney disease and emphysema (unpublished results). These manifestations of the absence of TAZ in lung and kidney are consistent with these organs serving as critical sites in the natural history of Py infection and transmission, and with a need for modulation of TAZ functions by the virus. The findings of high levels of expression of Sal2 in lung and kidney
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are also consistent with the need for the virus to have evolved a mechanism to overcome the inhibitory effects of this protein (Li et al. 2001).
D.
Determinants of Pathogenicity
Several Py mutants that have been well characterized in terms of their molecular biology and interactions with cells in culture have also been studied in the mouse. For some, the findings largely confirm results obtained in tissue culture. For others, the results are not at all in line with expectations. The latter findings raise new and interesting questions. They also challenge the general validity of cell transformation as a model for tumor induction (Benjamin 2001). In comparing cell transformation with tumor induction, several differences are apparent with respect to the state and expression of the viral genome. Present as integrated DNA in some tumors, viral DNA also exists as free, unintegrated copies in others. The latter may be present in high-copy numbers in cells of certain epithelial tumors. Some cells within these tumors express VP1 and are most likely undergoing productive and ultimately lytic infection. Others with high-copy free viral DNA are VP1 negative. The latter condition in which viral DNA is amplified without activation of late viral gene expression is unknown, or at least uncommon, in cultured mouse cells where, once viral DNA replication occurs, the late promoter is activated and synthesis of structural proteins begins (Talmage et al. 1992). To determine the effects of specific T antigen determinants on tumor induction requires that the virus be in a background with the appropriate VP1 type (see Section V above) to ensure efficient virus spread and with non-coding sequences that facilitate expression in various tissues (Freund et al. 1987). A single amino acid substitution in large T, in the appropriate virus background, has subtle effects on the tumor profile with respect to the frequency of tumors in the salivary gland and thymus (Freund, Calderone, et al. 1991). A much more pronounced effect is seen with a truncated middle T mutant that is defective in transformation (Carmichael et al. 1982). As expected, this mutant fails to induce tumors. Moreover, it is defective in its ability to replicate and persist, demonstrating that middle T is essential for productive infection in the intact host (Freund, Sotnikov, et al. 1992). The THR mutants give results in mice that are consistent with the known sites of their mutations and with their properties in cell culture. The mutant altered at the C-terminus of large T and unable to bind the Sal2 protein is defective in replication, both in culture and in the mouse. However, as expected from the fact that this mutant retains normal middle and small T antigens, it is able to induce subcutaneous fibrosarcomas at the site of virus inoculation. No tumors develop at other sites, due to the failure of the mutant to replicate and
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spread (Li et al. 2001). The THR mutant that fails to bind TAZ is affected in all three T antigens. It is both replication- and transformation-defective in cell culture and fails to induce any tumors in the mouse (Tian et al. 2004; Tian and Benjamin, unpublished results). The mutant 315YF is unable to activate the phosphatidylinositol 3-kinase pathway (Talmage et al. 1989) and is partially transformation-defective, inducing smaller foci on monolayers and smaller clones in soft agar (Carmichael et al. 1984). Tumors develop much more slowly in mice inoculated with this mutant compared to wild-type virus. Differences are also seen in the spectrum of tumor types and in the histological and biological properties of certain tumors (Freund, Dawe, et al. 1992). The delay in tumor growth may reflect a generally weaker transformed phenotype, or more specifically the inability to block apoptosis through the phosphatidylinositol 3-kinase/Akt pathway, or both. Large T mutants that are unable to bind pRb replicate well in the mouse and induce a broad array of tumors (Freund, Bronson, et al. 1992). These results are surprising, given the importance of pRb as a tumor suppressor and regulator of a G1 checkpoint. They are also surprising in view of the importance of pRb binding in productive infection and immortalization of cells in culture (Larose et al. 1991; Nemethova et al. 2004). These findings demonstrate that the ability of the virus to immortalize cells in culture is dispensable for tumor induction. This result is not in line with the predictions from studies of oncogene cooperation in cell transformation, in which an immortalizing function is thought to be essential. They are also not in concordance with studies of human cancers, which indicate a requirement for immortalization based on re-expression of telomerase (Shay and Wright 2004; Stewart and Weinberg 2000). Mice have significantly longer telomeres and shorter life spans than humans. However, when tested in Mus spretus, a subspecies of mouse with short telomeres (Zhu et al. 1998; Zijlmans et al. 1997), the pRb-binding mutants are still able to induce tumors (unpublished results). Induction of tumors by these mutants does not depend strictly on naturally cycling cells in newborn mice because the mutants also induce tumors as well as wild-type virus in adult irradiated animals (unpublished results). More surprising is the fact that a mutant that is defective in cell transformation in vitro is able to induce tumors efficiently. This finding was made with a middle T mutant blocked in signaling via the SHC-ras-MAP kinase pathway (Bronson et al. 1997). The results are particularly striking because, unlike pRb-binding mutants of large T that retain transforming ability, middle T mutants blocked in this pathway are clearly defective in transformation (Bronson et al. 1997; Campbell et al. 1994; Dilworth et al. 1994; Druker et al. 1992; Markland, Oostra, et al. 1986). The lack of concordance between transformation and tumor induction with this mutant demonstrates the importance of contributions of the host cell, and specifically differences
between established fibroblasts and various target cells in the intact host, most of which are epithelial. At some sites, such as skin and ovary, this mutant induces tumors more frequently and of larger size than the wild-type virus (Bronson et al. 1997). The likelihood of tumor development thus depends not only on functions brought in by the virus through its T antigens but also on the network of growth controls operating in particular target cells. The finding of abnormally large hair follicle tumors induced by this transformation-defective mutant suggests that activation of the Ras-MAP kinase pathway, though essential for transformation of fibroblasts, may exert a protective effect, acting against the abnormal proliferation of these specialized cells in the skin. Another example of a tissue-specific effect stemming from a mutation in middle T comes from studies in mice with a mutant altered at a serine phosphorylation site. Serine 257 of middle T is a phosphorylation-dependent binding site for 14-3-3, a chaperone-like protein with regulatory functions affecting various signaling pathways. Substitution of this serine results in the virus being unable to induce salivary gland tumors but able to induce tumors at all other sites (Cullere et al. 1998). The basis of these differences in tumor induction by different T antigen mutants and the reasons for lack of correlation with cell transformation in vitro are not understood.
VII.
VIRAL REGULATORY SEQUENCES
The regulatory region of Py consists of roughly 400–450 bp of non-coding DNA flanking the origin of replication. It includes the early and late viral promoters and multiple enhancer elements with effects on transcription and replication (de Villiers et al. 1984; Herbomel et al. 1984; Nilsson et al. 1991; Tyndall et al. 1981). Large T binding sites, variable in number between different wild-type virus strains (Deininger et al. 1979; Freund et al. 1987; Friedmann et al. 1979; Soeda et al. 1980), are critical cis-acting elements for replication and transcription. Large T acts positively on the early promoter at early times after infection and negatively at later times as the levels of large T increase (Farmerie and Folk 1984). Large T also acts to stimulate transcription from the late promoter at late times in productive infection (Kern et al. 1986). The Py enhancers contain multiple sites for the binding of host transcription factors (Jones et al. 1988). Binding sites for members of the AP-1 and ets families are present along with ones for a variety of other factors, making the non-coding region a potentially versatile cassette for expression of viral genes in a wide variety of tissues. Differences in enhancer sequences in different virus strains are correlated with differences in tissue-specific patterns of replication and persistence in the animal (Amalfitano et al. 1992; Rochford et al. 1990, 1992). Sequences in the origin region in different virus strains
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show considerable variation compared to the strongly conserved coding sequences. This is true among standard laboratory strains of virus (Freund et al. 1988, 1987) and also among isolates from feral mice (unpublished results). Alterations in the enhancer region allow Py to replicate in embryonal carcinoma cells in culture (Couture and Lehman 1993; Fujimura et al. 1981; Katinka et al. 1981; Sekikawa and Levine 1981; Trotot et al. 1994). The replication defect of hr-t mutants lacking the middle and small T antigens can be overcome by duplication of sequences in the region of the enhancer containing the PEA1 (AP-1) and PEA3 (ets) sites (Chen et al. 1995). Enhancer differences may also contribute to differences in tumor profiles, most likely reflecting the degree of adaptation to specific tissues and their transcription factors. Non-coding sequences contribute to the high tumor profile induced by certain strains of Py. As in the case of T antigen variants, the effects of different regulatory sequences are dependent on having a VP1 type that allows efficient virus spread (Freund et al. 1987). A specific contribution of noncoding sequences is conferred by a duplication of a 40 bp sequence in the early promoter region. Strains carrying two copies of this sequence induce a high frequency of large thymic epitheliomas, leading to early death. Strains with a single copy induce only microscopic thymic tumors that do not threaten the life of the host (Freund et al. 1988).
VIII. A.
HOST DETERMINANTS
Cell Receptors and Machinery of Virus Uptake
Oligosaccharide chains with terminal α2,3-linked sialic acid are found in both glycoproteins and glycolipids. Thus, in principle, either class of molecule may serve as functional receptors for the virus. Pretreatment of cells with inhibitors of protein N-glycosylation effectively inhibits infection, suggesting that any of a possibly broad class of sialoglycoproteins may serve as Py receptors (Chen and Benjamin 1997). However, sialyltransferases involved in the synthesis of carbohydrate chains on both glycoproteins and glycolipids may themselves be N-glycosylated and require such glycosylation to function (J. Paulson, personal communication). Various attempts have been made to identify specific glycoprotein receptors. Efforts based on screening monoclonal antibodies produced in hamsters against mouse cells for their ability to protect against infection proved unsuccessful (Bauer et al. 1999). MHC class I molecules have been implicated in the binding and infection by several different classes of virus, including SV40 (Breau et al. 1992). Expression of class I molecules is not essential for Py infection, however, as shown using cells from β2 microglobulin-deficient mice (Sanjuan et al. 1992). Glycoproteins provide ample binding sites for Py, but whether specific ones function to internalize the virus along a
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pathway leading to infection is unclear. Antibody to α4β1 integrin confers partial protection, suggesting that this integrin may play a role in a post-attachment step of infection (Caruso, Belloni, et al. 2003; Caruso, Caveldisi, et al. 2003). Virus bound to sialoglycoproteins may remain on the cell surface or be routed internally in a way that fails to lead to infection. Such nonproductive binding could contribute to the low specific infectivity of the virus. The latter has been measured at about 30–100 as the ratio of physical particles to plaque-forming units. Glycoproteins with O-linked oligosaccharide chains carry the branched chain sialic acid structure expected to function as pseudoreceptors for small plaque viruses (Haselbeck et al. 1990; Zimmer et al. 1995). Direct evidence has been obtained for sialic acid–containing glycolipids or gangliosides serving as receptors for both Py and SV40 (Gilbert and Benjamin 2004; Gilbert et al. 2005; Tsai et al. 2003). A rat glioma cell line with a defect in glycolipid biosynthesis (Sottocornola et al. 1998) is highly resistant to infection by both viruses. Incubation of these cells with specific gangliosides prior to exposure to virus renders them highly infectible. Two gangliosides confer infectability by Py. These are GD1a and GT1b, each carrying a terminal α2,3–linked sialic acid on the longer arm of its carbohydrate moiety. Modeling shows that these gangliosides interact with VP1 as predicted from the crystal structure. A different ganglioside, GM1, confers infectability by SV40 (Tsai et al. 2003). Independent confirmation that these gangliosides serve as receptors for Py and SV40 comes from studies of Py-resistant mouse cells. Mouse cell lines were screened for resistance to Py infection while retaining susceptibility to transfection with viral DNA. This screening procedure was designed to identify cells that manifest a defect in some early step of infection, prior to uncoating of the virus and initiation of viral gene expression. These mouse cells proved to be resistant to SV40 as well as to Py, and the addition of the same gangliosides restored infectability as with the rat glioma cells (Gilbert et al. 2005). The pathway of cell entry by the virus has been investigated by electron microscopy and more recently with fluorescently labeled virus particles and high resolution de-convolution microscopy. The virus enters cells via small uncoated micropinocytotic vesicles (Mackay and Consigli 1976; Mattern et al. 1966) and is conveyed to the endoplasmic reticulum ER as the likely site of disassembly in a manner similar to that described for SV40 (Norkin et al. 2002; Pelkmans et al. 2001). Like SV40 (Anderson et al. 1996; Pelkmans et al. 2001), Py is endocytosed in some cells via caveolae (Mannova and Forstova 2003; Richterova et al. 2001). In other cells, uptake of Py is ostensibly caveolae-independent based on the fact that cholesterol-sequestering drugs that disrupt caveolae and block SV40 entry have no effect on Py (Gilbert et al. 2003). Dynamin I, involved in uptake of other viruses and cargo via caveolae, is not required for uptake of Py (Gilbert and Benjamin 2000).
5. P O LY O M A
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Py can also enter and infect ganglioside-deficient cells in a pathway of basal infection that differs from the gangliosidesupplemented pathway. Binding sites for the virus are abundant on ganglioside-deficient cells. The virus may utilize sialoglycoprotein receptors and be taken up by a caveolae-independent mechanism before converging on the intracellular pathway to the ER (Gilbert and Benjamin 2004). The basal infection pathway may also rely on a low level of gangliosides that are not localized in lipid rafts and caveolae. At earliest times in the pathway of cell entry, the virus associates with caveolin-1. Mouse cells deficient in caveolin-1 and resistant to Py infection become more infectible following restoration with exogenous caveolin-1 (Gilbert et al. 2005). The virus, apparently still in vesicles, colocalizes with microtubules as an essential step prior to reaching the ER roughly 2 hours post-infection (Gilbert et al. 2003). Transient association is also seen with microfilaments (Gilbert and Benjamin 2004). Entry and infection by Py is prevented by microtubule-disrupting but not microtubule-stabilizing drugs. In contrast, actin filament–disrupting agents facilitate infection by Py (Gilbert and Benjamin 2004), similar to what has been reported for SV40 (Pelkmans et al. 2002). The pathway of virus uptake from the plasma membrane to the ER is remarkably similar to that of bacterial toxins, which utilize gangliosides as receptors and are conveyed to the ER, where they rearrange and exit into the cytoplasm (Lencer and Tsai 2003; Tsai et al. 2003). While the toxins pass through the Golgi, the viruses apparently bypass the Golgi and enter a different vesicular compartment termed the “caveosome” prior to reaching the endoplasmic reticulum (Gilbert and Benjamin 2004; Mannova and Forstova 2003; Pelkmans et al. 2001). Fluorescent bacterial toxins have been used as markers for ganglioside receptors in studies of virus-resistant and virussusceptible cell lines (Gilbert et al. 2005). The virus must eventually cross a cellular membrane in order to reach the nucleus, where transcription, replication, and assembly of progeny virus occur. A role of myristylated VP2, exposed during virus disassembly, is thought to be involved in this crucial step of cell entry (Sahli et al. 1993). Partially disassembled virus particles are thought to exit from the endoplasmic reticulum into the cytosol and enter the nucleus via nuclear pores. It is also conceivable that the virus is transported further back through the endoplasmic reticulum and crosses the inner nuclear membrane. Recent studies have identified three factors in the ER involved in virus disassembly and exit into the cytosol. Erp29, a chaperone-like protein related to protein disulphide isomerase (PDI) but lacking in isomerase activity, brings about an alteration leading to increased hydrophobicity of the virus particle. Incubation of purified virus with Erp29 brings about partial disassembly or rearrangement of the virus resulting in the ability of the altered particle to bind to liposomes (Magnuson et al. 2005). Expression of a dominant-negative form of Erp29 inhibits viral infection (Magnuson et al. 2005). Catalytically
active PDI itself also appears to be important. Down-regulation of PDI results in retention of Py in the ER and inhibition of infection (Gilbert et al. 2006). The requirement for PDI suggests a need to disrupt the ring of disulphide bonds that link VP1 molecules together at the base of the pentamer (see section V-A; Gilbert et al. 2006; Stehle and Harrison 1997). The derlin family of proteins function to eliminate misfolded proteins in the ER, facilitating their translocation into the cytosol and degradation by the proteasome (Lilley and Ploegh 2004). Among this family, derlin-2 specifically is important for infection by Py. Upon infection of cells depleted of functional derlin-2, the virus is retained in the ER and fails to initiate infection (Lilley et al. 2006). It thus appears that following partial disassembly, Py utilizes the “quality control” machinery of the cell to pass from the lumen of the ER into the cytosol. Some fraction of the altered particles escapes degradation and enters the nucleus. The discrete steps of the disassembly process, the nature of the subviral particle that escapes from the ER and how it is transported into the nucleus are not fully understood.
B.
Effects of the Host Genetic Background
Mice of different inbred strains vary greatly in their responses to Py. Table 5-4 lists the strains for which information is available (unpublished results; see also Gross 1983b). Newborn mice were inoculated subcutaneously or intraperitoneally with the PTA or other large plaque strain (see Sections V-B and V-C and Fig. 5-3. In most cases, mice were followed for over a year and examined for microscopic as well as overt tumor development. In highly susceptible strains, multiple tumors develop in essentially every animal within 3–4 months. Mice of highly resistant strains develop no tumors attributable to the virus, although some may develop spontaneous tumors, usually after one year. Some resistant mice are susceptible to the virus; that is, they develop widespread lytic lesions but fail to develop tumors (Freund, Dubensky, et al. 1992). Others are resistant to infection by the virus per se (Carroll et al. 1999). Differences in immune responses account for some but not all of the differences between highly susceptible and highly resistant strains. Many strains are intermediate between these extremes. Mice of these strains show reduced frequencies of tumors, varying between roughly between 20% and 80%. They also develop fewer tumors per affected animal, a restricted spectrum of tumor types, and delayed appearance of tumors compared to highly susceptible strains. Unusual responses have been noted in certain strains in which tumors develop at some sites but not at others, or in which a particular tumor type metastasizes regularly. Such strains present opportunities and challenges for understanding the genetic and physiological mechanisms underlying their unusual tissue-specific responses.
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L.
BENJAMIN
TABLE 5-4
SUSCEPTIBILITIES OF INBRED MICE TO TUMOR INDUCTION BY POLYOMA VIRUSa,b Highly susceptible
Intermediate
Resistant
AKR/J C3H/BiDaCr C58/J CBA/J CZECHII/Ei PERA/Ei (Peru-Atteck) RF/J ST/bJ SWR/J
A/J AKR.M/SnJ BALB/cByJ BALB/K BIO.BR C3H/HeJ C57BL/10J CAST/Ei M. m. castaneus CBA/CAJ DBA/2J MOLD/Rk M. m. molossinus MOLF/Ei M. m. molossinus MOLG/Dn M. m. molossinus Mus pahari/Ei Mus pahari PERC/Ei (Peru-Coppock) RIIIS/J SF/CamEi SJL/J SK/CamEi SKIVE/Ei SM/J SPRET/Ei Mus spretus Swiss (non-inbred) WSB/Ei
C57BL/6J C57Br/cdJ CE/J MA/MyJ P. leucopus (deer mouse - LL Stock)
a
Compilations from Gross, L. 1983b and unpublished results. Inbred strains may be of several different subspecies. Strains listed here without designation are considered to be M. musculus or M. mus domesticus or some mixture of the two. Strains that are clearly of a different subspecies are indicated. b
The earliest studies of tumor induction by Py focused on a few susceptible strains, notably C3H and AKR. Resistance to tumor induction in other strains such as C57BL6 and C57BR was also noted by early workers in the field (Eddy 1982; Gross 1983b; Stewart 1960). Variable results were sometimes reported by different labs ostensibly using the same mouse strains, possibly due to differences in the virus strains used. Early attempts to establish dominance-recessive relationships among resistant and susceptible mice and to determine the likely number of genes involved also met with variable results (Chang and Hildeman 1964; Jahkola 1965). The use of cloned virus together with adherence to standardized protocols of infection, gross examination, necropsy, and histological examination have allowed better definition of the different host responses. Two forms of resistance and two forms of susceptibility have been distinguished based on studies of certain highly susceptible and resistant strains. The genetic and physiological bases of these forms as currently understood are summarized in Table 5-5. Immune responses dictated by the major histocompatibility complex (MHC) play an important role in determining resistance. Early studies of resistant mice, mainly of the H-2b haplotype, showed that these mice become susceptible
following neonatal thymectomy, treatment with anti-lymphocyte serum, or other forms of immunosuppression (Allison and Law 1968; Law 1966; Law and Dawe 1960; Law et al. 1967; Ting and Law 1965). Cytolytic T lymphocytes (CTL) responses are the major mechanism underlying immune surveillance and rejection of Py tumors. The highly susceptible mouse strains used by early workers in the field were almost exclusively of the H-2k haplotype (Eddy 1982; Gross 1983b; Stewart 1960). Contributions of the MHC locus have been shown by studies of congenic strains. Substitution of H-2k on an otherwise resistant H-2b background confers partial susceptibility (Freund, Dubensky, et al. 1992). In crosses between susceptible and resistant mice of different MHC backgrounds, resistance is transmitted as a dominant or co-dominant trait as expected from the immune response contributed by the resistant parent. A different result was noted in a cross between MHCidentical strains. C57BR mice, though carrying the H-2k haplotype commonly found in susceptible strains, are highly resistant and mount an effective immune response to Py tumors. When crossed to highly susceptible and H-2k-identical C3H mice, susceptibility was found to be dominant (Lukacher et al. 1993). Analysis of F2 and backcross mice indicated that
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TABLE 5-5
MECHANISMS OF RESISTANCE AND SUSCEPTIBILITY TO POLYOMA VIRUS INDUCED TUMORS Resistance
Mechanisms
Strains
References
Immunological (radiation - sensitive)
T cell immunity
C57Br/cdJ C57BL/6J others
(Carroll et al. 1999), (Law 1966)
Non Immunological (radiation - resistant)
Unknown
(Carroll et al. 1999)
Susceptibility Lack of effector T cells Superantigen – dependent
MA/MyJ CE/J
Inhibitory NK receptor T cell deletion
C3H/HeN C3H/BiDaCr CBA/J others PERA/Ei CZECHII/Ei
(Moser, Pitot, et al. 1990), (Moser, Altman, et al. 2001) (Love et al. 2005), (Lukacher et al. 1993)
Superantigen – independent
Innate Type 2 cytokine response
the dominant susceptibility of C3H is conferred by a single gene acting in some manner to prevent or override the immune response of C57BR. This gene proved to be an endogenous superantigen encoded by a particular mouse mammary tumor provirus, mtv-7 SAG (Lukacher et al. 1995). Endogenous superantigens effectively delete specific subsets of T lymphocytes based on expression of Vβ segments in their T cell receptors. On an H-2k background, precursors of Py-specific CTLs are eliminated by the endogenous superantigen. Mtv-7 SAG is present in each of five susceptible mouse strains carrying H-2k and absent from each of five resistant mice that are also H-2k (Lukacher et al. 1995). That mtv-7 SAG and H-2k together are sufficient to confer susceptibility in an otherwise mixed background has been confirmed in crosses between resistant 129/SvJ mice (H-2b; Mtv-7 SAG-negative) and susceptible CBA/J mice (H-2k; Mtv-7 SAG-positive). Selecting only for H-2k and Mtv-7 in mixed progeny of this cross is sufficient to confer susceptibility (Dey et al. 2000). Mtv-7 SAG and H-2k are therefore effective co-determinants of susceptibility based on a mechanism of endogenous superantigen-mediated deletion of T cells. A distinctly different genetic form of susceptibility has been found in two strains of “wild-derived” inbred mice (Velupillai et al. 1999). These strains are as susceptible to tumor induction by Py as the most highly susceptible of the classical inbred strains. Unlike the latter however, these mice carry no detectable endogenous mouse mammary tumor proviruses and show no evidence of Vβ deletion in their T cell repertoire. One of these strains, PERA/Ei, bears the H-2k haplotype and is thus identical to resistant C57BR mice with respect to MHC type and in lacking Mtv-7. The susceptibility of PERA/Ei is dominant in crosses with C57BR, thus defining a superantigen-independent form of susceptibility. The basis of susceptibility in PERA/Ei mice does not lie in a failure to
(Ugi et al. 2002), (Velupillai et al. 2002)
process or present viral antigen, since tumor cells from PERA/Ei and F1 mice are killed efficiently by a CTL line from infected C57BR mice specific for an H-2k-restricted viral peptide (Velupillai et al. 1999). The innate immune response of PERA/Ei mice plays a key role in their susceptibility (Velupillai et al. 2002). While C57BR mice respond to Py infection with a type I cytokine response and develop effective CTL immunity, PERA/Ei mice show a type 2 response and fail to sustain such immunity. The polarization of T cell cytokine responses in these mice is dictated at the level of antigen-presenting cells (APCs). APCs from infected C57BR mice produce predominantly interleukin-12 and show upregulation of the costimulatory molecule B7.1. APCs from infected PERA/Ei and F1 mice produce mainly interleukin-10 with upregulation of B7.2. This difference in cytokine production is an important factor in determining their tumor responses as shown by the fact that administration of recombinant IL-12 to infected and normally susceptible F1 mice leads to complete resistance (Velupillai et al. 2002). Further results have shown that exposure of naive APCs to virus in vitro leads to the same cytokine responses as occurs in vivo. Virus-like particles consisting of shells of Py VP1 (Montross et al. 1991) trigger the same responses as whole virus, indicating that viral gene expression is not required to elicit the strain-specific cytokine responses (Velupillai et al. 2006). Single pentamers of VPI are ineffective indicating that engagement of multiple receptors on APCs is essential (Velupillai et al. 2006) The underlying basis for the differential cytokine responses to Py VP1 by APCs from these strains is not understood. The immunological basis of resistance in C57BR mice is typical of many resistant strains that mount effective CTL responses to Py tumors. A hallmark of mice with this common form of resistance is their sensitivity to irradiation. Mice that
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THOMAS
are highly susceptible as newborns acquire immunological resistance within a few days of birth, and their resistance as adults is also overcome by whole body irradiation (Law and Dawe 1960). Not all mice show this radiation-sensitive form of resistance, however. Two strains have been identified that remain resistant following irradiation, indicating an unusual and possibly non-immunological form of resistance (Carroll et al. 1999). MA/MyJ mice which display this radiation-resistant form of resistance to tumor induction are also resistant to infection by the usually virulent LID strain of virus (Bauer et al. 1995) (see Sections V-B and V-C above). This form of virus resistance manifested by the intact host is not seen in cells derived from these mice which are fully susceptible to lytic infection in vitro. Sera from MA/MyJ mice have no demonstrable hemagglutination inhibitory antibodies or other viral inhibitors, nor do they have unusually high levels of radiationresistant NK cells or circulating levels of antiviral interferons (Carroll et al. 1999). The basis of this form of resistance remains unknown.
IX.
TRANSGENIC MICE
Lines of transgenic mice carrying various wild-type and mutant forms of Py and SV40 T antigens have been established. Studies of these mice have greatly expanded the scope of investigations bearing on the oncogenic potential and mechanisms of action of the viral proteins. This is particularly true with respect to SV40. While able to transform mouse cells in culture, SV40 is not oncogenic in the mouse due to its failure to replicate and spread. Expression of T antigen transgenes can be directed to particular tissues by coupling to specific promoters. Expression in the animal may also be regulated in a time-dependent manner through use of promoters that are regulatable, for example, by addition of doxycycline to the drinking water or by other means. Once the effects of a promoter-T antigen construct have been established in a particular mouse background, crosses to different backgrounds or to mice carrying other transgenes or knockouts can be carried out to identify possible modifying genes or to investigate components of cellular pathways with which T antigens are known or suspected to interact. Tumors induced by T antigen transgenes, though they arise in animals of homogeneous background, may be heterogeneous and depend on stochastic somatic events. An extensive literature describes studies with T antigen transgenic mice. Several studies are noted here to indicate how this approach has been used to broaden the analysis of T antigen functions and to establish mouse models of specific kinds of cancer. T transgenic mice have also been used in studies of immune responses to endogenous transgenes and in relation to various aspects of tumor immunity (Schell et al. 2000; Schell et al. 1999; Staveley-O’Carroll et al. 2003; Tevethia et al.
L.
BENJAMIN
1992; Zheng et al. 2002). A transgenic mouse expressing human MHC class I has been used to define an HLAA2-restricted epitope from SV40 large T antigen (Schell et al. 2001).
A.
SV40 and JCV T Antigens
Key functions of SV40 large T antigen are based on its ability to bind and disrupt the actions of pRb and p53. These interactions lead to overriding the G1 checkpoint in the cell cycle and to blocking apoptosis. They are critical for both virus replication and transformation. When expressed as a transgene with elements of the natural viral enhancer, SV40 large T induces choroid plexus tumors (Palmiter et al. 1985). Transfer of this SV40 large T transgene to a different mouse background alters the level of T expression and time of appearance of choroid plexus tumors (Cho et al. 1989). Various heterologous promoters have been used to direct SV40 large T expression to liver (Sandgren et al. 1989), prostate (Geenberg et al. 1995), exocrine pancreas (Ornitz et al. 1987), testis (Peschon et al. 1992), bone and heart (Behringer et al. 1988), and lymphoid cells (Saenz Robles et al. 1994) and to drive tumor formation in these tissues. Osteogenic sarcomas develop late in life in mice expressing SV40 large T under the α-amylase promoter; tumor development in this system is accompanied by loss of CTL responses and acquisition of tolerance to specific large T epitopes (Schell et al. 2000). Mutant and truncated forms of SV40 large T have also been studied as transgenes. Choroid plexus tumors can be induced by an amino terminal fragment that interacts with pRb but not with p53. However, these tumors are slower growing than those induced by full-length large T, due most likely to the inability of the truncated form to counteract p53-dependent apoptosis (Symonds et al. 1994). The amino terminal fragment (residues 1–121) under control of the lymphotropic polyoma virus promoter and enhancer is unable to induce lymphoid tumors, again indicating a requirement for p53-binding (Saenz Robles et al. 1994). A similar fragment (residues 1–127) under control of the elastase-1 promoter induces pancreatic acinar tumors (Tevethia et al. 1997). Mice expressing wild-type SV40 large T under the control of the rat insulin promoter develop pancreatic islet cell carcinomas (Hanahan 1985). This so called “RIP-Tag” model has been exploited in several studies of SV40 large T functions and interactions with host pathways. The islet cell tumors develop through well-characterized stages of progression, including acquisition of an angiogenic phenotype (Folkman and Hanahan 1991) and upregulation of insulin-like growth factor-2 production (Christofori et al. 1995). Complexes of SV40 large T with p53 are evident in the earliest stages of tumor development (Efrat et al. 1987). Interestingly, p53, either by itself or bound to large T, may play a positive role in the development of these islet cell tumors, since the tumors are smaller on a p53 null
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background (Herzig et al. 1999). Independently established RIP-Tag lines and outcrosses to different mouse strains reveal effects of the host genetic background and of timing of expression of the transgene (Hager et al. 2004). Candidate modifying genes of the host that are either lost or amplified during tumor development have been identified (Hodgson et al. 2001). Loss of telomerase does little to affect tumor development in this system, although telomeres appear to be maintained in the tumors by a telomerase-independent mechanism (Argilla et al. 2004). The human neurotropic polyoma virus JCV is the etiological agent of the rare but fatal demyelinating disease progressive multifocal leukoencephalopathy. Questions have been raised about the possible association of JCV with certain human tumors (Khalili et al. 2001; White and Khalili 2004). Transgenic mice expressing the large T antigen of JCV have been shown to develop pituitary tumors and neurofibromatosislike tumors (Gordon et al. 2000; Shollar et al. 2004). The role of the onset and duration of T antigen expression required for tumor development has been investigated in another system in which the SV40 large T protein is expressed under control of a tetracycline-regulated promoter. In these studies, salivary gland hyperplasia develops in a large T–dependent manner. Initiation and early stages of tumor development are dependent on expression of the SV40 large T, such that silencing of its expression results in reversal of the hyperplasia. However, if large T is allowed to act for longer periods of time before being silenced, the hyperplasia persists, indicating that the viral oncogene is no longer needed (Ewald et al. 1996). This and similar systems should be useful for investigating events of tumor progression dependent on p53 inactivation or other possible mechanisms. Mice expressing the SV40 small T antigen along with large T, each directed by the mouse mammary tumor virus long terminal repeat, develop lung and kidney tumors in addition to lymphomas. This is in contrast to mice expressing large T by itself, which develop only lymphomas (Choi et al. 1988). This demonstration of cooperativity between the small and large T proteins in tumorigenesis extends the results on cooperation in cell transformation (Bikel et al. 1987). Small T may contribute to tumorigenesis by several mechanisms, including an anti-apoptotic effect, possibly via Akt, as indicated by studies of hepatocarcinogenesis in transgenic mice expressing large and small T antigens (Gillet et al. 2001). Another approach to regulating expression of T antigen transgenes has been developed using the “cre recombinase.” In this system, the recombinase is used to activate expression of “dormant” T antigen transgenes in a tissue-specific manner. Activation occurs following excision of sequences surrounding the inactive transgene. This system has been used to target expression of large and small T of SV40 and middle T of Py, leading to the development of specific tumors depending on the promoter for the recombinase (Politi, Kljuic, et al. 2004; Politi, Szabolcs, et al. 2004).
B.
Polyoma T Antigens
Polyoma T antigens have been established as transgenes, using viral regulatory sequences as well as heterologous promoters (Bautch et al. 1989; Rassoulzadegan et al. 1990). Attention has focused on the middle T antigen as the major viral transforming protein. Several studies using different promoters to drive middle T expression reported hemangiomas as the sole or major tumor type (Bautch et al. 1987; Gottlieb and Villarreal 2001; Kiefer et al. 1994; Williams et al. 1988). Interestingly, in transplantation experiments, endothelioma cells expressing middle T could induce hemangiomas by recruitment of normal host cells (Williams et al. 1989). When the entire Py early region was expressed as a multifunctional transgene, vascular tumors arose, but these were mainly lymphangiomas rather than hemangiomas. Fibrosarcomas and osteosarcomas were also found in some but not all transgenic mouse lineages (Wang and Bautch 1991). Interestingly, the tumor spectrum in these mice was less broad than found with highly tumorigenic strains of virus. In particular, no epithelial tumors were found. It thus appears that the virus gains access to a wider range of tissues through interaction with broadly expressed cell receptors and that the expression of T antigen transgene(s) is somehow restricted to specific lineages based either on the promoter or possibly the site of integration. Several studies have shown that T antigen transgenes are expressed in nontumor as well as tumor tissues, indicating that manifestations of T antigen expression are subject to tissue-specific factors or that tumor formation may depend on secondary events. Middle T transgenic mice using the MMTV promoter and enhancer to drive middle T expression have provided a wellstudied model of mammary tumorigenesis (Guy et al. 1992). The mammary adenocarcinomas that arise in these mice are capable of invasive growth and metastasis. The development of mammary tumors is greatly reduced when these transgenic mice are crossed to knockout mice lacking the c-src protooncogene (Guy et al. 1994). Late-appearing hyperplastic lesions in src-deficient mice express middle T in association with the srcrelated tyrosine kinase c-yes. These findings are consistent with c-src being the major target and partner of middle T in tumorigenesis, as found earlier for cell transformation. The progenitor cell(s) that give rise to tumors in MMTV-middle T transgenic mice have not been identified, although they apparently differ from the early progenitor cells found in Wnt-1-induced mammary tumors (Li, Welm, et al. 2003). That Py-induced mammary tumors are not entirely estrogen dependent is clear from the induction of these tumors in male as well as female mice (Freund, Dawe, et al. 1992). Ovariectomy prior to virus infection decreased the incidence of mammary tumors in young female athymic mice but had little effect if carried out at the time of or after virus infection, suggesting that the initiation of tumor formation requires ovarian hormones to maintain mammary epithelium as the target, but
128 that the tumors have little or no hormone dependence once they arise (Rondinelli et al. 1995). Progression of mammary tumors in female MMTV– middle T transgenic animals may be accompanied by loss of estrogen and progesterone receptors (Lin et al. 2003), although studies in the same system show tumor growth and angiogenesis to be estrogen dependent (Dabrosin et al. 2003). Mammary tumors in MMTV–middle T mice progress from carcinoma in situ to invasive carcinomas and metastasis through stages similar to those in human breast cancer. In addition to their histological similarities, these tumors show a number of molecular phenotypes found in human breast tumors (Lin et al. 2003). Activation of the matrix metalloproteinase-9 promoter is found in invasive but not preinvasive tumors in this system (Kupferman et al. 2000). When crossed to insulin receptor substrate (IRS-2)-deficient mice, tumor incidence is unaffected but the frequency of metastasis is reduced; tumors from these mice also failed to metastasize when transplanted to mammary fat pads of normal mice, indicating the dependence on endogenous expression of IRS-2 by the tumor cells themselves (Nagle et al. 2004). A role of tumor-associated macrophages in promoting progression has been shown by crossing MMTV–middle T mice with mice deficient in macrophage function (Lin and Pollard 2004). Blood vessels in mammary tumors show differences from vessels in normal tissues of the transgenic mice, as shown by the preferential homing of suitably marked transplanted bone marrow cells to the tumors (Dwenger et al. 2004). The MMTV–middle T transgenic mouse has been used in studies of immunotherapy, using adenoviral vectors expressing various cytokines (Emtage et al. 1999; Putzer et al. 1998). Middle T expression has been directed to the pancreas using the “RCAS-TVA” system (Lewis et al. 2003). Transgenic mice that are Ink4a/Arf null and express the receptor for subgroup A avian retroviruses in acinar cells of the pancreas are infected intraperitoneally with an avian retrovirus vector (Fisher et al. 1999) engineered to express Py middle T. These mice develop a range of histologically heterogeneous acinar and ductal tumors. Transgenic mice expressing Py large T have been established, but not all of these show signs of tumor development. However, mice expressing large T under the control of the insulin promoter develop β cell tumors, and others have been found to develop pituitary tumors (Bautch et al. 1989). An immortalizing rather than a transforming function has been ascribed to large T in astroglial cells from brains of large T transgenic mice; these mice have been used to develop established cell lines (Galiana et al. 1990). Independently established large T transgenic mice show neurological symptoms coupled with an apparent block in oligodendroglial differentiation and function (Baron-Van Evercooren et al. 1992). Testicular tumors involving Sertoli cells have also been described in Py large T transgenic mice under control of the viral enhancer and promoter (Lopez et al. 1999; Paquis-Flucklinger et al. 1993).
THOMAS
C.
L.
BENJAMIN
Use of Polyoma Virus Regulatory Sequences in Transgene Expression
The roles of noncoding sequences in polyoma viruses have been investigated through construction of chimeric transgenes with viral enhancer and promoter elements linked to various coding sequences. Different viral enhancers have been shown to have different effects in regulating transgene expression. Thus, enhancer/promoter sequences from a strain of Py that grows in embryonal carcinoma cells in vitro were able to drive expression of a bacterial reporter gene in early mouse embryos, while sequences from a standard Py strain that was unable to replicate in the same cells could not (Krippl et al. 1988). The growth of the human polyomavirus JCV is largely restricted to glial cells in vivo and in vitro. Mice expressing chimeric large T constructs in which the enhancer/promoter regions of JC and SV40 were exchanged showed pathologies to be determined in part by the enhancer regions (Feigenbaum et al. 1992).
X.
THE PNEUMOTROPIC VIRUS OF MICE
A polyomavirus that induces a fatal interstitial pneumonia in newborn mice has been isolated from both laboratory and wild mice (Greenlee 1979; Kilham and Murphy 1953). Originally designated the K or Kilham virus, it is now referred to as the murine pneumotropic virus or MptV. Early studies showed MptV to have a morphology essentially indistinguishable from that of Py and SV40 (Mattern et al. 1963). The viral DNA showed some features in common with that of Py but was clearly different in sequence and in terms of the antigenic properties of its T and V antigens (Bond et al. 1978; Law et al. 1979). The VP1 capsid protein of MPtV major is serologically distinct from that of Py, and MPtV virus-like particles show sialic acid–independent binding to cells (Tegerstedt et al. 2003). The MptV genome is around 4.8 kb, slightly smaller than that of Py, and unlike the latter, does not encode a middle T antigen (Mayer and Dories 1991). Its large and small T antigens show distinct homology with Py, but its noncoding region is quite distinct (Mayer and Dorries 1991). PCR cloning and sequencing of the enhancer region of MPtV from lung tissue of infected mice show heterogeneity in the viral regulatory sequences. Interestingly, these studies provide evidence for incorporation of host sequences into the viral enhancer (Zhang and Magnusson 2001, 2003). MPtV has thus far proven difficult to study in cell culture, although a quantitative assay based on immunofluorescent staining for V antigen has been developed using cultured mouse embryo cells (Greenlee et al. 1982). The virus can be successfully passaged in suckling mice, and its interactions and pathogenicity in newborn and adult mice have been studied. Newborn animals inoculated with the virus show extensive
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replication in vascular endothelium, primarily in the lung and liver but also in other organs (Greenlee 1979; Greenlee et al. 1994). Following immunosuppression of persistently infected adult mice, the virus is reactivated and, like Py, replicates primarily in renal tubular epithelium (Greenlee and Dodd 1984; Greenlee et al. 1991). Studies in adult athymic nude mice show that these mice become persistently infected and remain largely asymptomatic. B cell responses are important in controlling infection in these mice, and adoptive transfer of spleen cells from infected adult mice can protect newborns inoculated with normally fatal doses of virus (Greenlee 1986; Mokhtarian and Shah 1983). MPtV is not oncogenic in its natural host (Parsons 1963). Its T antigens have not been studied directly in terms of their transforming potential and interactions with cellular proteins. However, cultures of lung cells isolated from acutely infected mice give rise to transformed cells that express nuclear T antigen and are tumorigenic (Kanda and Takemoto 1984; Takemoto and Fabisch 1970). Lytic lesions are found in capillary endothelial cells in brains of neonatally infected mice (Ikeda et al. 1988). Infection of embryonic glial cells in culture followed by longterm cultivation gives rise to transformed cells (Greenlee and Law 1984, 1985).
XI.
PERSPECTIVES AND CHALLENGES
Polyomaviruses generally establish persistent silent infections in their natural hosts. In contrast to these usually benign natural infections is a wide range of pathological conditions that these viruses are capable of inducing. The broad pathogenic potential of polyomaviruses is particularly remarkable in view of the limited coding capacity of their genomes. The Py-mouse system presents a useful and challenging model in experimental cancer research. Infection of newborn mice of certain strains leads to the rapid development of solid tumors (Section II). Studies of this highly oncogenic virus derive advantages from the relative ease of propagation and manipulation of the virus in culture and from the availability of diverse inbred strains of mice with widely differing responses to the virus. The Py structural proteins provide a delivery system that operates over a broad range of cell types (Section V). Regulatory sequences in the viral DNA, by presenting binding sequences for cellular transcription factors, assure efficient expression of the virus in a variety of target cells (Section VII). The T antigens act by binding and altering the functions of key regulatory proteins of the cell, resulting in either lytic destruction or neoplastic transformation (Section VI). Two important directions for future work are apparent. One concerns further investigations of the T antigens and their cellular targets. The other focuses on identifying discrete effects of the host genetic background that result in resistance or susceptibility to disease.
129 Py tumors manifest features of early cancer development in dramatic fashion—viz., events of initiation leading to loss of growth control, angiogenesis, and rapid local tumor expansion (Sections III and IV). Interesting questions remain concerning the frequent presence in Py-infected mice of occult as well as overt tumors. This may reflect the elaboration of systemically acting anti-angiogenic factors by the large tumors (Hahnfeldt et al. 1999) or the absence of some local stochastic event necessary for the acquisition of an angiogenic phenotype by the microscopic tumors. Invasion and metastasis are generally not prominent features of Py tumors, but they do occur in certain mouse backgrounds and in some transgenic mice expressing Py T antigens (Section IX). Mechanisms of evasion of the host immune system can be studied using mice of different genetic backgrounds (Section IV-C and Section VIII). Other features of tumor induction by Py raise challenges because they run counter to generally accepted views on cancer development. These include the absence of a requirement for immortalizing function(s) and the apparent lack of dependence on p53-inactivation or other factors leading to genomic instability (Section VI-D). Do Py tumors arise in a manner dictated entirely by the T antigens and independent of cellular mutations? Does the failure of most primary Py tumors to become invasive and metastatic reflect the absence of a mechanism that drives genomic instability? Evidence indicates that Py tumors become locally invasive and metastatic upon serial transplantation, opening the way for investigations into the mechanism(s) of progression. Further challenges lie in understanding the functions of the T antigens as they affect tumor development in various tissues (Section VI). Protein kinase pathways, pathways of phospholipid synthesis and turnover (phosphatidylinositol-3 kinase and phospholipase C), and actions of the retinoblastoma and possibly other tumor suppressor proteins are normal cellular processes affected by the Py T antigens. Genetic alterations in these pathways are common in human cancers. Functions of the T antigens in cell transformation (Section IV) are commonly regarded as being essential for tumor induction. However, studies with Py mutants blocked in certain T antigencell protein interactions have shown that these functions associated with transformation in vitro are not required for induction of tumors in mice (Section VI-D). While established fibroblasts are generally used to define transforming functions, various cell types, predominantly epithelial, constitute targets of tumor induction in the animal. The effects of T antigen expression are manifested differently depending on the tissue. The outcome of infection thus depends on a superposition of T antigen functions and particular patterns of gene expression in different target cells (Section VI-D). Discordant findings in comparisons of cell transformation and tumor induction serve to emphasize that the actions of the T antigens are not fully understood and suggest the likelihood that additional cellular targets and pathways remain to be identified. The discovery of p53 as a binding partner of the SV40 large T antigen (Lane and Crawford 1979; Linzer and Levine 1979)
130 illustrates the importance of identifying cellular proteins that interact with polyomavirus T antigens. Screening procedures based directly on physical interaction should prove useful in identifying new cellular proteins that bind to the viral T antigens. A biological approach has been devised to uncover new targets and pathways of the T antigens. Host range mutants that have lost the ability to replicate in normal cells while retaining some ability to replicate in particular tumor cells have been selected (Section VI-C). One such “tumor host range” or THR mutant has been used to identify the homeotic transcription factor Sal2 as a target of the large T antigen. The functions of the Sal2 protein overlap in part with those of p53 with respect to pro-apoptotic and growth arrest properties. Its tissue distribution is similar in the mouse and human. The protein is abundantly expressed in surface epithelial cells of the normal ovary. Expression is frequently lost or reduced in human ovarian carcinomas and also in other tumor types derived from cells that normally express the protein (unpublished results). Cellular proteins targeted by the Py T antigens may have functions relevant to non-neoplastic diseases as well as to cancer. Studies of another Py THR mutant have led to the identification of the transcriptional co-activator TAZ as a binding target of all three T antigens. Py mutants that fail to bind TAZ are unable to replicate or to induce tumors. A knockout of TAZ in the mouse results in partial neonatal and perinatal lethality (unpublished results). Surviving mice do not develop any recognizable tumors but show severe abnormalities in both lung and kidney. Further studies on the infection process at the cell and molecular levels are needed. These include virus-receptor interactions, identification of cellular factors that mediate virus uptake and intracellular trafficking, steps of virus disassembly, mechanism of crossing a cell membrane, and nuclear entry (Sections V and VIII-A). By reason of their small size, polyomaviruses are restricted in their capacity as vehicles for gene delivery. Nevertheless, further work on the pathway(s) of cell and nuclear entry by Py is needed for the development of these viruses and others as vectors for gene or protein delivery. A difficult challenge lies in being able to narrow the naturally broad host range of Py dictated by ubiquitously expressed sialoglycolipid receptors to a more restricted and specific host range based on cell type–specific surface markers. Knowledge of the virus structure and its receptor binding properties is an important aid in engineering Py virus-like particles (Section V). The availability of various inbred strains of mice with well-defined genetic defects affecting essentially all major organ systems should provide useful models for virus-mediated gene and protein therapy. Studies of the genetics of the mouse in relation to Py infections have begun to reveal mechanisms that underlie susceptibility or resistance to tumor development (Section VIII-B). Several forms of susceptibility based on immune evasion and distinct forms of immunological and nonimmunological resistance have been documented. Some of the underlying mechanisms
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have been identified, but much remains unknown. Problems that await further investigation include the role of innate immune responses to the virus, host factors that predispose to invasion and metastasis of particular tumor types, and the genetic basis for resistance to tumor development in specific tissues. Identifying specific genes in the mouse that mediate resistance or susceptibility represent long-term goals. Progress toward these goals should be aided by the currently available and rapidly improving tools of mouse genomics and genetics. The mouse, natural host for two known viruses of the polyoma group (Section X), has also been used in studies of primate and human polyomaviruses. A mouse model of BK-induced nephropathy in kidney transplant patients is underway using the mouse Py virus (Section II, B). Transgenic mice expressing T antigens of SV40 and JCV have allowed investigations of these viral gene products expressed in selected cells in a wholeanimal setting (Section IX). These and similar transgenic systems using conditional and knock-in approaches should continue to be useful. The lingering question of the possible role of SV40 in certain human cancers is unlikely to be settled by the demonstration of viral sequences in the tumors themselves, as the virus is expected to be present far more frequently than any rare disease it might cause (Wong et al. 2002). However, further investigations may derive clues from work in the mouse with Py and SV40 (Sections IV-C 2 and VIII-B). This would entail determining whether patients in question show an HLA bias, whether their T cells respond to T antigen–derived epitopes defined in HLA transgenic mice (Schell et al. 2001), whether they show an innate type 2 cytokine response to SV40 capsid protein, or other immune mechanisms suggesting a specific infectious etiology.
ACKNOWLEDGMENTS The author gratefully acknowledges the help and advice of John Carroll, Jean Dahl, Deborah Hull, Aron Lukacher, Brian Schaffhausen, Satvir Tevethia, and Palanivel Velupillai.
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5. P O LY O M A
VIRUSES
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Chapter 6 Mouse Hepatitis Virus Stephen W. Barthold and Abigail L. Smith
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Natural History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Duration of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Vertical, Germplasm, and Embryonic Stem Cell Transmission . . . . . . . E. Host Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Properties of the Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Virus Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Virus Replication and Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Virus–Host Cell Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Species Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pathogenesis of Respiratory and Enterotropic MHV . . . . . . . . . . . . . . . B. Pathogenesis of Experimental Hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . C. Pathogenesis of Experimental Encephalitis and Demyelination . . . . . . . IV. Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Host Immune Response to Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Immune Response During Experimental Brain Disease . . . . . . . . . . . . . C. Passively Acquired Maternal Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . D. Immunity to Reinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Vaccination Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Immunomodulation by MHV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Isolation and Detection of MHV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Isolation and Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Serodiagnosis and Antigenic Interrelationships . . . . . . . . . . . . . . . . . . . . C. Mouse Antibody Production (MAP) Test . . . . . . . . . . . . . . . . . . . . . . . . D. Molecular Methods of Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Diagnostic Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Gross and Microscopic Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Immunohistochemistry and In Situ Hybridization . . . . . . . . . . . . . . . . . VII. Surveillance and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Control of MHV in Mouse Populations . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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S T E P H E N W. B A R T H O L D A N D A B I G A I L L . S M I T H
I.
INTRODUCTION
Since the last edition of this series, the mouse hepatitis virus (MHV) literature has burgeoned, but much of this work has had relatively little direct relevance to MHV as a naturally occurring pathogen in laboratory mouse populations. This chapter has therefore been a challenge, with an attempt to provide an overview of the important aspects of MHV experimental biology while emphasizing the virus as a naturally occurring mouse pathogen. Even this has been complicated, as it is impossible to generalize with this virus, due to marked differences in the biologic behavior of the myriad virus strains, plus the marked effects of a large number of host factors. MHV was initially discovered in 1949, and continues to be among the most prevalent infections in mouse populations to this day. It is now known that the MHV group is represented by numerous variants that are constantly mutating, and that these viruses can be biologically separated into respiratory (polytropic) MHVs and enterotropic MHVs, with distinctly different patterns of tissue tropism. This dichotomy is emphasized for the purpose of discussion, but biology is never absolute.
A.
History
An excellent review of the early history of MHV was written by Marcello Piazza (Piazza 1969). At that time, MHV was yet to be classified, but it fell within a group of agents with similar size, growth characteristics in cell culture, sensitivity to ether and other chemicals, antigenic cross-reactivity (serum neutralization and complement fixation), and pathogenic characteristics, including the ability to produce encephalitis and/or hepatitis when inoculated intracerebrally or intraperitoneally into mice. Not all isolates produced encephalitis, and there was growing recognition of other viruses, such as reoviruses, that also caused hepatoencephalitis when inoculated into mice. Thus, the appellation “mouse hepatitis virus” was born. At that time, enterotropic MHVs were not known to belong to the MHV group. The first MHV to be discovered was named “JHM” after J. Howard Mueller at Harvard University. The virus caused encephalitis and hepatitis (Cheever et al. 1949). The discovery was made during investigation of diarrheal disease in infant mice. Viral diarrhea was a recognized entity in mice during these early years, and multiple syndromes and etiologies were being identified. Among the enteric diseases that were recognized were epizootic diarrhea of infant mice (EDIM) and lethal intestinal virus of infant mice (LIVIM), with distinctly different patterns of disease (Kraft 1966). A number of early MHV isolates were discovered and studied in mice that were co-infected with LIVIM, but for many years LIVIM was considered to be a separate entity from MHV. LIVIM did not produce hepatitis or encephalitis, did not grow in MHV-susceptible cells in vitro, and produced a distinctive enteritis in young mice that was not
observed with known MHV isolates (Biggers et al. 1964). It was three decades after the first report of MHV-JHM in 1949 before it was recognized that the enteritis caused by LIVIMlike agents was due to enterotropic MHVs. It is now known that enterotropic MHVs belong to the MHV group (and may actually be the most common type of MHV), but they have distinct enterotropic characteristics that differentiate them from the respiratory (polytropic) MHV strains (Barthold et al. 1982; Broderson et al. 1976; Carthew 1977; Hierholzer et al. 1979; Ishida and Fujiwara 1979; Ishida, Taguchi, et al. 1978; Sugiyama and Amano 1980). Many of the early MHV isolations were made as a result of contamination of biological materials (serum, tissue, ascites tumors, leukemia virus stocks and tumor lines, cell lines, etc.), which correlated with their polytropic biologic behavior. Their stated tropisms frequently were the result of the investigators’ research interests (e.g., hepatotropism, neurotropism), and many have therefore been subjected to selective passage that favors a particular tissue tropism. The history of a few of the important prototype strains, which have undergone extensive passage in the laboratory, follows. All MHV isolates are related genetically and antigenically, but isolates can be differentiated by genetic sequencing, cross-serum neutralization, or with monoclonal antibodies. Genetic and antigenic relationships are not predictive of biologic behavior (tissue tropism, virulence, etc.). For example, MHV-JHM and MHV-S are relatively divergent genetically and antigenically, yet produce similar patterns of disease, and MHV-S and MHV-S/CDC are very closely related, but produce different patterns of disease (Barthold and Smith 1984; Hierholzer et al. 1979; Lai et al. 1983). Early isolates included MHV-JHM (also known as MHV-4, in spite of it being the first isolate), -1, -2 (-Pr), -3, -A59, EHF120, -H747, -S, -Braunsteiner and Friend, -Balb C, -SR1, -SR2, -SR3, and -SR4, among others (Piazza 1969). Some of these isolates have been maintained as “prototype” MHV strains for experimental and diagnostic purposes. A brief history of several of these prototype strains of MHV is provided, not only to set a foundation for where commonly studied strains of MHV originated but also to underscore their discovery as agents that not only caused disease in mice but also disrupted research. ●
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MHV-JHM (MHV-4) was isolated during studies of an outbreak of diarrheal disease in suckling Swiss mice. In addition to disseminated lesions, including hepatitis, mice exhibited flaccid paralysis, which prompted serial passage of brain material by intracerebral inoculation. Lesions were reproduced that were identical to those in the index mice (Cheever et al. 1949). MHV-JHM and all its various iterations (laboratory variants, substrains, and mutants) have been extensively utilized experimentally as a “neurotropic” MHV strain. MHV-1 was discovered in P (Parkes) mice during an outbreak in 1950 at the National Institute for Medical Research (Mill Hill, London). Mice that died had liver necrosis, and affected livers contained enterococci. Because enterococci rarely cause
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disease, the investigators reasoned that a second agent, possibly a virus, was responsible. Liver homogenates were filtered, and material caused hepatic disease after the fourth passage. Subsequently, the virus stock was shown to be contaminated with Eperythrozoon coccoides and the combination caused fatal hepatitis, whereas the virus alone caused only mild hepatitis (Gledhill 1956; Gledhill and Andrewes 1951). MHV-1 has not been extensively studied, but is often included in comparative genetic and antigenic studies among MHV strains. MHV-2 (MHV-Pr) was isolated from Princeton (PRI) mice during the course of leukemia studies (Nelson 1952a, b). It was noted that the weanling mice were not becoming leukemic and that some mice died during the 12th passage of the “leukemic” cells. Those mice had severe hepatitis, and passage of liver material in PRI mice resulted in the same syndrome noted in the index mice. This virus contributed to seminal work on host genetic resistance and susceptibility by Fred Bang. The in vivo susceptibility of different strains of mice was mirrored by the response of peritoneal or hepatic macrophage cultures derived from mice that were susceptible (PRI), resistant (C3H), or moderately susceptible (BSVS) (Bang and Warwick 1960). MHV-2 continues to be of interest as an MHV strain that is “hepatotropic” and nonfusigenic (at least in most stocks of MHV-2), and therefore does not induce the hallmark syncytia seen with other MHV strains during infection in vitro or in vivo (Keck, Soe, et al. 1988). MHV-3 was found during the course of efforts to transmit human viral hepatitis with human patient serum inoculated into Swiss Webster (then called VS) mice. Inoculated mice were subsequently administered a hypertonic solution of glucose. One mouse died and its liver was severely damaged. Serial passages of infectious material caused severe hepatitis in weanling mice. When additional mice were inoculated with the human material, they did not show clinical signs of hepatitis, so the authors concluded that the responsible agent was of mouse origin (Dick et al. 1956). MHV-3 has been extensively studied as a virulent “hepatotropic” MHV strain. MHV-A59 was discovered during the serial passage of the Moloney leukemia agent in BALB/c mice (Manaker et al. 1961). After 30 passages of pooled organ homogenates, the mice were hunched with ruffled hair coats. After two additional passages, livers of the mice were pale with white foci. MHV-A59 was isolated from the spleens and livers of these mice. MHV-A59 grows robustly in culture, has been studied as a prototype virus for virus receptor, hepatitis, and encephalitis research, and is a common strain for use as an antigen for serodiagnostics. MHV-S was isolated from newborn CD-1 Swiss mice that were cesarean derived and maintained within a strict barrier. Mice were noted to develop high mortality after contact with conventional mice of several stocks. Their livers were affected, and a pool of viscera, excluding intestine, yielded MHV-S. The virus was shown to be highly contagious and
present in the feces of various stocks of mice (Rowe et al. 1963). Weanling mice inoculated intraperitoneally with feces from these mice became paralyzed. Intraperitoneal or intracerebral inoculation of CD-1 mice with the virus resulted in hepatitis and encephalitis, but the virus did not kill mice after serial passage in cultured cells. Researchers reasoned that either the virus that was in feces may have become less virulent after multiple passages or the mortality in CD-1 mice was caused by a contaminating enteric agent, LIVIM (Kraft 1962), which did not replicate in cultured cells. MHV-S is now known as a relatively nonpathogenic MHV strain and not highly contagious. Thus, its early reported characteristics as a highly contagious enteric agent is probably attributable to co-infection with LIVIM, or perhaps artificial selection of a hepatotropic variant (MHV-S) that is a derivative of LIVIM. In addition to the above prototype MHV strains, a number of enterotropic MHV strains have been maintained and studied. The agent of LIVIM has never been isolated, but the realization that LIVIM was related to MHV occurred with the isolation of MHV-S/CDC. As the name implies, MHV-S/CDC was found to be serologically closely related to MHV-S. It was isolated from pooled intestinal homogenates during an outbreak of lethal enteritis in suckling mice manifesting features resembling those described for LIVIM. Virus propagated in cultured NCTC 1469 cells produced enteric disease commensurate with lesions that had been previously described for LIVIM in suckling mice (Broderson et al. 1976; Hierholzer et al. 1979). The realization that LIVIM was related to MHV occurred simultaneously in another laboratory (Carthew 1977). Although LIVIM is not available for analysis, it was clearly an enterotropic MHV, based upon descriptions of the pathology in infant mice (Biggers et al. 1964; Kraft 1962, 1966). Subsequent isolations of enterotropic MHV soon followed, including MHV-D (Ishida and Fujiwara 1979; Ishida, Taguchi, et al. 1978), MHV-DVIM (diarrhea virus of infant mice) (Sugiyama and Amano 1980), MHV-Y (Barthold et al. 1982), and MHV-RI (Barthold et al. 1985), among others. Like other MHV strains, these enterotropic MHV strains vary in their genetic and antigenic relatedness to each other. In other words, like respiratory MHV strains and isolates, “LIVIM” is not one virus.
B.
Natural History
The two basic MHV biotypes, respiratory or enterotropic, have primary tropism for either upper respiratory mucosa (respiratory MHV strains) or intestinal epithelium (enterotropic MHV strains). Respiratory strains, which replicate in nasal epithelium, need not disseminate to other organs to be successfully transmitted to other mice. However, nasal virus titers tend to be low and, thus, direct contact is needed for transmission. Enterotropic strains, which replicate in intestinal mucosal epithelium, also need not disseminate to other organs to be transmitted to
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other mice. Enterotropic MHV strains tend to be excreted in very high titer in feces, regardless of the age of the host, and thus tend to be highly contagious. The differences between respiratory and enterotropic MHVs are most clearly manifested when susceptible infant mice are inoculated oronasally. Under such circumstances, respiratory MHVs disseminate to multiple organs (polytropism), whereas enterotropic MHVs tend to be restricted to intestinal mucosa (enterotropism) (Barthold and Smith 1984). Respiratory strains, when afforded the opportunity to disseminate in susceptible hosts, have nonselective tropism for many tissues and thus are the strains that have been frequently associated with contamination of serum, tumors, cell lines, virus stocks, and other biologic material of mouse origin. MHV is extremely prevalent not only in laboratory mice but also among wild mice. Wild Mus domesticus in Australia are frequently infected, based upon serologic surveys. Similar surveys of several wild mouse populations along the East Coast of the United States (Maine to Virginia) have confirmed the ubiquity of MHV. Analysis of weaning-age, wild-trapped mice by histopathology has suggested that enterotropic MHV is present, but the biotypes of MHV among wild mice have not been otherwise characterized (Smith and Barthold, unpublished observations). Thus, both feral and wild mice pose a high risk of introducing MHV into laboratory mouse populations. It is emphasized that the MHV respiratory-enteric biotype dichotomy is only relative, with considerable overlap between the two groups. MHV-DVIM, for example, not only causes enteric infections but also disseminates to liver and other organs (Sugiyama and Amano 1980), whereas MHV-Y and MHV-RI are more restricted for intestinal mucosa (Barthold and Smith 1984; Barthold et al. 1982, 1985). Furthermore, studies with congenic B6 wild-type, B cell–deficient, and T cell–deficient mice have shown that enterotropic MHV-Y was restricted to intestine in wild-type and B cell–deficient mice, but disseminated and produced hepatitis in T cell–deficient mice (Compton, Ball-Goodrich, Paturzo, et al. 2004). Patterns of tissue tropism in naturally infected nude mice suggest that strictly enterotropic, enterotropic-polytropic (disseminated), and non-enterotropic-polytropic patterns of disease are apparent in nude mouse sentinels exposed to different enzootically infected mouse colonies (Homberger et al. 1998). These observations lead to conjecture about “which came first, the chicken or the egg”— enterotropic or respiratory MHV?
C.
Duration of Infection
Duration of MHV infection in mice is dependent upon MHV strain, route of inoculation, and host factors, including age, immunocompetence, passive immunity, genetic strain, and genetic alterations. Although persistent infection of the central nervous system can be induced experimentally in immunocompetent mice, especially following intracerebral inoculation, this is not likely to have practical significance under natural conditions.
Experimental studies, using both respiratory and enterotropic MHV, have shown that genetically susceptible BALB/c and resistant CD-1 immunocompetent mice effectively recovered from infection when inoculated oronasally with MHV-JHM, -1, or –S by 1 month. Treatment with hydrocortisone or cyclophosphamide while mice were still infected exacerbated infection, but had no effect after recovery and did not reactivate virus (Barthold and Smith 1990). Subsequent studies confirmed these findings, using intranasal MHV-JHM inoculation of genetically susceptible BALB/c and resistant SJL mice at 1, 3, 6, or 12 weeks of age. Virus titers peaked in various organs at 3–5 days after inoculation, and were subsequently cleared in SJL mice by day 10, regardless of age at inoculation. In contrast, in surviving BALB mice inoculated at 6 weeks of age, virus was cleared from most organs by 30 days and by day 60 in brain. Thus, age and genotype were significant factors, but both strains of mice completely recovered from infection if they survived the acute disease (Barthold and Smith 1987). Similar studies have been performed with enterotropic MHV, using BALB/c and SJL mice inoculated oronasally at 1, 3, or 12 weeks of age with MHV-Y. Most mice recovered by day 20, but low titers of virus were detectable in a few mice of all ages and both genotypes at day 30 (Barthold et al. 1993). The above studies suggest that most mice recover from MHV infection, but duration of infection may vary and last for weeks, even under experimental conditions. The practical significance of these findings relates to how long a mouse can transmit infectious virus. Studies of naturally infected transgenic, null mutant, and wild-type mice have indicated that soiled bedding from various types of MHV seropositive mice could transmit virus for at least 10 weeks. Most of the mice with persistent viral excretion were genetically altered mice, but they were phenotypically normal, most were heterozygous, and they were genetically engineered in loci that did not have expected immune effects (Smith et al. 2002). Transgenic mice with altered T cell function have been shown to transmit MHV for 2 years (Rehg et al. 2001), and transmission of apparently enterotropic MHV by B6-tumor necrosis factor (TNF) null mutant mice to sentinels was shown to last over 5 months (Pullium et al. 2003). In a controlled study that utilized BALB/c and B6 mice and B6 congenic B-cell (Igh6 null mutant) and T-cell (Tcr beta/delta null mutant) immunodeficient mice infected oronasally with enterotropic MHV-Y, virus was shown to be transmissible to sentinel mice exposed directly to infected mice or to soiled bedding. B6 wild-type mice transmitted virus for 2 weeks, BALB mice for 4 weeks, T cell–deficient B6 mice for at least 4 weeks (they were euthanized at that interval due to illness), and B cell–deficient B6 mice for over 3 months (Compton, Ball-Goodrich, Paturzo, et al. 2004). A number of studies have shown that interferon-γ null mutant mice become chronically infected with MHV (Compton et al. 2003; France et al. 1999; Kyuwa et al. 2002; Kyuwa, Tagawa, Machii, et al. 1998; Schijns et al. 1996). Considering the growing number of genetically altered mice and the already known unpredictability of virus shedding from
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such mice with or without known immune defects that are naturally infected with MHV (Pullium et al. 2003; Rehg et al. 2001; Smith et al. 2002), it is likely that a growing number of factors will be documented that influence MHV persistence. D.
Vertical, Germplasm, and Embryonic Stem Cell Transmission
In utero transmission of MHV from infected dams to fetuses has been demonstrated experimentally, but it is unlikely to occur under natural conditions in immunocompetent mice for a number of reasons, including the relatively short duration of infection during the breeding life of the dam and the need for coinciding viremic infection with pregnancy, which requires a nonimmune susceptible host and a virulent strain of polytropic virus. Transplacental infection has been demonstrated experimentally following intravenous inoculation of ICR dams with MHVJHM during all three trimesters of pregnancy (Katami et al. 1978). Similar studies have shown transplacental transmission during all three trimesters of pregnancy in BALB/c dams, but not CD-1 dams, inoculated intranasally with MHV-JHM. Less virulent MHV-S could also be transmitted in utero in BALB/c mice, but not as efficiently. Viral antigen was found throughout the placenta, and particularly at the maternal-fetal junction. In contrast, enterotropic MHV-Y, which does not disseminate hematogenously, did not infect BALB/c or CD-1 fetuses in utero. Furthermore, these studies demonstrated that the physiologic state of pregnancy favored higher titers of MHV-JHM in livers of the pregnant dams (Barthold et al. 1988). On the other hand, in utero transmission was not found in NMRI mice inoculated intraperitoneally with MHV-3, even among mice treated with prednisolone (Galanti et al. 1969; Piccinino et al. 1966). Even in the absence of infection of embryos, MHV infection during pregnancy has been shown to affect embryo genotype in heterozygous mice. MHV-infected mice produced more MHCheterozyous embryos than uninfected mice, suggesting selective promotion of certain MHC haplotypes during fertilization (Rulicke et al. 1998). The significance of vertical transmission lies not so much in the risk to the embryo, which if infected would likely die, but more in the risk of introducing MHV through contaminated embryos or germplasm into a virus-free colony during cesarean or embryo transfer rederivation. The zona pellucida affords significant protection to mouse embryos against MHV infection. Zona-intact embryos incubated with MHV for 48 hours as 2-cell embryos or for 1.5 hours as blastocysts were shown to resist infection, but embryos devoid of the zona pellucida were susceptible to infection. MHV was transmitted to foster dams when uninfected embryos were transferred in medium that was flushed from uterine horns of infected donor dams (Carthew et al. 1985). The danger of this happening is significantly increased when donor dams are immunodeficient and have disseminated infections with respiratory (polytropic) MHV.
In a recent study, disseminated MHV infection was detected in multiple organs of nude mice, including both male and female reproductive organs. Transplantation of ovaries from infected donors transmitted MHV to recipient immunocompetent and immunodeficient mice, but transmission was not observed with sperm from infected testes when used for in vitro fertilization (Scavizzi and Raspa 2004). Embryo transfer and in vitro fertilization with sperm from donors derived from MHV enzootically infected colonies (undefined virus or infection status of donors) have been used successfully for rederivation (Suzuki et al. 1996). Embryonic stem cells also pose a risk of MHV contamination and transmission to recipient dams through blastocyst injection. MHV-A59 and -2 have been shown to replicate in a number of murine embryonic cell lines derived from several genetic backgrounds, and both MHV types did so with no or minimal cytopathic effect (Kyuwa 1997). Notably, in contrast to a persistent carrier state in which only a few cells are infected, studies have shown that all of the ES cells developed a steadystate infection with MHV-2, in which A3-1 cells produced virus while also proliferating and differentiating into embryoid bodies (Okumura et al. 1996). These experimental studies underscore the importance of periodic testing of ES cell lines for adventitious pathogens. Liquid nitrogen storage of ES cells, embryos, and other germplasm can also potentially be a means of cross-contamination with MHV. Liquid nitrogen is not sterile, it can penetrate into cryotubes containing mouse embryos, and accidental transmission of other viruses under such circumstances has been documented. Inadvertent introduction of MHV-infected material into liquid nitrogen tanks is therefore quite possible. A recent study stored mouse embryos and MHV-NuU tubes in the same tanks for 6 or 12 months, with no evidence of MHV contamination of embryos (Kyuwa et al. 2003). A false sense of security could be obtained from such results, as the study utilized 2-cell embryos, which are protected by their zona pellucida, and enterotropic MHV-NuU, which is likely to have limited cell tropism.
E.
Host Range
It is important to emphasize that the most likely source of MHV in mouse colonies is mice (wild, feral, or captive) or mouse products (serum, tumor lines, etc.). Mice are not likely to be natural hosts for other coronaviruses. Nevertheless, the cross-reactive nature of coronavirus antibody in humans, rats, mice, and other species that occurs following natural infection with their respective coronaviruses within Group II (see Section II, A) has led to conjecture regarding the host species specificity of MHV. This has been reinforced by observations that two-way cross-species infections with coronaviruses can occur naturally and experimentally among dogs, cats, and pigs (Reynolds and Gawes 1979; Woods et al. 1981). Humans have been shown to naturally develop serum neutralizing antibody to MHV, which is generally considered to be virus strain–specific
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(Hartley et al. 1963). Intranasal inoculation of infant mice with MHV-S, MHV-Y, rat sialodacryoadenitis virus, human coronavirus OC43, and bovine coronavirus (comembers of Group II) all produced encephalitis in intranasally inoculated infant mice. Transmission of virus by direct contact to naive infant mice, however, occurred only with MHV and rat coronavirus, not with human or bovine coronaviruses. Furthermore, only mice infected by inoculation or contact with MHV developed MHVrelated patterns of infection (Barthold et al. 1990). Thus, even highly susceptible infant mice do not easily favor infection with non-mouse coronaviruses. When MHV was initially being characterized, it was experimentally inoculated intracerebrally into a wide variety of species. Syrian hamsters, rats, cotton rats, but not guinea pigs or rabbits, were found to be susceptible to MHV-JHM inoculation (Bailey et al. 1949; Cheever et al. 1949). MHV-JHM infection induced demyelinating encephalitis following intracerebral inoculation of African Green Monkeys and Owl Monkeys, and after intranasal and intravenous inoculation of Owl Monkeys (Cabirac et al. 1994; Kersting and Pette 1956; Murray et al. 1992). Rats have been used as a model system to investigate pathogenesis of demyelinating encephalitis following intracerebral inoculation with MHV-JHM, -3, and –A59 (Sorensen et al. 1982; Wege et al. 1984). Suckling rats can be asymptomatically infected with MHV-S following intranasal inoculation, with virus replication in nasal mucosa (Taguchi et al. 1979a).
II.
PROPERTIES OF THE VIRUS A.
Classification
The Coronavirus genus, which includes MHV, is within the family Coronaviridae. Members of this family are large enveloped viruses with plus-stranded (positive sense) RNA genomes. The family Coronaviridae contains Coronavirus and Torovirus genera, and is part of the order Nidovirales, which includes two other families, Arteriviridae and Roniviridae, but this classification is still in flux. Nidoviruses share similar replication strategies, but constituent families are significantly divergent, based upon gene sequences (Gonzalez et al. 2003). Members of the genus Coronavirus have the largest genomes of all of the RNA viruses. Three distinct groups of coronaviruses have been identified, each having members that share nucleotide sequence similarity and antigenic interrelationships. Antigenic Group I includes human coronavirus 229E, transmissible gastroenteritis virus of pigs, porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, and rabbit coronavirus. Group II includes MHV, rat coronavirus, human coronavirus OC43, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, rabbit enteric coronavirus, and turkey coronavirus. Group III includes infectious bronchitis virus of chickens (Lai and Holmes 2001).
A newly recognized human coronavirus NL63 is a member of Group I (van der Hoek et al. 2004). There are many other coronaviruses that infect a wide variety of species but have been insufficiently characterized for inclusion within these antigenic groups. Severe acute respiratory syndrome (SARS) virus, for example, is genetically distinct from other known coronaviruses, and is distantly related to Group II and, to a certain extent, Group III coronaviruses. It may represent an entirely new group of coronaviruses that is enzootic in wild animal species in China (Poon et al. 2004). Furthermore, as exemplified by MHV, each of these named coronaviruses actually represents a diverse group of viruses that infect a particular host species. Coronaviruses are often defined by their tissue tropism, but this varies among isolates from a single host species. However, there are common patterns of tissue tropism among all coronaviruses that, like MHV, have primary tropism for either the respiratory tract or the intestine and variable secondary tissue tropism. Enteric coronaviruses of other species, like enterotropic MHV, are associated with neonatal diarrhea in their respective hosts.
B.
Virus Structure
Coronaviruses were named because of their virion morphology, with their characteristic corona of spikes (peplomers) protruding from the surface. With negative-contrast electron microscopy, the images of coronaviruses do not reflect the way they appear in their native state, as the peplomers tend to fall off easily. Therefore, most images of MHV are partly “bald.” Native virions are so heavily covered with peplomers that they may not resemble the classical images that are generally depicted in texts and publications (Fig. 6-1A and 6-1B). The genome encodes nucleocapsid phosphoprotein (N), which forms tubular nucleocapsid strands that bind to viral RNA and lie within the lipoprotein envelope that is formed by budding from intracellular membranes (Sturman and Holmes 1985; Tooze and Tooze 1985). The envelopes contain two major viral glycoproteins: membrane (M) protein (formerly E1) and spike (S) protein (formerly E2) (Sturman and Holmes 1985). A third, envelope (E) small membrane protein (formerly sM), is involved in virus assembly (Raamsman et al. 2000; Yu et al. 1994). Some coronaviruses, particularly members of antigenic Group II, have an additional hemagglutinin-esterase (HE) envelope protein (formerly E3). Some, but not all, strains of MHV possess HE proteins (Yokomori et al. 1991, 1989). The M glycoprotein of MHV binds the membrane to the nucleocapsid within the virion (Sturman et al. 1980). Antibody to the external domain of M can neutralize the virus, but requires the presence of complement (Collins et al. 1982). The S glycoprotein represents the structural protein that creates the prominent spikes on the surface of the virion that form the characteristic “corona.” The S glycoprotein has three structural domains: an N-terminal external domain that is subdivided into
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A
B Fig. 6-1 Negative contrast electron micrographs of human coronavirus 229E (A) and MHV-S/CDC (B). The top micrograph represents the “classical” image of coronaviruses that have lost many of their peplomers during processing, whereas the bottom image depicts the way coronaviruses appear in their native state (Courtesy of F.A. Murphy, University of California, Davis).
S1 and S2, a transmembrane domain, and a short C-terminal cytoplasmic domain. A region between S1 and S2 may (but may not) contain a cluster of protease cleavage motifs. There is considerable diversity in length and sequence of S1 glycoproteins of different MHV strains. The S1 domain contains hypervariable regions that foster large deletions or insertions that are not essential for the structure of the spikes but contribute to antigenic variation, tissue tropism, and virulence (Banner et al. 1990; Daniel et al. 1993; Gallagher et al. 1990; Parker et al. 1989; Wang et al. 1992). The S1 glycoprotein binds to specific host cell receptors, induces neutralizing antibody, binds antibody, and elicits cell-mediated immunity. In addition, organotropism and virulence have been attributed to sequence differences in the S1 gene (Daniel et al. 1993; Gallagher 1997; Gallagher et al. 1990; Hasony and Macnaughton 1981; Holmes et al. 1986; Luytjes et al. 1989; Oleszak et al. 1992; Phillips et al. 1999, 2002;
Stauber et al. 1993; Welsh et al. 1986). S2 is involved in membrane fusion, and can mediate cell fusion without S1 interaction with MHV cell receptor (Krueger et al. 2000; Taguchi and Shimazaki 2000). S2 also has hypervariable regions within its coding sequence (Wang et al. 1992). Although the functions of S1 and S2 can be differentially defined experimentally, they act in concert for efficient infectivity and entry of the virus. MHV cell fusion is mediated through highly conserved heptad repeat 1 (HR1) and HR2 regions of the S protein that function as a class I fusion protein that shares structural and functional features with other viral fusion proteins (Xu et al. 2004). Despite distinct differences in biologic behavior among different MHV strains, attempts to identify characteristics that differentiate respiratory MHV from enterotropic MHV have been unrewarding. Enterotropic MHV strains are morphologically
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and antigenically indistinguishable from other MHV strains. Furthermore, there are no distinctive characteristics in the S genes or the gene products of enterotropic MHV strains that make them unique from other MHV strains (Kunita et al. 1995), nor are there distinctive differences in sequence of N or M genes (Homberger 1994, 1995). Nevertheless, because variation in S genes and their amino acid products has been proven to influence cell tropism and biologic activity of prototype MHV strains in various ways under experimental conditions (Dalziel et al. 1986; Fleming et al. 1986; Gallagher et al. 1990; Gombold et al. 1993; Ontiveros et al. 2003; Parker et al. 1989; Wang et al. 1992; Wege et al. 1988), it is likely that biotype is determined in some way by peplomeric structure or function.
C.
Virus Replication and Mutation
MHV genomic RNAs are 27 to 32 kilobases in size, and are capped and polyadenylated (Lai 1990; Spaan et al. 1988). They can function as messenger RNAs, and are infectious (Lai 1990). The gene order of all coronaviruses consists of polymerase (P), S, E, M, and then N, interspersed by a variable number and order of open reading frames that encode nonstructural proteins of unknown function, as well as a variably present HE protein (Lai and Holmes 2001). The plus-strand viral genomic RNA is transcribed into subgenomic and genomic length minus-strand RNAs, which serve as templates for synthesis of plus-strand viral mRNAs and genomic RNA. There are five to eight subgenomic mRNAs that are numbered in order of decreasing size and form a nested set with a common 3′ end. Although most of the mRNAs contain two or more open reading frames, only the open reading frame at the 5′ end is translated. All subgenomic mRNAs possess a common leader sequence at their 5′ ends that is identical to the leader sequence at the 5′ end of the genomic RNA (Lai 1990; Lai and Holmes 2001; Sturman and Holmes 1983). Viral replication takes place on late endosomal membranes within the cytoplasm of infected cells (van der Meer et al. 1999), with a latent period of only a few hours. Because MHV is an RNA virus, it is naturally prone to a high frequency of mutation. This is due to the high error frequency of RNA polymerases, but is accentuated by the size of the MHV genome. Thus, it has been calculated that MHV genomic RNA probably accumulates several point mutations with each round of replication. Plaque-purified MHV is therefore actually a quasispecies of heterogeneous viruses. These constantly arising variations impact upon antigenicity, pathogenicity, and virulence (Lai and Holmes 2001). The S gene, in particular, possesses hypermutable regions, resulting in mutants that differ significantly in biologic behavior (Dalziel et al. 1986; DasSarma et al. 2000; Fleming et al. 1986; Gallagher et al. 1990; Gombold et al. 1993; Ontiveros et al. 2003; Parker et al. 1989; Wang et al. 1992; Wege et al. 1988). This can be followed in vitro, but there is remarkably high incidence of deletion mutations in the S gene among field isolates of MHV
(Banner et al. 1990; Gallagher et al. 1990; Parker et al. 1989). Therefore, there are significant genetic and antigenic differences among stocks of a single prototype MHV strain (Lai et al. 1983). In addition to mutation, the MHV genome is prone to a high frequency of RNA recombination, and this is related to the segmented subgenomic RNAs that are a feature of coronaviral replication. This is believed to be due to discontinuous transcription and polymerase jumping (Lai 1992). The recombination frequency for the entire RNA genome has been calculated to approach 25% (Baric et al. 1990). Recombination among different MHV strains has been documented in cells that have been co-infected in vitro (Keck et al. 1987; Keck, Soe, et al. 1988; Lai et al. 1985; Makino et al. 1986), but has also been readily documented in experimental MHV infections in mice (Keck, Matsushima, et al. 1988). The variation in the number and order of nonstructural genes within coronavirus genomes suggests that some of these genes have been usurped from cellular mRNAs through nonhomologous recombination with coronavirus genomes (Lai and Holmes 2001). This is believed to be the explanation for the ancient acquisition of the HE gene among many Group II coronaviruses, which has sequence similarity to the influenza virus C hemagglutinin gene (Luytjes et al. 1988). Recombination probably represents the major means by which coronaviruses evolve into variants with novel host species specificity.
D.
Virus–Host Cell Interactions
Several different molecules can function as MHV receptors (Beauchemin et al. 1999; Chen et al. 1995; Dveksler et al. 1991; Nedellec et al. 1994; Williams et al. 1991), among which carcinoembryonic antigen-related cell adhesion molecule 1, or CEACAM1, is the most prevalent (Dveksler et al. 1993). CEACAM1 is an isoform of the mouse biliary glycoprotein 1 gene and has therefore also been referred to as Bgp1 or BgpA (as well as MHVR1, mmCGM1, C-CAM, and CD66a). CEACAM belongs to the Ig superfamily, and possesses four Ig-like loops, a transmembrane region, and a short intracytoplasmic domain. The N-terminal region of MHV S1 binds to the N-terminal Ig-like domain of CEACAM1 (Dveksler et al. 1993), resulting in conformational change in S2 that exposes a domain that initiates fusion with the host cell membrane (Gallagher and Buchmeier 2001). Genetic resistance and susceptibility to MHV-A59 infection is related to allelic variation of CEACAM1 in mice. The functional consequences of these allelic differences have been shown with L2, Sac-, 17CL-1, J774A-1, JLSV9, and primary BALB peritoneal macrophages, all of which are susceptible to MHVA59 infection and all of which express CEACAM1a, whereas SJL peritoneal macrophages, hamster BHK-21 cells, and cell lines from cats, dogs, monkeys, and humans were not susceptible and did not express CEACAM1a (Compton et al. 1992).
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A monoclonal antibody against CEACAM1a significantly reduced virus titers and clinical disease in MHV-A59 infected infant mice, but did not prevent infection (Smith, Cardellichio, et al. 1991). A, C3H, B6, SWR, DBA/2, AKR, CBA, and BALB/c mice, and probably most other inbred strains, are homozygous for the CEACAM1a allele (MHVR1, mmCGM1), whereas CEACAM1b (MHVR2, mmCGm2) is expressed in tissues of SJL mice, and both CEACAM1 alleles are expressed in tissues of outbred Swiss mice (Blau et al. 2001; Chen et al. 1995; Compton 1994; Dveksler et al. 1993; Knobler et al. 1981; Ohtsuka and Taguchi 1997; Smith et al. 1984; Yokomori and Lai 1992a). Differences in genetic susceptibility have been attributed to the fact that CEACAM1a has a receptor binding efficiency that is 300–500 times higher, with 10–30 times higher receptor functionality, compared to CEACAM1b (Ohtsuka et al. 1998, 1996; Rao et al. 1997). Other members of the CEA family, as well as human CEA-related glycoproteins, can also serve as alternate MHV receptors, including CEACAM2 (Chen et al. 1997, 1995; Dveksler et al. 1993; Hensley and Baric 1998; Nedellec et al. 1994; Yokomori and Lai 1994, 1992a). CEACAM2 also has Ig domains, but there is considerable divergence in amino acid sequences of the N-terminus of the protein compared to CEACAM1. CEACAM2 is expressed in a variety of tissues of BALB/c, SJL, C3H, and B6 mice, and has been shown to function as an alternate receptor for several strains of MHV when transfected into hamster BHK cells (Nedellec et al. 1994). In addition, there appear to be additional host-dependent stages for entry of MHV into cells that involve mechanisms other than CEACAM (Asanaka and Lai 1993; Koolen et al. 1983; Yokomori et al. 1993), thereby helping to explain the range of susceptibility among different mouse strains that can be shown at the cellular level. MHV interactions with CEACAMs as specific receptors provide interesting insight into virus-host interactions at the molecular level, but MHV does not necessarily respect such clear explanations. Many MHV strains are neurotropic and nonselectively and readily infect neurons, glia, ependyma, meninges, and vessels within the brain, yet there is a relative paucity of CEACAM molecules in the central nervous system, with the exception of low-level expression on microglia (Chen et al. 1995; Ramakrishna et al. 2004; Yokomori and Lai 1992a). In spite of this, neurons appear to be a key target and determinant of disease (Knobler et al. 1981). A possible explanation is that neurotropic viruses such as MHV-JHM are highly fusogenic and may spread within the central nervous system by S protein– mediated cell-to-cell fusion, rather than through specific cell receptors (Gallagher et al. 1992). Alternatively, non-CEACAM1 CEA-type receptors are also expressed in brain, such as a pregnancy-specific glycoprotein (PSG). When this receptor was expressed in COS-7 cells, which normally lack the MHV receptor, they were rendered susceptible to MHV-A59, -2, and –3, but, unexpectedly, not to neurotropic MHV-JHM (Chen et al. 1995).
SJL mice are reported to be remarkably resistant to infection with MHV-A59 and MHV-JHM because of their absence of CEACAM1a. In spite of this, the SJL CEACAM1b receptor is functional when expressed at high levels in hamster or human cells (Dveksler et al. 1991; Yokomori and Lai 1992b). Furthermore, SJL-derived cell lines have been shown to be susceptible to MHV-A59, but not MHV-JHM, and SJL mice express equivalent amounts of CEACAM1b compared to expression of CEACAM1a by B6 mice in brains and livers (Yokomori and Lai 1992b). When adult SJL mice were inoculated intracerebrally with MHV-JHM, virus titers and mortality were significantly lower than those in BALB/c mice (Ohtsuka and Taguchi 1997). SJL mice, when inoculated oronasally with MHV-JHM at 3, 6, or 12 weeks of age, do not support disseminated infection compared to BALB/c mice, which do (Barthold and Smith 1987). In contrast, SJL mice support equal levels of virus replication in intestinal tissue compared to BALB/c mice when infected with enterotropic MHV-Y at 1, 3, or 12 weeks of age (Barthold et al. 1993). Studies with enterotropic strains MHV-Y and -RI suggest that enterotropic MHV strains can use the same or different receptors for infection, and that receptors are probably not the primary determinant of the limited tissue distribution of enterotropic MHV strains (Compton 1994, 1998). Finally, the role of receptors in MHV infection and pathogenesis may vary by tissue type. Intranasal inoculation of MHV-JHM results in replication of virus in nasal tissue of 1-, 3-, 6-, or 12-week-old SJL mice, despite their resistance to disseminated infection to other tissues (Barthold and Smith 1987). Collectively, these observations emphasize that CEACAM or other known receptor expression is not the sole determinant of MHV strain–related virulence, virus organotropism, or mouse genetic susceptibility.
E.
Species Specificity
The host range of coronaviruses is believed to be determined largely by the interaction of the virion S protein with host cell receptors. Specific modification of the S protein of MHV-A59 can extend the host range of this virus to cells of other species (Thackray and Holmes 2004). Establishment of persistent MHV infections in various permissive cell lines can also result in emergence of host range variants that interface with phylogenetic orthologs of their normal receptor (Baric et al. 1999, 1997; Hensley et al. 1998; Schickli et al. 1997). This is likely mediated by selection for host cells that resist virus infection by down-regulating the expression of CEACAM1 (Chen and Baric 1996; Sawicki et al. 1995). In addition, it appears that different MHV strains can utilize more than one CEA-related molecule as alternate receptors, whereas some strains are more restricted in their receptor-ligand interactions (Chen et al. 1997, 1995). MHV-JHM variants, despite their apparent selectivity for mouse CEACAM1, can infect a human hepatocellular carcinoma cell line (Koetters et al. 1999).
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III. A.
PATHOGENESIS
Pathogenesis of Respiratory and Enterotropic MHV
Respiratory MHV strains initially replicate in nasal respiratory and olfactory epithelium as their primary targets. Virus replication was shown to take place in the nasal tissue of all mice experimentally infected with several prototype respiratory MHV strains (Barthold and Smith 1984), including genetically resistant SJL mice, regardless of the age at inoculation (Barthold and Smith 1987), and mice infected with low-virulence strains of MHV (Taguchi, Goto, et al. 1979). When respiratory MHVs of sufficient virulence infect hosts of sufficient susceptibility (infant mice, genetically susceptible mice, and immunodeficient mice), they disseminate through lymphatics to regional lymph nodes and by viremia to lungs, where they replicate in pulmonary vascular endothelium, then disseminate by secondary viremia to multiple organs, including lymphoid tissue (thymus, lymph nodes, and spleen), liver, bone marrow, brain, reproductive organs, and other tissues (Barthold et al. 1992; Barthold and Smith 1983, 1984, 1987, 1992). In addition to viremic dissemination, several strains of MHV have been shown to infect the brain by direct extension along olfactory tracts. Low-virulence virus, such as MHV-S, can gain access to the brain in adult, immunocompetent mice by this route in the absence of dissemination to other organs (Barthold 1988b; Barthold et al. 1986). Although the intestine becomes infected with respiratory MHV strains, viral replication is largely confined to gut-associated lymphoid tissue (Peyer’s patches) (Barthold and Smith 1984, 1987). This is in marked contrast to enterotropic MHVs, which selectively infect enterocytes (Barthold et al. 1993; Barthold and Smith 1984; Barthold et al. 1982). Respiratory MHV strains are nonselective in their tissue tropism, and are therefore “polytropic” when given the opportunity to disseminate in susceptible hosts. Relative organotropism of these viruses, such as neurotropism or hepatotropism, is often emphasized in the scientific literature and can be selected experimentally, but these viruses with “selective tissue tropism” still retain their ability to infect other organs in susceptible hosts. The polytropic behavior of these viruses explains how they were initially discovered through isolation from a myriad of cell cultures, transplantable tumors, tissue extracts, and other biological material and were likewise readily cultured in a variety of cell lines. In the authors’ experience, respiratory MHV strains tend to spread inefficiently from mouse to mouse. Others (Dick et al. 1956; Rowe et al. 1963) have reported this as well, even with virulent MHV-3 by direct contact among infant mice. Host age at the time of MHV infection is a critical determinant of disease severity, regardless of MHV strain or host genotype. Neonatal mice are highly susceptible to respiratory MHV strains, and usually succumb to disseminated infections with hepatitis and/or encephalitis. Clinical disease in mice infected with these viruses is due to hepatitis and/or encephalitis, and
therefore appears after several days of infection. Resistance to respiratory MHV increases at around 2 weeks of age, with significant differences in susceptibility among older mice, depending upon genotype (Barthold and Smith 1987; Hirano, Takenaka, et al. 1975; Suzuki et al. 1997; Taguchi et al. 1979b). SJL mice are highly resistant to infection with MHV-JHM and -A59, but nevertheless manifest age-related susceptibility. Neonatal, but not older, SJL mice develop disseminated infections when inoculated intranasally with MHV-JHM (Barthold and Smith 1987). Lymphoreticular function is one major factor (but not the only factor) in age-related susceptibility, and resistance can be reversed by immunosuppression (Bang 1978; Dupuy et al. 1975; Levy-Leblond and Dupuy 1977; Taguchi et al. 1979b; Tardieu et al. 1980). Disease susceptibility generally correlates with virus titer in various target organs following infection with respiratory MHV strains (Bang 1978; Barthold and Smith 1987; Hirano, Takenaka, et al. 1975; Suzuki et al. 1997; Taguchi et al. 1979b). An exception is the severity of hepatitis in some mouse strains that is mediated through procoagulant activity, rather than direct viral damage (see Section III, B). Enterotropic MHV strains, on the other hand, tend to be highly contagious. When introduced to a naive breeding population of mice, enterotropic MHV can result in epizootics with high mortality within a few days of introduction to a mouse population (Barthold et al. 1982; Hierholzer et al. 1979). Disease susceptibility to enterotropic MHV is also age-dependent, but is influenced predominantly by intestinal epithelial proliferative dynamics rather than susceptibility to disseminated infection (Biggers et al. 1964). Neonatal mice have slowly dividing intestinal mucosa, making them highly vulnerable to a rapidly cytolytic enterotropic MHV infection. Under these circumstances, mice die within a few days from severe enteritis, malabsorption, and dehydration (Barthold et al. 1993, 1982). As mice increase in age, intestinal mucosal turnover rates are more rapid, and the mice tend to cope better through replacement of damaged cells, resulting in less severe disease. When mice are infected at around 1–2 weeks of age, infection with enterotropic MHV has less severe consequences but will still produce significant damage that is repaired by mucosal proliferation. Adult mice are susceptible to infection, but manifest no clinical signs and have minimal lesions. Mice of all ages are susceptible to enterotropic MHV infections and support equivalent titers of virus, but disease severity is age-dependent (Barthold et al. 1993; Biggers et al. 1964). As with respiratory MHV strains, enterotropic MHV infection is influenced by host genetic background, but apparently via different mechanisms. For example, SJL mice are markedly resistant to infection with respiratory MHV-JHM and therefore develop minimal disease commensurate with low virus replication compared to BALB/c mice, which have high MHV-JHM titers and severe disease (Barthold and Smith 1987). In contrast, SJL mice infected with enterotropic MHV-Y sustain equivalent titers of virus in intestine compared to BALB/c mice, but have less severe disease (Barthold et al. 1993).
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B. Pathogenesis of Experimental Hepatitis MHV has been extensively studied as a model for viral hepatitis. The principal model utilizes the highly virulent MHV-3 strain in genetically susceptible (BALB/c and DBA/2), semisusceptible (C3H), and highly disease-resistant (A/J) mice (Levy et al. 1981; Virelizier and Allison 1976). As previously noted, susceptibility to MHV-induced disease is multifactorial and related to virus strain, host genotype, and host immunity. Furthermore, this model has shown that disease severity does not necessarily correlate with virus titer. Hepatitis in highly susceptible BALB/c mice reaches severe proportions due to thrombosis and coagulation necrosis that result from microcirculatory disturbance (MacPhee et al. 1985). This is due to induction of procoagulant activity by macrophages in susceptible mice, with minimal effect on cells from resistant A/J mice (Levy et al. 1981; Pope et al. 1995). Although virus infection is the initiator, disease severity correlates with macrophage activation and production of a procoagulant, rather than the effects of viral replication (Pope et al. 1995). MHV-3, being a polytropic virus, replicates not only in liver but also many other organs, yet severe disease with fibrin deposition and ischemic necrosis occurs principally in the liver (Ding et al. 1997; MacPhee et al. 1985). Both the initiation and the deposition of fibrin are associated with expression of a novel fibrinogen-like protein, Fgl2/ fibroleukin prothrombinase, by endothelial and Kupffer cells, but not hepatocytes, in the liver. High levels of fgl2 transcription have been found in liver, spleen, and lungs, which are all rich in reticuloendothelial cells, but only low levels of fgl2 were found in brains and kidneys of MHV-3 infected mice (Ding et al. 1997, 1998). Fibrin deposition and liver necrosis were markedly reduced in B6/129Sv/J hybrid mice with a targeted null mutation of fgl2, and peritoneal macrophages from such mice did not generate a procoagulant response when infected with MHV-3 (Marsden et al. 2003). Both B6 and 129 mice are MHV-3 susceptible mouse strains (MacPhee et al. 1985). It has been shown that the nucleocapsid (N) protein of MHV strains (MHV-3 and MHV-A59) that induce severe liver disease, but not the N protein from MHV strains (MHV-JHM and MHV-2) that do not induce severe hepatitis, causes transcription of fgl2 through host hepatic nuclear factor-4 alpha (Ning et al. 2003, 1999). Thus, host susceptibility to MHV-3 liver disease requires specific viral determinants and multiple host genetic determinants, including presence of the appropriate MHV receptor that determines susceptibility or resistance to infection and differential induction of fgl2 that determines susceptibility or resistance to procoagulant-induced hepatic necrosis. These can both be measured at the cellular level in macrophages from susceptible and resistant mice. Furthermore, host immunity is an important factor. Pre-existing antibody can protect susceptible mice from initiation of infection, but T lymphocytes from MHV-3 immunized susceptible BALB/c mice augment, whereas T lymphocytes from resistant A/J mice inhibit, procoagulant activity of macrophages from susceptible mice (Liu et al. 1998;
Pope et al. 1996). Resistance of A/J mouse cells to MHV-3 is not absolute. Treatment of MHV-3 resistant A/J mice with corticosteroids reduces their resistance to liver disease with concomitant elevation of fgl2 transcription and elevated procoagulant activity, and a similar effect can be shown with their macrophages (Fingerote et al. 1996). Genetic susceptibility of mice in the MHV-3 model has also been linked with defective nitric oxide production by BALB/c macrophages compared to A/J macrophages (Pope et al. 1998). Resistance of A/J mice to MHV-3 liver disease can also be attributed to increased induction of apoptosis in virally infected A/J mouse cells, resulting in reduced expression of fgl2 prothrombinase in apoptotic macrophages (Belyavsky et al. 1998). The virulent effects of MHV-3 in other tissues, including lymphoid tissues, also affect susceptibility to liver disease. Disseminated infection of susceptible B6 mice with MHV-3 results in generalized lymphocytopenia, with reduction of NK, B, and T cells (Dupuy et al. 1975; Lamontagne et al. 1989; Lehoux et al. 2004; LePrevost et al. 1975). C.
Pathogenesis of Experimental Encephalitis and Demyelination
Many strains of respiratory MHV have varying degrees of neurotropism, given the opportunity to enter the brain through experimental intracerebral inoculation, viremic dissemination, or extension through the olfactory tracts. One of the first published reports of naturally occurring MHV disease induced by an (at that time) unclassified “JHM” virus involved description of disseminated infection involving several organs, with encephalomyelitis and demylination arising from extension of infection through the olfactory tracts of mice (Bailey et al. 1949; Cheever et al. 1949). MHV infection of the central nervous system represents a commonly used model system, and MHVJHM remains the most frequently utilized “neurotropic” strain for such studies. Most such studies utilize intracerebral inoculation, but provide some insight into brain infection following natural routes of exposure. Most polytropic MHV strains, including prototype strains MHV-A59, -S, -1, -3, and -JHM, display neurotropism following intranasal inoculation of susceptible mice, including A, BALB/c, CBA, C3H/He, and C3H/Rv, but not SJL mice (Barnett and Perlman 1993; Barthold 1988b; Barthold et al. 1986; Barthold and Smith 1983, 1984, 1987; Goto et al. 1977; Ishida and Fujiwara 1979; Koolen et al. 1983; Lavi et al. 1986; Taguchi, Yamada, et al. 1979). MHV initially replicates in nasal and olfactory epithelium, with subsequent extension along olfactory nerves to the olfactory bulb (Fig. 6-2) and olfactory tracts within the brain (Barnett et al. 1993; Barnett and Perlman 1993; Barthold 1988b; Goto et al. 1977; Lavi et al. 1988; Schwob et al. 2001; Sun and Perlman 1995; Youngentob et al. 2001). Spread of virus into the brain can be ameliorated by surgical or chemical olfactory bulb ablation (Barnett and Perlman 1993).
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Fig. 6-2
Olfactory bulb of mouse inoculated intranasally with MHV-A59. Note syncytia within molecular layer (arrows).
Genetic resistance also affects extension into the olfactory tracts, as virus replicates in nasal epithelium of SJL mice but does not extend into brain (Barthold et al. 1986; Barthold and Smith 1987). Once virus gains access to the brain, there is retrograde spread along ramifications of the olfactory system and contiguous structures within the brain through cell-to-cell transmission (Fig. 6-3) (Barnett and Perlman 1993; Barthold 1988b; Goto et al. 1977). In addition, viremic dissemination to the central nervous system is often seen in infant mice (Barthold and Smith 1984), and immune-deficient strains of mice, such as nude, SCID, or RAG null mutant mice (Figs. 6-4, 6-5). Enterotropic MHV-Y infection of neonatal BALB/c mice can also cause encephalitis
through viremia (Barthold et al. 1993). Regardless of how MHV gains entry to the central nervous system, once present, MHV will cause an acute, rapidly fatal encephalitis, with extensive neuronolysis in most mice. If mice survive the acute encephalitis, infection can spread to the spinal cord through involvement of the ventral reticular nucleus and its neuronal and astrocytic projections to the white matter of the spinal cord (Sun and Perlman 1995). In such mice, demyelinating lesions are a prominent feature of disease, which may manifest clinically as posterior paresis (Lampert et al. 1973; Stohlman and Weiner 1981; Weiner 1973). Demyelination results from the direct cytolytic effect of MHV on oligodentroglia (Lampert et al. 1973; Weiner 1973), as well
Fig. 6-3 Immunohistochemical labeling of MHV antigen in neuron and glial cell in the brain of a mouse inoculated intranasally with MHV-JHM, depicting cell-to-cell spread of virus.
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Fig. 6-4
Diffuse encephalitis of the entorhinal cortex from a SCID mouse naturally infected with MHV following viremic dissemination to the brain.
as immune-mediated effects. Infection of immunosuppressed or immunodeficient mice has been shown to result in high titers of virus within the central nervous system, but no detectable demyelination (Lane et al. 1998, 2000; Wang et al. 1990). Athymic nude or neonatally thymectomized mice were shown to have demyelinating lesions following MHV infection, suggesting that T cells do not contribute to demyelination (Houtman and Fleming 1996; Sutherland et al. 1997). In contrast, others
Fig. 6-5
have shown that MHV-JHM-infected SCID or RAG1 null mutant mice did not develop demyelination, but that adoptive transfer of MHV-primed splenocytes depleted of CD4+ or CD8+ cells, but not both, contributed to development of demyelination and axonal damage (Dandekar et al. 2001; Lane et al. 2000; Wu et al. 2000). Furthermore, gamma/delta T cells, which are present in nude, but not RAG1 null mutant, mice, can substitute for alpha/beta T cells as effectors of demyelination (Dandekar and
Vasculitis in brain from an MHV-infected infant mouse with viremic dissemination to the brain.
154 Perlman 2002). This is overly simplistic, as others (Matthews et al. 2002) have shown that demyelination can occur in RAG1 null mutant mice infected with MHV-A59, and the authors have observed demyelinating lesions in SCID and RAG null mutant mice that are naturally infected with MHV (Fig. 6-6). Thus, demyelination can evolve from both direct viral damage and from indirect immune-mediated damage. B cells or antibodies do not appear to play a role in MHV-induced demyelination (Lin et al. 1999), whereas macrophages and microglia contribute to the lesion (Lane et al. 2000; Wu et al. 2000; Wu and Perlman 1999). In addition to T cells with MHV-specificity, CD8+, but not CD4+, T cells may contribute to demyelination in MHVinfected mice as “bystander” cells. These CD8+ cells are specific for non-MHV epitopes and are interferon-γ dependent (Dandekar et al. 2004; Haring and Perlman 2003), whereas the effects of MHV-specific CD4+ T cells on demyelination are accentuated in the absence of interferon-γ (Pewe et al. 2002). Persistent infection of the central nervous system can develop in mice infected with relatively nonvirulent variants of neurotropic MHV that do not kill the mice from encephalitis. MHV-JHM, for example, has tropism for both neurons and glia and causes severe encephalitis, whereas MHV-JHM variants with mutations and deletions in the S protein preferentially infect glial cells, but do not result in fatal encephalitis (Fazakerley et al. 1992; Fleming et al. 1986; Marten et al. 2000; Stohlman et al. 1982; Wang et al. 1992). Similar outcomes occur in infant mice that are infected with virulent virus but protected by maternal antibody. Under these circumstances, viral RNA and antigen can be detected in white matter of spinal cords (Perlman et al. 1988, 1990). During persistent infections, infectious virus cannot be readily recovered, and the majority of virus species are defective in one or more genes that are essential for growth or assembly (Adami et al. 1995; Fleming et al. 1995; Rowe et al. 1998). Low levels
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of infectious virus may also be present, especially in mice that are clinically symptomatic with hind limb paralysis. Under these circumstances, cytotoxic T cell–resistant escape mutants have been shown to possess mutation in regions of the S protein (Pewe et al. 1996). These variants have increased neurovirulence when inoculated into naive mice (Pewe et al. 1998).
IV. A.
IMMUNITY
Host Immune Response to Infection
Both cellular and humoral arms of the acquired immune response are important in controlling MHV disease and recovery from infection. A wide variety of lymphoreticular components, including macrophages, interferons, NK cells, B cells, T cells, and others, have been shown by many earlier investigators to influence the course of MHV infection (reviewed in Barthold 1986). The role of acquired immunity in controlling MHV infection has been vividly illustrated in globally immunodeficient SCID and RAG1 null mutant mice, which are susceptible to disseminated forms of natural MHV infection (Choi et al. 1999; Croy and Percy 1993) and experimental MHV-S, -JHM, and low virulence MHV-2cc infection (Houtman and Fleming 1996; Huang et al. 1996; Uetsuka et al. 1995). T cells are important determinants of MHV clearance and host immunity. Original descriptions of the nude mouse phenotype, which discussed the “pleiotropic” effects of the nude gene, featured MHV lesions (Flanagan 1966). T cell–deficient nude mice develop disseminated infections when infected with respiratory MHV strains, and die of progressive wasting syndrome. The severity and rate of the disease in nude mice is determined
Fig. 6-6 Demyelination of ventral spinal cord from a T cell receptor null mutant mouse naturally infected with MHV. Note vasculitis with endothelial syncytia of ventral vessels.
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by not only host factors but also virulence of the infecting virus (Hirano, Tamura, et al. 1975; Ishida, Tamura, et al. 1978; Liang et al. 1995; Sebesteny and Hill 1974; Tamura et al. 1977; Uetsuka et al. 1996a; Ward et al. 1977). Nude mice infected with enterotropic MHV also develop persistent infections, but wasting syndrome is often not apparent because of the more restricted tissue tropism of these viruses (Barthold et al. 1985; Homberger et al. 1998). The importance of T cells in controlling disseminated MHV infection in nude mice was initially proven with spleen cell transfer experiments, in which disease could not be controlled with transfer of cells treated with anti-theta serum (depleted of T cells), but could be controlled with cells treated with anti-IgG serum (depleted of B cells) (Fujiwara et al. 1978; Kai et al. 1981). Subsequent studies in B6 mice depleted of CD4+, CD8+, or both T cell subsets have shown that CD8+ cells were critical for control and recovery from MHV-JHM hepatitis (Kyuwa et al. 1996). In experimental studies with MHV-JHM and -A59, it has been shown that T cells are critical for virus clearance from the brain during the acute phase of infection, and B cells are important in preventing virus recrudescence during the chronic phase of infection (see below). In contrast, studies with B cell–deficient mice have shown that MHV-A59 virus in the liver, and the hepatitis that it causes, are cleared in the complete absence of B cells (Matthews et al. 2001). Organ specificity of B and T cells in host immune response to infection appears to also occur with enterotropic MHV. In contrast to wild-type immunocompetent mice, which cleared enterotropic MHV-Y infection, T cell– deficient mice developed persistent intestinal infections, as well as dissemination of virus to multiple organs; B cell–deficient mice also had persistent intestinal infection, but did not develop disseminated infection and eventually recovered from intestinal infection by 4 months (Compton, Ball-Goodrich, Paturzo, et al. 2004; Compton, Ball-Goodrich, Johnson, et al. 2004). Studies with interferon-γ and interferon-γ receptor null mutant mice have revealed interesting nuances on experimental pathogenesis of both respiratory and enterotropic strains of MHV. B6 interferon-γ receptor null mutant mice that were inoculated intraperitoneally with MHV-A59 developed more severe hepatitis compared to wild-type B6 mice (Schijns et al. 1996). This paralleled findings of more severe disease and higher virus titers in liver and spleen in BALB/c mice infected with MHV-JHM that were treated with anti-interferon-γ monoclonal antibody (Smith, Barthold, et al. 1991). B6 interferon-γ null mutant mice inoculated intraperitoneally with MHV-JHM developed persistent infections, with hepatitis in the acute phase that resolved. However, at around 2 weeks, mice developed progressively severe fibrinous peritonitis and pleuritis (Kyuwa, Tagawa, Shibata, et al. 1998). Naturally infected B6 interferon-γ null mutant mice were found to have fibrinous serositis without active hepatitis. In that case report, MHV antigen was demonstrated by immunoperoxidase in serosal inflammatory tissue, but not liver or intestine (France et al. 1999). In studies with interferon-γ deficient BALB/c and B6 mice, it was found that
BALB/c mice developed lethal necrotizing hepatitis by day 5, whereas B6 mice developed only mild hepatitis, and virus titers paralleled these findings (Kyuwa et al. 2002). Disseminated infections with hepatitis and serositis have also been observed in C3H-interferon-γ deficient mice naturally infected with MHV, and in BALB/c-interferon-γ deficient mice experimentally inoculated orally with enterotropic MHV-G (Compton et al. 2003). The role of type I interferon in MHV infection has also been explored, but not with genetically altered mice. Recombinant interferon-β and interferon-α/β administration has been shown to protect BALB/c and athymic nude mice against hepatitis when inoculated intraperitoneally with MHV-2 (Matsuyama et al. 2000; Uetsuka et al. 1996b). Furthermore, intranasal instillation of interferon-α/β was found to block olfactory neural extension of intranasally inoculated MHV-JHM into brains of BALB/c mice (Smith et al. 1987). B.
Immune Response During Experimental Brain Disease
MHV, and neurotropic MHV-JHM in particular (or variants thereof), has served extensively as a model for the study of host immune responses to the virus during acute and persistent infection of the central nervous system. In mice that survive the ravages of acute encephalitis, during which virtually all cell types within the brain can be infected, infection resolves into a nonproductive persistent infection with demyelination. MHV clearance from brain during the acute phase of infection requires CD8+ T cells (Lin et al. 1997; Parra et al. 1999; Stohlman et al. 1998, 1995, 1993), and antibody plays a minimal role during this stage of brain infection (Parra et al. 1999). Indeed, MHV-JHM was cleared from the central nervous system in B cell–deficient mice with kinetics similar to wild-type B6 mice (Lin et al. 1999). During acute infection, CD8+ T cells are the principal effectors for elimination of virus from astrocytes and microglia via a perforin-dependent mechanism (Lin et al. 1997), and from oligodendroglia via interferon-γ (Parra et al. 1999). CD4+ T cells help expand CD8+ T cells and facilitate their function (Bergmann et al. 2001; Stohlman et al. 1998), as well as participate directly through secretion of interferon-γ (Bergmann et al. 2001; Marten et al. 2001; Xue and Perlman 1997). A number of chemokines have been shown to be involved in the orchestration of MHV infection in the brain (Glass et al. 2002), and are strongly influenced by S protein characteristics of the infecting MHV strain (Rempel et al. 2004). Adoptive transfer experiments with MHV-JHM-specific CD4+ and CD8+ T cell clones demonstrated that both T cell clones protected mice from acute encephalitis, with the greatest effect induced with the CD8+ clone (Yamachuchi et al. 1991). Conflicting results have been found with central nervous system infection with MHV-A59 in CD8+ T cell–deficient (beta2 microglobulin null mutant) mice, which were able to clear acute central nervous system infection in most mice (Sutherland et al. 1997).
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Thus, caution is advised in generalizing about pathogenesis and host responses to MHV, which are likely to be both virus and mouse strain-specific. Antibody plays a complementary role in MHV clearance, although MHV can be cleared from most central nervous system cell types in B cell–deficient mice (Lin et al. 1999; Ramakrishna et al. 2002). Passive transfer of MHV-specific antibody by injection or through maternal milk before or during infection will prevent infection or attenuate severity of central nervous system disease (Buchmeier et al. 1984; Fleming et al. 1989; Kolb et al. 2001; Lecomte et al. 1987; Nakanaga et al. 1986; Perlman et al. 1987). A number of studies have shown the essential role of B cells and antibody in preventing MHV reactivation during persistent central nervous system infection and in complete clearance of infection (Lin et al. 1999; Mathews et al. 2001; Ramakrishna et al. 2002). There is gradual recruitment of B cells into brains of mice with persistent infection (Tschen et al. 2002). In mice with persistent central nervous system infections, the continued presence of neutralizing antibody is required to prevent virus recrudescence. Passive transfer of neutralizing (but not non-neutralizing) monoclonal antibodies directed at S protein into persistently infected, B cell–deficient mice has been shown to maintain persistent infection through virus neutralization and prevention of cell-cell fusion, with reduction of viral antigen in most cell types, except oligodendroglia, and reduction of demyelination (Ramakrishna et al. 2003). Such results must be interpreted cautiously, as B cell– deficient B6.Igh6
mice have impaired T cell responses to MHV-JHM (Bergmann et al. 2001). Furthermore, naive B cells also function as innate antiviral cytotoxic B cells (Welsh et al. 1986), and this effect is inhibited by antibody directed against the S protein (Morales et al. 2001). In mice with disruption of the JHD locus (absence of B cells and Ig) and syngeneic H-2d B cell–positive antibody-deficient mice, it was found that the absence of B cells or antibody had no effect upon CD4+ or CD8+ T cell responses or virus clearance during acute MHV-JHM infection of the central nervous system (Ramakrishna et al. 2002). In addition, these studies found altered tropism of MHV during reactivation during persistent infection, with virus selectively infecting oligodendroglia in B cell–positive antibodynegative mice, and astrocytes in B cell–deficient mice, which was regulated by virus-specific CD4+ T cells (Ramakrishna et al. 2002).
C.
Passively Acquired Maternal Immunity
The clinical effects of MHV infection among infant mice are profoundly different during epizootic and enzootic infection of mouse populations. Maternally derived passive immunity has been shown to confer strong protective activity to pups exposed to both respiratory and enterotropic MHVs under both natural and experimental conditions (Barthold et al. 1988; Gustafsson et al. 1996; Hierholzer et al. 1979; Homberger 1992;
Homberger and Barthold 1992; Ishida and Fujiwara 1982; Kolb et al. 2001; LePrevost et al. 1975; Manaker et al. 1961; Perlman et al. 1987; Pickel et al. 1985). IgG is selectively transferred from immune dams in utero through yolk sac receptors, and from colostrum for up to 2 weeks postnatally through intestinal receptors (Brambell and Hemmings 1960). In addition, IgA provides protection within the gut lumen. Cross-fostering experiments using naive and MHV-JHM infected mice have shown that detectable antibody to MHV-JHM was transferred via colostrum but not in utero, and that pups were capable of absorption through 2 weeks of age. IgG was detected in serum of pups suckling immune dams at 2 and 4 weeks postpartum, but not at 6 or more weeks. Pups nursing MHV-JHM immune dams were protected against both MHV-JHM and MHV-S challenge, but pups nursing MHV-S immune dams were protected against MHV-S, but not MHV-JHM, challenge. Thus, maternally derived passive immunity confers strong protection to pups against homologous respiratory MHV challenge and some degree of protection against heterologous strains of respiratory MHV (Barthold et al. 1988). Maternally derived passive immunity is also strongly protective against challenge with enterotropic MHV, but via significantly different mechanisms. In the above described cross-fostering studies, it was shown that passive immunity to challenge with MHV-JHM could be conferred by transfer of maternal IgG postnatally and was not dependent upon active ingestion of whey at the time of challenge exposure (Barthold et al. 1988). In similar cross-fostering studies with enterotropic MHV-Y, it was shown that there was placental transfer of IgG, but protection required active ingestion of whey containing IgA and IgG antibodies to MHV. If pups were separated from their immune dams, they were fully susceptible to MHV-Y infection, despite the presence of MHV-IgG in their sera. Both IgA and IgG were protective, and IgG alone was only partially protective. Maternally derived passive immunity did not diminish over the course of four consecutive litters, and pups remained seropositive for up to 10 weeks of age (Homberger 1992; Homberger and Barthold 1992). Cross-protective immunity against respiratory MHV or heterologous enterotropic MHV has not been thoroughly investigated, but it has been shown that mouse pups benefiting from maternal antibody against enterotropic MHV-Y or MHV-RI were protected against challenge with the homotypic MHV strain, but only partially protected against the heterotypic strain. Thus, like active immunity (below), passive immunity appears to be at least partially strain-specific (Homberger et al. 1992). The practical message is that during enzootic infections of a mouse population, pups are protected by maternal antibody during their critical pre-weaning period of susceptibility to MHV-related disease. Although maternal protection is waning against respiratory MHV, and absent against enterotropic MHV at weaning, age-related resistance factors, including genetic, immunologic, and intestinal proliferative activity, are capable of preventing severe disease once pups reach weaning age. Thus, epizootics of high mortality are seen among naive infant
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mice with highly contagious enterotropic MHV strains, which rapidly become enzootic with occult or only mild disease among pups subsequently born to immune dams (Barthold et al. 1982; Hierholzer et al. 1979). Respiratory MHV can potentially result in the same scenario, but these MHV strains appear to be less contagious, and such outbreaks have not been reported with respiratory MHV (excepting MHV-S, see Section I, A).
D.
Immunity to Reinfection
A fundamentally important concept in MHV biology is that immunocompetent mice recover from MHV infection and resist reinfection with MHV, but immunity is MHV strain-specific. Thus, seropositive mice that have recovered from MHV can potentially be actively infected with a novel strain of MHV, which may explain some of the mythology about MHV persistence and “latency” in mice. When susceptible BALB/c mice were initially infected intranasally with MHV-JHM and allowed to recover, they resisted intranasal challenge with MHV-JHM at 1, 6, and 12 months (Barthold and Smith 1989a). When BALB/c mice were initially exposed intranasally to MHV-JHM, MHV-S (antigenically disparate from MHV-JHM, but same biotype), or MHV-Y (antigenically related to MHV-S, and unrelated to MHV-JHM, but different biotype), then challenged intranasally with MHV-JHM or MHV-S after recovery (30 days), mice resisted challenge with the homotypic virus. However, mice immunized with MHV-S were fully susceptible to MHV-JHM, and mice immunized with MHV-Y were only partially protected against antigenically related but biologically different MHV-S (Barthold and Smith 1989b). In similar studies with BALB/c mice immunized by oral infection with enterotropic MHV-Y or MHV-RI, which are distinctly different by serum neutralization, the mice were resistant to reinfection with the homotypic MHV strain at 1 and 6 months. However, although they resisted challenge with the heterotypic strain at 1 month, they were susceptible to challenge infection with heterotypic virus at 6 months (Homberger et al. 1992). These studies demonstrate that challenge immunity against MHV is strong, but virus strain–specific. Further support for this finding is that homotypic vaccination–challenge results in near-normal post-challenge T cell mitogen responses, whereas heterotypic vaccination challenge yields profoundly depressed post-challenge T cell function (Smith et al. 1992). Considering the mutable nature of this virus within a population of mice, a scenario in which new mutants with novel antigenicity arise explains how MHV is likely to persist within a population.
E.
Vaccination Immunity
Vaccination has been attempted as a means of protecting mice against MHV infection; however, considering the strain
specificity of MHV immunity, the mutable nature of the virus, the generally mild clinical effects, and the perturbations (research variables) that are likely to be induced by vaccination, it is an unfruitful venture for MHV control, but a useful experimental model for development of vaccine strategies to coronaviruses. Earlier work demonstrated challenge immunity against MHV-3 in mice vaccinated with whole inactivated virus and S subcomponents, but not with M or N proteins (Hasony and Macnaughton 1981). Vaccines consisting of adenovirus expressing structural S and N proteins of MHV-A59 have been shown to protect against lethal challenge (but not infection) with MHV-A59 (Wesseling et al. 1993). Mice immunized intranasally or subcutaneously with hybrids of tobacco mosaic virus containing S epitopes were shown to be protected against challenge with MHV-JHM (Koo et al. 1999). Intramuscular injection of plasmid DNA containing N genes protected mice against disease, but not infection with MHV-JHM (Hayashi et al. 1998).
F.
Immunomodulation by MHV
A number of studies have documented significant effects on immune responses of MHV-infected mice through both direct and indirect effects of the virus (reviewed in Barthold 1986; Lindsey et al. 1991). Most MHV strains, including enterotropic MHVs, infect lymphoid tissues (Fig. 6-7) (Barthold and Smith 1984, 1992). A number of low-virulent MHV strains were shown to exhibit various tropisms for B6 and A/J mouse macrophages, B cells, and thymic stromal cells, but not T cells (Lamontagne and Jolicoeur 1994), whereas both T and B cells of B6 mice were permissive to MHV-3 infection (Lamontagne et al. 1989). Effects do not necessarily occur due to direct infection of immune cells. Severe, transient lymphocyte apoptosis in thymus, with only small numbers of stromal cells and few lymphocytes actively infected, has been documented in BALB/c mice inoculated with MHV-A59 (Godfraind et al. 1995). Oronasal inoculation of BALB/cByJ mice with MHV-JHM has been shown to result in significant but transient functional disturbances of both CD4+ and CD8+ T cells and antigen–presenting cells. Neither T cell subset was infected with virus, although B cells were transiently infected and adherent peritoneal cells contained viral antigen (deSouza and Smith 1991; deSouza et al. 1991). Splenic T cell proliferative responses were depressed in MHV-JHM infected BALB/cByJ, A/JCr, C3H/HeSnJ, but not C57BL/6NCr mice (Smith, Winograd, et al. 1991). Impairment of pre–B cells and B cells has also been documented in mice with experimentally induced chronic MHV-3 infections (Jolicoeur and Lamontagne 1995). Although the immunomodulatory effects are diverse, it has also been noted that MHV-JHM infection can induce self-reactive T cells 1 month after infection, and therefore potentially affect autoimmunity (Kyuwa et al. 1991). Possibly contrary or related to this observation is the finding that natural MHV infection
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Fig. 6-7
Immunofluorescent labeling of MHV antigen in lymph node from a mouse infected with MHV-3.
has been shown to significantly reduce the incidence of diabetes in non-obese diabetic (NOD) mice (Wilberz et al. 1991). MHV infection has also been shown to modulate the course of a variety of other infectious agents, including viruses, bacteria, and parasites (reviewed in Barthold 1986; Lindsey et al. 1991).
V. ISOLATION AND DETECTION OF MHV A.
Isolation and Propagation
In vitro growth of MHV serves several purposes, including isolation of the virus from naturally infected mice, propagation of prototype stocks of virus, plaque assays for quantification of infectious virus, and investigation of species and tissue specificity. Historically, MHV was isolated and propagated in primary mouse embryo explant cultures (Mosley 1961), newborn mouse kidney explant cultures (Starr and Pollard 1959), primary liver explant cultures (Gallily et al. 1964; Miyazaki et al. 1957; Paradisi and Piccinino 1968; Vainio 1961), and mouse peritoneal macrophage cultures (Shif and Bang 1966). Recognition of the hepatotropism of MHV led to the successful logic of using NCTC 1469 cells, derived from C3H mouse liver, as a stable cell line that supported the growth of MHV (Hobbs et al. 1957). Considering the polytropism of many MHV strains, it is not surprising that a wide variety of murine cell lines have been found to be MHV-permissive. Among the more commonly used cell lines are DBT astrocytoma cells, derived from CDF1 “delayed brain tumor” (Hirano et al. 1978); 17Cl-1 fibroblast cells, derived from BALB/c 3T3 cells (Sturman and Takemoto 1972);
L fibroblast cells from C3H mice (Starr and Pollard 1959); and various derivatives thereof, such as L2 and L2-Percy (Compton et al. 1995). The increasing recognition of tissue specificity of various MHV isolates, such as enterotropic MHV, has prompted the use of cell lines of specific cellular composition, such as CMT-93 cells, derived from a carcinogen-induced colonic tumor of C57BL origin (Franks and Hemmings 1978). Use of CMT-93 cells led to the successful growth of a highly enterotropic MHV-RI strain, which originally failed to grow in NCTC 1469 cells (Barthold et al. 1985). Interest in favoring the neurotropism of some MHV strains for experimental use has promulgated the use of DBT cells and highly specific cell lines, such as OBL21A, an olfactory bulb neuronal cell line derived from transformed neonatal mouse cells (Ryder et al. 1990). It has not been determined if growth in specific cell lines in vitro favors specific organotropism in vivo, but it is common practice. Recently, B16 cells, derived from a melanoma cell line of C57BL mice (Silagi 1969), and A3-1M cells, derived from 129/SvJ embryonic stem cells (Kyuwa 1997), have shown utility for isolation and propagation of a variety of MHV strains and isolates (Kyuwa et al. 2000). J774A.1 monocyte/macrophage cells, derived from DBA/2 mice (Yamaguchi et al. 1988), have also been found to have similar universal utility (Compton et al. 1995). Prototype MHV strains, which have been passaged many times both in vitro and in vivo, tend to grow well in a variety of cell lines, and this can be true of enterotropic MHV strains that have been likewise manipulated (Kyuwa et al. 2000). Passage history of various MHV strains, and the viral genetic variability that is likely to ensue, clearly influences their growth characteristics in vitro, explaining apparent discrepancies in results among
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different laboratories. In one study that compared the growth of enterotropic MHV-Y and MHV-RI in eight different cell lines (17Cl-1, CMT-93, J774A.1, L2p176, L2-Percy, NCTC 1469, N18, and WEHI-279), it was determined that these viruses consistently produced cytopathic effect only in J774A.1 cells. J774A.1 cells produced high titers of MHV-1, -3, -A59, -JHM, -S, and -DVIM as well, and thus were deemed optimal for growth of a variety of MHV strains (Compton et al. 1995). In contrast, another study examined susceptibility of six different cell lines (A3-1M, B16, CMT-93, DBT, IC-21, and J774A.1) to infection with eight MHV strains, including enterotropic strains MHV-RI, -Y, and -KU, and polytropic strains -A59, -JHM, and recently isolated (in DBT cells) strains KQ1E, KQ3E, and KQ6E. All MHV strains replicated and induced cytopathic effect in B16, DBT, IC-21, and J774A.1 cells, but produced little if any cytopathic effect in A3-1M or CMT-93 cells. B16 and IC-21 cells were deemed most suitable for propagation and isolation of all viruses tested. The study concluded that the kinetics of virus replication depended upon both the cell line and the virus strain, and that prototype viruses replicated better than new isolates (Kyuwa et al. 2000). The lesson learned from these studies is that successful isolation of MHV from naturally infected mice is not as easy as isolation of MHV from mice experimentally infected with prototype (passaged) strains of virus, and may require attempts with multiple cell lines for ultimate success. Enterotropic MHV isolations are a case in point: MHV-RI could not be isolated from naturally infected mice using NCTC 1469 liver cells but could be isolated with CMT-93 colon tumor cells (Barthold et al. 1985), whereas MHV-RI that had been subjected to in vitro passage grew better in NCTC 1469 cells than in CMT-93 cells (Compton et al. 1995). Wild-type MHV does not grow readily in cultured cells, and serial passage may be required before
Fig. 6-8
cytopathic effect is observed. The cytopathic effect, however, is very distinctive: Syncytia can arise by fusion of up to hundreds of cells whose membranes have fused to make “giant cells” with multiple nuclei (Fig. 6-8). The prototype MHV strains induce syncytia of different sizes. Some MHV mutants do not induce syncytia, but most strains of the virus do (Gallagher et al. 1991; Gombold et al. 1993). MHV-2 seems to be a naturally derived strain that is nonfusigenic (Keck, Soe, et al. 1988). MHV also causes acute cytolytic damage to susceptible cells. Some infected cells develop large cytoplasmic accumulations of nucleocapsid material in vitro (David-Ferreira and Manaker 1965), which can also be seen in vivo as poorly defined (and nondiagnostic) cytoplasmic inclusions (Barthold et al. 1982). It has been known for many years that MHV can cause persistent and inapparent infection of cultured cells with minimal cytopathic effect (Gledhill and Niven 1955). This generally follows an initial cytocidal infection, followed by a surviving population of cells, with only a fraction of cells producing infectious virus at any given time. Cells in persistently infected cultures become resistant to superinfection with MHV, with reduced expression of viral receptors. They also readily produce mutant variants of the virus that can have alterations in protease cleavage sites of the S glycoprotein, delayed penetration, and altered pathogenesis in vivo (Gombold et al. 1993; Holmes and Behnke 1981; Lamontagne and Dupuy 1984; Lucas et al. 1978). The exquisite susceptibility of neonatal mice to MHV infection offers an alternate approach to propagation of MHV or amplification of virus titers during attempts to isolate the agent. Regardless of MHV strain or tropism, intracerebral inoculation is highly effective and sensitive. Infant mice will generally die within 24 to 72 hours, depending upon dose and strain of the virus. By standardizing the volume and concentration of inoculum, and using serial (usually 10-fold) dilutions, the level of infectivity
NCTC 1469 cell culture infected with MHV, depicting pathognomonic cytopathic effect with syncytium formation.
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of both polytropic and enterotropic MHV can be effectively titrated using infant mice as a bioassay (Barthold et al. 1993; Barthold and Smith 1987). Following intracerebral inoculation, MHV readily disseminates, so that mice inoculated with enterotropic MHV will succumb from enteritis, and those infected with hepatotropic or neurotropic strains will succumb from hepatitis and/or encephalitis. This method has the ethical drawback of requiring live animals and the financial issue of being more expensive than culture for detecting and titrating live virus. Immunodeficient strains of mice can also be used to isolate and amplify MHV. For example, athymic nude mice have been utilized as sentinel mice in colonies of enzootically infected immunocompetent mice in order to isolate and characterize endemic wild-type MHVs in multiple populations of mice (Homberger et al. 1998).
B.
Serodiagnosis and Antigenic Interrelationships
Based on speed, cost, and sensitivity, serology is the best method for monitoring mouse populations for MHV infection. Infected mice seroconvert to all of the viral structural antigens of the virion, as well as to nonstructural antigens that may be expressed during infection. The immunodominant structural antigens are M, N, and S. Antigenicity of M and N proteins is highly cross-reactive among MHV strains, and S antigens tend
to be less cross-reactive and more strain-specific, as their gene sequences would predict. Historically, complement fixation was used to detect antibody to MHV, but it proved to be too insensitive for practical use (Peters and Collins Jr. 1983; Smith 1983). Hemagglutination inhibition has been used for detecting hemagglutinating antibody against MHV strains that possess HE protein in their virions, such as MHV-DVIM (Sugiyama and Amano 1980), but the HE gene is not present in most MHV strains (Yokomori et al. 1991). The enzyme-linked immunosorbent assay (ELISA) has proven to be very sensitive and specific for detecting MHV antibody in naturally infected mice and has an added advantage of detecting rat coronavirus antibody in rats, depending upon how the antigen is prepared (Carthew et al. 1981; Peters et al. 1979; Peters and Collins Jr. 1983; Smith 1983). The immunofluorescence assay (IFA), using MHV-infected cells as antigen, can also be used to detect antibody to MHV and rat coronaviruses (Smith 1983). It does not lend itself well to testing large numbers of samples, because it cannot be automated and relies on the human eye for interpretation. However, IFA is frequently used to confirm positive ELISA reactions. Furthermore, it offers an advantage of specificity, as the pattern of staining is distinctive. If cells are handled gently during slide preparation, clearly positive syncytia are pathognomonic for MHV (Fig. 6-9). Syncytia are fragile, and if handled too roughly, the antigen will leak from the cells and generate green debris
Fig. 6-9 Immunofluorescent labeling of MHV antigen in cultured cells, depicting pathognomic pattern of staining of syncytia in the indirect immunofluorescence assay.
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on the slide. ELISA relies on the use of purified virus or recombinant antigens. An alternative is to use infected cells as the antigen substrate (Smith and Winograd 1986). To minimize risk of aerosol transmission of MHV, cells can be chemically fixed to inactivate virus. Both ELISA and IFA methods can use multiple MHV strains as antigen simultaneously in order to improve sensitivity. Serum neutralization is also very sensitive, and offers the opportunity to examine strain specificity of antibody, since neutralizing antibody is directed at strain-specific antigens on the peplomers (S proteins). However, serum neutralization is cumbersome, time consuming, and expensive. Serum neutralization utilizes several prototype strains of MHV for comparison with new isolates, and the relative neutralizing activity of the test antibody to each virus strain can be compared. Because of the strain specificity of neutralizing antibody to MHV, this method can be used to examine the neutralizing profiles of sera from different mouse populations to determine if the same or unrelated MHV strains are involved, or to examine the profiles of isolates from different outbreaks within a population over the course of time (Barthold et al. 1982; Hierholzer et al. 1979; Smith et al. 1983). Two types of tests can be performed: either in vivo, usually with baby mice as a bioassay, or in vitro, using cell cultures. In addition, one can use a fixed virus dose with serially diluted antisera or dilutions of virus with fixed serum dilutions. The former is performed most frequently. Investigators and veterinarians frequently request typing of wild-type MHV isolates in animal colonies by serum neutralization, but this is to be discouraged because, aside from the expense, such information predicts absolutely nothing about the type, virulence, tropism, or identity of the isolate. For example, enterotropic strains MHV-Y and MHV-RI do not cross-neutralize and have different patterns of reactivity with five different prototype MHV strains (Homberger et al. 1992). Most MHV isolates will cross-react to some degree with one or more prototype viruses. The most frequent finding is reciprocal crossreactivity with one prototype strain, and one or two one-way reactions. For example, an isolate of Tettnang virus (EgArt1147), which was found to be MHV, showed reciprocal reactivity with MHV-JHM and one-way reactivity with MHV-3 and MHV-S (Smith et al. 1983). Enterotropic MHV-S/CDC reacted reciprocally with MHV-S and unilaterally with MHV-JHM (Hierholzer et al. 1979). Indeed, long before MHV was characterized as a coronavirus, interrelationships among many of the early isolates were demonstrated by serum neutralization (Cheever et al. 1949; Dick et al. 1956; Gledhill and Andrewes 1951; Manaker et al. 1961; Morris 1959; Nelson 1952a; Rowe et al. 1963). Serum neutralization can be a useful epidemiological tool, but it does not prove that an isolate is identical to a prototype strain, and does not predict or define biologic activity. Gene sequencing of variable regions of the genome can also be used for comparison among MHV isolates (Yamada et al. 2001) but also does not predict biological behavior.
C.
Mouse Antibody Production (MAP) Test
The MAP test was first utilized for detecting polyoma virus (Rowe et al. 1959), and the method has long been the mainstay for testing biological products for contamination with infectious agents. There are many permutations, but the basic test involves inoculating mice intraperitoneally and oronasally with biologic material to be tested. These may be sera or lysates of tumors or other cells. The mice are held in isolation for 21–35 days, and their sera are tested for antibody to the agent(s) of interest by ELISA or IFA. Although MAP testing is quite sensitive, and more sensitive than isolation of MHV in culture (deSouza and Smith 1989), the assay is time consuming and expensive. Several laboratories have developed what might be called “molecular MAP tests” that utilize direct polymerase chain reaction amplification for the same agents with equivalent sensitivity to the MAP test, but results are available in days rather than weeks (Bootz et al. 2003).
D.
Molecular Methods of Detection
Reverse transcriptase polymerase chain reaction (RT-PCR), targeting either conserved or variable regions of the MHV genome, has proven to be a useful diagnostic and epidemiologic tool. Caution is advised, as nonspecific amplification can and does occur, so use of appropriate controls and verification of size and sequence of the amplicon may be needed before condemnation of a mouse population. Initial application of RT-PCR for MHV detection showed that RT-PCR was more sensitive than infant mouse assay and equally sensitive as MAP testing. The approach used primers that amplified a conserved M gene sequence of 375 base pairs, and showed that all of the prototype and several field isolates of MHV could be detected using the chosen primers. This approach also allowed amplification of rat coronavirus, but not human coronavirus OC-43 or 229E (Homberger et al. 1991). During an MHV outbreak in athymic mice in Australia, histologic findings suggested MHV as the etiology, but serologic testing of nude and immunocompetent sentinel mice was negative. The use of nested primers directed at M gene sequences by RT-PCR resulted in amplification in the liver of an athymic mouse (Matthaei et al. 1998). Similarly, nested RT-PCR detected N gene RNA in feces of infected A/WySnJ mice. This approach, combined with sequencing the amplicons, is useful as an epidemiologic tool to determine whether one or more strains of MHV are circulating in different mouse facilities (Homberger et al. 1998; Oyanagi et al. 2004; Yamada et al. 2001). Attempts have been made to increase the specificity of RTPCR. Fluorogenic RT-PCR assays use an internal fluorogenic hybridization probe and thus have the added advantage of eliminating the need for post-PCR processing. Using primers specific to the highly conserved M gene, it has been possible to amplify sequences from several MHV and rat coronavirus strains.
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The assay was also successful when applied to infected tissues, feces, and swipes from cages containing infected animals (Besselsen et al. 2002).
VI. A.
DIAGNOSTIC PATHOLOGY Gross and Microscopic Pathology
Acute disseminated MHV infections, caused by respiratory MHV strains infecting susceptible hosts such as neonates, genetically susceptible mice, and immunodeficient mice, are characterized by multifocal areas of necrosis, leukocytic infiltration, and syncytium formation. Small white foci may be apparent on the capsular surface of the liver (Fig. 6-10). Thorough microscopic evaluation of tissues will reveal mild necrosis of nasal epithelium, focal interstitial pneumonia, and necrosis and syncytia in lymphoid organs (spleen, lymph nodes, Peyer’s patches, thymus) as well as bone marrow, mesothelium, and other organs. The liver is often involved, and necrotic lesions, when severe, can coalesce and be accompanied by hemorrhage with parenchymal collapse. Cells at the periphery of necrotic foci often have characteristic dense, marginated chromatin or multiple dense bodies (Fig. 6-11). Syncytia can arise from hepatocytes or other cells, such as endothelial cells. Hematogenous dissemination to the central nervous system is rare in adult immunocompetent mice, but can result in foci of vasculitis, encephalitis, and
Fig. 6-10 Multifocal hepatitis in a BALB/c mouse infected with MHV-JHM. Multiple white foci are visible on the capsular surface.
meningitis (Figs. 6-4, 6-5). Infection along olfactory neural pathways results in similar lesions, but present within the olfactory bulb (Fig. 6-2), and along olfactory tracts and the ventral brain (Barthold 1988a, 1997b; Percy and Barthold 2000; Piazza 1969). Hepatitis is accompanied by serum elevations in
Fig. 6-11 Focal necrotizing hepatitis in a mouse naturally infected with MHV. Note degenerating syncytia at periphery of lesion (arrow).
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Fig. 6-12 Perivascular lymphocytic infiltration in lung of a mouse recovered from prior infection with MHV.
liver enzymes, and the hemogram can reveal pancytopenia (Piazza, 1969). The full manifestations of acute MHV infection are usually interrupted by immune response, or death in infants. In the subacute phase of infection, in which virus is in the process of being cleared, liver lesions become granulomatous in character with mixed leukocyte infiltrates, portal tracts may be infiltrated with lymphocytes and hematopoeitic elements, lungs may develop perivascular and interstitial lymphocytic infiltrates (Fig. 6-12), spleens often enlarge from hematopoiesis and reactive lymphoid hyperplasia, lymph nodes may enlarge with
reactive hyperplasia, and areas of demyelination, spongiosis, and perivascular lymphoplasmacytic infiltrates (Fig. 6-13) may be found in the brain stem and spinal cord. During the recovery phase of infection, attempts to detect viral antigen or isolate virus are usually unrewarding. In contrast, the full manifestations of MHV infection are seen in immunodeficient mice, such as nude mice, especially those that are infected with relatively nonpathogenic strains of the virus that allow the mice to live while lesions fully and progressively evolve. These mice develop “wasting syndrome” with chronic weight loss when infected with relatively
Fig. 6-13 Brainstem (pons) from a mouse recovering from MHV-S encephalitis, with spongiosis and perivascular lymphoplasmacytic infiltration.
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nonpathogenic variants of MHV, and acute death when infected with more virulent strains. Under these circumstances, liver lesions are grossly apparent, with multiple white foci of necrosis that can be hemorrhagic and result in parenchymal collapse and irregular capsules (Fig. 6-14). Microscopic features include multiple foci of acute necrosis, parenchymal collapse, varying degrees of hemorrhage, fibrosis, and prominent syncytia that favor a definitive diagnosis (Fig. 6-15). Mice often have marked hematopoiesis in portal regions and spleen, and splenomegaly is often apparent. Examination of other organs reveals necrosis, leukocytic infiltration, and prominent syncytia, including lung (Fig. 6-16) and portal vessels, spleen, lymph nodes, and bone marrow. Vasculitis with thrombosis and endothelial syncytia are frequently found in vessels of the brain and spinal cord (Fig. 6-6), with foci of necrosis, syncytia, and demyelination (Barthold 1988a, 1997b; Ishida, Tamura, et al. 1978; Percy and Barthold 2000; Piazza 1969; Tamura et al. 1977; Ward et al. 1994). Mild serositis is often present, but can be florid, with granulomatous thickening of the serosa of the thoracic and abdominal viscera in interferon-γ null mutant mice (Compton et al. 2003; France et al. 1999; Kyuwa, Tagawa, et al. 1998). This serositis syndrome has also been observed in mutant mice with unanticipated immune deficiencies (unpublished) (Figs. 6-17, 6-18). A recent study found granulomatous serosititis in B6 T cell receptor beta/delta deficient mice experimentally infected
Fig. 6-14 Severe multifocal hepatitis virus in a SCID mouse naturally infected with MHV. Note numerous pale, as well as dark, hemorrhagic foci.
Fig. 6-15 Focal necrotizing hepatitis in a SCID mouse naturally infected with MHV. Note multiple prominent syncytia (arrows).
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Fig. 6-16 Endothelial syncytium in pulmonary vessel from a T cell receptor null mutant mouse naturally infected with MHV.
with enterotropic MHV-Y (Compton, Ball-Goodrich, Johnson, et al. 2004). The pathology of enterotropic MHV is markedly different, with lesions often restricted to intestinal mucosa. Because disease severity is determined by intestinal mucosal proliferative kinetics, lesions are most severe in infant mice, regardless of
Fig. 6-17 Florid granulomatous serositis in a genetically altered mouse with unanticipated immune deficiency. The serosa is markedly thickened with infiltration of histiocytes, many of which are forming syncytia (arrows).
immune competence. Neonates become dehydrated, and their stomachs are usually devoid of milk due to the severity of illness. Pre-weanling mice may continue to suckle, but the intestines may be dilated with fluid and gas, with yellow, sticky feces. Microscopic features include segmental regions of mucosal necrosis, enterocyte swelling and necrosis, villus attenuation, varying degrees of crypt hyperplasia, leukocytic infiltration of mucosa, and fibrinopurulent exudation into the gut lumen. The most significant diagnostic feature, however, is the presence of multinucleate enterocytic syncytia (Figs. 6-19, 6-20). These features characterize the neonatal disease, whereas older mice may have less necrosis and syncytia and more mucosal hyperplasia. Adult mice may have minimal lesions, excepting occasional syncytia during active infection. It is important to emphasize that all levels of intestine should be examined, as different strains of virus may preferentially infect different levels of gut, and lesions are often very segmental in distribution. Nevertheless, examination of the ascending colon is often the most fruitful region for identifying syncytia, including in adult mice (Fig. 6-21). Depending upon virus strain and host factors, enterotropic MHV may disseminate to other organs, including liver and brain, to varying degrees. Mesenteric lymph nodes often contain syncytia (Fig. 6-22), even in highly enterotropic MHV infections (Barthold 1997a; Barthold et al. 1993, 1982; Biggers et al. 1964; Percy and Barthold 2000). Infection of immunodeficient mice with enterotropic MHV can have variable outcomes. One report of natural infection of nude mice with enterotropic MHV-RI described marked proliferative typhlocolitis. Mice had grossly thickened large bowels and mesenteric lymph nodes (Fig. 6-23), with marked mucosal hyperplasia (Fig. 6-24). Experimental inoculation of nude mice with MHV-RI, however, resulted in only mild enteric lesions, without hyperplasia (Barthold et al. 1985). Examination of tissue from athymic nude mouse sentinels that were exposed to
Fig. 6-18 High-power field of serositis from lesion in Fig. 6-17, depicting prominent syncytia.
Fig. 6-19 Enterocytic necrosis, villus attenuation, and syncytium formation in small intestine of an infant mouse infected with enterotropic MHV-Y. Note shallow crypts that are typical of neonatal intestine.
Fig. 6-20 Cecum of an infant mouse infected with enterotropic MHV-Y. There is nearly complete destruction of intestinal epithelium, except for a few flattened enterocytes at the luminal interface. The lumen contains numerous neutrophilic leukocytes.
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naturally infected mice in a number of mouse populations has revealed very mild intestinal lesions, with syncytia in the ascending colon, but no hyperplasia (Fig. 6-25) (Homberger et al. 1998). Thus, the severe hyperplasia that was noted in the initial report may be atypical, and possibly due to co-infection with agents such as Helicobacter. Recent studies have shown more severe disease in MHV-Helicobacter dually infected mice (Compton et al. 2003). In the authors’ experience, the most common manifestation of enterotropic MHV infection in nude mice is subclinical infection, in which careful examination of ascending colonic mucosa is required to document infection.
B.
Immunohistochemistry and In Situ Hybridization
Formalin- and Bouin’s-fixed tissues with active MHV lesions can be subjected to immunohistochemistry (immunofluorescence, immunoperoxidase, and other markers) for confirmation of infection or as an experimental method for pathogenesis studies, using either direct or indirect methods. Serum from recovered, infected mice (either naturally or experimentally) lends itself well to indirect immunohistochemistry. Antigen retrieval methods, such as protease treatment or microwave, can enhance success with this approach (Barthold et al. 1986; Barthold and Smith 1984, 1987; Barthold et al. 1982, 1985; Brownstein and Barthold 1982). Alternatively, in situ hybridization has also been used to label N gene RNA in tissue sections, using either random-primed biotinylated or (32P)-labelled cDNA probes (Compton et al. 2003). It is emphasized, however, that none of these methods are practical unless mice are actively infected, with evidence of acute lesions.
Fig. 6-21 Multinucleate syncytia in surface mucosal epithelium of the ascending colon from an adult mouse infected with enterotropic MHV-Y.
Fig. 6-22 Multinucleate syncytium in mesenteric lymph node of an adult mouse infected with enterotropic MHV-Y.
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VII.
SURVEILLANCE AND CONTROL A.
Surveillance
Effective surveillance should incorporate a combination of diagnostic evaluation of clinically ill mice with periodic testing of sentinel mice AND testing of biological material (mousederived serum, monoclonal and polyclonal antibodies, cells, hybridomas, embryonic stem cells, tumors, virus stocks, etc.) that can be a source of MHV (Nicklas et al. 1993). The frequency and intensity (sample size, etc.) must be individually tailored to scientific, husbandry, financial, and risk conditions. Contact immunocompetent sentinels or sentinel mice exposed to soiled bedding are frequently used for surveillance of MHV and other agents within a mouse population. Under these circumstances, commercially available, pathogen-free, outbred Swiss mice are useful, as they are disease resistant and therefore do not amplify the infection, but readily seroconvert. Soiled bedding contact is most likely to detect the presence of enterotropic Fig. 6-23 Athymic nude mouse naturally infected with enterotropic MHV-RI. Note thickened cecum and colon and enlarged mesenteric lymph node (arrow).
Fig. 6-24 Cecal mucosa of the athymic nude mouse depicted in Fig. 6-23. There is marked mucosal hyperplasia with syncytium (arrow).
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Fig. 6-25 Syncytium of ascending colonic surface epithelium from an athymic sentinel mouse that was naturally infected with enterotropic MHV. Note lack of hyperplasia, compared to Fig. 6-24.
strains of MHV, but may fail to detect less contagious strains of respiratory MHV. When enterotropic MHV is introduced to a naive population of mice, seroconversion of the entire population is likely to occur within 14 days, which is nearly as rapid as seroconversion after experimental exposure (Homberger and Thomann 1994). The rate of seroconversion may be slower among mice infected with respiratory strains of MHV, which tend to be less contagious and require more direct contact. Nevertheless, the presence of a low percentage of positive sera from a sample population suggests nonspecific reactivity. Nonspecific reactions can also occur in some strains of mice with “sticky” sera, such as MRLLpr mice. Another consideration is the type of cage-level containment used for housing. Microisolator and individually ventilated caging offer advantages in prevention and control but complicate surveillance, which may require larger sample sizes. If possible, it is desirable to test indigenous mice in addition to, or instead of, sentinel mice, or to test sentinel mice placed directly in the cage with them. This will enhance detection of less contagious and more environmentally labile viruses, regardless of the agent. Immunodeficient animals may also require this approach, although it should be noted that athymic nude mice seroconvert to MHV, albeit variably. Direct sentinel mice can also be used effectively for detecting virus shedding from mice with uncharacterized or perturbed immune status, as has been shown with genetically altered mice (Pullium et al. 2003; Rehg et al. 2001). Finally, the use of immunodeficient mice, such as nude mice, can be used as a modified sentinel approach in order to detect active MHV, enhance isolation of MHV, or characterize the biotype of MHV that is enzootic within a population (Homberger et al. 1998). Caution is advised with
this approach, as immunodeficient mice will also serve to amplify and perpetuate infection within a population. Likewise, use of breeding populations of sentinel mice, with periodic introduction of naive neonates, can be used for detecting MHV, but will have the same amplifying effect. The ability to detect MHV in feces by RT-PCR in combination with serology permits following single animals over the course of time to determine whether the virus is cleared, thereby selecting nonshedding mice for future use, such as for rederivation (Casebolt et al. 1997; Compton, Ball-Goodrich, Paturzo, et al. 2004; Compton et al. 2003; Smith et al. 2002). This is advantageous when applied to genetically modified mice whose immune competence may not be known and whose virus shedding may be unpredictable. For example, RT-PCR detected MHV in the feces and ascending colons of immunocompetent mice after only 24 hours of contact with tumor necrosis factor–null mice (Pullium et al. 2003). Caution is advised, however, as the effectiveness of fecal RT-PCR testing has not been fully evaluated in mice infected with respiratory (non-enterotropic) MHV strains. RT-PCR has been used successfully to detect MHV sequences on gauze pads placed on the exhaust prefilters of ventilated cage racks containing MHV-infected mice (Compton, Ball-Goodrich, Johnson, et al. 2004). It has also been used to amplify MHV from filter dust in the ventilation ducts of animal rooms (Oyanagi et al. 2004).
B.
Control of MHV in Mouse Populations
Effective control and elimination of MHV from laboratory mouse populations requires global understanding of MHV biology. Critical points that have already been emphasized
170 include the acute nature of infection, recovery in fully immunocompetent mice, unpredictable or persistent infections in immunodeficient or genetically engineered mice, strain specificity of immunity, mutability of the virus, maternal antibody, contagiousness of the virus, and sources of introduction and reintroduction of the virus. MHV can be effectively eliminated from breeding populations of mice by temporary cessation of breeding (Weir et al. 1987), but this “burn-out” approach has been widely misunderstood and abused. Building upon the principle that seropositive mice are recovered or in the process of recovery, if seropositive breeders are selected and isolated from the infected colony and not allowed to breed so that new (susceptible) pups are not exposed, MHV-free (seropositive) breeding stock can be rederived. When dealing with genetically engineered mice, and as a general precaution, testing feces of founder mice for MHV shedding using PCR is also advised (Smith et al. 2002). Depending upon degree of risk, breeding should be ceased for a minimum of one month after verification of seropositive reactivity of breeding stock (longer if the mice are seronegative but from an infected colony); then breeding can be reactivated. Progeny will acquire maternal antibody from their seropositive dams and thus will be antibody-positive for several weeks, and then antibody titers will wane. These mice will serve as MHV-free founder animals for the newly derived colony, but caution is advised in assuring their MHVfree status. Direct contact, virus-free sentinel mice can enhance this process while rederivation is taking place. It is important to emphasize that this cannot be done with a “burn-out” approach on a whole population or room basis. It requires selecting the minimal number of breeding founders possible, isolating them from the infected colony of mice, isolating them from each other, and building up the rederived colony in isolation. The more mice that are being rederived, the greater the risk of continued infection or reinfection. The infected colony contains not only active virus but also new mutants that recirculate within the population because of the strain-specific nature of immunity to MHV. This approach can only be successful by working with the fewest possible founders, strict isolation of those founders, fully immunocompetent founders, assuring the virus-negative status of the founders, and expanding the rederived colony in a protected environment. Resumption of the same traffic patterns and husbandry practices, including placing the rederived mice in conventional housing, will result in failure. Rederivation can also be achieved through cesarean section, embryo transfer, or in vitro fertilization (see Section I, D for caveats). Rederivation of MHV-free mice can be readily accomplished with all of these methods. Xenogeneic tumor lines that are contaminated with MHV can be cleared of infection by transplantation and passage in athymic nude rats. For example, MHV contamination of a human tumor xenograft that was maintained in nude mice (the source of infection) was eliminated by passage in nude rats for 24, but not 12 days (Takakura et al. 2000). This approach, which takes advantage of
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the strict host-specificity of MHV, is not likely to be effective with MHV-infected mouse tumor lines. Isolation of founder mice that have been rederived in the above manner or maintenance of virus-free mice can be accomplished in a number of ways, including housing in isolators, microisolators, barriers, or even geographically isolated animal rooms away from other mice, again depending upon degree of risk. Microisolator cages, with strict attention to protocol, can effectively isolate mice and prevent infection (Barthold and Smith 1983). HEPA-filtered ventilated caging, with appropriate protocol, is also highly effective (Lipman et al. 1993). Facilities with two-corridor barrier design, strict traffic control, and protocol can also effectively prevent introduction of MHV, but the additional protection afforded by microisolator or ventilated caging is advised. Vermin control is mandatory, as is personnel traffic. If investigators insist upon unrestricted traffic in and out of their animal rooms, they must fully accept the consequences. Microisolator or ventilated caging in conventional animal facilities with unrestricted traffic patterns can be a suitable compromise, but with risk. Breeding stock that is not commercially available should always be cryopreserved or otherwise protected against catastrophic loss, especially immunodeficient stocks and genetically engineered mice from which MHV cannot be eliminated. A number of husbandry conditions contribute to introduction and maintenance of MHV in mouse facilities. Shipping mice by commercial carriers is a major risk factor, but MHV can be effectively excluded from mice with filtered shipping boxes (Orcutt et al. 2001). Feral mice are always a factor that requires control. Unauthorized introduction of mice from other facilities and institutions by naive or thoughtless investigators, students, or technicians is increasing in this age of the genetically altered mouse, where mouse strains are shared among investigators. Injection of mice with untested biological products of mouse origin, such as tumor lines, can also introduce MHV. Personnel traffic patterns and manipulation of mice with nonsterile instruments, such as use of forceps or the same gloves from cage to cage, are major risk factors. Mice from commercial vendors often arrive at weekly intervals and are placed in mouse rooms among MHV-infected mice. Even if the indigenous mouse population is nonbreeding, this pattern of weekly introductions provides a renewable source of susceptible mice that allows perpetuation of infection. Mixed quarantine of incoming groups of mice serves little purpose, and in fact can perpetuate and spread MHV among incoming mice. Breeding populations of mice provide a constant source of susceptible mice, even when protected by maternal antibody, with infection taking place after passive immunity wanes. The growing use of genetically altered mice, many of which do not behave in predictable ways in terms of MHV susceptibility and duration of infection (Smith et al. 2002), as well as immune-deficient mutants, can all foster perpetuation of infection in mouse facilities.
6 . M O U S E H E PAT I T I S V I R U S
ACKNOWLEDGEMENTS Supported in part by Public Health Service grant U42 RR14905, “Mutant Mouse Regional Resource Center,” from the National Center for Research Resources, National Institutes of Health.
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177 Tamura, T., Taguchi, F., Ueda, F., and Fujiwara, K. (1977). Persistent infection with mouse hepatitis virus of low virulence in nude mice. Microbiol Immunol 21, 683–691. Tardieu, M., Hery, C., and Dupuy, J.M. (1980). Neonatal susceptibility to MHV3 infection in mice. II. Role of natural effector marrow cells in transfer of resistance. J Immunol 124, 418–423. Thackray, L.B., and Holmes, K.V. (2004). Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus MHV-A59. Virology 324, 510–524. Tooze, J., and Tooze, S.A. (1985). Infection of AtT20 murine pituitary tumor cells by mouse hepatitis virus strain A59: virus budding is restricted to the Golgi region. Eur J Cell Biol 37, 203–212. Tschen, S., Bergmann, C.C., Ramakrishna, C., Morales, S., Atkinson, R., and Stohlman, S.A. (2002). Recruitment kinetics and composition of antibody secreting cells within the central nervous system following viral encephalomyelitis. J Immunol 168, 2922–2929. Uetsuka, K., Nakayama, H., and Goto, N. (1996a). Hepatitogenicity of three plaque purified variants of hepatotropic mouse hepatitis virus, MHV-2 in athymic nude mice. Exp Anim 45, 183–187. — — —. (1996b). Protective effect of recombinant interferon (IFN)-alpha/beta on MHV-2cc-induced chronic hepatitis in athymic nude mice. Exp Anim 45, 293–297. Uetsuka, K., Nakayama, H., Goto, N., and Fujiwara, K. (1995). Severe combined immunodeficiency (SCID) mouse hepatitis experimentally induced with low virulence mouse hepatitis virus. Adv Exp Med Biol 380, 105–107. Vainio, T. (1961). Studies on murine hepatitis virus (MHV3) in vitro. Proc Soc Exp Biol Med 107, 326–331. van der Hoek, L., Pyrc, K., Jebbink, W.F., et al. (2004). Identification of a new human coronavirus. Nat Med 10, 368–373. van der Meer, Y., Snijder, E.J., Dobbe, J.C., et al. (1999). Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication. J Virol 73, 7641–7657. Virelizier, J.L., and Allison, A.C. (1976). Correlation of persistent mouse hepatitis virus (MHV-3) infection with its effect on mouse macrophage cultures. Archiv Virol 50, 279–. Wang, F., Stohlman, S., and Fleming, J. (1990). Demyelination induced by murine hepatitis virus JHM (MHV-4) is immunologically mediated. J Neuroimmunol 30, 31–41. Wang, F.I., Fleming, J.O., and Lai, M.M. (1992). Sequence analysis of the spike glycoprotein gene of murine coronavirus variants: study of genetic sites affecting neuropathogenicity. Virology 186, 742–749. Ward, J.M., Collins Jr., M.J., and Parker, J.C. (1977). Naturally occurring mouse hepatitis virus infection in the nude mouse. Lab Anim Sci 27, 372–376. Ward, J.M., Fox, J.G., Anver, M.R., et al. (1994). Chronic active hepatitis and associated liver tumours in mice caused by persistent bacterial infection with a Helicobacter species. J Natl Cancer Inst 86, 1222–1227. Wege, H., Watanabe, R., and terMeulen, V. (1984). Coronavirus JHM infection of rats as a model for virus induced demyelinating encephalitis. Prog Clin Biol Res 146, 13–22. Wege, H., Winter, J., and Meyermann, R. (1988). The peplomer protein E2 of coronavirus JHM as a determinant of neurovirulence: definition of critical epitopes by variant analysis. J Gen Virol 69, 87–98. Weiner, L.P. (1973). Pathogenesis of demyelination induced by a mouse hepatitis virus (JHM virus). Arch Neurol 28, 298–303. Weir, E.C., Bhatt, P.N., Barthold, S.W., and Simack, P.A. (1987). Elimination of mouse hepatitis virus from a breeding colony by temporary cessation of breeding. Lab Anim Sci 37, 455–458. Welsh, R.M., Haspel, M.V., Parker, D.C., and Holmes, K.V. (1986). Natural cytotoxicity against mouse hepatitis virus-infected cells. II. A cytotoxic effector cell with a B lymphocyte phenotype. J Immunol 136, 1454–1460. Wesseling, J.G., Godeke, G.J., Schijns, V.E., et al. (1993). Mouse hepatitis virus spike and nucleocapsid proteins expressed by adenovirus vectors protect mice against a lethal infection. J Gen Virol 74, 2061–2069.
178 Wilberz, S., Partke, H.J., Dagnaes-Hansen, F., and Herberg, L. (1991). Persistent MHV (mouse hepatitis virus) infection reduces the incidence of diabetes mellitus in non-obese diabetic mice. Diabetologia 34, 2–5. Williams, R.K., Jiang, G.S., and Holmes, K.V. (1991). Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc Natl Acad Sci USA 88, 5533–5536. Woods, R.D., Cheville, N.F., and Gallagher, J.E. (1981). Lesions in the small intestine of newborn pigs inoculated with feline and canine coronavirus. Amer J Vet Res 42, 1163–1169. Wu, G.F., Dandekar, A.A., Pewe, L., and Perlman, S. (2000). CD4 and CD8 cells have redundant but not identical roles in virus-induced demyelination. J Immunol 165, 2278–2286. Wu, G.F., and Perlman, S. (1999). Macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus. J Virol 73, 8771–8780. Xu, Y., Liu, Y., Lou, Z., et al. (2004). Structural basis for coronavirus-mediated membrane fusion. J Biol Chem 279, 30514–30522. Xue, S., and Perlman, S. (1997). Antigen specificity of CD4 T cell response in the central nervous system of mice infected with mouse hepatitis virus. Virology 238, 68–78. Yamachuchi, K., Goto, N., Kyuwa, S., Hayami, M., and Toyoda, Y. (1991). Protection of mice from lethal coronavirus infection in the central nervous system by adoptive transfer of virus-specific T cell clones. J Neuroimmunol 32, 1–9. Yamada, Y.K., Yabe, M., Kyuwa, S., Nakamura, N., Takimoto, K., and Urano, T. (2001). Differentiation of mouse hepatitis viruses in animal facilities in
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Japan by use of nucleotide analysis of the nucleocapsid gene. Comp Med 51, 319–325. Yamaguchi, K., Kyuwa, S., Nakanaga, K., and Hayami, M. (1988). Establishment of cytotoxic T-cell clones specific for cells infected with mouse hepatitis virus. J Virol 62, 2505–2507. Yokomori, K., Asanaka, M., Stohlman, S.A., and Lai, M.M. (1993). A spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry. Virology 196, 45–56. Yokomori, K., Banner, L.R., and Lai, M.M. (1991). Heterogeneity of gene expression of the hemagglutinin-esterase (HE) protein of murine coronaviruses. Virology 183, 647–657. Yokomori, K., and Lai, M.M. (1994). Mouse hepatitis virus receptors: more than a single carcinoembryonic antigen. Arch Virol Suppl 9, 461–471. Yokomori, K., and Lai, M.M.C. (1992a). Mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors. 66, 6194–6199. — — —. (1992b). The receptor for mouse hepatitis virus in the resistant mouse strain SJL is functional: implications for the requirement of a second factor for viral infection. J Virol 66, 6931–6938. Yokomori, K., LaMonica, N., Makino, S., Shieh, C.K., and Lai, M.M. (1989). Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus. Virology 173, 683–691. Youngentob, S.L., Schwob, J.E., Saha, S., Manglapus, G., and Jubelt, B. (2001). Functional consequences following infection of the olfactory system by intranasal infusion of the olfactory bulb line variant (OBLV) of mouse hepatitis virus strain JHM. Chem Senses 26, 953–963. Yu, X., Bi, W., Weiss, S.R., and Leibowitz, J.L. (1994). Mouse hepatitis virus gene 5b protein is a new virion envelope protein. Virology 202, 1018–1023.
Chapter 7 Lymphocytic Choriomeningitis Virus Stephen W. Barthold and Abigail L. Smith
I. Introduction and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Properties of the Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Virion Structure and Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Virus Strains: Antigenic and Genetic Relationships . . . . . . . . . . . . . . . D. Virus Strains: Biologic Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. In Vitro Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mouse Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Natural History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Natural Infection of Laboratory Mice . . . . . . . . . . . . . . . . . . . . . . . . . . C. Routes of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Horizontal Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Vertical Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Host Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Zoonotic Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Disease Syndromes in Laboratory Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Immunopathogenesis of Prenatal, Neonatal, and Adult Infection. . . . . B. Disease in Prenatally and Neonatally Infected Mice . . . . . . . . . . . . . . . 1. Immune Complex Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Endocrine Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Disease in Experimentally Inoculated Adult Mice . . . . . . . . . . . . . . . . 1. Lymphocytic Choriomeningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Wasting Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Lymphoid Organ Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Hematopoietic Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Autoimmune Pancreatic and Neurologic Disease in LCMV-Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Major Histocompatibility (MHC) Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Experimental Induction of Persistence in Adult Mice . . . . . . . . . . . . . . 1. Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. CD8+ T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3. Memory CD8+ T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. CD4+ T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. B Cells and Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. NK Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Interferons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Effects on Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Contamination of Biologic Material . . . . . . . . . . . . . . . . . . . . . . . . . B. Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Effects of Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Behavioral Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Effect on Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Molecular Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Control, Prevention, and Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION AND HISTORY
Lymphocytic choriomeningitis virus (LCMV) is an important virus of the laboratory mouse from a number of perspectives. Foremost is its well-documented zoonotic potential for humans. Mus musculus and its various aboriginal and commensal species or subspecies represent the natural reservoir hosts for LCMV, with an intimate host-virus relationship. This relationship may involve subclinical persistent infection that is associated with minimal or undetectable levels of circulating antibody; thus, detection of infection can be a challenge. LCMV infects a wide variety of tissues, and infection of mice can have protean effects upon normal physiology and immune response, with deleterious impact upon research. The polytropism of LCMV and its noncytolytic course of infection contribute to a welldeserved reputation as a cryptic contaminant of tumors and cell lines. In addition, LCMV is now recognized as the prototype virus of the family Arenaviridae, and has been extensively studied as an experimental model system. As a result, the literature on LCMV is enormous. For more comprehensive, focused, or historical information, the reader is referred to several other reviews (Borrow and Oldstone 1997; Buchmeier, Welsh, et al. 1980; Buchmeier et al. 2001; Buchmeier and Zajac 1999; Hotchin 1962; Lehmann-Grube 1971, 1982; Lehmann-Grube et al. 1983; Oldstone 2002a). This chapter emphasizes the biology of LCMV as a naturally occurring infection of laboratory mice and the practical consequences of infection. Experimental studies, although artificial, provide insight into understanding the biology of natural infection, and are therefore reviewed. LCMV was initially discovered during investigation of the 1933 St. Louis encephalitis epidemic. It received its name at that time due to the nature of brain lesions seen in monkeys and mice inoculated with infectious material, but its biologic origin (monkey, mouse, or human) was initially not determined (Armstrong and Lillie 1934). Its direct association with human
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cases of aseptic meningitis was shortly realized (Findlay and Stern 1936; Rivers and Scott 1935). Human isolates were soon found to be identical to isolates from naturally infected laboratory mice in both the United States and Europe (Findlay and Stern 1936; Traub 1935). Erich Traub, a veterinarian at the Rockefeller Institute of Princeton, described the epizootiology of LCMV infection in a mouse colony, and documented several seminal features of LCMV biology, including in utero transmission, persistent subclinical infection, serologic response, and the relationship of disease and persistence to host immunity (Traub 1935, 1936a, 1936b, 1939). Interest in persistent LCMV infection of mice rose with the concept of immunological tolerance (Burnet 1955). Further recognition that the immune system was responsible for LCMV-related disease (Hotchin 1962; Rowe 1954) fostered a large and continuing body of research on mechanisms of host immunity, disease pathogenesis, and virus persistence, including the critical discovery that antigen recognition by CD8+ T cells occurs in a major histocompatibility (MHC) class I-restricted manner (Zinkernagel and Doherty 1974a, 1974b)
II.
PROPERTIES OF THE VIRUS A.
Classification
LCMV is a member of the family Arenaviridae, which includes a single genus, Arenavirus. Arenaviruses are split into Old World and New World groups that share genetic and antigenic relationships. New World arenaviruses cluster into three lineages: A (Flexal, Parana, Pichinde, Pirital, Tamiami, Whitewater Arroyo viruses); B (Guanarito, Junin, Machupo, Sabia, Amapari, Tacaribe viruses); and C (Latino, Oliveros viruses). Old World arenaviruses include LCMV, Ippy, Mobala,
7 . LY M P H O C Y T I C C H O R I O M E N I N G I T I S V I R U S
Mopeia, and Lassa viruses. Among the Old World viruses, LCMV is most closely related to New World arenaviruses (Bowen et al. 1997). Arenaviruses are generally associated with persistent infections of their reservoir rodent hosts; Mus musculus is the natural reservoir host for LCMV (Buchmeier et al. 2001; Lehmann-Grube 1971).
B.
Virion Structure and Replication
LCMV virions are quite pleomorphic, ranging from 400–300 nm in diameter. The virions encapsidate variable copy numbers of the genome, accounting for the size variation. Virions are enveloped with 7–10-nm T-shaped, tetrameric glycoprotein spikes on their surface (Neuman et al. 2005). Genomic RNA is configured in circular helical nucleocapsid structures that range in length from 400–1300 nm (Young and Howard 1983). A variable number of host ribosomes are typically incorporated within the virions (Farber and Rawls 1975), creating the appearance of grains of sand (Fig. 7-1), for which this group of viruses is named (Latin arenosus, for “sandy”) (Rowe et al. 1970). The name was suggested by Ernest Borden and chosen by a group of virologists at the Centers for Disease Control (Murphy 1975). Arenavirus genomes consist of two single-stranded negative-sense RNA molecules: L (long), approximately 7,200 bases, and S (short), approximately 3,400 bases, which encode
181 non-overlapping sequences. There are short regions of sequence at the 3′ termini of both L and S that are highly conserved among Old and New World arenaviruses (Auperin et al. 1982). In addition, there are inverted complementary sequences at the 5′ termini, which allow for intra- and intermolecular complexes that account for the size of the nucleocapsid (Salvato and Shimomaye 1989; Young and Howard 1983). The S segment encodes the major virus structural components, including the nucleoprotein (NP) and a glycoprotein (GP) precursor, GP-C. The mature glycoproteins GP-1 (44 kDa) and GP-2 (35 kDa) are produced by posttranslational proteolytic cleavage of a primary translation product, GP-C (Beyer et al. 2003; Buchmeier and Oldstone 1979; Kunz et al. 2003; Southern et al. 1987). The L segment encodes viral RNA– dependent RNA polymerase (L) and a small zinc-finger structural or regulatory protein (Z) that is required for virus budding (Perez et al. 2003; Salvato and Shimomaye 1989; Salvato et al. 1989). Arenaviruses have a unique “ambisense” mechanism of transcription (Fig. 7-2), in which the NP coding region is transcribed into a genomic complementary mRNA. In contrast, the GP-C coding region is transcribed into genomic-sense mRNA from antigenomic RNA, which requires replication of the virus to be effected. The L segment functions similarly, with Z encoded in the genomic sense (Salvato and Shimomaye 1989; Southern et al. 1987). Further details of arenavirus replication are reviewed elsewhere (Meyer et al. 2002).
Fig. 7-1 Electron micrograph of LCMV virions grown in Vero cells. Note the variable number of osmophilic granules (host cell ribosomes) within each virion. (courtesy of F. A. Murphy)
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Fig. 7-2 Schematic representation of the LCMV genomic coding regions and ambisense transcription strategy. The viral genomic RNA consists of L and S segments. The L segment contains Z and L coding regions, separated by a stem-loop structure. The S segment contains GPC and NP coding regions, separated by a stem-loop structure. The S segment is represented to depict the transcription strategy of GPC and NP genes. GPC mRNA can only be transcribed from an antigenomic RNA template that is generated during viral replication, whereas NP mRNA is transcribed directly from the genomic RNA template. A similar ambisense strategy is utilized for transcription of Z and L encoded in the L segment. GPC is cleaved into GP-1 and GP-2 following translation. (Adapted and modified from Southern 1996.)
LCMV binds to cellular receptors through the GP-1 glycoprotein, and GP-2 functions as a fusion protein (Borrow and Oldstone 1994). The principal cellular receptor for LCMV GP-1 and other (but not all) arenaviruses is a ubiquitously expressed 120- to 140-kilodalton glycoprotein, alpha-dystroglycan (Cao et al. 1998). Immunosuppressive variants of LCMV bind with high affinity to this receptor, but nonimmunosuppressive variants either do not bind to alpha-dystroglycan or do so with low affinity, and use alternative cellular receptors (Kunz et al. 2004; Smelt et al. 2001). Minor sequence variation in GP-1 confers a degree of tissue specificity to different LCMV strains or variants within the quasispecies population. For example, the presence of phenylalanine at residue 260 of GP-1 confers tropism for neuronal cells, whereas variants with a leucine at residue 260 have selective tropism for cells of the immune system (Ahmed et al. 1991; Dockter et al. 1996; Evans et al. 1994; Villarete et al. 1994). After attachment, virions enter cells via uncoated endocytic vesicles. Nucleocapsids are released into the cytoplasm by pH-dependent fusion of the virion and endosomal membranes. Within the endosome, GP-1 is dissociated from GP-2 upon acidification, which facilitates GP-2 mediated membrane fusion (DiSimone et al. 1994). Cleavage of GP-C into GP-1 and GP2 is not required for cell surface expression of infectious virus, but cell-to-cell transmission is markedly reduced without cleavage (Kunz et al. 2003). Replication and assembly of virions take place in the cytoplasm of the cell, with minimal cytolytic effect, and virions bud from the cell membrane. Virion assembly is imprecise, with variability in L:S genomic RNA ratios and
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genome ploidy, variable incorporation of viral transcripts, and variable incorporation of host cell ribosomes and other cytoplasmic components, which account for the pleomorphism of virions (Buchmeier et al. 2001; Neuman et al. 2005; Southern 1996). In addition to physical pleomorphism, LCMV replication results in a high degree of mutation, typical of RNA viruses in general, and the virus therefore exists as a diverse “quasi-species” population of variants, both in vitro and in vivo. Thus, clonal isolates, upon replication, diversify into a quasispecies population dominated by the parental virus, but with subpopulations of variants (mutants). This must be kept in mind when generalizing about “strain” characteristics, and is an important component of viral pathogenesis (discussed below).
C.
Virus Strains: Antigenic and Genetic Relationships
Commonly studied laboratory strains of LCMV include Armstrong (Arm or ARM) and its variants CA 1371 (Rowe et al. 1963), Arm E-350 (Armstrong and Lillie 1934), Arm 53b (Dutko and Oldstone 1983), Arm clone 13 (Cl 13) (Ahmed et al. 1984), WE (Rivers and Scott 1936), Traub (Volkert 1962), UBC (Hotchin and Weigand 1961), Albany (Hotchin and Weigand 1961), and Pasteur (Riviere et al. 1977). Although WE and UBC are sometimes referred to as separate strains, UBC is a derivative of WE (Hotchin and Weigand 1961). Derivatives of UBC include DOCILE (Docile or DOC) and AGGRESSIVE (Aggressive or AGG), which differ in their replication dynamics in vivo (Hotchin et al. 1962; Pfau et al. 1982). These terms are also used more generically when referring to biologic properties of LCMV strains. Different LCMV strains cannot be readily differentiated by conventional serological assays, peptide profiles, or morphology (Buchmeier, Lewicki, et al. 1980a; Rowe et al. 1970; Weibel et al. 1993). Serum neutralization has shown that LCMV is monotypic. Several different strains and isolates of LCMV have broad cross-reactivity (Weibel et al. 1993). Panels of monoclonal antibodies have shown that LCMV antibodies can be broadly cross-reactive not only among LCMV strains but also with other arenaviruses. However, some monoclonal antibodies can discriminate among strains of LCMV (Buchmeier, Lewicki, et al. 1980; Weber and Buchmeier 1988). Although RNA agar gel electrophoresis of S and L RNAs could not detect differences among several (Arm CA 1371, Arm E-350, WE, UBC, Traub, and Pasteur C1pV 76001) LCMV strains, RNase T1 fingerprint analysis could (Dutko and Oldstone 1983). Genome and amino acid sequence comparisons between Arm CA 1371 and Arm E-350 reveal very high homology (suggesting they may be the same strain), and Arm CA 1371 and WE are also related, whereas considerably more heterogeneity exists among Traub, Pasteur, and WE (UBC) strains (Dutko and Oldstone 1983; Southern and Bishop 1987).
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D.
Virus Strains: Biologic Differences
There are marked differences in the biological behavior of LCMV strains and variants. For example, Arm CA 1371 and Pasteur strains are lethal when inoculated into infant C3H mice, whereas Traub and WE strains do not kill C3H mice (Doyle et al. 1980; Lehmann-Grube 1971; Riviere et al. 1980; Volkert and Larsen 1965a). In other studies involving persistently infected C3H mice, Arm CA 1371, E-350, and Pasteur produced more severe disease than Traub and WE strains (Oldstone et al. 1985). Thus, although Armstrong and WE strains are closely related genetically, they differ significantly biologically. Indeed, minute genetic differences in LCMV can result in marked biologic effects. Clonal variants of the WE strain have significantly different abilities to induce growth hormone deficiency syndrome in persistently infected mice, including variants with a single amino acid difference in GP-1 (BuesaGomez et al. 1996; Teng, Borrow, et al. 1996). Likewise, single amino acid changes in ARM 1371 and WE GP-1 result in clonal variants that suppress the cytotoxic T-lymphocyte response and establishment of persistence (Hunziker, Recher, et al. 2002; Salvato et al. 1991). These studies have given rise to a growing lexicon of clonal substrains of LCMV, including Arm 53b, Arm c13, Arm 4, Arm 5, WE c54, WE c2.2, and WE c2.5, among others (Buesa-Gomez et al. 1996; Salvato et al. 1991; Teng, Borrow, et al. 1996; Wright et al. 1989), and reassortant viruses that possess alternate S or L genomes, resulting in names such as WE c2.5/ARM, ARM/WE c2.5, ARM/WE, and WE/ARM, with the L genome designated first and the S genome second, (Teng, Borrow, et al. 1996). Although creation of L/S reassortant viruses should be straightforward by simply co-infecting target cells, it is complicated by the variance in ploidy among LCMV virions. These clonal variants and reassortants have proven to be very useful for precise investigation of pathogenesis. The disease that results from LCMV infection is dependent upon virus strain (or variant), dose, route of inoculation, host age, host strain, and host immunocompetence (reviewed in Lehmann-Grube 1971). “Aggressive,” “docile,” “neurotropic,” and “viscerotropic” monikers are the result of studies in which intracerebral passage of LCMV reduced the “tolerizing” capability of the virus and enhanced “neurotropism.” “Neurotropic” strains derived from this process were termed “aggressive” because they caused illness and mortality. In contrast, wild-type viruses, especially when inoculated intraperitoneally, were more apt to induce immunosuppression, and were deemed “docile” or “viscerotropic” (Hotchin 1962; Hotchin and Weigand 1961). These terms no doubt evolved from Shwarzman, who initially used the terms “viscerotropic” and “meningoencephalotropic” (Shwartzman 1946). “Aggressive” and “neurotropic” viruses are nonimmunosuppressive variants that can be effectively eliminated by the host immune response, thereby causing immune-mediated brain or liver disease, whereas “docile” and
“viscerotropic” viruses are ones that are immunosuppressive, and thereby reduce immune-mediated brain or liver disease and mortality. Because of the quasispecies nature of LCMV, these virus characteristics can be modified by the route of inoculation, dose, and host factors.
III.
GROWTH IN VIVO AND IN VITRO A.
In Vitro Propagation
Propagation of LCMV in vitro is readily accomplished in several common cell lines derived from a number of host species, including BHK21 (hamster), L (mouse), and Vero (African Green Monkey) cells. The indiscriminate host range of LCMV is exemplified by early success at LCMV propagation in cells derived from humans, monkeys, pigs, cattle, guinea pigs, rabbits, mice, chicks, and even ticks (Lehmann-Grube 1971). Virus replication does, however, vary in different cell lines and with different LCMV isolates (Lehmann-Grube 1971). For example, embryo cells derived from SWR mice produce more LCMV than embryo cells from C3H mice (Oldstone et al. 1969), which correlates with genetic susceptibility and resistance (respectively) to LCMV challenge of mice of these strains (Oldstone and Dixon 1968). Generally, LCMV infection of cells is noncytolytic, so that cytopathic effect is at best subtle. Plaque assays can be performed with cell monolayers that are overlayed with agarose, which allows visualization of plaques (Buchmeier et al. 1978; Hotchin et al. 1971). A modification of this assay utilizes immunocytochemical staining, which provides the advantage of detecting poorly or non-plaquing LCMV isolates (Battegay et al. 1991). Immunoflourescence staining of LCMV-infected cells reveals punctate patterns of antigen in the cytoplasm of infected cells (Fig. 7-3). LCMV-infected cells in culture undergo cycles of acute infection, during which there is ample virus replication, and persistent infection, in which there is a reduction in infectious virus production. During the persistent phase, there can be an increase in S RNA relative to L RNA, down-regulation of viral glycoprotein expression, production of truncated GP-C, and increased levels of NP (Bruns et al. 1990; Oldstone and Buchmeier 1982; Southern 1996). Truncated genomic RNAs accumulate over time, and become incorporated into defective interfering (DI) particles that may have short deletions at the termini of both S and L RNAs. DI particles may also lack one or both genomic RNAs, GP-1 and GP-2 can be absent or nonglycosylated, and GP-C and NP are often altered in size. DI particles are replication-competent, but fail to transcribe, and compete with competent viral genomes for viral proteins (Meyer and Southern 1997). During passage of persistently
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Fig. 7-3 Immunofluorescent staining of LCMV antigen in cultured Vero cells infected with LCMV. Note the typical punctate cytoplasmic pattern of antigen in infected cells.
infected cells, the production of infectious virus and DIs tend to rise and fall synchronously. Thus, DI particles are not a requisite for persistence, but are associated with this state (LehmannGrube et al. 1983; Meyer and Southern 1997; Southern 1996). The significance of DI particles in vivo is not known.
B.
Mouse Bioassays
Virus isolation or detection historically has been accomplished by intracerebral inoculation of adult mice. The speed of detection can be increased by immunofluorescence staining of brain sections before the advent of clinical signs (Lewis et al. 1976). LCMV infectivity can be quantified with LD50 or ID50 assays, using 0.02–0.03 ml of serially diluted virus. Because the mice are immunocompetent, infection results in immunemediated lymphocytic choriomeningitis and death after 5–7 days. This method is fraught with inaccuracy, in that there is an asymmetrical dose response curve due to fewer mice dying in the most concentrated dose range. It is also well established that a significant amount of intracerebrally inoculated material becomes hematogenous; thus, infectious virus in terminally diluted samples may not stay in the brain, thereby skewing the least concentrated dose range (Cairns 1950; Mims 1960). Accuracy can be improved by re-challenge of surviving mice with a dose that causes 100% mortality in non-immune animals, and adding those mice that survive the second challenge (due to immune resistance) to the mortality observed on the first round. This, in effect, converts the LD50 to an ID50 assay. With this combined method, similar ID50 results are obtained when mice are initially inoculated intraperitoneally, subcutaneously, or intraperitoneally, and survivors are challenged intracerebrally. The advantage of intracerebral inoculation
in the first round is the higher rate of mortality, which reduces the number of mice needed for subsequent challenge (Lehmann-Grube 1971). Intracerebral LD50 and ID50 bioassays rely on mortality as an endpoint, which may be straightforward, but poses an animal welfare issue. ID50s can be readily determined by noncerebral inoculation of serially diluted material into outbred, immunocompetent mice, followed by assays for seroconversion (mouse antibody production test). The advantage of mouse infectivity assays is that they are 2- to 10-fold more sensitive than in vitro growth assays (Lehmann-Grube et al. 1985). A more recent permutation of a mouse bioassay involves combined interferon (IFN)-α/β receptor and IFN-γ receptor null mice (AG129 mice), which are exquisitely susceptible to LCMV infection. Such mice can be inoculated intraperitoneally, and then tissues (blood, spleen, kidney, lung) can be tested by plaque assay 4 days later. This assay requires individual housing of the AG129 mice because of the efficiency of virus transmission among these mice (Ciurea et al. 1999; vandenBroek et al. 1995).
IV. A.
EPIZOOTIOLOGY Natural History
It is generally accepted that the natural reservoir host for LCMV is Mus musculus and its subspecies. Recent surveys have revealed LCMV infection among several subspecies of Mus musculus (M. m. castaneus, M. m. gansuensis, and M. m. homourus) in China (Morita et al. 1996) and among Mus domesticus populations in Australia (Moro et al. 2003; Smith et al. 1993).
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Infection of wild mice is well documented in Europe, the United States, Canada, South America, and Japan (reviewed in Lehmann-Grube 1971). This worldwide distribution of LCMV in wild mice is likely due to a long and intimate association between the virus and the mouse, but infection of mouse populations is remarkably sporadic. Enzootic infection of a population is maintained through highly efficient vertical transmission of virus from the persistently infected dam to the fetus (Haas 1954; Lehmann-Grube 1964; Traub 1960). Mouse populations can apparently eliminate the virus through extinction of infected carriers, selective advantage of uninfected mice, failure of maternal transmission in some mice, or recovery of a few mice that subsequently serve as founders for new populations. Variable numbers of LCMV-uninfected pups may be born to actively infected dams, especially in their first litters (Lehmann-Grube 1964; Seamer 1965; Traub 1939, 1975). In utero and postnatal transmission of maternal antibody may also play a role in protecting some fetuses and neonates from infection. In an experimental study, 10–14-day-old mice that received maternal antibody through nursing were protected from lethal LCMV challenge. Using monoclonal antibodies, the protective antibodies were immunoglobulin IgG2a, while IgG1 antibodies mapping to the same epitope were not protective (Baldridge and Buchmeier 1992). Thus, if or when persistently infected dams develop late serum neutralizing (IgG2a) antibodies, effective maternal protection is likely to increase (discussed below). Surveys of wild or feral mouse populations indicate that LCMV infection tends to be focally restricted to specific populations of mice and is not ubiquitous. A serologic survey for LCMV infection of house mouse populations in Baltimore, Maryland, was undertaken, testing almost 500 mice trapped over a 5-year period (1984–1989). Nine percent were seropositive, with infected animals obtained from six of eight sampling sites (Childs et al. 1992). The prevalence varied considerably from 3.9% to 13.4%. In residential areas, positive mice were found clustered within city blocks and households. During the course of a serologic survey of feral mice (Mus domesticus) in southeastern Australia in 1989, 1991, and 1994, a focus of LCMV infection was identified in northeastern New South Wales. Thirty to fifty percent of mice sampled were seropositive (A.L. Smith, unpublished; Smith et al. 1993).
B.
Natural Infection of Laboratory Mice
When LCMV was initially discovered in laboratory mice, investigation revealed that it was not widespread among laboratory mice (Traub 1936a). Serologic surveys within the last two decades have revealed the presence, albeit at low prevalence, of LCMV infections in colonies of laboratory mice, rats, and hamsters (Kraft and Meyer 1990; Lindsey et al. 1991; Sato and Miyata 1986; Smith et al. 1984; Jacoby and Lindsey 1998). LCMV has been commonly associated with contamination of
rodent transplantable tumor lines (Bhatt et al. 1986; Collins and Parker 1972). The rising interest in wild mice for investigation of the genome may pose an increased risk of introduction of LCMV into laboratory mouse populations.
C. 1.
Routes of Transmission
Horizontal Transmission
Mice can be experimentally infected with LCMV through a number of routes, including intracerebral and intraperitoneal routes that are commonly used for experimental studies, but also by dermal scarification, intradermal, subcutaneous, intravenous, intranasal, and intragastric routes (Hotchin et al. 1962; Hotchin and Benson 1963; Lehmann-Grube 1964; Lillie and Armstrong 1945; Rai et al. 1996, 1997; Skinner and Knight 1979; Traub 1936a; Volkert and Larsen 1965b; Weigand and Hotchin 1961). Natural transmission can occur through aerosol exposure, since indirect contact is sufficient for transmission and intranasal inoculation is an efficient means of infection (Lillie and Armstrong 1945; Traub 1936a, 1939). Transmission by direct contact is more efficient, and is likely to include respiratory, oral, and cutaneous routes. Studies that examined the distribution of LCMV antigen or nucleic acid in various tissues of congenitally infected mice or persistently infected adult mice have revealed infection of brain, retina, nasal mucosa, pulmonary interstitium and bronchiolar epithelium, liver and bile ducts, pancreatic acini and islets, pituitary, thyroid, parathyroid, adrenal medulla and cortex, spleen, thymus, lymph nodes, bone marrow, peripheral blood, testes, ovaries, placenta, uterus, renal glomeruli and tubules, urinary bladder transitional epithelium, lacrimal glands, salivary glands, tongue, esophagus, stomach, intestine, epidermis, and hair follicles (Fazakerley et al. 1991; Mims 1966; Rai et al. 1997; Wilsnack and Rowe 1964). Significant amounts of virus are present in urine of congenitally infected mice (Traub 1936a). Fresh urine from persistently infected mice readily infected excoriated skin of guinea pigs, hamsters, and rabbits (Skinner and Knight 1979), and bite wounds have been implicated as a means of transmission (Skinner et al. 1977). Following gastric inoculation, viral nucleic acid was identified in gastric mucosa, with subsequent dissemination to multiple tissues. Closed ileal loop inoculation revealed virus in the intestinal M cells, providing evidence for ingestion as another effective route of infection (Rai et al. 1997).
2.
Vertical Transmission
The status of mice as a persistently infected natural reservoir host of LCMV is highly dependent upon in utero transmission from dam to fetus, and birth of asymptomatic pups that grow to reproductive adulthood and continue the cycle with subsequent infected generations. Intrauterine transmission is highly efficient (Traub 1936a, 1936b, 1939). Infection of fetuses occurs
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during early pregnancy and in fact, ova may be infected prior to implantation (Mims and Subrahmanyan 1966; Traub 1960). Nearly every cell in the fetus can be infected during the first two weeks of gestation. LCMV antigen has been confirmed in uterine mucosa, placenta, and reproductive cells of female carriers, including germinal epithelium, follicles, and ova (Mims and Subrahmanyan 1966; Wilsnack and Rowe 1964). Virus can also be transmitted by the male through infection of the female during mating. In one study, viral nucleic acid was confirmed in spermatogonia but not mature spermatozoa (Fazakerley et al. 1991), whereas antigen has been seen in a proportion of spermatozoa (Lehmann-Grube 1982). However, there is no evidence of infection by spermatozoa through fertilization (Skinner and Knight 1969, 1973; Traub 1960).
D.
Host Range
Aside from mice and humans, a number of other captive domestic and wild animal species are naturally susceptible to infection and disease, and experimental studies have confirmed these observations. Guinea pigs have been noted to be highly susceptible to LCMV infection, and can develop interstitial pneumonia following indirect (presumably aerosol) exposure to enzootically infected mice (Traub 1936a, 1936b). Naturally infected guinea pigs can also be persistently infected without clinical disease (Jungeblut and Kodza 1962). Experimental inoculation of guinea pigs results in varied responses (resistance to lethal), depending upon LCMV strain (Djavani et al. 1998; Dutko and Oldstone 1983; Peters et al. 1987; Volkert and Larsen 1965b). Rats develop neurologic disease following intracerebral inoculation (Cole et al. 1971a; Monjan et al. 1973; Volkert and Larsen 1965b), and serosurveys indicate natural infection of rat colonies (Lindsey et al. 1991; Sato and Miyata 1986). Syrian hamsters are highly susceptible to persistent natural infections, and their zoonotic hazard is discussed below. Experimental infection of hamsters of different ages (fetal, newborn, and young adult) has been well described (Parker et al. 1976; Volkert and Larsen 1965b). In early LCMV studies, monkeys were inoculated intracerebrally, and developed neurologic disease (Armstrong and Lillie 1934). Intravenous, intragastric, and aerosol inoculation of rhesus and cynomolgus macaques with LCMV-WE resulted in hemorrhagic fever and hepatitis (Lukashevich et al. 2002, 2003; Peters et al. 1987). Notably, LCMV was readily transmitted through aerosol exposure in the same room from LCMV-WE persistently infected laboratory mice to cynomolgus monkeys, and both cynomolgus and rhesus monkeys were highly susceptible to experimental aerosol inoculation, with high mortality (Peters et al. 1987). LCMV is strikingly pathogenic for New World callitrichids (marmosets, tamarins, and Goeldi’s monkeys). In these species, a highly fatal hepatitis ensues following ingestion of infected infant mice (Asper et al. 2001; Montali et al. 1993, 1995).
In addition to the species described above, a wide variety of other captive domestic and wild species have been experimentally inoculated with LCMV: rabbits, embryonated and hatched chicks, pigeons, canaries, dogs, cats, ferrets, pigs, horses, cows, bulls, calves, hedgehogs, voles (Microtus, Evotomys), cotton rats (Sigmodon), and several species of nonhuman primates, including chimpanzees (Lehmann-Grube 1971; Skinner and Knight 1979). The logic for performing these studies and the selection of species is dubious, and confirmation of infection in many of these studies was equivocal. Unsubstantiated claims of LCMV isolations from arthropods (including cockroaches), chinchillas, foxes, sheep, and goats have also been reported (Lehmann-Grube 1971). Serologic evidence of LCMV infection in wild rodents has been documented among Microtus pennsylvanicus in Manitoba (Descoteaux 1992), Microtus arvalis, Clethrionomys glareolus, Apodemus sylvaticus, A. flavicollis, and A. microps in Britain (Kaplan et al. 1980), and Sciurus carolinensis in North Wales (Greenwood and Sanchez 2002). Serosurveys of Peromyscus leucopus trapped in forest preserves in Cook County, Illinois, in 1996 and 1997 (A.L. Smith, unpublished) and southern Wisconsin were negative (Burgess et al. 1990).
E.
Zoonotic Disease
Several arenaviruses are associated with severe disease in humans, and others are less pathogenic (Buchmeier et al. 2001). The most notable disease syndromes are hemorrhagic fevers caused by Old World arenaviruses, including Lassa virus (Lassa fever) and the New World lineage B arenaviruses, including Junin virus (Argentine hemorrhagic fever), Machupo virus (Bolivian hemorrhagic fever), Guanarito virus (Venezuelan hemorrhagic fever), and Sabia virus (Brazilian hemorrhagic fever). Hemorrhagic fevers feature generalized petechial hemorrhages, pulmonary edema, thrombocytopenia, focal necrosis of multiple organs including liver, kidney and adrenal, and lymphoid depletion, among other lesions (Walker et al. 1982; Walker and Murphy 1987). LCMV infection in humans is characterized by fever, myalgia, headache, photophobia, nausea, and variable involvement of testes and salivary glands. Some patients have cough and chest pain, and possibly pneumonia. Other manifestations may include rash, arthritis, and myocarditis (Baum et al. 1966; Biggar et al. 1975; Duncan et al. 1951; Lehmann-Grube 1971; Lewis and Utz 1961; Vanzee et al. 1975). LCMV infection is rarely fatal, so autopsy material is limited. One fatal case revealed nonsuppurative meningitis and perivasculitis, with parenchymal gliosis. Antigen was present in meninges and cortical neurons (Warkel et al. 1973). Intrauterine infections have been well documented in humans, resulting in chorioretinitis, hydrocephalus, microencephaly, macroencephaly, intracranial calcifications, mental retardation, seizures, and fetal death (Barton and Hyndman 2000; Jahrling and Peters 1992;
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Wright et al. 1997). Typically, these infections result from maternal contact with infected mice in the home (Barton and Hyndman 2000; Barton et al. 1995, 2002). LCMV has also been reported to cause fatal hemorrhagic fever–like syndromes in humans (Smadel et al. 1942), and hemorrhagic fever–like lesions appear in monkeys experimentally infected with LCMV-WE (Lukashevich et al. 2002, 2003; Peters et al. 1987). In addition, neurologic disease is quite common in patients with Bolivian and Argentinian hemorrhagic fevers (Peters et al. 1987), emphasizing commonality in pathogenesis among arenaviruses. All of the arenaviruses that are known to infect humans, including LCMV, are transmitted from rodent reservoirs to humans by contact with rodent excretions, especially urine and saliva (Salas et al. 1991). LCMV infection of humans is quite common. In a survey of hospitalized patients with aseptic meningitis, over 10% of cases were due to LCMV, especially during the winter, when mice tend to move indoors (Adair et al. 1953; Barton and Hyndman 2000; Meyer et al. 1960). In 1991, nearly 5% of tested inner-city Baltimore residents had antibody to LCMV (Childs et al. 1991). Data on the incidence of LCMV infection in the United States are incomplete, but suggest that the rate has been declining in the latter half of the last century. Sera from age-stratified samples of hospitalized patients in Birmingham, Alabama, had an overall incidence of 3.5% positive, but the prevalence was only 0.3% among patients who were less than 30 years of age, and was 5.4% among those older than 30 years. Socioeconomic status was not associated with positive results (Park et al. 1997b). A remarkably high prevalence of LCMV seroconversion (37.5% of 56 samples) has been reported among humans in Bratislava, Slovakia (Reiserova et al. 1999). In a serologic survey of feral mice (Mus domesticus) in southeastern Australia, a focus of LCMV infection was identified in northeastern New South Wales, in which approximately half of sampled mice were seropositive. Because of zoonotic concerns, testing of nearly 200 human sera obtained from a local public medical clinic did not yield any positive results (A.L. Smith and G.R. Singleton, unpublished; Smith et al. 1993). Thus, factors other than the presence of infected mice in a geographic region determine the risk to the human population. Pet mice have been associated with human infections (Duncan et al. 1951), but pet hamsters pose an even higher risk for human infection (Ackermann 1973; Biggar et al. 1975; Deibel et al. 1975; Hirsch et al. 1974). Childhood infections result from contact with infected rodents in the school setting or in the home (Barton and Hyndman 2000). Hamsters are uniquely susceptible to persistent LCMV infection, and shed copious amounts of virus in their urine (Parker et al. 1976). LCMV-infected laboratory hamsters, and their transplantable tumors, pose a major risk to laboratory personnel, and have been responsible for several outbreaks among laboratory personnel in research institutions (Baum et al. 1966; Lewis Jr. et al. 1965), as well as outbreaks among hospital personnel (Hinman et al. 1975; Hotchin et al. 1974). Recently, LCMV
infection with severe illness was documented in four organ transplant recipients who had received transplants from a common donor. The source of infection was suspected to be a hamster in the donor’s home (Centers for Disease Control and Prevention 2005). Traub’s earliest studies on the epizootiology of LCMV demonstrated seropositivity among animal handlers working with persistently infected laboratory mice (Traub 1936a, 1939). The risk of infection of humans through exposure to LCMVinfected laboratory mice and tumors is apparently low, but this is likely an effect of whether infection of mice is acute or persistent. Two epizootics of LCMV in several mouse colonies did not result in human exposure (Smith et al. 1984), nor did exposure of laboratory personnel to a number of infected tumor lines and mice (Bhatt et al. 1986). Caution is advised, however, as persistently infected C57BL/6 mice readily transmitted the virulent LCMV-WE strain to cynomolgus monkeys through aerosol exposure. Rhesus monkeys were more susceptible than cynomolgus monkeys to experimental aerosol transmission, resulting in a high rate of mortality (Peters et al. 1987). Risk can be increased when nude mice are infected. Infection has been reported among animal handlers at a cancer research center working with infected nude mice, including mice inoculated with a number of contaminated transplantable tumor cell lines (Dykewicz et al. 1992). Thus, differences in infectivity that are observed under natural conditions are likely to be a reflection of variation in virus virulence and the nature of mouse infection (acute or persistent).
V.
DISEASE SYNDROMES IN LABORATORY MICE A.
Immunopathogenesis of Prenatal, Neonatal, and Adult Infection
Natural infections of wild-mouse populations depend upon vertical transmission of virus from the persistently infected dam to the fetus, and maintenance of infection in the population by persistently infected mice from generation to generation. This can occur under laboratory conditions, but is now rare. Congenitally infected mice tend to be subclinically infected, but eventually develop immune-mediated disease, known as “late disease.” When LCMV gains entry to mice with immature immune systems through in utero exposure or during the very early neonatal period, infection is lifelong. Mice manifest a selective lifelong defect in CD8+ T cell responsiveness that is restricted to LCMV (immune tolerance), but can mount normal CD8+ T cell responses to unrelated viruses, including vaccinia, herpes simplex, and influenza viruses. There is generalized LCMV infection in most tissues, including thymus (Southern et al. 1984). The presence of LCMV antigens in the immature thymus results
188 in negative selection of LCMV-reactive T cells (King et al. 1992; Pircher et al. 1989). Asymptomatic persistent infection resulting from prenatal or neonatal exposure was originally described by Traub as a feature of LCMV epizootiology in naturally infected mice, and it was noted at that time that the mice had no detectable serum antibody to the virus (Traub 1935, 1936a, 1936b, 1939). At the time, a novel concept in immunology was becoming popular, known as “immunologic tolerance” in regard to tissue engraftment (Billingham et al. 1953). Burnet speculated that LCMV might also induce tolerance by having either low antigenicity (which was not correct) or low intrinsic pathogenicity (which was correct) (Burnet 1955; Burnet and Fenner 1949). The concept was subsequently proven (Hotchin and Cinitis 1958; Traub 1960; Volkert 1962). As work proceeded, it was shown that “tolerance” could be induced by inoculation of neonatal mice, but only during the first few hours of life (Hotchin 1962; Hotchin and Cinitis 1958; Hotchin et al. 1962; Hotchin and Weigand 1961; Volkert and Larsen 1965b; Weigand and Hotchin 1961). A state of total immune tolerance to LCMV following prenatal or neonatal exposure is actually not present. Although such mice have selective deletion of LCMV-specific T cells, they develop LCMV-specific antibody. The antibody is undetectable because it is complexed with excess circulating antigen and is also deposited in tissues, particularly renal glomeruli (Oldstone and Dixon 1967). This condition has therefore been referred to as “split tolerance,” which is initially relatively innocuous to the host, but with time, mice develop antigen-antibody immune complex disease with glomerulonephritis, arteritis, and other complications. Thus, congenital (in utero) infection, which is part of the natural life cycle of the virus in its reservoir host (the mouse), is conducive to successful reproduction with transmission of virus among generations. Mice develop disease at an age in which their contributions to the population are no longer vital. In contrast, natural or experimental infection of adult, immunocompetent mice follows a distinctly different course. Although experimental inoculation of adult mice has little relevance to natural infection, it provides important insight into the host-virus relationship. Adult mice can develop a wide variety of disease manifestations, depending upon the balance between the host and the virus. When naturally exposed to the virus or experimentally inoculated by natural routes, adult mice develop acute, short-term, and subclinical infections with recovery and seroconversion. Experimental inoculation of virus into immunologically responsive mice can result in disseminated infection, but the host immune response results in CD8+ T cell-mediated disease of multiple organs, most notably the brain (when inoculated intracerebrally) and liver (when inoculated intraperitoneally or intravenously). Virus strains that are classified as “aggressive” are ones that are associated with such disease, but it is the host immune response, not the virus, that is responsible for pathogenesis. In contrast, inoculation of adult mice with high doses of virus, and with viral strains that are
S T E P H E N W. B A R T H O L D A N D A B I G A I L L . S M I T H
“docile,” “viscerotropic,” or lymphocytotropic, results in profound immunosuppression and persistent infection. These viruses are “docile” because the immunosuppression precludes development of immune-mediated disease. Thus, persistence can be induced by two distinctly different mechanisms, depending upon whether mice are exposed prenatally or neonatally, or infected as adults. The concept of “immunologic tolerance” can thus be applied to the former (prenatal or neonatal) situation, which most closely parallels the natural epizootiology of LCMV in mouse populations, and “immunologic exhaustion” to the latter (experimental-adult) situation. In both types of persistent infection, the CD8+ T cell is centrally important, but for different reasons. The differences can be illustrated by the outcome of adoptive transfer of CD8+ T cells into persistently infected mice that were exposed as neonates or as adults. Such treatment of neonatally infected mice results in abrogation of tolerance and clearance of infection (Jamieson et al. 1991), whereas adoptive transfer of CD8+ T cells into mice infected as adults results in rapid exhaustion of the T cells with failure to clear infection (Planz et al. 1997; Tishon et al. 1995). There are significant morphologic differences in persistently infected mice exposed as neonates compared to adults. The levels of Thy1.2+, CD4+, and CD8+ lymphocytes were identical in age- and sex-matched SWR/J and BALB/cByJ mice that were infected at birth compared to uninfected mice, but mice infected as adults had marked Thy1.2+, CD4+, and CD8+ lymphopenia compared to controls (Tishon et al. 1993). Spleens of neonatally infected mice are histologically normal, whereas the white pulp of spleens from mice infected as adults has severe destruction and scarring of lymphoid tissue (spleen, thymus, and lymph nodes) (Tishon et al. 1993).
B.
Disease in Prenatally and Neonatally Infected Mice
In utero or early neonatal exposure results in lifelong infection characterized by a state of high viremia, disseminated infection, and no detectable circulating antibody. In the absence of a host immune response, infection by LCMV can be endured with relatively few overt effects. Infection is not without longterm consequences, as infected mice develop immune-mediated “late disease” (or “late onset disease”) as they age (Hotchin 1962; Hotchin and Cinitis 1958; Traub 1939, 1960, 1975; Volkert and Larsen 1965b). The onset of late disease depends upon the genetic background of the mouse, and also probably on the virus strain. Among SWR/J, B10D2, NZB, NZB/W, AKR, A, and C3H mice inoculated as neonates, SWR carriers had the highest virus titers and AKR the least, which corresponded with the onset and severity of disease. Viral antigen could be visualized in all tissues, but the greatest concentrations were found in brain, liver, and kidney. Chronic disease was characterized as proteinuria in NZB, SWR, and NZB/W strains, and early mortality in NZB and NZB/W mice. Mice developed chronic glomerulonephritis, focal hepatic necrosis,
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generalized lymphoid proliferation, and widespread interstitial lymphoid cell infiltrates in multiple tissues, including liver, kidney, lungs, pancreas, adrenal, omental fat, heart, spleen, thymus, and (rarely) brain. Lesions appeared by 2 months in SWR mice, and were prevalent in all strains, except C3H mice, at 1 year. Vertical transmission of LCMV is not totally innocuous to the fetus and neonate. In utero exposure has been associated with a variable degree of fetal mortality, especially in primiparous dams. Original descriptions of LCMV epizootiology described mortality among mice following the first introduction of the virus into the population, which became inapparent once the virus became enzootic. When LCMV was first introduced to the colony, disease was most apparent among pups. These observations suggest that maternal antibody may influence clinical disease, even in congenitally infected mice. Fetal death is common, and litters born to carrier mice are smaller than litters born to uninfected dams. Maternal cannibalism is also higher (Mims 1968, 1970; Seamer 1965; Traub 1936a, 1936b, 1939). 1.
Immune Complex Disease
Mice that are prenatally or neonatally infected with LCMV are CD8+ T cell tolerant, but produce antibody to the virus (Oldstone and Dixon 1967, 1969). However, the antibody is predominantly IgG1 and non-neutralizing (Baldridge and Buchmeier 1992; Thomsen et al. 1985). Mice have high levels of circulating virus and antigen that are complexed with antibody and complement (Oldstone and Dixon 1969). These immune complexes are deposited in various tissues, but particularly the renal glomeruli (Fig. 7-4), arteries, and choroid
189 plexus (Buchmeier, Welsh, et al. 1980). Although only a small fraction of circulating antibody is LCMV-specific, the immunoglobulins that are deposited in tissues are predominantly LCMV-specific, and bind to the GPs or NP of the virus (Buchmeier and Oldstone 1978; Oldstone et al. 1980). Immune complex deposition leads to a cascade of tissuedamaging events, including glomerulonephritis (Fig. 7-5), arteritis, and infiltration of polymorphonuclear leukocytes, plasma cells, and lymphocytes in tissues (Fig. 7-6) (Accinni et al. 1978; Oldstone and Dixon 1969, 1970). These sites also have local production of interferon (IFN), which plays a role in glomerular damage that can be abrogated with anti-IFN antibodies (Gresser et al. 1978; Riviere et al. 1980; Woodrow et al. 1982). Acquisition of maternal antibody to LCMV or passive transfer of LCMV antibodies will accelerate the evolution of glomerulonephritis and arteritis (Oldstone and Dixon 1970, 1972). Susceptibility to immune complex disease is genetically determined, but polygenic (Oldstone et al. 1983; Tishon et al. 1991). Mouse strains that are most susceptible to immune complex disease, such as SWR mice, are those that generate high levels of antibody and have heavier deposition of immune complexes in their glomeruli (Oldstone et al. 1983). Antibody could be detected earliest and at the highest titer in strains with early disease, such as SWR (Oldstone and Dixon 1969). Notably, high- and low-antibody responders that are susceptible or resistant to immune complex disease, such as SWR and BALB/WEHI mice, respectively, that are infected with LCMVARM may have equivalent viral loads (Oldstone et al. 1983). In addition, SWR mice infected with LCMV-Traub develop low levels of antibody and minimal disease, despite the fact that they have equivalent viral loads as SWR mice infected with LCMV-ARM that develop severe disease (Tishon et al. 1991).
Fig. 7-4 Immunofluorescent staining of immunoglobulin deposition in glomeruli of the kidney from a LCMV neonatally infected mouse with immune-complex glomerulonephritis. (Adapted from Hirsch et al. 1968 with permission of F.A. Murphy.)
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Fig. 7-5 Glomerular hypercellularity with basement membrane thickening and adhesions in a LCMV neonatally infected mouse with immune-complex glomerulonephritis. (From Hirsch et al. 1968 with permission of F.A. Murphy.)
Mouse strains with high levels of endogenous IFN are also prone to more severe glomerulonephritis, such as C3H mice. BALB/c mice are low IFN producers and have less severe disease, whereas Swiss mice are intermediate (Riviere et al. 1980; Woodrow et al. 1982). Thus, “late disease” can be attributed to the combined effects of immune-complex glomerulonephritis and progressive disseminated immune-mediated lymphocytic infiltration of infected
organs due to the presence of LCMV-specific antibody and the gradual breakdown of T cell tolerance, respectively. “Runt” mice can also occur when an intermediate response is effected between the neonatal response and the adult response (Hotchin and Weigand 1961). Driving the outcome in this direction has been induced by transient treatment of neonatal mice with antithymocyte serum at the time of, and within several days after, LCMV infection. Under these circumstances, mice develop high
Fig. 7-6 Periarteriolar infiltration of mixed mononuclear leukocytes in the kidney of a LCMV neonatally infected mouse with immune-complex disease. (From Hirsch et al. 1968 with permission of F.A. Murphy.)
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191
Fig. 7-7 Six-week-old ICR mouse was inoculated with LCMV at birth and treated with anti-thymocyte serum at 3-day intervals. The mouse is severely runted (less than 20 gm body weight) with alopecia and stiff tail. (From Hirsch et al. 1968 with permission of F.A. Murphy.)
titers of both circulating antibody and virus, with development of glomerulonephritis and lymphocytic infiltrates in multiple organs (Hirsch et al. 1968). Mice treated in this way have marked retardation of growth, alopecia, oily skin, joint stiffness, and ataxia (runt disease), thereby manifesting earlier onset of “late disease” (Fig. 7-7). 2.
Endocrine Disorders
C3H/St mice infected as neonates with LCMV-ARM develop a growth hormone deficiency syndrome and severe hypoglycemia that are apparent by 5–7 days of age. By 15 days, mice are approximately half the size of uninfected controls, and most mice die by 30 days of age. The syndrome is due to LCMV infection of growth hormone–producing cells in the anterior pituitary, in which there is significant reduction in growth hormone transcription, but no microscopic evidence of cell damage (Oldstone et al. 1982). Other mouse strains such as CBA/N are also susceptible, but BALB/WEHI and SWR/J mice are not. The syndrome can be induced by other LCMV strains, such as LCMV E-350, and to a lesser extent, LCMV-Pasteur, but LCMV-WE and -Traub have minimal effect. Disease susceptibility is not MHC-linked and appears to be polygenic (Riviere et al. 1985). In keeping with the quasispecies nature of LCMV, although LCMV-WE infects few growth hormone–producing cells, there are variants within LCMV-WE that can be isolated and cloned with the diseaseproducing phenotype (Buesa-Gomez et al. 1996). An excess of disease-negative clones within a quasispecies population prevented the syndrome. The syndrome is correlated with the extent to which growth hormone–producing cells are infected (Teng, Oldstone, et al. 1996). Susceptibility is not linked to the alpha-dystroglycan gene on chromosome 9, but rather maps to a locus on chromosome 17 (Bureau et al. 2001). In susceptible
strains of mice infected with LCMV-ARM or E-350, over 90% of the growth hormone–producing cells of the anterior pituitary contain antigen and replicating virus, but less than 15% of these cells are affected in mice infected with WE and Traub strains (Oldstone et al. 1982). The viral factor is due to viral genetic determinants within the S gene (Oldstone et al. 1985), and genetic sequencing of viral variants with this phenotype has revealed a single amino acid change in the GP-1 of these variants (Teng, Borrow, et al. 1996). The functional lesion in these mice is selective down-regulation of growth hormone transcription, with minimal effect on transcription of other genes, such as thyroid-stimulating hormone, actin, and others (Oldstone 2002a, 2002b). The viral component that seems to mediate LCMV-related selective dysfunction of growth hormone is the viral NP. In transgenic mice in which the growth hormone promoter was used to express LCMV NP in pituitary cells, the syndrome was reproduced (Oldstone 2002b). Another endocrine disorder is diabetes, in which persistently infected mice have hyperglycemia and abnormal glucose tolerance. This syndrome is due to infection of beta cells of the pancreatic islets. Adult mice that were infected as neonates have minimal histologic damage to the pancreas, but mice inoculated as adults develop perivascular inflammatory infiltrates in the islets. Sub-diabetogenic doses of streptozotocin enhanced the virus-induced diabetes (Oldstone et al. 1984; Rodriquez et al. 1985; Tishon and Oldstone 1987). A less well-studied syndrome that develops in persistently infected mice is thyroid dysfunction. Several mouse strains, including BALB/WEHI, C3H/St, and SWR, when persistently infected following prenatal or neonatal exposure to several LCMV strains, including Arm, Traub, Pasteur, and WE, have decreased levels of thyroxine and reduced thryoglobulin mRNA expression in their thyroid glands. Mice have infection of follicular epithelium with the virus, in the absence of lesions (Klavinskis et al. 1988).
192 C.
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Disease in Experimentally Inoculated Adult Mice
Experimental inoculation of adult mice can result in immunemediated pathology in many organs. A thorough and still timely description of the pathology associated with experimental inoculation of mice with different LCMV isolates by various routes provides insight into the acute effects of infection (Lillie and Armstrong 1945). These descriptions were made before there was a known association with the immune response, but correlated findings with those noted by others in early studies (Armstrong and Lillie 1934; Findlay and Stern 1936; Lepine and Sautter 1936; Rivers and Scott 1936; Traub 1936b). Gross pathology included marked splenomegaly, lymphadenomegaly, pale yellow livers, pale kidneys, varying degrees of pleural and peritoneal exudates (regardless of route of inoculation), pulmonary edema, atelectasis and hemorrhage, and several less consistent findings. Microscopically, necrotizing inflammation was noted in liver, lymphoid organs (thymus, spleen, and lymph nodes), bone marrow, and salivary glands. Fatty change of hepatocytes and proximal convoluted tubules of the kidneys were noted, as well as interstitial and perivascular lymphocytic infiltrates in numerous organs, including muscle, heart, salivary glands, thyroid, parathyroid, adrenal, lungs, intestine, pancreas, periadrenal fat, kidneys, liver, testes, ovaries, and uterus. The hallmark lesion, lymphocytic choriomeningitis, was of course also described. Brain lesions generally occurred only in mice inoculated intracerebrally, but mild focal lesions could be inconsistently found in mice inoculated by other routes. The lesions found in various organs were nearly all consistent with the acute phase of infection that evolves with host immune response, but this relationship was unknown at the time. Although it was noted that most mice developed lymphoid hyperplasia in the spleen, occasional mice were also noted to have frank necrosis of the lymphoid follicles. Unbeknownst to the researchers at the time,
they were noting the immunosuppressive course of infection that occurs under circumstances that could later be consistently induced experimentally with high doses and immunosuppressive variants of virus. Somewhat later, Rowe implicated immune-mediated pathogenesis of LCMV disease (Rowe 1954). He and others were finding that various forms of immunosuppression could abrogate LCMV disease in mice, particularly neurologic disease following intracerebral inoculation. These included neonatal thymectomy (Rowe et al. 1963), irradiation (Rowe 1956; Hotchin and Cinitis 1958), cortisone (Hotchin and Cinitis 1958; Hotchin and Weigand 1961), cyclophosphamide (Gilden et al. 1972), and alpha-methopterin (Haas and Stewart 1956). Before the role of CD8+ T cells in pathogenesis was defined, Hotchin and Weigand prophetically noted that disease was “due to the immunological conflict between the host and the agent, analogous to a delayed type hypersensitivity reaction (Hotchin and Weigand 1961).” Sequential studies were performed in mice inoculated intracerebrally with the UBC strain, demonstrating the temporal relationship of disease that paralleled the immune response. Lesions were similar to those previously described in brain, spinal cord, liver, bone marrow, spleen, pancreas, and other tissues, including polyserositis (Fig. 7-8). The temporal evolution of lesions, which generally peaked at 7–10 days, then waned, could be ameliorated by prior X-irradiation of the mice (Collins et al. 1961). 1.
Lymphocytic Choriomeningitis
The extensively studied hallmark lesion of LCMV infection of mice (and other species) is immune-mediated lymphocytic inflammation of the meninges (Fig. 7-9), ependyma, and choroid plexus (reviewed in Lehmann-Grube 1971; Buchmeier, Welsh, et al. 1980). Despite the emphasis, the lesion of lymphocytic
Fig. 7-8 Inflammation of the serosa of the liver from a mouse infected with LCMV. Polyserositis is a common feature of LCMV infection, regardless of route of inoculation.
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Fig. 7-9
Lymphocytic meningitis from a mouse inoculated intracerebrally with LCMV. (Courtesy of F.A. Murphy.)
choriomeningitis is rare or nonexistent in mice infected by any other route than intracerebral inoculation (Lillie and Armstrong 1945). Nevertheless, lymphocytic choriomeningitis has been the major focus of experimental studies since the initial discovery of the virus (Armstrong and Lillie 1934; Findlay and Stern 1936; Rivers and Scott 1936). Clinical signs arise within 4–5 days after intracerebral inoculation, followed by irritability, seizures, and death by 6–8 days. Seizures and mortality can be reduced or delayed with anticonvulsant drug treatment, allowing morphologic lesions to become more advanced (Camenga et al. 1977). There is a strong correlation between the evolution of lesions and brain electrographic abnormalities, which begin as slow waves, followed by paroxysms of electrical discharges during seizures (Chastel et al. 1978).
The pattern of inflammation corresponds to the distribution of virus within the brain and spinal cord (Cole et al. 1971b; Mims 1960; Nathanson et al. 1975; Schwendemann et al. 1983; Wilsnack and Rowe 1964). In persistently infected mice inoculated intracerebrally as neonates, viral antigen is widespread in the parenchyma, including neurons and glia, with minimal or no involvement of the meninges and ependyma. In contrast, in mice inoculated as adults, antigen is present in meninges, ependyma (Fig. 7-10), and choroid plexus, but not the parenchyma (Gilden et al. 1972; Mims and Subrahmanyan 1966; Oldstone and Dixon 1969; Rodriquez et al. 1983). Treatment of mice inoculated as adults with cyclophosphamide results in extension of antigen from the meninges into the parenchyma (Gilden et al. 1972).
Fig. 7-10 Immunofluorescent staining of LCMV antigen in the ependyma of a mouse at 8-days after intracerebral inoculation with LCMV. (Courtesy of F.A. Murphy.)
194 The major effector of lymphocytic choriomeningitis is the CD8+ T cell, which recognizes LCMV antigen in the context of MHC class I (Zinkernagel and Doherty 1974a). Adoptive transfer of lymphocyte subsets (Dixon et al. 1987; Doherty et al. 1988), monoclonal antibody depletion of lymphocyte subsets (Leist et al. 1987; Moskophidis et al. 1987), or intracerebral inoculation of virus-specific T cell clones (Baenziger et al. 1986; Byrne and Oldstone 1986; Klavinskis et al. 1989) have all implicated the CD8+ T cell. In spite of the importance of CD8+ T cells in the pathogenesis of lymphocytic choriomeningitis, disease can occur in mice that are CD8+ T cell–deficient, indicating a contributing role of CD4+ T cells (Fung-Leung et al. 1991; Lehmann-Grube et al. 1993; Quinn et al. 1993). The events involved in development of disease have been carefully studied (reviewed in Doherty et al. 1990). In brief, CD8+ T cells leave the circulation and enter the brain following recognition of endothelial cells that present viral antigens and MHC class I at the blood-brain barrier. Relatively few virusspecific CD8+ T cells appear to be required in breaking the blood-brain barrier (Andersen et al. 1991; Marker et al. 1984). At this stage, the blood-brain barrier becomes dysfunctional without ultrastructural changes (Walker et al. 1975). Once the barrier is breached, there is extravasation of monocytes and nonspecific CD8+ T cells, in addition to specific CD8+ T cells (Allan and Doherty 1985; Walker et al. 1975). NK cells are also present, and few, if any CD4+ T cells. Studies in beige mice or mice depleted of NK cells indicate that NK cells contribute to but are not required for disease (Allan and Doherty 1986). “Bystander” CD8+ T cells of an irrelevant specificity are present in the infiltrates, but their contribution to disease is relatively minor (McGavern and Truong 2004). CD3 delta null mice, which possess 10% of the normal complement of T cells, mount a weak response to LCMV that suppresses the virus in peripheral tissue, but not the brain. These mice do not develop overt choriomeningitis (Kappes et al. 2000). IFN-α transgenic mice, which express IFN-α chronically from astrocytes, have significantly increased survival rates after intracerebral inoculation, with markedly reduced virus replication in brain, normal peripheral CD8+ T cell responses, and less severe immune-mediated neurologic disease (Akwa et al. 1998). 2.
Wasting Disease
Intracerebral, but not intraperitoneal or intravenous, inoculation of mice that are deficient in CD8+ T cells, including beta2-microglobulin-deficient, perforin-deficient, and CD8+ T cell–depleted mice, results in severe weight loss. This syndrome has been termed “wasting disease.” It is not due to the direct effects of the virus, but requires virus-specific CD4+ T cells (Doherty et al. 1993; Fung-Leung et al. 1991; Hildeman and Muller 2000; Kagi et al. 1994; Lehmann-Grube et al. 1993; Quinn et al. 1993). Mice lose 25%–30% of their body weight due to anorexia, but regain the weight by 40–50 days (Doherty et al. 1993). In studies using various null mutant mice
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and adoptive transfer methods of CD4+ and CD8+ T cells from wild-type and null mutant mice, the specific role of CD4+ T cells was confirmed, as well as the pro-inflammatory cytokines IFN-γ, IL-1, and IL-6, but not TNF-alpha. Alpha-melanocyte-stimulating hormone, which is produced in the hypothalamus, was also implicated, but not other hypothalamic appetite-suppressing neuropeptides (leptin, neuromedin U, or neuropeptide Y) (Kamperschroer and Quinn 2002). Albeit somewhat confusing, the term “wasting disease” has also been applied to describe a very different syndrome that represents the often fatal CD8+ T cell–mediated disease that follows infection of IFN-γ null mice with virulent LCMV-Docile or -Traub (Nansen et al. 1999; Ou et al. 2001). 3.
Lymphoid Organ Pathology
In their original descriptions of LCMV-related pathology, Lillie and Armstrong noted that most mice developed splenomegaly, lymphadenomegaly, and lymphoid hyperplasia, but they also noted that some mice had severe necrosis of the splenic white pulp and lymphocytic necrosis in the thymus and lymph nodes (Lillie and Armstrong 1945). These early observations were describing the different forms of response in mice experimentally inoculated with LCMV resulting in either immune-mediated recovery or exhaustion. Prenatal or neonatal infection results in negligible pathology of lymphoid organs. In adult mice, inoculation of LCMV strains or variants (viscerotropic, docile, or lymphocytotropic) with strong affinity for the alpha-dystroglycan receptor on dendritic cells may result in severe immune-mediated necrosis of the splenic white pulp (Figs. 7-11 and 7-12), thymus (Fig. 7-13), and T cell regions of lymph nodes (Borrow et al. 1995; Fazakerley et al. 1992; Smelt et al. 2001). A similar outcome is favored with high doses of virus or certain routes of inoculation, such as intraperitoneal, rather than intracerebral. This severe necrotizing lesion, resulting in global T cell exhaustion and establishment of persistent infection, requires the combination of virus tropism for the alpha-dystroglycan receptor, the presence of dendritic cells with that receptor in the marginal zones of the spleen, spread of virus from dendritic cells to the T cell–rich white pulp, and CD8+ T cell–mediated immune attack on the infected target cells (Muller et al. 2002). 4.
Hepatitis
Necrotizing and inflammatory lesions of the liver were noted in many early reports following experimental inoculation of mice with LCMV (see above). Collins and others (Collins et al. 1961) noted the severity of liver disease, and suggested that it might serve as a model for immune-mediated hepatitis. The model has been established, with defined histology, elevations of alanine aminotransferase, aspartate aminotransferase, glutamate dehydrogenase, and alkaline phosphatase, as well as cellular immune responses. As with neurologic disease, the evolution of
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Fig. 7-11 Spleen of a mouse experimentally inoculated with wild-type viscerotropic LCMV, depicting severe immune-mediated lymphocytic necrosis of the white pulp.
LCMV-related hepatitis is dependent upon virus strain, route of inoculation, and mouse genotype. LCMV-WE causes severe hepatitis, whereas LCMV-Arm does not, and intravenous or intraperitoneal inoculation does, but subcutaneous inoculation does not. Swiss- and A-strain mice are more susceptible than C57BL/6 or CBA mice, and BALB/c and DBA/2 are least susceptible. Hepatitis requires CD8+ T cells, and mice depleted or deficient in B cells develop more severe disease (Zinkernagel et al. 1986). The liver model essentially parallels the intracerebral inoculation model with CD8+ T cell–mediated disease, heralded by infiltration of lymphocytes as acquired immunity develops (Fig. 7-14), followed by immune-mediated parenchymal damage (Fig. 7-15). Describing the lesion is a challenge,
in that the distribution is panacinar but not uniform, with hepatocyte necrosis, swelling, fatty change, and infiltration of the parenchyma with lymphocytes. In an early publication, the hepatitis was described as “here and there the liver cells had undergone necrosis” (Findlay and Stern 1936). The recovery phase is represented by a return to normal acinar structure, but with infiltrates of predominantly lymphocytes in the portal regions. In studies involving C57BL/6 mice inoculated intraperitoneally, hepatitis evolved in three phases, with virus titers rising to day 7, then declining. The initial infiltrating cells were NK cells. By days 5 to 7, livers were pale with panacinar necrosis and infiltration of CD8+ T cells. By day 10, the hepatitis was waning, with infiltration of CD4+ T cells in portal regions. Infiltrates contained predominantly T cells, but also
Fig. 7-12 Higher power of splenic white pulp necrosis from Fig. 7-11.
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Fig. 7-13 Severe immune-mediated lymphocytic depletion of the thymic cortex and medulla of a mouse experimentally inoculated with wild-type viscerotropic LCMV.
macrophages, NK cells, B cells, plasma cells, and neutrophils. Viral antigen was initially seen in Kupffer and endothelial cells, followed by hepatocytes and bile ductular epithelium. Hepatitis was associated with up-regulation of both MHC class I and class II antigens (Lohler et al. 1994). In studies with C3H mice inoculated with “non-hepatotropic” LCMV-Arm and “hepatotropic” LCMV-WE, it was also found that NK cells were the initial infiltrating cell, followed by CD8+ T cells, but the latter cell type did not appear in athymic nude mice (McIntyre and Welsh 1986). Depletion of NK cells had no effect on severity of hepatitis (Bukowski et al. 1983).
5.
Hematopoietic Disorders
Although peripheral blood and bone marrow were generally not examined during early studies, bone marrow necrosis and lymphocytic infiltration were noted by Lillie and Armstrong (Lillie and Armstrong 1945). A common phenomenon is marked but transient peripheral blood pancytopenia in LCMV-infected mice, which appears to be associated with IFN-α/β (Bro-Jorgensen 1978; Broomhall et al. 1987; Silberman et al. 1978; Thomsen et al. 1986). C57BL/6, CBA, C3H/HeJ mice, and, to a lesser extent, DBA/2 and BALB/c mice were shown to develop transient pancytopenia during the first week after low-dose
Fig. 7-14 Liver from a mouse experimentally inoculated with wild-type viscerotropic LCMV, depicting infiltration of sinusoids with lymphocytes during the early evolution of host immunity.
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Fig. 7-15 Liver from a mouse experimentally inoculated with wild-type viscerotropic LCMV, depicting parenchymal necrosis, typical of immune-mediated hepatitis.
LCMV-Arm, -WE, or -Docile infection, and high doses of virus induced severe pancytopenia in all strains. Susceptibility correlated with IFN-α/β response (high responders had lower cell counts than low responders). IFN-γ deficient mice exhibited reduced peripheral blood values, whereas values in IFN-γ receptor and IFN-α/β deficient mice were unaffected compared to wild-type mice. Antigen was present in bone marrow stromal cells of wild type and IFN-γ receptor deficient mice, but was extensively present in megakaryocytes and myeloid precursors, but not erythroid precursors in IFN-α/β deficient mice. The transient depression of hematopoeisis was found not to be associated with CD8+ T cell responses, cytolysis, or secreted IFN-γ from virally induced NK cells, but rather was a direct effect of IFN-α/β, which suppresses virus replication (Binder et al. 1997). In addition to the transient effects of IFN-α/β, C3HeB/FeJ mice develop an autoimmune hemolytic anemia when infected with LCMV. When inoculated with LCMV-DOC, there is transient polyclonal activation of B cells, hypergammaglobulinemia, pancytopenia, and markedly increased erythrocyte-elutable autoantibodies that are not LCMV-specific (Broomhall et al. 1987; Stellrecht and Vella 1992; Vella and Pfau 1991). Immunoglobulins eluted from circulating red cells are predominantly IgG2a, but other subclasses are present. The target antigen corresponds to murine membrane Band 3 protein (Mazza et al. 1997). The anemia is due to peripheral erythrocyte destruction, with no loss in erythropoietic capability, although mice also manifest thrombocytopenia and leukopenia, which are related to bone marrow inhibition (Broomhall et al. 1987). Depletion of CD4+ cells in C3H mice blocks the development of autoantibodies and anemia (Coutelier et al. 1994). In contrast, B10.BR mice develop anemia and hypergammaglobulinemia, but their anemia is due to suppression of erythropoiesis rather than hemolysis (Stellrecht and Vella 1992).
6. Autoimmune Pancreatic and Neurologic Disease in LCMV-Transgenic Mice
Transgenic mice have been created that express LCMV NP or GPs under control of tissue-specific promoters. These models utilize the rat insulin promoter, which stimulates expression of viral antigen in the islets of Langerhans, or the myelin basic protein promoter, which stimulates expression of viral antigen in oligodendroglia. These transgenic mice do not develop autoimmune disease spontaneously, but when they are infected with LCMV, which stimulates a strong CD8+ T cell response, they develop immune-mediated insulitis or demyelinating disease, respectively (Evans et al. 1996; Ohashi et al. 1991; Oldstone et al. 1991).
VI.
MAJOR HISTOCOMPATIBILITY (MHC) LOCUS
A significant and often emphasized determinant of host susceptibility to LCMV is the MHC class I (H-2) locus. The association of MHC class I haplotype with susceptibility to LCMV-mediated disease was initially demonstrated in adult mice by determining the LD50 following intracerebral inoculation of different strains (SWR/J, C3H/HeJ, SWRC3HF1, and C3H.Q). Mice with the H-2q/q or H-2q/k haplotype (SWR, SWRC3HF1, and C3H.Q) were susceptible to disease, and mice of the H-2k/k haplotype (C3H/HeJ) were resistant. Disease susceptibility was associated with inflammation of the leptomeninges and choroid plexus, but did not correlate with virus titers in brain, which were equivalent in C3H/HeJ (H-2k) and C3H.Q (H-2q) mice (Oldstone et al. 1973; Oldstone and Dixon, 1968). Seemingly conflicting results arose after intracerebral inoculation of neonatal mice with H-2b, H-2d, and
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H-2q backgrounds, which are resistant to disease, and mice with H-2k background, which had high mortality (Doyle et al. 1980). The difference in findings was due to the age at inoculation, as neonates are immunologically immature, and therefore do not develop immune-mediated lymphocytic choriomeningitis. Disease susceptibility that evolves over time in inbred mice infected as neonates has been linked to the generation of high levels of antiviral antibody that result in immune-complex disease. SWR make 50-fold more NP and GP specific antibody and 7-fold higher C-binding immune complexes than BALB/ WEHI mice. Both strains have similar virus loads, but SWR have heavier immune complex deposits in tissues, with earlier onset and more severe glomerulonephritis (Oldstone et al. 1983). This varies with virus strain, as SWR mice infected as neonates with LCMV-ARM or E-350 have high levels of immune complexes, somewhat less with LCMV-WE and Pasteur, and less with the Traub strain of LCMV (Tishon et al. 1991). An important breakthrough in immunology research, as well as in understanding LCMV pathogenesis, involved the observation that LCMV-specific induction of CD8+ T cells required LCMV antigens in the context of MHC class I matched target cells (Zinkernagel and Doherty 1974a, 1974b). CD8+ T cells bear antigen-specific receptors that recognize specific viral peptides bound by host cell MHC class I molecules. The viral epitopes that are presented to T cells in different mouse strains with different MHC class I haplotypes differ. Thus, haplotype is an important genetic determinant of susceptibility to infection and immune-mediated disease. Both immune-mediated lymphocytic choriomeningitis and hepatitis have been utilized to study H-2 linkage with susceptibility. For example, C57BL (H-2b) mice mount a CD8+ T cell response against three distinct viral epitopes that map to GP-1, GP-2, and NP, whereas BALB (H-2d) mice mount a response that is directed against a GP-1 epitope and a highly immunodominant NP epitope (reviewed in Borrow and Oldstone 1997). In B10 (H-2b) and B10 congenic mice with differing haplotypes (H-2q, H-2k), severe immune-mediated hepatitis occurred in mice with the susceptible haplotype (H-2q). In this model, it was shown that outcome was influenced by virus isolate, virus dose, and the H-2 locus. An earlier and more rapid CD8+ T cell response, as seen in H-2q high responders, results in more limited CD8+ T cell–mediated liver damage. Higher doses of virus, or rapid replication and dissemination of more virulent virus (such as Docile compared with Aggressive) before efficient T cell– mediated immunity, result in more disseminated infection and more target cells that will be affected by CD8+ T cells. However, these generalities are further impacted by variants of virus that initiate but soon abrogate the CD8+ T cell response, thereby causing a state of anergy and no disease (Leist et al. 1989; McIntyre and Welsh 1986; Pfau et al. 1982; Zinkernagel et al. 1985, 1986). The H-2d locus has been further defined in mice with various allelic variants of H-2d that have lower and slower CD8+ T cell responses (Moskophidis, Lechners, et al. 1994).
A.
Experimental Induction of Persistence in Adult Mice
The host immune response faces significant challenges in eliminating LCMV, requiring CD8+ T cells as the major effector of virus elimination, but also CD4+ T cells and neutralizing antibodies for complete recovery. Immunocompetent mice can generally recover from infection when exposed by natural routes, but persistent infection can be readily induced when mice are inoculated with high doses of immunosuppressive (docile, viscerotropic, or lymphocytotropic) LCMV strains. LCMV employs multifaceted strategies for immune evasion, involving various arms of the innate and acquired immune responses, and is thus readily adapted toward establishment of persistent infections. LCMV has been extensively studied as an experimental model for viral persistence, and although much of that work utilizes artificial routes of inoculation and experimentally selected immunosuppressive virus variants, these studies provide insight into the natural epizootiology of LCMV. Persistent infections are induced under conditions that favor high-dose exposure to virus or infection with immunosuppressive variants. Persistence may also be favored under conditions of low viral growth. Infection of C57BL/6 mice with low doses of LCMV-ARM, -WE/ARM reassortant, -WE, -Traub, and -DOC all induced CD8+ T cell responses, but CD8+ T cell responses ranged from low to high, depending upon growth rate of the infecting virus (listed in order from low to high). Thus, “underwhelming” the immune response may also favor persistence, as occurs with chronic hepatitis virus B and C infections (Bocharov et al. 2004). Although these studies have not been extended to determine if persistent infection ensues, they are likely to lead to further inquiry of this phenomenon. It should be emphasized that the acute vs. persistent infection scenario is not absolute, and in fact represents a spectrum of response, depending upon factors that include virus strain, virus dose, route of inoculation, mouse strain, and mouse immunocompetence. The general dogma is that adult immunocompetent mice that are exposed to LCMV mount immune responses that effectively clear infection. Chronically infected mice can, under some circumstances, recover from infection. However, there is evidence that infectious LCMV can be recovered at very low levels in various tissues following recovery in immunocompetent mice (Ciurea et al. 1999). Furthermore, within a single inbred mouse strain, C57BL/6, the course of infection and subsequent induction or exhaustion of CD8+ T cells and/or immune-mediated disease depend upon virus strain, antigen distribution and tropism, and host response (Moskophidis et al. 1995). DBA/2 mice are susceptible to LCMV because they have low levels of CD8+ T cells compared to other mouse strains. They are able to initially clear virus from the blood, but virus reemerges after 90 days (Hunziker, Ciurea, et al. 2003). LCMV can induce nonspecific immunosuppression in experimentally inoculated adult mice by directly infecting several cell types of the immune system (dendritic cells, lymphocytes,
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and macrophages), or by indirect effects on different components of the global immune response (Borrow et al. 1991, 1995; Doyle and Oldstone 1978; Sevilla et al. 2000, 2003, 2004; Tishon et al. 1993). In contrast to mice that are persistently infected following exposure as fetuses or neonates, which have impaired immune responses to LCMV (tolerance), adult mice with persistent LCMV have severely impaired immune responses to all antigens (exhaustion). 1.
Dendritic Cells
A major mechanism of LCMV immunosuppression is mediated through preferential targeting of dendritic cells, which express high levels of the viral receptor alpha-dystroglycan. Immunosuppressive LCMV strains bind the alpha-dystroglycan receptor at high affinity, whereas viruses that bind at low affinity are cleared by a robust CD8+ T cell response (Sevilla et al. 2000). The major difference between immunosuppressive and nonimmunosuppressive variants of LCMV is infection of dendritic cells. Dendritic cell infection results in impaired expression of MHC class I and class II molecules and costimulatory molecules on both splenic and lymphoid dendritic cells; therefore, dendritic cells fail to stimulate T cell proliferation, and also fail to migrate to lymph nodes to initiate T cell responses. Persistent infection maintains this suppression of dendritic cell function. Furthermore, immunosuppressive strains of LCMV infect bone marrow precursors, inhibiting development of dendritic cells in both the spleen and the lymph nodes (Sevilla et al. 2000, 2003, 2004). Thus, persistence is favored by LCMV strains with high affinity for alpha-dystroglycan. Immunosuppressive LCMV strains Cl 13, WE, and Traub bind to alpha-dystroglycan at higher affinity than other strains, including ARM, E-350, and nonimmunosuppressive variants of LCMV that are isolated from the central nervous system (Kunz et al. 2001; Sevilla et al. 2000; Smelt et al. 2001). 2.
CD8+ T Cells
Immune function is further impaired nonspecifically by virusspecific CD8+ T cell–mediated destruction of infected cells, including macrophages and dendritic cells (Odermatt et al. 1991). The immunosuppressive effect is amplified when LCMV-infected dendritic cells in the marginal zones of the splenic white pulp serve as a conduit for invasion and subsequent immunemediated destruction of the adjacent T cell–dependent white pulp (Fig. 7-11 and 7-12). Similar events take place in lymph nodes, in association with follicular dendritic cells (Borrow et al. 1995; Odermatt et al. 1991). LCMV also infects the epithelialreticular cells of the thymic medulla, and to a lesser extent, the cortex, with CD8+ T cell–mediated destruction of infected cells (Fig. 7-13) (Gossman et al. 1991). The adaptive immune response of the murine host to LCMV infection varies with the virus strain, virus dose, and mouse genotype. When inoculated with a low dose of nonimmunosuppressive
199 LCMV, mice respond with a strong and broadly directed virusspecific CD8+ T cell response, with effective clearance of the virus. Following recovery, the majority of antigen-specific CD8+ T cells undergo apoptosis, with retention of a population of memory T cells. The critical importance of CD8+ T cells is underscored in LCMV-infected, perforin-deficient mice, which cannot eliminate infection (Fuller and Zajac 2003; Kagi et al. 1994; Walsh et al. 1994; Zhou et al. 2002). In contrast, high doses of virus or immunosuppressive strains of virus inoculated into adult mice result in persistent disseminated infections with production of high levels of antigen. This results in a transient CD8+ T cell response in which CD8+ cells are induced, proliferate, and initially function as antiviral effectors, but progressively lose their killing ability (Moskophidis et al. 1993b). Functionally defective CD8+ T cells survive, with loss of ability to produce IL-2, followed by loss of TNF-α, and then IFN-γ production (Fuller and Zajac 2003). With time, CD8+ T cells are progressively exhausted, thereby favoring virus persistence (Fuller and Zajac 2003; Ou et al. 2001; Zajac et al. 1998; Zhou et al. 2002). During early infection, CD8+ T cells with effective antiviral activity may reach peripheral tissues and escape the clonal exhaustion in lymphoid tissues. These cells survive, but are unable to control infection and are eventually eliminated (Zhou et al. 2004). A subpopulation of CD8+ T cells, which coexpress the inhibitory MHC class I NK cell receptor Ly49G2, also participate in the acute-virus specific T cell response, but lyse a more restricted range of targets than conventional CD8+ T cells (Peacock et al. 2000; Peacock and Welsh 2004). The mechanism for depletion of CD8+ T cells during persistent infection is due to emergence and selection of virus variants that are immunosuppressive (Ahmed and Oldstone 1988; Ahmed et al. 1984; Borrow et al. 1995; Dockter et al. 1996; Evans et al. 1994; Sevilla et al. 2000). Persistence is also associated with changes in the viral epitope hierarchy due to contraction of certain epitope-specific CD8+ T cells (Fuller and Zajac 2003), and the hierarchy of epitope-specific responses can also be influenced by changes in viral tropism for different cell types that present viral antigen (Butz and Bevan 1998). Immunosuppressive LCMV variants have alterations in their GP-1 that confer high affinity for the alpha-dystroglycan receptor (Sevilla et al. 2000). A single amino acid change in GP-1 is sufficient to confer this biotype (Salvato et al. 1991). Emergence of immunosuppressive variants dominates over nonimmunosuppressive parental virus in several tissues, especially variants derived from lymphoid tissue (Dockter et al. 1996; Evans et al. 1994). Lymphoid tissue apparently creates a selective environment, in contrast to nervous tissue. When viral clones derived from the nervous system are intravenously inoculated into adult mice, the mice recover, and therefore, these clones are biologically similar to the parental virus, whereas the overwhelming majority of variants derived from lymphoid tissue cause generalized immunosuppression (Tishon et al. 1993). In spite of the strong tendency for development of immunosuppressive variants during infection, there is also a reversion of
200 acquired mutations back to wild-type following elimination of immunological selective pressure. Revertants do not necessarily have “repair” of their original mutation but may have secondary mutations in other regions of the viral genome, or the mutants are simply eclipsed by the quasispecies nature of the virus population in the host (Salvato et al. 1991). These observations suggest that the parental virus strain tends to be conserved as a virus with optimal replication fitness (Hunziker, Ciurea, et al. 2003). 3.
Memory CD8+ T Cells
Memory CD8+ T cells are capable of perforin-dependent killing of LCMV-infected target cells. Although resting CD8+ T cells are poorly cytolytic and express little or no perforin or granzyme, they possess nearly equally rapid cytolytic capability as effector CD8+ T cells against intravenously injected target cells when the effector or memory CD8+ T cells are adoptively transferred into LCMV-immune mice. It is therefore likely that memory CD8+ T cells can rapidly up regulate perforin and granzyme in the presence of antigen (Barber et al. 2003). However, memory CD8+ T cells are seriously perturbed during chronic LCMV infection. Memory CD8+ T cells from persistently infected mice fail to persist when removed from antigen, and are incapable of antigen-independent homeostasis that is maintained by IL-7 and IL-15. In contrast, memory CD8+ T cells from mice that have recovered from acute LCMV infection behave like normal memory CD8+ T cells and are maintained in the absence of LCMV antigen. There are fewer LCMV-specific memory CD8+ T cells in the liver and lung of persistently infected mice compared to memory CD8+ T cells from mice that recovered from acute infection. Furthermore, because of their unresponsiveness to IL-7 and IL-15, memory CD8+ T cells in persistently infected mice are likely to be outcompeted for limited space by other memory T cells in lymphoid organs (Wherry et al. 2004). 4.
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Mutations arose within the immunodominant MHC class II– restricted LCMV GP-1 region. Transgenic LCMV-specific CD4+ T cells, when adoptively transferred into persistently infected mice, selected for CD4+ T cell epitope escape mutants of the virus (Ciurea et al. 2001). When mice are infected with high doses of rapidly replicating and invasive LCMV, such as LCMV-Traub, CD4+ cells are necessary to prevent exhaustion of CD8+ T cells, but CD4+ T cells have only a minor role in clearance of less invasive strains during acute infection and in the long-term control of slowly replicating LCMV strains such as LCMV-Arm (Ahmed et al. 1988; Battegay et al. 1994; Matloubian et al. 1994). Depletion of CD4+ T cells at the time of inoculation with LCMV has minimal effect upon the proliferation or functional inactivation of CD8+ T cells during acute infection, but CD4+ T cells are necessary for maintenance of CD8+ T cell activation and survival of CD8+ T cells. When virus-specific CD8+ T cells are adoptively transferred into persistently infected recipients, the CD8+ T cells are rapidly exhausted (Planz et al. 1997; Tishon et al. 1995), whereas co-transfusion of mice with immune CD4+ T cells or B cells prevents CD8+ T cell exhaustion (Hunziker, Klenerman, et al. 2002). Adoptive transfer of unprimed CD8+ memory T cells into persistently infected mice results in induction and stimulation of the CD8+ T cells by widely distributed antigen, with clonal exhaustion of the T cells before virus can be completely eliminated (Moskophidis, Laine, et al. 1993). In the absence of CD4+ T cells, mice mount virus-specific CD8+ responses, but the CD8+ T cells rapidly lose their antiviral functions, and persistent infection is favored (Battegay et al. 1994; Matloubian et al. 1994; Zhou et al. 2004). This is mediated through the role of CD4+ T cells in initiation of the virus-specific neutralizing antibody response (Hunziker et al. 2002a; Ou et al. 2001). Thus, CD4+ T cell–deficient mice are prone to persistent LCMV infection and cannot sustain a CD8+ memory response (Battegay et al. 1994; Christensen et al. 1994; Matloubian et al. 1994; vonHerrath et al. 1996).
CD4+ T Cells
Although the major effector of adaptive host immunity to LCMV is the CD8+ T cell, CD4+ T cells also play a critical role. CD4+ T cells are involved in virus clearance through a number of mechanisms, including induction, differentiation, and maintenance of CD8+ T cells (Kalams and Walker 1998; Oxenius et al. 1998), secretion of antiviral factors IFN-γ, TNF-alpha, and granzyme A (Baldridge et al. 1997; Guidotti et al. 1999; Oxenius et al. 1998; Planz et al. 1997; Thomsen et al. 1996), and regulation of neutralizing antibody responses. Persistent LCMV infection produces not only CD8+ T cell escape mutants of the virus but also CD4+ T cell escape mutants. Mutations in viral CD4+ T cell epitopes are selected during polyclonal T cell response conditions. This has been demonstrated in perforindeficient C57BL/6 mice infected with a low dose of LCMV-WE. In contrast to a high-viremia scenario, such mice controlled but did not eliminate infection, allowing persistent T-helper responses.
5.
B Cells and Antibody
B cell function is also perturbed by CD8+ T cell destruction, resulting in delayed or reduced neutralizing antibody responses, which further favors persistence and evolution of neutralizing antibody-resistant mutants (Planz et al. 1996). During the early, acute stages of LCMV infection, there is a pronounced nonspecific polyclonal hypergammaglobulinemia that favors survival of the virus. This phenomenon is characterized by up to 10-fold elevations in total immunoglobulin levels, of which greater than 90% are non-LCMV reactive antibodies. This is the result of switching natural IgM specificities to IgG, and is dependent on help from CD4+ T cells that specifically recognize LCMV peptides presented with MHC class II molecules on infected B cells (Coutelier et al. 1994; Hunziker, Recher, et al. 2003; Recher, Lang, et al. 2004). The hypergammaglobulinemia results in a distracted humoral response to LCMV, with impaired and
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delayed formation of virus-specific neutralizing antibodies. Reduction of virus-specific CD4+ T cells results in a reduced polyclonal B cell response and a more effective and specific virus-neutralizing antibody response (Recher, Lang, et al. 2004). Virus neutralizing antibodies are important for controlling LCMV infection, and are essential for maintenance of memory CD8+ T cells. The neutralizing antibody response appears late in infection, and is often low titered. The early CD8+ T cell response is correlated with the absence of neutralizing antibodies. This is due, in part, to CD8+ T cell destruction of infected B cells (Battegay et al. 1993; Planz et al. 1996). In addition, because of the polyclonal B cell activation that is induced during early infection, virus-specific CD4+ T cells impair the induction of neutralizing antibody responses (Recher, Lang, et al. 2004). When neutralizing antibodies are passively administered prior to or during early infection, they are effective at eliminating virus (Seiler et al. 1998; Wright and Buchmeier 1991). The neutralizing antibodies that arise during the late stage of infection prevent CD8+ T cell exhaustion and the emergence of immunosuppressive escape variants by limiting viral replication (Bachmann et al. 2004; Baldridge et al. 1997). Antibody that develops in nonpersistent infections is primarily IgG2a, which is neutralizing, whereas antibody in persistently infected mice is low affinity IgG1 antibody (Thomsen et al. 1985), which is not protective (Baldridge and Buchmeier, 1992). Serum-neutralizing antibodies are directed at epitopes of the GP-1 virion protein (Parekh and Buchmeier 1986). CD40-CD40L interaction has been found to maintain the CD8+ T cell memory response by induction of virus-specific neutralizing antibodies (Bachmann et al. 2004). CD40- or CD40L-deficient mice develop CD8+ T cell responses during early infection comparable to wild-type mice, but CD8+ T cell responses wane over time without CD40-CD40L or CD4+ T cell help, and virus replicates to high titer in these mice (Borrow et al. 1996, 1998; Thomsen et al. 1998). This effect can be abrogated in CD40- or CD40L-deficient mice by repetitive passive immunization with LCMV-specific neutralizing antibodies (Bachmann et al. 2004). Likewise, B cell–deficient mice initially respond to LCMV infection similarly to B cell– competent mice, but eventually fail to control infection (Brundler et al. 1996; Planz et al. 1997; Thomsen et al. 1996). LCMV neutralizing antibody escape mutants arise in persistently infected mice (Ciurea et al. 2000; Hunziker, Ciurea, et al. 2003), which in turn favor higher virus titers in the host and the generation of LCMV immunosuppressive variants with high affinity for alpha-dystroglycan and CD8+ T cell exhaustion. The CD8+ T cell response is inversely proportional to the neutralizing antibody response during LCMV infection. When CD8+ T cell responses are robust and effective, neutralizing antibodies arise late in infection and often at low titer, when there are waning LCMV CD8+ T cells (Bachmann et al. 2004; Baldridge et al. 1997). In mice depleted of CD8+ T cells, there is delayed but efficient control of LCMV by neutralizing antibody responses, but neutralization-resistant LCMV variants arise
under these conditions (Ciurea et al. 2000). When these viral variants are inoculated into naive CD8+ T cell–deficient mice of the same genotype, the mice develop an antibody response against the escape variant, indicating that within the original host, the escape variants have not evaded the genetically possible B cell repertoire. In addition, the new host can generate neutralizing antibody responses against not only the escape variant but also the parental virus, albeit at lower titer. In spite of this, the neutralizing response of CD8+ T cell–depleted mice to neutralization-resistant variants tends to be markedly decreased compared to responses to parental virus (Ciurea et al. 2000). Neutralizing antibody escape mutants do not require a total absence of CD8+ T cells to be generated. DBA/2 mice have low CD8+ T cells compared with other inbred strains. When inoculated with high doses of LCMV-WE, these mice transiently control LCMV-WE infection and clear viremia within 30–50 days, but virus reemerges after 90 days. Emergent variants are neutralization resistant, and contain single-point mutations in GP-1, representing escape mutant serotypes unique to individual mice. When naive mice were inoculated with parental or escape variants, they mounted neutralizing antibody responses with relatively high titers to the inoculated variant (private response), as well as lower titered responses to the parental and other variants (public responses). In summary, low-dose infection of DBA/2 mice resulted in effective CD8+ T cell responses, early and complete virus clearance, and low titered neutralizing antibody responses with less cross-reactivity. In contrast, high-dose infection favored virus persistence, CD8+ T cell anergy, and generation of neutralizing antibody escape mutants (Hunziker, Ciurea, et al. 2003). This phenomenon is likely due to the quasispecies nature of LCMV during infection of the host. Under conditions that favor high virus titers (such as CD8+ T cell deficiency), the replicating virus generates many mutants that stimulate both public and private responses, even though the initially introduced virus may have been clonal (private) (Recher, Hunziker, et al. 2004). 6.
NK Cells
Natural killer (NK) cells are engaged during the first few days of LCMV infection, followed by the adaptive CD8+ T cell response (Welsh Jr. 1978). In addition, LCMV infection stimulates and depletes NK T cells, which utilize the MHC class I–like CD1d1 molecule and are important in the induction of innate immunity, including NK cells (Hobbs et al. 2001). However, infection of CD1d1-deficient mice, which lack NK T cells, resulted in normal NK cell and CD8+ T cell responses, and mice were able to clear LCMV infection (Spence et al. 2001). 7.
Interferons
Interferons exert pleiotropic effects on the innate and adaptive immune response, including stimulation of MHC class I and II expression, regulatory functions in the immune response,
202 activation of macrophages and NK cells, augmentation of dendritic cell responses, and promotion of proliferation and survival of activated lymphocytes. Thus, impaired IFN-α/β or IFN-γ production is likely to influence the complex dynamics of LCMV infection. The ability of different LCMV strains, such as Docile and Arm Cl 13, to induce rapidly disseminated infections correlates with their relative resistance to IFN-α/β and IFN-γ, whereas slow-growing LCMV strains WE, Aggressive, and Arm are IFN-sensitive (Moskophidis, Battegay, et al. 1994). Marked differences in liver disease susceptibility have been noted following inoculation of suckling BALB, Swiss, and C3H mice (listed in order of increasing susceptibility). Susceptibility was associated with levels of IFN, with more severe disease in mice with higher levels of IFN. Treatment of C3H mice with IFN antiserum reduced disease severity, and administration of IFN to BALB mice increased disease susceptibility (Riviere et al. 1977, 1980). Parallel results were found with the severity of glomerulonephritis (Woodrow et al. 1982). IFN-γ null mice infected with LCMV-Docile or -Traub develop CD8+ T cell–mediated wasting disease with impaired silencing of CD8+ T cell responses and exacerbation of immune complex disease (Nansen et al. 1999; Ou et al. 2001). In contrast, infection of IFN-γ null mice with the less virulent LCMV-Arm does not lead to wasting disease, but rather results in persistent infection without CD8+ T cell exhaustion, with enhanced CD8+ T cell activity (Bartholdy et al. 2000). Under these circumstances, CD4+ T cells are essential for maintaining effective CD8+ T cell control of LCMV-Arm. Depletion of CD4+ T cells results in wasting syndrome and death (Christensen et al. 2001). During the acute phase of infection, clonal expansion and differentiation of LCMV-specific CD8+ T cells take place relatively unaffected in the absence of IFN-γ (Lohman and Welsh 1998; Moskophidis, Battergay, et al. 1994a; Nansen et al. 1999; Tishon et al. 1995; vandenBroek et al. 1995). Also, during the acute phase of infection, activated T cells have enhanced capacity to undergo apoptosis upon T cell receptor ligation, resulting in a transient immunodeficiency. IFN-γ receptor deficient mice mount a T cell response to LCMV but do not undergo the transient immunodeficiency, and their immune response results in effective but delayed clearance of virus (Lohman and Welsh 1998). This may explain why the effect of IFN-γ during acute infection depends upon the invasiveness of the virus strain. Infection of IFN-γ deficient mice with rapidly invasive LCMV-Traub results in CD8+ T cell–mediated wasting syndrome, whereas infection of mice with a low dose of this virus strain caused persistent infection. The absence of IFN-γ had no effect on mice inoculated with slowly invasive LCMV-Arm (Nansen et al. 1999). During the recovery phase of infection in mice depleted of IFN-γ with IFN-γ antiserum, there is inhibition of virus clearance due to a reduced CD8+ T cell response (Utermohlen et al. 1996; Willie et al. 1989). In IFN-γ deficient mice, CD8+ T cells cannot clear infection and are permanently activated by the continued presence of virus, but do not become exhausted or depleted (Bartholdy et al. 2000;
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VonHerrath et al. 1997). IFN-γ is important for terminating persistent infection following adoptive transfer immunotherapy in mice exposed as neonates (Planz et al. 1997; Tishon et al. 1995). Somewhat similar effects occur with IFN-α/β. In IFN-α/β receptor deficient mice, LCMV-WE infection results in a persistent infection due to an absence of CD8+ T cells, and elimination of LCMV-Arm can occur, but in a delayed manner (Cousens et al. 1999; vandenBroek et al. 1995). Mice that are dually deficient for IFN-α/β receptor and IFN-γ receptor are highly susceptible to persistent LCMV infection, and do not develop a CD8+ T cell response to infection (vandenBroek et al. 1995).
VII.
EFFECTS ON RESEARCH
Aside from the obvious undesirability of using mice infected with a zoonotic agent, mice exposed to or infected with LCMV have no value as experimental animals, unless they are being studied specifically for research on LCMV biology.
A.
Contamination of Biologic Material
Perhaps the most common effect of LCMV on research is its presence as an adventitious agent in primary cell lines, serially propagated cell lines, and transplantable tumors of both mouse and hamster origin (Bhatt et al. 1986; Bowen et al. 1975; Collins and Parker 1972; Dykewicz et al. 1992; Hinman et al. 1975; Mahy et al. 1991; Nicklas et al. 1993; Smith et al. 1984; Stewart and Haas 1956; vanderZeijst et al. 1983). LCMV has been shown to contaminate human cell lines as well (Simon et al. 1982), and sublines of Toxoplasma gondii, one of which was implicated in infection of a laboratory worker (Grimwood 1985). LCMV has also been found to contaminate leukemia virus stocks (Collins and Parker 1972) and was a contaminant during efforts to adapt human poliomyelitis virus to mice (Wenner 1948). The zoonotic implications of LCMV contamination of cell lines, tumors, and biologic products are major reasons for implementation of institutional surveillance programs that include such material.
B.
Immunosuppression
In addition to the nonspecific immunosuppressive effects of experimentally induced persistent LCMV infection, adult immunocompetent mice that are infected but recover from LCMV infection manifest a transient immunosuppression within 2 to 3 days after infection, which peaks at 7 days and then gradually returns to normal over the course of several weeks (Bro-Jorgenson and Volkert 1974; Butz and Southern 1994; Guttler et al. 1975; Jacobs and Cole 1976; Lehmann-Grube et al. 1972; Mims and Wainwright 1968; Saron et al. 1990,
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1991; Su et al. 1991). This effect is similar to the transient immuno suppression that occurs during the acute immune response to many other viruses. It is believed that during induction of virus-specific immune responses, antigen-nonspecific, “bystander” T cells are activated and proliferate in response to cytokine signaling, and are therefore sensitized to undergo apoptotic cell death upon antigen recognition. Long-lasting immunosuppression can also occur under circumstances in which the virus infects dendritic cells, with depletion of these cells by CD8+ T cells (Borrow et al. 1995; Leist et al. 1988). Under these circumstances, mice may have competent T and B cells, but are immunosuppressed because of impaired antigen presentation (Althage et al. 1992; Borrow et al. 1995; Odermatt et al. 1991). This is associated with necrosis of lymphoid architecture in the spleen and lymphoid tissues (Althage et al. 1992; Lehmann-Grube and Lohler 1981; Moskophidis et al. 1992; Odermatt et al. 1991). The severity of the immune suppression may vary depending upon route of inoculation, virus strain, dose, and host genotype (Althage et al. 1992; Borrow et al. 1995; Odermatt et al. 1991; Roost et al. 1988), and the duration may vary from weeks to months (Ruedi et al. 1990). Recovery is dependent upon immune clearance of the virus, with repopulation of lymphoid organs. Under experimental conditions that favor immunologic exhaustion and persistent infection in adult mice, the suppression is life long (reviewed in Borrow and Oldstone 1997).
C.
Effects of Immunsuppression
Infected mice may have suppressed responses not to only LCMV (Moskophidis et al. 1992) but also to experimental infection with other viruses (Hotchin 1962; Mims and Wainwright 1968; Padnos and Molomut 1973; Roost et al. 1988; Ruedi et al. 1990; Tishon et al. 1993; Youn and Barski, 1966), Eperythrozoon coccoides (Mims and Wainwright 1968), bacterial endotoxin (Hotchin 1962), opportunistic pathogens (Wu-Hsieh et al. 1988), and tumors (Kohler et al. 1990; Padnos and Molomut 1973). The immunosuppressive effects of LCMV infection have also been shown to delay rejection of both skin (Lehmann-Grube et al. 1972) and tumor allografts (Guttler et al. 1975). Mice that are persistently infected with LCMV by natural routes of exposure may have increased susceptibility to the development of lymphomas as they age compared with uninfected mice (Skinner et al. 1980; Traub 1962).
D.
Behavioral Effects
Although there are no reports pertaining to naturally infected mice, experimental persistent LCMV infections have been reported to induce behavioral abnormalities in mice. Persistently infected mice showed increased latency when subjected to openfield tests, decreases in the level required to elicit a startle
response, and decreased locomotor activity in a running wheel (Hotchin and Seegal 1977). A subsequent report indicated that such mice had deficits in acquisition of Y-maze discrimination. Injection of the cholinergic antagonist scopolamine disrupted the performance of trained infected mice more than control performance, suggesting that cholinergic dysfunction accounted for at least a portion of the learning deficit. Persistently infected mice were also hypoactive during first exposure to a locomotor testing apparatus (Gold et al. 1994). Infection resulted in longterm learning deficits, altered synaptic plasticity, and impaired cognitive function. These effects appeared to be due to abnormal GAP-43 expression in the hippocampus, where virus expression was high (delaTorre et al. 1996).
E.
Effect on Autoimmune Diseases
LCMV infection modulates the course of disease in mice that are genetically susceptible to autoimmune diseases. NZBNZWF1 mice develop anti-DNA and -RNA antibodies, resulting in autoimmune glomerulonephritis. When NZBNZWF1 mice or NZB mice are persistently infected with LCMV, their autoimmune disease is significantly accelerated (Dixon et al. 1971; Tonietti et al. 1970). In contrast, autoimmune insulitis of non-obese diabetic (NOD) mice can be prevented by infection of neonatal mice with lymphocytotropic LCMV-Arm 53b (Oldstone 1988, 1990). The effect appears to be due to depletion of LCMV-infected CD4+ T cells, yet the mice retain normal CD4+ T cell responses to other antigens (Oldstone 1990). The active element maps to the small RNA segment of the viral genome (Oldstone et al. 1990). This effect is LCMV strain–specific, as LCMV-Arm 53b, Traub, WE, and Pasteur appear to prevent diabetes in NOD mice, while other strains do not (Oldstone et al. 1990).
VIII. A.
DIAGNOSIS Serology
Historically, the complement fixation (CF) and indirect immunofluorescent antibody (IFA) tests have been used for LCMV serologic diagnosis in rodents and also humans. The CF test, which uses as antigen infected cell culture lysates or guinea pig spleen homogenate (Brown and Kirk 1969), is now generally acknowledged to be insensitive for both rodent and human serodiagnosis (Barton and Hyndman 2000; LehmannGrube et al. 1979; Lewis et al. 1975; Thacker et al. 1982). The neutralization test (NT), performed with cultured cells, has been used extensively; however, it is labor intensive and expensive, and neutralizing antibody may be very slow to develop or may not develop at all (Cohen et al. 1966;
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Lehmann-Grube et al. 1979; Volkert et al. 1964). The sensitivity of the NT may be enhanced by incubation of the virus-serum mixture at 4°C (Lewis et al. 1975) and by the addition of complement to the virus-serum mixture (Webster and Kirk 1974). The IFA test remains one of the tests of choice. It is both sensitive and specific, as well as relatively rapid to perform and interpret (Lewis et al. 1975). In contrast to CF or NT tests, which detect antibody directed toward the NP or GP-1 antigens of the virus, respectively, IFA detects antibodies that are reactive to all viral antigens, although heavily skewed to NP. The specificity of a reaction can be shown by comparative absorption of test sera with infected versus control antigen, either in the form of mouse brain homogenates (Lewis and Clayton 1969) or cultured cell lysates (Smith et al. 1984). Furthermore, IFA offers the advantage of a built-in test of specificity, in that LCMV antigen can be visualized as typical punctate fluorescence in the cytoplasm of infected cells (Fig. 7-3). Enzyme-linked immunosorbent assays (ELISAs) have also been reported for detection of antibody in both human and mouse sera (Homberger et al. 1995; Turkovic and Ljubicic 1992), including an ELISA that utilizes recombinant NP antigen generated in a baculovirus expression system that obviated the need to work with infectious virus to prepare the antigen reagent. Compared with IFA, the recombinant NP ELISA was 100% specific and 95% sensitive (Homberger et al. 1995).
B.
Molecular Detection
Reverse transcriptase-polymerase chain reaction (RT-PCR), both single-stage and nested, have been developed for the detection of LCMV RNA in patient specimens. Targeting the GP-C and N genes of the virus, the assay can detect less than one median tissue culture infectious dose of LCMV (Park et al. 1997a). Although nested RT-PCR was quite sensitive, intraperitoneal inoculation of IFN-α/β and -γ receptor-null (AG129) mice detected even lower concentrations of virus (Ciurea et al. 1999). In a recent report, the sensitivity of the mouse antibody production (MAP) test was comparable to conventional RT-PCR and real-time RT-PCR (Bootz et al. 2003). A fluorogenic nuclease RT-PCR assay has been developed that targeted sequences in a unique region of the N gene. Although the assay had the advantage of eliminating the post-PCR processing and could be used to detect viral RNA in infected tissues, feces, and swipes from cages housing experimentally infected mice, it was 100-fold less sensitive than the MAP test (Besselsen et al. 2003). Further work, perhaps targeting other regions of the LCMV genome, may yield more promising results.
IX.
CONTROL, PREVENTION, AND SAFETY
Because of its zoonotic potential, but also because of its effects on research, LCMV is an unacceptable pathogen in
mouse populations and biological products. This virus alone justifies institutional support for infectious disease quality assurance surveillance of mouse populations to protect personnel, mice, and research. The presence of LCMV requires prompt action when infected mice are identified within a colony. For reasons discussed above, serologic surveillance is not optimal for this virus and should be approached with adequate sample sizes of adult mice, which will be most apt to have detectable antibody. The significance of infection in an adult, immunocompetent mouse population, in which mice are likely to recover from infection, is different than infection in a breeding population of mice, which may be prone to vertical transmission of the virus with persistence, increased virus shedding, and no detectable antibody. Furthermore, immune-deficient mice should be considered an especially high risk. Under these circumstances, adult sentinel mice may be a useful means of surveillance. Sentinel programs that utilize breeding mice or indigenous mice may preclude detection. If the mice are readily available commercially or from colleagues, quarantine with complete depopulation and thorough decontamination are recommended. If the mice are invaluable and/or unique, rederivation should be considered as an option, keeping in mind the fact that LCMV infects female germ cells, and may contaminate sperm. This approach requires extreme caution, as donor mice and offspring, if persistently infected, are likely to be seronegative. Under these circumstances, nucleic acid detection by PCR is warranted. Caretakers or research personnel who have been in contact with LCMV-infected mice or other rodents should be monitored for illness and tested serologically. Pregnant women should never work in areas at risk for LCMV infection of mice or contamination of biological products. The chapter on LCMV in the last edition of this series stated that prevention is as simple as preventing access by wild or feral mice (Lehmann-Grube 1982). While this is an extremely important aspect of prevention of LCMV (and other infectious diseases of mice), prevention also requires screening of transplantable tumors and mouse- or hamster-derived cell lines and commercially purchased mouse serum prior to injection into mice. All such material should be tested as an institutional occupational safety requirement. The improvement in molecular diagnostic methods facilitates this testing and costs the investigator very little time compared to the MAP test that has been historically used. It also obviates the need for intracerebral inoculation of adult mice, a technique that causes distress to the mice and exposes the handler to undue hazard. It is also noteworthy to mention the extent of mouse-related research with LCMV that involves persistent infection, infection of immune-deficient mice, and immunosuppressive variants of the virus. This may increase with biodefense-related research that may utilize LCMV as a prototype virus for its more virulent arenavirus relatives. Thus, iatrogenic introduction of experimental LCMV into mouse facilities is a risk factor that may be on the rise. Because of its zoonotic risk, the risk of aerosol exposure, and the potential for excretion from various routes, LCMV and infected mice require CDC Biosafety Level 3 (BSL3)
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containment facilities, equipment, and protocols. Such work should be reviewed and supervised by the Institutional Biosafety Committee as well as the Institutional Animal Care and Use Committee.
ACKNOWLEDGMENTS Supported in part by Public Health Service grant U42 RR14905, “UCD Mutant Mouse Regional Resource Center,” from the National Center for Research Resources, National Institutes of Health. A.L.S. was supported by a Fulbright Short-Term Senior Scholar Award for studies performed in Australia. The contribution of images for this publication by F.A. Murphy, DVM, PhD, University of California at Davis is appreciated. More importantly, S.W.B. is deeply appreciative of Dr. Murphy’s guidance, friendship, and support.
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213 Wright, R., Johnson, D., Neumann, M., et al. (1997). Congenital lymphocytic choriomeningitis virus syndrome: a disease that mimics congenital toxoplasmosis or cytomegalovirus infection. Pediatrics 100, E9 1–6. Wu-Hsieh, B., Howard, D., and Ahmed, R. (1988). Virus-induced immunosuppression: a murine model of susceptibility to opportunistic infection. J Infect Dis 158, 232–235. Youn, J., and Barski, G. (1966). Interference between lymphocytic choriomeningitis and Rauscher leukemia in mice. J Natl Cancer Inst 37, 381–388. Young, P., and Howard, C. (1983). Fine structure of Pichinde virus nucleocapsids. J Gen Virol 64, 833–842. Zajac, A., Blattman, J., Murali-Krishna, K., et al. (1998). Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med 188, 2205–2213. Zhou, S., Ou, R., Huang, L., and Moskophidis, D. (2002). Critical role for perforin-, Fas/FasL-, and TNFR1-mediated cytotoxic pathways in downregulation of antigen-specific T cells during persistent viral infection. J Virol 76, 829–840. Zhou, S., Ou, R., Huang, L., Price, G., and Moskophidis, D. (2004). Differential tissue-specific regulation of antiviral CD8+ T-cell immune responses during chronic viral infection. J Virol 78, 3578–3600. Zinkernagel, R., and Doherty, P. (1974a). Immunological surveillance against altered self components by sensitized T lymphocytes in lymphocytic choriomeningitis. Nature 251, 547–548. — — — (1974b). Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 248, 701–702. Zinkernagel, R., Haenseler, E., Leist, T., Cerny, H., Hengartner, H., and Althage, A. (1986) T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay J Exp Med 164, 1075–1092. Zinkernagel, R., Leist, T., Hengartner, H., and Althage, A. (1985). Susceptibility to lymphocytic choriomeningitis virus isolates correlates directly with early and high cytotoxic T cell activity, as well as with footpad swelling reaction, and all three are regulated by H-2D. J Exp Med 162, 2125–2141.
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Chapter 8 Lactate DehydrogenaseElevating Virus Jean-Paul Coutelier and Margo A. Brinton
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. History of Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Properties of the Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Physicochemical Properties and Structure . . . . . . . . . . . . . . . . . . . . . . . D. Intracellular Viral RNA Synthesis and Viral Replication . . . . . . . . . . . E. Susceptibility to Temperature and Various Chemical Agents . . . . . . . . IV. Virus Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Quasispecies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Genetic and Antigenic Relationships with Other Viruses . . . . . . . . . . . V. Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Site of Virus Replication In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Kinetics of Replication In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. LDV Replication in Primary Cell Cultures . . . . . . . . . . . . . . . . . . . . . . D. LDV Replication in Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Characteristics of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Impaired Serum Enzyme Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Morphological Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Persistence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Interferons and Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Cellular Innate Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. T Lymphocyte Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Antibody Response and Immune Complexes . . . . . . . . . . . . . . . . . . . . H. Modulation of Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Polioencephalomyelitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. LDV and Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Host Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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VIII. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. LDH Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Detection of LDV RNA, Virions, or Antigens . . . . . . . . . . . . . . . . . . . . C. Detection of Anti-LDV Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
Lactate dehydrogenase-elevating virus (LDV) is a mouse arterivirus, unusual in its extreme host specificity and its persistence in the circulation of the infected host that naturally infects wild mice. Although probably not as frequent in laboratory mouse colonies as it used to be, LDV infection may affect experimental results, primarily through its effects on the host immune responses. On the other hand, because of its unique properties, LDV infections serve as a good animal model for viral persistence, virally induced immunomodulation, and pathogenic infection of neurons in the central nervous system.
II.
HISTORY OF ISOLATION
During a search for methods that could be used in the early diagnosis of tumors, Riley and Wroblewski (1960) found that, following the inoculation of mice with Ehrlich carcinoma cells, a 5- to 10-fold increase in lactate dehydrogenase (LDH) levels in the serum occurred before detectable tumor growth. This early rise in LDH levels observed in the tumor-bearing mice was duplicated in normal mice by the injection of serum from the mice with tumors (Riley et al. 1960); serum from these infected mice subsequently raised the levels of LDH in other normal mice. These results suggested the presence of an infectious agent. That this agent is a virus was indicated by its ability to pass through a bacteria-retaining filter and by its susceptibility to inactivation at 70°C. Riley (1968) subsequently found that this virus (LDV) was a contaminant of 26 murine tumors that had been maintained by serial transplantation in mice. However, that LDV is not a necessary component of tumor lines was indicated by the finding that many spontaneous or induced tumors were not contaminated with LDV (Riley 1961, 1962; Crispens 1963; Mundy and Williams 1961; Notkins et al. 1962). Although LDV has been referred to in the literature by a number of names, including “Riley virus,” “virus enzymeelevating factor,” and “lactic dehydrogenase virus,” it is now commonly referred to as “lactate dehydrogenase-elevating virus.”
III.
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PROPERTIES OF THE VIRUS A.
Classification
LDV was first classified as a togavirus (Fenner 1977). Based on its virion morphology, genomic structure and conserved sequence motifs, and replication strategy, LDV has now been reclassified as an arterivirus, together with porcine reproductive and respiratory syndrome virus (PRRSV), equine arteritis virus, and simian hemorrhagic fever virus (Kuo et al. 1991; de Vries et al. 1992; Plagemann and Moennig 1992; Meulenberg et al. 1993, 1994; Godeny et al. 1993, 1995; Conzelmann et al. 1993). Arteriviruses are related to coronaviruses, toroviruses, and roniviruses. These virus families form the order Nidovirales (Cavanagh 1997). Among the arteriviruses, PRRSV and LDV are the most closely related. It has been hypothesized that PRRSV may have evolved from LDV following infection of wild boars that fed on infected mice (Plagemann 2003).
B.
Morphology
LDV is a spherical enveloped virus. Its surface appears smooth in electron micrographs with no obvious spike projections (Horzinek and Wielink 1975). The carbohydrate moiety of the LDV glycoprotein is thought to be located on the outer surface of the viral envelope, since LDV can be precipitated by concanavalin A (Brinton-Darnell and Plagemann 1975). The average virion diameter of LDV is 50–55 nm (Fig. 8-1) (Rowson and Mahy 1975). Unfixed LDV is very susceptible to distortion by the usual procedures of negative staining. Some preparations contain a few virions with a diameter of about 80 nm; these large particles may contain two nucleocapsids (Horzinek and Wielink 1975; Brinton-Darnell and Plagemann 1975). The diameter of the nucleocapsid of LDV is approximately 30–35 nm.
C.
Physicochemical Properties and Structure
In glycerol gradients, LDV has a sedimentation coefficient of about 200 S, whereas its nucleocapsid sediments at 176 S
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Fig. 8-1 A positively stained thin section of LDV pelleted from mouse plasma that was collected 24 hours after infection (Brinton-Darnell and Plageman, 1975).
(Horzinek and Wielink 1975). In sucrose gradients, LDV has an unusually low density (1.13–1.14 gm/ml) (Michaelides and Schlesinger 1973; Horzinek and Wielink 1975; Brinton-Darnell and Plagemann 1975). The genome of LDV is a 48 S RNA molecule with a molecular weight of 5–6 × 106 Da and a length of about 14,000 nucleotides (Godeny et al. 1993). That the RNA is sensitive to ribonuclease (RNase) digestion shows that it is single-stranded. This RNA contains nine open reading frames (ORFs) (Fig. 8-2)
(Kuo et al. 1991, 1992; Godeny et al. 1993; Chen et al. 1993). ORF 1 produces two polyproteins, 1a and 1b, that contain conserved protease, RNA polymerase and RNA helicase motifs, and a number of transmembrane domains (Godeny et al. 1993; Faaberg and Plagemann 1996). ORFs 2a through 7 are overlapping and encode structural proteins. Purified viral RNA is infectious when injected into mice intracerebrally, a route that minimizes the chance of RNA digestion by ribonucleases (Notkins and Scheele 1963; Rowson et al. 1968; Darnell and Plagemann 1972; Niwa et al. 1973). LDV virions likely contain six envelope proteins (Snijder et al. 2003). All but two of these proteins are minor components. The major envelope proteins are GP5 (formerly VP3) and M (formerly VP2). GP5 is a 199 amino acid protein. However, after polyacrylamide gel electrophoresis (PAGE), its molecular weight cannot be estimated with accuracy because it appears as a wide heterogeneous band of 24–44 kDa due to glycosylation (Michaelides and Schlesinger 1973; Brinton-Darnell and Plagemann 1975). GP5 is encoded by ORF 5 and is composed of a short, N-glycosylated ectodomain that contains the major antigenic epitopes and the single neutralization epitope of the virus (Plagemann 2001b), and a longer endodomain (Chen et al. 1993; Faaberg and Plagemann 1995). The other major envelope protein, M, is a nonglycosylated integral membrane protein of 18 kDa, is encoded by ORF 6, and is apparently nonantigenic (Godeny et al. 1993; Chen et al. 1993). GP5 and M form heterodimers linked by disulfide bonds that are essential for viral infectivity (Faaberg, Even, et al. 1995a). The 13–15 kDa nucleocapsid protein, N (formerly VP1), is encoded by ORF 7 (Godeny et al. 1990, 1993; Chen et al. 1993). The molecular ratios of N, M, and GP5 within LDV virions have been estimated to be 3–5:1:1 by Michaelides and Schlesinger (1973) and 2–3:1:1 by Brinton-Darnell and Plagemann (1975).
ORF1b
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Fig. 8-2 LDV genome structure. The nonstructural polyprotein open reading frames (ORFs) 1a and 1b are followed by the E protein gene, three genes encoding minor virion glycoproteins (ORFs 2b, 3, and 4), and the genes encoding the major structural proteins, GP5, M, and N. The cleavage sites in the nonstructural polyprotein are indicated by arrowheads. Black arrowheads indicate sites cleaved by the nsp4 serine protease (S). Cleavage sites for the papainlike proteases (P) and the nsp2 cysteine protease (C) are indicated as white arrows. Four highly conserved domains in ORF 1b are indicated. These are the RNA-dependent RNA polymerase (R), a putative zinc finger (Z), an RNA helicase (H), and a nidovirus-specific domain (X) of unknown function.
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D.
Intracellular Viral RNA Synthesis and Viral Replication
No continuous cell line has yet been found that is permissive to LDV infection. The synthesis of LDV RNA has been studied in cultures of peritoneal exudate cells in the presence of actinomycin D. Single-stranded 48 S genome RNA and RNase-resistant 27 S replicative form RNA can be detected in infected cells within 3–5 hrs after infection. The time period required for a completed genome to appear extracellularly within a mature virion is about 1.5 hrs at 37°C. Virions begin to be released 5–6 hr after infection (Brinton-Darnell et al. 1975). The synthesis of intracellular viral RNA is completely inhibited by the addition of cycloheximide to the culture medium 0.5–1 hr after infection, indicating that protein synthesis is necessary for the initiation of viral replication. LDV replication involves the formation of a 3′ terminal nested set of subgenomic mRNAs in infected cells (Kuo et al. 1991). Negative-stranded subgenomic RNAs have also been detected in infected macrophages (Chen et al. 1994). The production and accumulation of LDV proteins as well as virus maturation occurs in perinuclear regions. LDV matures by budding from the cytoplasm into intracytoplasmic vesicles
(Fig. 8-3) (Brinton-Darnell et al. 1975; Tong et al. 1977; Ritzi et al. 1982; Plagemann et al. 1995). The virions are thought to be released extracellularly by fusion of these vesicles with the plasma membrane.
E.
Susceptibility to Temperature and Various Chemical Agents
LDV is most stable in undiluted mouse plasma. However, upon purification or dilution, the virions become more sensitive to inactivation by heating, variation in the salt content of the suspending medium, and treatment with various chemicals. LDV in mouse plasma can be stored indefinitely at −70°C without loss of titer (Notkins and Shochat 1963). However, storage at 4°C results in a loss of infectivity with a decrease in titer of about 3.5 logs in 32 days (Riley 1968). At room temperature, virus-containing plasma or feces retain their infectivity undiminished for about 24 hr. Infectivity is lost more rapidly at higher temperatures. It has been reported that LDV infectivity can be completely inactivated by heating at 58°C for 1 hr (Bailey et al. 1963; Rowson et al. 1966). Virus suspended in
Fig. 8-3 Mature LDV particles within intracytoplasmic vesicles and immature particles in the process of budding into these vesicles. Thin sections of cultured mouse peritoneal exudate cells 12 hr after infection with LDV (Brinton-Darnell et al. 1975).
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tissue culture fluid is more susceptible to heating at 37°C than is virus in mouse plasma (Evans and Salaman 1965; Riley 1968). LDV infectivity is also strongly reduced by ultrasonic treatment (Cafruny et al. 1995). LDV is fairly stable between pH 6 and pH 8 but is inactivated at pH 3 (Riley 1968; Crispens 1965a). The virus is inactivated by lipid solvents such as ether, butanol, and chloroform (Notkins and Shochat 1963; Crispens 1965b; Mahy et al. 1966). LDV is extremely sensitive to detergent treatment; a brief incubation with a nonionic detergent, such as 0.01% Nonidet P40 or Triton X-100, is sufficient for the disruption of the virus (Brinton-Darnell and Plagemann 1975). LDV is resistant to digestion with trypsin or papain (Crispens 1965a, 1965b).
IV.
VIRUS STRAINS A.
Quasispecies
The etiological agent of a polioencephalomyelitis that develops in some mouse strains (see below) was identified as LDV (Martinez et al. 1980). However, various LDV isolates differ in their ability to induce the disease, as well as by a few oligonucleotides detected by RNA fingerprinting, although their physicochemical characteristics and their structural protein patterns are indistinguishable (Martinez et al. 1980; Contag, Retzel, et al. 1986b; Brinton, Gavin, and Fernandez 1986). Most virus pools obtained from infected mice consist of populations of different virus variants (quasispecies), and most virus pools contain both neuropathogenic and non-neuropathogenic LDV isolates (Chen et al. 1997; Chen, Li, et al. 1998a). Homologous recombination between LDV quasispecies may occur at a high frequency in infected mice (Li et al. 1999). Subsequent sequence analysis of the RNA of biologically cloned neurovirulent (LDV-C, LDV-v) and non-neurovirulent (LDV-P, LDV-vx) LDV isolates revealed divergence, especially in the first three ORFs (Kuo et al. 1992; Palmer et al. 1995). Particular differences in the ORF 5 sequence result in differential glycosylation of the GP5 envelope protein of these isolates (Faaberg, Palmer, et al. 1995). Reduced glycosylation of GP5 and sensitivity to antibody neutralization correlate with neuropathogenicity in particular strains of immunosuppressed mice (Chen, Li, et al., 1998b; Chen et al. 2000). Non-neuropathogenic and antibody-resistant virions predominate in chronically infected mice (Monteyne and Coutelier 1994; Chen et al. 1999; Plagemann et al. 2001).
B.
Genetic and Antigenic Relationships with Other Viruses
Analyses of the sequence and structure of the LDV genome indicated homology with the genomes of other arteriviruses
(de Vries et al. 1992; Meulenberg et al. 1993, 1994, 1995; Godeny et al. 1993, 1995; Chen et al. 1993; Conzelmann et al. 1993), but not with those of flaviviruses or togaviruses (Godeny et al. 1989), with which LDV was previously classified. In addition, antigenic cross-reactivity between the primary envelope glycoproteins of LDV and PRRSV was found using antiviral polyclonal and monoclonal antibodies (Plagemann et al. 2002).
V.
GROWTH IN VIVO AND IN VITRO A.
Site of Virus Replication In Vivo
That LDV replicates in macrophages has been suggested by a number of different investigations. The replication of LDV is normal in mice subjected to whole-body irradiation (duBuy and Johnson 1966; M. A. Brinton, unpublished observations), which only slightly reduces macrophage number. Using an indirect immunofluorescent technique, two laboratories have demonstrated the presence of cells containing LDV-specific antigen in sections of the spleen and liver (Porter et al. 1969; Rowson and Michaels 1973). Antigen-containing cells were not observed in sections of kidney, lung, thymus, or salivary gland. The maximum number of stained cells (1500–5000 per 6–8 nm2) were observed in spleen sections between 18 and 24 hr after infection. These stained cells contained a nucleus and were located in the red pulp, which suggests that they were monocytes or macrophages. A reduction in the number of nucleated cells in the spleen was observed 36 hr after infection, and only about 200 cells in a 6-8 mm2 section showed positive staining. In the liver sections, staining was confined to Kupffer cells (Porter et al. 1969). Electron microscopic examination of the spleens and lymph nodes of infected mice revealed increasing numbers of virus particles 12 hrs after infection that were in close association with the plasmalemma of reticular cells located in the marginal zone of lymphoid nodules in the spleen and medulla of the mesenteric lymph nodes (Snodgrass et al. 1972). In attempts to locate a target organ, a number of investigators have removed various tissues from infected animals and assessed the titer of virus each contained (Bailey and Monroe 1972; duBuy and Johnson 1966; Plagemann et al. 1963). Such experiments are complicated by the fact the LDV is found in very high titers in the blood. Titers in the spleen, lymph nodes, liver, and thymus are similar to those in the serum, whereas such tissues as kidney, lung, small intestine, pancreas, and brain contain less virus than the serum. Perfusion of the spleen or lymph nodes with saline before titration did not reduce the virus titer significantly (duBuy and Johnson 1966). K. E. K. Rowson (1964, unpublished observations) looked at tissue titers soon after infection and was unable to demonstrate the appearance of virus in any organ prior to its appearance in the serum. These experiments are consistent with the hypothesis that LDV replicates in macrophagelike cells and that such cells are present in many tissues as well
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as in the bloodstream. Although LDV replicates in spleen macrophages, this organ does not represent the major source of circulating virions, since LDV titers in the blood are not affected by splenectomy (Chan et al. 1989). Neuropathogenic LDV strains have been shown by immunofluorescent analyses, in situ hybridization, and electron microscopy to replicate in anterior horn neurons (Stroop et al. 1985; Brinton, Gavin, and Weibel, et al. 1986; Contag, Chan, et al. 1986). However, viral RNA is detected in these motor neurons only 10 days after infection, while in the same animals, infection of macrophages in lymphoid organs and of cells in the leptomeninges occurs one day after virus inoculation (Anderson et al. 1995).
B.
Kinetics of Replication In Vivo
LDV replicates rapidly in mice after infection, reaching an unusually high titer in the serum of 1010–1011 infectious doses 50% per ml (ID50/ml) 12–14 hr after infection (Riley 1974; Notkins 1965a). Few other known mammalian viruses replicate as efficiently. The titer subsequently drops to about 107 ID50/ml 72–96 hr after infection; thereafter, there is a further gradual decrease until a stable level of 105 ID50/ml is reached approximately 2 weeks after infection (Fig. 8-4). The efficiency of LDV replication in mice is demonstrated by the fact that the injection of as little as 10 ID50 LDV yields serum titers of 1010 ID50/ml 24 hr after infection (M. A. Brinton, unpublished observations; Rowson and Mahy 1975). It has been suggested that the cause of the rapid decrease in titer that occurs after the first day of infection is due to the death of infected target cells and the resulting scarcity of infectible cells (see also the next section). Treatment of mice with dexamethasone before viral inoculation enhances both the number of infected cells and the level of
viremia (Cafruny et al. 2003), supporting the hypothesis that LDV replication is only limited by the availability of target cells.
C.
LDV Replication in Primary Cell Cultures
In vitro, LDV replicates only in primary cultures of normal mouse tissue (duBuy and Johnson 1966; Evans 1967; Plagemann and Swim 1966a, 1966b; Yaffe 1962). Primary cultures of murine spleen, bone marrow, and embryo fibroblasts propagate LDV (Evans and Salaman 1965; Anderson et al. 1965, 1966; Brinton-Darnell and Plagemann 1975). It seems likely that monocytes or macrophages in these cultures are the cells in which LDV replicates, since cultures of adult peritoneal exudate cells, which are rich in macrophages, yield the highest titers of virus. In cultures of peritoneal macrophages, the initial lag period lasts about 5 hours, and a maximum titer of virus of 108.5 ID50/ml of culture fluid is observed about 16 hours after infection. The production of LDV decreases by about 99% 1–2 days after infection regardless of the age of the culture at the time of infection (Brinton-Darnell et al. 1975; Ritzi et al. 1982). Although a small number of infected cultures may continue to shed virus at a low level, usually the replication of infectious LDV ceases completely and these cultures become resistant to superinfection with LDV. Moreover, infected cells disappear quickly, indicating that LDV replication is cytocidal (Ritzi et al. 1982; Onyekaba, Harty, and Plagemann 1989). When cultures are infected on successive days after seeding, the virus yield decreases progressively with increasing age of the culture at the time of infection (Evans and Salaman 1965; Brinton-Darnell et al. 1975). When peritoneal macrophages are cultured in the presence of macrophage colony-stimulating factor, a substance present in L cell–conditioned medium (Virolainen and Defendi 1967; Stewart et al. 1975), they retain
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LDV infectivity titers and LDH activity levels in the plasma of mice at various times after infection with LDV (Notkins and Schochat 1963).
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their susceptibility to infection with LDV for several weeks (Lagwinska et al. 1975; Stueckemann et al. 1982). Even in primary peritoneal exudate cell cultures, only a small proportion of cells seems to be infected. Although 95% of the cells in cultures prepared from starch-stimulated mice are capable of phagocytosis of latex particles, only 6–20% of these cells, when analyzed autoradiographically, showed evidence of a productive LDV infection (Tong et al. 1977). Autoradiographic analysis and electron microscopic examination of thin sections of infected macrophage peritoneal cultures revealed virions in about 3–8% of the cells (Ritzi et al. 1982). However, when macrophages are obtained from 1- to 2-week-old mice, up to 80% of cells are permissive to LDV replication (Onyekaba, Harty, and Plagemann 1989). Together, these observations suggest that LDV replicates in a subpopulation of macrophages and that virus replication in these cells leads to cell death. Persistence of infection depends on renewal of this cell subpopulation, and this appears to be facilitated by treatment of the cultures with macrophage colony-stimulating factor. Occasionally, peritoneal macrophage cultures were found to produce very low yields of virus after infection with LDV (M. A. Brinton, unpublished data). This is thought to have been due to an inapparent mouse hepatitis virus (MHV) infection in the mice from which the macrophages were harvested. MHV infections have been shown to alter macrophage ectoenzyme phenotypes and host resistance to a second virus infection (Dempsey et al. 1986).
D.
LDV Replication in Cell Lines
Although primary peritoneal exudate cultures support LDV replication, cultures of SV40-transformed macrophages and other murine macrophage cell lines do not (Brinton-Darnell et al. 1975; M. A. Brinton, unpublished observations). Neither primary cultures of rat (Evans 1964) nor human peritoneal macrophages support LDV replication. A number of other cells, such as suckling hamster kidney cells (Tennant and Ward 1962), murine tumor cell lines (Yaffe 1962; Plagemann and Swim 1966a), HeLa cells (Plagemann and Swim 1966a), and Rhesus kidney cells (Evans and Salaman 1975), have also been found not to replicate LDV. Only a few LDV-permissive cells have been detected in some clones of cell lines derived from macrophages of 1- to 2-week-old mice (Onyekaba et al. 1989). One group of investigators has reported that mouse, rat, mink, rabbit, and human cell lines infected with ecotropic, dual-tropic, amphotropic, and xenotropic murine leukemia viruses (MuLV) are permissive to LDV (Inada and Yamazaki 1991; Inada 1993). The ability to infect MuLV-infected cell lines was higher for neurovirulent isolates of LDV than for non-neurovirulent ones, whereas infectivity for macrophages is similar for all LDV strains. This difference correlates with differences in adsorption to the cells, rather than in differences in viral replication, since
transfected LDV RNAs from both neurovirulent and nonneurovirulent isolates replicated equally well in MuLV-infected and non-infected cells (Inada and Yamazaki 1991; Inada et al. 1993). E.
Receptor
The identity of the cell receptor(s) for LDV is not known. Pseudotype virions containing LDV RNA and the envelope proteins of mouse hepatitis virus, a coronavirus, could infect cells that are usually resistant to LDV infection (Even and Plagemann 1995), but pseudotype virions containing LDV RNA and the envelope proteins of Sindbis, a togavirus, could not (Lagwinska et al. 1975). These data suggest that the absence of a suitable receptor for LDV on the cell surface represents the major restrictive element to LDV infectivity. This hypothesis was also supported by the efficient infection by LDV of usually resistant cell lines after LDV RNA transfection (Inada and Yamazaki 1991; Inada et al. 1993). Analysis of the binding of labeled purified LDV virions has shown that one or several trypsin-sensitive molecules, expressed on a macrophage subpopulation and distinct from Fc and C3 receptors, were responsible for LDV attachment to their target cells (Kowalchyk and Plagemann 1985). A decrease in the number of LDV-infected cells detected by immunofluorescence after treatment of cells with anti-Ia antibodies have suggested to some authors a correlation between the expression of Ia antigens by macrophages and their permissiveness for LDV infection (Inada and Mims 1984, 1985a, 1985b, 1987). However, these results could not be confirmed by another group (Buxton et al. 1988), and there is still controversy about the putative role of Ia as the major receptor for LDV. On the other hand, Fc receptors can enhance LDV infectivity for macrophages when the virus has been complexed by antibodies (Cafruny and Plagemann 1982b; Inada and Mims 1985b).
VI. A.
CHARACTERISTICS OF INFECTION Impaired Serum Enzyme Clearance
Increased plasma levels of LDH in mice after LDV infection were responsible for the original discovery of the virus, for its name, and are utilized for the assay of virus infectivity titers. Although there are five naturally occurring LDH isozymes in mouse plasma, only isozyme LDH V has been found in increased amounts in mice infected with LDV (Plagemann et al. 1963; Warnock 1964). As can be seen in Table 8-1, the levels of a number of other serum enzymes are also elevated after LDV infection. Enzyme levels begin to rise by 24 hr after infection, and the maximum increase is observed after 72–96 hr. Although enzyme levels fall somewhat during the next 2 weeks
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TABLE 8-1
PLASMA ENZYMES ELEVATED IN PLASMA OF MICE INFECTED WITH LDV Enzyme
Degree of elevation
Lactate dehydrogenase Isocitrate dehydrogenase Malate dehydrogenase Phosphoglucose isomerase Glutathione reductase Aspartate transaminase Glutamate-oxalacetate transaminase Alanine transaminase Acid phosphatase Alkaline phosphatase Aldolase α-Glycerophosphate Glucose-6-phosphate dehydrogenase Glutamate pyruvate transaminase Leucine aminopeptidase
8- to 11-fold 5- to 8- fold 2- to 3-fold 2- to 3-fold 2- to 3-fold 2- to 3-fold 2- to 3-fold Slight None None None None None None None
after infection, elevated levels persist in infected animals (Fig. 8-4) (Rowson et al. 1963; Notkins 1965a). Observed plasma levels represent a balance between influx and clearance of enzymes. Normally, an increase in the plasma levels of tissue enzymes is the result of cell damage; however, with LDV infection, enzyme levels are permanently raised without evidence of significant tissue damage. Moreover, no increase in LDH levels is observed in LDV-infected cultures of peritoneal exudate cells. The in vivo increase in LDH levels after LDV infection appears to result mainly from a decreased rate of enzyme clearance. The clearance rate has been shown to be significantly reduced in infected animals as compared to uninfected ones (Mahy 1964; Bailey et al. 1964; Notkins and Scheele 1964; Notkins 1965b; Riley et al. 1965; Mahy et al. 1965a, 1965b). The clearance of many plasma enzymes is due to receptormediated endocytosis and involves cells of the reticuloendothelial system, including liver Kupffer cells and other resident macrophages. Increases in the levels of these enzymes likely correspond to cytocidal infection of these cells by LDV (Smit et al. 1990). Interestingly, the SJL/J mouse strain displays a uniquely higher (by about 2-fold) elevation of LDH after LDV infection (Crispens 1971) that is under the control of a recessive Mendelian gene (Crispens 1972). However, the rate of clearance of exogenously injected LDH V was similar in LDV-infected SJL/J and Swiss mice (Brinton and Plagemann 1977), and the reason for the increased LDH levels in LDVinfected SJL mice is not known. B.
Morphological Changes
LDV infection usually causes no obvious clinical disease. A slight but significant splenomegaly has been reported to occur by 24 hours after infection (Notkins 1965a). A transient
fall in the total white blood cell count begins at about 24 hours after LDV infection and lasts for one day (Riley 1968). LDV induces a transitory decrease in thymus weight, starting 24 hours after infection. By 3–4 days after infection, thymus weight, which has decreased by about 40%, then increases again, and by the seventh day exceeds that of the controls (Proffitt and Congdon 1970; Santisteban et al. 1972). Adrenalectomy prior to infection with LDV prevents this involution of the thymus, suggesting that LDV infection causes an increase in the levels of circulating adrenal cortical hormones, which in turn are responsible for cellular loss in the thymic-dependent areas (Santisteban et al. 1972). Although a significant number of immune complex deposits have been observed in the kidney glomeruli of chronically infected mice, only mild subclinical lesions develop (Porter and Porter 1971) and no symptoms of an immune complex disease are displayed. Significant pathology is induced by LDV infection only in synergy with other pathogenic mechanisms.
C.
Persistence
Although LDV infection results in lifelong viremia, during which the average titer of infectivity in the plasma is 105 ID50/ml (Fig. 8-4), infected mice live a normal life span (Notkins 1965a). LDV first replicates in a macrophage subpopulation present in most tissues, and especially in lymph nodes, spleen, and skin (Anderson, Rowland, et al. 1995). At later times after infection, virus is found in the same tissues, as well as in the liver and testis. Persistence of the virus in the blood is thought to be due to the existence in most viral pools of LDV quasispecies that resist neutralization by antiviral antibodies (Monteyne and Coutelier 1994; Chen et al. 1997). As a consequence of the development of an antiviral immune response in the infected host, these resistant LDV quasispecies outcompete antibody-sensitive ones and are predominantly found in chronically infected mice (Plagemann et al. 2001).
D.
Interferons and Cytokines
Although LDV appears to be a poor inducer of type I interferon (α/β-interferon) in vitro (Evans 1970; Notkins 1971a; Yamazaki and Notkins 1973; duBuy et al. 1973), proliferating cells may produce IFN when infected with LDV (Lagwinska et al. 1975). LDV replication is sensitive to the action of interferon in primary mouse embryo cultures (Notkins 1971a; Yamazaki and Notkins 1973), but not in peritoneal exudate cultures (Stueckemann and Plagemann 1978). In vivo, α/β-interferon is produced at high levels about 24 hr after LDV infection (Baron et al. 1964, 1966; duBuy and Johnson 1965; Evans and Riley 1968; duBuy et al. 1973; Koi et al. 1981), and it may be involved in bringing about the decline in plasma viral titers that begins about 24 hr after
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infection, as suggested by in vivo experiments that utilized the interferon inducer statolon (Crispens 1970). Production of type II interferon (γ-interferon) has also been reported in the early hours and days after LDV infection (Plagemann et al. 1995; Markine-Goriaynoff et al. 2002). However, LDV-induced γ-interferon does not control the replication of the virus (Cafruny et al. 1999; Markine-Goriaynoff et al. 2002). LDV permissiveness is reduced in macrophages obtained from mice treated in vivo with γ-interferon, but not in cells incubated in vitro with the same cytokine (Cafruny et al. 1994). Finally, LDV infection is followed by an early and transient burst of pro-inflammatory cytokines (Fig. 8-5), including interleukins 6 (Markine-Goriaynoff et al. 2001), 12 (Coutelier et al. 1995), 15, and 18 (J. P. Coutelier, unpublished results). The effects, if any, of these cytokines on LDV replication or pathogenesis are not yet known.
increased phagocytic activity (Stevenson et al. 1980; Meite et al. 2000). However, the ability of these cells to present antigens appears to be impaired (Isakov et al. 1982a, 1982b). Analyses of the number of activated macrophages and of the kinetics of activation indicate that the functions of non-infected cells are stimulated by LDV infection. LDV infection activates natural killer (NK) cells via the secretion of α/β-interferon (Koi et al. 1981; Leclercq et al. 1987), which in turn leads to an increased production of γ-interferon (Markine-Goriaynoff et al. 2002). A strong enhancement of the cytolytic activity of NK cells is also observed after LDV infection (Koi et al. 1981; Leclercq et al. 1987; Markine-Goriaynoff et al. 2002). However, this activation of innate immune cells fails to control LDV replication in vivo (Markine-Goriaynoff et al. 2002).
F. E.
T Lymphocyte Response
Cellular Innate Immune Response
The importance of the innate immune system in antiviral defense has been demonstrated during the past few years. Like other viruses, LDV strongly activates cells involved in the innate immune system. Activation of macrophages by LDV leads to the early production of the pro-inflammatory cytokines listed above (Coutelier et al. 1995; Markine-Goriaynoff et al. 2001). Macrophages from LDV-infected mice display an increased potential to produce nitric oxide (Rowland, Butz, et al. 1994). These macrophages also express enhanced levels of Fc and complement receptors (Lussenhop et al. 1982), and have
Cytolytic T lymphocytes able to recognize and lyse cells infected with LDV or expressing LDV proteins are activated in the course of an LDV infection (Even et al. 1995). Whether these anti-LDV cytolytic T cells disappear, due to clonal exhaustion, or persist in chronically infected mice is disputed (Even et al. 1995; van den Broek et al. 1997). Stimulation of anti-LDV T helper lymphocytes in the course of infection has been demonstrated by the T-dependence of anti-LDV antibody production (Coutelier et al. 1986) and by the production of antihapten antibodies in mice infected with LDV and challenged with an LDV-hapten complex (van den Broek et al. 1997).
120
Relative cytokine message (%)
120 100 100 80 80 60 60 40 40 20 20 0 IL-6
0 0
6
IL-12 12
18
IL-15 24
48
IL-18 72
Time after infection (hours) Fig. 8-5 Pro-inflammatory cytokine expression after LDV infection. Expression of IL-6, IL-12, IL-15, and IL-18 messenger RNA was monitored by RT-PCR at different times after LDV infection in the spleen of CBA/Ht mice.
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However, these anti-LDV T lymphocyte responses have no effect on the control of virus replication (Onyekaba, Harty, Even, et al. 1989). It has also been reported that LDV infection induces the activation of suppressor T cells able to control the induction of a delayed-type hypersensitivity response to the virus (Inada and Mims 1986a).
G.
between sensitive and resistant quasispecies (Chen et al. 2000; Plagemann 2001b). Neutralization results from the binding of multiple antibody molecules, leading to disruption of the virions (Plagemann et al. 1992). However, prevention of LDV-induced polioencephalomyelitis by antibodies (see below) may rely on other mechanisms, since this prevention is particularly elicited by antibodies of the IgG2a isotype (Markine-Goriaynoff and Coutelier 2002).
Antibody Response and Immune Complexes
LDV is antigenic, and xenoimmune antisera, with neutralizing activity, can be obtained after immunization of rabbits with viral particles (Cafruny and Plagemann 1982b). Anti-LDV antibodies can also be elicited after immunization of mice with inactivated LDV or purified nucleocapsid (Coutelier et al. 1986; Cafruny, Chan, et al. 1986; Harty and Plagemann 1988). In infected mice, anti-LDV antibodies are produced as early as 6–10 days after infection (Porter et al. 1969; Cafruny, Chan, et al. 1986). This antiviral antibody production depends on the presence of functional helper T lymphocytes (Coutelier et al. 1986), but only partially on the secretion of γ-interferon (Cafruny et al. 1999; Markine-Goriaynoff et al. 2000). In contrast to antibodies elicited by immunization with the inactivated virus, which are primarily of the IgG1 isotype, most anti-LDV antibodies secreted in infected mice belong to the IgG2a subclass (Coutelier et al. 1986, 1987). While most of these IgG2 antibodies recognize the major envelope glycoprotein, GP5 (VP3), some of them react with the nucleocapsid, N (VP1), but none of them react with the minor non-glycosylated envelope protein M (VP2) (Coutelier et al. 1986; Cafruny, Chan, et al. 1986). Neutralizing activity for these antiviral antibodies has been demonstrated, but is incomplete (Notkins, Mahar, et al. 1966; Notkins, Mergenhagen, et al. 1966; Rowson et al. 1966; Coutelier and Van Snick 1988). As a result, LDV persists in the circulation of infected mice as infectious immune complexes. These complexes can be further neutralized to some extent by the addition of anti-mouse immunoglobulin antibodies (duBuy and Johnson 1965; Notkins, Mahar, et al. 1966; Notkins, Mergenhagen, et al. 1966; Notkins et al. 1968; Cafruny and Plagemann 1982a; McDonald 1982). Injection of anti-LDV antibodies prevents transplacental transmission of the virus to the fetus (Broen et al. 1992) and polioencephalomyelitis (see below), but not persistence of infection in normal mice. This viral persistence in the presence of antiviral antibodies is due to the existence of LDV quasispecies that escape neutralization (Monteyne and Coutelier 1994; Chen et al. 1997). The mechanisms of viral neutralization have been analyzed with monoclonal anti-LDV antibodies derived from infected or immunized mice (Harty et al. 1987; Harty and Plagemann 1988; Coutelier and Van Snick 1988). The major neutralization epitope recognized by these antibodies is located on a short hydrophilic segment in the center of the ectodomain of GP5 (VP3) (Li et al. 1998; Plagemann 2001b). Neutralization is impaired by glycans that flank this epitope and that differ
H.
Modulation of Immune Responses
A depression of cellular immune responses, as indicated by a longer survival of skin or thyroid allografts and a decreased intensity of the graft-versus-host reaction follows infection with LDV (Howard et al. 1969; Isakov et al. 1981). Contact sensitization to 2,4-dinitro-1-fluorobenzene is inhibited (Michaelides and Simms 1977). The proliferative response of T lymphocytes from LDV-infected mice to concanavalin A or anti-CD3 antibody is decreased (Rowland et al. 1994). This depression of T lymphocyte responses could not be attributed to T cell depletion, nor to nitric oxide production by macrophages (Michaelides and Simms 1977b; Rowland, Butz, et al. 1994). However, some of the effects of LDV on T lymphocyte functions, such as depression of secretion of Th2 cytokines, including interleukins 4, 9, and 13, may be triggered by γ-interferon production (Monteyne et al. 1993; Monteyne, Renauld, et al. 1997; Morimoto et al. 1999, 2003). This bias in T helper differentiation may lead to inhibition of antiparasite and allergic immune responses in mice infected with LDV (Morimoto et al. 1999, 2003). LDV infection suppresses other cellular functions as well. Inhibition of endotoxin-induced inflammation in LDV-infected animals, as measured by the footpad swelling reaction, is mediated by type I interferon (Heremans et al. 1987). The capacity of macrophages from LDV-infected mice to present antigens is decreased (Isakov et al. 1982a, 1982b), although their ability to bind and phagocytose antigens may be enhanced (Michaelides and Simms 1979; Meite et al. 2000). Stimulation of the formation of germinal centers and an increased level of γ-globulin that does not react with viral antigens have been observed in mice infected with LDV (Notkins, Mahar, et al. 1966; Notkins, Mergenhagen, et al. 1966; Coutelier and Van Snick 1985; Rowland, Even, et al. 1994; Anderson, Rowland, et al. 1995). N-glycans expressed on the GP5 (VP3) protein of LDV are responsible for this nonspecific activation of B lymphocytes (Plagemann et al. 2000). The resulting hypergammaglobulinemia is restricted to the IgG2a and IgG2b subclasses (Isakov et al. 1981; Coutelier and Van Snick 1985; Coutelier et al. 1988; Hovinen et al. 1990) and depends on the presence of functional T helper lymphocytes (Coutelier, Coulie, et al. 1990; Li et al. 1990) and on the secretion of interleukin-6, but not of γ-interferon (Markine-Goriaynoff et al. 2000, 2001). This enhanced immunoglobulin response is regulated differentially from the antiviral antibody response and may represent the first
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humoral defense against the virus. This first humoral response consists of secretion of so-called natural Igs that may have a protective effect before specific antiviral antibodies (including IgM) are produced (Ochsenbein et al. 1999). Moreover, LDV infection may modulate antibody responses elicited against nonviral antigens. Most often, this modulation results in a moderate adjuvant effect on these responses, with a shift of their isotypic distribution toward the IgG2a subclass (Notkins, Mergenhagen, et al. 1966; Mergenhagen et al. 1967; Michaelides and Simms 1977a, 1980; Isakov et al. 1982c; Coutelier et al. 1988). However, depending on the mouse strain, on the immunizing antigen, and on the time of infection, LDV may sometimes inhibit concomitant antibody responses (Oldstone et al. 1974; Michaelides and Simms 1980). The repertoire of the antibodies elicited against the immunizing protein may then be changed, resulting in a modification in the proportion of antibodies reacting with either native or cryptic epitopes (Gomez et al. 1997, 2003).
I.
Autoimmunity
LDV infection can affect the outcome of concomitant autoimmune diseases, probably via modulation of the immune responses in the infected host. LDV infection in New Zealand (NZ) mice leads to a decrease in the production of antibodies directed against nuclear antigen and red blood cells. In (NZB x NZW)F1 hybrid mice, which display a high incidence of autoantibodies and a severe immune complex glomerulonephritis with autoimmune hemolytic anemia, LDV infection decreases the amount of antibody synthesized by 4- to 5- fold and significantly reduces the incidence of mortality (Oldstone and Dixon 1972). LDV also suppresses the development of experimental allergic encephalomyelitis induced in SJL/J mice by immunization with spinal cord homogenate (Inada and Mims 1986b). It also prevents the onset of spontaneous diabetes in NOD mice (Takei et al. 1992). The mechanism by which LDV infection suppresses the development of these autoimmune diseases is not known. LDV infection triggers the spontaneous production of widely different autoantibodies, possibly as a result of polyclonal B lymphocyte activation. These autoantibodies react with various tissues (Cafruny and Hovinen 1988a) and with Golgi apparatus antigens (Weiland et al. 1987; Grossmann et al. 1989), but not with mouse immunoglobulins (Coutelier, Van der Logt, et al. 1990). In CBA/Ht, but not in BALB/c mice, LDV infection induces the secretion of autoantibodies that preferentially recognize cryptic autoantigens (Gomez et al. 2000). LDV-elicited production of autoantibodies reacting with intermediate filaments may be partly due to antigenic mimicry, since these autoantibodies cross-react with the viral GP5 (VP3) protein (Grossmann and Weiland 1991). Because of its adjuvant effect, LDV infection enhances the production of anti-erythrocyte autoantibodies in mice immunized with rat red
blood cells (Verdonck et al. 1994). Finally, through activation of the phagocytic activity of macrophages, LDV strongly increases the pathogenicity of unrelated anti-erythrocyte and anti-platelet autoantibodies, leading to severe anemia and thrombocytopenia, respectively (Meite et al. 2000; Musaji et al. 2004).
J.
Polioencephalomyelitis
During studies of the immune response of C58 mice to leukemia cells of the syngeneic Ib line, it was found that polioencephalomyelitis develops after injection of tumor cells either in old C58 mice that become spontaneously immunosuppressed between 6 and 12 months, or in young (3-month-old) experimentally immunosuppressed C58 animals (Murphy et al. 1970, 1983; Duffey et al. 1976; Anderson, Even, et al. 1995). The etiologic agent of this polioencephalomyelitis has been identified as LDV (Martinez et al. 1980; Nawrocki et al. 1980). The disease is characterized by early replication of the virus in cells of the leptomeninges, followed by dissemination into the central nervous system and cytolytic infection of motor neurons of the anterior horn of the spinal cord (Kascsak et al. 1983; Stroop et al. 1985; Brinton, Gavin, and Weibel 1986; Anderson et al. 1995b). Lysis of motor neurons leads to progressive paralysis, respiratory failure, and ultimately the death of the infected animals (Schlenker et al. 2001). LDV-induced polioencephalomyelitis may serve as a model for human amyotrophic lateral sclerosis (Murphy et al. 1983; Sillevis-Smitt and de Jong 1989). Polioencephalomyelitis is not equally induced by all LDV isolates (Murphy et al. 1983). Neuropathogenic and nonneuropathogenic isolates coexist in most LDV pools as quasispecies (Chen et al. 1997; Chen, Li, et al., 1998a; Li et al. 1999; Plagemann et al. 2001) and differ by the number of polylactosaminoglycan chains attached to the ectodomain of their major envelope glycoprotein, GP5 (VP3) (Faaberg, Palmer, et al. 1995; Chen, Li, et al., 1998b; Chen et al. 2000). The LDV that was isolated from C58 tumor-bearing mice replicated efficiently in 13 strains of mice tested but induced paralysis only in two, AKR and C58 (Duffey et al. 1976; Martinez 1979). Preliminary studies suggested that control of susceptibility to LDV-induced paralysis might be multigenic and that a gene(s) of the H-2 complex might be involved (Martinez 1979). However, it now appears that the Fv-1 gene, rather than the H-2 haplotype, controls susceptibility to LDVinduced polioencephalomyelitis (Pease et al. 1982; Murphy et al. 1983; Stroop and Brinton 1983). Mouse strains susceptible to the LDV-induced disease are homozygous for the Fv-1n allele, whereas inheritance of a Fv-1b allele confers resistance (Murphy et al. 1983). Moreover, mice with the Fv-1nr allele, which differs from the Fv-1n allele by a single nucleotide, are also resistant to LDV-induced polioencephalomyelitis (Monteyne et al. 2000).
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The two susceptible mouse strains, C58 and AKR, show a high incidence of spontaneous leukemia, and coinfection with endogenous N-tropic MuLV, which is controlled by the Fv-1 gene, appears to be required for the development of LDVinduced polioencephalomyelitis (Pease and Murphy 1980; Pease et al. 1982). In situ hybridization analyses suggested that expression of this MuLV in motor neurons is required for their infection by LDV, suggesting that there is a correlation between Fv-1 alleles, MuLV expression, and development of polioencephalomyelitis after LDV infection (Contag and Plagemann 1988, 1989; Anderson et al. 1995a). However, the precise mechanism by which these parameters differentially affect susceptibility to LDV-induced polioencephalomyelitis is not currently known. Finally, a state of immunosuppression is necessary for the induction of paralysis by LDV in C58 and AKR mice. Also, sensitization with spinal cord tissue may enhance the disease (Stroop and Brinton 1985). Both CD8+ and CD4+ T lymphocytes are required to prevent the development of the polioencephalomyelitis (Bentley and Morris 1982; Bentley et al. 1983; Monteyne, Meite, et al. 1997). Treatment of immunosuppressed mice with IFN-γ protected them against the paralytic disease (Cafruny et al. 1997). Finally, although suppression of the antibody response is not the only prerequisite for the disease (Cafruny, Stranke, et al. 1986), passive transfer of anti-LDV antibodies prevents or delays the progression of this polioencephalomyelitis (Murphy et al. 1983; Harty, Chan, Contag, et al. 1987; Harty and Plagemann 1990). This protective effect is maximal for antibodies of the IgG2a isotype (MarkineGoriaynoff and Coutelier 2002) and correlates with the high sensitivity of neuropathogenic LDV quasispecies to neutralization by antiviral antibodies (Chen et al. 1999).
K.
2 months. Since the development of this type of tumor is not affected by immunosuppression (Michelich et al. 1977), it seems unlikely that such a delay in FB tumorigenesis is mediated by the effect that LDV infection has on the cellular immune response. The effect of LDV on FB tumorigenesis may well be mediated through LDV-induced alterations in the monocyte population or in the functions of macrophages (Mahy et al. 1965, 1967; Riley 1974). These cells seem to be intricately involved in the multistage process of FB tumorigenesis (Brand et al. 1975). Finally, LDV-infected mice are protected against the growth of syngeneic Sac tumor cells through the secretion of antibodies reacting with tumor cell surface antigens (Weiland et al. 1990).
VII.
EPIZOOTIOLOGY
A.
Host Specificity
LDV is unusual in its extreme host specificity. To date, LDV is known to infect only mice. However, it has been hypothesized that LDV may have infected wild boars that ate infected mice and that this led to the evolution of PRRSV (Plagemann 2003). Viruses with biological properties identical to those of LDV have been isolated from small groups of wild mice in Australia (Pope 1961), Germany (Georgii and Kirschenhofer 1965), the United States (Pope and Rowe 1964; Li et al. 2000), and England (Rowson 1963; Field and Adams 1968). However, nothing is known about the incidence of LDV infection in wild mouse populations. In the laboratory, the virus replicates rapidly in all strains of mice so far tested, producing a persistent infection.
LDV and Tumors B.
LDV has been found in association with more than 50 different transplanted murine tumors (Riley 1968). It is widely accepted that this association is due to chance contamination of a tumor suspension by the blood of an LDV-infected host and that this then leads to the infection of each sequential tumor host with LDV. When mice receive an injection of tumor cells or tumor virus during the first week after LDV infection, an enhancement of the growth and oncogenicity of some tumors is observed. This phenomenon appears to be related to the transient depression in cellular immunity that occurs during the first few days after LDV infection (Howard et al. 1969; Michaelides and Schlesinger 1974). In contrast, it has been reported that LDV infection delays foreign body (FB) tumorigenesis (Brinton-Darnell and Brand 1977). Mice that received subcutaneous implants of unplasticized vinyl chloride-vinyl acetate copolymer films 2 weeks after LDV infection developed FB tumors at the same 100% incidence as uninfected mice, but at a rate that was slower by
Transmission
Although LDV replicates rapidly once it enters its host, it is not readily transferred from one mouse to another by natural means. No insect vector for this virus has been identified. Mice infected with LDV have been found to excrete the virus in their feces, urine, milk, and probably saliva (Plagemann et al. 1963; Notkins 1965a; Crispens 1964a, 1964b). However, the oral, ocular, and vaginal mucosal barriers to LDV transmission are relatively effective (Cafruny and Hovinen 1988b), but these barriers are more effective against free viruses than against LDV-infected macrophages (Cafruny and Bradley 1996). In the laboratory, LDV is rarely transferred between mice, even when they are housed in the same cage. However, it has been reported that, when fighting males were housed in the same cage, virus was apparently transferred via saliva to an open wound (Notkins 1963). In addition, when normal males were housed with infected males whose incisors had been removed, the virus was again transmitted at a high rate, suggesting that
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the ingestion of blood and/or tissue of infected animals can lead to infection. Infected females infrequently transmit the virus to their young. Passage of the virus from the mother to the fetus is maximal between 24 and 72 hours after infection of the mother, and between 12.5 and 15 days of gestation (Haven et al. 1996). The major mode of transmission of LDV among laboratory mice has most likely been via experimental procedures. Since the virus remains at high titers in the blood throughout the lifetime of an infected mouse, the transfer of serum or tissue from one mouse to another can result in the transfer of virus. The use of the same needle for the injection of several mice can also inadvertently spread an infection.
VIII. A.
DIAGNOSIS LDH Assay
LDH plasma levels increase in all mice infected with LDV. Maximum levels are observed 4 days after infection, but elevated levels persist in chronically infected mice (Fig. 8-4). The most common method for detecting LDV infection is to measure this plasma LDH increase. Small samples of plasma can be obtained from the retro-orbital sinus of mice with heparinized capillary tubes. If plasma samples have a reddish color after centrifugation, they should be discarded, since high levels of cytoplasmic LDH from lysed red blood cells give false positive results. LDH activity is assayed in a coupled reaction with nicotinamide-adenine dinucleotide (NAD). The activity of LDH can be assessed by measuring either the increase or the decrease in NADH with time: LDH ⎯⎯⎯ ⎯⎯ → pyruvate + NADH Lactate + NAD + ← ⎯
To measure the disappearance of NADH, the reaction mixture containing 2.6 ml 0.5 M phosphate buffer, pH 7.4, 0.2 ml plasma, and 0.1 ml NADH (2.5 mg/ml) is mixed thoroughly in a cuvette, and then 0.1 ml sodium pyruvate (2.5 mg/ml) is added. The decrease in optical density is measured during a 1- to 2-min period at 340 nm. One conventional unit of LDH activity produces a decrease in optical density of 0.001/min/ml plasma. One conventional unit/ml is equal to 0.5 IU. Normal plasma levels of LDH for mice are 400–800 conventional units/ml, whereas levels in LDV-infected mice can range from 1,800–16,000 units/ml. Information on other methods used to measure LDH activity can be found in the review by Rowson and Mahy (1975). If a sample of mouse plasma is found to contain an elevated level of LDH by the above assay, proof that this increase in LDH was caused by LDV infection can be obtained by intraperitoneal injection of serial dilutions of this plasma into normal mice. Two or more mice are used per dilution, and
a sample of their plasma is then obtained on the fourth day after injection and is in turn assayed for LDH activity. A titer of about 105 ID50/ml is normal for plasma obtained from a mouse chronically infected with LDV.
B.
Detection of LDV RNA, Virions, or Antigens
Contamination by LDV can be detected by amplification of LDV RNA by reverse transcription/polymerase chain reaction (RT/PCR). By using specific primers, for instance corresponding to the LDV ORF 7, it is possible to detect contamination of mice, of cell lines, or of other biological material, including parasites, without false positive results from closely related arteriviruses such as PRRSV or equine arteritis virus (van der Logt et al. 1994). With careful selection of the primers, RT-PCR amplification can be used as a means of detecting LDV quasispecies (Chen and Plagemann 1997; Goto et al. 1998). On the other hand, degenerate primers located in conserved replicase regions can be used to amplify LDV sequences as well as those from other arteriviruses in samples from other host species (Chen and Plagemann 1995). Several other assays have been developed to detect the presence of either LDV RNA, proteins, or virions in various materials. Viral RNA can be detected on tissue sections by in situ hybridization (Brinton, Gavin, and Weibel 1986). Immunofluorescent studies with anti-LDV antibodies and electron microscopy can be used to detect the presence of LDV antigens and virions in tissue sections (Brinton, Gavin, and Weibel 1986; Stroop et al. 1985). LDV grown in primary mouse peritoneal macrophage cultures in the presence of [5-3H]uridine is detectable by autoradiography in sucrose density gradient fractions (Brinton-Darnell and Plagemann 1975; Tong et al. 1977). After in vivo virus amplification followed by purification of virus from plasma by centrifugation on sucrose gradients, the major LDV structural proteins can be detected by silver nitrate staining after electrophoresis on SDS-polyacrylamide gels (Coutelier et al. 1986; Heremans et al. 1987). Finally, LDV antigens can be quantitated by radioimmunoassay (M. A. Brinton and T. G. Tachovsky, unpublished observations) or by a particle-counting immunoassay based on the agglutination of latex beads coated with anti-LDV monoclonal antibodies (Markine-Goriaynoff et al. 2002).
C.
Detection of Anti-LDV Antibody
Measurement of anti-LDV antibody by an enzyme-linked immunosorbent assay (ELISA) has become the method of choice for detecting mice chronically infected with LDV. Using plates coated with purified LDV virions, specific antiviral antibodies can be detected in the serum of infected mice by 1 or 3 weeks post infection (Cafruny and Plagemann 1982a; Coutelier et al. 1986, 1987; Cafruny, Chan, et al. 1986). However, the B lymphocyte
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polyclonal activation triggered by an LDV infection leads to the production of antibodies that are not specific for the virus but that bind to ELISA plates not coated with viral antigens (Cafruny, Heruth, et al. 1986; Hu et al. 1992). Specific anti-LDV antibodies can also be assessed by indirect fluorescent antibody staining of infected cells or tissues or by a virus neutralization assay (Cafruny, Chan, et al. 1986), but neither of these methods is used routinely for the detection of infected mice.
IX.
CONTROL AND PREVENTION
Because LDV is not very contagious, can escape antiviral immune responses, and infects only mice, it is unlikely that advanced methods of control and prevention of LDV infection, including a vaccine, will be developed. In the case of contamination in animal facilities, LDV-infected mice can be easily detected and euthanized. If a tumor cell line is injected into a mouse with a chronic LDV infection during serial transplantation, LDV will then be transferred with the tumor tissue at each subsequent passage (Riley 1968). However, since LDV does not replicate in tumor cells in culture, nor in laboratory animal species other than the mouse, it can be eliminated either by passage of the tumor cells in another rodent species or by growing the tumor cells in tissue culture (Plagemann and Swim 1963, 1966b). The tumor cells must be maintained in culture for several passages in order to eliminate any macrophages or macrophage precursor cells that may be present. Thereafter, the tumor cells can again be maintained in mice. It would be wise to check the LDH levels of recipient mice before injection of tumor cells. Plasma LDH levels should also be assayed 4 days after injection of the cured tumor cells to check that all LDV virions have indeed been eliminated. Likewise, pools of other viruses prepared in mice can be freed of LDV by passage in cultures of continuous cell lines derived from other animal species.
X.
CONCLUSION
Although much has been learned about the biology of LDV since its discovery in 1960 by Riley et al. many questions about this interesting virus remain unanswered. The inability of LDV to infect in transformed cell lines and to cause a detectable cytopathic effect in cell cultures in which it does replicate, such as mouse peritoneal exudate cells, represents technical obstacles to the further analysis of this virus. Identification of the cell receptor utilized by LDV would constitute a major advance, since this might lead to the creation of a cell line permissive to LDV infection. Whereas LDV is a natural and sometimes pathogenic infectious agent of the mouse, it does not currently represent a major threat to animal facilities. The virus is not very contagious and
there are a number of easy methods available to detect infected mice. However, additional viruses able to produce chronic infections that are accompanied by no obvious clinical symptoms in most of their hosts may well exist in nature. There is no reason to suppose that such viruses would induce an increase in the host’s serum enzyme levels, the indicator that led to the fortuitous discovery of LDV. The detection of these viruses might well prove difficult. However, such viruses may indeed be responsible for certain effects, that either are currently attributed to known infectious agents or remain unexplained. Further research on LDV infections might thus provide valuable information in fields such as complex infectious diseases, autoimmunity, immunopathology, and oncology. Moreover, further study of the interaction between relatively “silent” viruses such as LDV and their host species should lead to new insights into the mechanisms by which viruses can establish and maintain relatively harmless, persistent infections in their hosts. ACKNOWLEDGMENTS The authors would like to thank Lydia Woltz for word processing, William Davis and Gertrud Radu for assistance with graphics, and Gertrud Radu for proofreading the manuscript.
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233 Pope, J. H., and Rowe, W. P. (1964). Identification of WM1 as LDH virus, and its recovery from wild mice in Maryland. Proc. Soc. Exp. Biol. Med. 116, 1015–1019. Porter, D. D., and Porter, H. G. (1971). Deposition of immune complexes in the kidneys of mice infected with lactic dehydrogenase virus. J. Immunol. 106, 1264–1266. Porter, D. D., Porter, H. G., and Deerhake, B. B. (1969). Immunofluorescence assay for antigen and antibody in lactic dehydrogenase virus infection of mice. J. Immunol. 102, 431–436. Proffitt, M. R., and Congdon, C. C. (1970). The effect of a large dose of LDH virus on mouse lymphatic tissue. Fed. Proc., Fed. Am. Soc. Exp. Biol. 29, 559. Riley, V. (1968). Lactate dehydrogenase in the normal and malignant state in mice and the influence of a benign enzyme-elevating virus. Methods Cancer Res. 4, 493–618. — — —. (1974). Persistence and other characteristics of the lactate dehydrogenase elevating virus (LDH-virus). Prog. Med. Virol. 18, 198–213. — — —. (1962). Role of viruses in glycolysis of tumors and hosts. Fed. Proc., Fed. Am. Soc. Exp. Biol. 21, 21–87. — — —. (1961). Virus-tumor synergism. Science 134, 666–668. Riley, V., Lilly, F., Huerto, E., and Bardell, D. (1960). Transmissible agent associated with 26 types of experimental mouse neoplasms. Science 132, 545–547. Riley, V., Loveless, J. D., Fitzmaurice, M. A., and Siler, W. M. (1965). Mechanism of lactate dehydrogenase (LDH) elevation in virus-infected hosts. Life Sci. 4, 487–507. Riley, V., and Wroblewski, F. (1960). Serial lactic dehydrogenase activity in plasma of mice with growing or regressing tumors. Science 132, 151–152. Ritzi, D. M., Holth, M., Smith, M. S., et al. (1982). Replication of lactate dehydrogenase-elevating virus in macrophages. 1. Evidence for cytocidal replication. J. Gen. Virol. 59, 245–262. Rowland, R. R. R., Butz, E. A., and Plagemann, P. G. W. (1994). Nitric oxide production by splenic macrophages is not responsible for T cell suppression during acute infection with lactate dehydrogenase-elevating virus. J. Immunol. 152, 5785–5795. Rowland, R. R. R., Even, C., Anderson, G. W., Chen, Z., Hu, B., and Plagemann, P. G. W. (1994). Neonatal infection of mice with lactate dehydrogenase-elevating virus results in suppression of humoral antiviral immune response but does not alter the course of viraemia or the polyclonal activation of B cells and immune complex formation. J. Gen. Virol. 75, 1071–1081. Rowson, K. E. K. (1963). Riley virus in wild mice, effect of drugs on replication of Riley viruses. Br. Emp. Cancer Campaign Annu. Rep. 41, 222–223. Rowson, K. E. K., Adams, D. H., and Salaman, M. H. (1963). Riley’s enzymes elevating virus: a study of the infection in mice and its relation to virusinduced leukaemia. Acta Unio Int. Contra Cancrum 19, 404–406. Rowson, K. E. K., and Mahy, B. W. J. (1975). Lactic dehydrogenase virus. Virol. Monogr. 13, 1–121. Rowson, K. E. K., Mahy, B. W. J., and Bendinelli, M. (1966). Riley virus neutralizing activity in the plasma of infected mice with persistent viraemia. Virology 28, 775–778. Rowson, K. E. K., and Michaels, L. (1973). Lactic dehydrogenase (LDH) virus and its localization by immunofluorescence. J. Med. Microbiol. 6, xi. Rowson, K. E. K., Parr, I. B., and Alper, T. (1968). Radiation target size of Riley virus. Virology 36, 157–159. Santisteran, G. A., Riley, V., and Fitzmaurice, M. A. (1972). Thymolytic and adrenal cortical responses to the LDH-elevating virus. Proc. Soc. Exp. Biol. Med. 139, 202–206. Schlenker, E. H., Jones, Q. A., Rowland, R. R. R., Steffen-Bien, M., and Cafruny, W. A. (2001). Age-dependent poliomyelitis in mice is associated with respiratory failure and viral replication in the central nervous system and lung. J. Neurovirol. 7, 265–271. Sillevis Smitt, P. A. E., and de Jong, J. M. B. M. (1989). Animal models of amyotrophic lateral sclerosis and the spinal muscular atrophies. J. Neurol. Sci. 91, 231–258.
234 Smit, M. J., Duursma, A. M., Koudstaal, J., Hardonk, M. J., and Bouma, J. M. (1990). Infection of mice with lactate dehydrogenase-elevating virus destroys the subpopulation of Kupffer cells involved in receptor-mediated endocytosis of lactate dehydrogenase and other enzymes. Hepatology 12, 1192–1199. Snijder, E. J., Brinton, M. A., Faaberg, K. S., et al. (2003). Arteriviridae. In Virus taxonomy, eighth report of the international committee on taxonomy of viruses, M. H. V. Regenmortal, C. M. Fauquet, and D. H. L. Bishop, eds., Springer-Verlag, Wien, New York. Snodgrass, M. J., Lowrey, D. S., and Hanna, M. G., Jr. (1972). Changes induced by lactic dehydrogenase virus in thymus and thymus-dependent areas of lymphatic tissue. J. Immunol. 108, 877–892. Stevenson, M. M., Rees, J. C., and Meltzer, M. S. (1980). Macrophage function in tumor-bearing mice: evidence for lactic dehydrogenase-elevating virusassociated changes. J. Immunol. 124, 2892–2899. Stewart, C. C., Lin, H. S., and Adles, C. (1975). Proliferation and colonyforming ability of peritoneal exudate cells in liquid culture. J. Exp. Med. 141, 1114–1132. Stroop, W. G., and Brinton, M. A. (1985). Enhancement of encephalomyeloradiculitis in mice sensitized with spinal cord tissue and infected with lactate dehydrogenase-elevating virus. J. Neuroimmunol. 8, 79–92. — — —. (1983). Mouse strain-specific central nervous system lesions associated with lactate dehydrogenase-elevating virus infection. Lab. Invest. 49, 334–345. Stroop, W. G., Weibel, J., Schaefer, D., and Brinton, M. A. (1985). Ultrastructural and immunofluorescent studies of acute and chronic lactate dehydrogenase elevating virus-induced nonparalytic poliomyelitis in mice. Proc. Soc. Exp. Biol. Med. 178, 261–274. Stueckemann, J. A., Holth, M., Swart, W. J., et al. (1982). Replication of lactate dehydrogenase-elevating virus in macrophages. 2. Mechanism of persistent infection in mice and cell culture. J. Gen. Virol. 59, 263–272. Stueckemann, J., and Plagemann, P. G. W. (1978). Persistent infection of mouse peritoneal exudate cells by lactate dehydrogenase-elevating virus (LDV) in vitro. In ICN-UCLA symposia on molecular and cellular biology, p. 247, Academic Press, New York.
JEAN-PAUL COUTELIER AND MARGO A. BRINTON
Takei, I., Asaba, Y., Kasatani, T., et al. (1992). Suppression of development of diabetes in NOD mice by lactate dehydrogenase virus infection. J. Autoimmun. 5, 665–673. Tennant, R. W., and Ward, T. G. (1962). Pneumonia virus of mice (PVM) in cell culture. Proc. Soc. Exp. Biol. Med. 111, 395–398. Tong, S. L., Stueckemann, J., and Plagemann, P. G. W. (1977). Autoradiographic method for detection of lactate dehydrogenase-elevating virus-infected cells in primary mouse macrophage cultures. J. Virol. 22, 219–227. van den Broek, M. F., Sporri, R., Even, C., et al. (1997). Lactate dehydrogenaseelevating virus (LDV): lifelong coexistence of virus and LDV-specific immunity. J. Immunol. 159, 1585–1588. van der Logt, J. T. M., Kissing, J., and Melchers, W. J. (1994). Enzymatic amplification of lactate dehydrogenase-elevating virus. J. Clin. Microbiol. 32, 2003–2006. Verdonck, E., Pfau, C. J., Gonzalez, M.-D., Masson, P. L., and Coutelier, J.-P. (1994). Influence of viral infection on anti-erythrocyte autoantibody response after immunization of mice with rat red blood cells. Autoimmunity 17, 73–81. Virolainen, M., and Defendi, V. (1967). Dependence of macrophage growth in vitro upon interaction with other cells. In Growth regulating substances for animal cells in culture, V. Defendi and M. Stoker, eds., pp. 67–83, Wistar Inst. Press, Philadelphia. Warnock, M. L. (1964). Isozymic patterns in organs of mice infected with LDH agent. Proc. Soc. Exp. Biol. Med. 115, 448–452. Weiland, E., Grossmann, A., Thiel, H. J., and Weiland, F. (1990). Lactate dehydrogenase-elevating virus induces antibodies reactive with a surface antigen of aetiologically unrelated murine cell transformants. J. Gen. Virol. 71, 1233–1236. Weiland, E., Weiland, F., and Grossmann, A. (1987). Lactate dehydrogenaseelevating virus induces anti-Golgi apparatus antibodies. J. Gen. Virol. 68, 1983–1991. Yaffe, D. (1962). The distribution and in vitro propagation of an agent causing high plasma lactic dehydrogenase activity. Cancer Res. 22, 573–580. Yamazaki, S., and Notkins, A. L. (1973). Inhibition of replication of lactic dehydrogenase virus by actinomycin. J. Virol. 11, 473–478.
Chapter 9 Reoviridae Richard L. Ward, Monica M. McNeal, Mary B. Farone, and Anthony L. Farone
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Rotavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties of Rotavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Structure of the Rotavirus Particle . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Stability of the Virus Particle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Virus Strains and Antigenic Relationships . . . . . . . . . . . . . . . . . . . . . . 1. Rotavirus Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Electropherotypes of Rotavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Serotypes of Rotavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Strains of Murine Rotavirus (EDIM) . . . . . . . . . . . . . . . . . . . . . . . . D. Growth of Rotavirus In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Rotavirus Replication In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Pathogenesis of Rotavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mechanisms of Diarrhea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Age Restrictions for Rotavirus Disease . . . . . . . . . . . . . . . . . . . . . . F. Epizootiology—Host Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Reoviridae (Orthoreovirus; Mammalian Reovirus) . . . . . . . . . . . . . . . . . . . A. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties of Reovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Structure of Reovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Stability of Reovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Reovirus Strains and Antigenic Relationships . . . . . . . . . . . . . . . . . . . . 1. Reovirus Serotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Reovirus Reassortants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Reovirus Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Growth of Reoviruses In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Growth of Reoviruses In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Entry into Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cardiorespiratory System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4. Hepatobiliary System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Endocrine System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
Prior to the development of pathogen-free mouse colonies and the use of laminar-flow hoods with sterile, filtered air in which to handle mice and barrier facilities in which to house them, most if not all mouse colonies were contaminated with a variety of pathogens including reoviruses and rotaviruses. Today, the potential disruptions and anomalies in experimental outcomes due to unwanted microbial infections in mouse colonies can be avoided but at a much increased expense compared to that required for conventional housing. Even today, mouse colonies become contaminated with these viruses (McNeal et al. 2004) and, once this occurs, it is extremely difficult to eliminate them due to the large numbers shed in feces, their stability in the environment, and the ease with which they establish a new infection after consumption by naive animals. Once it became possible to routinely retain mice free of either reovirus or rotavirus contamination, they could be used for studies in which these viruses were purposely administered under controlled environments. These studies have yielded numerous seminal findings regarding the pathogenesis and immunology of these viruses. The studies with reovirus have been conducted almost solely with human strains that readily infect mice and other animal species. In contrast, studies with rotavirus in mice have been performed primarily with homologous EDIM strains (a generic designation for murine rotaviruses that will be fully defined later in this chapter) and with nonhuman heterologous strains, particularly rhesus rotavirus (RRV). In both cases, it is assumed, and has so far been found to be generally true, that the results found with these strains of rotavirus in mouse models are similar to those found with homologous rotaviruses in larger mammals and humans. Thus, rotavirus studies in mouse models have been invaluable in understanding the biology of these important viruses.
II.
ROTAVIRUS A.
History
Rotaviruses are the primary cause of severe gastroenteritis in young children. They are also major etiologic agents of severe
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diarrhea in young animals of many species and cause large economic losses within the livestock industry due to their associated morbidity and mortality. The first rotavirus to be described, based on pathology and epidemiology, was a murine rotavirus that had been classified under the general description as the agent responsible for “epizootic diarrhea of infant mice,” hence the name EDIM (Cheever and Mueller 1947; Pappenheimer and Enders 1947). This name is a term used for all murine rotaviruses, and today 8 different EDIM strains have been isolated (McNeal et al. 2004). A detailed description of murine rotaviruses is provided in Section II, C, 4. Murine rotaviruses were the first to be visualized by electron microscopy (Adams and Kraft 1963, 1967). Virus particles with similar morphologies were subsequently observed in diarrheal feces of many species, primarily in the young. Ten years after murine rotavirus was first visualized in 1963, Bishop et al. (1973) observed the presence of a virus with this morphology in duodenal mucosal biopsies of children with acute nonbacterial gastroenteritis. These viruses were subsequently assigned to the genus Rotavirus because of their wheel-like appearance (Latin rota, wheel) and placed in the family Reoviridae, which contains double-stranded RNA viruses with segmented genomes. Since that time, a great amount of information has been obtained on the structure of rotaviruses, their replication strategies, their ability to induce disease, and the host responses that resolve their infections and prevent subsequent illnesses. The use of murine rotaviruses has been crucial in a number of these types of studies, particularly those involving the use of mice as models for human rotavirus disease and its prevention. Although this chapter primarily concerns murine rotaviruses, much of what is known about these strains has been learned from studies with other rotaviruses. This information will be presented as appropriate within the different sections of this chapter. The history of murine rotaviruses can be generally divided into two phases, neither of which is mutually exclusive. During the early discovery phase, which dates back to the late 1940s, many of the reports on this group of viruses concerned the fact that they were common contaminants of conventional mouse colonies and routinely caused extensive morbidity in these colonies, which had serious economic impact. Today, when colonies containing mice that are immunosuppressed by genetic alteration become contaminated, the outcome can be extremely costly, both regarding the loss of mice and the efforts required to eliminate
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the problem through decontamination of the facility. Rotaviruses are spread between animals or humans by the fecal-oral route, which is highly efficient since these viruses survive well in the environment and are shed in quantities exceeding 1011 particles per gram of fecal matter. Since rotaviruses are typically species-specific, heterologous strains have not been found as contaminants in mouse colonies. However, once murine rotaviruses become endemic within a mouse colony, the only reliable method to eliminate them, if it is necessary to retain the animals, is a long period of burnout followed by complete disinfection of the facility, cages, food, water, and bedding. The burnout period must be at least several weeks because even in immunologically normal mice the virus can be shed for up to 2 weeks in neonates (McNeal et al. 2004) and mouse-to-mouse spread can continue until all mice in the room have been infected. Burnout is not possible with many of the immunologically compromised animal strains because, once infected, they can shed the virus indefinitely. The only reliable method to prevent contamination of mouse colonies is to work with rotavirus-free mice under BL2 conditions. The second phase in the history of murine rotaviruses concerns their use in experimentation, particularly for studies
on pathogenesis and immunity. These studies began in earnest in the early 1980s and culminated with the development of the adult mouse model for studies on active immunity in 1990 (Ward et al. 1990). Today, this is the primary model used to define mechanisms of protection after rotavirus vaccination and to evaluate the potential of new rotavirus vaccine candidates. B. 1.
Properties of Rotavirus
Structure of the Rotavirus Particle
A computer-generated image of the rotavirus particle obtained by cryo-electron microscopy (Fig. 9-1) showed that it is ∼100 nm in diameter and has a capsid composed of three concentric protein layers (Shaw et al. 1993; Prasad and Chiu 1994). The outer layer contains the VP7 glycoprotein (780 molecules/virion) and 60 dimers of the VP4 protein, the latter of which forms spike-like projections that extend through and 11–12 nm beyond the VP7 layer (Prasad et al. 1990; Shaw et al. 1993; Prasad and Chiu 1994; Yeager et al. 1994). The VP4 protein is anchored to the intermediate layer of the particle, composed of 780 molecules of the VP6 protein. The innermost layer contains 120 molecules
Fig. 9-1 Computer-generated image of the triple-layered rotavirus particle obtained by cryoelectron microscopy. The cutaway diagram shows the outer capsid composed of VP4 spikes and a VP7 shell, an intermediate VP6 layer, and an inner VP2 layer surrounding the core containing the 11 double-stranded RNA segments and VP1 and VP3 proteins. (Photograph courtesy of B. V. V. Prasad, Baylor College of Medicine, Houston, Texas.)
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of the VP2 protein that interact with 12 molecules each of the viral transcriptase (VP1) and guanylyltransferase (VP3), along with the 11 segments of double-stranded RNA genome. These segments encode the 6 structural proteins of the virus as well as 6 nonstructural proteins designated NSP1-NSP6 (Table 9-1). The smallest segment encodes both NSP5 and NSP6. The functions of all proteins have been examined but in some cases remain poorly understood. 2.
Stability of the Virus Particle
As already noted, the infectivity of rotavirus is retained, sometimes under considerable stress, outside of the body, a property typical of enteric viruses, whose transmission is by the fecal-oral route. This fact, coupled with the findings that rotaviruses are shed in quantities of >1011 particles per gram of fecal matter, makes these viruses highly transmissible within members of a species, including humans. Almost every child has at least one rotavirus infection by the age of 3 years. This occurs independently of regional and national cultural practices and public health standards. Initial studies on the heat stability of rotavirus performed with the murine EDIM strain found that about 50% of its infectivity, as an intestinal filtrate, was lost after 24 hrs at 4°C or 1 hr at 37°C (Cheever and Mueller 1947; Kraft 1957, 1962). Heat treatment at 60°C for 30 min destroyed most but not all infectious virus, while retention at 70°C for 30 min abolished all infectivity. Later it was shown that the titer of the simian strain SA11 was reduced 50% after 15 min at 50°C and by >4 orders of magnitude during this time at 55°C (Ward and Ashley 1980). A recent study indicated that after only 10 min
at 60°C, the titer of rotavirus was reduced by at least 7 orders of magnitude (O’Mahony et al. 2000). When animal or human rotaviruses in clarified viral lysates are stored at −70°C, no loss of infectivity has been observed over periods of >10 years (R. Ward, unpublished observations). Because rotaviruses must survive the highly acidic environment of the stomach during their transport to the intestine, they are expected to be relatively stable at low pH. This expectation was supported by data showing that the infectivity of SA11 was reduced <20% during 60 min at pH 4 when held at 21°C and was unaffected during this period at 4°C (Ward and Ashley 1980). At pH 2, however, the titers of this virus decreased >99.99% and 99.8% when held for 60 min at 21°C and 4°C, respectively. Others have reported that rotaviruses retain infectivity in acidic environments as low as pH 3 (Estes 2001). Therefore, rotavirus is acid resistant, but the low pH of the stomach would still be expected to destroy most of the virus if held for an extended time without buffer or other protection. It has also been reported that ionic detergents destabilize rotavirus and that the destabilizing effects of these detergents are augmented by low pH and increased temperature (Ward and Ashley 1980). Interestingly, non-ionic detergents enhance the infectivity of rotavirus, presumably by disrupting viral aggregates or dissociating virus from particulates that may block its infectivity (Ward and Ashley 1980). As is typical of non-enveloped viruses, rotaviruses are stable in the presence of highly hydrophobic organic liquids. Much and Zajac (1972) reported that purified EDIM is not inactivated during 60 min at 4°C in either 20% ether, 5% chloroform, or 0.1% sodium deoxycholate. In another study with EDIM, it was
TABLE 9-1
SIZES OF ROTAVIRUS GENE SEGMENTS AND PROPERTIES OF ENCODED PROTEINS Number of base pairs
Encoded protein
1 2 3 4
3300 2700 2600 2360
VP1 VP2 VP3 VP4
12.5 10.2 9.8 8.7
5
1600
NSP1
5.9
6
1360
VP6
4.5
7
1100
NSP3
3.5
8 9 10
1060 1060 750
NSP2 VP7 NSP4
3.7 3.7 2.0
11
660
NSP5
2.2
NSP6
1.2
RNA segment
Molecular weight of protein (×10–4)
Properties of protein Inner core protein, RNA binding, RNA transcriptase Inner capsid protein, RNA binding Inner core protein, guanylyl transferase Outer capsid protein, HA, NP, sialic acid binding, fusogenic protein Nonstructural protein, RNA binding, contains zinc fingers, host range determinant (?) Intermediate capsid protein, group and subgroup antigen Nonstructural protein, RNA binding, translational control Nonstructural protein, RNA binding, NTPase Outer capsid glycoprotein, NP Nonstructural glycoprotein, transmembrane protein, enterotoxin Nonstructural protein, phosphorylated, O-glycosylated Nonstructural protein, interacts with NSP5
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9. REOVIRIDAE
noted that the virus titer was not significantly reduced when held in ether or 0.1% sodium deoxycholate for 24 hr at 4°C (Kraft 1962). Other rotaviruses have also been found to be resistant to these organic compounds (Estes 2001). Finally, it should be noted that rotaviruses are resistant to the high concentrations of proteolytic enzymes released into the intestinal tract (Estes 2001). In fact, the infectivity of the virus is greatly enhanced by proteolytic cleavage of the VP4 protein into its VP5 and VP8 subunits. In spite of their resistance to inactivation, rotaviruses are readily destroyed by disinfectants such as phenols, chlorine, and ethanol (Estes 2001). Furthermore, the infectivity of these viruses is lost after treatment with calcium-chelating agents such as EDTA and EGTA. Infectivity of rotavirus is dependent on the presence of the outer capsid proteins, VP4 and VP7, and calcium is needed for the stabilization and retention of these proteins on the particle surface (Estes 2001). The doublelayered particles produced by treatment with chelating agents can be further reduced to single-layered particles by treatment with chaotropic agents such as sodium thiocyanate or high concentrations of calcium.
C.
Virus Strains and Antigenic Relationships
1. Rotavirus Groups
In addition to their distinctive morphologies, rotaviruses were found to share a group antigen (Kapikian et al. 1976; Woode et al. 1976) that was later determined to be the highly conserved VP6 protein that comprises the intermediate capsid layer. In 1980, particles that were indistinguishable morphologically from established rotavirus strains but lacked the common group antigen were discovered in pigs (Bridger 1980; Saif et al. 1980). This subsequently led to the identification of rotaviruses belonging to 6 additional groups (B to G) based on a common group antigen, with the original rotavirus strains classified as group A (Saif and Jiang 1994). Because VP6 is so highly conserved and is also the most immunogenic protein of the virus, immunological reagents developed to detect rotaviruses by binding to antibodies made in infected or hyperimmunized animals (e.g., EIA assays) will detect all strains of group A rotavirus, including those found to contaminate mouse colonies. Although rotaviruses belonging to groups A to C have been associated with human diseases, the vast majority of illness appears to be due to group A strains. All murine rotaviruses that have been isolated and characterized belong to group A. Therefore, non–group A rotaviruses will not be discussed further in this chapter.
2.
Electropherotypes of Rotavirus
In order to understand the niche filled by murine strains within the population of group A rotaviruses, it is important to describe the methods used for rotavirus classification. A variety
of classification schemes have been used to characterize rotaviruses for epidemiologic purposes. Each scheme, however, is intertwined with a unique property of viruses with segmented genomes, that is, the ability to form reassortants. During the rotavirus replication cycle, newly formed plus-sense viral mRNAs are free within the viroplasm prior to incorporation into replication intermediates in the first stages of virion assembly (Ramig and Ward 1991). Co-infection of cells with more than one virus permits reassortment of the mRNAs from both parents. If co-infection is between different strains of virus, reassortment of mRNAs results in progeny that are genetic mosaics of the coinfecting strains. These new strains, or reassortants, are identified by their specific array of genome segments, usually through their electrophoretic mobilities during polyacrylamide gel electrophoresis (i.e., electropherotypes). The properties of the new virus strains depend on which segments are inherited from which parent and the functional behavior of each particular combination of segments and their protein products. Rotavirus reassortants form readily in cell culture and in co-infected experimental animals, which at least partially is responsible for the variety of rotavirus strains found in nature. There are, however, severe limitations within strain combinations that are capable of forming stable reassortants, limitations that appear to be associated with the degree of genetic variation between strains. 3.
Serotypes of Rotavirus
Both outer capsid proteins of rotavirus, VP4 and VP7, contain neutralization epitopes, and, thereby, both are involved in serotype determination. Originally, serotyping was based solely on differences in the VP7 protein because animals hyperimmunized with rotaviruses develop most neutralizing antibody to this protein. Cross-neutralization studies conducted with these hyperimmune sera readily separated the strains into VP7 serotypes (Wyeth et al. 1982; Hoshino et al. 1984). When it was found later that VP4 could, in some cases, be the dominant neutralization protein (Ward et al. 1988, 1993), a dual serotyping scheme was required. Although VP7 serotypes could be determined readily by cross-neutralization studies, this was more difficult for VP4 (Tanaguchi et al. 1988; Gorziglia et al. 1990; Padilla-Noriega et al. 1992; Snodgrass et al. 1992). Therefore, two numeric systems were devised to classify the VP4 protein in rotavirus strains. One is based on comparative nucleic acid hybridization and sequence analyses (genotypes), and the second is based on neutralization (serotypes) using antisera against baculovirus-expressed VP4 proteins or reassortants with specific VP4 genes. Rotavirus classification based on VP4 and VP7 is designated P and G types to describe the protease sensitivity and glycosylated structure of these two proteins, respectively. Until very recently, 14 G types and 22 P types had been identified (Rao 2000). However, a new strain of rotavirus was recently obtained from asymptomatic rhesus macaques, whose VP4 protein shares <87% amino acid homology with that of any representative P genotype and has been tentatively classified as
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genotype P[23] (McNeal et al. 2005). Rotaviruses belonging to 11 G serotypes and 7 P genotypes have been isolated from humans (Gerna et al. 1993; Kapikian et al. 2001). In contrast, the variety has been much more limited within murine rotaviruses, where only 1 G serotype (G3) and 2 P genotypes (P[16] and P[20]) have been identified and only 1 strain belonging to the P[20] genotype has been identified (Estes 2001). 4.
Strains of Murine Rotavirus (EDIM)
The term “EDIM” is generic for murine rotaviruses as the agents of “epizootic diarrhea of infant mice.” Several EDIM strains have been identified and have been assigned specific names (Burns et al. 1995). The EDIM strain was originally isolated by Kraft (1957) and visualized by Adams and Kraft (1963). However, when other investigators obtained this virus from J. Wolf they assigned it a more specific name (i.e., EW: E for “EDIM” and W for “Wolf”) (Wolf, Cukor, et al. 1981; Greenberg et al. 1986). Our laboratory has retained the original generic name for this strain of murine rotavirus. Thus, we have called it EDIM. Therefore, our designation of EDIM and the EW designation of others refer to an EDIM strain of the same origin. In addition to these strains, 7 other strains of murine rotavirus have been isolated and characterized, the most recent of which was obtained as a contaminant within the conventional mouse colony at Cincinnati Children’s Hospital and named EMcN (McNeal et al. 2004).
Based on nucleotide sequence analysis, all 9 strains are G3[P16] except EHP, which has been classified as G3[P20] (Estes 2001). From these sequence comparisons, it appears that both the VP4 and VP7 genes of these rotavirus strains co-evolved such that they are approximately equidistant from one another in almost all cases. One exception is that the VP4 gene of EHP is unique and, therefore, this virus strain probably arose through a reassortment event since its VP7 gene appears to have coevolved with that of the other murine strains (McNeal et al. 2004). Another exception is that both proteins from the EC and EL strains differ from one another by ≤1%, while at least 1 of these 2 proteins from all other paired comparisons (except EDIM and EW) differ by ≥3.5%. This suggests that the EL and EC strains separated more recently. The final exception is that the VP4 and VP7 proteins of EDIM and EW strains differed by only 3 and 1 amino acids, respectively, an expected result based on their common origin. Phylogenetic analyses provide a graphic summary of the relationships between the VP4 and VP7 proteins of these 9 murine rotavirus strains (Fig. 9-2).
D.
Growth of Rotavirus In Vitro
The cells most commonly used for in vitro growth of group A rotaviruses are a line of kidney cells called MA104 derived from an African green monkey. However, rotaviruses grow well in other monkey kidney cell lines as well as in primary monkey
EB
EDIM
EB-2
EW
EDIM
EC
EW EL EC EMcN EL EB EMcN YR-1
YR-1
EHP
EHP
1.00
A
1.00
B
Fig. 9-2 Phylogenetic analyses of the VP4 and VP7 proteins of EMcN and other strains of murine rotavirus. Phylogenetic trees of VP4 (A) and VP7 (B) proteins of EMcN and the other murine strains were constructed as described in McNeal et al. (2004). The line lengths in the trees denote the number of amino acid substitutions present in the sequences, and the scale represents 1 substitution per 100 amino acid residues.
9. REOVIRIDAE
kidney cells. Transformed intestinal cell lines of human origin have also been used for growth of human rotaviruses. Even so, many cell types are not susceptible to rotavirus infection, and rotaviruses of human and murine origin appear to be among the most fastidious. Prior to the initiation of in vitro replication, rotaviruses are activated by cleavage of their outer capsid VP4 proteins by trypsin-like proteases into proteins VP5 and VP8, which remain virus-associated. After attachment to receptors on the cytoplasmic membrane via association with protein VP8 (Denisova et al. 1999; Ludert et al. 1996; Mendez et al. 1996; Rugeri and Greenberg 1991; Zarate et al. 2000), the activated virion either passes directly through this membrane or is taken within a vesicle into the cytoplasm (Suzuki et al. 1985; Kaljot et al. 1988; Fukuhara et al. 1988). Either during membrane penetration (Cuadras et al. 1997) or soon thereafter, the outer capsid proteins are removed, thus stimulating the RNA-dependent RNA polymerase (i.e., the VP1 transcriptase) associated with the inner shell to synthesize the 11 viral mRNAs that are capped by VP3 and subsequently translated into viral proteins. Once viral proteins accumulate within the cytoplasm, large inclusions or viroplasms are formed in which the assembly of virion precursors is initiated (Aponte et al. 1996). The earliest particles detected contain the complete complement of singlestranded plus-sense RNAs (i.e., one molecule of each mRNA) together with VP1, VP3, NSP1, NSP2, NSP3, and NSP5 (Gallegos and Patton 1989; Patton 1994). This initial replication intermediate then loses its NSP1 proteins and becomes a core replication intermediate with the addition of VP2. Still later, core replication intermediates become double-layered particles with the addition of VP6 and the loss of most of the remaining nonstructural proteins. During these assembly steps, an RNA polymerase (replicase) associated with the replication intermediates uses the single-stranded mRNAs within the particles as a template for minus-strand synthesis and formation of the double-stranded RNA genome segments. The double-layered particles then become transiently associated with the transmembrane protein NSP4, perhaps also along with VP4, and bud into the rough endoplasmic reticulum. The other rotavirus glycoprotein, VP7, becomes sequestered within the rough endoplasmic reticulum, where it is added to complete the formation of mature viral particles. These mature viruses accumulate within the lumen of the rough endoplasmic reticulum until cell lysis occurs. In cell culture, maximum production of infectious rotaviruses is found at approximately 12 hrs after infection is initiated.
E. 1.
Rotavirus Replication In Vivo
Pathogenesis of Rotavirus
After fecal-oral transmission of rotavirus, infection is initiated in the upper intestine and typically leads to a series of histologic and physiologic changes. These changes have been examined
241 extensively, particularly through experimental infections of animals. Studies in calves revealed that rotavirus infection causes the villus epithelium to change from columnar to cuboidal, which results in shortening and stunting of the villi (Pearson et al. 1978). The cells at the villus tips become denuded, while in the underlying lamina propria, the numbers of reticulum-like cells increase and mononuclear cell infiltration is observed. The infection starts at the proximal end of the small intestine and advances distally. Similar observations have been made after rotavirus infection of piglets (Pearson and McNulty 1979). The pathology of murine rotavirus infection was also examined in several studies, and the results are similar, but certainly not identical, to those found in larger animals. Furthermore, unlike the outcome found in most young animals and humans, the severity of rotavirus diarrhea in mice is relatively mild. This is true even in immunocompromised animals, which sometimes shed rotavirus indefinitely. Many of these pathogenesis studies in mice have been conducted with heterologous rotavirus strains that require orders of magnitude more rotavirus because of their restricted replication in mice. The histologic changes induced by these heterologous strains are similar to those found after murine rotavirus infection, even though viral replication is limited after oral inoculation with these viruses. A very recent report on the pathogenesis of EDIM in neonatal mice revealed that only the mature enterocytes at the tips of the intestinal villi become infected, which within 1 day results in histological changes characterized by vacuolization of these infected cells, swelling of the villus tips, constriction of the bases, and nuclei that are irregularly positioned within the cells (Boshuizen et al. 2003). Diarrhea begins within 1 day after EDIM inoculation and sometimes persists until the mice are 14 days of age (McNeal et al. 2004). No diarrhea can be induced by rotavirus, even in naive mice, after the animals reach 2 weeks of age. By day 2 after virus inoculation, when vacuolated cells are still abundant, very few rotavirus infected cells are found in the villus epithelium (Boshuizen et al. 2003). Crypt cell movement to villus tips is greatly accelerated in EDIM-infected mice and, by day 4 after inoculation, these become targeted for a new round of rotavirus replication. By day 7 after EDIM inoculation, no rotavirus is found in any part of the small intestinal epithelium (Boshuizen et al. 2003) or lumen (McNeal et al. 2004). Studies with the mouse model have also revealed a potential hazard associated with the possible use of heterologous viruses as vaccine candidates. Although live rotaviruses have been found in the blood of mice following oral infection with murine rotaviruses (Blutt et al. 2003), actual replication of the homologous murine strains may be restricted to the intestine. In contrast, oral inoculation of neonatal mice with a simian rotavirus resulted in its spread to the liver and induction of hepatitis (Uhnoo et al. 1990). Because of evidence of abnormal liver function during natural rotavirus infection in humans (Kovacs et al. 1986; Grimwood et al. 1988), this observation has caused concern over the use of animal strains as vaccine candidates. No significant alteration of liver function has so far been associated with
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any rotavirus vaccine candidate after extensive investigations, even though many candidates have been derived from animal rotavirus strains. However, use of the same simian rotavirus strain that caused hepatitis in mice, in combination with 3 monoreassortants of this virus as a vaccine in infants (RotashieldTM), was found to increase the incidence of intussusception in the week following the first dose of vaccine (Murphy et al. 2001). This necessitated the removal of the licensed vaccine from the market in the United States. The molecular basis for pathogenicity has not been established. Offit et al. (1986) reported that the virulence of reassortants generated between heterologous rotaviruses and tested in a mouse model correlated with the presence of the VP4 protein from the more virulent virus. Neither rotavirus strain used in his study (a simian and a bovine strain) replicated efficiently in mice, however, which suggested that the observation may have limited applicability. A later study with murine-simian rotavirus reassortants revealed no association between the VP4 protein and virulence (Broome et al. 1993). In that study, the strongest association between virulence and a gene product was with NSP1, a nonstructural protein. Associations between virulence and specific gene segments also were examined in piglets. Virulence variants that appeared to differ only in their VP4 genes were isolated from the feces of an infected pig (Bridger et al. 1992; Burke et al. 1994). To eliminate the possibility that virulence was determined by other gene products, the VP4 gene of the virulant strain was transferred into the avirulent strain by reassortment. This caused the avirulent strain to become virulent (Bridger, Tauscher et al. 1998). In another study with reassortants between a virulent porcine virus and a human strain attenuated for piglets, it was found that the porcine rotavirus genes encoding VP3, VP4, VP7, and NSP4 all were required for virulence in piglets (Hoshino et al. 1995). Whether either of these observations has general applicability or pertains only to a limited combination of rotavirus strains because of specific interactions between their proteins remains to be determined. It was reported that passage of a porcine rotavirus in piglets dramatically increased its virulence (Bridger et al. 1992), whereas passage of a virulent porcine rotavirus in cell culture attenuated this virus (Tzipori et al. 1989). Thus, the association between specific rotavirus genes and virulence can be altered readily by natural selection through mutation.
the conclusion that destruction of the villus tip cells causes carbohydrate maladsorption and osmotic diarrhea (Graham et al. 1984). In mice it has been reported that carbohydrate maladsorption did not occur as in piglets and, therefore, that crypt cell secretions may be the cause of fluid loss (Collins et al. 1988). Additional studies in animals and humans concerning changes in the adsorption of macromolecules across the intestinal surface after rotavirus infection have revealed no general pattern. The importance of virus replication for induction of rotavirus diarrhea has been challenged. It was reported that inoculation of mice with a large number of inactivated particles from a heterologous rotavirus also resulted in diarrhea (Shaw et al. 1995). The authors suggested that rotavirus attachment or entry into cells was sufficient to induce diarrhea in this model and that the mechanism of rotavirus-induced diarrhea was consistent with a viral toxin-like effect exerted during virus-cell contact. Diarrhea also has been induced in infant mice and rats by intraperitoneal inoculation with the rotavirus NSP4 protein as well as with a 22–amino acid peptide derived from this protein (Ball et al. 1996). It was observed that this protein and its peptide caused an increase in Ca2+ concentration in insect cells when added exogenously (Tian et al. 1995). In previous experiments, it was found that NSP4 and its peptide can increase the levels of intracellular Ca++ (Tian et al. 1994) by activating a calcium-dependent signal transduction pathway that mobilizes transport of this ion from the endoplasmic reticulum (Dong et al. 1997). Further reports suggest that NSP4 possesses membrane destabilization activity (Tian et al. 1996; Browne et al. 2000) that may result from increased intracellular Ca++ concentrations resulting in cytoskeleton disorganization and cell death (Perez et al. 1998, 1999; Brunet et al. 2000). Thus, binding NSP4 to intestinal epithelium after its release from infected cells may contribute to altered ion transport and diarrhea. Whether this is a major mechanism of diarrhea occurring after rotavirus infection remains to be determined. Some additional studies in mice support a role for NSP4 as a cause of diarrhea (Horie et al. 1999; Zhang et al. 1998), while other studies indicate that mutations in NSP4 are not responsible for attenuation of rotavirus in either mice or humans (Ward, Mason, et al. 1997; Angel et al. 1998; Lee et al. 2000), thus questioning its importance as a cause of diarrhea in nature.
2.
3.
Mechanisms of Diarrhea
Although rotaviruses cause severe diarrhea in numerous species, including humans, the mechanisms responsible have not been determined and may be due to multiple factors. An early study in piglets indicated that glucose-mediated sodium adsorption was diminished by rotavirus infection (Davidson et al. 1977). The authors concluded that retarded differentiation of uninfected enterocytes that migrated at an accelerated rate from the crypts after the virus had invaded villus cells was responsible for adsorptive abnormalities. Another study with piglets led to
Immunity
The immunological effectors that prevent rotavirus disease have been partially identified, particularly through studies with animal models, but in humans remain poorly understood. Because rotaviruses replicate in intestinal enterocytes, resulting in the associated gastrointestinal symptoms, it is generally assumed that effector mechanisms must be active at the intestinal mucosa. The most obvious immunological effector is secretory IgA. Following infection of mice with a high dose of heterologous rotavirus, up to 50% of all IgA cells in the lamina propria of the
243
9. REOVIRIDAE
intestine can be rotavirus-specific (Shaw et al. 1993). Furthermore, protection against rotavirus infection in orally immunized mice correlates with levels of intestinal (stool) and serum rotavirus IgA but not serum rotavirus IgG (McNeal et al. 1994; Feng et al. 1994). In humans, titers of serum rotavirus IgG and IgA as well as intestinal rotavirus IgA correlate with protection following natural infection. However, the titer of any isotype of rotavirus-specific antibody could not be consistently correlated with protection after either natural infection or vaccination. Thus, the possibility remains that rotavirus antibody is merely an indicator of protection and not the actual effector. The most obvious mechanism of protection by antibody is by virus neutralization. Passive protection has been definitively linked with the consumption of neutralizing antibody in both animal and human studies. Evidence that active immunity induced by oral immunization with live rotavirus or natural rotavirus infection is due to neutralizing antibody is varied (Chiba et al. 1986; Hoshino et al. 1988; Ward, Clemens, et al. 1992). For example, initial vaccine trials with both bovine and simian rotaviruses suggested that protection developed in the absence of neutralizing antibody to the circulating human rotavirus strains. Protection was, however, inconsistent, and subsequent vaccine trials with a rhesus rotavirus (RRV, simian) strain suggested that protection may be serotype-specific (Flores et al. 1987; Santosham et al. 1991). These results led to the development of bovine and simian rotavirus vaccine strains containing genes for human rotavirus neutralization proteins, which have been or are currently being evaluated in infants. Even in these trials, the relationship between serum neutralizing antibody titers and protection is inconsistent, and protection is much greater than serotype-specific neutralizing antibody responses to the circulating human rotavirus strains (Ward, Knowlton, et al. 1997). Most data from animal studies indicate that classical neutralization is not the only mechanism of protection. The most immunogenic protein is VP6, which does not appear to stimulate neutralizing antibody responses. Evidence, however, suggests that IgA antibodies directed at VP6 are protective by as yet incompletely understood mechanisms (Burns et al. 1996; Feng et al. 2002). Vaccination with either virus-like particles (VLPs) that lack the outer capsid proteins and thus do not induce neutralizing antibody, or a chimeric VP6 protein can also elicit protective immunity against infection in adult mice (O’Neal et al. 1997; Choi et al. 1999). Passive protection against murine rotavirus disease in neonatal mice has also been produced by adoptive transfer of CD8+ T cells from spleens of mice previously infected (orally) with either homologous or heterologous rotavirus strains (Offit and Dudzik 1990). Similarly, CD8+ splenic or intraepithelial lymphocytes from rotavirus-infected mice can eliminate chronic rotavirus shedding in severe combined (SCID) immunodeficiency mice (Dharakul et al. 1990). Thus, at least passive protection against rotavirus disease and resolution of rotavirus shedding can be promulgated with cytotoxic T cells.
An adult mouse model of rotavirus infection has been particularly useful in examining the mechanisms of active immunity against rotavirus in mice (Ward et al. 1990). Since adult mice become infected with rotavirus but do not develop disease, this model uses protection against infection as its endpoint. According to this model, protection against live oral murine rotavirus infection is not correlated with either serum or intestinal neutralizing antibody titers against the challenge virus (Ward, McNeal, et al. 1992). However, it is correlated with total serum and stool rotavirus IgA titers (McNeal et al. 1994; Feng et al. 1994, 1997) as well as high titers of rotavirus-specific IgA at the intestinal mucosa surface (Moser et al. 1998). Subsequently, the use of B cell–deficient mice that cannot produce antibody has shown that long-term protection against rotavirus infection after a previous rotavirus infection depends at least partially on antibody (Franco and Greenberg 1995; McNeal et al. 1995). Even after parenteral immunization, migration of antigenpresenting cells from the peripheral lymphoid tissues to the gut-associated lymphoid tissues may contribute to mucosal IgA responses and protection (Coffin et al. 1999). Although protection in this model is typically associated with rotavirus IgA, genetically modified mice that cannot produce IgA are also protected after live virus immunization, presumably due to increased titers of rotavirus IgG (O’Neal et al. 2000). Recent studies have also demonstrated the importance of integrin-mediated B cell homing to the intestine for their antirotaviral effectiveness (Kuklin et al. 2001). Resolution of rotavirus shedding and protection against subsequent rotavirus infection of mice has also been associated with rotavirus-specific CD8+ T cells. Depletion of CD8+ cells in B cell–deficient mice prior to oral inoculation with live murine rotavirus prevents resolution of the initial infection (Franco and Greenberg 1995; McNeal et al. 1995). Thus, cytotoxic T cells appear to be critical for the initial resolution of virus shedding when antibody is not present. In fully immunocompetent mice, however, CD8+ cell depletion merely delays the resolution of the shedding that occurs with the appearance of antibody. More recently, it was shown that intranasal or oral inoculation of mice with a chimeric VP6 protein, or even a 14–amino acid peptide of VP6, along with an effective adjuvant consistently elicited >95% reductions in rotavirus shedding after challenge (Choi et al. 1999, 2000). CD4+ T cells were subsequently found to be the only lymphocytes required to elicit this protection (McNeal et al. 2002). Therefore, B, CD8+, and CD4+ T cells have all been identified as effectors of protection against rotavirus shedding in mice, and the relative importance of each appears to be dependent on the immunogen and the method of immunization. A summary of the major findings on immune mechanisms in this adult mouse model are listed in Table 9-2. 4.
Age Restrictions for Rotavirus Disease
Age restrictions associated with rotavirus disease vary greatly between humans and animals. In animals, rotavirus illness
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TABLE 9-2
EFFECTOR MECHANISMS OF RESOLUTION AND PROTECTION IDENTIFIED IN THE ADULT MOUSE MODEL Mouse strain
Immunization
Outcome
Reference
BALB/c (normal)
JHD (B cell–deficient)
Oral, live murine rotaviruses
JHD BALB/c
Oral, live murine rotavirus Intramuscular with live murine rotavirus Oral, live murine rotavirus Adoptive transfer of immune B or CD8+ T cells into chronically shedding Rag2-deficient mice Intranasal immunization with VP6
Protection correlates with serum rotavirus IgA Protection correlates with intestinal rotavirus IgA Resolution dependent on CD8+ cells; protection primarily dependent on antibody Partial protection related to CD8+ cells Intestinal IgA production after parenteral immunization IgA not required for protection B but not CD8+ T cells require α4β7 homing receptor
McNeal et al. 1994
BALB/c
Oral, several live homologous and heterologous rotaviruses Oral, live RRV, EDIM
IgA −/− β7 −/−
JHD J chain −/−
Intranasal immunization with 2/6 VLPs
CD4+ cells are only lymphocytes required for protection Reduced protection in absence of transcytosed IgA, IgM
appears to be limited to the first days or weeks of life. Mice, even immunocompromised animals, are susceptible to rotavirus disease for only their first 15 days of life but can experience a rotavirus infection for their entire lifetime (McNeal and Ward 1995). Similarly, piglets and calves are most susceptible to rotavirus diarrhea during their first days of life. In contrast, severe human rotavirus disease is most common between 6 and 24 months of age, but milder rotavirus illnesses occur throughout our lifetimes. Possibly, nonimmunologic, age-dependent changes occur within the intestine and at least partially account for age-related changes in the sensitivity to rotavirus infection and disease in both animals and humans, such as the observed decrease in virus-specific receptors on enterocytes between suckling and adult mice (Riepenhoff-Talty et al. 1982). A similar suggestion has been made for calves (Varshney et al. 1995). This also may explain why human infants are more susceptible to rotavirus illnesses than older children or adults. It has been reported that the onset of rotavirus disease in infants coincided with the decline of maternal antibody titers to low concentrations (Zheng et al. 1989). Therefore, the commonly asymptomatic nature of neonatal rotavirus infections in humans may be due to protection from transplacental antibody that may persist for the first months of life. Animals typically have little transplacental maternal antibody, which could explain why their period of greatest susceptibility to rotavirus disease is immediately after birth, prior to the development of active immunity.
F.
Epizootiology—Host Range
Rotaviruses have an extremely wide host range, but natural cross-species infections may be rare, particularly those
Feng et al. 1994; Moser et al. 1998 Franco and Greenberg 1995; McNeal et al. 1995 Franco et al. 1997 Coffin et al. 1999 O’Neal et al. 2000 Kuklin et al. 2000, 2001
McNeal et al. 2002 Schwartz-Cornil et al. 2002
between animals and humans. However, a number of human isolates appear to be animal strains or animal-human rotavirus reassortants, as determined by genogroup and sequence analyses. The importance of these strains in human disease may be limited. It has been suggested, however, that once adapted to replication in humans, such strains may become important human pathogens (Nakagomi and Nakagomi 1993). The property of host restriction has been utilized extensively to develop rotavirus vaccines for humans from naturally attenuated bovine and simian rotaviruses. Oral immunization of infants with these experimental live virus vaccines has resulted in low levels of intestinal replication and partial protection against human rotavirus illnesses (Vesikari et al. 1985; Clark et al. 1988; Bernstein et al. 1995; Rennels et al. 1996; Ward et al. 1998). Thus, the barrier of host restriction can be bypassed sufficiently under these controlled conditions to permit the development of protective immune responses in a heterologous host. Experimental studies in animals have shown that intestinal replication of rotaviruses in heterologous species generally is limited, and, if shedding of progeny viruses is detectable, it often occurs only when animals are inoculated with very high doses of the heterologous viruses (Feng et al. 1994; Ciarlet et al. 1998). The basis for host range restriction is unknown and probably involves the collective properties of at least several genes. When reassortants between a murine and a simian rotavirus were used in a mouse model, however, a significant linkage to host range restriction was associated with gene 5 encoding NSP1 (Broome et al. 1993). Other studies also report nonrandom selection of gene 5 in progeny after co-infection of cells in culture (Graham et al. 1987) and in mice (Gombold and Ramig 1986), thus suggesting a possible growth advantage associated with this gene. This gene also shows a high amount of sequence divergence between
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rotaviruses of different species, which supports its possible role in host restriction. It should be noted, however, that in a recent study where the NSP1 gene from a bovine rotavirus that produces an abortive infection in pigs was substituted in a porcine rotavirus that replicates productively in pigs, the new reassortant still demonstrated productive replication in piglets (Bridger, Dhaliwal, et al. 1998). Thus, NSP1 is not the only determinant of host range.
G.
Diagnosis
In initial studies, electron microscopy was used for identification of rotavirus because of the large number of particles present in stools (often >1011) and the characteristic appearance of rotavirus. This technique has been largely replaced by enzyme immunoassay–based and latex agglutination tests, for which kits are available commercially. Both have good sensitivity when compared with electron microscopy. One problem that has been noted in the past was false positive results in neonatal stools with certain ELISA kits (Christie et al. 1983; Ratnam et al. 1984; Troonen 1984). Inclusion of a rotavirusnegative capture antibody as a control in these kits should eliminate these false positive reactions. Rotavirus also can be grown in tissue culture, although the methods used for routine viral cultures from humans do not detect rotavirus. The serotype (G type) of cultured strains can be identified using monoclonal antibodies. Genotypes can be used as surrogates for serotypes using DNA probes (Larralde and Flores 1990; Steele et al. 1993) and by reverse transcription/ polymerase chain reaction. Electrophoresis of extracted RNA also can be used to identify rotavirus by its characteristic 11 segments and is used to define electropherotypes. All these methods have proved useful as epidemiologic tools and in vaccine studies. Rotavirus infection, both symptomatic and asymptomatic, can also be identified by changes in rotavirus antibody. ELISA assays are used most commonly to measure serum IgM, IgA, and IgG levels, as well as stool and intestinal antibodies. Specific neutralizing titers also can be measured for each serotype of rotavirus by plaque reduction or focus reduction assays. One ELISA–based antigen reduction neutralization assay has been found to be better suited for the analyses of large numbers of specimens (Knowlton et al. 1991). Serologic detection of infection is more difficult in the first months of life for humans because of the presence of maternal antibodies. Detection of IgA, which does not cross the placenta, has been used as a marker for previous infection in the first months of life. In animals that have little transplacental maternal antibody, changes in rotavirus antibody titer is generally a reliable indicator of infection.
rotavirus illness in humans. Therefore, vaccines are being developed to prevent these illnesses. Based on the belief that protection from rotavirus is best achieved by inducing local intestinal immune responses and the finding that natural rotavirus infections induce at least partial protection against subsequent rotavirus disease, vaccine efforts have been primarily directed at the development of live-attenuated, orally deliverable rotavirus vaccines. Most of these efforts have concentrated on the use of animal rotavirus strains that are naturally attenuated for humans and stimulate largely heterotypic immune responses [e.g., RIT 4237 (bovine), WC3 (bovine), RRV (simian)]. More recently, human rotavirus genes have been introduced into these animal strains by creating reassortant viruses to increase their serotypic relatedness to human rotaviruses (e.g., RRV-TV, WC3TV) (Kapikian et al. 2001). Human rotaviruses have also been developed as vaccine candidates. Most are neonatal strains that may be naturally attenuated. However, the most extensively evaluated human rotavirus vaccine candidate is strain 89-12, a G1[P8] obtained from the stool of a symptomatic child and attenuated by multiple cell culture passages (Bernstein et al. 1999). Subunit and DNA vaccines, various expression vectors, synthetic peptides, and virus-like particles (VLPs) produced from baculovirus-expressed rotavirus capsid proteins are also being considered as alternative vaccine candidates. In every animal model of active and passive immunity examined, nonreplicating VLPs have been reported to be safe, highly immunogenic, and capable of inducing at least some immunity (O’Neal et al. 1997; Cialet et al. 1998; Yuan et al. 2001; Coste et al. 2000). Intranasal or oral inoculation of a chimeric VP6 protein along with a mucosal adjuvant has also been shown to provide excellent protection against rotavirus shedding in the adult mouse model (Choi et al. 1999). Although mice have been successfully used to develop and test new vaccine candidates, these vaccines will not be used in mice colonies to prevent rotavirus infection. Therefore, the only method to prevent these infections is by preventing exposure. As already noted, this can be accomplished by the use of BL2 conditions. However, once rotavirus has contaminated a conventional mouse colony, it requires great effort to eliminate it. The saying that “an ounce of prevention is worth a pound of cure” could have been formulated with protection of mouse colonies from rotavirus contamination in mind.
III.
REOVIRIDAE (ORTHOREOVIRUS; MAMMALIAN REOVIRUS) A.
H.
History
Control and Prevention
Nonspecific supportive measures such as oral or intravenous rehydration have been the only methods available to overcome
The mammalian reoviruses have been shown to cause debilitating and fatal disease in neonatal and immunodeficient mice. Their pathogenesis has been studied extensively using the mouse
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as a model system to investigate viral pathogenesis, particularly since these viruses are neurotropic and spread via the oral route from the gastrointestinal tract through the bloodstream to the nervous system and other body sites, including the heart, liver, and lungs. The first mammalian reovirus was isolated in 1951 from the feces of an Australian aboriginal child with a history of cough, fever, vomiting, hypertrophic tonsils, and bilateral pneumonia. When a preparation of the fecal sample was injected intraperitoneally, mice developed a syndrome of encephalitis, jaundice, peritonitis, alopecia, and emaciation, and their fur appeared matted and oily. This virus isolate was initially called hepatoencephalomyelitis virus (Stanley et al. 1953). As part of an effort to identify viral causes of human and veterinary diseases, a second reovirus was isolated from a rectal swab of a healthy child (“Lang”) from Cincinnati, Ohio, in 1953 (Ramos-Alvarez and Sabin 1954). This virus became known as reovirus serotype 1 strain Lang and is the prototypic virus for serotype 1. Two additional mammalian reoviruses were isolated from children with diarrhea. One isolate from a child (“Jones”) was initially referred to as “D5” because it was the fifth unclassifiable isolate from a child with diarrheal illness. This isolate became the prototype for reovirus serotype 2, known as reovirus serotype 2 strain Jones (Sabin 1959). The other isolate from a child (“Dearing”) became the prototype for reovirus serotype 3 and is referred to as reovirus serotype 3 strain Dearing (Ramos-Alvarez and Sabin 1954). These viruses were initially classified as ECHO type 10 viruses, because they were recovered from human feces using primate cell cultures, they were not originally (albeit erroneously) found to be pathogenic for the usual laboratory animals, and they were not related to any previously described virus group (Rosen 1965). In 1957, another virus was recovered from the rectal swab of an institutionalized infant (“Abney”) in Washington, D.C. This isolate became the second prototype strain for reovirus type 3 (strain Abney) (Rosen et al. 1960). The term “reovirus” was introduced in 1959, when Albert Sabin proposed the grouping of these viruses into the respiratory enteric orphan viruses, because the first members were found to inhabit both the respiratory and enteric tracts of humans and animals but were orphans in the sense that they were not associated with any disease (Sabin 1959). The discovery in 1962 that the genomes of these reoviruses were composed of segments of double-stranded RNA (dsRNA) confirmed their classification as a separate, unique group of viruses (Gomatos and Tamm 1962; Gomatos et al. 1962). Reoviruses are ubiquitous in nature, with exceptionally wide geographic and host ranges. Reoviruses are commonly found in river water and stagnant water, and there does not appear to be any seasonality to their transmission (Adams et al. 1982; Ridinger et al. 1982; Matsuura et al. 1988). Because the concentration of reovirus has been reported to parallel numbers of Escherichia coli, it has been suggested that fecal contamination may be a source of reovirus in water supplies (Matsuura et al. 1993).
In 1961, Hartley et al. showed that reovirus serotype 3 could be found naturally in wild mice populations. Additionally, reoviruses have been recovered from insects, crustaceans, birds, wallabies (quokkas), rats, cats, dogs, horses, sheep, cattle, Macaca monkeys, Cercopithecus monkeys, chimpanzees, and humans (Hull et al. 1956; Sabin 1959, 1960; Rosen 1962; Cook 1963; Lou and Wenner 1963; Malherbe et al. 1963; Rosen, Abinanti, et al. 1963; Stanley and Leak 1963a; Stanley, Leak, Grieve, et al. 1964; Simpson et al. 1965; Scott et al. 1970; McFerran et al. 1973, 1976; Lester and Kalter 1980). Serological studies have identified antibodies to reovirus in trout, reptiles, chickens, marsupials, bats, voles, guinea pigs, rabbits, swine, goats, horses, and several genera of New World monkeys (Rosen, Abinanti, et al. 1963; Stanley and Leak 1963b; Stanley, Leak, Grieve, et al. 1964; Sturm et al. 1980; Goto et al. 1981; Lazarowicz et al. 1982; Lamontagne et al. 1985; Descoteaux et al. 1986). Viral isolations and antibody levels have shown that reovirus serotype 3 commonly infects laboratory mouse and rat colonies (Hartley et al. 1961; Kraft et al. 1986; Lussier et al. 1986), and although reovirus serotype 1 has been isolated from both wild and laboratory mouse colonies, it appears to be less common than serotype 3 (Nelson and Tarnowski 1960; Hartley et al. 1961; Cook 1963; Chastel et al. 1985). In addition to mice and rats, other laboratory animals can be infected with reoviruses. Pathological manifestations following experimental infection have been observed in dogs, Macaca monkeys, and chimpanzees and primarily involve the respiratory, gastrointestinal, and nervous systems (Stanley et al. 1954; Hull et al. 1956; Sabin 1960; Lou and Wenner 1963). No pathological symptoms were found in guinea pigs, ferrets, or rabbits (Rosen 1960; Rosen and Abinanti 1960; Lou and Wenner 1963). Natural infections of cats, dogs, pigs, sheep, cattle, horses, and several genera of nonhuman primates can result in conjunctivitis, respiratory and diarrheal illnesses, and rare cases of encephalitis (Hull et al. 1956; Abinanti 1963; Lou and Wenner 1963; Malherbe et al. 1963; Lamont et al. 1968; McFerran and Conner 1970; McFerran et al. 1973; Belak and Palfi 1974). In nonhuman primates, cases of hepatitis, biliary atresia, and meningitis have also been reported (Sabin 1959; Thien and Scheid 1981; Rosenberg et al. 1983). In humans, the reoviruses have been associated with gastroenteritis and upper respiratory infections, particularly in infants and children (Ramos-Alvarez and Sabin 1954, 1958; RamosAlvarez 1957; Rosen et al. 1960; Jackson et al. 1961; Rosen, Evans, et al. 1963). There are also possible associations of the virus with infant biliary atresia and exanthema in children (Lerner et al. 1947; Tillotson and Lerner 1967; Bangaru et al. 1980; Morecki et al. 1982; Glaser et al. 1984). Isolated cases of reovirus involvement in meningioencephalitis, pneumonitis, keraconjunctivitis, and myocarditis have also been reported (Krainer and Aronson 1959; Nelson and Tarnowski 1960; Joske et al. 1964; Tillotson and Lerner 1967; Jarudi et al. 1973).
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B. 1.
Properties of Reovirus
Structure of Reovirus
The non-enveloped, dsRNA viruses are currently grouped into six families: Reoviridae, Birnaviridae, Totiviridae, Partitiviridae, Hypoviridae, and Cystoviridae. Only viruses in the Reoviridae and Birnaviridae are known to infect vertebrates, and only the Reoviridae are known to infect mammals (Nibert and Schiff 2001). There are currently nine recognized genera in the Reoviridae. Of these, four genera, Orthoreovirus, Rotavirus, Orbivirus, and Coltivirus, infect mammals. Neither the Orthoreovirus or Rotavirus genera have been shown to be transmitted by insects, although other dsRNA viruses have (Simpson et al. 1965). Orthoreovirus includes the prototype viruses for the Reoviridae, the nonfusogenic mammalian orthoreoviruses, or more commonly, the mammalian reoviruses. These viruses will be referred to as reoviruses for the remainder of the chapter. The distinction between the nonfusogenic and fusogenic orthoreoviruses refers to the ability of the latter to fuse infected cells into large multinucleated syncytia (Ni and Ramig 1993). The fusogenic orthoreoviruses include the avian and reptilian orthoreoviruses (Kawamura et al. 1965; Vieler et al. 1994; Lamirande et al. 1999) as well as two viruses isolated from an Australian flying fox and at a baboon research colony in Texas (Gard and Compans 1970; Wilcox and Compans 1982; Leland et al. 2000). The genomes of all of the Reoviridae contain 10–12 segments of dsRNA. Each segment uses one of the complementary strands to encode 1–3 proteins, and the viral mRNAs are synthesized by virally encoded enzymes. The structure and replication strategies of reoviruses have been extensively reviewed (Nibert and Schiff 2001). Reovirus has a genome of 10 dsRNA segments and encodes 12 proteins (Table 9-3). Reoviruses also have the
capacity to form genomic reassortants (Fields and Joklik 1969; Wenske et al. 1985). When cells are co-infected with related isolates, the progeny viruses can contain homologous gene segments from either of the parent viruses. Although reassortment within the avian orthoreoviruses has been reported (Schnitzer 1985), reassortment between the mammalian and avian groups has not. No reassortment has yet been demonstrated in the fusogenic reptilian or fusogenic mammalian orthoreoviruses. The capacity for reassortment among the reoviruses may be an important part of their evolution (Chappell et al. 1994; Kedl et al. 1995; Goral et al. 1996). The reovirus dsRNA segments are grouped into 3 classes based on molecular weight, large (L: 3.8–3.9 kbp), medium (M: 2.2–2.3 kbp), and small (S: 1.2–1.5 kbp). The dsRNA segments are enclosed within 2 concentric icosahedrally (5:3:2) symmetric protein capsids, the outer capsid and core. Each segment encodes 1 protein, with the exceptions of the S1 gene segment, which encodes 2 proteins using alternative reading frames (Ernst and Shatkin 1985; Jacobs and Samuel 1985; Sarkar et al. 1985), and the M3 segment, which encodes µNS and µNSC, a second protein formed by alternative translation initiation at a downstream AUG in the M3 mRNA in the same open reading frame as µNS (Wiener et al. 1989; McCutcheon et al. 1999). There are 8 structural and 4 non-virion-associated proteins. The proteins are referred to by size, such that proteins encoded by the L segments are designated l, proteins encoded by the M segments are designated m, and proteins encoded by S segments are designated s (Table 9-3) (Nibert and Schiff 2001). Viral particles (Fig. 9-3) measure 85 nm in diameter (Harvey et al. 1981; Metcalf et al. 1991; Dryden et al. 1993). The outer capsid is primarily composed of σ3 and µ1C, a cleavage product of µ1. Protein µ1C also has a crucial role in initiating host cell
TABLE 9-3
SIZES OF REOVIRUS GENE SEGMENTS AND PROPERTIES OF ENCODED PROTEINS RNA segment
No. base pairsa
Mass of protein (kDa) 142 145 143 83 76
1416
λ3 λ2 λ1 µ2 µ1/µ1c µNS µNSC σ1
1331 1198 1196
σ1s σ2 σNS σ3
14 47 41 41
L1 L2 L3 M1 M2 M3
3854 3916 3901 2304 2203 2241
S1
S2 S3 S4 a
Encoded proteins
Genome sizes refer to reovirus serotype 3 Dearing.
49
Location/properties of protein core; RNA-dependent RNA polymerase core spike; guanylyltransferase, methyltransferase core; RNA-dependent RNA polymerase core; binds RNA, particle assembly outer capsid; penetration, transcriptase activation nonstructural; role in secondary transcription nonstructural outer capsid; cell attachment, hemagglutinin nonstructural core; binds dsRNA nonstructural; binds ssRNA outer capsid; binds dsRNA, translation
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TABLE 9-4
SELECTED VACCINE STUDIES Vaccine
Country
RIT 4237
Finland Gambia Peru USA (Philadelphia, PA) USA (Cincinnati, OH) Central African Republic Venezuela Finland Venezuela USA USA Finland Venezuela USA USA
WC3
RRV
RRV TV
WC3 TV 89-12
Number of subjects
Number of doses
178 185 391 104 206 472 247 200 320 898 1187 2273 2207 417 215
1 3 3 1 1 2 1 1 1 3 3 3 3 3 2
Percentage* protection (Overall/Severe disease) 50/58 0/37 40/75 43/89 17/41 0/36 68/100 38/67 64/90 69/73 54/69 66/91 48/88 73/73 89/100
* Measured in the first year after vaccination
membrane penetration (Nibert and Fields 1992; Lucia-Jandris et al. 1993; Tosteson 1993). Protein λ2 is a homopentamer that extends from the core to the outer capsid and forms the 12 turretlike spikes at each of the 12 vertices of the virus (Metcalf et al. 1991; Dryden et al. 1993). Trimers of protein σ1 interact with λ2 at each vertex and may have 2 different conformations. They may either extend from the virus as an elongated fiber or have
a more folded and retracted form (Strong et al. 1991; Coombs 1998). Protein σ1 is also the cell-attachment protein and hemagglutinin (Weiner et al. 1977, 1978; Weiner, Powers, et al. 1980; Lee et al. 1981b; Burstin et al. 1982; Epstein et al. 1984; Armstrong et al. 1984; Masri et al. 1986; Pelletier et al. 1987; Yeung et al. 1987; Banerjea et al. 1988; Furlong et al. 1988; Fraser et al. 1990).
Fig. 9-3 Electron micrograph of purified reovirus serotype 3. Uranyl acetate negative stain using the pseudoreplica technique. Scale bar = 100 nm. (Micrograph republished from “The Mouse in Biomedical Research,” 1st edition. Original micrograph courtesy of Erskine Palmer, Centers for Disease Control and Prevention, Atlanta, Georgia.)
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The reovirus core is composed of the proteins λ1, σ 2, λ3, and µ2 and contains the segmented genome. During infection, reoviruses are partially uncoated to form an infectious subvirion particle (ISVP). The uncoating of virions to ISVPs involves the removal of outer capsid protein σ3, additional cleavage of the µ1C protein, and extension of σ1 outward from the surface (Nibert and Schiff 2001). Removal of σ1 and the µ1 cleavage fragments and a conformational change in λ2 result in cores with open channels through the turrets at the 12 vertices. Diameters for ISVPs and core particles have been measured at 80 nm and 60 nm, respectively (Metcalf et al. 1991; Dryden et al. 1993). The buoyant densities of the virions, ISVPs, and cores in CsCl (g/cm2) are 1.36, 1.38, and 1.43, respectively (Nibert and Schiff 2001). 2.
Stability of Reovirus
Reoviruses are stable at 4°C and room temperature, while higher temperatures result in loss of infectivity. In a study by Rhim et al. (1961), a reovirus serotype 1 was found to have a half-life of 19 hours at 37°C, while Gomatos et al. (1962) found a half-life of 157 minutes for reovirus serotype 3 at 37°C. Prolonged storage at 4°C can result in a gradual loss of infectivity. Reovirus T2J is the most thermolabile, T3D is intermediate, and T1L is the most thermostable (Drayna and Fields 1982a, 1982b; Jané-Valbuena et al. 1999). Reoviruses are also stable as aerosols, especially under conditions of high humidity (Adams et al. 1982). Ultraviolet light inactivates reoviruses, although multiplicity reactivation can occur because of the segmented genomes. The segmented genomes may allow viral reassortment in suspensions of high virus concentration and therefore high multiplicities of infection (Rauth 1965; McClain and Spendlove 1966). Because reoviruses lack a lipid envelope, they are resistant to treatment with lipid solvents such as ether (Sabin 1959; Gomatos et al. 1962). Virions are also resistant to treatment with 1% hydrogen peroxide, 1% phenol, and 3% formaldehyde (Stanley et al. 1953; Drayna and Fields 1982a, 1982b). Although chloroform treatment may inhibit hemagglutination, it does not destroy infectivity of the virus (Rozee and Leers 1967). Most reoviruses are inactivated by 95% ethanol, although ethanol-resistant mutants of reovirus type 3 have been generated in vitro (Wessner and Fields 1993). Sodium hypochlorite (800 ppm chlorine) effectively inactivates the virus after brief exposure (Nibert and Schiff 2001). Ionic detergents, such as those found in wastewater sludge, destabilize reoviruses by decreasing the temperature at which they are inactivated; however, nonionic detergents do not affect reovirus stability (Ward and Ashley 1977, 1978). Chelating agents do not decrease viral infectivity, although Mg2+ has been reported to enhance viral stability when heated for 1 hr at 50°C (Wallis et al. 1964). Reovirus growth is optimal between pH 6.8–7.5, and viral yield drops sharply below pH 6.8 (Fields and Eagle 1973). Although the virus is stable at a pH as low as 2.2 (Stanley et al. 1953), extremely alkaline pH (pH 11) has been shown to decrease infectivity of some serotypes due to loss of the σ1 protein (Drayna and Fields 1982b). Reoviruses are also resistant to the effects
of antibiotics commonly employed in tissue culture, including aminoglycosides and penicillin (Stanley 1974; Rosen 1979). Reovirus infectivity is destroyed by the photodynamic interaction of the virus with the dye neutral red, which is commonly used in plaque assays of the virus (Ramig and Fields 1977b). The physical and chemical properties of reovirus have implications for storage of specimens and disinfection procedures. Reoviruses can be stable at 4°C and room temperature. Because infectivity is gradually lost, however, optimal storage should be at −70°C to preserve any virus. The use of ultraviolet light in surgical areas or biosafety cabinets may be sufficient to eradicate the virus, but should be avoided if isolation of reoviruses is desired. Disinfection of potentially contaminated material or equipment can be performed with either 95% ethanol or sodium hypochlorite, with preferable sequential application of both solutions. Other chemical compounds that may affect reovirus infection and replication include the mutagens, nitrous acid, nitrosoguanidine, and proflavin (Ikegami and Gomatos 1968; Fields and Joklik 1969), which have been used to select for temperature-sensitive mutants with altered hemagglutination and pathogenesis (see below; Fields and Raine 1972). Viral replication is not affected by low concentrations of the cellular DNA synthesis–inhibiting drugs 5-fluordeoxyuridine (0.25 µg/ml), 5-bromodeoxyuridine (100 µg/ml), mitomycin C (1 µg/ml), and cytosine arabinose (10 µg/ml), although higher levels did lower the viral yields from infected cells (Gomatos et al. 1962; Silagi 1965; Shatkin 1969). Host RNA synthesis in infected cells is sensitive to low concentrations of actinomycin D, whereas virus yields are unaffected by the same concentrations (Kudo and Graham 1966; Shatkin 1965; Rada and Shatkin 1967). Virus yields from infected cells are reduced by azauridine, and cordycepin can block viral RNA synthesis (Rada and Shatkin 1967; Ramig and Fields 1977a). Ethidium bromide does not appear to affect viral transcription or translation, but blocks dsRNA synthesis, resulting in a high yield of empty particles (Lai et al. 1973). Synthesis of reovirus proteins is blocked by cyclohexamide and puromycin (Watanabe et al. 1968). Inhibition of reovirus replication has been reported with the antiviral compounds neplanocin A (De Clercq et al. 1989), acivicin (Keast and Vasquez 1992), cicloxolone sodium (Dargan et al. 1992), cyclopentenylcytosine (De Clercq 1993), and ribavirin (Connolly and Dermody 2002). A recent study by Hermann and Coombs (2004) has reported that mycophenolic acid, which is clinically used to prevent rejection of transplanted organs but also has antiviral activity, can inhibit reovirus replication in cell culture. C. 1.
Reovirus Strains and Antigenic Relationships
Reovirus Serotypes
Reoviruses are grouped into 3 serotypes, 1, 2, and 3, based on neutralization (NT) and hemagglutination inhibition tests (HAI) (Sabin 1959; Rosen 1960). The cell attachment protein, σ1, is the major serotype-specific antigen recognized
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in NT and HAI tests using type-specific polyclonal antisera (Weiner and Fields 1977; Weiner et al. 1978). Monoclonal antibodies specific for serotypes 1 (5C6) and 3 (9BG5) are also available for serotyping (Virgin et al. 1991; Williams et al. 1991). For reoviruses, the terms “strain” and “clone” have been used interchangeably to designate the origin of the virus isolate, such as in the prototypic reovirus serotype 3 strain Dearing. No distinction has been made between animal and human strains of the same serotype by antigenic or other properties (Rosen 1962), and there is little evidence for restrictions to the range of mammalian species in which a given isolate can replicate. In a study by Hrdy et al. (1979), from nearly 100 isolates collected from humans, cattle, and mice, 30 different circulating strains of reovirus were identified. All three serotypes of reovirus can agglutinate human erythrocytes. Human type O erythrocytes are the preferred substrate for reovirus serotype 1, although hemagglutination occurs with types A, B, and AB blood cells. Reovirus serotype 3 strains produce more efficient hemagglutination with bovine red blood cells (Rosen 1960; Halonen 1961; Eggers et al. 1962; Dermody et al. 1990). The reovirus receptors on erythrocytes are αanomeric sialic acid residues on glycophorin molecules (Paul and Lee 1987; Paul et al. 1989). Pre-treatment of blood cells with neuraminidase, sialyated oligosaccharides, and brain gangliosides abolishes hemagglutination (Gentsch and Pacitti 1987; Paul and Lee 1987). Hemagglutination can be inhibited by Vibrio cholerae filtrate (RDE) and normal mouse, rat, and rabbit serum (Gomatos and Tamm 1962, Schmidt et al. 1964). Monoclonal antibodies against the σ1, σ3, and µ1 proteins can all produce HAI (Hayes et al. 1981; Lee et al. 1981a; Burstin et al. 1982; Virgin et al. 1991; Tyler et al. 1993). The prototypic viruses from each of the 3 serotypes are morphologically similar, but differ in the nucleotide sequences of their cognate dsRNA segments and in their migration patterns of their proteins in polyacrylamide gel electrophoresis (Cashdollar et al. 1985; Wiener and Joklik 1988, 1989; Coombs 1998). The greatest divergence between the 3 serotypes occurs at the S1 gene that encodes protein σ1. At the amino acid level, the σ1 proteins of strains serotype 1 Lang and serotype 2 Jones are about 48% identical (about 28% at the sequence level), while these 2 strains only share about 25% identity with strain serotype 3 Dearing (less than 10% at the sequence level) (Cashdollar et al. 1985; Duncan et al. 1990). Despite this divergence, there are 5 highly conserved domains of 22–34 amino acids in all 3 serotypes (Duncan et al. 1990). The other gene products of reovirus all share greater than 72%–97% identity at the sequence level, with the gene products of serotype 1 being more closely related to the cognate genes of serotype 3, with the S1 gene segment being the exception. Reovirus infection results in the production of both NT and HAI antibodies in the host. The use of monoclonal antibodies for specific proteins monospecific σ1 antisera suggests that antibodies directed against σ1, σ3, and λ2 can all neutralize infectivity, with the σ3 and λ2 antibodies neutralizing
independent of serotype (although some antibodies showed strain-specific patterns), and the σ1 antibodies being serotypespecific (Hayes et al. 1981; Lee et al. 1981a; Burstin et al. 1982; Tyler et al. 1993). The neutralizing epitope on protein σ1 appears to be different from the hemagglutinating epitope and in separate, distinct regions of the protein (Burstin et al. 1982; Spriggs, Kaye, et al. 1983; Nibert et al. 1990). The mammalian reoviruses also all share a common complement-fixing antigen, although the viral protein responsible for this property has not been identified (Sabin 1959). 2.
Reovirus Reassortants
As with rotaviruses, co-infection of a cell with 2 reovirus strains gives rise to progeny that contain different combinations of parental genome segments. This phenomenon is different from recombination, in which distinct portions of the same genome segment are derived from separate parental viruses. Recombination events have not been demonstrated for reoviruses. Reoviruses containing the different combinations of parental genome segments are termed reassortant viruses. Reassortant viruses are analyzed by the different mobilities of their genome segments (electropherotypes) during polyacrylamide gel electrophoresis. Reassortment of genome segments occurs not only in cultured cells but also in mice (Wenske et al. 1985). The formation of reassortants does not appear to be a random process, with only 3%–20% of viral progeny forming reassortants during co-infection (Fields and Joklik 1969). Some studies have suggested that not all genome segments or protein products from 2 different strains can be productively paired (Joklik and Roner 1995; Nibert et al. 1996). Reassortant viruses have been particularly valuable in the study of reovirus pathogenesis. Reassortment between serotypes, particularly the most divergent genome segment, S1, has led to the determination of many of the biologic properties of reoviruses, such as spread via the neural or hematogenous route, to be discussed later in this chapter. 3.
Reovirus Mutants
Although the prototype viruses of the 3 serotypes have been extensively passaged in culture, sequencing of genome segments by different groups has revealed remarkable consistency (Nibert and Schiff 2001). This may be the result of tight constraints on mutations tolerated in the viral proteins that must retain the capacity to stably assemble and infect cells. The relative lack of mutations in reoviruses with different passage histories is an important property of reovirus and has contributed to the success of pathogenesis studies, which would be complicated by the rapid accumulation of mutations during tissue culture passage in different laboratories. However, several types of reovirus mutants have been selected for in the laboratory. Temperature-sensitive mutants of reovirus have been isolated using chemical mutagenesis and serial high-multiplicity of
9. REOVIRIDAE
infection passage stocks (Ikegami and Gomatos 1968; Fields and Joklik 1969; Ahmed et al. 1980). The resulting mutants grow normally at 31°C but not at 40°C. Some of these mutants produced different patterns of infection in rats compared to the wild-type strains (Fields and Raine 1972). Serial passage at high multiplicities of infection can also result in deletion mutants. Genome segments L1 and L3 are the most commonly deleted, but L2 and M1 deletions are also seen (Ahmed and Fields 1981; Nonoyama and Graham 1970). Some of these defective viruses have the ability to interfere with growth of wild-type virus (defective interference) and the capacity to establish persistent infections (Nonoyama et al. 1970; Spandidos and Graham 1975). Ethanol-resistant mutants have also been isolated by selection (Wessner and Fields 1993). These mutants exhibited increased thermostability and a decreased capacity to permeablize cells (Hooper and Fields 1996). Mutants obtained after passage in cells or animals have also been isolated. These include smallplaque mutants and cold-sensitive mutants (Ahmed and Graham 1977; Ahmed et al. 1983). Mutants with the ability to establish persistent infections and mutant viruses with altered binding capacities, organ tropisms, and virulence have also been selected (Spriggs and Fields 1982; Sherry et al. 1989; Dermody et al. 1993; Haller, Barkon, Li, et al. 1995; Chappell et al. 1997; Wetzel et al. 1997). These mutants have been valuable in assigning specific biological functions to reovirus genome segments (Spriggs, Bronson, et al. 1983; Kaye et al. 1986; Chappell et al. 1997; Rodgers et al. 1998).
D.
Growth of Reoviruses In Vitro
Reoviruses grow and produce cytopathic effects in a wide variety of cultured cell lines. Mouse L929 fibroblasts are the most commonly employed cell line for experimental work, including viral growth, purification, and plaque assay. Madin-Darby bovine kidney (MDBK), rhesus monkey kidney (LLC-MK2), and human embryonic intestinal (intestinal 407) cells are also sensitive lines for reovirus, although there is variation in the kinetics of viral multiplication, the time of appearance of infectious virus, and the rate of host cell death (Franklin 1961; Rhim and Melnick 1961a, 1961b; Ridinger et al. 1982). Golden et al. (2002) have found that the addition of proteases to the cell culture medium during infection can promote viral growth in transformed, nontransformed, and primary cell lines that normally restrict virus infection. The cytopathic effects in infected cells are characterized by a granular appearance of the cells that do not readily slough off from the surface of tissue culture flasks. Cells develop cytoplasmic inclusions, or “viral factories,” that typically begin as dense granular material scattered in the cytoplasm that coalesces and moves toward the nucleus to form the dense perinuclear inclusions (Gomatos et al. 1962; Gomatos and Stoeckenius 1964). In L929 cells, the prototype strains of serotypes 1 and 3 usually form plaques 1–3 mm in diameter, while the serotype 2 strain plaques are usually less than 0.5 mm.
251 The typical yield of virus is 1000 plaque-forming units per cell, with a particle-to-PFU ratio of 50:1 to 200:1 (Jané-Valbuena et al. 1999). Most virus produced during infection remains cell associated, and physical disruption of the cells by sonication or several cycles of freeze-thawing is required for complete recovery of the virus during assay procedures. Studies of reovirus growth in L929 cells shows a one-step growth curve with an eclipse phase lasting 6–10 hrs, followed by viral growth that peaks 15–18 hrs after infection (Jané-Valbuena et al. 1999). In addition to cultured cells, Stanley et al. (1954) showed that reovirus serotype 3 can multiply in the chorioallantois of 12-dayold chick embryos without causing death of the embryo. Pocks were observed on the membrane but with succeeding passages, the pocks could no longer be observed, although oral inoculation of suckling mice with the chorioallantoic suspensions resulted in active disease. Reovirus infection of host cells begins with binding of the virion to susceptible host cells by the σ1 attachment protein. Although the σ1 protein has been studied in detail, the cellular receptors to which it binds are not clearly understood. Both reovirus serotypes 1 (Lang) and 3 (Dearing) have been shown to bind to junctional adhesion molecule 1 (JAM1) using a domain in the globular head region of the protein (Barton et al. 2001). JAM1 serves as the receptor for both prototype and field-isolate strains of all 3 reovirus serotypes (Forrest and Dermody 2003) and thus does not explain the serotype-dependent differences in reovirus tropism, suggesting that coreceptors may influence reovirus pathogenesis, although serotype-specific differences may occur following viral entry. JAM1-null mice are currently being used to assess the role of this molecule in reovirus pathogenesis (Forrest and Dermody 2003). Reovirus serotype 3 binds to α-linked sialic acid residues via a domain in the elongated tail region (Chappell et al. 2000). The serotype 1 σ1 also binds to cell surface carbohydrate, but this molecule has not been identified. After attachment to cellular receptors, virions are delivered by receptor-mediated endocytosis to an endocytic compartment, where the viral outer capsid is uncoated by pHdependent proteases. Uncoating of the outer capsid leads to the generation of ISVPs, which penetrate the endosomes leading to the delivery of viral core particles or dsRNA into the cytoplasm of the cell (Nibert and Schiff 2001). Extracellular generation of ISVPs can occur naturally in the lumen of the gut such that the ISVP may be capable of directly penetrating the cell membrane (Borsa et al. 1979; Bodkin et al. 1989). The use of proteases such as chymotrypsin has been shown to enhance reovirus infectivity through the extracellular generation of ISVPs (Spendlove et al. 1970; Joklik 1972). Removal of the outer capsid protein σ3 appears to be a prerequisite for entry into cells. This results in the exposure of hydrophobic domains of µ1C that appear capable of interacting with membranes to mediate entry (Virgin et al. 1994). Upon entry into the cytoplasm, the remaining outer capsid proteins are removed and the λ2 pentamers undergo a conformational change to open the channels to the virus core (Dryden et al. 1993). The uncoated core particles are
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transcriptionally active and produce the viral mRNA, which is extruded through the channels of the modified λ2 spikes. These mRNAs also serve as the templates for synthesis of the genome dsRNA segments. After synthesis of the viral structural proteins and genome segments, progeny virions are assembled and released concomitant with lysis of the host cell. The replication cycle occurs entirely in the cytoplasm, although the nucleus has been shown to be necessary for efficient replication because enucleated cells exhibit decreased viral yield (Nibert and Schiff 2001). Cells infected with reovirus produce large, perinuclear cytoplasmic inclusions. Microtubules and intermediate filaments are associated with these inclusions (Gomatos et al. 1962; Dales 1963; Fields et al. 1971; Sharpe et al. 1982). Reovirus infection also inhibits host cell DNA, RNA, and protein synthesis (Ensminger and Tamm 1969; Shaw and Cox 1973; Sharpe and Fields 1981, 1982). Cells infected with reovirus exhibit changes in growth factor receptor number and signaling capacity (Verdin et al. 1986; Strong et al. 1993). Reoviruses can induce apoptosis in both cultured cells and in vivo (Oberhaus et al. 1997; Clark et al. 2000; Connolly et al. 2000; DeBiasi et al. 2001). Reovirus serotype 3 (Dearing) induces apoptosis to a greater extent than serotype 1 (Lang) in several cell lines, and reassortant studies have shown that these differences are due primarily to the S1 gene, although the M2 gene, which encodes µ1, contributes to the magnitude of the apoptotic response, which suggests that viral disassembly may be important to induce apoptosis (Tyler et al. 1995; Rodgers et al. 1997; Connolly et al. 2001; Connolly and Dermody 2002). Blocking sialic acid residues and JAM1 molecules has shown that binding to both molecules is required for the induction of apoptosis (Barton et al. 2001; Connolly et al. 2001). A critical component of the apoptosis induction is transcription factor NF-κB. Apoptosis is blocked in cells in which NF-κB is inhibited (Connolly et al. 2000). Inhibitors of the calcium-dependent protease calpain also block reovirus-induced apoptosis (DiBiasi et al. 1999), although it is not known how the calpain and NF-κB pathways couple to cause apoptosis. Although reovirus infections are usually cytocidal, persistent infection occurs in cell culture and in SCID mice (>100 days) (Taber et al. 1976; Ahmed and Graham 1977; Montgomery et al. 1991; Haller, Barkon, Vogler, et al. 1995). Cell cultures persistently infected with reoviruses produce high titers of virus for long periods of time. Establishment of persistently infected cultures occurs when infection is initiated with viral stocks passaged serially at high multiplicities of infection (Ahmed and Graham 1977; Ahmed and Fields 1982; Dermody et al. 1993). Such stocks contain larger numbers of viral mutants that facilitate establishment of persistence. Mutations in the L2 and S4 segments appear to be the most important for the generation of persistence (Ahmed and Fields 1982; Brown et al. 1983). The capacity of cells to maintain persistent infection may be linked to cellular resistance to reovirus-induced inhibition of cellular protein synthesis or resistance of the host cell to one or more steps in entry
of the mutant virus, suggesting a co-evolution event to maintain the persistent state (Duncan et al. 1978; Danis et al. 1993; Dermody et al. 1993). Because reoviruses have dsRNA genomes, they are potent inducers of interferon (Long and Burke 1971; Lai and Joklik 1973; Henderson and Joklik 1978). However, because the dsRNA remains encased in the viral core during virus replication, this may prevent the inhibition of viral protein synthesis by interferon, as suggested by ultraviolet treatment of viral particles, which destabilizes the viral core and results in the release of genomic RNA and an increase in interferon production (Henderson and Joklik 1978). Despite activation of antiviral enzymes by interferon in infected cells, reovirus can still replicate efficiently (Danis et al. 1997), although reovirus strains can differ in the extent to which their replication is inhibited by interferon (Jacobs and Ferguson 1991). The role of interferon induction in vivo by reovirus and its role in pathogenesis has not been characterized. Another interesting observation in cultured cells has been that transformed cells are more susceptible to reovirus-induced cytolysis than nontransformed cells (Hashiro et al. 1977; Duncan et al. 1978). Evidence suggests there may be involvement of epidermal growth factor (EGF) receptor-linked signal transduction pathways or involvement of ras signaling pathways that render the transformed cells susceptible to reovirus infections (Strong et al. 1993, 1998). Such observations have led to the use of reoviruses as immunotherapeutic agents for tumors in both mouse and human systems (Bryson and Cox 1986; Williams et al. 1986; Strong and Lee 1996; Hirasawa et al. 2003).
E. 1.
Growth of Reoviruses In Vivo
Entry into Hosts
Although mammalian reoviruses are not considered human pathogens, reovirus infection of animals can lead to severe pathology. In the neonatal mouse, which serves as the model for experimental pathology, symptoms include acute pancreatitis, diarrhea, encephalitis, hepatobiliary jaundice, myocarditis, oily hair syndrome, pulmonary edema, respiratory distress, and runting. As implied by their name, reoviruses enter into their hosts by both respiratory and enteric routes. After inoculation into the upper gastrointestinal tract of mice, reoviruses adhere selectively to surface projections and stunted microvilli on the luminal surface of M cells (Wolf, Rubin, et al. 1981; Bass et al. 1988). Binding to M cells in the intestine requires conversion of virions to ISVPs by intestinal serine proteases (Amerongen et al. 1994). Electron microscopy indicates that the particles are then endocytosed by M cells and transported across the cells within cytoplasmic vesicles (Wolf et al. 1981; Wolf et al. 1987). Viral particles that reach the submucosal tissues can either spread to adjacent intestinal tissue by infecting the basal surface of intestinal epithelial cells or disseminate within the host by using
9. REOVIRIDAE
neural, lymphoid, or hematogenous pathways (Rubin et al. 1985, 1987; Weiner et al. 1988; Kaufmann et al. 1991; Morrison et al. 1991). Branski et al. (1980b) also found that suckling mice infected with reovirus type 3 Dearing did not exhibit any significant changes in intestinal morphology or enzymatic activity but by day 6, the villi of brush border epithelial cells were shortened with lymphangiectatic lesions and there was a mild mononuclear cell infiltration. The activity of several intestinal enzymes, including lactase and enterokinase, were significantly decreased, indicating that reoviruses can directly affect brush border cells and enzymes. The intestinal contents of infected neonatal mice may also appear lemon yellow in color (Stanley et al. 1953). The respiratory tract contains aggregates of lymphoid tissue that are analogous to Peyer’s patches of the gastrointestinal tract. M cells are found within the epithelial cell layer overlying this bronchial-associated lymphoid tissue. Immediately after intratracheal inoculation of reovirus serotype 1 Lang into adult rats, the virus preferentially binds to the apical surface of the bronchial M cells (Morin et al. 1994). Within 30–60 minutes, the virus is present within membrane-bound cytoplasmic vesicles and subsequently in the intercellular space on the basal surface of the M cells. The reovirus serotypes 1 (Lang) and 3 (Dearing) show marked differences in their pathogenicity after inoculation into suckling mice. Serotype 1 grows well in epithelial cells of the ileum, with little replication in the remainder of the small and large intestine, whereas serotype 3 does not (Rubin et al. 1985; Keroack and Fields 1986). Reovirus serotype 3 Dearing, however, is much more virulent after intramuscular, intraperitoneal, or intracerebral inoculation than after peroral inoculation (Virgin et al. 1997). Reovirus serotype 1 is also taken up by Kupffer’s cells and subsequently excreted in the bile (Rubin et al. 1986). When perorally inoculated into newborn mice, serotype 1 Lang could be detected up to 8 days after inoculation, whereas serotype 3 Dearing could only be detected 2 days postinoculation. Larger numbers of virus were shed from the gastrointestinal tracts of serotype 1–infected mice. Reassortant studies have shown that the S1 (encoding protein σ1) and the L2 (encoding the core spike λ2) determine the capacity to survive in the gut. The increased susceptibility of protein σ1 of serotype 3 strains to intraluminal proteases may contribute to the reduced virulence (Chappell et al. 1998). The L2 segment also determines the degree of viral shedding (Keroack and Fields 1986; Bodkin and Fields 1989). 2.
Central Nervous System
After peroral inoculation into newborn mice, reoviruses spread to extraintestinal organs and the central nervous system. In newborn mice, a strain of reovirus serotype 3 (clone 9) that is highly infectious after peroral inoculation spreads from the intestinal tract into mononuclear cells of ileal Peyer’s patches, through neurons of the myenteric plexus adjacent to Peyer’s patches, and then ultimately through the vagus nerve to brain-stem neurons of the
253 vagal dorsal motor nucleus (Morrison et al. 1991). Reovirus serotype 1 Lang follows a different pathway. After transport across M cells, serotype 1 can be sequentially detected in Peyer’s patches, mesenteric lymph nodes, and the spleen, a pattern consistent with local lymphatic spread and bloodstream invasion (Kaufmann et al. 1983). Spread of reovirus to the central nervous system after intramuscular injection has shown that serotype 3 Dearing spreads to the spinal cord through the nerves, resulting in the development of lethal encephalitis (Tyler et al. 1986). Infected neurons could be detected by immunohistochemical staining as early as 19 hrs after inoculation (Flamand et al. 1991). The spread of the virus can be prevented by surgical disruption of the nerve between the site of inoculation and the spinal cord. The use of selective inhibitors of the fast and slow components of axonal transport indicate that the neural spread of reovirus serotype 3 occurs by the microtubule-associated system of fast axonal transport (Tyler et al. 1986). Conversely, intramuscular injection of reovirus serotype 1 Lang results in spread to the spinal cord through the bloodstream. Viral antigen is detectable within the endothelium of blood vessels, while only a limited amount of neural spread occurs (Flamand et al. 1991). Spread of serotype 1 is not significantly inhibited by nerve section or by inhibitors of axonal transport. Reassortant studies indicate the S1 gene segment to be an important determinant of viral spread from muscle to the central nervous system (Tyler et al. 1986). Intracerebral inoculation into newborn mice with serotype 3 results in lethal meningoencephalitis, with necrosis and an inflammatory response that is concentrated in the cortex, limbic system, thalamus, basal ganglia, cerebellum, brain stem, and spinal cord (Margolis et al. 1971; Weiner, Greene, et al. 1980; Weiner, Powers, et al. 1980). Incoordination with tremors and paralysis may occur in the mice just before death. Inclusion bodies can be seen in infected neurons and in later stages of infection appear to fill the cytoplasm and dendrites, leaving only the encircled nucleus as an identifiable cellular structure (Margolis et al. 1971). In situ assays have indicated that cells were apoptotic in the areas of the brain in which infected cells and tissue damage were observed (Oberhaus et al. 1997). Different serotype 3 strains differ in their virulence and the peak titer they achieve in the brain (Hrdy et al. 1982). After intracerebral inoculation of serotype 1 Lang or serotype 2 Jones, no significant neuronal injury occurs in newborn mice (Flamand et al. 1991). Instead, reovirus serotype 1 Lang infects ependymal cells lining the ventricles and produces ependymitis with associated hydrocephalus, due to ependymal cell sloughing and obstruction of the aqueduct of Sylvius, and blockage of cerebrospinal fluid outflow from the fourth ventricle (Kilham and Margolis 1969; Margolis and Kilham 1969). Reassortant analysis indicates the M2 gene encoding outer capsid protein µ1 is a major determinant of neurovirulence for serotype 3. Adult, immunocompetent mice are resistant to lethal infection, but adult SCID mice are killed by reovirus. Although neonatal mice infected with reovirus serotype 3 die of meningoencephalitis, adult SCID mice
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contract hepatitis or myocarditis (Sherry et al. 1989; Haller, Barkon, Vogler, et al. 1995). Intramuscular or intracranial inoculation of neonatal mice with reovirus serotype 3 Dearing typically results in titers of 108–109 PFU per brain, while titers in the brains of adult SCID mice are only 103–104 PFU despite the lack of functional immunity in the mice (Haller, Barkon, Vogler, et al. 1995). These findings suggest that innate immunity and tissue maturation may be important determinants of reovirus tropism and pathogenesis. 3.
Cardiorespiratory System
Reoviruses also produce myocarditis in neonatal mice and adult SCID mice following injection in the hindlimb (Sherry et al. 1989; Haller, Barkon, Vogler, et al. 1995). Although the efficiency with which different serotypes and strains induce myocarditis varies, serotype 1 strains generally cause greater viral injury than serotype 3. Myocarditic potential also inversely correlates with the amount of IFN-β induced and the sensitivity of the virus to its action (Sherry et al. 1998). Reovirus-induced myocarditis is characterized by a mild inflammatory infiltrate, with a marked necrosis as evidenced by large, white calcified lesions covering the surface of the heart occurring as early as 5 days after injection (Sherry and Fields 1989). Microscopically, there is muscle fiber necrosis and the myocardial cells appear swollen and fragmented, with a loss of their cross-striations and the development of pyknosis and karyorrhexis. Electron microscopy shows edema of the tubular system and sarcoplasmic reticulum, enlargement of the Golgi apparatus, and an increase in the number of lysosomes (Hassan et al. 1965; Goller et al. 1986). At the extreme stage, there may be extensive areas of coagulative necrosis composed mostly of nuclear and cytoplasmic debris from degenerating cells. The absence of a significant inflammatory response suggests that direct viral injury accounts for the observed disease. This is supported by the development of myocarditis in SCID mice, demonstrating that neither antiviral immunity nor autoimmunity are required for the damage. Sherry et al. (1998) have shown that induction of and sensitivity to IFN-α/β may be determinants of myocarditic potential (Noah et al. 1999). As with other reovirus studies, reassortant viruses have been used to determine the gene segments important for myocarditis. These studies have shown that viral core proteins (M1, L2, and S2 gene products) as well as the cell attachment protein σ1 may contribute to myocarditis induction (Sherry and Fields 1989; Sherry et al. 1998). Studies have also shown that the cysteine protease calpain, which has been implicated in apoptosis, increases in reovirus-infected myocardiocytes, and histopathological evidence of myocardial injury is significantly reduced after treatment with a calpain inhibitor (DeBiasi et al. 2001). Reovirus produces pneumonia in adult rats following intratracheal inoculation. No signs of respiratory disease could be detected by intranasal or intravenous injection (Morin et al. 1994). In the rats inoculated with reovirus serotype 1 Lang or
reovirus serotype 3 Dearing, an influx of leukocytes into alveolar spaces could be seen as early as 3 days after inoculation. There was extensive epithelial damage in the alveoli in response to infection, as indicated by the type II alveolar epithelial cell hyperplasia. Inflammation in the airways was minimal and no hemorrhage was observed. Reovirus serotype 3 infection differed from serotype 1 in that the serotype 3–inoculated rats had a greater neutrophil influx. Subsequent studies have shown virusmediated inflammatory cytokine induction may be responsible for the neutrophil influx into the lungs (Farone et al. 1996). Patchy areas of alveolar hemorrhage have been seen in the lungs of newborn mice inoculated by all three serotypes of reovirus. There is associated pulmonary edema and an inflammatory infiltrate that may take the form of dense aggregates resembling granulomas (Tyler and Fields 1990). 4.
Hepatobiliary System
Infection with all 3 reovirus serotypes can produce hepatobiliary infection in mice following intraperitoneal, peroral, or intravenous inoculation. Reovirus infection of hepatocytes results in cell death and diffuse hepatitis, and reovirus serotype 3 infection of bile duct epithelial cells can result in chronic obstructive jaundice and biliary atresia (Stanley et al. 1953; George et al. 1990; Haller, Barkon, Vogler, et al. 1995; Wilson et al. 1994). Mice may have marked abdominal distension with ascites, and in reovirus serotype 3–infected mice, jaundice may be apparent in the ears, feet, nose, and tail. The hepatic injury induced by all three serotypes is usually visible as small (1–3 mm), depressed, yellow lesions on the surface of the liver. Ultrastructurally, it appears the virus reaches the liver leukocytes and is engulfed by Kupffer cells (Papadimitriou 1965). The virus enters hepatocytes by phagocytosis (Papadimitriou 1968). Although minimal changes are observed before day 4 postinoculation, over the next several days, hepatic necrosis begins in the midzonal and centrilobular regions and then progresses to produce more widespread zones of coagulative necrosis and hemorrhage (Walters et al. 1963; Papadimitriou 1968; Stanley and Joske 1975a, 1975b). By day 7 postinoculation, hepatocytes show a wide range of morphological changes, including eosinophilic degeneration, ballooning, atrophy, and necrosis. Hyperplasia of the Kupffer cells also occurs as the debris from necrotic cells is phagocytized, and the sinusoids may become congested with erythrocytes and debris. By day 14, many of the necrotic cells lyse, although new necrotic foci continue to develop (Walters et al. 1963, 1965; Stanley et al. 1964; Papdimitriou 1965, 1966). Stanley, Leak, Walters, et al. (1964) continued to find necrotic foci at 4 weeks after inoculation in reovirus serotype 3–infected animals. Reovirus serotype 1 Lang, but not reovirus serotype 3 Dearing, was secreted in the bile in concentrations higher than those in the blood following intravenous inoculation (Rubin et al. 1986). In neonatal mice, reovirus serotype 3 strains Dearing and Abney inoculated perorally are associated with the development
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of oily hair effect (OHE), a syndrome linked to replication of the virus in the intrahepatic bile duct epithelium, biliary obstruction, and fat malabsorption, with feces containing as much as 29% fat (Stanley et al. 1953, 1954). In the chronic phase, mice appear wasted with jaundice, OHE, and areas of alopecia, although infectious virus could not be recovered from the tissues during this chronic stage (Stanley, Leak, Walters, et al. 1964). The efficiency of viral replication does not appear to be an important determinant of which viral strains induce OHE (Haller, Barker, Li, et al. 1995). This is in contrast to the fact that hepatitis can occur in adult SCID mice, suggesting that viral injury to the liver, like that to the heart, is a result of viral replication and not inflammation. Mice infected with reovirus serotype 1 strains have also been reported to develop OHE and steatorrhea, although these symptoms are not associated with infection of the liver but may result from injury to the exocrine pancreas (Onodera et al. 1981). In a study by Barton et al. (2003), reovirus serotype 3 strains that differed only in their capacity to bind sialic acid as a coreceptor (T3SA+ and T3SA−) were inoculated perorally into newborn mice. By day 4, the T3SA+ strain titers in the spleen, liver, and brain were approximately 50-fold greater than T3SAtiters. By day 12, the titers of both strains were equivalent. Both strains produced equivalent titers in the intestine at days 4-12 after inoculation. Mice infected with T3SA+ but not T3SA− developed
jaundice, steatorrhea, and oily fur. Liver sections from animals infected with the T3SA+ demonstrate a marked inflammatory response concentrated in the portal areas, while T3SA− animals showed mild inflammatory infiltrates. Immunohistochemical staining revealed viral antigens in bile duct epithelial cells in the T3SA+ animals, whereas viral antigens in mice infected with T3SA− was localized to the hepatocytes (Fig. 9-4). These findings suggest that the sialic-acid binding specificity of reovirus can dramatically alter disease in the host. Reovirus infection of newborn mice shows a remarkable similarity to infantile biliary atresia and biliary disease in liver transplantation patients. Although several studies have attempted to correlate reovirus seropositivity with biliary atresia (Morecki et al. 1982; Dussaix et al. 1984; Brown 1990), the results have been conflicting. However, one study by Tyler et al. (1998) found reovirus RNA in 50%–78% of patients with obstructive liver disease, compared to less than 12% of control samples. 5.
Endocrine System
Reoviruses of all 3 serotypes are capable of pancreatic injury in newborn mice (Onodera et al. 1978; Branski et al. 1980a). There is progressive injury to the acinar cells of the exocrine pancreas, the pancreatic ducts become clogged with debris from the necrotic cells, and there is an inflammatory infiltrate.
A
B
C
D
Fig. 9-4 Reovirus localization in bile duct epithelial cells. ND4 Swiss Webster mice, 2–3 days old, were inoculated perorally with 2.5 × 103 PFU of reovirus either with or without the ability to bind sialic acid (T3SA+ or T3SA−). Six days after inoculation, liver tissue was harvested, embedded in paraffin, thin-sectioned, and stained for reovirus antigen using rabbit anti-reovirus serum and horseradish peroxidase. Dark brown staining indicates reovirus antigen. Liver tissue of mice infected with T3SA− (A and B) showed antigen-positive hepatocytes in the parenchyma but not in the bile duct epithelial cells. Nearly every bile duct cell in mice infected with T3SA+ (C and D) showed strong reovirus antigen staining. Photomicrographs from two separate mice are shown at 400× final magnification. (Photomicrographs are republished from Barton et al. 2003.)
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The salivary glands show a similar pattern of acinar necrosis (Stanley, Leak, Walters, et al. 1964). Pancreatic enzyme activity is also affected, with decreases in amylase and lipase activities and increases in trypsin and chymotrypsin activities (Branski et al. 1980a). Although virus can be seen in the pancreatic acinar cells, Papdimitriou and Walters (1967) concluded that the principal cause of acinar degeneration is ductal obstruction. Both reovirus serotypes 1 (Lang) and 3 (Dearing) can infect and injure the endocrine areas of the pancreas (Onodera et al. 1978). In vivo, the islets of Langerhans show areas of focal, coagulative necrosis and a mononuclear cell infiltrate. The infected mice develop autoantibodies to insulin, and serum insulin levels also drop such that mice develop abnormalities in glucose tolerance. A mild thyroiditis has been described following infection with serotype 1 Lang. Autoantibodies against thyroglobulin and thyroid microcosmal antigens can be detected in the serum (Srinivasappa et al. 1988). Reovirus serotype 1 Lang also infects the growth hormone–producing cells of the anterior pituitary and is associated with decreased circulating growth hormone (Onodera et al. 1981). This may contribute to the “runting” syndrome observed in reovirus-infected mice (Stanley and Leak 1963a). 6.
Immune Response
Both natural and experimental infections with reoviruses result in the induction of antibody-mediated (humoral) and cellmediated immune responses. In normal adult mice infected with serotype 1 (Lang), clearance of the mucosal infection occurs within about 10 days (Major and Cuff 1996). Type-specific and non-type-specific H-2d-restricted cytotoxic T lymphocytes appear within the first week of intraperitoneal, peroral, or intranasal infection and persist for more than 3 months (Thompson et al. 1996; Virgin et al. 1997). Many different epitopes on reovirus structural and nonstructural proteins are recognized by the cytotoxic T lymphocytes (London et al. 1989; Hogan et al. 1991; Hoffman et al. 1996). Peroral infection with reoviruses results in increases in virus-specific CD4+ and CD8+ T lymphocytes. The cytokine profiles of the reovirus-specific CD4+ helper T (TH) cells indicate that the TH1 responses are greater than the TH2, with an associated increase in IFN-γ (Fan et al. 1998). Similar results have been found in experimental reovirus serotype 2–induced diabetes (Hayashi et al. 1998). Virus-specific CD8+ cytotoxic T lymphocytes generated within the intraepithelial lymphocyte population express the α/β T cell receptor and are capable of major histocompatibility class I–restricted lysis of virus-infected target cells (London et al. 1987; Chen et al. 1997). Fulton et al. (2004) have found that the route of infection can influence selection or expansion of virus-specific CD8+ T cells. The antibody-mediated response in mice depends on the route of reovirus inoculation and the H-2 haplotype of the infected animals (Major and Cuff 1996). Serum antibody titers are comparable after peroral and intradermal inoculation of reovirus serotype 1 (Lang), with both routes of infection resulting in IgG2a and IgG2b (Major and Cuff 1996). Intradermal infection also
resulted in high levels of IgG1. These findings indicate that oral reovirus infections induce predominant TH1 responses and that parenteral responses may be mixed TH1 and TH2. After peroral inoculation, mice develop an IgA response in Peyer’s patches, while IgG2a is predominant in the peripheral lymph nodes following intradermal inoculation (Major and Cuff 1996). Differential cytokine induction in the tissues after oral or parenteral infection may be responsible for differences in the immunoglobulin classes (Mathers and Cuff 2004). Oral inoculation of neonatal mice with reovirus also induces IgA in the intestinal tract, although this response is inhibited by maternal antireovirus antibodies through suckling (Periwal et al. 1997). Monoclonal antibodies of the IgA isotype directed against the cell attachment protein σ1 of reovirus serotype 1 Lang prevented Peyer’s patch infection in adult mice, whereas monoclonal IgA antibodies to the other viral proteins did not (Hutchings et al. 2004). Intestinal reovirus infection has also been studied in SCID mice deficient in all antigen-specific lymphocytes and in mice lacking CD8+ T cells or B cells (George et al. 1990; Taterka et al. 1995; Barkon et al. 1996; Major and Cuff 1997). Although adult immunocompetent mice are not susceptible to reovirus disease, adult SCID mice inoculated orally with reovirus develop lethal infection (George et al. 1990). In these studies, passive transfer of reovirus-specific immune or normal lymphocytes protected SCID mice against reovirus infection. Mice lacking CD8+ T cells clear infection normally, whereas mice lacking B cells and antibody have delayed clearance (Barkon et al. 1996; Major and Cuff 1997). Neonatal mice depleted of CD4+ cells, CD8+ cells, or both show enhanced pathology in multiple tissues (Virgin and Tyler 1991). These studies indicate the importance of both antigen-specific lymphocytes and antibody in intestinal infection and clearance of reovirus. Both polyclonal antisera and monoclonal antibodies against protein σ1 (neutralizing and non-neutralizing) can protect mice against lethal challenge with reovirus (Virgin et al. 1988; Tyler et al. 1989, 1993). Monoclonal antibodies against each of the other outer capsid proteins are also protective (Tyler et al. 1993). Studies on the effects of protective monoclonal antibodies on pathogenesis of reoviruses indicate that antibody-mediated protection inhibited the neural spread of reovirus serotype 3 without significantly inhibiting replication of virus at the inoculation site (Tyler et al. 1989, 1993). Antibody can also protect against reovirus serotype 1 Lang–induced hydrocephalus and serotype 3–induced myocarditis (Sherry et al. 1993; Tyler et al. 1993). Antibodies have been used prophylactically to prevent disease and as treatment for established nervous system disease (Virgin et al. 1988). The adoptive transfer of immune spleen cells protects neonatal mice from lethal infection with either perorally or intramuscularly administered reovirus serotype 3 (strains clone 9 and Dearing, respectively) (Virgin and Tyler 1991). Both CD4+ and CD8+ cells were required for maximal protection. The adoptive transfer of Peyer’s patches was also protective for adult SCID and neonatal mice (George et al. 1990;
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Cuff et al. 1991). The immune cells must be given either before or within 28 h after viral inoculation and fail to protect when reovirus is given intracerebrally. Reoviruses can induce IFNs, tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein-2 (MIP-2) (Henderson and Joklik 1978; Farone et al. 1993, 1996). They also increase cytokine mRNA expression for interleukins 4, 10, and 12 (Mathers and Cuff 1996). Reovirus serotype 3 Dearing induced higher levels of mRNA for TNF-α and MIP-2 than serotype 1 Lang in alveolar macrophages from infected rats (Farone et al. 1996). Although reoviruses are susceptible to IFN-β, strains differ widely in their sensitivity, with reovirus serotype 3 being more sensitive than serotype 1 (Jacobs and Ferguson 1991). Reovirus has also been shown to induce autoimmunity or immunosuppression in mice, which may result from functional alterations in T cell populations (Hauser et al. 1987; Onodera et al. 1991). Infection of mice with reovirus serotype 1 Lang also results in the induction of autoantibodies against a variety of endocrine tissues and hormones (Onodera et al. 1981; Onodera and Awaya 1990).
For mice, there does not appear to be any evidence that any mouse strain is more sensitive to infection with reovirus than others, with the exception of SCID or other immunodeficient mice. The acute disease does affect mainly neonatal and weanling mice, with chronic disease occurring rarely in mice over 28 days old. There is no indication that either sex is more or less susceptible to infection, although one study found that the incidence of complement-fixing antibodies was greater in females than males in a colony (Parker et al. 1976). This difference may be attributed to continual exposure of females to infected litters. In a study by Barthold et al. (1993), oronasal infection of mice with the 3 serotypes revealed that even weanling mice were comparatively resistant to infection. Uniform transmission of the virus to cage mates or mothers of infants did not occur, indicating low contagiousness of all three serotypes following oronasal inoculation. Infection of neonates may be influenced by birth order. In one study, 130 of 800 first litters were affected, whereas laterpariety litters were rarely infected (Cook 1963). While it is possible that maternal antibodies may play a role in this protective response, these studies were not performed.
G. F.
Diagnosis
Epizootiology
The host range of mammalian reoviruses has been discussed previously in the history of reoviruses. The virus has been found in over 60 species, and antibodies to reoviruses have been found in all tested mammals (Stanley, Leak, Grieve, et al. 1964; Stanley 1974). The virus is ubiquitous in nature and is found in river water, standing water, and untreated sewage. There does not appear to be a seasonality to transmission of reoviruses (Rosen et al. 1960; Matsuura et al. 1988), although one study obtained the most reovirus isolates in the winter months (Gelfland 1959) and most dairy cattle infections have been observed in the fall and winter months (Rosen and Abianti 1963). Serological prevalence studies indicate that by the time children reach 5 years of age, more than 70% have been exposed to the reoviruses (Lerner et al. 1947; Leers and Royce 1956). Serological testing has also indicated that as many as 13% of mouse colonies may be infected at any given time (Kraft and Meyer 1986). Reoviruses and or reovirus antibodies have been isolated from both humans and animals in North and South America, Europe, Asia, Africa, and Australia (Rosen 1962; Stanley and Leak 1963b). Because reovirus isolates from different species, including humans, cannot be distinguished from one another and strains originally isolated from humans can infect mice, cross-species transmission is indicated, although direct transmission from one species to another in nature has not been demonstrated. Because reoviruses are shed in the feces, the fecal-oral route of transmission is considered the primary route of spread. Although reoviruses have been found in nasal and conjunctival secretions of felines infected with reovirus, there are no reports on the transmission of reovirus by the respiratory route in mice.
The observation of OHE in mice, although not pathognomic for reovirus, may be indicative of infection and the need for further diagnosis. Sentinel animals in a mouse colony should be considered for diagnostic purposes, as infections could alter tissue or immune response studies. Diagnosis of mammalian reovirus infections is based on isolation of virus from tissues or body fluids, detection of viral antigen in the infected material, or serological identification (Tyler and Fields 1988). For isolation of reovirus from specimens, monkey kidney cells have been used (Stanley 1974). Early diagnostic procedures often employed electron microscopy of tissue specimens (Spence et al. 1977); however, the availability of polyclonal and monoclonal antibodies now makes immunochemistry more feasible. Once isolated in culture, the presence of viral antigen can be confirmed by immunofluorescence or immunocytochemical staining. These techniques can also be used for the detection of viral antigen in tissues or serum (Bangaru et al. 1980; Morecki et al. 1982; Glaser et al. 1984). Determination of serotype or strain of a viral isolate can also be based upon the capacity of a panel of type-specific antisera to inhibit viral hemagglutination by the isolate (Rosen 1979). ELISA techniques have been used for reovirus antibody detection in serum (Kraft and Meyer 1986; Richardson et al. 1988; Selb and Weber 1994). Although serum has been used for hemagglutination inhibition and indirect immunofluorescence tests, ELISA technology is currently used by most commercial diagnostic laboratories. Because reovirus serotype 3 strains most commonly affect mouse colonies, some commercial laboratories may use only reovirus serotype 3 antigen for their assays. However, diagnostic testing should involve antigens for all
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3 reovirus serotypes to assess the possibility that serotype 1 or 2 may be infecting the mice. This is especially important as these serotypes generally cause less morbidity and mortality in neonatal mice and, thus, infections initially may be inapparent (Barthold et al. 1993). The availability of nucleotide sequences for the RNA segments of all three serotypes can also provide the opportunity for molecular-based diagnostic techniques such as in situ hybridization or polymerase chain reaction (PCR), specifically, real-time PCR. A diagnostic procedure using fluorgenic nuclease reverse transcriptase PCR has already been developed for reoviruses. This assay combines reverse transcriptase PCR with an internal fluoregenic probe. Using reovirus serotype 3–specific probes, this assay has successfully detected both the Dearing and Abney strains of reovirus serotype 3 in tissues of mice experimentally infected with reovirus, but did not detect serotypes 1 or 2 or other viruses in the family Reoviridae (Uchiyama and Besselsen 2003). H.
Control and Prevention
These viruses are stable infectious agents that survive for long periods of time at and below room temperature. Because they lack a lipid envelope, they are resistant to lipid solvents and to many disinfectants, including 1% hydrogen peroxide, 1% phenol, and 3% formaldehyde (Stanley et al. 1953; Drayna and Fields 1982a, 1982b). Ionic and nonionic detergents are also ineffective at completely inactivating the virus (Ward and Ashley 1977, 1978). Sequential application of 95% ethanol and sodium hypochlorite (800 ppm) is the recommended disinfection procedure. Several antiviral compounds have been effective at preventing virus infection in tissue culture, although these have not been studied extensively in vivo. In mouse colonies, cesarean delivery and barrier maintenance have been used for the control and prevention of reovirus infections. REFERENCES Abinanti, F. R. (1963). Respiratory disease of cattle and observations of reovirus infections in cattle. Am. Rev. Respir. Dis. 88, 290–304. Adams, D. J., Spendlove, J. C., Spendlove, R. S., and Barnett, B. B. (1982). Aerosol stability of infectious and potentially infectious reovirus particles. Appl. Environ. Microbiol. 44, 903–908. Adams, W. R., and Kraft, L. M. (1963). Epidemic diarrhea of infant mice. Identification of the etiologic agent. Science 141, 359–360. — — —. (1967). Electron microscopic study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus). Am. J. Pathol. 51, 39–60. Ahmed, R., Chakraborty, P. R., Graham, A. F., Ramig, R. F., and Fields, B. N. (1980). Genetic variation during persistent reovirus infection: presence of extragenically suppressed temperature-sensitive lesions in wild-type virus isolated from persistently infected L cells. J. Virol. 34, 383–389. Ahmed R., and Fields B. N. (1981). Reassortment of genome segments between reovirus defective interfering particles and infectious virus: construction of temperature-sensitive and attentuated viruses by rescue of mutations from DI particles. Virology 111, 351–363.
— — —. (1982). Role of the S4 gene in the establishment of persistent reovirus infection in L cells. Cell 28, 605–612. — — —. (1977). Persistent infections in L cells with temperature-sensitive mutants of reovirus. J. Virol. 23, 250–262. Ahmed R., Kauffman, R. S., and Fields, B. N. (1983). Genetic variation during persistent reovirus infection: isolation of cold-sensitive and temperaturesensitive mutants from persistently infected L cells. Virology 131, 71–78. Amerogen, H. M., Wilson, G. A., Fields, B. N., and Neutra, M. R. (1994). Proteolytic processing of reovirus is required for adherence to intestinal M cells. J. Virol. 68, 8428–8432. Angel, J., Tang, B., Feng, N., Greenburg, H. B., and Bass, D. (1998). Studies of the role for NSP4 in the pathogenesis of homologous murine rotavirus diarrhea. J. Infect. Dis. 177, 455–458. Aponte, C., Poncet, D., and Cohen, J. (1996). Recovery and characterization of a replicase complex in rotavirus-infected cells by using a monoclonal antibody against NSP2. J. Virol. 70, 985–991. Ball, J. M., Tian, P., Zeng, C. Q.-Y., Morris, A. P., and Estes, M. K. (1996). Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272, 101–104. Banerjea, A. C., Brechling, K. A., Ray, C. A., Erikson, H., Pickup D. J., and Joklik, W.K. (1988). High-level synthesis of biologically active reovirus protein σ1 in a mammalian expression vector system. Virology 67, 601–612. Bangaru, B., Morecki, R., Glaser, J. H., Gartner, L. M., and Horwitz, M. S. (1980). Comparative studies of biliary atresia in human newborn and reovirusinduced cholangitis in weanling mice. Lab. Invest. 43, 456–462. Barkon, M., Haller, B. L., and Virgin, H. W., IV. (1996). Circulating immunoglobulin G can play a critical role in clearance of intestinal reovirus infection. J. Virol. 70, 1109–1116. Barthold, S. W., Smith, A. L., and Bhatt, P. N. (1993). Infectivity, disease patterns, and serologic profiles of reovirus serotypes 1, 2, and 3 in infant and weanling mice. Lab. Anim. Sci. 43, 425–430. Barton, E. S., Forrest, J. C., Connolly, J. L., et al. (2001). Junction adhesion molecule is the receptor for reovirus. Cell 104, 441–451. Barton, E. S., Youree, B. E., Ebert, D. H., et al. (2003). Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease. J. Clin. Invest. 111, 1823–1833. Bass, D. M., Trier, J. S., Dambrauskas, R., and Wolf, J. L. (1988). Reovirus type 1 infection of small intestinal epithelium in suckling mice and its effect on M cells. Lab. Invest. 58, 226–235. Belak, S., and Palfi, V. (1974). Isolation of reovirus type 1 from lambs showing respiratory and intestinal symptoms. Arch. Gesamte. Virusforsch. 44, 177–183. Bernstein, D. I., Glass, R. I., Rodgers, G., Davidson, B. L., and Sack, D. A. (1995). Evaluation of rhesus rotavirus monovalent and tetravalent reassortant vaccines in U.S. children. J. Amer. Med. Asso. 273, 1191–1196. Bernstein, D. I., Sack, D. A., Rothstein, E., et al. (1999). Efficacy of live, attenuated, human rotavirus vaccine 89–12 in infants: a randomized placebocontrolled trial. Lancet 354, 287–290. Bishop, R. F., Davidson, G. P., Holmes, I. H., and Ruck, B. J. (1973). Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet 2, 1281–1283. Blutt, S. E., Kirkwood, C. D., Parreno, V., et al. (2003). Rotavirus antigenaemia and viraemia: a common event? Lancet 362, 1445–1449. Bodkin, D. K., and Fields, B. N. (1989). Growth and survival of reovirus in intestinal tissue: role of the L2 and S1 genes. J. Virol. 63, 1188–1193. Borsa, J., Morash, B. D., Sargent, M. D., Copps, T. P., Lievaart, F. A., and Szekely, J. G. (1979). Two modes of entry of reovirus particles into L cells. J. Gen. Virol. 45, 161–170. Boshuizen, J. A., Reimerink, J. H. J., Korteland-van Male, A. M., et al. (2003). Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice. J. Virol. 77, 13005–13016. Branksi, D., Lebenthal, E., Faden, H. S., Hatch, T. F., and Krasner, J. (1980a). Reovirus type 3 infection in a suckling mouse: the effects on pancreatic structure and enzyme content. Pediatr. Res. 14, 8–11. — — —. (l980b). Small intestinal epithelial brush border enzymatic changes in suckling mice infected with reovirus type 3. Pediatr. Res. 14, 803–805.
9. REOVIRIDAE
Bridger, J. C. (1980). Detection by electrol microscopy of caliciviruses, astroviruses and rotavirus-like particles in the faeces of piglets with diarrhoea. Vet. Rec. 107, 532. Bridger, J. C., Burke, B., Beards, G. M., and Desselberger, U. (1992). The pathogenicity of two porcine rotaviruses differing in their vitro growth characteristics and gene 4. J. Gen. Virol. 73, 3011–3015. Bridger, J. C., Dhaliwal, W., Adamson, M. J. V., and Howard, C. R. (1998). Determinants of rotavirus host range restriction—a heterologous bovine NSP1 gene does not affect replication kinetics in the pig. Virology 245, 47–52. Bridger, J. C., Tauscher, G. I., and Desselberger, U. (1998). Viral determinants of rotavirus pathogenicity in pigs: evidence that the fourth gene of a porcine rotavirus confers diarrhea in the homologous host. J. Virol. 72, 6929–6931. Broome, R. L., Vo, P. T., Ward, R. L., Clark, H. F., and Greenberg, H. B. (1993). Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restrictions and virulence. J. Virol. 67, 2448–2455. Brown, E. G., Nibert, M. L., and Fields, B. N. (1983). The L2 gene of reovirus serotype 3 controls the capacity to interfere, accumulate deletions and establish persistent infection. In Double-stranded RNA viruses, R. W. Compans and D. H. L. Bishop, eds., pp. 275–287. Elsevier, New York. Brown, W. R. (1990). Lack of conformation of the association of reovirus 3 and biliary atresia: methodological differences. Hepatology 12, 1254–1255. Browne, E. P., Bellamy, A. R., and Taylor, J. A. (2000). Membrane-destabilizing activity of rotavirus NSP4 is mediated by a membrane-proximal amphipathic domain. J. Gen. Virol. 81, 1955–1959. Brunet, J.-P., Jourdan, N., Cotte-Lafitte, J., et al. (2000). Rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration. J. Virol. 74, 10801–10806. Bryson, J. S., and Cox, D. C. (1986). Characteristics of reovirus-mediated chemoimmunotherapy of murine L1210 leukemia. Cancer Immunol. Immunother. 26, 132–138. Burke, B., McCrae, M. A., and Desselberger, U. (1994). Sequence analysis of two porcine rotaviruses differing in growth in vitro and in pathogenicity: distinct VP4 sequences and conservation of NS53, VP6 and VP7 genes. J. Gen. Virol. 75, 2205–2212. Burns, J. W., Krishnaney, A. A., Vo, P. T., Rouse, R. V., Anderson, L. J., and Greenburg, H. B. (1995). Analyses of homologous rotavirus infection in the mouse model. Virology 207, 143–153. Burns, J. W., Siadat-Pajouh, M., Krishnaney, A. A., and Greenberg, H. B. (1996). Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity. Science 272, 104–107. Burstin, S. J., Spriggs, D. R., and Fields, B. N. (1982). Evidence for functional domains on the reovirus type 3 hemagglutinin. Virology 117, 146–155. Cashdollar, L. W., Chmelo, R. A., Wiener, J. R., and Joklik, W. K. (1985). Sequences of the S1 genes of the three serotypes of reovirus. Proc. Natl. Acad. Sci. USA 82, 24–28. Chappell, J. D., Barton, E. S., Smith, T. H., et al. (1998). Cleavage susceptibility of reovirus attachment protein sigma 1 during proteolytic disassembly of virions is determined by a sequence polymorphism in the sigma l neck. J. Virol. 72, 8205–8213. Chappell, J. D., Duong, J. L., Wright, B. W., and Dermondy, T. S. (2000). Identification of carbohydrate binding domains in the attachment protein of type 1 and type 3 reoviruses. J. Virol. 74, 8472–8479. Chappell, J. D., Goral, M. I., Rodgers, S. E., dePamphilis, C. W., and Dermody, T. S. (1994). Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature and their termini are predicted to form a panhandle motif. J. Virol. 68, 750–756. Chappell, J. D., Gunn, V. L., Wetzel, J. D., Baer, G. S., and Dermody, T. S. (1997). Mutations in type 3 reovirus domain of the viral attachment protein σ1. J. Virol. 71, 1834–1841. Chastel, C., Quillien, M. C., Couatarmanach, A., LeLay, G., and Beaucournu, J. C. (1985). Reovirus type 1 and small wild mammals in a mixed focus of virus infections in France. Acta Virol. (Praha) 29, 318–323.
259 Cheever, F. S., and Mueller, J. H. (1947). Epidemic diarrheal disease of suckling mice. I. Manifestations, epidemiology, and attempts to transmit the disease. J. Exp. Med. 85, 405–416. Chen, D., Lee, F., Cebra, J. J., and Rubin, D. H. (1997). Predominant T-cell receptor Vbeta usage of intraepithelial lymphocytes during the immune response to enteric reovirus infection. J. Virol. 71, 3431–3436. Chiba, S., Yokoyama, T., Nakata, S., et al. (1986). Protective effect of naturally acquired homotypic and heterotypic rotavirus antibodies. Lancet 2, 417–421. Choi, A. H. C., Basu, M., McNeal, M. M., Clements, J. D., and Ward, R. L. (1999). Antibody independent protection against rotavirus infection of mice stimulated by intranasal immunization with chimeric VP4 or VP6 protein. J. Virol. 73, 7574–7581. Choi, A. H. C., Basu, M., McNeal, M. M., et al. (2000). Functional mapping of protective domains and epitopes in the rotavirus VP6 protein. J. Virol. 74, 11574–11580. Chrystie, I. L., Totterdell, B. M., and Banatvala, J. E. (1983). False positive rotazyme tests on faecal samples from babies. Lancet 2, 1028. Ciarlet, M., Crawford, S. E., Barone, C., Bertolotti-Ciarlet, A., Estes, M. K., and Conner, M. E. (1998). Subunit rotavirus vaccine administered parenterally to rabbits induces active protective immunity. J. Virol. 72, 9233–9246. Ciarlet, M., Estes, M. K., Barone, C., Ramig, R. F., and Conner, M. E. (1998). Analysis of host range restriction determinants in the rabbit model: comparison of homologous and heterologous rotavirus infections. J. Virol. 72, 2341–2351. Clark, H. F., Borian, F. E., Bell, L. M., Modesto, K., Gouvea, V., and Plotkin, S. A. (1988). Protective effect of WC3 vaccine against rotavirus diarrhea in infants during a predominantly serotype 1 rotavirus season. J. Infect. Dis. 158, 570–587. Clarke, P., Meintzer, S. M., Gibson, S., et al. (2000). Reovirus-induced apoptosis is mediated by TRAIL. J. Virol. 74, 8135–8139. Coffin, S. E., Clark, S. L., Bos, N. A., Brubaker, J. O., and Offit, P. A. (1999). Migration of antigen-presenting B cells from peripheral to mucosal lymphoid tissues may induce intestinal antigen-specific IgA following parental immunization. J. Immunol. 163, 3064–3070. Collins, J., Starkey, W. G., Wallis, T. S., et al. (1988). Intestinal enzyme profiles in normal and rotavirus-infected mice. J. Pediatr. Gastroenterol. Nutr. 7, 272. Connolly, J. L., and Dermody, T. S. (2002). Virion disassembly is required for apoptosis induced by reovirus. J. Virol. 76, 1632–1641. Connolly, J. L., Rodgers, S. E., Clarke, P., et al. (2000). Reovirus-induced apoptosis requires activation of transcription factor NF-kappaB. J. Virol. 74, 2981–2989. Cook, I. (1963). Reovirus type 3 infection in laboratory mice. Aust. J. Exp. Biol. 41, 651–660. Coombs, K. M. (1996). Identification and characterization of a double-stranded RNA reovirus temperature-sensitive mutant defective in minor core protein µ2. J. Virol. 70, 4237–4245. — — —. (1998). Stoichiometry of reovirus structural proteins in virus, ISVP, and core particles. Virology 243, 218–228. Coste, A., Sirard, J. C., Johansen, K., Cohen, J., and Kraehenbuhl, J. P. (2000). Nasal immunization of mice with virus-like particles protects offspring against rotavirus diarrhea. J. Virol. 74, 8966–8971. Cuadras, M. A., Arias, C. F., and Lopez, S. (1997). Rotaviruses induce an early membrane permeabilization of MA104 cells and do not require a low intracellular Ca2+ concentration to initiate their replication cycle. J. Virol. 71, 9065–9074. Cuff, C. F., Cebra, C. K., Lavi, E., Molowitz, E. H., Rubin, D. H., and Cebra, J. J. (1991). Protection of neonatal mice from fatal reovirus infection by immune serum and gut derived lymphocytes. Adv. Exp. Med. Biol. 310, 307–315. Dales, S. (1962). Association between the spindle apparatus and reovirus. Proc. Natl. Acad. Sci. USA 50, 268–275. Danis, C., Mabrouk, T., Faure, M., and Lemay, G. (1997). Interferon has no protective effect during acute or persistent reovirus infection of SCI fibroblasts. Virus Res. 51, 139–149. Danis, C., Mabrouk, T., Garzon, S., and Lemay, G. (1993). Establishment of persistent reovirus infection in SCI cells: absence of protein synthesis and
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Strong, J. E., Tang, D., and Lee, P. W. K. (1993). Evidence that the epidermal growth factor receptor on host cells confers reovirus infection efficiency. Virology 197, 405–411. Sturm, R. T., Lang, G. H., and Mitchell, W. R. (1980). Prevalence of reovirus 1, 2 and 3 antibodies in Ontario racehorses. Can. Vet. J. 21, 206–209. Suzuki, H., Kitaoka, S., Konno, T., Sato, T., and Ishida, N. (1985). Two modes of human rotavirus entry into MA104 cells. Arch. Virol. 85, 25–34. Taber, R., Alexander, V., and Whitford, W. (1976). Persistent reovirus infection of CHO cells resulting in virus resistance. J. Virol. 17, 513–524. Taniguchi, K., Maloy, W., Nishikawa, K., et al. (1988). Identification of crossreactive and serotype 2 specific neutralization epitopes on VP3 of human rotavirus. J. Virol. 62, 2421–2426. Taterka, J., Cebra, J. J., and Rubin, D. H. (1995). Characterization of cytotoxic cells from reovirus-infected SClD mice: activated cells express natural killerand lymphokine-activated killer-like activity but fail to clear infection. J. Virol. 69, 3910–3914. Theil, K. W., Bohl, E. H., Cross, R. F., Kohler, E. M., and Agnes, A. G. (1978). Pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs. Am J. Vet. Res. 39, 213–220. Thein, P., and Scheid, R. (1981). Reoviral infections. In Viral zoonoses, CRC handbook series in zoonoses, J. H. Steele, ed., vol. 2, pp. 191–216. CRC Press, Boca Raton, Florida. Thompson, A. H., London, L., Bellum, S. C., Hamamdzic, D., Harley, R. A., and London, S. D. (1996). Respiratory-mucosal lymphocyte populations induced by reovirus serotype 1 infection. Cell Immunol. 169, 278–287. Tian, P., Ball, J. M., Zeng, C. Q. Y., and Estes, M. K. (1996). The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity. J. Virol. 70, 6973–6981. Tian, P., Estes, M. K., Hu, Y., Ball, J. M., Zeng, G. G., and Schilling, W. P. (1995). The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum. J. Virol. 69, 5763–5772. Tillotson, J. R., and Lerner, A. M. (1967). Reovirus type 3 associated with fatal pneumonia. N. Engl. J. Med. 276, 1060–1063. Tosteson, M. T., Nibert, M. L., and Fields, B. N. (1993). Ion channels induced in lipid bilayers by subvirion particles of the nonenveloped mammalian reoviruses. Proc. Natl. Acad. Sci. USA 90, 10549–10552. Troonen, H. (1984). False positive rotazyme results. Lancet 1, 345. Tyler, K. L., and Fields, B. N. (1988). Laboratory diagnosis of infectious disease: principles and practice. In Reoviridae: the reoviruses, viral, rickettsial and chlamydial diseases, E. H. Lennette, P. Halonen, and F. A. Murphy, eds., vol. 2, pp. 353–374. Springer-Verlag, NewYork. — — —. (1990). Reoviruses. In Fields virology, B. N. Fields and D. M. Knipe, eds., pp. 1307–1328. Tyler, K. L., Mann, M. A., Fields, B. N., and Virgin, H. W. (1993). Protective anti-reovirus monoclonal antibodies and their effects on viral pathogenesis. J. Virol. 67, 3446–3453. Tyler, K. L., McPhee, D. A., and Fields, B. N. (1986). Distinct pathways of viral spread in the host determined by reovirus Sl gene segment. Science 233, 770–774. Tyler, K. L., Sekel, R. J., Oberhaus, S. M., et al. (1998). Detection of reovirus RNA in hepatobiliary tissues from patients with extrahepatic biliary atresia and choledochal cysts. Hepatology 27, 1475–1482. Tyler, K. L., Virgin, H. W., Bassel-Duby, R., and Fields, B. N. (1989). Antibody inhibits defined stages in the pathogenesis of reovirus serotype 3 infection of the central nervous system. J. Exp. Med. 170, 887–900. Tzipori, S., Unicomb, L., Bishop, R., Montenaro, J., and Vaelioja, L.M. (1989). Studies on attenuation of rotavirus: a comparison in piglets between virulent virus and its attenuated derivative. Arch. Virol. 109, 197–205. Uchiyama, A., and Besselsen, D. G. (2003). Detection of reovirus type 3 by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction. Laboratory Animals 37, 352–359. Uhnoo, I., Riepenhoff-Talty, M., Dharakul, T., et al. (1990). Extramucosal spread and development of hepatitis in immunodeficient and normal mice infected with rhesus rotavirus. J. Virol. 64, 361–368.
Varshney, K. C., Bridger, J. C., Parson, K. R., Cook, R., Teucher, J. and Hall, G. A. (1995). The lesions of rotavirus infection in 1- and 10-day-old gnotobiotic calves. Vet. Pathol. 32, 619–627. Verdin, E. M., Maratos-Flier, E., Carpentier, J. L., and Kahn, C. R. (1986). Persistent infection with a nontransforming RNA virus leads to impaired growth factor receptors and response. J. Cell. Physiol. 128, 457–465. Vesikari, T., Isolauri, E., Delem, A., et al. (1985). Clinical efficacy of the RIT 4237 live attenuated bovine rotavirus vaccine in infants vaccinated before a rotavirus epidemic. J. Pediatr. 107, 189–194. Vieler, E., Baumgartner, W., Herbst, W., and Kohler, G. (1994). Characterization of a reovirus isolate from a rattlesnake, Crotalus viridis, with neurological dysfunction. Arch. Virol. 138, 341–344. Virgin, H. W., IV, Bassel-Duby, R., Fields, B. N., and Tyler, K. L. (1998). Antibody protects against lethal infection with the neurally spreading reovirus type 3 (Dearing). J. Virol. 62, 4594–4604. Virgin, H. W., IV, Mann, M. A., Fields, B. N., and Tyler, K.L. (1991). Monoclonal antibodies to reovirus reveal structure/function relationships between capsid proteins and genetics of susceptibility to antibody action. J. Virol. 65, 6772–6781. Virgin, H. W., IV, Mann, M. A., and Tyler, K. L. (1994). Protective antibodies inhibit reovirus internalization and uncoating by intracellular proteases. J. Virol. 68, 6719–6729. Virgin, H. W., IV, and Tyler, K. L. (1991). The role of immune cells in protection against and control of reovirus infection in neonatal mice. J. Virol. 65, 5157–5164. Virgin, H. W., IV, Tyler, K. L., and Dermody, T. S. (1997). Reovirus. In Viral pathogenesis, N. Nathanson, ed., pp. 669–699. Lippincott-Raven, Philadelphia. Wallis, C., Smith, K. O., and Melnick, J. L. (1964). Reovirus activation by heating and inactivation by cooling in MgCl2 solutions. Virology 22, 608–619. Walters, M. N., Joske, R. A., Leak, P. J., and Stanley, N. F. (1963). Murine infection with reovirus: I. Pathology of the acute phase. Br. J. Exp. Pathol. 44, 427–436. Walters, M. N., Leak, P. J., Joske, R. A., Stanley, N. F., and Perret, D. H. (1965). Murine infection with reovirus: III. Pathology of infection with types I and II. Br. J. Exp. Pathol. 46, 200–212. Ward, J. M., Collins, M. J., Jr., and Parker, J. C. (1977). Naturally occurring mouse hepatitis virus infection in the nude mouse. Lab. Anim. Sci. 27, 372–376. Ward, R. L., and Ashley, C. S. (1977). Discovery of an agent in wastewater sludge that reduces the heat required to inactivate reovirus. Appl. Environ. Microbiol. 34, 681–688. — — —. (1980). Effects of wastewater sludge and its detergents on the stability of rotavirus. Appl. Environ. Microbiol. 39, 1154–1158. — — —. (1978). Identification of detergents as components of wastewater sludge that modify the thermal stability of reovirus and enteroviruses. Appl. Environ. Microbiol. 36, 889–897. Ward, R. L., Clemens, J. D., Knowlton, D. R., et al. (1992). Evidence that protection against rotavirus diarrhea after natural infection is not dependent on serotype-specific neutralizing antibody. J. Infect. Dis. 166, 1251–1257. Ward, R., Knowlton, D., Schiff, G. M., Hoshino, Y., and Greenberg, H. B. (1988). Relative concentrations of serum neutralizing antibody to VP3 and VP7 proteins in adult infected with a human rotavirus. J. Virol. 62, 1543–1549. Ward, R.L., Dinsmore, A.M., Goldberg, G., Sander, D.S., Rappaport, R.S., and Zito, E.T. (1998). Shedding of rotavirus following administration of the tetravalent rhesus rotavirus vaccine. Pediatr. Infect. Dis. J. 17, 386–390. Ward, R. L., Knowlton, D. R., Zito, E. T., Davidson, B. L., Rappaport, R., and Mack, M. E. for the United States Rotavirus Efficacy Group. (1997). Serological correlates of immunity in a tetravalent reassortant rotavirus vaccine trial. J. Infect. Dis. 176, 570–577. Ward, R. L., Mason, B. B., Bernstein, D. I., et al. (1997). Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene. J. Virol. 71, 6267–6270. Ward, R. L., McNeal, M. M., Sander, D. S., Greenberg, H. B., and Bernstein, D. I. (1993). Immunodominance of the VP4 neutralization protein of rotavirus in protective natural infections of young children. J. Virol. 67, 464–468.
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Chapter 10 Retroelements in the Mouse Herbert C. Morse III
I. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Retroviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. MuLV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mouse Mammary Tumor Viruses (MMTV) . . . . . . . . . . . . . . . . . . . . . C. Intracisternal A Particles (IAP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. VL30 Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Early Transposon (ETn)-Related Elements . . . . . . . . . . . . . . . . . . . . . F. Mammalian Apparent LTR-Retrotransposons (MaLR) . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
OVERVIEW
The inbred mouse has become a progressively more powerful tool for understanding aspects of biology too numerous to list. The ability of investigators to create new strains through innovative breeding schemes involving conventional inbred strains and the ability to manipulate the genome to generate transgenic, knockout, and knock-in mice as well as variants induced by ENU mutagenesis and other approaches has grown exponentially. This has led to an explosive growth in the numbers of strains, including many with complex genetic backgrounds. Infections of the mice or cells from them with replicationdefective murine leukemia viruses (MuLV) containing genes of interest is a widely used approach for enriching these studies, adding yet another layer of complexity. Ideally, then, those involved in studying the normative biology and pathobiology of these mice will be well versed in natural history of strains contributing to the crosses. This should include an understanding of the role played by endogenous retroelements in THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
269 270 271 274 276 276 276 276 276
the occurrence of different neoplasms, their effects on the immune system, and their potential for inducing somatic and germline mutations. The scope of this information has been amplified many fold by the sequencing of the mouse genome (Waterson et al. 2002). Using much of this data, now well in hand, this chapter provides a current perspective on the nature of retroelements in the germline of many inbred strains and some wild-caught mice and their variations among inbred strains, as well as factors influencing their spread and effects. The role of MuLV in development of spontaneous lymphomas and leukemias has recently been reviewed for the characteristics of these neoplasms (Kogan et al. 2002; Morse et al. 2002) and will not be covered in this chapter. Retroelements comprise a remarkably rich collection of molecular species that have as a unifying theme the copying of RNA into DNA as part of the life cycle. The critical element in this process is the protein, reverse transcriptase (RT), most often encoded by the retroelement itself. Retroelements can move within the genome by a “cut-and-paste” process involving an RNA intermediate. In this course of action, the original element Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
269
270 is retained in situ and transcribed. The transcript is reverse transcribed by RT into a DNA copy that is then inserted into the genome in a new location. As a result of being copied repeatedly into new genomic locations over millions of years, retroelements have accumulated in vast numbers in mammalian genomes. Indeed, in the mouse, retrotransposons now account for over 37% of the total genome (Waterson et al. 2002), in contrast to the ~1% that is composed of protein-coding sequences. Retrotransposons can be subdivided into autonomous and nonautonomous elements based on whether they have open reading frames (ORF) encoding proteins required for retroposition (Table 10-1). Autonomous retrotransposons can be further subdivided into those with and without long terminal repeats (LTR). The LTR class accounts for about 10% of the mouse genome (Fig. 10-1). Retroviruses are LTR transposons in which overlapping ORF for their group-specific antigen (gag), protease (pro), polymerase (pol), and envelope (env) are flanked by LTR. Some LTR retrotransposons, such as mouse intracisternal A-particles, are quite similar to retroviruses but, most often, have no ORF and lack an intact env gene. The non-LTR class of autonomous retroelements includes long interspersed nucleotide sequences (LINE) represented by L1 elements in mice. LINE alone account for ~20% of the mouse genome. Mammalian genomes also contain large numbers of nonautonomous retroelements, sometimes termed retrotranscripts. These elements encode no proteins and thus are dependent on the RT-competent autonomous elements for their mobility. Some contain LTR, while others do not. Early transposons (ETn) contain nonretroviral sequences between the LTR, while mammalian
HERBERT C. MORSE III
TABLE 10-1
CLASSIFICATION OF MOUSE RETROELEMENTS Autonomous
Nonautonomous
LTR-containing Retrovirus IAP Non-LTR LINE
LTR-containing ETn MaLR Non-LTR SINE
LTR-retrotransposons (MaLR), the largest class of mouse retroviral-like elements, have ORF that do not appear to encode proteins but also are framed by LTR. The best-known nonautonomous sequences are the short interspersed nuclear elements (SINE), which comprise just over 8% of the mouse genome and have the B1 elements as the largest subclass. Processed pseudogenes are included among other nonautonomous retroelements.
II.
RETROVIRUSES
The genomes of inbred strains of mice contain multiple types of endogenous retroviral elements, some of which are shown in Fig. 10-1. The two most well-known groups are the C-type murine leukemia viruses (MuLV) and the B-type mouse mammary tumor viruses (MMTV). These types differ from the rest in having closely related relatives that occur exogenously and are passed through the milk rather than as Mendelian genetic elements.
Fig. 10-1 Structures of the major families of autonomous and nonautonomous LTR-containing retroelements found in the mouse germline. Only typical structures are indicated. Regions coding functional proteins or likely to encode variants of them are indicated above the genomes. ETn have small regions of homology to ORF of other retroelements but have not been associated with protein products. LTR include U3 (blue) and U5 (yellow) specific regions.
271
10. RETROELEMENTS IN THE MOUSE
A.
MuLV
TABLE 10-2
Infectious MuLV derived from endogenous proviruses can be subdivided into three classes based on their host range characteristics, i.e., the cell types from mice or other species they can infect, as well as other structural features (Table 10-2). The hostrange phenotype is determined by the characteristics of the env genes and the specificity of receptors on target cell populations. Ecotropic MuLV are infectious for mouse cells but not for cells of other species. Xenotropic MuLV have the opposite host-range characteristics, being infectious for non-mouse but not for mouse cells. MuLV designated mink cell focus-inducing (MCF) or polytropic are infectious for mouse and non-mouse cells. Another class of MuLV, termed amphotropic, has the same host range as polytropic MuLV but has not been found in the mouse genome (Hartley and Rowe 1976; Rasheed et al. 1976). These viruses have only been recovered from some demes of wild-caught mice and never from laboratory mice, and passage occurs through the milk. In addition, a class of ecotropic MuLV distinct from that of inbred
HOST RANGE AND RECEPTORS FOR ENDOGENOUS MULV AND MMTV Name
Tropism
Receptor Reference
Ecotropic MuLV Xenotropic MuLV Polytropic MuLV Amphotropic MuLV MMTV
Mouse only Non-mouse only Mouse and non-mouse Mouse and non-mouse Mouse and non-mouse
Slc7a1 Xpr1 Xpr1 Pit2 Tfr1
Albritton et al. 1989 Tailor et al. 1999 Tailor et al. 1999 Miller et al. 1994 Ross et al. 2002
mice also occurs in wild mice, is passaged via milk, and causes B cell lymphomas and, independently, a fatal motor neuron disease with hind-limb paralysis. Proviruses that encode infectious ecotropic and xenotropic MuLV are found in the genomes of some strains of mice but not others (Table 10-3). Using type-specific molecular probes, it was shown that the common inbred strains carry between zero
TABLE 10-3
RETROVIRAL CONTENT OF SOME INBRED STRAINS MuLV Ecotropic NFS, FVB, NZB, 129, C57L, AU, BUB, CBA/Ca, CBA/N, RIII, SWR A, BALB/c, CBA/J, C3H, SEC/1, SM, MTG C57BL/6, C57BL/Ks, C57BL/10, C57BR/cd DBA/1, DBA/2, BDP LG LP ST/b DA HRS, SEA, CWD MA/My SJL
Number of proviruses
0 1 1 1 1 1 1 1 2 2 2
AKR 3
RF C58 I/Ln PL Xenotropic A, BDP, NFS, NZC, SEA, 129 AKR, BALB/c, C57BL/6, C57BL/10, C57L, C58 MA/My NZB DBA/3, C3H a
Unk, unknown. Both AKR/J and AKR/N strains. c AKR/N but not AKR/J. b
3 >3 >3 >3 0 1 2 2 Unk
Designation
Chromosome
Phenotype
Emv1 Emv2 Emv3 Emv4 Emv5 Emv6 Emv7 Emv1, 3 Emv8, 9 Emv9 Emv10 Emv11 b Emv12 b Emv13 c Emv14 b Emv1 Emv16, 17 Emv26 C58V2 Unnamed Unnamed
5 8 9 Unka Unk Unk Unk 5, 9 Unk Unk Unk 7 16 7 11 5 1 8 Unk Unk Unk
Low Low Low Unk Unk Unk Unk Int-High Unk Low Non High High Non Non Low High High High High High
Bxv1 Bxv1 Mxv1 Nzv1 Nzv2 Unnamed
1 1 Unk Unk Unk Unk
Low Low Low High Low Low
272 and five copies of ecotropic MuLV env genes, most of which are associated with full-length proviruses that can be expressed as infectious virus (Chattopadhyay et al. 1974; Jenkins et al. 1982; Kozak and Rowe 1982). Most strains carry more than 30 copies of env genes for xenotropic MuLV, but few of these proviruses can produce infectious virus (Kozak et al. 1984). Endogenous proviruses encoding polytropic sequences are all defective. Many of these sequences are very similar to the genes that encode the amino-terminal regions of the SU envelope protein found in recombinant polytropic MuLV (Frankel and Coffin 1994; Khan et al. 1982; Stoye and Coffin 1988). Using oligonucleotide probes of genomic DNA, Coffin and coworkers identified two subgroups designated polytropic (PT) and modified polytropic (mPT), with a major distinguishing feature being a 27-bp deletion in the SU-encoding sequences of the mPT family. More recent studies of the NFS/N strain identify potential donor sequences that phylogenetically appear to represent an intermediate between xenotropic MuLV and distinct groupings of mPT and other previously defined PT (Evans et al. 2003). The host range of polytropic env sequences was uncovered as a consequence of recombinations between infectious ecotropic MuLV and both xenotropic and polytropic viral sequences, giving rise to infectious polytropic/MCF MuLV (Chattopadhyay et al. 1981; Quint et al. 1984; Hoggan et al. 1986; Stoye and Coffin 1987). Ecotropic, xenotropic, and both subsets of defective polytropic MuLV behave as quite stable genetic elements; however, their locations within the genome are generally strain-specific. Consequently, two different strains of mice may produce ecotropic MuLV at high levels as a consequence of expression from loci located on two different chromosomes. Nonetheless, the germline content of ecotropic, xenotropic, and polytropic MuLV has been shown to undergo gain or loss due to reinsertions or deletions in germ cells (Buckler et al. 1982; Jenkins and Copeland 1985). Novel, germline proviral insertions for xenotropic or polytropic MuLV have been identified but once (Frankel et al. 1990), while there are multiple instances of ecotropic virus reinsertions. Loss of germline proviruses seems to occur more frequently than gain. Proviral reinsertion is an inherently mutagenic event that is silent if it occurs in intergenic regions where the insertion of promoter and enhancer sequences within the LTR cannot influence the expression of distant genes; however, insertion into or in proximity to a host gene can alter its function or disable it completely (Table 10-4; Fig. 10-2). Two mutations in the mouse have been unequivocally associated with proviral insertions: dilute (d), caused by an ecotropic virus (Jenkins et al. 1981), and hairless (hr), caused by a polytropic virus (Stoye et al. 1988). Definitive evidence that the insertions were truly mutagenic was provided by studies of revertants that had lost the provirus. Retinal degeneration (rd) is associated with insertion of a xenotropic virus (Bowes et al. 1993), but the presence of other changes in the locus makes it uncertain whether the rd mutation can be ascribed to the viral insertion alone.
HERBERT C. MORSE III
TABLE 10-4
INSERTIONAL MUTAGENESIS OF MOUSE GENES BY RETROTRANSPOSONS Agent Retroviruses Ecotropic Polytropic Xenotropic IAPa IAP
Locus
Allele or disease
Myo5a hr Pde6b
Dilute—d Jenkins et al. 1981 Hairless—hr Stoye et al. 1988 Retinal degeneration—rd Bowes et al. 1993
a
Agouti—Aiapy Avy, Aiy Mahoganoid - md Mahogany—mg Friend virus resistance— Fv1nr Pale ear—ep Vibrator—vb Albino—cm10R Eyes absent—Eya1bor Fused—Fu Fu-kb Epidermolysis bullosa— Lamb3IAP Reeler—Relnrl-Alb2 β-glucuronidase— Gusmps-2J
IAP IAP IAP
Mgrn1 Atrn Fv1
IAP IAP IAP IAP IAP
Hps1 Pitpn Tyr Eya1 Axin
IAP
LamB3
IAP IAP
Reln Gus
ETnb ETnII or MusD Lep
Obese—ob2J
Loftus et al. 1997
ETnII
Adcy1
Barrelless—brl
ETnII ETnII ETnII
Fign Foxn1 Gli3
b
Royaux et al. 1997 Gwynn et al. 1998
Npc1
Albino—c3Bc Lymphoproliferation—lpr Cataract—Cat-Fr
a
Gardner et al. 1997 Hamilton et al. 1997 Wu et al. 1997 Johnson et al. 1999 Zeng et al. 1997 Vasicek et al. 1997 Kuster et al. 1997
Fidget—fignfi Nude—nu-Bc Polycactily Nagoya— Pdn Cacng2 Stargazer—stg Stg-3J Hkl Downcast anemia—Hkldea T Brachury—TWis
Tyr Fas Mip
ETnII ETnII MaLR MaLR
Michaud et al. 1994 Duhl et al. 1994 Phan et al. 2002 Gunn et al. 2001 Best et al. 1996
Moon and Friedman 1997 Hofmann et al. 1998 Adachi et al. 1993 Shiels and Bassnet 1996 Abdel-Majid et al. 1998 Cox et al. 2000 Hofmann et al. 1998 Thien and Ruther 1999 Letts et al. 1998 Letts et al. 2003 Peters et al. 2001 Hermann et al. 1990
ETnII ETnII ETnII
ETnII
Reference
Nieman Pick—m1N
IAP, intracisternal A particles. ETn, early transposons.
The acquisition of new proviral sequences seems to require infection of germ cells rather than direct DNA transposition (Lock et al. 1988). For this to occur, the infecting virus must overcome several barriers. The first level is binding of the virion envelope to an appropriate receptor. The absence of a receptor might seem an absolute barrier, yet there are multiple proviral copies of xenotropic MuLV, suggesting that the receptor was
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10. RETROELEMENTS IN THE MOUSE
A.
Promoter insertion
B.
Enhancer insertion
C.
Read-through transcription
D.
Alteration of 3' sequences
Fig. 10-2 Insertional mutagenesis by retroviruses. A canonical normal gene with four exons, shown at the top, can be mutated as a consequence of proviral insertion in different locations and transcriptional orientations. If the gene is a protooncogene, the resulting changes in gene expression can contribute to oncogenesis. (A) Intronic insertion resulting in promoter substitution and transcription of the gene initiated from the 3′ LTR. Reversion occurs by homologous recombination between the proviral LTR, leaving a solo LTR and restoring expression. Reversion to expression of a wild-type protein has been shown to occur in mice with the dilute (d) mutation caused by insertion of an ecotropic provirus into the locus. (B) Insertion 5′ to the gene resulting in activation by enhancer sequences in the LTR. Enhancer activation can occur with insertions in either transcriptional orientation both 5′ and 3′ to the gene and at considerable distances in some cases. (C) Gene activation through the generation of read-through transcripts. (D) Insertion 3′ to the gene resulting in activation by alteration of noncoding sequences.
lost or mutated at some time in the past, perhaps as a mechanism to prevent “colonization” of the germline by retroviruses of this class. Studies of the polytropic/xenotropic receptor gene Xpr1 associated a polymorphism of the locus Sxv with resistance of cells of the common inbred strains to infection with xenotropic MuLV (Kozak 1985b), providing one explanation for the lack of continuing spread of these agents; however, xenotropic transcripts may have been packaged by a virion envelope capable of receptor binding, a “Trojan horse” approach to evading restriction at the level of receptors. Resistance at the level of virion binding to a receptor can also be mediated by receptor interference—competitive blocking of virion binding by occupancy of the receptor by an env gene product from the same class of virus. Fv4 is a mouse gene normally limited to some Asian and Californian wild-caught mice that dominantly confers resistance to infection with ecotropic MuLV (Suzuki 1975; Gardner et al. 1980). Sequence studies showed that the Fv4 gene is a truncated ecotropic MuLV sequence including the 3′ portion of the pol region, the entire env gene, and the 3′ portion of the LTR (Ikeda et al. 1985).
Binding of the Fv4-encoded env to the ecotropic MuLV receptor is at least one mechanism contributing to resistance to infection. Another resistance gene, the Rmcf locus of DBA/2 mice, restricts infectivity with polytropic MuLV (Hartley et al. 1983). Recent studies identified a defective provirus with partially deleted pol and gag genes, the 5′ half of env, and the 5′ LTR that maps with resistance (Jung et al. 2002). The fact that the env gene is quite similar to the env genes of modified polytropic proviruses suggests Rmcf may also mediate resistance through an interference mechanism. A second barrier to infection is provided by the Fv1 gene. This gene was initially identified during a screen of inbred mice for susceptibility to disease induction by the Friend erythroleukemia virus complex (Lilly 1967) and was later shown to regulate infection with naturally occurring MuLV as well (Pincus et al. 1971). Alleles at Fv1 are codominantly expressed both in vivo and in vitro and include two major variants, Fv1n and Fv1b (Table 10-5). Mice homozygous for Fv1n are permissive for infection with a subset of ecotropic MuLV termed N-tropic, while mice homozygous for Fv1b are permissive for
274
HERBERT C. MORSE III
TABLE 10-5
FV1 GENOTYPES OF COMMON INBRED STRAINS AND SOME WILD-CAUGHT MICEa Fv1 allele Strains n nr nr-like b o
AKR, BUB, CBA/Ca, CBA/N, CE, CFW, C3H/He, C57BR/cd, C57L, C58, LP, MA/My, NFS, P, PL, RF, SJL, SM, ST/b, SWR NZB, NZW, 129, DBA1, DBA/2 M. m. musculus, M. m. domesticus A, AL, BALB/c, BDP, C57BL/6, C57BL/Ks, C57BL/Ka, C57BL/10, FVB, I, LG, RIIIS M. molossinus, M. spretus, M. cervicolor, M. castaneous
a Summarized from Jolicoeur 1979; Kozak 1985a; Z. Naghashafar and J. W. Hartley, unpublished observations.
another virus subset termed B-tropic MuLV. Fv1n/b heterozygotes are restrictive for replication of both types of MuLV. A third allele, termed Fv1nr, a variant of Fv1n and dominant to that allele, is found in a more limited set of strains including RF, 129, NZB, and NZW. Finally, a null allele, designated Fv10, is present in wild mice fully sensitive to all virus strains (Hartley and Rowe 1975; Kozak et al. 1985a). Studies on the restriction imposed by Fv1 showed that such restriction does not affect entry virus into cells or formation of double-stranded DNA but inhibits the nuclear entry and integration of newly synthesized DNA into the host genome (Jolicoeur and Rassart 1980). It does this by targeting a small region near the center of the capsid protein that remains with the proviral DNA in a preintegration complex (DesGroseillers and Jolicoeur 1983; Kozak and Chakraborti 1996). A probable explanation for how Fv1 works was suggested by the molecular characterization of the gene by Best et al. (1996). They found that Fv1 was encoded by a small intronless ORF with sequence similarity to the HERV-L family of human endogenous retroviruses. Based on its position in this sequence, Fv1 appears to be a gag-related gene with limited similarities to other gag genes. The known high affinity of Gag proteins for one another makes it likely that GAG/Fv1-GAG/MuLV interactions account for the specific block to N- and B-tropic viruses. A large number of other genes affects spread of MuLV in cell nonautonomous manners by regulating the immune responses to these agents. Several of these genes map to the major histocompatibility complex (MHC), while others are non-MHC linked. The major manifestations of endogenous MuLV expression in inbred strains of mice are cancer and, to some extent, autoimmunity (Krieg et al. 1992). In particular, high-level expression of ecotropic MuLV from birth is responsible for the development of leukemias and lymphomas in various strains of mice. Historically, strains AKR (Cole and Furth 1941) and C58 (Richter and MacDowell 1930) were bred to develop high incidences of thymic lymphomas. These lymphomas of AKR mice were found to contain viruses that could be passaged in cell-free extracts to induce similar lymphomas in other strains (Gross 1951). Electron microscopic studies of affected tissues revealed
the presence of C-type viruses that were later shown to comprise a mixture of ecotropic and polytropic/MCF MuLV. Studies of viruses of the two types purified from these mixtures showed that ecotropic MuLV was not the direct causative agent (Cloyd et al. 1980) but rather the polytropic/MCF viruses (Hartley et al. 1977). This virus class was shown to develop from a series of genetic substitutions of ecotropic viral sequences with sequences from endogenous xenotropic and polytropic MuLV (Chattopahdyay et al. 1982; Khan 1984; Stoye et al. 1991). Surprisingly, polytropic/MCF MuLV recovered from B cell–lineage lymphomas were not demonstrably pathogenic, demonstrating that this virus class comprises two distinct types with respect to their lymphomagenicity (Cloyd et al. 1980). The mechanism by which pathogenic polytropic/MCF MuLV contribute to T cell lymphoma development is through proviral insertional mutagenesis (reviewed in Jonkers and Berns 1996; Mikkers and Berns 2003), mechanistically the same process that led to germline mutation of the dilute and hairless genes described above. Here, as part of the normal process of infection by highly expressed MuLV, a provirus inserts in the proximity of a gene, altering its expression. If the affected gene influences cell growth or death, the targeted cell may have a survival advantage, leading to the outgrowth of a clonal tumor with all cells containing a copy of the virus at precisely the same site. The same mechanism appears to hold for ecotropic MuLV and the pathogenesis of B cell–lineage lymphomas (Justice et al. 1994; Hansen and Justice 1999; Suzuki et al. 2002). Once a target site is identified, even if the proximate gene is not known, a probe generated from flanking cellular DNA can be used to study other lymphomas to see whether the genomic structure of that region is altered in a similar manner. A probe that detects a site targeted by a provirus in more than one tumor is said to have identified a common integration site (CIS). The identification of CIS was, until quite recently, a very laborious process, and the identification of linked genes affected by the insertions a sometimes heroic undertaking. Over the last several years, however, the use of high-throughput techniques for cloning and sequencing host-viral DNA junction fragments and the availability of the mouse genome sequence have markedly simplified the process of identifying CIS and linked genes that are candidate cancer genes (Lund et al. 2002; Mikkers et al. 2002; Suzuki et al. 2002). These studies have uncovered several hundred CIS. The task for some time will be to determine whether the linked genes truly contribute to transformation. The use of transgenic mice to drive overexpression in selected cell types and the use of knockout mice to determine the effects of absent expression will lead to the generation of many new strains of mice.
B.
Mouse Mammary Tumor Viruses (MMTV)
MMTV is the second class of autonomous retrotransposons that encode RT. They hold a special place in the pantheon of
275
10. RETROELEMENTS IN THE MOUSE
mouse retroviruses, as they were the first class shown to be involved in development of cancer (Staff of the Roscoe B. Jackson Memorial Laboratory 1933; Bittner 1936). The structure of MMTV is quite similar to that of MuLV (Fig. 10-1) but is distinct in that the LTR encodes a protein known as a super antigen (Sag) that is intimately involved in the pathogenesis of the virus. There are more than 50 endogenous MMTV loci, designated Mtv1 to Mtv56, most of which have been mapped to specific chromosomal locations in different inbred strains (Kozak et al. 1987); related sequences are found in some but not all wildcaught mice (Callahan et al. 1982; Cohen et al. 1982; Imai et al. 1994). The strain distribution and chromosomal locations of some of the loci in commonly used inbred strains are listed in Table 10-6. Due to mutations in transcriptional regulatory sequences of coding domains, the majority of endogenous MMTV do not produce infectious virus. Only Mtv1 and Mtv2 as endogenous MMTV encode full-length proviruses that are expressed as infectious virus. Strains of mice negative for these two loci can also produce infectious virus that is passaged when newborns suckle on the milk of viremic mothers. The cycle of exogenous virus infection can be interrupted by fostering pups on virus-negative mothers. In pups suckled on virus-positive mothers, virus transits the M cell layer of Peyer’s patches. The exact sequence of events that follow is still being investigated, but current studies suggest that dendritic cells may first bind and be activated by virus through the toll-like receptor 4 (TLR4) (Burzyn et al. 2004), leading to their infection (Martin et al. 2002; Vacheron et al. 2002), as well as activation and then a second round of infection involving B cells. Both endogenous and exogenous MMTV have an ORF in the LTR that encodes a Sag (Choi et al. 1991). These proteins, presented in the context of MHC class II molecules on
dendritic and B cells, are recognized by T cells that bear particular T cell receptor β chains specific for the MHC class II-Sag pairing, resulting in massive T cell proliferative responses (reviewed in Herman et al. 1991; Czarneski et al. 2003). Because proliferation of the target cells is required for integration of the provirus into cellular DNA, expression of the Sag is indispensable for virus transmission. After the initial activation of T cells, there is a steady deletion of reactive cells in all infected mice (Marrack et al. 1991). Following circulation through the blood, immune cells infect dividing cells of the mammary gland, where the virus causes breast cancers through the process of insertional mutagenesis (Nusse 1988). There are several ways in which this process is subverted. First, mice with I-E-negative MHC class II haplotypes are relatively resistant to MMTV infection and tumorigenesis, as only the I-E-positive haplotypes are well suited to presentation of Sag to cognate T cells (Pucillo et al. 1993; Wrona and Dudley 1996). Second, mice with endogenous Sag sequences similar to those of the infecting virus will have deleted T cells bearing normally reactive β chains in the thymus and periphery (Dyson et al. 1991; Frankel et al. 1991; Woodland et al. 1991). In the absence of reactive T cells, infection fails to induce the striking proliferative response needed to drive infection in large numbers of cells. Third, in I/LnJ mice, sustained production of virus-neutralizing antibodies results in impaired mammary gland infection (Purdy et al. 2003). Finally, recent studies showed that the persistence of wild-type MMTV in C3H/HeN mice reflects the ability of the virus to undermine the innate immune system (Jude et al. 2003). In this strain, binding of the virus to TLR4 induced expression of the immunosuppressive cytokine IL-10. Thus, subversion of both innate and acquired immunity are survival mechanisms for different MMTV in genetically distinct strains of mice.
TABLE 10-6
STRAIN DISTRIBUTION, CHROMOSOMAL LOCATION, AND Vβ SPECIFICITY OF SOME MMTV PROVIRAL LOCIa Locus designation Mtv1 Mtv2 Mtv3 Mtv6 Mtv7 Mtv8 Mtv9 Mtv11 Mtv13 Mtv14 Mtv17 Mtv21 Mtv27 Mtv28 Mtv29 a Data
Chromosomal location 7 18 11 16 1 6 12 14 4 4 4 8 1 X 6
Strain(s) DBA, C3H/He, C3H/An GRS GRS BALB/c, CBA, A, C3H, DBA C3H/B1, C3H/K1, DBA, NFS, GRS DBA/2, BALB/c, STS/A, RIIIS, CBA, AKR, A, GRS, C3H, NFS, C57BL BALB/c, CBA, C57BL, AKR, NZB, C3H/Bi, C3H/Ki DBA/2, C3H/He, C3H/An DBA/2, NFS, A DBA/2, C3H, GRS, CBA, CE, NZB, NFS DBA/2, C57BL, AKR, NZB, C3H/BI, C3H/KI, GRS, NFS, C58 BR6 NZB NZB SJL
Vβ specificity 3, 5 14, 15 3, 5, 17a1 3, 5, 17a2 6, 7, 8.1, 9 5, 11, 17a1 5, 11, 17a1 5, 11 3, 5
3, 5
summarized from Kozak et al. 1987; Acha-Orbea et al. 1993; Simpson et al. 1993; Tomonari et al. 1993; http://www.informatics.jax.org
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HERBERT C. MORSE III
C.
Intracisternal A Particles (IAP)
This class of retroviral agents does not encode replicationcompetent viruses but is active and relatively abundant in the mouse genome. Their importance to mouse biology is indicated by that fact that nearly 15% of all spontaneous mutations have an allele with an IAP or ETn insertion. IAP include full-length class I forms comprising at least four subclasses with recognizable gag and pol genes but with most lacking env genes. Class II forms, comprising three subclasses, resemble class I forms but have undergone any of a series of deletions (Shen-Ong and Cole 1982). Together, IAP are present at about 1,000–2,000 copies per cell (Kuff and Lueders 1988). Instead of being assembled at the cell membrane like MuLV and MMTV, IAP are assembled on the endoplasmic reticulum and bud into the cisternae rather than the exterior of the cell. IAP are expressed in a variety of tissues, with some elements exhibiting tissue-restricted expression. This includes a subset expressed selectively in T and B lymphocytes. IAP are expressed at particularly high levels in plasmacytomas, neoplasms of mature plasma cells. Surprisingly, the IAP members expressed in plasmacytomas are distinct from those expressed in normal lymphocytes (Lueders et al. 1993).
D.
VL30 Elements
The name for this class derives from the original finding that retroviruses propagated on mouse cells contained the expected 35-38S MuLV RNA species but also an unexpected 30S RNA similar to but clearly distinct from retroviral genome RNA—thus, “virus-like 30S,” morphing to VL30. These elements have attracted interest because of their ability to retrotranspose extracellularly through formation of a pseudovirion complex when provided with replicative MuLV functions in trans. This generates a MuLV envelope with VL30 RNA (Carter et al. 1986). Their distribution and variations in the mouse label this family as one that has evolved and amplified as a consequence of this activity through successive rounds of duplicative retrotransposition and high-frequency recombination (Keshet et al. 1991; French and Norton 1997). Although this activity is most likely to result in recurring mutagenesis perhaps contributing to neoplasia, definitive evidence for an important pathobiologic or evolutionary role for VL30 sequences has not been reported. The LTR of VL30 have a typical U3-R-U5 structure. While most VL30 elements are composed of two LTR flanking an internal region, the mouse genome also contains a number of VL30 “solo” LTR. Unlike other classes of LTR retroelements, solo LTR are present in lower numbers than intact VL30. The internal portion of the VL30 genome has small regions with sequence homology to pol- and gag-like genes; the presence of multiple stop codons in all three reading frames makes it unlikely that it encodes any functional proteins.
E.
Early Transposon (ETn)-Related Elements
ETn-related elements, first discovered and characterized in the 1980s (Brulet et al. 1983, 1985), can be divided into three subsets. ETnI and ETnII differ in the 3′ half of the LTR and the 5′ end of the internal region (Shell et al. 1990). Noncoding sequences of unknown origin occupy the rest of the internal regions, except for a short stretch of varying content belonging to a retroviral pol gene. The third family member is the recently described MusD family (Mager and Freeman 2000). MusD retroviral elements contain regions similar to gag, pro, and pol, suggesting that they may provide the proteins necessary for transposition of ETnII elements in trans. Analyses of the C57BL/6 genome database have indicated the presence of approximately 200 ETnI sequences, 40 for ETnII, and 90 for MusD (Baust et al. 2003). In addition, there are nearly 500 ETnI- and 150 MusD-related solitary LTR. ETn are expressed primarily in embryonic tissues between days 3.5 and 7.5 of embryogenesis (Brulet et al. 1985).
F.
Mammalian Apparent LTR-Retrotransposons (MaLR)
With over 380,000 copies in the genome, MaLR are the single most successful nonautonomous LTR element. They comprise three subsets, including MERVL, MT, and ORR1 (Smit 1993). As noted previously, these transposons have structural similarities to retroviruses, but an internal open reading frame flanked by LTR, when present, has no similarities to retroviral proteins.
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Chapter 11 Sendai Virus and Pneumonia Virus of Mice (PVM) David G. Brownstein
I. II. III. IV. V.
VI.
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VIII. IX. X. XI. XII. XIII. XIV.
XV.
Sendai Virus: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties of the Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Virus Strains and Antigenic Relationships . . . . . . . . . . . . . . . . . . . . . . . . Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Growth in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Growth in Eggs and Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vivo Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Age, Gender, and Mouse Strain Dependence . . . . . . . . . . . . . . . . 2. Gross Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Microscopic Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Host Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Geographical Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mode(s) of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pneumonia Virus of Mice (PVM): Introduction . . . . . . . . . . . . . . . . . . . . History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties of the Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Virus Strains and Antigenic Relationships . . . . . . . . . . . . . . . . . . . . . . . . Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Growth in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Growth in Eggs and Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vivo Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Age, Gender, and Mouse Strain Dependence . . . . . . . . . . . . . . . . 2. Gross Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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D AV I D G . B R O W N S T E I N
3. Microscopic Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVI. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Host Range and Geographical Distribution . . . . . . . . . . . . . . . . . . . B. Mode(s) of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVII. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVIII. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
SENDAI VIRUS: INTRODUCTION
With the exception of mouse hepatitis virus, no adventitious infection of rodents has had the impact that Sendai virus has had over the past half century on the implementation of techniques to prevent and eliminate infectious diseases in laboratory mice. As one of a few naturally occurring murine viruses that regularly elicits clinically significant disease in its epizootic form and a highly contagious infection, Sendai virus has been a driving force behind universal implementation of procedures and practices to eliminate and prevent natural infections in mice. It has not just been for its nuisance value, however, that Sendai virus infection of mice has been investigated in some detail. It is also because it is a naturally occurring Respirovirus of mice and therefore an excellent paradigm for a viral genus that causes significant morbidity in many mammalian species, including humans. This has been a major impetus for most of the detailed investigations of Sendai virus epizootiology, pathogenesis, immunology, and molecular virology.
II.
302 302 302 302 303 303 304 304
in several reports in 1957 (Gerngros 1957; Zhdanov et al. 1957; Gorbunova et al. 1957). Doubts as to the human origin of Sendai virus, referred to as “hemagglutinating virus of the mouse” (HVM) in 1953 (Fukai and Suzuki 1955) and “hemagglutinating virus of Japan” (HVJ) in 1955 (Ishida and Homma 1978), were raised when it was determined that laboratory mice in Japan were ubiquitously infected with a virus with properties identical to that of the hemagglutinating virus recovered from mice inoculated with human material (Fukumi et al. 1954). During the early 1950s Sendai virus was also isolated from hamsters in Japan (Matsumoto et al. 1954). Much of the early confusion as to the host origin of Sendai virus stemmed from the close relationship between Sendai virus and human parainfluenza 1 virus. The initial isolation of human parainfluenza 1 virus was reported in 1958 (Chanock et al. 1958), and its close relationship with Sendai virus appeared in several reports soon after (Fukumi and Nishikawa 1961; Chanock et al. 1963; Stark and Heath 1967). Today there is no evidence to support humans as natural hosts of Sendai virus or that Sendai virus originated from humans. The consensus opinion is that rodents are natural hosts of Sendai virus.
HISTORY
The initial recovery, characterization, and descriptions of Sendai virus occurred in Japan and slightly later in the Soviet Union in the 1950s, during attempts to isolate human respiratory viruses from clinical materials inoculated into laboratory mice. Several excellent reviews of this early history of Sendai virus have been published (Ishida and Homma 1978; Parker and Richter 1982). Sendai virus was first described in 1953 in a series of reports on a virus recovered from mice that had been inoculated with suspensions of pneumonic lungs from human infants at Tohoku University Hospital in Sendai, Japan (Kuroya et al. 1953; Sano et al. 1953; Noda 1953). The virus that was recovered grew in embryonated hen’s eggs and agglutinated a variety of erythrocytes. It was initially referred to as “newborn virus pneumonitis (type Sendai)” and was interpreted as being of human origin, in spite of the fact that virus with similar properties was recovered from mice that had not been inoculated with human lung suspensions. A virus recovered in mice inoculated with materials from humans with respiratory illness in the Soviet Union with the same characteristics as the Japanese virus was described
III.
PROPERTIES OF THE VIRUS
The International Committee on Taxonomy of Viruses classifies Sendai virus as follows: Order: Mononegavirales (nonsegmented, linear, negativestranded RNA genome) Family: Paramyxoviridae (15–16 kb genome, pleomorphic virions) Subfamily: Paramyxovirinae (genome encodes 6–7 transcripts) Genus: Respirovirus (genome encodes neuraminidase and C protein) Species: Parainfluenza 1 Some of the properties of Sendai virus are listed in Table 11-1. The single-stranded, negative-sense RNA genome, containing 15,384 nucleotides, is tightly associated with over 2000 copies of the major nucleocapsid protein (NP), 517–524 amino acids in length (Morgan et al. 1984; Neubert et al. 1991). The virally encoded polymerase is packaged in the virion and is also
11. SENDAI VIRUS AND PNEUMONIA VIRUS OF MICE (PVM)
TABLE 11-1
PROPERTIES OF SENDAI VIRUS 1. Size 150–250 nm diameter 2. Morphology Enveloped roughly spherical (Fig. 11-1) to filamentous 3. Nucleic acid Nonsegmented, linear, negative-stranded RNA 4. Encoded mRNAs from 3′ to 5′ NP, nucleocapsid P/C/V, nucleocapsid-associated and nonstructural accessory proteins M, internal envelope F, external envelope fusion glycoprotein HN, external envelope hemagglutinin-neuraminidase glycoprotein L, nucleocapsid-associated major polymerase protein 5. Replication site Cytoplasm 6. Site of virion formation Plasma membrane 7. Stability at room temperature 99% infectivity lost by 48 hours in 2% protein suspension; complete inactivation between 5 and 14 days (Birkum-Petersen 1959; Dick and Mogabgab 1962; Watanabe et al. 1989) 8. Agglutinates erythrocytes from: humans (type O), guinea pig, rat, mouse, hamster, rabbit, cow, sheep, monkey, dog, chicken, and pigeon
associated with the nucleocapsid. It consists of a major subunit, the L protein, 2228 amino acids in length (Morgan and Rakestraw 1986), and the smaller P protein, 568 amino acids in length (Giorgi et al. 1983; Neubert 1989). Two glycosylated proteins and one nonglycosylated protein are associated with the virion envelope. The two glycosylated proteins, HN and F, are transmembrane external envelope components. HN, the
283
hemagglutinin-neuraminidase is 575 amino acids in length (Shioda et al. 1986; Neubert and Willenbrink 1990). The hemagglutinin mediates virus attachment and hemagglutination (Scheid and Choppin 1974; Scheid et al. 1980). The hemagglutinin portion of HN binds to slight variations on two sialyloligosaccharide sequences that are components of membrane gangliosides and glycoproteins, and these serve as the viral receptors (Holmgren, Elwing, et al. 1980;Holmgren, Svennercholm, et al. 1980; Wu et al. 1980; Markwell and Paulson, 1980; Markwell et al. 1981; Epand et al. 1995; Bitzer et al. 1997). Cellular receptors for Sendai virus are widely distributed in mouse tissues and therefore do not account for the respiratory tropism of the virus (Ito et al. 1983). The neuraminidase removes sialic acid to facilitate internalization of virions and is on a site separate from the hemagglutinin on the HN glycoprotein (Portner 1981). F, the fusion glycoprotein, is 565 amino acids in length (Shioda et al. 1986). It mediates penetration of the host cell by fusion of the viral envelope to the plasma membrane, syncytium formation, and is responsible for hemolytic activity. The monomeric fusion glycoprotein is translated as an inactive molecule, F0. The biologically active form is generated by one or more specific host proteases that cleave F0 into two disulfide-linked subunits, F1 and F2 (Ohuchi and Homma 1976; Hsu et al. 1981). This is an important event, and the specificity and distribution of these host proteases partially underlies the respiratory tropism of this virus, as discussed in Section VI, B (Tashiro and Homma 1983b; Tashiro et al. 1992; Kido et al. 1999). The F0 of wildtype Sendai virus is cleaved at the carboxy terminal side of arginine 116 in the motif Gly-Val-Pro-Gln-Ser-Arg (Hsu et al. 1987; Kido et al. 1997, 1999) by specific proteases that include
Fig. 11-1 Spherical Sendai virion amid cilia of bronchiolar epithelium. (×90,000)
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Fig. 11-2 Apical surface of alveolar type II epithelium. Multiple nascent Sendai virions are budding into the alveolar lumen. Cross-sections of regularly spaced ribonucleoproteins are visible along the inner layer of the plasma membrane, having bound to migrated M protein. (×60,000)
tryptase Clara (Kido et al. 1992), ectopic anionic trypsin I (Towatari et al. 2002), and mini-plasmin (Murakami et al. 2001) in the murine respiratory tract; clotting factor Xa in hen amniotic and allantoic fluid (Gotoh et al. 1990; Muramatsu and Homma 1980; Appleyard and Davis 1983); and trypsin, subtilisin, and thermolysin (Scheid and Choppin 1974; Ogura et al. 1982). Most susceptible continuous cell lines lack the requisite protease for F0 activation. These cell lines therefore limit virus replication to a single cycle, since the nascent progeny virus is inactive (Kido et al. 1992; Sakai et al. 1993; Kido et al. 1996, 1997). The nonglycosylated internal envelope protein, M, is 348 amino acids in length (Blumberg et al. 1984; Hidaka et al. 1984; Willenbrink and Neubert 1990). It is the rate-limiting protein in virion assembly (Portner and Kingsbury 1976). M protein migrates to the plasma membrane, where it forms orthogonal crystalline aggregates within the inner lipid leaflet that serve as attachment sites for ribonucleoproteins (Fig. 11-2) destined for incorporation into virions (Yoshida et al. 1976; Buechi and Bachi 1982). It therefore only appears at the apical plasma membrane where virions are forming (Kristensson and Orvell 1983). M protein also binds the cytoplasmic tails of the two glycoproteins HN and F (Sanderson et al. 1993). M protein synthesis is deficient relative to ribonucleoproteins synthesis about 2-fold (Portner and Kingsbury 1976). Redundant ribonucleoproteins, not committed to virion formation, accumulate in large numbers in the cytosol (Fig. 11-3) and serve as viral RNA transcriptive complexes (Portner and Kingsbury 1976). The P/C/V gene encodes the 568 amino acid phosphorylated nucleocapsid-associated protein, P, mentioned previously, as well as a nested set of small basic accessory proteins, C′, C, Y1, and Y2 (Curran and Kolakofsky 1987; Dillon and Gupta 1989; Curran and Kolakofsky 1990). These accessory proteins, 175 to
215 amino acids in length, are carboxyl-coterminal and are initiated at different start codons within the P gene (Kurotani et al. 1998). They are nonessential for virus replication in vitro but play important roles in vivo, as discussed in Section VI B.
IV.
VIRUS STRAINS AND ANTIGENIC RELATIONSHIPS
All strains of Sendai virus are considered antigenically homologous and of a single serotype. Nevertheless, antigenic variation at the epitope level has been detected in 13 strains of Sendai virus isolated from various sources in the 1950s and after 1976, based on reactivity to a panel of monoclonal antibodies (Yamaguchi et al. 1989). Six strains isolated after 1978 lacked reactivity with an HN-specific monoclonal antibody to which strains recovered earlier did react. Two strains recovered after 1976 did not react with an F-specific monoclonal antibody to which strains recovered earlier did react. This type of antigenic variability has little impact on overall serotype specificity. Sendai virus strain variations relative to virulence for mice have been reported (Yamaguchi et al. 1988). A 1000-fold difference in median lethal doses of Sendai virus were found in five strains (MN, Z, KN, Mol, and Hm) recovered from laboratory rodents using 3-week-old Jc1-ICR mice. Commonly used laboratory strains of Sendai virus are: strain 52 (ATCC VR-1050 from Kuroya et al. 1953), Fushimi (Kuroya et al. 1953), Akitsugu (Misao et al. 1954), MN (Fukumi et al. 1954), Z (Fukai and Suzuki 1955), KN (Yamaguchi et al. 1988) 771076 (Brownstein et al. 1981), Hamamatsu (Sugita et al. 1974), Enders (Hou et al. 1992a), HVJ-W (Kimura et al. 1979), Mol (Itoh et al. 1989), and SeVM (Ohita) (Itoh et al. 1997).
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11. SENDAI VIRUS AND PNEUMONIA VIRUS OF MICE (PVM)
Fig. 11-3 Cytosol of alveolar type II cell. Redundant ribonucleoproteins are accumulating in non-membrane-bound aggregates. (×96,000)
The passage history for many of these strains is incomplete; most have been extensively passaged in hen’s eggs (Sugita et al. 1974). The entire genomes of some of these strains have been sequenced, including Hamatsu, Z, Harris and Fushimi, Ohita-M1 and E0 (Fujii et al. 2002, 2001; Sakaguchi et al. 1994; Itoh et al. 1997). Host range, temperature-sensitive, protease activation, and bipolar budding mutant strains of Sendai virus have been recovered from persistently infected cell cultures. Some of these have been entirely sequenced (Itoh et al. 1997; Tashiro et al. 1999; Fujii et al. 2002). Protease activation mutant strains, such as ts- fl, F1-R, TR-2, and TR-5, have amino acid exchanges at or near the cleavage site of the F protein, which alters the specificity of the requisite protease. F1-R also has two mutations in the matrix (M) protein, at residues 128 and 210, which promote bipolar budding in polarized epithelial cells and mouse bronchial epithelium (Tashiro and Seto 1997). F1-R is a pantropic strain due in part to its bipolar budding and protease activation phenotypes. Recently developed technology to recover infectious viruses of the order Mononegavirales from cDNA has allowed development of recombinant strains of Sendai virus (Kato et al. 1996; Leyrer et al. 1998). Most of these engineered strains have deletions in the accessory proteins encoded by the P/C/V gene. These deletant strains show protein synthesis and growth patterns in cell cultures similar to those of wild-type virus but are strongly attenuated in mice for reasons discussed in Section VI, B, 4.
(Kato et al. 1997; Gotoh et al. 1999; Garcin et al. 1999; Kurotani et al. 1998; Delenda et al. 1998; Garcin et al. 2001; Strahle et al. 2003; Fukuhara et al. 2002).
V.
GROWTH IN VIVO AND IN VITRO A.
Growth in Mice
Under natural conditions, Sendai virus infection is acute and restricted primarily to respiratory epithelium. Early investigators demonstrated Sendai viral antigen in macrophages in natural infection, and isolated mouse macrophages do support replication (Mims and Murphy 1973; Mills 1979). After intranasal inoculation of immunocompetent mice, virus can first be isolated from the lungs between 48 and 72 hours post-inoculation (Fig. 11-4). Virus titers rise rapidly on day 3 as indicated by differences of 2 or 3 logs in titers between individual mice. From day 4 to peak virus titers on day 6 or 7, virus growth is slower with tight clustering of titers between individual mice. Titers begin to decline on day 7 or 8 with a rapid decline on day 9, again reflected in the disparity of titers among individual mice. The decline in titers corresponds with maturation of the adaptive immune response. Transient low-level viremia can occur, notably during intervals of peak virus titers in the lungs, which
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8
Virus Titer (Log10 PFU/0.1 Gram Lung)
7 6 5 4 3 2 1 <0.8 0
1
2
3
6 7 8 9 4 5 Days After Intranasal Inoculation
10
11
12
13
Fig. 11-4 Sendai virus titers in the lungs of young adult BALB/c mice after intranasal infection.
accounts for brief periods of virus recovery from nonrespiratory organs. Somewhat longer phases of Sendai virus replication have been reported, in which infectious virus could be recovered from the lungs up to 14 days after intranasal inoculation depending on virus strain, dose of virus, volume of inoculum, and strain of mouse (Parker and Reynolds 1968; Sawicki 1962; van Nunen and van der Veen 1967; Robinson et al. 1968; Appell et al. 1971; Parker et al.1978).
B.
Growth in Eggs and Cell Culture
Sendai virus grows readily in embryonated hen’s eggs, which are commonly and widely used. Fertile 8- to 13- day-old eggs are inoculated in the amniotic or allantoic cavity and incubated at 35°–38° C for 48–72 hours and the appropriate fluid harvested. Because amniotic and allantoic fluids contain blood clotting factor Xa, an arginine-specific serine protease capable of activating Sendai virus F0 glycoprotein, multiple cycles of replication are supported and high virus yields are attained (Muramatsu and Homma 1980; Appleyard and Davis 1983). Egg passage attenuates Sendai virus virulence for mice compared to mouse lung-passaged virus (Kiyotani et al. 2001; Fujii et al. 2002). This attenuation has been linked to mutations in the leader, the HN gene, and the L gene (Fujii et al. 2002). Primary and secondary cultures of rhesus monkey (Fukumi et al. 1959; Fukumi and Nishikawa 1961; Chanock and Parrott 1965) and
cynomolgus monkey (Shibuta et al. 1971; Shibuta 1972) and calf (Nagata et al. 1965) kidney cells support multiple virus replication cycles, exhibit characteristic cytopathic changes, and are useful in plaque assays and plaque purification (Shibuta et al. 1971). Infected cells elongate, with ends becoming long and slender; their nuclei also elongate. Syncytia formation is common (Parker and Richter 1982). Serial passage of monkey kidney cells lose their ability to support multiple-cycle replication due to loss of trypsin-like proteolytic activity necessary for activation of F0 (Silver et al. 1978). Serial cell lines BHK-21, BSC-1, and Vero support single-cycle replication and can be used in fluorescent focus (Smith 1986) and hemadsorption assays (Brownstein et al. 1981). A sensitive plaque assay for Sendai virus uses an established cell line from rhesus monkey kidney, LLCMK2, with added trypsin at a concentration of 5–10 µg/ml to allow multiple-cycle replication (Sugita et al. 1974). This assay has been shown to be as sensitive as primary monkey kidney cells in plaque assays of Sendai virus (Sugita et al. 1974; Silver et al. 1978; Frank et al. 1979). A plaque assay using a clonal line of porcine kidney cells, PS-Y15, has also been reported, with sensitivity comparable to egg titration (Ito 1976). Sendai virus readily persists in continuous cell cultures. The virus recovered usually has characteristics that differ from the original wild-type virus. These include temperature sensitivity of M or P synthesis (Portner et al. 1974; Kimura et al. 1979; Yoshida et al. 1979; Ogura et al. 1981), protease specificity for activation of F0 (Ogura et al. 1982; Tashiro, Seto, Choosakul,
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Hegemann, et al. 1992), weak cytopathogenicity (Kimura et al. 1975), and defective interfering viruses (Roux and Holland 1980). Although there is clearly a selective advantage for these variant strains in persistently infected cell cultures, it is unclear whether they are necessary for persistence. Antigenic variants arise at about 100-fold greater frequency in persistently infected cultures than in lytically infected cultures (Ogura et al. 1982). Viral persistence in cell cultures is also influenced by host cell metabolism, since virion assembly and nucleic acid and protein synthesis are governed by the phase of the cell cycle. Confluent cultures support less virus replication than growing cultures (Ogura, Sato, and Hanato 1984). Host cells may also selectively suppress the synthesis of specific structural proteins, as has been shown for M protein in rat glial cells (Ogura, Sato, and Tanaka 1984).
VI.
IN VIVO INFECTION A.
Clinical Disease
Acute signs elicited by Sendai virus can include chattering, piloerection, polypnea, rapid weight loss, depression, fetal resorption, prolonged gestation, and death in neonates, suckling, and aged mice and at any age in mice of genetically susceptible strains (129, DBA, A). Growth is often poor in infected weanling and young adult mice. Athymic nude (nu/nu) mice respond to persistent Sendai virus infection by developing a chronic progressive wasting disease accompanied by progressive polypnea, hyperpnea, and depression (Ward et al. 1976).
B.
Pathogenesis
Under conditions of natural infection, Sendai virus causes a descending infection, usually restricted to the mucociliary epithelium of the conducting airways (Fig. 11-5). Depending on the airway segments that receive the initial exposure, this can involve epithelium of nasal passages, trachea, bronchi, and bronchioles, or multiple levels (Degre and Midtvedt 1971; Blandford and Heath 1972; Heath 1979; Carthew and Sparrow 1980a; Brownstein et al. 1981). Sendai virus that descends to the level of terminal bronchioles can spread to alveolar epithelium, notably alveolar type II cells, which supports more limited virus replication than respiratory epithelium (Fig. 11-6). The virus also targets ciliated epithelium of the middle ear under conditions of natural infection, presumably by ascent of the Eustachian tube. Intranasally inoculated Sendai virus can access the central nervous system via olfactory neurons and persist in a non-infectious form (Mori et al. 1995). The Sendai virus nucleoprotein gene can be detected in the olfactory bulb for at least 168 days using the nested polymerase chain reaction (Mori et al. 1995). Less sensitive techniques have found no evidence of Sendai virus invasion of olfactory bulbs after intranasal inoculation (Lundh et al. 1987). This targeted infection of respiratory epithelium is due in part to viral adaptation to apical budding from respiratory epithelium and to activating respiratory proteases (Tashiro and Homma 1983; Tashiro and Seto 1997; Tashiro et al. 1999). Mutant strains of Sendai virus have been recovered from persistently infected cell cultures that exhibit altered organ tropism. Mutations that affect sites of virion assembly and activating protease specificity are important determinants of organ tropism. F1-R is a pantropic mutant strain of Sendai virus capable of causing systemic infections (Tashiro, Pritzer, et al. 1988).
Fig. 11-5 Expression of Sendai virus antigens in lungs after intranasal inoculation. Virus replication is confined to bronchiolar epithelium. (Avidin-biotin immunoperoxidase, ×170)
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Fig. 11-6 Expression of Sendai virus antigens in lungs after intranasal inoculation. Virus replication has spread from a terminal bronchiole (arrow) to numerous alveolar type II cells. (Avidin-biotin immunoperoxidase, ×170)
It exhibits both apical and basolateral budding, attributable to two mutations in the viral M gene (Tashiro et al. 1996, 1999). In wild-type Sendai virus, apical budding from polarized respiratory epithelium is controlled by M protein and facilitates virus spread to susceptible surface epithelium (Tashiro, Yamakawa, Tobita, Klenk, et al. 1990; Tashiro, Yamakawa, Tobita, Seto, et al. 1990; Tashiro et al. 1996; Okada et al. 1998). The F1-R mutant strain of Sendai virus causes a generalized infection after intranasal inoculation of mice, partly because of basolateral budding, which allows systemic spread, and partly because of a change in the activating protease specificity of its F glycoprotein to ubiquitous proteases found in tissues outside of the respiratory tract (Tashiro et al. 1988; Tashiro, Yamakawa, Tobita, Seto, et al. 1990; Tashiro, Seto, Choosakul, Hegemann, et al. 1992; Tashiro and Seto 1997; Tashiro et al. 1999). The mutable F glycoprotein of wild-type Sendai virus is specifically cleaved by one or more endogenous respiratory argininespecific serine proteases. The first to be identified was tryptase Clara, which, as the name implies, has been localised to nonciliated secretory cells (Clara cells) of terminal conducting airways in rats (Kido et al. 1992; Sakai et al. 1993). In rats, the subcellular location of tryptase Clara is secretory granules of Clara cells as well as extracellular airway secretions (Kido et al. 1996; Sakai et al. 1993). Sendai virus infection promotes the secretion of tryptase Clara (Sakai et al. 1994). Anti-tryptase Clara antibody inhibits Sendai viral activation in vitro in rat lung block cultures and in vivo in infected rats when the enzyme-specific antibody is administered intranasally (Tashiro, Yokogoshi, et al. 1992). Mini-plasmin is another serine protease recently identified in rodent lungs that is capable of activating F0. Mini-plasmin has been localized to epithelium of pre-terminal bronchioles and may serve to activate nascent virus infecting larger airways
(Murakami et al. 2001). A third serine protease found in rat lungs and capable of activating fusion glycoprotein is ectopic anionic trypsin I. This enzyme is localized to peribronchiolar stromal cells and is much less likely to be accessed by apical budding virus (Towatari et al. 2002). Mice inoculated intranasally with Sendai virus grown in LLC-MK2 cells in which fusion glycoprotein is uncleaved will develop patent infections, but the process is inefficient and delayed (Kiyotani et al. 1993). This attests to the small amounts of soluble activating proteases in mouse respiratory secretions. 1.
Age, Gender, and Mouse Strain Dependence
Infant and aging mice have been shown to be more susceptible to the lethal effects of Sendai virus than young adult mice. This susceptibility correlates with greater permissiveness for virus growth, including higher titers and delayed clearance (Sawiki 1961; Jacoby et al. 1994). Inbred strains of mice vary widely in their susceptibility to Sendai virus–induced morbidity, mortality, and respiratory disease. Parker et al. (1978) tested 19 inbred and 4 outbred Swiss strains of mice for susceptibility to lethal Sendai virus infection after intranasal challenge (Table 11-2). Among the strains of mice tested, there was a continuum of susceptibility based on LD50 values, with a 20,000-fold difference in susceptibility to lethal infection between the most susceptible 129/ReJ strain and the most resistant SJL/J strains. Genetic factors account for most of the variations in susceptibility to lethal Sendai virus infection between strains of mice. Conventional genetic analyses of crosses between resistant C57BL/6J and susceptible DBA/2J mice showed that resistance is a multifactorial autosomal dominant trait determined by loci that do not map to the major histocompatibility complex
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(Brownstein 1983). Genetic susceptibility to Sendai virus is expressed as clinical severity and the degree of parenchymal lung inflammatory injury (Parker et al. 1978; Brownstein et al. 1981; Itoh et al. 1991). Genetic differences in the degree of parenchymal lung injury correlates with the extent to which virus replication extends to alveolar lining cells from primary replication sites in the epithelium of conducting airways (Brownstein et al. 1981). There have been conflicting studies concerning whether susceptibility to Sendai virus pneumonia is also expressed through differences in lung virus burdens. Significant differences in lung virus burdens have been reported between C57BL/6J and DBA/2J mice after aerosol exposure with Sendai virus grown in mouse lungs (Fig. 11-7) but not after intranasal inoculation (Brownstein et al. 1981; Brownstein and Winkler 1986). After aerosol exposure, lung virus titers were 10- to 300-fold higher in DBA/2J than in C57BL/6J mice beginning 3 days after infection, with both strains clearing virus at the same time. In another study, susceptible 129/J mice had 10- to 30,000-fold higher lung virus burdens than C57BL/6 J mice after intranasal inoculation of Sendai virus grown in hen’s eggs (Mo, et al. 1995). In reciprocal bone marrow radiation chimeras between H-2bcompatible C57BL/6J and 129/J mice, higher lung virus burdens were expressed in irradiated, unreconstituted 129 versus C57BL/6 mice and in reconstituted 129 versus C57BL/6 mice regardless of the strain of bone marrow cell origin. These results
TABLE 11-2
SUSCEPTIBILITY OF INBRED AND OUTBRED STRAINS OF MICE TO LETHAL SENDAI VIRUS INFECTIONa Mouse strain
LD50 (log10 TCID50)
129/ReJ 129/J DBA/1J C3H/Bi DBA/2J DBA/2 A/HeJ A/J SWR/J Swiss (NIH) C57L/J C57BL/10Sn C3HeB/FeJ BALB/cJ C57BL/6 Swiss (Life Sciences) C58/J AKR/J Swiss (National Laboratory Animal Co.) Swiss (Microbiological Associates) C57BL/6J RF/J SJL/J a
0.5 0.6 1.3 1.4 1.6 2.0 2.5 2.5 2.7 2.7 2.7 2.8 2.8 3.0 3.0 3.1 3.2 3.4 3.4 4.4 4.4 5.0 5.0
Modified from Parker et al. 1978.
6
Lung Virus Titer (Log10 FFU/0.1 G)
5
4
3
2
1
0 3
4
5
6
7
8
9
10
11
12
Days After Infection Fig. 11-7 Sendai virus titers in lungs of DBA/2J mice (open circles) and C57BL/6J mice after aerosol infection. (Modified from Brownstein and Winkler 1986.)
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showed that different virus burdens were expressed at the level of radiorestant lung cells, presumably epithelium (Mo, Sangster, et al. 1995). A polymorphism in mucociliary transport between C57BL/6J and DBA/2J mice has been genetically linked with resistance/susceptibility to lethal Sendai virus infection (Brownstein 1987). By contrast, Breider et al. (1987) reported no differences in lung Sendai virus burdens between resistant C57BL/6NCr and susceptible DBA/2NCr mice after intranasal inoculation with egg-passaged strain 52 Sendai virus. Itoh et al. (1989) also found no differences in Sendai virus growth in the lungs of resistant BALB/cA and susceptible DBA/2N mice.
2.
Gross Changes
Sendai virus infection causes pathological changes that range from mild rhinitis to severe bronchointerstitial pneumonia. Gross changes are generally only observable in cases of severe bronchointerstitial pneumonia and not when infection is confined to conducting airways. When present, gross lung changes vary from discrete plum-colored to grey areas of consolidation in the hilar region of the lungs, cranial lung lobes, or multifocally (Fig. 11-8A). These changes appear on about day 6 of infection and reach peak severity on days 10–13, followed by gradual resolution if animals survive. More severe cases, such as those encountered in genetically susceptible strains of mice (Fig. 11-8B) or immunoincompetent mice (Fig. 11-8C), may be associated with uniform lung consolidation and failure of lungs to collapse at post mortem.
A
B
3. Microscopic Changes
During the latter stages of the log phase of virus growth (days 4 to 6), Sendai virus causes mild to moderate cytopathic changes in infected respiratory epithelium. These changes include cellular hypertrophy with accumulation of redundant ribonucleoproteins in the cytoplasm and irregular hyperplasia, leading to foci of disorganized epithelium, often with piling and papillary projections (Fig. 11-9). Cytoplasmic ribonucleoprotein accumulations can be visualized by light microscopy in hematoxylin and eosin-stained sections as poorly delineated, non-membrane-bound, homogeneous eosinophilic areas in the cytoplasm of hypertrophied cells (Fig. 11-9). These cytopathic changes are usually accompanied by mild acute inflammatory changes in affected airways. The decline in virus titers is attended by the appearance of a local nonsuppurative inflammatory response that heralds the arrival of the virus-specific cytotoxic T cells (Tc) and CD4+ helper T cells (Th). This inflammatory response is localized to the mucosa, submucosa, and adventitia of infected airways and is rich in lymphocytes (Fig. 11-10). Respiratory epithelium that expresses Sendai virus antigens with Tc specificity undergoes Tc-triggered apoptosis, leading to erosion and desquamation of epithelium, often in sheets (Fig. 11-10). A characteristic feature of Sendai virus infection is the non-uniformity of airway changes; some airways are spared while others are extensively altered. If the infection reaches the level of lung parenchyma with virus infection radiating from infected terminal bronchioles into alveolar epithelium, then the inflammatory response follows the infection into the alveoli, causing well-circumscribed
C
Fig. 11-8 Gross changes in lungs, 13 (A and B) and 21 (C) days after intranasal inoculation. (A) The lungs of the genetically resistant C57BL/6 mouse contain multifocal discrete areas of consolidation, signifying inflammation confined to terminal bronchioles. (B) The lungs of the genetically susceptible DBA/2 mouse are uniformly consolidated due to extensive involvement of lung parenchyma. (C) The lungs of the athymic nude mouse exhibit confluent areas of grey consolidation, reflecting a more chronic clinical course.
11. SENDAI VIRUS AND PNEUMONIA VIRUS OF MICE (PVM)
291
Fig. 11-9 Cytopathic changes in bronchiolar epithelium caused by Sendai virus during latter stage of log phase of Sendai virus growth. Cytoplasmic ribonucleoprotein accumulations are just visible as homogeneous non-membrane-bound inclusions (arrow). (H&E, ×680)
areas of alveolar inflammation. Initially, there is an exudative phase lasting several days in which pleomorphic lymphocytes, foamy macrophages, and neutrophils accumulate in alveolar spaces and in thickened, congested alveolar septa (Fig. 11-11). This is followed by localization of the inflammatory response to alveolar walls with a classical bronchointerstitial distribution. By the end of the second week of infection, the peribronchial and peribronchiolar lymphoid collars are enlarged and densely populated with lymphocytes (Fig. 11-12). Regenerating respiratory epithelium is present in denuded airways. At the alveolar
level, inflammation if present is localized to alveolar septa, which are thickened by mononuclear cells and contain hyperplastic type II pneumocytes. The pattern is that of a classical bronchointerstitial pneumonia (Fig. 11-13). If alveolar injury has been severe, with widespread destruction of pneumocytes, then fibrin deposits in alveoli may become organized, leading to fibrosing alveolitis (Fig. 11-14) and bronchiolitis oblitterans. Squamous metaplasia in bronchioles and alveoli is also a common sequelae of severe Sendai virus pneumonia (Fig. 11-15).
Fig. 11-10 Lung from C57BL/6 mouse during early phase of declining virus titers. There is a lymphocyte-rich inflammatory infiltrate in the wall of the bronchiole, and sheets of apoptotic bronchiolar epithelium are desquamating into the lumen. (H&E, ×170)
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Fig.11-11 Lung from DBA/2 mouse during exudative phase of alveolar inflammation. The alveolar spaces adjacent to a terminal bronchiole are filled with mixed inflammatory cells. (H&E, ×170)
Histopatholgical changes in the lungs of persistently infected athymic nude mice resemble those of severe infection of euthymic mice, with some notable differences. First, bronchiolar epithelial apoptosis does not occur due to an absence of virus-specific Tc. The result is a persistence of infected mucociliary epithelium that manifests continuing viral-induced cytopathology. This includes hypertrophy, hyperplasia, and non-membrane-bound cytoplasmic inclusions. In addition, intranuclear inclusion bodies have been reported (Ward et al. 1976). These inclusions are crystalline arrays of viral nucleocapsids, which are presumably translocated to the nucleus from their normal subcellular domocile in
the cytosol. Secondly, there is a notable absence of lymphoid cells in the interstitial infiltrate in the lungs. 4.
Immune Response
INNATE IMMUNITY During the phase of virus replication, a series of innate followed by virus-specific immune defences are mobilized. The best-studied of the innate defence mechanisms is the type I interferon (IFN) response. Sendai virus is a potent inducer of type I IFN in the mouse (but not in the rat), and type I IFN induction in the lung closely emulates the kinetics of
Fig. 11-12 Lung from C57BL/6 mouse immediately after virus clearance. A thick collar of lymphoid cells envelops a previously infected bronchiole. (H&E, ×170)
11. SENDAI VIRUS AND PNEUMONIA VIRUS OF MICE (PVM)
293
Fig. 11-13 Bronchointerstitial pneumonia in a DBA/2 mouse soon after virus clearance. Alveolar walls adjacent to terminal bronchioles are thickened by mononuclear inflammatory cells and hyperplastic alveolar type II cells. (H&E, ×170)
virus replication in the lung (Anderson et al. 1980; Brownstein and Winkler 1986) (Fig. 11-16). Peak lung IFN titers of about 104–105 international units per gram of lung are attained, which then declines with virus titers (Brownstein and Winkler 1986). Sendai virus induces type I IFN by a variety of mechanisms. In addition to the classical induction by double-stranded RNA of viral replicative intermediates after penetration of host cells, the viral HN glycoprotein, functioning as a lectin, can trigger its production (Ito and Hosaka 1983). Anti-viral effects of type I and type II IFN responses are effectively antagonized by Sendai virus. This can be demonstrated in vivo by the inability of pretreatment and concurrent treatment
with polyclonal antibody to type I IFN to increase susceptibility to lethal Sendai virus infection in mice (D. G. Brownstein, unpublished observation; Breider et al. 1987). This clinical observation can be explained by recent experiments showing that products of the viral P/C/V gene, which encodes a nested set of carboxyl-coterminal nonstructural proteins initiated at different start codons (C′, C, Y1, and Y2), interdict IFN signaling in infected cells to prevent cellular mobilization of IFN-induced antiviral defenses (Garcin et al. 1999; Gotoh et al. 1999). These proteins, referred to collectively as the C proteins, suppress IFN-stimulated tyrosine phosphorylation of signal transducers and activators of transcription (STATs) and subsequent
Fig. 11-14 Fibrosing alveolitis in a DBA/2 mouse after Sendai virus infection. (H&E, ×170)
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Fig. 11-15 Alveolar squamous metaplasia in a DBA/2 mouse that survived severe Sendai virus pneumonia. (H&E, ×170)
I IFN signaling, and GAF transcription is essential for type II IFN signaling. All four C proteins effectively interdict IFN signaling through the Jak/Stat pathway (Kato et al. 2001; Garcin et al. 2000, 2002). Recombinant Sendai viruses carrying null mutations to P/C/V genes and mutant Sendai viruses selected
6
6
5
5 Virus titer
4
4
3
3 Interferon titer
2
2
1
1
0
0 3
4
5
6 7 8 9 Days After Infection
10
11
Lung Interferon Titer (Log10 IU/0.01 G)
Lung Virus Titer (Log10 FFU/0.1 G)
formation of IFN-stimulated gene factor 3 transcription complex (ISGF3) and (GAF) transcription complexes at an early phase of infection (Garcin et al. 1999, 2002; Gotoh et al. 1999, 2001, 2002, 2003; Komatsu et al. 2000; Young et al. 2000; Takeuchi et al. 2001). ISGF3 transcription is essential for type
12
Fig. 11-16 Type 1 interferon (IFN) titers and Sendai virus titers in the lungs of C57BL/6 mice after intranasal inoculation. IFN titers closely follow the kinetics of virus replication.
11. SENDAI VIRUS AND PNEUMONIA VIRUS OF MICE (PVM)
TABLE 11-3
DAY 5 SENDAI VIRUS TITERS IN LUNG AND NK CELL ACTIVITY IN SPLEENS OF C57BL/6J MICE TREATED WITH NORMAL RABBIT SERUM AND RABBIT ANTI-ASIALO GM1 SERUMa Treatmentb Normal rabbit serum
Rabbit anti-asialo GM1
NK cell activityc in spleen (LU/106 spleen cells) 2.69 2.29 2.12 – –
Lung virus titer (log10 FFU/0.1 g) 4.38 4.82 4.48 4.56 4.52
aBrownstein
(unpublished observation). 6-week-old female mice were infected with 15,000 FFU Sendai virus intranasally under anesthesia at the same time they were administered 150 µl of a 1:3 dilution of normal rabbit serum or rabbit anti-asialo GM1. c NK cell activity by chromium relase assay using YAC1 cells. b
by passage in cell culture that contain mutated P/C/V genes are avirulent for mice because, unlike wild-type virus, they are sensitive to IFN-stimulated cellular antiviral mechanisms (Garcin et al. 1997; Kato et al. 1997; Itoh et al. 1997; Delenda et al. 1998; Kurotani et al. 1998; Nagai 1999; Fukuhara et al. 2002). Sendai virus infection also stimulates local and systemic natural killer (NK) cell activity (Anderson et al. 1977, 1979; Anderson 1979). NK cell depletion of mice prior to and during Sendai virus infection with monoclonal anti-NK1.1 and polyclonal anti-asialo GM1 antibodies fails to increase susceptibility or alter virus replication kinetics in the lungs (Table 11-3). This suggests that the Sendai virus genome may encode proteins that also interdict non-IFN innate defenses. This result and interpretation is contradicted by a study by Kast et al. (1990) in which NK cell activation of C57BL/6 mice by pretreatment with monoclonal anti-CD3 antibody protected mice from lethal Sendai virus infection. Products of the Sendai virus P/C/V gene have been shown to suppress expression of IFN-independent genes involved in innate defenses such as interleukins 6 and 8, based on a study using cDNA arrays to measure expression of various cellular genes in mice infected with wild-type and mutant Sendai virus (Strahle et al. 2003). Toll-like receptors (TLR) do not appear to be important in innate defense against Sendai virus infection. Mice with targeted mutations of TLR4, which has been implicated in innate immunity to respiratory syncytial virus, are as resistant to Sendai virus as wild-type mice (van der Sluijs et al. 2003). Complement appears to play a deleterious rather than a protective role in Sendai virus infection, based on evidence that decomplementation of CBA and DBA mice with purified cobra venom factor immediately prior to infection improves survival from 1LD50 of virus inoculated intranasally (Finnie et al. 1982). Similarly, tumour necrosis factor (TNF)-α appears to play more of a deleterious than a beneficial role in resistance
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to Sendai virus infection. Passive immunization of mice against TNF- α protects mice from lethal Sendai virus infection (Iwai et al. 1998). ADAPTIVE IMMUNITY During the course of Sendai virus infection, cellular and humoral immune responses are initiated, which ultimately lead to virus elimination while producing immunopathic lung injury. Sendai virus–specific, MHC class I–restricted CD8+ α β–cytotoxic T (Tc) cells are the dominant effectors of Sendai virus clearance during primary infection. MHC class II–restricted CD4+ α β−T cell responses are generally subordinate to class I–restricted responses and provide an alternative but delayed and less effective clearance mechanism. Both are required, however, to effectively deal with potentially lethal Sendai virus infection (Iwai et al. 1988, 1989). C57BL/6 mice that are homozygous for a disruption of the H-2I-Ab class II MHC glycoprotein gene generate normal Sendai virus–specific CD8+ Tc but fail to make virus-specific immunoglobulin G class antibodies. When exposed to sublethal doses of Sendai virus, clearance of virus is similar to that of normal mice (Hou et al. 1995). C57BL/6 that are homozygous for a disruption of the β2 microglobulin gene lack class I MHC glycoproteins and fail to develop Sendai virus–specific restricted CD8+ α β−Tc but have normal virus-specific, class II MHC–restricted CD4+ α β−T cell responses, including humoral immune responses. These mice have delayed clearance of sublethal doses of Sendai virus and eventually clear the infection. Depletion of CD4+ cells in these mice results in total failure to clear the infection and death (Hou, Doherty, et al. 1992). This indicates that class I MHC–restricted CD8+ α β–cytotoxic T cells play a dominant role in the recovery of C57BL/6 mice from sublethal Sendai virus infection but that class II MHC–restricted CD4+ α β–T cells provide an alternative mechanism, presumably by way of producing virus-specific antibody-forming cells, for viral clearance, albeit not as efficient at clearing a primary infection. CD4+ α β–T cells are the major source of cytokines found in bronchoalveolar lavage fluid during acute Sendai virus infection, including interleukin (IL) 2, IL-4, IL-6, IL-10, and IFN-γ (Mo, Sarawar, et al. 1995). C57BL/6 mice that are congenic for a mutation in the class I MHC gene H-2Kb fail to generate Sendai virus–specific CD8+ α β–Tc but develop normal B cell responses. These mice are 10-fold more susceptible to the lethal effects of Sendai virus than normal C57BL/6 mice but are 30-fold more resistant than athymic (nu/nu) C57BL/6 mice to lethal infection (Kast et al. 1986). This indicates that class I MHC–restricted CD8+ α β–Tc cells and class II MHC–restricted CD4+ α β−T cells cooperate when mice are infected with potentially lethal doses of Sendai virus. Sendai virus–specific, class I MHC–restricted CD8+ α β–Tc cell responses are normal in C57BL/6 and BALB/c mice with targeted disruptions of the IFN-γ gene and the H-2I-Ab class II MHC glycoprotein gene that is required for production of CD4+ T cell help (Hou et al. 1995; Mo et al. 1997; Tripp et al. 1997).
296 In C57BL/6 mice, Sendai virus–specific CD8+ α β–Tc lyse target cells expressing NP but not those expressing F, HN, M, or P/C/V. Thus, in this strain, only NP among these antigens is a significant MHC class I target (Cole, Hogg, et al. 1994). The dominant epitope of NP based on assays of synthetic peptides is residues 324–332 (Cole, Hogg, et al. 1994). By contrast, the HN glycoprotein of Sendai virus provides most of the class II epitopes that elicit CD4+ T cell responses in this mouse strain (Cole, Katz, et al. 1994). The CD8+ α β−T cell response requires syngeneity with the K and/or D regions of the major histocompatibility complex (H-2). In most inbred mouse strains, sygeneity at K rather than D is required. In BALB/c, DBA, and A strain mice, however, syngeneity at either is sufficient (Kurrle et al. 1978; de Waal et al. 1983). C57BL/6 mice also develop an early γδ-T cell response in the lungs that peaks and declines coincident with lung virus titers. These cells comprise 5%–20% of lymphoid cells recovered from bronchoalveolar lavage populations in C57BL/6 mice during the course of Sendai virus infection (Hou, Katz, et al. 1992). Sendai virus–induced γδ-T cells are specific for endogenous 65-kDa heat shock protein expressed by macrophages in response to infection (Hou, Katz, et al. 1992; Ogasawara et al. 1994). The role played by γδ-T cells in virus clearance and immunopathic lung injury is currently not known. Following intranasal Sendai virus infection of C57BL/6 and 129/Sv mice, Sendai virus–specific antibody forming cells in mediastinal and superficial cervical and mediastinal lymph nodes show a peak of IgM-producing cells on about day 7 and then IgG- and IgA-producing cells at about day 10 after infection (Sangster et al. 1995). In C57BL/6 mice, IgG isotypes are evenly distributed among IgG1, IgG2a, IgG2b, and IgG3. By contrast, IgG2a is the predominant IgG isotype in 129/Sv mice, an inherent proclivity of 129 lymphoid cells (Mo, Sangster, et al. 1995; Sangster et al. 1995). Approximately 90% of plasma cells recovered from the respiratory tract of C57BL/6 mice after Sendai virus infection by a natural route produce antibodies with specificity for at least 5 H-2I-Ab-restricted epitopes in the virus hemagglutininneuraminidase (HN). The majority of the remaining plasma cells have specificity for F glycoprotein, which contains at least 4 MHC class II–restricted epitopes (Orvell and Grandien 1982; Cole et al. 1994). Some epitopes on Sendai virus HN proteins induce antibodies that neutralize infectivity as measured by inhibition of plaque formation, hemadsorption, cytopathic effects, or end point dilution in embryonated eggs (Orvell and Grandien 1982). Anti-F antibodies have little or no neutralizing capacity except as measured by plaque reduction in Vero cells (Orvell and Norby 1977). Mice passively administered polyclonal or monoclonal antibodies to Sendai HN or F proteins are resistant to challenge with Sendai virus (Orvell and Norrby 1977; Orvell and Grandien 1982). Lung virus titers in mice passively immunized with anti-HN and anti-F antibodies prior
D AV I D G . B R O W N S T E I N
to infection are reduced regardless of their ability (i) to inhibit hemagglutination, neuraminidase activity, hemolysis, or fusion, (ii) to neutralize in vitro, or (iii) to fix complement (Orvell and Grandien 1982). Immunoglobulin A anti-Sendai virus HN monoclonal antibodies neutralize virus in vitro and provide passive protection against Sendai virus infection (Mazanec et al. 1987). Induction of CD4+ T helper cells requires class II MHC compatibility in the H-2IA region between T cells and antigen-presenting cells (Ertl 1981). Sendai virus–specific memory CD4+ T cells tend to distribute more to the spleen than to the regional lymph nodes of the respiratory tract 2–3 months after infection in C57BL/6 mice (Ewing et al. 1995; Topham and Doherty 1998). Serum antibodies to Sendai virus first appear 10–12 days after experimental infection (Blandford et al. 1971; van der Veen et al. 1970; Parker et al. 1979; Brownstein et al. 1981). Titers usually rise during the first 4–6 weeks of infection and then plateau (Parker et al. 1979). Passive maternal immunization of suckling mice with Sendai virus–specific antibodies of IgG1 and IgG2 isotypes provides solid protection against infection in suckling mice (Iida et al. 1973). Transplacental and colostral routes are apparently involved in passive immunization (Iida et al. 1973).
VII.
EPIZOOTIOLOGY A.
Host Range
Naturally occurring Sendai virus infections with confirmatory virus isolation have been reported in laboratory mice, rats, and hamsters. Serological evidence of Sendai virus infection has been reported in guinea pigs, which are indeed susceptible to experimental infection (Takakura et al. 1988). The recent recovery and characterization of caviid parainfluenza virus 3 and guinea pig parainfluenza virus 3 from naturally infected guinea pigs raises the possibility and probability that some or all serologic reactivity to Sendai virus is heterologous reactivity to these closely related Respiroviruses (Simmons et al. 2002).
B.
Geographical Distribution
Sendai virus infection of laboratory mice is distributed worldwide, having been reported in most developed regions of Asia, Australia, Europe, and the Americas. Rising prevalence rates were reported by Parker and Richter (1982) in several regions between 1968 (44% of colonies tested) and 1979 (85% of colonies tested). Since then, prevalence rates have declined due to improvements in and more universal implementation of surveillance and barrier housing.
11. SENDAI VIRUS AND PNEUMONIA VIRUS OF MICE (PVM)
C.
Mode(s) of Transmission
Sendai virus is highly contagious and is spread by direct contact with infected respiratory secretions and by aerosol transmission (Iida 1972). Experimentally infected mice most readily transmit infection between days 4–9 and rarely before or after this interval (van der Veen 1970). Airborne transmission is highly variable depending on numbers of transmitters, relative humidity, air flow, and distance to susceptible individuals. The risk of aerosol transmission to naive mice is proportional to the number of transmitters (van der Veen et al. 1970). High relative humidity (60%–70% versus 30%–45%) increases airborne transmission rates significantly (van der Veen et al. 1972). Higher transmission rates occur at 5–10 air exchanges per hour than at 20 air changes per hour (van der Veen 1972). Natural Sendai virus infections follow two basic epizootiological patterns that are typical for acute limited viral infections. (1) Enzootic infection is the common self-perpetuating pattern occurring in breeding colonies or in colonies in which susceptible mice are regularly introduced. Adult resident mice have active immunity due to prior infection. Neonatal mice have passive protection from maternal antibodies. Mice become susceptible to infection between 4–8 weeks of age as maternal protection wanes. Infection is maintained in the colony by the continuous supply of susceptible young mice becoming infected by the slightly older, acutely infected mice. Enzootic infection of mouse breeding colonies is frequently subclinical, in part because partial protection is often afforded to the susceptible population by waning passive immunity. Some mild clinical manifestations may be evident, however, in actively infected individuals. In the open colony setting, newly introduced naive mice become infected by recently introduced mice, and these infected individuals more frequently exhibit clinical disease due to the absence of passive antibodies. (2) Epizootic infection is the acute, often explosive, infection that occurs when Sendai virus is introduced into a naive colony. Infection rapidly spreads through the entire population and is much more likely to elicit clinical disease than enzootic infection. Clinical manifestations are nevertheless highly variable, depending on host (strain, age, genomic modification, intercurrent infections, and health) and environmental factors (temperature, humidity, air flow, husbandry).
VIII.
DIAGNOSIS
The diagnosis of Sendai virus infection is not difficult. The infection proceeds through predictable overlapping phases, during which certain diagnostic procedures are appropriate. No single diagnostic test can detect the infection during all phases. Virus isolation is the most definitive but also the most labor-intensive means of diagnosing Sendai virus infection, but
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infectious virus is often not present when animals become clinically ill. Animals selected for virus isolation should therefore be the least clinically affected individuals in contact with sick animals and preferably seronegative for Sendai virus. Sterile lung suspensions or nasal washes may be used. Sendai virus can be detected by its cytopathic effects on primary or secondary rhesus or cynomolgous monkey kidney cells. Virus can also be detected in susceptible but nonpermissive serial cell lines such as BHK-21, L929, BSC-1, LLC-Mk2, HeLa, and RK-13 by plaque formation using trypsin overlays, visualization of viral antigens using immunofluorescence, or hemadsorption. Virus can also be recovered by inoculation into the amniotic or allantoic sac of 8- to 10-day-old embryonated hen’s eggs. Histopathology is the least specific and also the least sensitive test employed. Many cases of Sendai virus infection do not involve sufficient respiratory injury to allow a presumptive diagnosis of Sendai virus infection to be made on the basis of histopathology. Since the principal site of viral replication is the respiratory epithelium, lesions, when present, usually consist of transient erosive or proliferative rhinitis, laryngotracheitis, or bronchitis-bronchiolitis. When the secondary sites of viral replication become involved, alveolar type II and to a lesser extent type I cells, a characteristic bronchointerstitial pneumonia develops that may lead to multifocal adenomatous change, squamous metaplasia or, in severe cases, organizing alveolitis. Such lesions are most likely to be detected in animals susceptible by virtue of age (sucklings and aged), heredity (inbred strains such as 129 and DBA), immune suppression, or debility. The application of immunohistochemistry to fixed tissue specimens provides a relatively simple means of combining histopathology and histochemistry, thereby extending the sensitivity and specificity of fixed tissue evaluation. Hall and Ward (1984) compared a variety of tissue fixatives and two immunocytochemical techniques for demonstrating Sendai virus antigens in mouse lungs. They concluded that the avidinbiotin-peroxidase complex technique was superior and could be applied to lungs fixed with 10% neutral formalin, Bouin’s, B-5, or Zenker’s fixatives. Failure to demonstrate viral antigens in lesions does not, however, preclude a Sendai virus etiology, since viral antigens are cleared before the resolution of lesions in most cases. Reverse transcriptase-polymerase chain reaction assays are useful for detection of Sendai virus genomes during the phase of active virus replication and beyond (Wagner et al. 2003). The fluorogenic nuclease RT-PCR assay that combines RTPCR with an internal fluorogenic hybridization probe can detect as little as 10 fg of Sendai virus RNA. Detection of serum antibodies is the most widely used technique for diagnosing Sendai virus infection, with numerous kits commercially available. Early serologic assays for Sendai virus, which relied on biological activity, included complement fixing, hemagglutination inhibition, hemadsorption inhibition, hemolysis inhibiting, tissue culture neutralization, and agar
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gel precipitation. These have been superseded by more sensitive binding antibody assays, notably ELISA (Ertl et al. 1979) and indirect fluorescent antibody assays (Smith 1986; Lucas et al. 1987). Serum ELISA and complement fixing antibodies to Sendai virus first appear 10–12 days (van der Veen et al. 1970; Parker et al. 1979; Brownstein et al. 1981) after experimental infection. HAI antibodies appear slightly later (Parker et al. 1979). Rising titers in serially bled animals can be interpreted to indicate recent exposure. The ELISA successfully detects anti-Sendai virus antibody in infected athymic (nu/nu) mice. By contrast the CF, HAI, and neutralizing antibody assays are generally negative (Ueda et al. 1977; Iwai et al. 1979, 1984). Using the ELISA, Iwai et al. (1984) were able to demonstrate Sendai virus–specific antibodies of IgM, IgG1, IgG2a, IgG2b, and IgG3 classes in athymic (nu/nu) mice after experimental inoculation.
IX.
CONTROL AND PREVENTION
Sendai virus is highly contagious, being transmitted by direct contact with infected secretions and by aerosol. Prevention requires strictly enforced barrier operations. Replacement stocks must be from Sendai virus–free sources as demonstrated by regular serologic surveillance. Tumor lines must be assessed for contamination using MAP tests, virus isolation, or PCR. The most effective way to eliminate Sendai virus infection from a mouse colony is to cull the entire colony and obtain clean replacement stock. Sendai virus is labile in the environment and rapidly inactivated outside the murine host. Caesarian rederivation and embryo transfer can be used to obtain virusfree progeny from infected or exposed dams (Carthew et al. 1983; Okamoto et al. 1990). These are transferred to clean foster mothers or embryo recipients under strict antiseptic and barrier conditions. A strict breeding moratorium of at least 8 weeks can eliminate enzootic Sendai virus infection by eliminating the temporal cycle of susceptible weanlings becoming infected from older, actively infected adolescents. Vaccination has been touted in the past as a means of prevention and control of Sendai virus infection. There are currently few advocates of this approach, given the general trend toward barrier control of Sendai virus and other rodent viruses. Killed vaccines (Fukumi and Takeuchi 1975; Nedrud et al. 1987; Tsukui et al. 1982), temperature-sensitive mutant strains (Kimura et al. 1979), and protease-activation mutant strains (Tashiro and Homma 1985; Tashiro et al. 1988; Maru et al. 1992; Wang et al. 1994) have been tested experimentally. Vaccination may prevent or inhibit replication of wild-type virus depending on the type of vaccine, the immunization protocol, and the strain and age of mice, including their immunocompetence. Justification of vaccination as a means of preventing and controlling Sendai virus infection needs to be judged on a case-by-case basis, and consideration should be
given to the possible interfering effects of vaccination on experimental mice.
X.
PNEUMONIA VIRUS OF MICE (PVM): INTRODUCTION
Pneumonia virus of mice (PVM) has always been overshadowed by its more pathogenic paramyxovirus relative, Sendai virus. Although PVM was recovered and described over a decade before Sendai virus under similar circumstances, it has remained more obscure due to its less ubiquitous distribution, lower contagion, and generally more benign symptomatology. Nevertheless, PVM is an important respiratory virus, in part because it is an opportunistic pathogen in immunologically compromised mice and because it represents the only naturally occurring Pneumovirus of mice. The human Pneumovirus, respiratory syncytial virus, is an important respiratory pathogen of children. Studies of PVM infection in mice are beginning to elucidate aspects of Pneumovirus pathogenesis and immunobiology that are directly relevant to this important human disease.
XI.
HISTORY
PVM was first described in 1939–1940, 14 years before initial descriptions of Sendai virus (Horsfall and Hahn 1939, 1940). Like Sendai virus, PVM was identified during attempts to recover human respiratory viruses from clinical materials by inoculating them into laboratory mice (Horsfall and Hahn 1939, 1940; Mills and Dochez 1944, 1945). Serially passaged lung homogenates from inoculated and uninoculated mice led to high rates of lung consolidation and mortality. The virus that was recovered agglutinated a narrower spectrum of erythrocytes than Sendai virus and did not grow in embryonated hen’s eggs. These somewhat primitive studies failed to delineate the role played by PVM in the lung consolidation that was observed in these experiments, as laboratory mice undoubtedly harbored various other respiratory agents.
XII.
PROPERTIES OF THE VIRUS
The International Committee on Taxonomy of Viruses classifies PVM as follows: Order: Mononegavirales (nonsegmented, linear, negativestranded RNA genome) Family: Paramyxoviridae (15–16 kb genome, pleomorphic virions) Subfamily: Pneumovirinae (genome encodes 10 transcripts) Genus: Pneumovirus (genome encodes NS1 and NS2)
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11. SENDAI VIRUS AND PNEUMONIA VIRUS OF MICE (PVM)
Some of the properties of PVM are listed in Table 11-4. The single-stranded, negative-sense RNA genome containing approximately 15,300 nucleotides (Chambers et al. 1990) is tightly associated with the major nucleocapsid protein, N, 393 amino acids in length (Barr et al. 1991). The virally encoded polymerase is packaged in the virion and is associated with the nucleocapsid. It consists of a major subunit, the L protein (2150 amino acids), and the smaller P protein (295 amino acids) (Barr et al. 1994). The matrix protein, M, is 257 amino acids in length (Easton and Chambers 1997). Two glycosylated proteins are associated with the virion envelope, G and F. The G protein is a 290 amino acid surface glycoprotein that mediates attachment (Randhawa et al. 1995). It lacks neuraminidase but mediates hemagglutination. F, the fusion glycoprotein, is 537 amino acids in length (Chambers et al. 1992). It mediates fusion of the viral envelope with the plasma membrane of the host cell. The fusion glycoprotein of PVM, like that of Sendai virus, is translated as an inactive molecule, F0, which requires cleavage by a furin-like host protease to become biologically active (Ling and Pringle 1982; Chambers et al. 1992). The 113 amino acid NS1 protein and the 156 amino acid NS2 protein are nonessential for viral replication but confer advantages in cell cultures and possibly in vivo (Chambers et al. 1991). In the closely related bovine respiratory syncytial virus, there is evidence that NS1 and NS2 antagonize interferon-mediated
TABLE 11-4
PROPERTIES OF PVM 1. Size 100 nm diameter, up to 3 µm in length (filaments), 80–200 nm in diameter (spheres) 2. Morphology Pleomorphic, usually filaments (Fig. 11-17) less commonly spheres (Compans et al. 1967; Berthiaume et al. 1974) 3. Nucleic acid Nonsegmented, linear, negative-stranded RNA 4. Encoded mRNAs from 3′ to 5′ NS1, nonstructural protein NS2, nonstructural protein N, nucleoprotein P, phosphoprotein polymerase co-factor M, matrix protein SH, small hydrophobic protein G, attachment glycoprotein F, fusion glycoprotein M2, transcription factor L, large nucleocapsid-associated protein, major polymerase subunit 5. Replication site Cytoplasm 6. Site of virion formation Plasma membrane 7. Stability at room temperature 99% infectivity lost by 48 hours in 2% protein suspension; complete inactivation between 5 and 14 days (Horsfall and Hahn 1939) 8. Agglutinates erythrocytes from: mouse, rat, and hamster (Mills and Dochez 1944, 1945; Curnen and Horsfall 1946)
Fig. 11-17 Filamentous forms of PVM (*). Note terminal swelling, a common feature of filamentous forms. (×60,000)
antiviral responses (Schlender et al. 2000). They may therefore have roles similar to those of the Sendai virus C proteins but have low homology with their RSV counterparts (Chambers et al. 1991). The M2 gene product is a 176 amino acid protein that functions as an elongation and anti-termination factor (Ahmadian et al. 1999). The function of the small (92 amino acids) hydrophobic protein, SH, is currently not known (Easton and Chambers 1997).
XIII.
VIRUS STRAINS AND ANTIGENIC RELATIONSHIPS
All PVM strains are considered antigenically homologous (Horsfall and Hahn 1939, 1940; Pearson and Eaton 1940). Continually mouse-passaged strain J3666 is virulent for mice (Cook et al. 1998). Strain 15, continually passaged in BHK-21 cells, is apathogenic for mice (Randhawa et al. 1995). Tissue culture passage of PVM rapidly diminishes virulence for mice (Harter and Choppin 1967). PVM is antigenically distinct from human and ruminant respiratory syncytial viruses. It does, however, share an identical genomic organization and nucleotide sequence homology in several genes, notably N and M2 (Berthiaume et al. 1974; Chambers et al. 1990; Barr et al. 1991; Ahmadian et al. 1999). There is weak immunologic cross-reactivity between PVM and RSV P and N proteins (Ling and Pringle 1989).
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XIV.
XV.
GROWTH IN VIVO AND IN VITRO A.
A.
Growth in Mice
Under natural conditions, PVM infection is acute and restricted to respiratory epithelium in immunocompetent mice. After intranasal inoculation of young adult BALB/c mice, infectious virus can first be isolated from the lungs 48 hours post-inoculation (Fig. 11-18). Virus titers rise to a peak on day 5, decline on days 6–8, and are undetectable on or after day 10 (Cook et al. 1998). Older studies in outbred mice showed more extended virus replication kinetics, with peak virus titers achieved 6–8 days after intranasal inoculation, declining titers beginning on day 9, and no detectable virus by day 14 (Horsfall and Ginsberg 1951).
B.
IN VIVO INFECTION
Growth in Eggs and Cell Culture
PVM does not grow in embryonated hen’s eggs. PVM replicates in primary hamster kidney cell culture (Tennant and Ward 1962), hamster embryo (Reed et al. 1975), and continuous cell lines BHK-21 (Harter and Choppin 1967) and Vero (Berthiaume et al. 1974). In Vero cells, cytopathic effects are slowly progressive without syncytia formation. Cytoplasmic inclusions appear after 24 hours (Parker and Richter 1982).
Clinical Disease
Clinical disease has not been associated with naturally occurring PVM infection in immunocompetent mice. Experimental infection of 6-week-old BALB/c with the pathogenic mouse lung–passaged J3666 strain of PVM (Rockefeller Institute, New York) has been described as causing the following clinical signs: ruffled fur, piloerection, depression, deep breathing, labored breathing, tremors, lethargy, emaciation, cyanosis of tail and ears, and death (Cook et al. 1998). After experimental inoculation, signs of illness first appear between days 3–5, with labored breathing, wasting, and cyanosis at their greatest between days 6–9. The onset and severity of clinical disease is related to the amount of virus and the volume of intranasal inoculum. Chronic wasting and fatal pneumonia in athymic nude (nu/nu) mice naturally infected with PVM has been described (Richter et al. 1988; Weir et al. 1988). B.
Pathogenesis
PVM infection is restricted to the respiratory tract. Virus distribution is determined by the amount of virus and the volume of the inoculum. Under conditions of natural infection with relatively small intranasal doses of virus, spotty replication restricted to nasal epithelium is likely to be the extent of the
10
8
6 Log10 pfu/g
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2
0
1
2
3
4
5 6 7 8 Days Post-Infection
9
10
11
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Fig 11-18 PVM titers in the lungs of young adult BALB/c mice after intranasal infection with the pathogenic J3666 strain. (Modified from Cook et al. 1998, with permission.)
11. SENDAI VIRUS AND PNEUMONIA VIRUS OF MICE (PVM)
301
Fig. 11-19 Demonstration of PVM antigens in nasal turbinate from a BALB/c mouse after intranasal infection. The apical cytoplasm of pseudostratified ciliated epithelial cells contain viral antigens. (Indirect immunofluorescence, ×680)
infection (Fig. 11-19). Under experimental conditions with larger inoculums, virus replication occurs in alveolar lining cells, possibly alveolar macrophages, and to a lesser extent bronchiolar epithelium (Fig. 11-20). 1.
Age, Gender, and Mouse Strain Dependence
There are no published reports on the effects of age, gender, or mouse strain on susceptibility to PVM-induced respiratory disease.
2. Gross Changes
Gross lung changes are unlikely to be encountered in natural PVM infections of immunocompetent mice. Naturally infected athymic nude (nu/nu) mice sustain chronic weight loss and progressive pulmonary consolidation. Lungs are initially diffusely or multifocally dark red, firm, and fail to collapse. Later, they develop a reticulated interstitial pattern somewhat reminiscent of pneumocystosis (Richter et al. 1988; Weir et al. 1988).
Fig. 11-20 Expression of PVM antigens in lung after intranasal infection. Viral antigens are present in large alveolar cells, most probably type II pneumocytes. (Avidin-biotin immunoperoxidase, ×680)
302 3.
Microscopic Changes
Naturally occurring PVM infection is more likely to cause histopathologic changes in the nasal passages than in the lungs. Mild transient exfoliative rhinitis with sparse suppurative exudate and mixed inflammatory infiltrates of the nasal mucosa has been described (Smith et al. 1984) (Fig. 11-21). Experimentally produced PVM pneumonia in BALB/c mice with the pathogenic J3666 strain consists of mild necrotizing terminal bronchiolitis and nonsuppurative interstitial pneumonia (Cook et al. 1998). Lungs of outbred mice experimentally infected with low mouse-passaged wild-type PVM have mild patchy interstitial pneumonia and variable perivascular lymphoid infiltrates (Fig. 11-22). It is in immune incompetent mice (nu/nu and scid) that the most dramatic microscopic lung changes occur (Fig. 11-23). These consist of monocytic exudates (alveolar histiocytosis), intra-alveolar hemorrhage and fibrin deposition, septal edema, fibrosis, and mononuclear cell infiltration (Richter et al. 1988, Weir et al. 1988) (Fig. 11-23).
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BALB/c and C57BL/6 mice infected intranasally with the continually mouse-passaged and virulent J3666 strain of PVM mount an immediate acute inflammatory response containing 12%–14% eosinophils as early as day 3 post-inoculation (Domachowske et al. 2000, 2002). Local production of the CC chemokine MIP-1α is the apparent chemoattractant for eosinophils. C57BL/6 mice with a targeted disruption of the gene encoding MIP-1α (MIP-1α −/−) fail to recruit eosinophils and recruit fewer neutrophils during the acute phase of PVM infection. Lung virus titers during the acute phase are 10-fold higher in MIP-1α −/− mice than in wild-type mice (Domachoske et al. 2000), supporting an antiviral role for eosinophils or neutrophils during the acute phase of PVM infection.
XVI. A.
4.
Immune Response
There have been relatively few investigations of the immunobiology of PVM infection. As the only known naturally occurring pneumovirus infection in mice, and given its close relationship to clinically significant respiratory syncytial viruses of humans and ruminants, this is surprising. Recent investigations have targeted the innate phase of the immune response to PVM infection. Clearly, the chronic lethal disease that PVM infection elicits in athymic (nu/nu) mice signals the preeminent role played by T cells in the adaptive response that leads to virus clearance and resolution.
EPIZOOTIOLOGY
Host Range and Geographical Distribution
The natural host range of PVM infection consists of mice, rats (Rattus and Sigmodon), and hamsters (Parker and Richter 1982). Serological surveys of infections in rodent colonies carried out in the 1960s and 1970s were reviewed by Parker and Richter (1982) and indicated that PVM was prevalent and geographically dispersed, including in North America, Europe, and Asia. More recent surveys suggest that the prevalence of PVM has declined (Gannon and Carthew 1980; Lussier and Descorteaux 1986). This decline is attributable to increased awareness and serological monitoring of rodent colonies and improvements in rodent barrier facilities and their more universal implementation.
Fig. 11-21 Nasal cavity after intranasal infection with PVM. The lumen contains a mucopurulent exudates. Surface epithelium is attenuated, and there are mixed inflammatory cells in the lamina propria and traversing the epithelium. (H&E, ×170)
303
11. SENDAI VIRUS AND PNEUMONIA VIRUS OF MICE (PVM)
Fig.11-22 Lung changes in outbred Swiss mouse after intranasal inoculation of low mouse-passaged wild-type PVM. Note perivascular lymphoid infiltrates. (H&E, ×85)
B.
Mode(s) of Transmission
Epizootiological evidence indicates that PVM infection has low transmissibility and is probably spread by direct contact with infected respiratory secretions. PVM is rapidly inactivated at room temperature, and aerosol transmission is apparently not significant. The low contagiousness of PVM is indicated by the frequent finding of focal seropositive animals within a colony. In most enzootically infected colonies, fewer than 25% of mice
are seropositive for PVM (Tennant et al. 1966). There have been no published reports on experimental transmission of PVM.
XVII.
DIAGNOSIS
Like Sendai virus, PVM infection proceeds through predictable overlapping phases, during which certain diagnostic procedures
Fig. 11-23 Lung changes in athymic nu/nu mouse with spontaneous PVM pneumonia. There is diffuse nonsuppurative inflammation, localized primarily to alveolar walls. (H&E, ×170)
304
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are appropriate. No single diagnostic test can detect the infection during all phases. Virus isolation is the most definitive means of diagnosing PVM infection, but it is complicated by the usually low prevalence of infection in a colony and the absence of clinical signs. Animals selected for virus isolation should be seronegative for PVM, but a majority of these are likely to be uninfected. Sterile lung suspensions or nasal washes may be used. BHK-21 and Vero cells are sensitive to PVM with subsequent demonstration of typical CPE, hemagglutinins, IFA, or hemadsorption. A plaque assay using BHK-21 cells with proteolytic enzyme in the overlay medium has been described (Shimonaski and Carne 1970). Histopathology is the least specific and also the least sensitive test employed. Most cases of PVM infection in mice do not have identifiable lung lesions (interstitial pneumonia). Mild rhinitis may be the only recognizable lesion. Athymic nude (nu/nu) mice develop a characteristic and severe diffuse interstitial pneumonia. Reverse transcriptase-polymerase chain reaction assays are useful for detection of PVM genomes during the phase of active virus replication and beyond (Wagner et al. 2003). Detection of serum antibodies is the most widely used technique for diagnosing PVM infection, with numerous kits commercially available. Serological tests include: HAI, CF, HadI, neutralization, IFA, and ELISA. Infected mice usually seroconvert by HAI and CF 9–10 days after infection. CF antibody titers decline after 2 weeks, whereas HAI antibody persists at high titers for at least 4 months (Tennant et al. 1966). For routine monitoring, ELISA is the most sensitive of the serologic tests available (London et al. 1983).
XVIII.
CONTROL AND PREVENTION
Caesarian derivation or embryo transfer and barrier maintenance effectively eliminate and prevent PVM infection. Because enzootic PVM infection in a colony is usually focal, it is often possible to remove individual breeding pairs to isolators, select those that remain free of serum antibodies to PVM, and use these breeders to repopulate a PVM-free colony. Alternatively, PVM seropositive breeding pairs that have recovered from acute PVM infection can be isolated and will produce seronegative progeny. A strict breeding moratorium cannot eliminate PVM infection because of the focal smouldering nature of enzootic PVM infection.
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Tashiro, M., McQueen, N.L., and Seto, J.T. (1999). Determinants of organ tropism of sendai virus. Front. Biosci. 4, D642–D645. Tashiro, M., McQueen, N.L., Seto, J.T., Klenk, H.D., and Rott, R. (1996). Involvement of the mutated M protein in altered budding polarity of a pantropic mutant, F1-R, of Sendai virus. J. Virol. 70, 5990–5997. Tashiro, M., Pritzer, E., Khoshnan, M.A., et al. (1988). Characterization of a pantropic variant of Sendai virus derived from a host range mutant. Virol. 165, 577–583. Tashiro, M, and Seto, J. T. (1997). Determinants of organ tropism of Sendai virus. Front. Biosci. 2, D588–D591. Tashiro, M., Seto, J.T., Choosakul, S., Hegemann, H., Klenk, H.D., and Rott R. (1992a). Changes in specific cleavability of the Sendai virus fusion protein: implications for pathogenicity in mice. J. Gen. Virol. 73, 1575–1579. Tashiro, M., Seto, J.T., Choosakul, S., Yamakawa, M., Klenk, H.D., and Rott, R. (1992b). Budding site of Sendai virus in polarized epithelial cells is one of the determinants for tropism and pathogenicity in mice. Virol. 187, 413–422. Tashiro, M., Yamakawa, M., Tobita, K., Klenk, H.D., Rott, R., and Seto, J.T. (1990a). Organ tropism of Sendai virus in mice: proteolytic activation of the fusion glycoprotein in mouse organs and budding site at the bronchial epithelium. J. Virol. 64, 3627–3634. Tashiro, M., Yamakawa, M., Tobita, K., Seto, J.T., Klenk, H.D., and Rott, R. (1990b). Altered budding site of a pantropic mutant of Sendai virus, F1-R, in polarized epithelial cells. J. Virol. 64, 4672–4677. Tashiro, M., Yokogoshi, Y., Tobita, K., Seto, J.T., Rott, R., and Kido, H. (1992c). Tryptase Clara, an activating protease for Sendai virus in rat lungs, is involved in pneumopathogenicity. J. Virol. 66, 7211–7216. Tennant, R. W., and Ward, T. G. (1962). Pneumonia virus of mice (PVM) in cell culture. Proc. Soc. Exp. Biol. Med. 111, 395–398. Tennant, R.W., Parker, J.C., and Ward, T.G. (1966). Respiratory virus infections of mice. Natl. Cancer Inst. Monogr. 20, 93–104. Topham, D.J., and Doherty, P.C. (1998). Longitudinal analysis of the acute Sendai virus-specific CD4+ T cell response and memory. J. Immunol. 161, 4530–4535. Towatari, T., Ide, M., Ohba, K., et al. (2002). Identification of ectopic anionic trypsin I in rat lungs potentiating pneumotrpic virus infectivity and increased enzyme level after virus infection. Eur. J. Biochem. 269, 2613–2621. Tripp, R.A., Sangster, M.Y., and Doherty, P.C. (1997). The cytotoxic T-lymphocyte response to Sendai virus is unimpaired in the absence of gamma interferon. J. Virol. 71, 1906–1910. Tsukui, M., Ito, H., Tada, M., Nakata, M., Miyajima, H., and Fujiwara, K. (1982). Protective effects of inactivated virus vaccine on Sendai virus infection in rats. Lab. Anim. Sci. 32, 143–146. Ueda, K., Tamura, T., Machii, K., and Fujiwara, K. (1977). An outbreak of Sendai virus infection in a nude mouse colony. Proc. Int. Workshop Nude Mice 2, 61–69. van der Sluijs, K.F., van Elden, L., Nijhuis, M., et al. (2003). Toll-like receptor 4 is not involved in host defense against respiratory tract infection with Sendai virus. Immunol. Lett. 89, 201–206. van der Veen, J., Poort, Y., and Birchfield, D.J. (1970). Experimental transmission of Sendai virus infection in mice. Arch. Gesamte Virusforsch. 31, 237–246.
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Chapter 12 Cardioviruses: Encephalomyocarditis Virus and Theiler’s Murine Encephalomyelitis Virus Howard L. Lipton, A.S. Manoj Kumar, and Shannon Hertzler
I. Introduction and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Biophysical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Viral Genome and Gene Products . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Virion Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Cardiovirus Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Antigenic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Growth In Vitro and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Clinical Disease and Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Group A (EMCV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Group B (TMEV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Natural Infection: Oral Route . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Experimental Infection: I.C. Inoculation . . . . . . . . . . . . . . . . . . VI. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Group A (EMCV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Group B (TMEV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION AND HISTORY
The cardiovirus genus in the family Picornaviridae consists of two groups, encephalomyocarditis virus (EMCV, group A) and Theiler’s murine encephalomyelitis virus (TMEV, group B) (Table 12-1). EMCV were first described in the early 1940s as THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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an infection of laboratory rodents (MM strain) (Jungeblut and Dalldorf 1943) but were later isolated in Florida from a captive chimpanzee with acute fatal myocarditis (R strain) (Helwig and Schmidt 1945) and in Uganda from a paralyzed rhesus monkey (Mengo 37A strain) (Dick et al. 1948). Other subsequent reports of EMCV isolations from rodents as well as outbreaks Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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TABLE 12-1
CLASSIFICATION OF CARDIOVIRUSES INTO TWO GROUPS Group A (EMCV)
Group B (TMEV)
Columbia SK EMCV, R strain (Helwig and Schmidt 1945) Mengo (Dick et al. 1948) MM (Jungeblut and Dalldorf 1943) Maus-Elberfeld (ME)
GDVII (Theiler and Gard 1940a)a FA (Theiler and Gard 1940a)a Ask-1 (Casals 1963)a DA (Daniels et al. 1952)b BeAn 8386 (Rozhon et al. 1983)b TO 1-5 (Theiler 1937)b Yale (Melnick and Riordan 1947)b WW (Wroblewska et al. 1977)b TO (B15) (Feltz et al. 1953)b Vie 415H (Casals 1963)b VL 761293 (Casals 1963)b MHG (Hemelt et al. 1974)c NGS910c (Ohsawa et al. 2003)
after peripheral routes of inoculation; (4) TMEV, but not EMCV, cause persistent infections in the CNS; and (5) 5′ untranslated sequences of EMCV contain long homopolymeric poly(C) tracts that are absent in TMEV. TMEV infection poses a greater epizootic threat for colonybred mice and rats used in biomedical research. In fact, outbreaks of EMCV infection are unknown in mouse and rat colonies, but experimental infections are usually fatal and provide experimental animal models for myocarditis and diabetes mellitus. Thus, in the context of cardiovirus-induced clinical diseases, this chapter focuses more on TMEV.
II. A.
PROPERTIES
Biophysical Properties
aHigh-neurovirulence
group. bLow-neurovirulence group. cRat TMEV.
of myocarditis in pigs led to the designation of EMCV as cardioviruses. TMEV, named for Max Theiler, who in the early 1930s isolated the virus from mice with spontaneous paralysis (Theiler 1934), are well known to biomedical investigators because TMEV is a common cause of infections in mice that are not housed under barrier conditions. Outbreaks of TMEV infection in colonyraised rats are much less common. In nature, TMEV infection is widespread in feral Mus musculus, and to a lesser extent in certain species of voles (Lipton et al. 2001). Virus transmission occurs by the fecal-oral route, with TMEV causing inapparent infections of the intestine, although even inapparent infections may perturb immune and other physiologic host functions. On rare occasions, estimated at 1 in 500–1,000 infections (Olitsky 1939), TMEV spread to the central nervous system (CNS), causing cytolytic infection of spinal cord anterior horn cells and flaccid hind-limb paralysis, that is, mouse poliomyelitis (Theiler 1941). The route of viral spread to the CNS after natural infection is unknown but may occur during viremia, when infected macrophages presumably cross the blood-brain-barrier, or by centripetal spread of virus through retrograde axoplasmic flow. Experimental manipulation of acutely infected mice can also disseminate TMEV to the CNS. The relationship of TMEV to EMCV was first recognized after the genomes of TMEV strains were sequenced in the late 1980s (Pevear et al. 1987; Ohara et al. 1988). The viral genes encoded by the two groups share >50% amino acid identity and the atomic virion structures are very similar. However, several features distinguish the groups: (1) EMCV have a wide host range, including invertebrates, birds, and mammals (even humans), whereas TMEV have a narrow host range of mice, rats, and voles; (2) EMCV more often infect rats, while TMEV predominately infect mice; (3) EMCV are more virulent, especially
Cardiovirus particles are 28 nm in diameter with icosahedral symmetry. The capsids are compact and relatively impermeable to electron-dense salts, precluding elucidation of substructure by electron microscopy. Virions have a sedimentation coefficient of 150S by velocity centrifugation in sucrose and a buoyant density of 1.34 g/ml by isopycnic centrifugation in cesium salts. Virion RNA sediments as a 35S species. Cardioviruses are more resistant than lipid-containing viruses to chemical and physical agents. Although laboratory disinfectants such as 70% alcohol and 5% Lysol inactivate cardioviruses, 0.3% formaldehyde, 0.1 NHyCl, and bleach are preferable. Cardioviruses are insensitive to chloroform, ether, deoxycholate, and other detergents that inactive lipid-containing viruses. They are also stable at freezing temperatures for years and do not lose titer at refrigerator temperatures for several weeks; however, UV radiation and exposure to temperatures above 50°C rapidly inactivate infectivity. Cardioviruses are not susceptible to acid pH (3–5), a feature that distinguishes them from non-enterically transmitted picornaviruses. Cardioviruses are also thermolabile at 37°C at pH 5–7 in the presence of dilute halide ions, revealing a biphasic curve of pH stability (Spier 1962). Since investigators other than virologists may use phosphate-buffered saline (PBS) as a diluent for viruses, it is important to note that EMCV and low-neurovirulence TMEV strains are inactivated in PBS, pH 7.2 at 37°C (Spier et al. 1962). B.
Viral Genome and Gene Products
The genetic component of cardioviruses is a single-stranded RNA molecule, 7.4–8.1 kilobases (kb) in size and of positiveor message-sense polarity; thus, viral RNA is infectious. A 20amino acid basic protein, VPg, is covalently linked to the 5′ end of the RNA (the 5′ terminus lacks a cap structure), and a poly(A) tail of heterogeneous length is present at the 3′ end. The RNA consists of a long 5′ untranslated region, an open
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reading frame, and a short 3′ untranslated region. The 5′ untranslated region contains a stable 450-base segment of secondary structure to which ribosomes bind (internal ribsome entry site, IRES) for initiation of translation at an immediate downstream AUG codon located at the end of the IRES (Jang et al. 1988). Picornaviral IRESs have been categorized into five types; those of cardioviruses and aphthoviruses are type 2 IRESs. Group A cardioviruses are also distinguished by the presence of long homopolymer poly (C) tracts (60–350 bases) within their 5′ untranslated sequences. Truncation of the Mengo virus poly (C) track dramatically attenuates pathogenicity for mice (Duke et al. 1990; Palmenberg and Osorio 1994). Although TMEV lack a poly (C) track in the 5′ untranslated region, they have a pyrimidine-rich sequence in an analogous location. The specific mechanism of poly (C)-mediated pathogenesis is not known, but one possibility is by the abrogation of dsRNA-dependent protein kinase (PKR)-mediated antiviral activity (A.C. Palmenberg, personal communication). The cardiovirus open reading frame encodes a large polyprotein, exemplified in Fig. 12-1 by the polyprotein of the TMEV BeAn 8386 strain. The BeAn polyprotein initiates at the AUG codon at nucleotide 1065 and extends for 6909 nucleotides or 2303 codons, terminating at the single UGA triplet at nucleotide 7974. The final gene products are generated by co- and posttranslational proteolytic cleavages in the polyprotein while nascent on the ribosome, primarily by a virally encoded protease, 3Cpro. An exception is the first or primary cleavage after the 2A sequence, which is autocatalytic and a prerequisite for P1 processing (Hahn and Palmenberg 1996). Processing of the polyprotein is common to all picornaviruses and follows a standard L-4-3-4 arrangement with a single leader protein (L), four capsid polypeptides in part 1 (P1), three nonstructural polypeptides in P2, and four nonstructural polypeptides in P3 (Rueckert and Wimmer 1984) (Fig. 12-1). The first protein translated from the positive-strand RNA genome is the L protein, which has sequences consistent with N-terminal zinc-binding motifs, centrally located tyrosine kinase phosphorylation sites, and
C-terminal, acid-rich domains (Dvorak et al. 2001). The TMEV L protein has been reported to influence host range (Kong et al. 1994; Chen et al. 1995), and the L protein of both cardiovirus groups has been shown to inhibit production of interferon (IFN)-α/β, in part at the transcriptional level (van Pesch et al. 2001; Zoll et al. 2002). Delhaye et al. (2004) reported that the TMEV L protein interferes with cytoplasmicto-nuclear trafficking of IFN regulatory factor-3, which binds to the promoters of immediate-early IFN genes to activate their transcription. The four structural or capsid proteins are arranged in P1 as 1A, 1B, 1C, and 1D (Fig. 12-1). The original designation for capsid proteins was virus protein (VP) 1 to 4, based on molecular size as determined by SDS-polyacrylamide gel electrophoresis. Functions are known for two P2 polypeptides. Cardioviral 2A protein has a 19-amino acid C-terminal processing cassette that results in primary cleavage of the polyprotein between proteins 2A and 2B (Batson and Rundell 1991; Hahn and Palmenberg 1996). Protein 2A has a nuclear localization motif KRvRPFRLP, common to many yeast ribosomal proteins, that has been shown to target cellular nuclei, particularly nucleoli (Aminev et al. 2004). A role for protein 2A in host protein shutoff has also been proposed (Aminev et al. 2004). Cardioviral 2C is considered a second RNA-dependent polymerase; it has binding sites for nucleotides and cellular membranes involved in viral RNA replication. In common with other picornaviruses, the four mature P3 polypeptides are protein 3B or VPg; protein 3C, a chymotrypsin-like cysteine protease responsible for most of the Q-G dipeptide cleavages in the polyprotein; and protein 3D, an RNA-dependent RNA polymerase.
C.
Virion Structure
The three-dimensional structures of Mengo, BeAn, DA, and GDVII viruses have been determined by X-ray crystallography at high resolution (Luo et al. 1987, 1992, 1996; Grant et al. 1992).
Fig. 12-1 Representative cardiovirus genome. TMEV BeAn strain RNA showing the 5′ untranslated region containing a stable 450-base segment of secondary structure into which ribosomes bind (internal ribsome entry site, IRES) to initiate translation, a long open reading frame (encoding a long polyprotein that gives rise to 11 final gene products, shown just below the RNA), and a short 3′ untranslated region and poly(A) tail of heterogeneous length. A 20-amino acid basic protein, VPg, is covalently linked to the 5′ end of the RNA (the 5′ terminus lacks a cap structure). Posttranslational processing of the polyprotein follows a standard L-4-3-4 arrangement, i.e., a single leader protein (L), four capsid proteins in part 1 (P1), three nonstructural proteins in P2, and the four nonstructural proteins in P3.
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the Cα chain of >1.5 Å. There are also two amino acid deletions at the top of VP1 loop II in DA. The differences between GDVII and the low-neurovirulence strains in VP2 puff B might induce conformational changes in the VP2 puff and account for the differences in sialic acid binding ability of the two neurovirulence groups (see below).
The picornavirus shell structure is composed of 12 pentamers, each pentamer consisting of five protomers, and each protomer containing the four capsid proteins (Fig. 12-2A). VP1, VP2, and VP3 are exposed on the virion surface, which has a 25Å-deep receptor binding depression that spans residues at the junction of VP1 and VP3 (Fig. 12-2A), while VP4 is a short internal polypeptide exposed to the RNA. The folding motif of the three major capsid proteins (VP1, VP2, and VP3) consists of a wedgeshaped, eight-stranded antiparallel β-barrel (Fig. 12-2B). The structures of Mengo virus and TMEV are very similar except for the loops and C-termini on the outer surface, which have very different conformations. The TMEV high-neurovirulence GDVII and the low-neurovirulence BeAn and DA strains also show overall structural similarity, except in residues 170–173 in VP2 puff B and in residues 59 and 60 on the VP3 knob, which mainly involve side chains and a root-mean-square deviation of
D.
Cardiovirus Receptors
Virus-receptor interactions usually involve multiple steps to promote viral entry and infection. The initial step is often the lowaffinity binding to an attachment factor, that is, a co-receptor, which affects docking and accumulation of the virus on the cell surface. This interaction is followed by binding to a high-affinity entry receptor. Viral receptors and co-receptors have been
VP1
VP2
A
VP3
C
B
Fig. 12-2 A) Diagram of the icosahedral shell structure of cardioviruses. The virion is composed of 12 pentamers, each of which has five protomers, each protomer composed of four capsid proteins VP1, VP2, VP3 (exposed on the virion surface), and VP4 (buried beneath VP2 next to the virion RNA). The virion surface contains 25Å-deep receptor binding depressions (oval hatched areas) that span residues at the junction of VP1 and VP3. A single pentamer and one of its protomers are highlighted by darker lines. B) Ribbon drawings of VP1, VP2, and VP3 of BeAn virus showing an eight-stranded antiparallel β-barrel folding motif commonly found in the structural proteins of spherical viruses. The termini and prominent surface loops are labeled, as are the three immunodominant CD4+ T cell epitopes (red) (VP1233-244 in βH, VP274-86 in βC, and VP324-37 at the N-terminus) and one dominant (VP3159-166 in βG) and two subdominant (VP111-20 in the N-terminus and VP3173-181 between βC and βD) CD8+ T cell epitopes (blue) that have been mapped in susceptible SJL mice. C) Four neutralizing immunogenic sites mapped in Mengo virus are shown on a pentamer containing five protomers. Neutralizing immunogenic site 3 is located on VP1 loop II, site 4 on the VP1 first corner, site on on the VP2 first corner and puff A, and site 2 on the VP3 knob.
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identified for a number of picornaviruses (Rossmann et al. 2002). Since picornaviruses do not have an envelope that can fuse to the lipid bilayer of the cell membrane, virus entry and initiation of infection requires binding to a protein entry receptor. The presence of a depression on the cardiovirus surface, analogous to the canyon on polioviruses and rhinoviruses, strongly suggests that cardioviruses also require a protein entry receptor. Several picornavirus receptors belong to the immunoglobulin superfamily. Huber (Huber 1994) demonstrated that an immunoglobulin superfamily member, VCAM-1, acts as a receptor for EMCV on murine vascular endothelial cells. A TMEV protein entry receptor has not yet been identified, but members of both neurovirulence groups bind to a 34-kD membrane protein in a ligand binding assay (Kilpatrick and Lipton 1991). In addition, low-neurovirulence DA and BeAn use sialic acid (α2,3-linked N-acetylneuramic acid) on an N-linked glycoprotein as a co-receptor (Fotiadis et al. 1991; Shah and Lipton 2002), whereas the high-neurovirulence GDVII uses the proteoglycan heparan sulfate as a co-receptor (Reddi and Lipton 2002).
III.
ANTIGENIC PROPERTIES
Cardioviruses form two distinct serological groups, based on lack of cross-reaction by neutralization (Kerr 1952; Casals 1963). However, the extensive amino acid sequence identity of the two groups leads to cross-reaction in enzyme-linked immunoadsorbent assay (ELISA), complement fixation, and hemagglutination-inhibition tests (Shaw 1956; Calisher and Rowe 1966; Lipton 1978; Dick 1949; Warren et al. 1949). Although both groups agglutinate erythrocytes at 4°C, group A viruses agglutinate sheep and human type O erythrocytes, whereas group B viruses agglutinate only human type O erythrocytes. Neutralizing monoclonal antibodies (mAb) generated in mice have identified four neutralizing immunogenic sites on the surface of Mengo virus that are analogous to B cell epitopes on other picornaviruses (Boege et al. 1991; Kobasa et al. 1995). The sites are located on VP1 loop II (site 3), the VP1 first corner (site 4), the VP2 first corner and puff A (site 1), and the VP3 knob (site 2) (Kobasa et al. 1995) (Fig. 12-2C). Although systemic mapping of neutralizing immunogenic sites on TMEV has not been performed, neutralizing mAb have been obtained that react with VP1 residue 100 (equivalent to site 3 on Mengo virus), VP2 residue 143 (equivalent to site 1), and with a trypsin cleavage fragment at the C-terminus of VP1 (Nitayaphan et al. 1985a, 1985b; Zurbriggen and Fujinami 1989; Sato et al. 1996). The latter site is probably equivalent to site 2 since the VP1 C-terminus forms a hook near the three-fold axis on top of the VP3 knob. Escape mutant viruses at these sites displayed attenuated pathogenic phenotypes in mice (Zurbriggen and Fujinami 1989; Sato et al. 1996).
IV.
GROWTH IN VITRO AND IN VIVO
Cardioviruses are highly lytic for mammalian cells in culture, leading to cytopathology, that is, cell rounding and detachment from monolayers. In one-step growth kinetic experiments at 37°C, the cardiovirus life cycle is completed in ~12 hours, and virus yields range from 500–10,000 pfu/cell. EMCV replicate to higher titers and produce larger plaques than the high-neurovirulence GDVII virus on cell monolayers. The low-neurovirulence TMEV produce smaller plaques and are highly membrane-associated as compared to the high-neurovirulence strains, so that only a portion of the total viral yield reaches the supernatant. (High-neurovirulence strains release 90% of infectious virus into the supernatant [Friedmann and Lipton 1980].) Electron microscopy has shown that lowneurovirulence BeAn virions assemble single file between two unit membranes in the cytoplasm that extend to and open onto the cell surface, which explains their cell association (Friedmann and Lipton 1980). The nature of the interaction between TMEV and these unit membranes remains to be elucidated. Both groups of cardioviruses have been reported to trigger the apopototic pathway in mammalian cells. For TMEV, the apoptotic response depends on cell permissiveness for infection; cells in which virus replication is restricted undergo apoptosis, whereas necrotic cell death predominates in permissive, productively infected cells (Jelachich and Lipton 1996, 1999). TMEV infection is also highly restricted in murine macrophage cell lines and peritoneal and bone marrow-derived macrophages, as well as in some primate cell lines such as BSC-1 cells (Levy et al. 1992; Jelachich et al. 1995; Jelachich and Lipton 1996; Obuchi et al. 1997).
V.
CLINICAL DISEASE AND PATHOGENESIS A.
Group A (EMCV)
EMCV have been adapted by in vivo passage for increased neurovirulence, and adult mice and rats develop encephalomyelitis and/or myocarditis within a few days postinfection (PI); involvement of the brain is more likely after intracerebral (i.c.) inoculation and the heart, after peripheral inoculation (Tesh and Wallace 1978). Viremia and widespread virus replication in most tissues leads to damage of brain and/or myocardium (Kilham et al. 1955; Friedman and Maenza 1968; Campbell et al. 1970). Encephalitic animals appear unkempt, with ruffled fur and hunched posture, followed by flaccid paralysis and death. Craighead et al. (1966) generated EMCV variants that caused either encephalitis (E variant) or myocarditis (M variant), depending upon serial brain-to-brain or heartto-heart passage, respectively. Acute interstitial myocarditis ultimately leads to extensive mural and valvular endocardial
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damage (Burch et al. 1981). The M variant also replicates in pancreatic beta cells in inbred strains of mice, resulting in diabetes mellitus (Craighead and McClane 1968; Notkins 1977). Beta cells from susceptible mice support virus replication better than those from resistant mice, suggesting that genetic differences in viral susceptibility at the level of the beta cell determines susceptibility to diabetes (Notkins 1977).
B. 1.
Group B (TMEV)
Natural Infection: Oral Route
TMEVs are enteroviruses transmitted by the fecal-oral route. In natural infections, infectious virus has been isolated from intestine and mesenteric lymph nodes of mice (Gard 1944); viremia and spread of virus to systemic organs and occasionally to the CNS may occur. Brownstein et al. (1989) found that the duration of virus excretion varied from 49 to 154 days after feeding. In 1953, a highly CNS invasive mutant TMEV, TO (B15) was described that produced poliomyelitis in infected mice, but no information was given as to whether CNS virus persistence or demyelinating disease developed (Feltz et al. 1953). To date, it remains unclear whether a natural route of infection results in demyelination. There are no reports other than those above concern the pathogenesis of TMEV infection in adult mice by feeding. This is unfortunate since such experiments might provide insight into the sites of virus replication in the intestine and how TMEV invades the CNS. 2.
Experimental Infection: I.C. Inoculation
TMEVs have been divided into two groups based on neurovirulence following i.c. inoculation (Table 12-2). High-neurovirulence strains, such as GDVII and FA, produce a rapidly fatal TABLE 12-2
TWO TMEV NEUROVIRULENCE GROUPS BASED ON INFECTION AFTER I.C. INOCULATION Characteristic
High neurovirulence
Low neurovirulence
Disease
Encephalitis
Incubation perioda Target cellb
1–10 days Neurons
PFU/LD50c Persistent CNS infection Ts phenotyped Co-receptor use
1–10 No No Heparan sulfate
Poliomyelitis/demyelinating disease 7–20 days/>30 days Macrophages, oligodendrocytes >105 Yes Yes α2,3-linked sialic acid
a
Incubation period depends upon titer of virus inoculum. Predominant cell in which TMEV replicates in the CNS. c Plaque-forming units/median 50% lethal dose. d Temperature-sensitive phenotype: High-neurovirulence TMEV are naturally restricted in their ability to replicate in cells at ≥39.8°C. b
encephalitis, whereas the low-neurovirulence strains, such as BeAn and DA, produce a persistent infection in the CNS of mice that results in mononuclear cell inflammation and demyelination (Lipton 1975; Lehrich et al. 1976). Infection of mice with the low-neurovirulence TMEV provides a highly relevant experimental analog for multiple sclerosis in humans. A. HIGH-NEUROVIRULENCE TMEV The incubation period after i.c. inoculation of mice with the high-neurovirulence GDVII and FA strains ranges from several to 14 days, depending upon virus dose. Infected mice show typical signs of encephalitis, including weight loss, inactivity, ruffled fur, hunched posture, and proximal hind-limb paralysis. Convulsions do not occur nor can they be induced by physical means. Simas et al. (1995) found a consistent pattern of infection in the CNS, including cerebral cortex (predominately cortical layers IV, V, and VI), pyramidal neurons of the hippocampus, basal ganglia, hypothalamus, substantia nigra, pons, and spinal cord gray matter. No virus was detected in the corpus callosum, cerebellum, dentate gyrus, or molecular layer of the hippocampus. GDVII infects neurons predominately but also some astrocytes (Simas et al. 1995; Martinat et al. 1999). GDVII viral spread in peripheral nerves to the CNS by microtubule-associated fast axonal transport has been reported (Martinat et al. 1999). Interestingly, attenuated GDVII virus does not persist in the CNS of mice (Lipton et al. 1998; Jarousse et al. 1999; Pilipenko et al. 1999), suggesting the absence of a determinant for persistence from the GDVII genome. B. LOW-NEUROVIRULENCE TMEV Following i.c. inoculation of brain-derived virus stocks, low-neurovirulence strains produce a classic biphasic CNS disease pattern, that is, an early phase of poliomyelitis followed by demyelinating disease in surviving mice (Fig. 12-3). Although Theiler (1934) found that some infected mice surviving poliomyelitis were “virus carriers,” a phrase used to indicate that infectious virus was present in the spinal cord for at least 150 days PI, the demyelinating pathology was only recognized 20 years later by Daniels et al. (1952). Adaptation of low-neurovirulence TMEV to growth in cell culture attenuates the early poliomyelitis phase, and mice no longer develop acute flaccid hind-limb paralysis, although mild pathological changes of poliomyelitis are still evident. Upon i.c. inoculation, the cell culture–adapted virus also enters the peripheral circulation, culminating in viremia and infection of visceral organs. Virus is cleared from extraneural organs by 7 to 14 days PI, except for somewhat longer replication in the intestine. In the CNS, virus replication takes place mostly in the brain and spinal cord gray matter (Fig. 12-3), primarily in neurons and to a lesser extent in glia, leading to microglial proliferation and destruction of anterior horn neurons. While infectious virus is cleared from the brain, persistent infection in spinal cord white matter evolves over the lifetime of the mouse (Fig. 12-3). Infected mice develop spastic paralysis, manifested initially by a waddling and unstable gait and tremulousness that leads to spastic paralysis with extensor spasms of the hind limbs and neurogenic bladder with urinary incontinence.
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Polio
Infectious virus titer (PFU)
Demyelination
Brain
106
Spinal Cord
Spinal Cord Brain
Viral RNA copy numbers
109
Virus-Specific Immune Responses: Neutralizing Abs
CD4 T cell prlf
0
30 Days Postinfection
180
Fig. 12-3 Summary of the pathogenesis of BeAn virus infection in susceptible SJL mice inoculated i.c. with ∼106 pfu of virus. The onset and duration of pathological changes of acute poliomyelitis and active inflammatory demyelination are shown at the top, and the days postinfection (PI) at the bottom. Increasing severity of demyelination is displayed in a stepwise fashion, with maximal involvement at ~90 days PI. The temporal kinetics of viral replication (infectious virus in PFU), viral RNA replication (genome copy numbers), and virus-specific neutralizing antibody and CD4+ T cell proliferative (prlf) responses are shown. The curves for virus replication and viral RNA replication are for spinal cord; the red lines indicate the kinetics in brain.
Beginning 14–21 days PI, mononuclear inflammatory cells, mostly lymphocytes, are observed in the spinal cord leptomeninges and white matter, followed by an influx of macrophages. Primary demyelination occurs in macrophage-rich areas and is the pathological substrate of neurological disease. ●
●
Sites of TMEV persistence. TMEV RNA and antigens have been found mainly in macrophages (Lipton et al. 1995; PenaRossi et al. 1997), and to a lesser extent in glia, particularly oligodendrocytes but also astrocytes (Aubert et al. 1987). TMEV replication in macrophages is highly restricted at the levels of RNA replication and virion assembly, consistent with mechanisms of persistence of cytolytic RNA viruses (Trottier et al. 2001; Jelachich and Lipton 1999). In contrast, oligodendrocytes are productively infected, possibly becoming infected as a byproduct of macrophage infection. An essential role for macrophages in TMEV persistence was demonstrated by abrogation of DA virus persistence in 70% of mice depleted of peripheral macrophages with mannosylated liposomes during infection (Pena-Rossi et al. 1997). High viral RNA copy numbers during persistent infection. Historically, TMEV persistence in susceptible mice has been determined by the recovery of infectious virus from spinal cords. Results of infectivity assays have led to the belief that TMEV persists at only low levels in the CNS, on the order
of 102 to 103 pfu per spinal cord. In contrast, between 108 and 1010 viral RNA copies have been detected in spinal cords of infected mice, even as late as 6 months PI (Trottier et al. 2001) (Fig. 12-3). The high abundance of viral genomes during persistence is also characteristic of HIV-1 and hepatitis C infections. The disparity between viral genome load and infectious virus during TMEV persistence in vivo remains unexplained, but probably reflects clearance of infectious virus by virus-specific antibodies and CD4+ T cells, as well as restricted production of infectious virus in CNS macrophages, the predominant cell type supporting TMEV replication (Clatch et al. 1985; PenaRossi et al. 1997). CD8+ T cells do not appear to limit TMEV replication during persistent infection in susceptible SJL mice (Kang et al. 2002). ●
Transition from acute to persistent infection. The mechanism(s) underlying the transition of TMEV infection from an acute neuronal stage in gray matter to a chronic macrophage and glial infection in the white matter is not well understood. Possible explanations might rest in differences between acute and chronic viral tropism or in the induction of virus-specific host immune responses. Ostensibly, the immune response is able to clear TMEV from the gray matter (brain and spinal cord)
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but not the white matter (spinal cord) (Njenga et al. 1997). Viral genome equivalent levels in spinal cord increase steadily to high levels between days 13 and 33 days PI, which is critical for maximum proinflammatory Th1 cytokine and chemokine mRNA expression (Trottier et al. 2004). Thus, active viral replication is necessary to reach and sustain high viral genome loads and levels of proinflammatory Th1 cytokine/chemokine expression that drive progression of the demyelinating disease. Host genetic influences. Susceptibility to TMEV-induced demyelinating disease is genetically determined. Susceptible mice include the SJL, DBA/2, C3H/He, SWR, and PLJ strains, while the BALB/c, C57/BL/6 (B6), A, and 129 strains are resistant. Several investigators have demonstrated that the major histocompatibility complex (MHC) class I locus H-2D in B6 mice provides strong resistance to disease, including F1 hybrids of susceptible strains and B6 mice. A possible mechanism of H-2D resistance via regulatory CD8+ T cells is discussed below. Bureau et al. (1992) demonstrated various degrees of susceptibility to persistent infection of inbred strains of mice, indicating that multiple genes are involved. A number of potential chromosomal loci influencing susceptibility to demyelinating disease have been identified, but specific genes remain to be determined (reviewed in Brahic and Bureau 1998; Melvold and Miller 2004). Host virus–specific immune responses. Infected mice produce virus-specific neutralizing antibodies by 7 days PI that peak at 1 to 2 months PI and remain elevated for the life of the host (Fig. 12-3). The principal antiviral IgG is IgG2a, indicative of a predominant virus-specific Th1 CD4+ T cell response (Peterson et al. 1992). In fact, substantial levels of virus-specific Th1 CD4+ T cell responses are observed by 7 to 14 days PI, rising thereafter (Clatch et al. 1986, 1985), and result in production of Th1 cytokines, such as IFN-γ by T cells and tumor necrosis factor-α by macrophages (Begolka et al. 1998). Immunodominant CD4+ T cell epitopes in SJL mice have been identified in each of the major surface virus proteins, VP1233-244, VP274-86, and VP324-37 (Gerety, Karpus, et al. 1994; Yauch et al. 1995; Yauch and Kim 1994) (Fig. 12-2B). Immunization of infected SJL mice with peptides of either the VP1233-244 or VP274-86 epitope potentiate demyelinating disease, supporting an immunopathological role for CD4+ T cells in demyelination (also see below) (Gerety, Rundell, et al. 1994).
Genetic studies have shown that resistance to TMEVinduced demyelinating disease is linked to the H-2D MHC class I locus (Lipton and Melvold 1984; Clatch et al. 1985; Rodriguez et al. 1986), suggesting a role for CD8+ T cells in susceptibility. Virus-specific CD8+ cytotoxic lymphocytes (CTLs), especially those specific for the immunodominant VP2121-130 epitope, are associated with acute viral clearance in resistant B6 mice (Dethlefs, Escrion, et al. 1997; Borson
et al. 1997; Mendez-Fernandez et al. 2003). In contrast to the early appearance of and rapid increase in CTL activity in resistant B6 mice, CTL activity was found to occur later and remain at low levels in susceptible SJL mice, suggesting that viral persistence in SJL mice might be due to insufficient virus-specific CTL activity (Dethlefs, Brahic, et al. 1997). On the other hand, Kang et al. (2002) found that not only do SJL mice generate CD8+ T cell responses but CNS-infiltrating CD8+ T cells in SJL mice are fully functional virus-specific effector cells. While the total number of virus-specific CNS-infiltrating CD8+ T cells is significantly higher in resistant B6 mice than in susceptible SJL mice at day 8 PI, the converse is seen at later times (>day 30 PI) (Lyman et al. 2004). Thus, TMEV CNS persistence may not be due to lack of virus-specific CTL activity. MHC class I– restricted T cells may also influence susceptibility through CD8+ T cell regulatory activity (Nicholson et al. 1994, 1996). In that case, susceptible SJL mice should generate less efficient CD8+ regulation of virus-specific CD4+ T cells compared to resistant B6 mice, which in fact has been shown for susceptible compared to resistant BALB/c substrains (Karls et al. 2002). Despite neutralizing antibody and virus-specific CD4+ and CD8+ responses that are temporally associated with a decline in acute CNS virus titers, TMEV are somehow able to evade immune clearance and persist in the spinal cord. Moreover, CNS persistence in susceptible SJL mice requires active virus replication to reach and maintain very high levels of viral genome copies, as pointed out previously, and is not merely a result of failure of viral clearance. ●
Mechanism(s) of demyelination. Myelin breakdown during persistent infection is multifactoral, although immune-mediated damage to myelin has been emphasized. Administration of immunomodulating agents, induction of tolerance to TMEV, use of mice rendered deficient in T cell subsets and specific inflammatory molecules, and immunization with immunodominant CD4+ T cell peptides during TMEV infection have shown varying degrees of protection against or potentiation of the demyelinating disease. Collectively, these data indicate that in susceptible mouse strains, MHC class II–restricted TMEV-specific Th1 T cells produce IL-2 and IFN-γ, leading to recruitment of monocytes into the CNS and their differentiation into macrophages that produce TNFα, neutral proteinases, and other molecules that nonspecifically mediate demyelination. In addition, oligodendrocytes, the myelin-maintaining cells, are productively infected and also contribute to myelin breakdown.
VI.
EPIZOOTIOLOGY
TMEV infection is ubiquitous and worldwide in its distribution in colony-bred and feral mice, but infection of colony-bred
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rats is rare (Descoteaux et al. 1977; Lipton et al. 2001). An exception may be Australia, where antibodies to TMEV were not found in any of a large number of Mus domesticus trapped in the southeastern part of the country (Smith et al. 1993). It is intriguing to speculate that TMEV do not exist on the Australian side of Wallace’s line. In contrast, EMCV infection is probably as prevalent in feral rats as TMEV infection is in feral mice (Tesh and Wallace 1978). Epizootics of TMEV infection in colony-bred mice have been described, but those of EMCV infection in rats have not. Although cardioviruses can infect hamsters and guinea pigs, these animals are an unlikely source of infection of barrier colony-raised mice or rats.
A.
Group A (EMCV)
Although this group of viruses is highly infectious for rodents by experimental inoculation, contradictory results on fecal shedding of virus and rodent-to-rodent transmission have been reported. While rodents are clearly susceptible to infection by the oral route, they do not excrete substantial amounts of virus in the feces and contact transmission is sporadic (Kilham et al. 1955, 1956; Vanella et al. 1956; Tesh and Wallace 1978), yet fecal-oral transmission is probably the natural route of spread. Are rodents the natural reservoir? Causey et al. (1962) described an apparent epizootic of EMCV in forest animals in Brazil, in which EMCV were isolated from sentinel mice, spiney rats and other rodents. Tesh and Wallace (1978) found that the prevalence of EMCV-neutralizing antibodies among mongoose, pigs, and cows was comparable to that in wild rats. Those investigators suggested that rodents of the genus Rattus may be indicators of virus activity and a dead-end host in the overall ecology of these viruses. Virus isolations from mosquitoes and ticks have been reported; however, attempts to transmit EMCV experimentally with mosquitoes were unsuccessful (Murnane et al. 1960). EMCV have also been responsible for disease in pigs and humans. Murane et al. (1960) first reported acute fatal myocarditis in piglets caused by EMCV in Panama, and outbreaks of myocarditis were subsequently recorded in herds of domestic pigs in Europe and on other continents (reviewed in Knowles et al. 1998). Neutralizing antibodies were found in a substantial portion of normal pigs in England (Sangar et al. 1977). Based on evidence of pig-to-pig transmission and EMCV genomic analysis of myocarditis outbreaks in Europe, transport of pigs from one geographic location to another has been implicated in the spread of disease (Knowles et al. 1998). EMCV have also been associated with reproductive failure in pigs. Occasional cases of aseptic meningitis and an epidemic of a mild febrile illness (“3-day fever”) in humans in the Philippines due to EMCV were reported during World War II; however, human cases of EMCV-induced myocarditis have not been reported to our knowledge.
B.
Group B (TMEV)
TMEV infection is acquired shortly after weaning, with all nonimmune mice eventually becoming infected (Olitsky 1939; Theiler and Gard 1940a; Dean 1951). Except in enzootic situations where suckling mice are protected by maternal antibody, virus can be recovered from feces at the time of weaning. Isolation becomes irregular as mice reach 1 to 2 months of age, in parallel with increasing humoral immunity (Olitsky 1940; Dean 1951). Because 53 days is the longest period that virus has been recovered from feces, persistence in the intestine is not a likely mechanism for TMEV maintenance in mouse colonies. The initial TMEV isolations in the early 1930s were from brains and spinal cords of colony-bred mice that had developed spontaneous flaccid limb paralysis (mouse poliomyelitis) (Theiler 1934, 1937). Thus, TMEV occasionally spread beyond the intestine to the CNS, but the incidence of spontaneous paralysis in mouse colonies was estimated at only 1 in 1,000–5,000 mice (Theiler and Gard 1940b). In the only large TMEV epidemic, 150 of 240 weanling mice died during an outbreak of acute encephalitis; a GDVII-like virus was isolated from the CNS of six affected mice (Thompson et al. 1951). More recently, a spontaneous TMEV outbreak was reported in neonates that developed fatal poliomyelitis in a colony of mice with severe combined immunodeficiency (Rozengurt and Sanchez 1993). There have been two reports of TMEV isolations in colony-bred rats.
VII.
DIAGNOSIS
The diagnosis of EMCV or TMEV in clinical as well as symptomless infections depends upon positive serology, virus isolation, or RT-PCR amplification of viral RNA. Serum should be collected, heat-inactivated at 56°C for 30 min, and stored at −20°C. The relative ease of conducting ELISA makes it the serological test of choice; however, EMCV and TMEV crossreact in ELISA, so the two groups of viruses may not be distinguishable and neutralization tests might also be needed. Neutralizing titers of ≥20 and ELISA titers of ≥500 are indicative of prior infection. When paired sera are available, a four-fold rise in IgG antibody titer is diagnostic of recent infection. Commercial ELISA tests for TMEV are available and are widely used, since the neutralization test is more labor intensive. EMCV are most likely to be recovered from blood, brain, heart, pancreas, and intestine or feces, and TMEV from brain, spinal cord, and intestine or feces. Specimens for virus isolation should be removed under sterile conditions, transferred to polyethylene tubes, snap-frozen in dry ice and ethanol or liquid nitrogen, and stored at −20°C or preferably at −70°C. Recovery of virus from clarified 10% suspensions of tissue homogenates or fecal extracts is indicated by development of
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typical picornavirus cytopathic effect in HeLa (EMCV) or BHK-21 and L929 (TMEV) cell monolayers but not in control monolayers. Proof that cytopathology is due to cardiovirus infection is obtained by immunofluorescent antibody staining of the cells using commercially available antisera. If virus recovery in cell culture is negative, i.c. inoculation of 1 or 2 litters of 1-day-old suckling mice provides an alternative approach; failure to thrive or death of mice over a 14-day period PI suggests cardiovirus infection. Passage of clarified 10% suckling mouse brain homogenates on cell monolayers is needed to confirm a cardiovirus isolate. RT-PCR provides further confirmation of the cardiovirus species; because the 5′ noncoding nucleotide sequences are conserved, primers can be designed that amplify all cardioviruses, but group-specific primers can also be used.
VIII.
CONTROL AND PREVENTION
The greater prevalence of TMEV than EMCV in mice has made TMEV a more serious concern to biomedical researchers. Since cardioviruses are easily spread by the fecal-oral route, the major aim is to prevent introduction of non-barrier colony– raised or feral mice and rats, or contaminated bedding from an outside source, into a clean (barrier conditions) mouse colony. Thus, strict adherence to standard biological containment measures and barrier conditions is advisable. Based on present knowledge, it seems unlikely that either EMCV or TMEV readily infect rabbits, hamsters, or guinea pigs, so these animals are unlikely to introduce cardioviruses into a mouse colony. There is no economic incentive for development of an EMCV or TMEV vaccine for mice or rats. Finally, the likelihood of cardiovirus contamination of biologics (primary cell lines, sera, etc.) is probably low even when derived from non-barrier colony–reared mice. However, picornavirus infections of mammalian cells over time (“carrier cultures”) will be soon revealed, since persistently infected cells go through crises with widespread cytopathology resulting.
ACKNOWLEDGMENTS This work was supported by NIH grants NS 21913 and NS23349.
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Chapter 13 Chlamydial Diseases Roger G. Rank
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History of Chlamydia muridarum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Infection of Mice with Chlamydial Agents of Human Disease . . . . . . . 1. Infection of Mice with Human Biovars of Chlamydia trachomatis . 2. Infection of Mice with Other Biovars . . . . . . . . . . . . . . . . . . . . . . . III. Properties of Chlamydiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Morphology and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Developmental Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. In Vitro Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Taxonomy and Molecular and Antigenic Relationships of MoPn . . . . . . . . V. Clinical Disease, Pathogenesis, and Immunology . . . . . . . . . . . . . . . . . . . . A. Basic Mechanisms of Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. MoPn Respiratory Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Infection Course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pathologic Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Immunity and Immune Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . C. Genital Tract Infection (MoPn and Human C. trachomatis) . . . . . . . . . 1. Infection Course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pathologic Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Immunity and Immune Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . D. Chlamydia pneumoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Infection Course and Pathologic Response . . . . . . . . . . . . . . . . . . . 2. Immunity and Immune Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . E. Chlamydia psittaci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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I.
INTRODUCTION
Chlamydiae are a unique group of bacteria in that they are obligate intracellular bacteria with two distinct morphologic forms. There is a wide host range for chlamydiae, although individual species maintain a relatively high degree of host specificity. One species, Chlamydia muridarum, has been isolated from the mouse and is indeed a natural parasite of the mouse. C. muridarum is a member of the order Chlamydiales, an ancient and widely occurring taxon. All known Chlamydiales are, like C. muridarum, obligate intracellular parasites. However, mice can also be infected with all of the human oculo-genital strains of Chlamydia trachomatis, several different biovars of Chlamydia psittaci, and C. pneumoniae, a natural human respiratory pathogen. C. muridarum has been studied extensively as a model for human chlamydial respiratory and chlamydial genital tract infections; thus, this chapter will discuss the natural history of the mouse-specific organism but also present an in-depth discussion of the use of this organism as a model for human chlamydial disease. In addition, since mice can be infected with chlamydiae not naturally found in mice, mice have been used as experimental models for these other infections. These infection model systems will also be discussed because of their importance in our study of chlamydial infections.
II. A.
HISTORY
History of Chlamydia muridarum
As with many organisms, C. muridarum was discovered by serendipity. In order to diagnose viral diseases, investigators would inoculate pharyngeal washings from patients either intracerebrally or intranasally into laboratory mice to recover the etiologic agent. Usually, serial passages of lung homogenates were inoculated intranasally into mice to “amplify” the infection. The first likely observation of a natural chlamydial infection in laboratory mice was made by Dochez and colleagues in 1937 when, while trying to adapt the virus of the common cold to Albino Swiss mice, they changed mouse vendors and found a dramatic change in the virulence of the virus, with 80% of the mice dying compared to only 10% previously (Dochez et al. 1937). They evaluated normal mice from the vendor and found that 17 of 50 mice had some degree of lung pathology. Passage of a homogenate of the lung lesions to other mice resulted in disease, although housing the infected mice with normal mice did not result in aerosol transmission. Mortality of the mice only resulted following four to seven consecutive intranasal passages of lung homogenates, but the virulence of these resulting passages was quite high, with death occurring within 48 hours. The “virus” was filterable. Thus, while the studies were not conclusive as to the nature of the organism, subsequent experiments
by others would suggest that they may have indeed isolated a chlamydia of mice. In a similar series of experiments, while studying influenza in 1938, Gordon noted that serial passage of lungs from 5 to 10 g mice, originally inoculated with pharyngeal washings from a patient, resulted in an interstitial pneumonia in the recipient animals; however, he also noted the same disease in mice serially inoculated with lung homogenates from normal control mice (Gordon et al. 1938). When he examined the pathology, he found that there were differences in the disease process caused by influenza and this new agent. In addition, neutralizing serum for influenza did not neutralize this infection. His conclusion was that he had isolated a new “virus” from mice that produced disease similar to influenza. This organism was later confirmed to be a “virus” belonging to the psittacosis/lymphogranuloma venereum group (Hilleman and Gordon 1944). About the same time, Gönnert, working in Germany with ectromelia virus, made similar observations in lungs of mice receiving serial passages of lung homogenates (Gönnert 1941a). Several passages were required to produce clinical disease in the mice. The infection was lethal for mice, although the time to death could be delayed by the dilution of the lung homogenates. He performed an extensive evaluation of the histopathology and morphology of the organism in the lung and concluded that it was a new agent of murine bronchopneumonia. He noted that the organism produced an intracellular inclusion and was remarkably similar to the causative agent of lymphogranuloma venereum and trachoma (Gönnert 1941b). Finally, Nigg reported in 1942 about the isolation of yet another murine respiratory “virus,” by serial passage of lung homogenates in Albino Swiss mice (Nigg 1942). Three separate isolates were obtained from mice inoculated with throat washings from three different cases of influenza. She also isolated two strains of the “virus” from normal mice that had received serial passages of lung homogenates from uninoculated mice, thus suggesting that this was an endogenous infection in laboratory mice. In a subsequent paper, Nigg and Eaton presented a detailed characterization of the “mouse pneumonitis virus (MoPn)” and the disease process (Nigg and Eaton 1944). They suggested that the virus was of mouse origin because they had failed to neutralize MoPn in yolk sacs with convalescent sera from the patients from whom the throat washings were obtained. To confirm the murine origin of the organism, they serially passaged lung homogenates from normal mice obtained from two different mouse breeders. The organism was isolated from two series of passages from one breeder but could not be isolated from mice obtained from the other breeder, even after passaging homogenates for one year. Generally, mice died within 24 hours of inoculation of heavily infected lung homogenate, but death could be delayed up to 20 days by dilution. The infectious agent was found to be a filterable “virus,” but Giemsa and Macchiavello staining of impression smears from
13. CHLAMYDIAL DISEASES
lungs demonstrated structures very closely resembling the elementary bodies of what was then considered to be the viruses causing lymphogranuloma venereum (LGV), meningopneumonitis, and psittacosis, all now recognized as chlamydial agents. They were able to culture the organism in the yolk sacs of embryonated eggs and find the elementary bodies by Giemsa-staining. Interestingly, they found that the organism was infectious for hamsters and produced pneumonia similar to that seen in mice; however, they could not demonstrate infection of ferrets or rabbits. That the organism was related to agents of LGV and psittacosis was demonstrated by incubating sera from patients with LGV with yolk sac preparations of MoPn and finding moderate to strong complement fixation reactions while normal human serum was essentially negative. There was no evidence of crossreaction with influenza and lymphocytic choriomeningitis virus. Conversely, sera from animals immunized with LGV and meningopneumonitis recognized the MoPn agent. Thus, the data strongly indicated that this agent was a new mouse virus that was related to the viruses of LGV and psittacosis. The mouse pneumonitis organism was later purified from mouse lung tissue by Gogolak using differential centrifugation (Gogolak 1953b). They used these preparations for visualization with electron microscopy and observed that the particles were compatible in size and structure with other members of the psittacosis-lymphogranuloma group, measuring about 462 µm in diameter. That the organism was more closely related to the agent of LGV than the agent of psittacosis was suggested by the crossreaction with LGV immune serum. However, it was clearly not the same organism, because neutralization tests conducted by Hilleman indicated that anti-LGV serum would not neutralize MoPn, nor would anti-MoPn serum neutralize LGV (Hilleman 1945). Moreover, MoPn was found to be susceptible to sulfonamides in vitro and in vivo as are other members of C. trachomatis, while members of the C. psittaci are resistant, although there are exceptions to this general rule (Rake et al. 1942). Thus, the early data linked the murine organism to the LGV and agents of the trachoma-inclusion conjunctivitis (TRIC), now classified as C. trachomatis. There has been controversy in the chlamydia field regarding the origin of MoPn, with many mistakenly believing that the organism in mice was actually derived from humans. The independent isolates from each of the three laboratories described above definitively indicate that MoPn was an actual murine isolate. Recent genomic studies have demonstrated that MoPn is quite distinct from any of the natural human chlamydiae (Read et al. 2000). The basis of the controversy is that no one has isolated MoPn from laboratory mice since those early studies. This is not surprising because mice today are produced in well-controlled facilities, in contrast to facilities 60 years ago. Feeding the controversy is the absence of any report documenting isolation of MoPn from wild mice, but it is not clear whether anyone has even tried to isolate chlamydiae from
327 wild mice. The only reports regarding the isolation of chlamydiae from mice are those mentioned here, in which laboratory mice were the sources of the organism. That the mice used in the studies by Gordon, Nigg, and Gönnert appeared to be healthy and that the infection could only be demonstrated by multiple blind serial passages suggest that the MoPn was present at very low levels in the lungs of the mice. It is also interesting to note that the mice used in the studies by Nigg and Gordon, both as donors of lung tissue and as recipients, were all young mice, 3 to 5 weeks of age. First, this suggests that the infection is persistent in mice at low levels and generally does not produce overt disease. Second, it is also possible that it may be transient in the respiratory tract, since it was found in young mice. Because the organism was isolated from the respiratory tract, it was assumed that transmission was via the respiratory route. Karr infected mice intranasally and housed them with normal mice in the same cage and then determined whether the infection spread to the normal animals (Karr 1943). She found no evidence that the normal mice became infected, suggesting that contact or close association with infected animals did not result in transmission. She then added infected lung material to the drinking water of uninfected mice and, on two separate days, added an infected mouse carcass to the cage instead of the standard chow. She found that MoPn could be isolated from the lungs of some of the mice by serial passage. Some animals did not become infected, and it was apparent that the more “feedings” given with mouse carcasses, the greater was the likelihood that the mice would be infected. Therefore, the data suggested that transmission of MoPn is via the oral route and that the animals aspirate organisms into the respiratory tract, where a low-level infection is established. In a similar study, Cotter and colleagues investigated potential dissemination of MoPn from the genital tract to other sites (Cotter, Ramsey, et al. 1997). They housed uninfected mice with infected mice in the same cage. Interestingly, when they cultured the mesenteric lymph nodes from the uninfected mice, they were able to isolate MoPn from the nodes but not from other tissues. Since mesenteric lymph nodes drain the gut, they hypothesized that the mice had acquired the infection by the oral route. Thus, these data support those of Karr suggesting that oral transmission is possible. Therefore, based on the data presented by Karr and Cotter and on the biology of other chlamydial species, this author would hypothesize that the primary target tissue of MoPn is the gut and that it is spread in nature via the fecal-oral route. It is well known that mice are coprophagic, so I would suggest that young mice ingest feces from infected mice and then aspirate some organisms into the respiratory tract. The number of organisms aspirated would undoubtedly be quite small, so that a low-level or subclinical infection is established that eventually resolves. This explanation would fit the data obtained by the early researchers and provide a rational explanation for persistence of the organism in a mouse colony. Moreover, a
328 chlamydial species closely related to MoPn has been isolated from the ileum of hamsters (Stills 1991; Fox et al. 1993). This organism, which has been designated the SFPD strain of C. trachomatis, is remarkably similar to MoPn (Zhang et al. 1993). Zhang found that the sequence of the major outer membrane protein in the SFPD strain had 91% identity to MoPn in contrast to 80%–83% homology with the human C. trachomatis biovars and 69%–70% homology with C. psittaci strains. Detailed descriptions of the pathologic response to MoPn respiratory infection with emphasis on the developmental cycle of the organism were first reported by Weiss (Weiss 1949). Weiss observed elementary bodies shortly after instillation of the organism and, after a period of 5–7 hours, could detect initial or reticulate bodies. By 13 hours, the formation of an inclusion membrane was seen that continued to expand in size up to 24–36 hours. This elegant microscopic study of the in vivo growth of the organism indicated a developmental cycle of about 36 hours. Later, also using the Chicago isolate, Gogolak presented a detailed analysis of the histopathologic response to the organism in the lung (Gogolak 1953a). Gross lesions could be detected by 3 days after intranasal infection, but microscopic analysis revealed a diffuse and generalized cellular response as early as 90 minutes after infection. The pathologic response described by Gogolak was similar to that reported by Weiss, but more extensive with regard to the overall description of the lung pathology. It is apparent that a number of chlamydial strains were isolated from laboratory mice in the mid-20th century, but only two strains have been passed down over the years and are currently available in various chlamydial research laboratories. The original isolate by Clara Nigg (Nigg 1942) was deposited in the American Type Culture Collection and is the strain whose genome has been recently sequenced (Read et al. 2000). This strain was originally referred to as the Atherton II strain but now is universally referred to as the Nigg strain. The strain isolated by Gordon was originally referred to as the Chicago strain because it was isolated at the University of Chicago (Gordon et al. 1938). Emilio Weiss, a student at the University of Chicago, published an extensive description of the infection and resultant pathology of the Chicago strain in the lungs of mice and compared it to the feline pneumonitis (Baker 1944) and the ferret meningopneumonitis agents (Francis and Magill 1938) of C. psittaci (Weiss 1949). He also described the growth of MoPn in yolk sacs. This strain was later given to others and became known as the Weiss strain. Thus, currently, both the Weiss and Nigg strains of MoPn are used interchangeably and have been assumed to be identical. In fact, Hilleman and Gordon reported that immune serum produced by immunizing roosters with the Chicago (Weiss) strain was able to neutralize the Atherton II (Nigg) strain equally as well as the Chicago strain, suggesting that they were antigenically similar (Hilleman 1945; Hilleman and Gordon 1944). However, recent data by Ramsey and his colleagues suggest that these strains may actually be phenotypically distinct (K.H. Ramsey, T. Darville, R.G. Rank,
ROGER G. RANK
unpublished data). This will be covered more extensively in Section IV. While both Weiss and Nigg strains were used in a variety of in vitro studies over the years that described the basic biology and antigenicity of chlamydiae, there was little additional work on the study of MoPn infection in the mouse or its use as a model for human disease. As will be discussed below, there were a number of studies using human strains of Chlamydia in the mouse, but MoPn was not used, most likely because it was of less interest, being a murine parasite and not a pathogen of humans, until 1981. That year, Barron and colleagues published a report of an experiment in which they successfully infected female mice in the genital tract by intravaginal instillation of the yolk sac–grown MoPn (Nigg strain) (Barron et al. 1981). Organisms could be observed on Giemsa-stained scrapings of the vagina and were also identified by light microscopy and electron microscopy in the superficial epithelial cells in the exocervix. They later characterized the course of the infection and found that the infection lasted approximately 20 days and then resolved (Barron et al. 1984). IgM antibody to MoPn was detected in serum by 7 days after infection and IgG appeared in both serum and genital tract secretions by 15 days after infection. Animals had also developed a delayedtype hypersensitivity response when assessed on day 30 after infection. Barron proposed that this model could be used as an animal model for chlamydial genital infection, and it has subsequently become the most widely used animal model for the study of chlamydial genital disease. Also in 1981, because of the then-recently recognized role of C. trachomatis in respiratory infections in the newborn, Williams reported on the infection of immunologically intact and congenitally athymic nude mice in respiratory tract with tissue culture–grown MoPn (Weiss strain) (Williams et al. 1981). Mice were inoculated intranasally with a dose determined to infect but not be lethal in normal mice. The infection resolved about 5 days after infection in normal mice but did not resolve in nude mice, with the majority dying by day 30. This model of respiratory infection continues to be used for the study of the disease and host response in the lung, as well as a screening system for testing possible chlamydial vaccine candidates. B.
Infection of Mice with Chlamydial Agents of Human Disease
While MoPn remains the only member of the Chlamydia genus that has been truly identified as a mouse pathogen, the mouse has been used for many years to study both C. trachomatis and C. psittaci, derived from humans and animals, respectively. The use of mice for the study of chlamydial diseases was based more on the intrinsic interest in the ability of the organism to elicit disease rather than on actually using mice to model the disease. The mouse was also used to demonstrate antigen specificity by neutralization tests in vivo. Only the very aggressive
13. CHLAMYDIAL DISEASES
strains of C. trachomatis were recognized as being able to infect mice. These included the agent causing lymphogranuloma venereum (LGV) and the so called “fast” egg-killing strains of C. trachomatis isolated from cases of trachoma, which are serologically identical to LGV serovar 2 and most likely the result of laboratory contamination in which fast-growing strains (LGV) overgrow slow-growing strains (trachoma) in culture (Wang and Grayston 1971). LGV was considered the only strain of importance with regard to genital tract disease, as it was not recognized until the 1970s that other, less aggressive trachoma biovars of C. trachomatis could cause genital tract infection. The major research emphasis until the 1970s was on chlamydiae as the causative agent of trachoma and inclusion conjunctivitis. Since mice did not develop ocular disease by infection with chlamydiae, they could not be used as a model for trachoma. 1.
Infection of Mice with Human Biovars of Chlamydia trachomatis
All known chlamydial strains are able to infect mice when mice are inoculated with material having high infectivity (Storz 1971). Findlay and his group first demonstrated that LGV could successfully infect mice in 1938 by inoculating a human isolate intracerebrally (Findlay et al. 1938). The infection resulted in the death of most of the animals in 2–4 days. In 1940, Shaffer and colleagues inoculated two LGV strains intranasally into 10 g Albino Swiss mice to evaluate the course of infection (Shaffer et al. 1940). Within 48–72 hours, the mice became ill with signs of marked respiratory disease and with some mice dying within this time period. The researchers observed neutrophils and mononuclear cells within the walls of the alveoli, and filling the alveoli, as well as being able to visualize chlamydiae. They were also able to recover organisms by inoculation into yolk sacs. As interest in trachoma increased, individuals began to utilize the mouse to study the TRIC agents. T’ang originally isolated the agent producing trachoma in yolk sacs, but was unable to infect mice, pigeons, hens, rabbits, or guinea pigs with the organism (T’ang et al. 1957). Bernkopf first inoculated a trachoma virus intracerebrally into suckling mice, primarily as a means to evaluate antibody via a neutralization test (Bernkopf 1959). Hurst and Reeve also were able to infect mice intracerebrally with yolk sac–adapted trachoma strains (Hurst and Reeve 1960). They treated 5-week-old mice with cortisone and then inoculated the mice with the yolk sac preparation. They were able to carry the organism through 4 passages at 5–7 day intervals and were able to infect mice with the original T’ang strain. Later, Wang and Grayston described a neutralization test that they developed to provide a means for serologic identification of the trachoma agent (Wang and Grayston 1963). Mice were immunized with low doses of yolk sac–grown organisms and after a sufficient period of time were challenged intravenously with a lethal dose of the test organism. The number of mice surviving the infection as a result of neutralization of the organism was determined.
329 Graham reintroduced mice as a model for respiratory infection (Graham 1965, 1967). She inoculated mice intranasally with the “fast” egg-killing strains of C. trachomatis biovars and was able to produce a respiratory infection with high concentrations that resulted in pulmonary consolidation and death of the animal (Graham 1967). Most animals died 2–3 days after infection. At the peak of infection, organisms were found in the bronchiolar epithelium amidst a strong acute inflammatory reaction, which later gave way to a dominant mononuclear response. By immunizing mice with different biovars and then challenging the mice by the intranasal route, Graham was able to demonstrate that protection was generally strain-specific. Watkins and MacKenzie also inoculated mice intranasally with a “fast” egg-killing strain, TE55 (Watkins and MacKenzie 1963). TE-55 is also a “fast-growing” trachoma strain that is derived from T’ang’s original trachoma isolated PK-2 (Peking-2). Sixweek-old Albino Swiss mice were inoculated intranasally with yolk sac–cultured organisms. Mice became infected by 1 day after inoculation and some remained infected as long as 28 days, as determined by histopathologic examination and reisolation chlamydiae in yolk sacs. As in MoPn, an acute inflammatory response was observed in the lungs, giving way to a mononuclear infiltrate. The authors noted that the degree of inflammation appeared to be out of proportion with the number of inclusions seen and speculated as to the nature of the disease process. Some years later with interest precipitated by the observation of chlamydial pneumonia of the newborn, investigators began to reexamine the murine models for chlamydial respiratory infection. Kuo and Chen infected mice with one LGV strain (serovar L2), two ocular strains (serovar B and C), and two genital tract strains (serovars D and G) (Kuo and Chen 1980; Chen and Kuo 1980). They inoculated 4- to 5-week-old mice intranasally with the various strains of chlamydiae and found that all strains were able to infect the mice, although the LGV strain was more virulent than the genital or ocular strains. Receiving an inoculum of 3 × 107 inclusion forming units (IFU)/ml, 44% of the infected mice died, whereas none of the other strains receiving the same dose died from the infection. The maximum yield of organisms from LGV-infected mice was also 2 logs higher than that obtained with the other strains. Generally the infections elicited an acute inflammatory response in the lung from days 1–3, with mononuclear cells appearing after day 3. The lungs returned to normal between days 10–14. All animals responded with serum antibody and were positive for delayed-type hypersensitivity when inoculated in the footpad with chlamydial antigen. Harrison et al. (Harrison et al. 1982) reported similar findings upon intranasal infection of mice with C. trachomatis serovar H. That human strains of chlamydiae could infect the genital tract was first demonstrated by Tuffrey and Taylor-Robinson when they inoculated an aggressive human strain of C. trachomatis into the vaginal lumen or injected organisms directly into the uterus (Tuffrey and Taylor-Robinson 1981). They also treated
330 mice with progesterone to stabilize the epithelium in the genital tract. It is not clear exactly which serovar was used in this study. Ito and colleagues inoculated mice in the genital tract with C. trachomatis serovar H (Ito et al. 1984). They found that high doses of organisms (in excess of 108 IFU) were required to infect the animals, although even at these doses only a maximum of 50% of the mice could be infected. The ability to infect the mice was highly dependent upon the stage of the estrous cycle on the day of inoculation. The infection course for those animals which did become infected varied from 2–10 days. Infected animals also responded with antibody production. Subsequently, Ito inoculated mice with seven different oculo-genital serovars (D, E, F, G, H, I, and K) of C. trachomatis intravaginally (Ito, Jr. et al. 1990). Mice were treated with progesterone this time to facilitate the infection. Of interest, significant differences in the length and intensity of infections with the various serovars were found, suggesting that the mouse model could be of value in determining mechanisms of pathogenesis. 2.
Infection of Mice with Other Biovars
Other chlamydiae have been inoculated into mice and have been found to be infectious. However, just as with the trachoma/ lymphogranuloma venereum group, mice were originally used to isolate organisms from human tissue. Initially, in the study of psittacosis, researchers inoculated patient or animal material into parrots, since they were the primary host of the psittacosis agent. In 1930, however, both Krumwiede and colleagues (Krumwiede et al. 1930) and Gordon (Gordon 1930) independently inoculated tissue from infected birds subcutaneously or intraperitoneally into laboratory mice and found that the mice became ill and died. Passage of spleens or blood from the infected mice was able to transmit the infection when inoculated into naive mice. Gordon was also able to infect the mice by the intracerebral route. Shortly thereafter, Rivers and colleagues characterized the disease in the mouse (Rivers and Berry 1931). They reported that intraperitoneal inoculation of the psittacosis virus into mice produced lesions primarily in the spleen and liver. The spleen was enlarged and had focal areas of necrosis. The liver also had a focal eosinophilic necrosis. Interestingly, mice that had recovered from the infection were not immune to reinfection. Bedson and Bland also infected mice with C. psittaci derived from infected parrots and observed two different forms of the organism on impression smears of spleens (Bedson and Bland 1932). They found larger, weaker staining forms inside mononuclear cells, generally early in the infection, at 15 hours and 24–30 hours after infection of the mice. They saw few elementary bodies at these early times, which became quite numerous when the animals became moribund at 48–72 hours after infection. Tissue culture experiments supported these in vivo observations in mice. They suggested that these two forms represented two stages in a developmental cycle, obviously a seminal observation regarding the biology of chlamydiae.
ROGER G. RANK
There was little use of the mouse infected with C. psittaci as an actual model for disease until Byrne infected mice intraperitoneally with the 6BC strain of C. psittaci as a model for the study of immunosuppression and the protective immune response (Byrne, Guagliardi, et al. 1988). Using this model, he developed some of the basic concepts of the role of gamma interferon in the control of chlamydial infections (Byrne and Faubion 1982; Byrne and Krueger 1983). The latest chlamydia to be identified as a cause of human disease was C. pneumoniae. Kuo and his colleagues inoculated different strains of mice intranasally with C. pneumoniae and characterized the course of the infection and the resulting pathology (Yang et al. 1993). Just as in other chlamydial respiratory infections, they noted an initial acute inflammatory response in the alveolar spaces, followed later by a chronic mononuclear response. Organisms were isolated up to 42 days after infection. Mortality from the infection varied according to the strain of mouse employed. It was later learned that C. pneumoniae was also associated with coronary artery disease in humans. The same laboratory evaluated the ability of C. pneumoniae infection via intranasal infection to seed organisms to the aorta of the mouse (Moazed et al. 1997). They observed that if they inoculated C57BL/6 mice on an atherogenic diet or Apo E knockout mice with C. pneumoniae that they could detect the organism in the aorta, thereby establishing a murine model for the human condition.
III.
PROPERTIES OF CHLAMYDIAE
Chlamydiae are obligate intracellular bacteria that are unique in that they have a developmental cycle with two distinct phases: the elementary body or the nonreplicating infectious phase, and the reticulate body or the noninfectious replicating stage. While generally host species–specific, chlamydiae in nature can be found infecting a wide range of mammals, including humans, mice, guinea pigs, sheep, cattle, cats, hamsters, swine, and koala bears, and birds, including budgerigars, parakeets, parrots, cockatiel, pigeons, doves, and turkeys (Tanner et al. 1999).
A.
Morphology and Structure
Morphologically, the elementary body is approximately 0.3 µm in diameter and somewhat spherical in shape, while the reticulate body is larger at 1 µm in diameter and more amoeboid in shape. When visualized by electron microscopy, the elementary body has a dense nuclear area containing the DNA and a less dense cytoplasm containing ribosomes. A unique morphologic component of the elementary body is the presence of hexagonally arrayed projections on the surface with a range of 13–30 projections and a mean of 22 projections
331
13. CHLAMYDIAL DISEASES
per elementary body (Matsumoto 1988). It is now hypothesized that these projections may actually be the type III secretion apparatus originally described by Hsia and Bavoil (Hsia et al. 1997). In contrast, reticulate bodies have a more homogeneous distribution of ribosomes and DNA fibers. Neither stage of the organism stains well with the Gram stain, but they can be identified by aniline-based stains such as the Giemsa stain (Fig. 13-1). The Macchiavello stain has also classically been used to stain chlamydiae. Chlamydiae have a small genome, with only about 1 × 106 base pairs. This has facilitated sequencing of the genome and, as of this writing, 13 different chlamydial strains have been sequenced. One of the major stumbling blocks to chlamydial research, however, has been the total lack of the ability to manipulate the genome so that genes can be deleted or inserted, an important prerequisite to establishing the function or role of many of the genes and open reading frames. Structurally, chlamydiae resemble Gram-negative bacteria in that they have a trilaminar outer membrane. The main structural component of the outer membrane is the major outer membrane protein (MOMP), a 39–41-kD protein with multiple cysteine residues providing disulfide cross-linking, which is thought to provide the structural integrity of the outer membrane. MOMP comprises about 60% of the outer membrane complex. MOMP is composed of essentially four loops, the end of each being surface-exposed. There is greater amino acid diversity in the surface-exposed or variable domains, probably accounting for the antigenic variation among the C. trachomatis serovars. There are also genus-specific and species-specific epitopes associated with MOMP (Stephens et al. 1988). MOMP is the immunodominant protein in all chlamydial strains except for
C. pneumoniae and, as such, has been identified as a major target antigen for the protective immune response. Antibodies to MOMP are generally capable of neutralizing chlamydiae and in both murine and guinea pig studies have been capable of eliciting a protective immune response (Rank 1999). Other structural components of the outer membrane include a 60-kD cysteine-rich molecule, OmcB, and a lipopolysaccharide (LPS) moiety. The LPS has mild endotoxic activity and resembles the “deep rough” (Re) mutant LPS of enteric bacteria (Nurminen et al. 1984). It is important to recognize that chlamydiae are differentiated from most other bacteria by the absence of peptidoglycan in the outer membrane. While much of the biosynthetic pathway is coded for by the genome, no one has been able to detect its presence or that of muramic acid. Another set of proteins has been described that is associated with the outer membrane and appears to be variable in its surface exposure. The polymorphic outer membrane proteins (POMPs) are high-molecular-weight molecules (≥90 kD) coded for by the pmp genes. Their expression and number is variable, depending on the species of Chlamydia. They are also quite variable in their antigenicity, and it is not clear what their function is; nevertheless, they remain a major focus of current research.
B.
Developmental Cycle
A biphasic developmental cycle in which one form (elementary body) is infectious but nonreplicating and the other (reticulate body) is not infectious and restricted to the intracellular environment as the replicating form is a unique characteristic
Fig. 13-1 An MoPn inclusion (arrow) in a epithelial cell on a scraping from the mouse genital tract.
332 of chlamydiae. The cycle is initiated by the attachment and internalization of the elementary body to the host cell. The mechanism of attachment has been a topic of great discussion over the years and is still not entirely clear. While there are likely several different mechanisms employed by this organism, the one that is most feasible and accepted by the majority of researchers involves a heparin sulfate–mediated attachment mechanism. Stephens has demonstrated that a sulphated glycosaminoglycan (GAG) appears to bridge yet-unidentified molecules on the surfaces of the host cell and elementary body in a trimolecular mechanism of attachment (Zhang and Stephens 1992). Since heparin sulfate–containing proteoglycan is present on the surface of the host cells, the mechanism would involve the displacement by molecular mimicry of host heparin sulfate by the chlamydial GAG. Data suggest that uptake of the organisms by the cell may be through either pinocytotic or phagocytic mechanisms. As the elementary body is taken up into the cell, a tightly associated vesicle is formed around it within 2 hours of uptake. Key to the intracellular survival of chlamydiae is the inability of lysosomes to fuse with the chlamydial vacuole, so that the organisms are not killed, although the mechanism by which this is prevented is not understood. Chlamydiae initiate protein synthesis soon after uptake and mediate the directed transport of the elementary bodies to the per-Golgi region of the cell. This facilitates the intersection of chlamydiae with the exocytic pathway of the Golgi, which delivers sphingolipids from the Golgi to the plasma membrane (Scidmore et al. 1996). Thus, the chlamydial vesicle or inclusion membrane acquires sphingolipids from the Golgi apparatus through an interaction of the chlamydiae with this exocytic pathway. During this time the elementary body differentiates into the reticulate body, and the reticulate body begins to divide by binary fission. As the reticulate bodies actively metabolize and divide, the inclusion expands in size until it occupies much of the host cell. Recently, it has become apparent that there is direct interaction between the metabolic active reticulate bodies and the host cell. Hsia and Bavoil found evidence for a type III secretion apparatus in chlamydiae, similar to that seen in other enteric bacteria (Hsia et al. 1997). The structure of the type III secretion apparatus bears a remarkable resemblance to the projections on the external surface of chlamydiae. Matsumoto observed by electron microscopy that these spike-like structures apparently penetrate the inclusion membrane and appear to establish a connection between the reticulate body and the host cell (Matsumoto 1988). It is quite possible that these structures may be the conduit for information exchange between the organism and the host cell. Clear definition of molecules that are transported via the type III secretion apparatus has not been made, but it is obvious that there is communication between the organism and the host. Evidence for some form of interaction is apparent from the observation that chlamydiae are able to interrupt the signaling pathways that lead to the induction of apoptosis early in the
ROGER G. RANK
developmental cycle (Fan et al. 1998; Perfettini et al. 2002). However, toward the end of the developmental cycle, there is an up regulation of apoptosis in the cell. Therefore, it would appear that the organism, in order to protect itself as it replicates, has evolved a mechanism to prevent the death of the cell (Byrne and Ojcius 2004). However, at the end of its cycle, it would be advantageous for the cell to die through nonnecrotic mechanisms so that the organisms can be released with a minimum of inflammation. As the inclusion continues to develop, eventually the reticulate bodies begin to de-differentiate back to elementary bodies. The mechanism by which this occurs is also unclear. The favored theory is that as the reticulate bodies begin to become crowded and are forced off the inclusion membrane, they lose contact with the membrane, and this signals the organism that it should begin to shut down its metabolic processes and differentiate into the elementary body. The chlamydial developmental cycle is completed on the average after about 36–48 hours. Release of the organisms may be either by lysis of the cell or by host cell death by apoptotic mechanisms.
C.
Metabolism
The availability of the genome sequence for several species of chlamydiae has made possible a more thorough understanding of the metabolic capabilities of the organism. A thorough analysis of the genome of C. trachomatis serovar D was presented by McClarty previously and will only be summarized here (McClarty 1999). Not surprisingly, chlamydiae have been found to have the full complement of genes necessary to carry out DNA replication and repair, as well as the necessary genes for rRNA, tRNA, and ribosomal proteins so that protein synthesis can be carried out. Because chlamydiae are obligate intracellular parasites, it is reasonable to assume that they could derive much of what they need from the host cytoplasm, given that they have the appropriate transport machinery. Accordingly, the C. trachomatis genome contains few genes for the biosynthesis of amino acids and a good number of amino acid transporters. Many of the amino acid biosynthetic pathways that are present are truncated and produce intermediates that are used for other biosynthetic pathways. They are, however, lacking the de novo and salvage pathways for nucleotide synthesis, so it would appear that these must be obtained from the host cell. Chlamydiae have been traditionally referred to as “energy parasites” because it was thought that they could not produce their own energy intermediates. This concept was supported by studies that showed that fragments of energy pathways were present but lacked the complete pathways for the generation of ATP. Hatch added to this concept by finding an ATP/ADP translocase that could theoretically allow the organism to obtain its ATP from the host cell (Hatch et al. 1982). However, in
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what was a great surprise, the genome showed that chlamydiae have retained energy-generating capabilities. Although incapable of using glucose because of the absence of a hexokinase (Vender and Moulder 1967), C. trachomatis can direct glucose6-phosphage into the Emden-Meyerhoff-Parnas pathway with the net production of 4 ATP. The TCA cycle is incomplete by lacking three important early enzymes, citrate synthase, aconitase, and isocitrate dehydrogenase, which are necessary for the entrance of acetyl-CoA into the pathway, but it could appear to be functional if supplied by an alternate source of carbon. Similarly, respiration also occurs in chlamydiae, with oxygen as the terminal electron acceptor in the respiratory chain. They appear to have both a redox-driven Na++ pump and an H+ pump in their respiratory chain. The role of the ATP/ADP translocase is not clear, but what is clear is that chlamydiae are far more capable of producing their own energy than previously thought. Thus, rather than energy being the limiting factor forcing the organism toward intracellular parasitism, it is more likely the absence of de novo or salvage pathways for nucleotide biosynthesis that forces the organism into this lifestyle.
D.
In Vitro Culture
Chlamydiae are obligate intracellular parasites and, as such, are not capable of growing in a cell-free medium. Initially, culture of chlamydiae was performed exclusively in embryonated hen’s eggs. The yolk sacs were harvested, and enriched suspensions of elementary bodies produced. Since the mid1960s, the optimal method of culturing chlamydiae has been in tissue culture (Gordon and Quan 1965). Despite the strong host specificity in animals, chlamydial species are quite promiscuous in tissue culture, being able to infect and grow in a wide range of cell lines. The most commonly used cell lines include fibroblast lines, McCoy and L cells, and epithelial cell lines such as HeLa, Hep2, and BGM cells; however, many different cell types have been successfully used. Generally, monolayers are infected by the addition of a chlamydial suspension to the culture with mild agitation at 15-minute intervals. The supernatant is then removed and fresh medium added and the cultures incubated for 48–72 hours, depending on the inoculating dose. Optimal culture of chlamydiae requires additional glucose and cycloheximide to prevent host cell replication. Chlamydiae may be quantified by adding serial dilutions of the stock or specimen to monolayers of cells in either 96 or 24 well plates or single Shell vials and then centrifuging to facilitate attachment prior to an incubation period (Schachter and Dawson 1978). The media is changed and the plates are incubated for 36–72 hours, depending on the chlamydial species. The monolayer is stained with a fluorescein-labeled antibody and the number of inclusions determined. The readout is the number of inclusion-forming units (IFU), since one cannot be certain that individual inclusions are not the result of fusion of inclusions following
infection with more than a single elementary body. Chlamydiae are easily stored in a sucrose-phosphate buffer at −70°C.
IV.
TAXONOMY AND MOLECULAR AND
ANTIGENIC RELATIONSHIPS OF MoPn As is evident from the discussion of the history of the organism, it was first thought that chlamydiae were viruses. However, molecular evaluation of rRNA sequences has confirmed that chlamydiae are indeed eubacteria, but distantly related to other eubacterial orders (Weisburg et al. 1986). Chlamydiae have their own order, Chlamydiales, which includes the family Chlamydiaeceae (Moulder et al. 1984). Until recently, there was a single genus, Chlamydia, with three species, C. trachomatis, C. psittaci, and C. pneumoniae. However, a new taxonomic scheme has been proposed in which two genuses, Chlamydia and Chlamydophila, have been designated with multiple new species designations based on the host animal species (Everett et al. 1999). This has created much debate among researchers in the chlamydial field, and both schemes are currently being used (Schachter et al. 2001). Nevertheless, there is rapidly developing acceptance of a third scheme in which the genus name, Chlamydia, is being retained for all chlamydiae but the new species names will be used (Kalayoglu and Byrne 2004). In the context of this review, C. trachomatis has been divided into three biovars: oculo-genital biovars with serovars A-K, the lymphogranuloma venereum biovars with serovars L1-L3, and the mouse pneumonitis biovar. In the new taxonomy, MoPn is still recognized as being closely related to C. trachomatis but is now referred to as Chlamydia muridarum. At the phylogenetic level, the oculo-genital biovars are more closely related to the LGV biovars than they are to MoPn (Moulder 1988). In DNA thermostability studies, Weiss reported a 30%–60% relatedness between MoPn and the other C. trachomatis biovars, while the relationship between the oculo-genital and LGV biovars was near 100% (Weiss et al. 1970). Over the last several years, a number of chlamydial genomes have been sequenced, including the Nigg strain of MoPn which was sequenced in 2000. Read and colleagues compared the sequence of MoPn to C. trachomatis serovar D and C. pneumoniae (Read et al. 2000). Genome analysis showed an average difference in orthologous genes of 10% but, despite this, there was a remarkable gene similarity between MoPn and serovar D. There was also a strong similarity between MoPn and C. pneumoniae, although the differences were greater than between MoPn and C. trachomatis. There is one segment of the chlamydial genome, called the plasticity zone, that shows greater genetic reorganization. It is suggested that the differences in this zone are significant enough to account for differences in pathogenesis of the chlamydial strains. A unique feature of MoPn is the presence of a gene that codes for a putative toxin with a 53% overall similarity to a toxin encoded by the
334 Eschericha coli 0157:H7 virulence plasmid. The N-terminal end of the MoPn and 0157:H7 toxins have some similarity to the large clostridial toxins (LCT) (Read et al. 2000; Belland et al. 2001). The gene is also present in C. trachomatis serovar D, but there are numerous frameshift mutations in it compared to MoPn. Whether it has a role in pathogenesis has not been determined. Because of remarkable similarity in gene content and order between MoPn and serovar D, it is difficult to deduce reasons for host specificity and differences in pathogenesis. An important difference between the two has been the finding that serovar D contains tryptophan biosynthesis genes not found in MoPn. Since one of the major effector mechanisms employed by the host against chlamydial infections is IFN-γ, which modulates the depletion of intracellular tryptophan and induces a bacteriostatic effect on chlamydiae, the absence of these genes in MoPn may make it more susceptible to IFN-γ and less likely to produce a persistent infection (Wood et al. 2003; FehlnerGardiner et al. 2002). Initially, studies suggested that MoPn was quite different antigenically from the oculo-genital and LGV strains, with little cross-reactivity with antibodies directed against the human strains (Caldwell et al. 1975). Moreover, monoclonal antibodies that recognized a number of human C. trachomatis biovars failed to react with MoPn by immunofluorescence or ELISA (Stephens et al. 1982). However, Stephens later noted that MoPn does indeed share species-specific determinants on MOMP with the human serovars, but that the MoPn MOMP epitope recognized by monoclonal sera was not surface exposed (Stephens and Kuo 1984). By sequencing MOMP from various strains, Zhang identified relationships among the various chlamydial species (Zhang et al. 1993). He found that MoPn was much more closely related to the hamster SFPD strain of C. trachomatis than to the human strains, but the sequence was even more dissimilar with the C. psittaci and C. pneumoniae strains. Thus, it appeared that phylogenetically, MoPn and the hamster SFPD strain were clearly classified as C. trachomatis but were different from the human C. trachomatis strains. To date, the hamster SFPD genome has not been sequenced, so further relationships cannot be deduced, although it is likely that the hamster SFPD strain is also C. muridarum. From a practical viewpoint of protection against infection, Ramsey observed that a primary MoPn or serovar E genital tract infection could protect mice against challenge with the heterotypic organism (Ramsey et al. 1999). Therefore, these data indicate that there is sufficient immunologic cross-reactivity between MoPn and human C. trachomatis that the host does not recognize the two biovars as being remarkably different. Only a single strain of MoPn has been traditionally recognized, although it is clear based on a historical review of the literature that there are two separate isolates in existence, both derived from the lungs of laboratory mice (Nigg 1942; Gordon et al. 1938). A direct comparison of the two isolates by respiratory infection of mice and in vitro culture in HeLa cells has
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suggested that there are subtle differences between the two and that they may be two different variants. Ramsey has observed that the Weiss strain has a lower LD50 for respiratory infection in mice than does the Nigg strain (K.H. Ramsey, personal communication). When the growth curve of the two strains was evaluated in vitro, it was noted that, although the multiplicity of infection was the same for the two variants, a significantly higher yield of elementary bodies was produced by the Weiss strain compared to the Nigg strain (R.G. Rank, T. Darville, and K.H. Ramsey, unpublished data). Although there was no obvious difference in the course of genital infection, mice infected with either the Weiss or Nigg strain were solidly immune to challenge with the Nigg strain; however, they routinely became infected when challenged with the Weiss strain (K.H. Ramsey, personal communication). Thus, these data suggest that there is a difference in pathogenic potential between the two isolates and that they may actually represent two distinct variants of MoPn.
V.
CLINICAL DISEASE, PATHOGENESIS, AND IMMUNOLOGY
The natural site of infection for MoPn appears to be the lung, although one cannot rule out the gastrointestinal tract, as discussed earlier. Nevertheless, as has been discussed above, the mouse may be utilized as a model for human diseases at other sites, in particular the genital tract. Thus, in this section, general pathogenic mechanisms will be discussed, followed by a description of the various parameters of disease and immunity for each organ site and chlamydial species because there are specific issues associated with each. The emphasis of research in the mouse in recent years has been on the basic immune mechanisms operative in chlamydial infection.
A.
Basic Mechanisms of Pathogenesis
Recent studies have made it clear that there is a dynamic relationship between the chlamydial organism and its host cell. The organism has obviously evolved mechanisms by which it is able to derive what it needs from the host cell, but it has learned to do so in a manner in which it is orchestrating host cell functions. One example described above is the regulation of the apoptotic pathway in the host. Although we are far from understanding the complexities of this particular host-parasite interaction, it is already apparent that chlamydiae cause the induction of cellular events in the host cell that have implications for both the survival of the parasite and the well-being of the host in general. As will be discussed below, the host’s innate and adaptive immune response to chlamydiae are responsible for the production of pathology and disease. However, much of the host response is initiated by the interaction of the organism
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with the host cell, such that the organism may actually dictate the nature of the host response directed against it. Thus far, it appears that disease is primarily the result of the host’s innate and adaptive immune response, although cytotoxicity has been demonstrated in vitro in association with a cytotoxin gene in MoPn (Belland et al. 2001). Nevertheless, there has been no definitive evidence of the effect of a toxin or other pathogenspecific effector molecules in vivo. It seems clear at this point that the acute inflammatory response is elicited upon infection of the host and that proinflammatory cytokines and chemokines are responsible for eliciting the response. However, recent studies have addressed the specific chlamydial components that initiate the response. Ingalls first demonstrated that incubation of chlamydial elementary bodies or chlamydial LPS with whole blood was able to elicit a TNF-α response, although the intensity of the response was much lower than when Salmonella LPS was used (Ingalls et al. 1995). In addition, she showed that chlamydial LPS could induce the translocation of NFκB in a Chinese hamster ovarian fibroblast line transfected with CD14, suggesting that LPS might be an initiating factor in the host inflammatory response. Rasmussen and colleagues later reported that infection of cervical and colonic epithelial cells with either C. trachomatis or C. psittaci up-regulated mRNA expression and secretion of the proinflammatory cytokines, IL-8, GROα, GM-CSF, and IL-6 (Rasmussen et al. 1997). IL-8 expression was seen as early as 2 hours after infection, and NFκB translocation was detected as early as 30 minutes after infection (Rasmussen et al. 1998). That these responses only occurred after infection with viable organisms and not by incubation of cells with inactivated organisms indicated that the process was initiated by a specific host/organism interaction. It is quite possible that the mechanism may relate to signals passed to the host cytoplasm via the type III secretion apparatus, but this remains to be determined. Recently, there has been a great deal of focus on pattern recognition receptors such as the toll receptors. These structures are able to recognize molecules common to many organisms such as LPS, peptidoglycan, and heat shock proteins, among others. Interaction of the molecule with a given toll receptor can initiate a signaling pathway in the cell that results in the production of proinflammatory cytokines. It has been suggested that LPS and hsp60 may be important molecules in the initiation of the acute inflammatory reaction through their interaction with CD14 and possibly the toll 4 receptor, since that receptor binds both molecules (Kol et al. 2000; Bulut et al. 2002). However, in an elegant study, Darville infected toll 4 knockout mice intravaginally and found no difference in the degree of the resulting pathology (Darville et al. 2003). In contrast, toll 2 knockout mice had a significantly decreased pathologic response and reduced level of TNF-α. Interestingly, there was no difference in the infection course in either set of animals. Thus, the data strongly suggest that the acute inflammatory pathologic process in chlamydial genital infections is mediated through the interaction of the organism with the toll 2 receptor,
ruling out a role for both LPS and hsp60. The identity of the stimulating molecule, however, is not yet known. As the infection progresses, the adaptive immune response becomes activated and one finds an influx of mononuclear cells, particularly T cells and macrophages, to the local site. In the MoPn infection of the mouse, this is mainly CD4 cells, but in the guinea pig and subhuman primates, there are equal numbers of CD4 and CD8 cells (Kelly and Rank 1997; Rank et al. 2003; Van Voorhis et al. 1996; Whittum-Hudson et al. 1986). These cells and the cytokines they secrete are critical for the resolution of infection, but it cannot be ruled out that they also contribute to the induction of pathology. It is very apparent that the mononuclear response is more intense upon reinfection, producing disease of increased severity (Tuffrey et al. 1990; Rank et al. 1995; Wolner-Hanssen et al. 1986). There has been much debate as to which chlamydial antigen(s) induce this pathologic response. Serologic studies in humans and experiments in the guinea pig ocular model suggested a possible role for chlamydial hsp60 as the target antigen responsible for the cell-mediated pathologic response (Wagar et al. 1990; Morrison et al. 1989). While the molecule is indeed very immunogenic, studies found that guinea pigs that were immunized with hsp60 and then challenged actually had less severe disease, suggesting that sensitization with hsp60 does not elicit a more severe response or that, alternatively, anti-hsp60 antibodies bind to the antigen and block the response (Rank et al. 1994). Other studies in the guinea pig suggested that the enhanced pathology was really associated with multiple infections and that animals with higher titers of antibody were those that had greater exposure to the antigen (Rank et al. 1995). There has been no definitive role for hsp60 as a virulence factor described in the murine models. It is more likely that there are several antigens associated with chlamydiae that induce a pathologic T cell response.
B.
MoPn Respiratory Infection
It is clear from the early publications that MoPn produces a subclinical respiratory infection in nature, because the early researchers could only detect the organism by serial passage of lung homogenates, in effect, by increasing the titer of organisms with each successive passage. However, the vast majority of the literature on MoPn is derived from studies in which mice were inoculated with unnaturally high numbers of the organism. Therefore, our view of the natural history of the organism in its native host may be distorted and have little bearing on the natural host-parasite relationship. Nevertheless, the accumulated studies do paint a comprehensive picture of MoPn respiratory disease and in particular of the host response to this organism. 1.
Infection Course
It is likely that low doses of organisms will be infectious via the respiratory route, although the only data are anecdotal.
336 That mice were clearly infected in the lung with homogenates from donor mice although no pathology was apparent suggests that low doses elicit a subclinical infection. Intranasal infection of mice with 104 IFU or less will result in infection of the mice, and the majority will recover from the infection. Larger numbers of organisms will be lethal for mice. It has been difficult to map out the kinetics of the infection because of the necessity to euthanize the mice for quantification of the organisms in the lung. However, Williams et al. (1981) reported that after infection with 102 to 103 IFU, animals were positive in the lung for chlamydiae on day 3 but had resolved the infection by day 7. Infection doses higher than 103 result in the death of the majority of the animals by 20 days after infection (Williams, Schachter, Coalson, et al. 1984). Gogolak reported a linear relationship between the number of elementary bodies inoculated and the day of death (Gogolak 1953c). Animals infected intranasally with 4 × 109 elementary bodies died on the average by 8 days after infection, whereas mice infected with only a log fewer elementary bodies survived, on the average, until day 12. Animals that recover from infection are immune to reinfection. Clearly, in the determination of the course of MoPn respiratory infection, one must ideally sacrifice different groups of animals at various times after infection. However, one can gain some degree of information about the course of the infection by weighing the animals at various intervals. Particularly in infections with higher doses of organisms, a loss of weight is related to the degree of pathologic response to the infection. 2.
Pathologic Response
Several reports have been written describing the pathologic response to MoPn in the lung following intranasal inoculation (Gogolak 1953a; Weiss 1949; Williams, Schachter, Coalson, et al. 1984). Weiss characterized the infection by concentrating on the local growth of the organism and the cells that it infected (Weiss 1949). MoPn can be found in the alveoli as early as 30 minutes after inoculation of a nonlethal dose. Over the next 30–36 hours, the organism can be found in inclusions in the alveolar cells. The inclusions are constantly increasing in size over this initial time period. Between 30–36 hours, it is apparent that some of the inclusions are breaking up, releasing elementary bodies into the alveolar space. After 36 hours, the infection loses its synchronicity, and numerous examples of all stages can be detected in the alveolus through 7 days after infection, gradually decreasing from 10–13 days, and disappearing at 21 days. Weiss reported that occasionally organisms could be found in free macrophages in the alveoli. Organisms could also be detected in the epithelial cells of the bronchioles as early as 24 hours after infection, with heavy infection of most cells by 3 days after infection. The columnar cells of the terminal bronchioles were the most common cells infected, although low cuboidal cells of the respiratory bronchioles were all infected.
ROGER G. RANK
In contrast to local growth described by Weiss, Gogolak emphasized the cellular response to MoPn infection in the lung as a whole (Gogolak 1953a). He first observed a cellular response to infection at 44 hours after intranasal inoculation. The response was characterized by an accumulation of heterophils in the infected alveoli, although there was clearly a stronger inflammatory response than had been suspected based on the numbers of organisms present. By 3 days after infection, the infection was generally focal in nature, with a sharp demarcation between normal tissue and inflamed tissue. The inflammatory exudate at day 3 was found to contain primarily a mononuclear infiltrate, consisting of lymphocytes, monocytes, and macrophages. The alveolar structure is obscured in the inflamed site and one finds extravasation of erythrocytes, but in heavy infections the inflammatory sites coalesce, probably resulting in loss of function of the affected tissue. As early as 4 days after infection, large portions of the lung, even those not directly associated with the infectious process, are no longer normal in appearance. The alveolar spaces contain a large number of macrophages, and the bronchioles become inflamed with both heterophils and a small number of mononuclear cells. Obviously, compromising the lung to this extent can lead to death of the animal. In animals receiving lower doses, the infection begins to wane and the mononuclear infiltrate diminishes as well. Since mice that have recovered from a primary MoPn respiratory infection have a markedly diminished infection course upon reinfection, it is obvious that there are likely differences in the histopathologic responses between a primary infection and challenge infection. Williams and his colleagues published a detailed analysis by light and electron microscopy of the histopathologic response in both nonimmune BALB/c mice and mice that had recovered from an infection (Coalson et al. 1987). By 2 hours after intranasal infection with 5 × 104 – 5 × 105 IFU (5 × 104 was lethal to 50% of the nonimmune mice), they found only normal tissue in naive animals but found peribronchiolar and perivascular collections of intermixed monocytes, lymphocytes, and plasma cells in immune mice. At 10 hours after infection, small numbers of heterophils were seen in the lumina of small bronchioles and surrounding alveolar spaces and scattered within the alveolar walls in both immune and nonimmune groups. By 24 hours, heterophil accumulations were found at the respiratory bronchiolar/alveolar duct sites in the nonimmune group but not in the immune group. Nevertheless, heterophils were evident in the inflammatory exudates of both groups. The nonimmune mice developed a confluent bronchopneumonia with heterophils and alveolar macrophages predominating by 5 days, whereas the perivascularperibronchiolar pattern of inflammation persisted in the immune mice, although the heterophil response was diminished. By 14–21 days, nonimmune mice developed persistent nodules of chronic inflammatory cells at airway and vascular sites. These contained primarily lymphoblasts and plasma cells, with fewer numbers of lymphocytes. Moreover, the mice had persistent
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focal alveolar abnormalities consisting of edema, some heterophils, plasma cells, and alveolar macrophages. The immune mice showed nodules of abundant lymphocytes, with fewer plasma cells and lymphoblasts and only rare sites of active alveolitis. Upon electron microscopic examination at 10 hours after infection, reticulate bodies could be found in the endosomes of type I alveolar epithelial cells in both naive and immune groups (Coalson et al. 1987). Elementary bodies were easily observed in the nonimmune group but were infrequent in the immune group. At 24 hours, large cytoplasmic inclusions were seen in the Type I cells of naive mice with all stages of the chlamydial developmental cycle. No organisms were seen in the immune group, but activated alveolar macrophages were abundant. The number of organisms continued to increase in the nonimmune group by 48 hours, with many inclusions either ruptured or found loose in the alveolar spaces. Many elementary bodies were observed free in the alveoli or in phagosomes of heterophils and macrophages. Focal epithelial disruption and scattered fibrin stands were present. As earlier, no inclusions were detected in the tissue from immune mice, although many alveolar macrophages were recognized with secondary lysosomes. A similar picture was still present at 5 days after infection. At 14 days, large numbers of plasma cells, lymphocytes, and monocytes were seen in the interstitium of both groups. Alveolar spaces contained many lipid-filled macrophages but few heterophils. No intact organisms were observed at this point in time. Plasma cells with widely dilated endoplasmic reticulum were apparent in both groups at 21 days. The data from these histopathologic analyses clearly indicated a two-stage pathologic process in a primary infection: The first is the induction of an acute inflammatory response through the production of proinflammatory cytokines via activation of NFκB and the toll receptor pathway, and the second is the adaptive response with an influx of lymphocytes, monocytes, plasma cells, and macrophages. In infections with sublethal inoculating doses, the combined innate and adaptive response is able to resolve the infection. Nevertheless, both acute and chronic inflammatory responses are the immediate cause of the pathology and may compromise lung function through the destruction of lung tissue. That mice receiving very low doses of infectious organisms, as apparently occurs in nature, are able to recover from infection while showing no obvious clinical signs of the disease suggests that the clinical and pathologic response is dependent upon the number of organisms and the resultant host response. In the case of a low inoculum, the early heterophil response may be able to contain the infection until the adaptive immune response becomes activated. However, as the inoculating dose is increased, the initial acute inflammatory response and subsequent mononuclear response will be greater and be more likely to cause increased pathology and clinical disease. The host response upon reinfection is also dose dependent and involves an initial acute inflammatory response but a more vigorous influx of mononuclear cells.
3.
Immunity and Immune Mechanisms
Whether the immune response in mice is sufficient to resolve a MoPn respiratory infection is strictly dependent upon the inoculating dose. Animals infected with low doses develop both antibody and cell-mediated responses and are able to recover from the infection. Recovery from infection does result in immunity to reinfection, although that immunity does not provide complete resistance to reinfection (Williams et al. 1997). Thus, immune animals that are given a fresh challenge become reinfected, but the resultant infection is significantly decreased in intensity and duration of infection. The immunity is also sufficient to prevent death from challenge with a lethal dose of MoPn. Not surprisingly, MoPn respiratory infection elicits both an antibody and cell-mediated immune response. Mice develop MoPn-specific IgM, IgG, and IgA antibodies in serum and both IgG and IgA in bronchial lavage specimens as a result of infection (Williams et al. 1982; Williams, Schachter, Weiner, et al. 1984). The presence of cell-mediated immunity was demonstrated by the response of splenic T cells to chlamydial antigen in a lymphocyte proliferation assay and the ability of chlamydial antigen to elicit a delayed-type hypersensitivity response when injected into the pinna of the ear (Williams, Schachter, Coalson, et al. 1984). To investigate the role of both antibody and T cells in the murine host response to MoPn, Williams and colleagues infected congenitally athymic nude mice intranasally with MoPn and followed the course of the infection (Williams et al. 1981). Nude mice were quite susceptible to the infection and died of fulminate pneumonia from nonlethal doses; therefore, it was obvious that T cells were essential for resolution of MoPn infection. Restoration of T cells by either thymic graft or adoptive transfer of splenic T cells was able to restore the protective response (Williams, Schachter, Coalson, et al. 1984; Williams et al. 1981). While it was clear that T cells were essential for the protective host response, these experiments could not differentiate whether antibody or T cell effector mechanisms or both were the essential components of the response. The role of antibody was first evaluated by inoculating immune serum or bronchial lavage fluid intranasally into nude or immunologically normal mice prior to challenge with a lethal dose (Williams, Schachter, Weiner, et al. 1984). The treatment was effective in bringing about the resolution of the infection in 70% of the normal mice, in contrast to none of the mice receiving nonimmune serum. However, in the nude mice, while the antibody delayed the time until death, it did not prevent the animals from dying from the infection. Similarly, intravenous injection of immune serum delayed death in nude mice but did not resolve the infection (Williams et al. 1982). Williams et al. also opsonized chlamydial elementary bodies with immune IgG or IgA prior to intranasal inoculation. This procedure prolonged survival, with both IgG and IgA fractions working equally as well (Williams, Schachter, Weiner, et al. 1984). These data suggested that antibody may play a role in immunity to
338 MoPn but is not the sole immune effector in the protective response. However, in the most definitive experiment, Williams depleted mice of B cells by treatment of mice with anti-IgM beginning on the day of birth and then several times a week for the life of the animal (Williams et al. 1987). When B cell–deficient mice were infected intranasally with MoPn, the mice recovered from the infection comparable to immunologically intact mice. With the advent of transgenic mice, Williams infected IgH−/− B cell–deficient mice with MoPn and found that they were as able to resolve infections as the controls but had a small but significant increase in number of organisms in comparison to normal controls upon reinfection (Williams et al. 1997). Thus, these data strongly indicate that antibody or B cells are not essential for recovery from a MoPn respiratory infection. To further investigate the role of cell-mediated immunity, Williams injected various populations of immune spleen cells into infected nude mice and observed that mice receiving a T cell–enriched population had a better survival rate than animals receiving a B cell–enriched population. Separation of the immune spleen cells into CD4- or CD8-enriched populations did not restore immunity to the mice as readily as an unseparated splenic preparation. Another study to determine the role of cell-mediated immunity was initiated by Williams, in which he gave mice a sublethal injection of Histoplasma capsulatum in order to activate macrophages (Williams, Schachter, Coalson, et al. 1984). The prior injection of this intracellular yeast served to activate macrophages in the mice and increased the resistance of nude mice to MoPn challenge. Conversely, prior immunization of normal and nude mice with MoPn increased their resistance to histoplasma challenge, indicating that MoPn is able to activate macrophages, even by nonspecific mechanisms. A role for CD4 T cells was confirmed when Magee infected mice deficient in MHC class II–bearing cells intranasally with MoPn (Magee et al. 1995). The knockout mice had prolonged infections with increased mortality in comparison to control mice and were more susceptible to reinfection, with higher numbers of organisms being isolated from the lungs. These data were supported by experiments in which CD4 or CD8 cells were depleted by treatment of mice with antibodies to each of the cell phenotypes (Magee et al. 1995). Mice in which CD4 cells were depleted had a higher mortality rate in comparison to mice lacking CD8 cells (Magee et al. 1995). Moreover, mice functionally deficient in CD4 cells (MHC Class II knockouts) were more susceptible than mice functionally deficient in CD8 cells (β2-microglobulin knockouts). Upon reinfection, CD4-deficient mice had higher levels of infection than did CD8-deficient mice, although there was a modest increase in infection in the CD8 knockout mice (Williams et al. 1997). Magee and colleagues also reported that SCID mice were unable to resolve a MoPn respiratory infection; however, the adoptive transfer of a CD4 T cell clone into the mice was able to significantly reduce the mortality associated with the
ROGER G. RANK
infection (Magee et al. 1993). Therefore, it is clear from these data that the CD4 cell is the crucial element of the protective immune response in MoPn respiratory infection. That CD4 cells are essential for the resolution of and resistance to MoPn respiratory infection would suggest a cytokine-mediated effector mechanism. Although cytotoxic activity of CD8 cells against chlamydiae has been demonstrated in vitro, there has been no evidence for a T cell–mediated cytotoxic mechanism in vivo (Beatty and Stephens 1994; Starnbach et al. 1994). Byrne and colleagues originally demonstrated that supernatants derived from the culture of splenic or bronchial lavage cells from mice infected intranasally with MoPn were able to inhibit the growth of chlamydiae in vitro, whereas comparable supernatants from nude mice were ineffective (Byrne et al. 1987). Because the activity was labile at low pH, they concluded that the cytokine responsible for the inhibition was gamma interferon (IFN-γ). Importantly, they noted that the activity could be abolished by elimination of cells with antibody to L3T4 (CD4) but not antibody to Lyt 2.2 (CD8) or asialo GM-1 (NK cells). In a follow-up report, they showed that IFN-γ produced by CD4 cells as a result of respiratory infection was cytotoxic for chlamydiae in vitro (Byrne, Grubbs, et al. 1988). However, they did note some reduction in IFN-γ production by CD8 cells late after infection, but not at the time of resolution of the infection. That IFN-γ was required for resolution of the infection was confirmed by Williams and his group, when they depleted IFN-γ from mice by treatment with anti-IFN-γ and found that the mortality of mice following MoPn respiratory infection was significantly increased (Williams et al. 1988). Yang and colleagues later quantified IFN-γ and IL-10 from spleen cells taken from mice infected with MoPn and incubated with MoPn antigen in vitro. They found that high levels of IFN-γ and only minimal IL-10 were produced, suggesting a Th1 response. Moreover, they reported that the dominant antibody response was that of IgG2a, which is indicative of a Th1 response. These findings indicate that MoPn respiratory infection induces a Th1 cytokine response in mice. The adoptive transfer into SCID mice of a CD4 Th1 T cell clone derived from spleens of mice having resolved a MoPn genital infection was able to significantly reduce mortality, therefore confirming that Th1 cells are critical for the protective immune response to chlamydial respiratory infection (Magee et al. 1993; Igietseme et al. 1993). In contrast, resistance to infection in IL-4 knockout mice was not affected, suggesting that the Th2 response is not critical in immunity to MoPn (Williams et al. 1997). While it is likely that the bulk of IFN-γ in MoPn infections is derived from CD4 cells, there is also evidence for IFN-γ production by NK cells. Both nude mice and SCID mice were found to have detectable levels of IFN-γ in bronchial secretions (Williams et al. 1993). When nude mice were treated with antiIFN-γ, respiratory disease elicited by MoPn was exacerbated. Similarly, Williams observed that CD4 knockout mice also had comparable levels of IFN-γ in bronchial secretions to that
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found in intact mice early in infection and that the early stages of the infection were no different with regard to kinetics than controls (Williams et al. 1997). These data suggest that non–T cell sources of IFN-γ are important in the early control of chlamydial infection. That other cytokines may play a role in the protective immune response to chlamydiae has also been investigated. IL-6 knockout mice were more susceptible to MoPn respiratory infection than control mice (Williams et al. 1998). Interestingly, treatment with anti-IL-10 diminished the enhanced disease and was associated with a concomitant increase in IFN-γ levels. Thus, IL-6 may play some role in the generation of a Th1 response. It is also of interest that IFN-γ knockout mice, having resolved a MoPn infection, were actually more resistant to a secondary infection than control mice (Williams et al. 1997). This enhanced resistance correlated with a significant increase in TNF-α and GM-CSF levels in bronchial lavages. Treatment of the mice with anti-TNF-α abolished this enhanced protection, suggesting a possible role of TNF-α in the response to challenge infection. C.
Genital Tract Infection (MoPn and Human C. trachomatis)
Since the recognition that chlamydiae are the major cause of sexually transmitted infections, the mouse:MoPn genital tract model, developed and characterized by Barron, has become the most widely used experimental model for the study of chlamydial genital tract disease (Barron et al. 1981). There are clearly differences from other models of chlamydial genital tract disease, including the guinea pig and the subhuman primate, and one must consider this caveat in the application of the data obtained in the mouse to the disease and immune process in humans. Whereas the guinea pig and subhuman primate are better models for the study of the pathologic processes because of the remarkable similarity of the disease to that in humans, the mouse is the better model for the study of the basic immune mechanisms, particularly cell-mediated immunity. Most of the studies on genital infection in the mouse have employed MoPn because it is a natural parasite of the mouse, albeit that the mouse genital tract is not the primary site of MoPn infection and the disease induced in the mouse by MoPn is more pronounced than that produced by the human C. trachomatis serovars. In addition, the vast majority of the studies on chlamydial genital infection have been in the female because of the ease of infection and also because of the relevance to human disease in females. 1.
Infection Course
MoPn is highly infectious when inoculated intravaginally. Investigators have used doses as low as 102 IFU and successfully infected female mice by the instillation of MoPn into
339 the vagina. However, the caveat of intravaginal inoculation is that MoPn is dependent upon the state of the host reproductive hormones. Mice cannot be easily infected when they are in estrus, most likely because the mucus and defoliated cells act as a mechanical block against attachment of the bacterium to its target tissue. We have noted that mice treated with estradiol cannot be easily infected, whereas essentially 100% of ovariectomized mice can be infected in the genital tract (R.G. Rank, unpublished data). Ito also reported that mice could not be infected during estrus or the early part of metestrus with human chlamydial serovars (Ito et al. 1984). Thus, to ensure infection, mice can be inoculated intravaginally on 3 consecutive days, since mice will only be in estrus for 1 day (Rank 1994). Alternatively, researchers have treated mice with progesterone in the form of Depo-Provera (2.5 mg subcutaneously) 7 or 10 days prior to infection. Progesterone has the effect of forcing the mouse into a state of anestrus, thereby providing a stable, more uniform epithelium that is readily susceptible to chlamydial infection. It is advantageous because mice need only be inoculated on a single day, and all mice will become infected. Progesterone does increase the intensity of MoPn infections but does not affect the length of the infection (Cotter, Miranpuri, et al. 1997). Moreover, there has been no evidence that progesterone alters the host immune response to the infection. While progesterone treatment is not required to infect mice in the genital tract with MoPn, it greatly facilitates the infection of mice with the human serovars. Tuffrey found that she could establish infections in mice more readily with prior progesterone treatment (Tuffrey and Taylor-Robinson 1981). Ito made the same observations using several different serovars of C. trachomatis (Ito, Jr. et al. 1990). While intravaginal infection is the method of choice because it mimics the route of infection in human disease, some investigators have infected mice by direct inoculation of the organisms into the uterine horn or the ovarian bursa in order to elicit endometritis and salpingitis in a high percentage of animals (Tuffrey et al. 1982; Pal et al. 1993; Swenson et al. 1983). However, one must be careful how such data are interpreted because of the large number of organisms inoculated into the organ and the potential for spillage of organisms into the peritoneum, thus stimulating the host immune response by a route other than the mucosal route. Upon intravaginal inoculation of MoPn, organisms can be isolated from cervico-vaginal swabs in tissue culture as early as 3 days later. Generally, MoPn can be isolated from the genital tract for about 3 weeks after infection (Barron et al. 1984). While the target tissue is the exocervix (Barron et al. 1981), organisms readily ascend the genital tract to the endometrium and the oviducts within 6 or 7 days of intravaginal inoculation (Cotter, Miranpuri, et al. 1997). Darville also reported that MoPn and serovar E could be found in the upper genital tract by 7 days after infection (Darville et al. 1997). C. trachomatis serovars are much more variable depending on the serovar used, with some infections resolving in 7 days (serovar H)
340 while others can still be isolated up to 9 weeks after infection (serovar D) (Ito, Jr. et al. 1990). Another major variable affecting the course of the infection is the strain of mouse utilized. de la Maza and colleagues observed that MoPn genital infection lasted longer in C3H/HeN mice than in BALB/c or C57Bl/6 mice (de la Maza et al. 1994). They also noted differences in the pathologic response and resultant infertility in the mice, with the response in C57BL/6 mice being less severe than the other two strains. Darville and colleagues compared the kinetics of MoPn genital infection in C3H/HeN and C57Bl/6 mice and found that the infection in C3H mice was significantly more intense and more prolonged than in C57BL/6mice (Darville et al. 1997). Infections in C3H mice lasted about 7 weeks, whereas infections in C57BL/6 mice resolved about 4 weeks after infection. This same difference was reflected in the number of animals in which organisms could be isolated from the upper genital tract. The strain of mouse also influences the course of MoPn infection in mice infected via the respiratory route, with the infection being more severe in BALB/c mice compared to C57BL/6 mice (Yang et al. 1996). Recently, Pal has demonstrated that male mice can be infected in the genital tract with MoPn (Pal et al. 2004). Mice were inoculated in the urethra with varying doses of MoPn, with levels as low as 103 IFU proving infectious. Organisms could be isolated from the urethra, bladder, epididymides, and testes. The infection consistently lasted about 4 weeks in the urethra, with some animals still positive at 7 weeks after inoculation. Organisms could also be detected in the bladder up to 4–7 weeks after infection. 2.
Pathologic Response
Barron first reported that MoPn infection of female mice appeared to be restricted to the exocervix, and organisms were found only in superficial mucus-secreting columnar-type cells overlying the keratinized squamous epithelium (Barron et al. 1981). The infection was accompanied by an acute inflammatory response that was most intense in the superficial epithelium, with less intense inflammation in the subjacent submucosa. It later became apparent that the infection could ascend the genital tract in the mouse. de la Maza described the histopathologic response in the endometrium and oviduct following intravaginal inoculation of MoPn (de la Maza et al. 1994). At 9 and 16 days after infection, they noted an acute inflammatory infiltrate permeating the wall of the uterus in BALB/c, C57, and C3H mice. A comparable response was seen in the oviduct, with a heavy acute inflammatory infiltrate through all layers of the oviduct, including the serosal surface. By days 37 and 49, there was a progressive replacement of the acute inflammatory response, with a chronic inflammatory reaction composed mainly of lymphocytes and plasma cells. In some animals, predominantly the C3H mice, they reported unilateral or bilateral hydrosalpinx. The hydrosalpinx was characterized by a marked
ROGER G. RANK
dilatation of the lumen of the oviduct with flattening of the fimbria and atrophy of the mucosal layer. A similar pathologic response was noted when mice were inoculated directly into the ovarian bursa with MoPn (Swenson et al. 1983). Using the intrabursal injection route, Patton reported a mixed neutrophil and mononuclear cell response in the lumen of the oviducts as well as the mucosal and submucosal tissues on days 5–8 after infection (Patton et al. 1989). They further stained frozen sections of oviductal tissue with labeled antibodies to T and B cells and found a 3:1 ratio of T cells to B cells. Finally, Patton evaluated the tissues by transmission and scanning electron microscopy. A key feature identified by scanning electron microscopy was that the loops of the oviducts were distended, and there was widespread deciliation of the epithelium with evidence of mucosal cell surface erosion. Many ciliated cells had broken away from the basement membrane of the mucosal surface. When the lymphocyte population in the genital tract was assessed, it was found that CD4 cells were the predominant T cell phenotype, with a CD4:CD8 ratio of approximately 2:1 (Kelly and Rank 1997; Morrison and Morrison 2000). The CD4 cells appeared in the genital tract as early as 7 days after infection and peaked approximately 21 days after infection. B cells were also present, although less numerous. The T cell response is clearly associated with the protective immune response to chlamydiae (see below), but there is also evidence in both the mouse and other animal models for a role for T cells in the production of pathology. Tuffrey reported that repeated inoculation of chlamydiae into the oviduct or uterine horn resulted in exacerbated disease, analogous to a delayed-type hypersensitivity response (Tuffrey et al. 1990). The pathologic response in the genital tract is most likely the summation of both an acute inflammatory response elicited through cytokine and chemokine release by infected cells and from an influx of T cells and macrophages, producing tissue damage by cellmediated immune mechanisms. Recent studies have suggested that at least one of the initiating factors in the development of the pathologic reaction is the production of proinflammatory cytokines via the activation of a toll pathway. It was reported by Darville and colleagues that mice lacking the toll 4 receptor had no change in the degree of pathology following MoPn genital infection (Darville et al. 2003). In contrast, mice deficient in the toll 2 receptor had a significant reduction in the acute inflammatory response, thereby suggesting that stimulation of this pathway by a yet-to-be-determined molecule on chlamydiae is a key element in the initiation of the acute inflammatory response in the genital tract. Pathology also varies greatly with the strain of mouse employed. Darville and colleagues did a comprehensive study of the pathologic response in the upper genital tract of both C3H and C57BL/6 mice infected with either MoPn or C. trachomatis serovar E (Darville et al. 1997). They observed that serovar E could be isolated from the upper genital tracts of both groups of mice by day 7 after infection and that acute
13. CHLAMYDIAL DISEASES
inflammation could be detected as early as day 3 in the uterine horn in C57BL/ 6 mice infected with either MoPn or serovar E. In general, C57BL/ 6 mice had stronger acute inflammatory responses in the uterine horns than did C3H mice with both MoPn and serovar E. The chronic or lymphocytic infiltrates in the uterine horns were also slightly more intense in C57BL/6 mice. Interestingly, serovar E did not cause a pathologic reaction in the oviducts of C57BL/ 6 mice but did elicit a modest neutrophil and lymphocytic response in the oviducts of C3H mice. However, MoPn elicited much more pronounced acute and chronic inflammatory responses in the oviducts of both groups of mice. These data indicated that the acute inflammatory response is critical for the development of upper tract disease and that there are significant differences in pathologic response, depending upon the strain of mouse and the chlamydial biovars. Upper tract disease in the mouse produces sufficient damage such that the mice become infertile. Swenson originally reported that direct inoculation of the ovarian bursa would produce sufficient damage to render the mice infertile when mated (Swenson and Schachter 1984). de la Maza later demonstrated that intravaginal inoculation of mice would also significantly reduce the fertility rate and the litter size (de la Maza et al. 1994). Thus, the mouse has become a useful model to determine the effect of immunization with various vaccine candidates on the prevention of infertility, the major morbidity associated with chlamydial genital tract infections. 3.
Immunity and Immune Mechanisms
The murine model of chlamydial genital infection has generated an extensive literature on the basic immune mechanisms elicited by the infection, with the majority of the work concentrating on the role of T cells and more recently on the innate host response. The model has also been used extensively for the evaluation of various vaccine candidates and immunization strategies. The host responses to chlamydial genital infections including the mouse model have been reviewed in great detail elsewhere, so only the major features of the immune response with emphasis on the key immune mechanisms will be covered in this chapter (Rank 1999; Morrison and Caldwell 2002). The host response to murine genital infection is very similar to that described above for the lung model. That an effective protective immune response to MoPn is mounted is evidenced by the resolution of the infection within several weeks of the onset of infection and the development of immunity to challenge infection. However, solid immunity to reinfection is shortlived. Ramsey reported that mice were immune to challenge infection for about 100 days but then became susceptible to reinfection (Ramsey et al. 1989), although the level and length of the challenge infections were diminished. All mice developed a strong antibody response in serum and genital secretions and cell-mediated immune response as a result of the infection (Barron et al. 1984; Ramsey et al. 1989).
341 While it was obvious that an exuberant immune response was mounted by the mouse to chlamydial genital infection, measurements of humoral and cell-mediated responses alone did not provide any definitive information about the mechanism of protective immunity. Rank and colleagues initially infected nude mice in the genital tract with MoPn and found that, in contrast to heterozygote immunologically intact littermates, the nude mice were unable to resolve the infection (Rank et al. 1985). In fact, chlamydiae could be continually isolated from the mice for 265 days after infection. The mice had no obvious clinical signs of infection and appeared quite healthy throughout the entire course of the experiment. Similar results were seen when nude mice were infected with C. trachomatis serovar E and H and followed with isolations for over one year (T. Darville, and R.G. Rank, unpublished data). Interestingly, none of the mice had developed obvious gross pathology when they were autopsied. These data clearly demonstrated that, just as in the lung, T cells were essential for resolution of a chlamydial genital infection. Because previous studies in the guinea pig indicated an important role for antibody in the resolution of and resistance to chlamydial genital infection and because T cells are essential for T cell–dependent antibody production (Rank and Barron 1983; Rank et al. 1979), mice made B cell deficient by treatment with anti-IgM were infected in the genital tract with MoPn (Ramsey et al. 1988). Surprisingly, all of the B cell– deficient mice recovered from the infection exactly as did the control immunologically normal mice. There was no difference in the course or length of the infection. Moreover, upon challenge, the mice were found to be resistant to reinfection. These data were later supported by studies in which B cell–knockout mice were infected with MoPn (Su et al. 1997). However, the B cell–knockout mice, while demonstrating immunity to reinfection, did have more organisms than the normal control mice. Similar data were reported in a study on MoPn genital infection of Fc receptor–knockout mice (FcR−/−) mice (Moore et al. 2002). The primary infection was indistinguishable from the control mice but upon reinfection, the deficient mice had more severe upper tract pathology than the controls. The authors noted that the T cell response was decreased in the FcR−/− mice. Thus, the data indicate that antibody and B cells are not required for the resolution of a primary chlamydial genital tract infection in the mouse but may play a yet-undefined role in immunity to reinfection. That humoral immune mechanisms are apparently less effective in murine chlamydial genital infections is in marked contrast to studies in the guinea pig model, in which antibody is essential for the protective immune response in both a primary and challenge infection. Because of the conflicting data in two animal models, it is unclear which more accurately represents the protective response in humans. That T cells alone were essential for resolution of chlamydial genital infection was confirmed by Ramsey and colleagues, who produced MoPn-specific T cell lines from infected mice and adoptively transferred them into nude mice prior to MoPn genital infection (Ramsey and Rank 1991). They observed
342 that a T cell line enriched for CD4 cells was more effective at clearing the infection than was a line containing both CD4 and CD8 cells. Landers also concluded that CD4 cells were important in the resolution of chlamydial genital infection by depleting CD4 cells from mice by treatment with anti-L3T4 (CD4) antisera (Landers et al. 1991). The mice had increased numbers of organisms in the oviducts and an increased incidence of hydrosalpinx. Su and Caldwell also supported the role of CD4 T cells in resolution of MoPn genital infection through adoptive transfer experiments (Su and Caldwell 1995). Mice receiving CD4 cells enriched from immune spleens were more effective in producing a protective immune response than were CD8- or B cell–enriched immune spleen cell preparations. Igietseme further supported a critical role for T cells in the protective response by deriving a CD4 T cell clone from the spleen of a mouse infected genitally with MoPn (Igietseme et al. 1993). The cell was found to produce large amounts of IFN-γ and was classified as a Th1 cell. Upon its adoptive transfer into infected nude mice, 80% of the mice resolved their infections compared to none of the control mice. When CD8 T cell clones were transferred into nude mice, one clone elicited a protective response in about 50% of the mice, while another clone was ineffective (Igietseme et al. 1994). Morrison and colleagues confirmed these data by determining the course of MoPn infection in the genital tract of transgenic mice lacking MHC–class II bearing cells (CD4-deficient) or mice lacking β2-microglobulin (CD8-deficient) (Morrison et al. 1995). Class II–deficient mice were unable to resolve the infections, whereas β2-microglobulin deficient mice recovered from the infection similar to control mice. Taken together, these data have demonstrated that protective immunity in murine chlamydial genital infection is dependent upon CD4 T cells. To determine whether the dominant CD4 response in the genital tract was Th1 or Th2 mediated, Cain and Rank determined the cytokine profile of genital tract T cells by assessing the number of cells secreting IFN-γ (Th1 cells) or IL-4 (Th2 cells) by ELISPOT analysis (Cain and Rank 1995). The majority of the cells appearing in the genital tract following infection were found to be Th1 cells. The antibody response was also indicative of a Th1 response, with the dominant immunoglobulin subclass being IgG2a. The protective role of Th1 cells was supported by studies in which mice were immunized by intranasal, intravaginal, oral, or subcutaneous routes with live MoPn and then challenged in the genital tract 50 days later with viable chlamydiae (Kelly et al. 1996). One week after challenge, the number of Th1 and Th2 cells was determined in the genital tracts. Mucosal immunization induced a Th1 response, while the subcutaneous immunization elicited a Th2 response. The resultant infection in the subcutaneously immunized mice did not resolve as quickly as the mucosally immunized mice. Thus, it appeared that a Th2 cell cannot elicit a protective response. A CD4 Th2 T cell clone derived from subcutaneously immunized animals was also not able to cure MoPn infection in nude mice (Hawkins et al. 2002).
ROGER G. RANK
As in the lung, the important T cell effector mechanism in the genital tract is the secretion of IFN-γ and its ability to inhibit the replication of chlamydiae (Byrne et al. 1986). Rank treated infected mice with anti-IFN-γ, and observed that the infection was more prolonged than in control mice (Rank et al. 1992). In addition, the passive administration of recombinant IFN-γ into infected nude mice was able to bring about resolution of the infection in some mice. It was also noted that there was a correlation between the amount of IFN-γ produced by T cell clones and their ability to cure the infection in nude mice (Igietseme et al. 1993, 1994). Using IFN-γ knockout mice, two separate groups of investigators observed that, although the early part of MoPn infection in the genital tract was unaffected, the infection was significantly prolonged (Perry et al. 1997; Cotter, Ramsey, et al. 1997). As stated in the previous section, the course of the infection and pathologic response is dependent to some extent upon the strain of mouse being used. The different infection course and pathologic responses have been found to relate to variances in the host immune response in different strains. Darville studied the host response to MoPn genital infection in C3H, BALB/c, and C57BL/6 mice (Darville et al. 2001). In general, all mice developed Th1 dominant adaptive responses. However, they observed significant differences in the cytokine profiles of each strain early in the infection course. C57BL/6 mice, which resolve the infection more quickly, had higher and more prolonged IFN-γ levels but lower IL-10 levels in comparison to the other two strains. A marked predominance of MIP-1α over MCP-1 was observed only in the C57BL/6 mice and was thus associated with the increased Th1 response in those mice. TNF-α levels were also higher early in the infection course in C57BL/6 mice, suggesting a possible role for TNF-α in the reduction of the MoPn infection in mice (Darville et al. 1997).
D. Chlamydia pneumoniae C. pneumoniae, which was isolated in Taiwan in 1965 from the conjunctiva of a child and was unclassified until 1989 (Grayston et al. 1989), was first recognized in 1985 as a human chlamydial pathogen causing a community-acquired pneumonia (Saikku et al. 1985). It was later discovered that there was an association between a serological response to C. pneumoniae and the presence of coronary artery disease (Saikku et al. 1988). Further studies have documented the presence of chlamydial antigen and DNA in coronary lesions as well as the isolation of chlamydiae from a lesion (Campbell et al. 1995; Jackson et al. 1997; Kuo et al. 1995, 1993). Both respiratory infections with C. pneumoniae and coronary artery disease associated with C. pneumoniae infection have been modeled in the mouse and have yielded a great deal of information regarding the disease process and host immune response.
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1.
Infection Course and Pathologic Response
Yang and colleagues first demonstrated that C. pneumoniae could infect mice and produce a pneumonia analogous to that seen in humans (Yang et al. 1993). They inoculated several different strains of mice intranasally with relatively high doses of the organism and found a great disparity among the susceptibility of the various strains, with C57BL/6 mice being the most susceptible, with 50% mortality. Outbred mice were the most resistant with no mortality, although the majority of the mice were infected. Chlamydiae could be isolated from lung tissue of Swiss Webster mice up to 42 days after infection. Upon a second intranasal inoculation at either 70 or 100 days after the primary infection, about 30% of the mice became reinfected but had lower yields of organisms, indicating the development of partial immunity to reinfection. The pathologic response in the lung was similar to that seen in the lung with MoPn and was characterized as an interstitial pneumonitis. Gross pathology showed a patchy distribution of areas of consolidation during the first 2 weeks following intranasal inoculation. As early as 2–4 days after infection, there was extensive infiltration of PMNs, with exudate in alveolar spaces and bronchial lumens. Infiltration became more severe by days 7–11, with mixed mononuclear cells and PMNs. The infiltrate changed to predominantly mononuclear cells after day 15. Mild inflammation with foci of mononuclear cells could still be detected 60 days after infection. An important feature was the persistence of perivascular and peribronchial lymphoid cell accumulations from day 11 through day 60. After it became recognized that C. pneumoniae was associated with coronary artery disease, the mouse was investigated as a possible model for this disease state. That C. pneumoniae could be disseminated systemically to organs other than the lung was demonstrated by Yang and colleagues, when they were able to detect chlamydiae in the lung and spleen and peritoneal macrophages after either intranasal or intravenous infection (Yang et al. 1995). Moazed and colleagues, working under the hypothesis that pre-existing coronary artery disease might be necessary for C. pneumoniae to localize at the site of atheroma, chose to evaluate the course of infection in apoE-deficient mice, which spontaneously develop atherosclerotic lesions similar to those in humans (Moazed et al. 1997). The apoEdeficient and the parent strain C57BL/6 mice were infected intranasally with the AR-39 strain of C. pneumoniae, and animals were euthanized at various times after infection for pathological examination. A characteristic pneumonia resulted in both groups of mice. Chlamydiae could be isolated from the lung, spleen, and aorta as early as 1 week after infection in apoE-deficient mice and detected by PCR for as long as 20 weeks in the heart and 28 weeks in the aorta, several weeks after the lungs and spleen had become negative. Chlamydial DNA could be found in the aorta of normal C57BL/6 mice no longer than 14 days after infection and could not be found in the heart at any time.
Pneumonitis in apoE-deficient mice was similar to normal mice and resolved by 16 weeks after infection (Moazed et al. 1997). Atherosclerotic lesions appeared about 2 weeks after inoculation, with the presence of macrophages adhering to the endothelium. Foam cell lesions were seen from 4–8 weeks after infection and progressed to early atheromas by 16 weeks. Mature atheromas with subendothelial foam cell migration were present by 20 weeks. Chlamydial antigen could be detected 16 and 20 weeks after inoculation and appeared to be confined to foam cells in both atheromas and the subendothelium. While apoE-deficient mice developed atherosclerotic lesions as a result of the genetic defect, respiratory infection with C. pneumoniae significantly accelerated the progression of the disease (Moazed et al. 1999). C. pneumoniae seems to have a predilection for atheromas, since only 35% of apoE−/− mice infected at 8 weeks of age developed infection of the aorta, while 100% had infected aortas when they were inoculated at 16 weeks of age, when the atherosclerotic lesions were well-formed (Campbell et al. 2000). The ability to produce persistent atherosclerotic disease also appeared to be a function of C. pneumoniae but not other chlamydiae. Intranasal infection with C. trachomatis resulted in the detection of organisms in the aorta, but they persisted no more than 1 week, in contrast to the greater than 20 weeks for the apoE−/−. Even though the apoE−/− model is highly susceptible to enhanced atherosclerotic disease caused by C. pneumoniae, atherosclerotic disease can be induced in normal C57BL/6 mice as well after a single intranasal inoculation or, more readily, after multiple inoculations (Campbell et al. 2000). 2.
Immunity and Immune Mechanisms
Swiss Webster mice infected with C. pneumoniae via the respiratory route develop partial immunity to reinfection, with about 50% of the mice becoming reinfected (Yang et al. 1993; Penttila et al. 1998). Nevertheless, isolations from the lung of the challenged mice are considerably lower than uninfected controls. Not surprisingly, the mice develop both an antibody and cell-mediated immune response. The serum IgG antibody response in both normal and apoE−/− mice becomes positive about 10 days after intranasal infection, peaks at about 28 days, and then decreases rather dramatically by day 60. There has been no association with antibody and the protective response thus far (Rottenberg et al. 1999). As in MoPn respiratory infection, the cell-mediated immune response is the key cell type in the resolution of infection and resistance to reinfection in the murine model of C. pneumoniae respiratory disease. It is clear that a large number of T cells make up the infiltrating lymphocyte population in the lung and that both CD4 and CD8 cells can be found in the tissue (Penttila et al. 1998). Rottenberg and colleagues examined the relative roles of CD4 and CD8 cells in the protective immune response using genetically deficient mice (Rottenberg et al. 1999). A higher number of chlamydiae were observed in
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mice deficient in CD8 cells when compared to mice deficient in CD4 cells. In contrast, the CD4-deficient mice did not have significantly more organisms than did the C57BL/6 immunologically intact controls. The same phenomenon was also found in BALB/c mice; thus, the importance of CD8 cells, at least in the early part of infections, crosses strain lines. However, there was evidence that late in the infection course and in reinfection studies that CD4 also played a protective role. In support of protective roles for both CD4 and CD8 cells, adoptive transfer of either population into RAG-1−/− mice was able to restore the protective response (Rothfuchs et al. 2004). In contrast to MoPn respiratory infection, the enhanced susceptibility of CD8-deficient mice indicates that these cells may be playing a critical role in immunity to C. pneumoniae respiratory infection (Rottenberg et al. 1999). Thus, the adaptive response to C. pneumoniae employs both CD4- and CD8-mediated protective mechanisms. That T cells may be participating in the pathologic response was also suggested. Rottenberg observed that SCID mice reconstituted with CD4 T cells were more susceptible to C. pneumoniae infection, suggesting that CD4 cells may contribute to the disease process early in C. pneumoniae infections (Rottenberg et al. 1999). The effector mechanisms employed by the host to control C. pneumoniae infections do not appear to be different from other chlamydial infections of the mouse in which IFN-γ plays a significant role. Infection of IFN-γR−/− mice demonstrated increased levels of organisms, so that it is probable that the primary T cell effector mechanism is the bacteriostactic function of IFN-γ. A possible role for nitrous oxide intermediates in the IFN-γR−/− mice was suggested by a decrease in iNOS levels. Infection of iNOS -/- mice resulted in increased numbers of organisms but not to the same extent as in IFN-γR−/− mice. Rottenberg subsequently reported that IFN-γ and IL-12 were important in the early control of C. pneumoniae infection as key components of the innate response, in addition to being effector mechanisms in the adaptive response (Rottenberg et al. 2000). Elimination of NK cells reduced the levels of IFN-γ early in the infection but did not increase the susceptibility of the mice to infection and, therefore, did not appear essential for the protective innate response (Rothfuchs et al. 2004). However, production of IFN-γ by macrophages did appear to be an important factor in the innate response. That IFN-γ was a key effector mechanism for CD4 and CD8 T cells was demonstrated by the inability of IFN-γ−/− CD4 or CD8 cells to restore immunity to functionally T cell–deficient animals (Rothfuchs et al. 2004). Direct cytotoxic mechanisms by CD8 cells were ruled out because there was no difference in the infection course in perforin-deficient mice (Rottenberg et al. 1999).
E.
Chlamydia psittaci
C. psittaci encompasses a large group of organisms with many different animal hosts. Because they are by nature parasites
of animals, most of the research on these organisms has been performed in the host species. As discussed in the section on the history of chlamydiae, the mouse was used to define the etiologic agent of the disease process, but has been little used as a model for the diseases themselves. C. psittaci is a systemic disease in the mouse when inoculated by intraperitoneal injection and can be lethal, depending on the size of the inoculating dose (Byrne, Guagliardi, et al. 1988) and the strain of mouse (Byrne et al. 1990). Interestingly, just as in MoPn infections, C57BL/6 mice are more resistant to infection than C3H, BALB/c, or A/J strains. If mice are infected with a low dose of C. psittaci 6BC, they are able to recover from the infection and are immune to subsequent challenge (Byrne, Guagliardi, et al. 1988). The mechanism of resistance appears to be through the production of IFN-γ. In fact, this model was used to establish that IFN-γ is bacteriostatic for chlamydiae and is an important effector mechanism against this organism (Byrne and Faubion 1982).
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13. CHLAMYDIAL DISEASES
Rank, R. G., Bowlin, A. K., Reed, R. L., and Darville, T. (2003). Characterization of chlamydial genital infection resulting from sexual transmission from male to female guinea pigs and determination of infectious dose. Infect. Immun. 71, 6148–6154. Rank, R. G., Dascher, C., Bowlin, A. K., and Bavoil, P. M. (1994). Effect of immunization with recombinant HSP60 on the course of GPIC ocular infection. In Chlamydial infections, J. Orfila, ed., pp. Rank, R. G., Ramsey, K. H., Pack, E. A., and Williams, D. M. (1992). Effect of gamma interferon on resolution of murine chlamydial genital infection. Infect. Immun. 60, 4427–4429. Rank, R. G., Sanders, M. M., and Patton, D. L. (1995). Increased incidence of oviduct pathology in the guinea pig after repeat vaginal inoculation with the chlamydial agent of guinea pig inclusion conjunctivitis. J. Sex. Transm. Dis. 22, 48–54. Rank, R. G., Soderberg, L. S. F., and Barron, A. L. (1985). Chronic chlamydial genital infection in congenitally athymic nude mice. Infect. Immun. 48, 847–849. Rank, R. G., White, H. J., and Barron, A. L. (1979). Humoral immunity in the resolution of genital infection in female guinea pigs infected with the agent of guinea pig inclusion conjunctivitis. Infect. Immun. 26, 573–579. Rasmussen, S. J., Eckmann, L., Quayle, A. J., et al. (1997). Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in chlamydial pathogenesis. J. Clin. Invest. 99, 77–87. Rasmussen, S. J., Sen, C. K., and Stephens, R. S. (1998). Temporal expression of interleukin-8 in Chlamydia-infected epithelial cells. In Proceedings of the ninth international symposium on human chlamydial infection, R. S. Stephens, G. I. Byrne, G. Christiansen, et al. eds., pp. 411–414. International Chlamydia Symposium, San Francisco. Read, T. D., Brunham, R. C., Shen, C., et al. (2000). Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res. 28, 1397–1406. Rivers, T. M., and Berry, G. P. (1931). Psittacosis: II. Experimentally induced infections in mice. J. Exper. Med. 54, 105. Rothfuchs, A. G., Kreuger, M. R., Wigzell, H., and Rottenberg, M. E. (2004). Macrophages, CD4(+) or CD8(+) cells are each sufficient for protection against Chlamydia pneumoniae infection through their ability to secrete IFN-gamma. J. Immunol. 172, 2407–2415. Rottenberg, M. E., Gigliotti, R. A., Gigliotti, D., et al. (2000). Regulation and role of IFN-gamma in the innate resistance to infection with Chlamydia pneumoniae. J. Immunol. 164, 4812–4818. Rottenberg, M. E., Rothfuchs, A. C., Gigliotti, D., Svanholm, C., Bandholtz, L., and Wigzell, H. (1999). Role of innate and adaptive immunity in the outcome of primary infection with Chlamydia pneumoniae, as analyzed in genetically modified mice. J. Immunol. 162, 2829–2836. Saikku, P., Leinonen, M., Mattila, K., et al. (1988). Serological evidence of an association of a novel chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet 2, 983–986. Saikku, P., Wang, S. P., Kleemola, M., Brander, E., Rusanen, E., and Grayston, J. T. (1985). An epidemic of mild pneumonia due to an unusual strain of Chlamydia psittaci. J. Infect. Dis. 151, 832–839. Schachter, J., and Dawson, C. R. (1978). Human chlamydial infections. PSG Publishing Company, Littleton, MA. Schachter, J., Stephens, R. S., Timms, P., et al. (2001). Radical changes to chlamydial taxonomy are not necessary just yet. Int. J. Syst. Evol. Microbiol. 51, 249, 251–249, 253. Scidmore, M. A., Fischer, E. R., and Hackstadt, T. (1996). Sphingolipids and glycoproteins are differentially trafficked to the Chlamydia trachomatis inclusion. J. Cell Biol. 134, 363–374. Shaffer, M. F., Rake, G., and McKee, C. M. (1940). Agent of lymphogranuloma venereum in the lungs of mice. Proc. Soc. Exp. Biol. Med. 44, 408–410. Starnbach, M. N., Bevan, M. J., and Lampe, M. F. (1994). Protective cytotoxic T lymphocytes are induced during murine infection with Chlamydia trachomatis. J. Immunol. 153, 5183–5189.
347 Stephens, R. S., and Kuo, C. C. (1984). Chlamydia trachomatis species-specific epitope detected on mouse biovar outer membrane protein. Infect. Immun. 45, 790–791. Stephens, R. S., Tam, M. R., Kuo, C. C., and Nowinski, R. C. (1982). Monoclonal antibodies to Chlamydia trachomatis: antibody specificities and antigen characterization. J. Immunol. 128, 1083–1089. Stephens, R. S., Wagar, E. A., and Schoolnik, G. K. (1988). High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis. J Exper. Med. 167, 817–831. Stills, H. F. (1991). Isolation of an intracellular bacterium from hamsters (Mesocricetus auratus) with proliferative ileitis and reproduction of the disease with a pure culture. Infect. Immun. 59, 3227–3236. Storz, J. (1971). Cultivation of Chlamydiae. In Chlamydia and Chlamydiainduced diseases, J. Storz, ed., pp. 71–88. Charles C. Thomas, Springfield, IL. Su, H., and Caldwell, H. D. (1995). CD4+ T cells play a significant role in adoptive immunity to Chlamydia trachomatis infection of the mouse genital tract. Infect. Immun. 63, 3302–3308. Su, H., Feilzer, K., Caldwell, H. D., and Morrison, R. P. (1997). Chlamydia trachomatis genital tract infection of antibody-deficient gene knockout mice. Infect. Immun. 65, 1993–1999. Swenson, C. E., Donegan, E., and Schachter, J. (1983). Chlamydia trachomatisinduced salpingitis in mice. J. Infect. Dis. 148, 1101–1107. Swenson, C. E., and Schachter, J. (1984). Infertility as a consequence of chlamydial infection of the upper genital tract in female mice. Sex. Transm. Dis. 11, 64–67. T’ang, F. F., Chang, H. L., Huang, Y. T., and Wang, K. C. (1957). Studies on the etiology of trachoma with special reference to isolation of the virus in chick embryo. Chin. Med. J. (Peking) 75, 429–446. Tanner, M. A., Harris, J. K., and Pace, N. R. (1999). Molecular phylogeny of Chlamydia and relatives. In Chlamydia: intracellular biology, pathogenesis, and immunity. R. S. Stephens, ed., pp. 1–8. American Society for Microbiology, Washington, DC. Tuffrey, M., Alexander, F., and Taylor-Robinson, D. (1990). Severity of salpingitis in mice after primary and repeated inoculation with a human strain of Chlamydia trachomatis. J. Exp. Path. 71, 403–410. Tuffrey, M., Falder, P., and Taylor-Robinson, D. (1982). Genital tract infection and disease in nude and immunologically competent mice after inoculation of a human strain of Chlamydia trachomatis. Br. J. Exp. Pathol. 63, 539–546. Tuffrey, M., and Taylor-Robinson, D. (1981). Progesterone as a key factor in the development of a mouse model for genital-tract infection with Chlamydia trachomatis. FEMS Microbiol. Let. 12, 111–115. Van Voorhis, W. C., Barrett, L. K., Sweeney, Y. T. C., Kuo, C. C., and Patton, D. L. (1996). Analysis of lymphocyte phenotype and cytokine activity in the inflammatory infiltrates of the upper genital tract of female macaques infected with Chlamydia trachomatis. J. Infect. Dis. 174, 647–650. Vender, J., and Moulder, J. W. (1967). Initial step in catabolism of glucose by the meningopneumonitis agent. J. Bacteriol. 94, 867–869. Wagar, E. A., Schachter, J., Bavoil, P., and Stephens, R. S. (1990). Differential human serologic response to two 60,000 molecular weight Chlamydia trachomatis antigens. J. Infect. Dis. 162, 922–927. Wang, S. P., and Grayston, J. T. (1963). Classification of trachoma virus strains by protection of mice from toxic death. J. Immunol. 90, 849. — — —. (1971). Classification of TRIC and related strains with microimmunofluorescence. In Trachoma and related disorders caused by chlamydial agents, R. L. Nichols, ed., pp. 305–321. Excerpta Medica. Watkins, J. F., and MacKenzie, A. M. R. (1963). Pulmonary infection of adult white mice with the TE 55 strain of trachoma virus. J. Gen. Microbiol. 30, 43–52. Weisburg, W. G., Hatch, T. P., and Woese, C. R. (1986). Eubacterial origin of chlamydiae. J. Bacteriol. 167, 570–574. Weiss, E. (1949). The extracellular development of agents of the psittacosislymphogranuloma group. I. (Chlamydiozoaceae). J. Infect. Dis. 84, 125. Weiss, E., Schramck, S., Wilson, N. N., and Newman, L. W. (1970). Deoxyribonucleic acid heterogenicity between human and murine strains of Chlamydia trachomatis. Infect. Immun. 2, 24–48.
348 Whittum-Hudson, J. A., Taylor, H. R., Farazdaghi, M., and Prendergast, R. A. (1986). Immunohistochemical study of the local inflammatory response to chlamydial ocular infection. Invest. Ophthalmol. Vis. Sci. 27, 64–69. Williams, D. M., Byrne, G. I., Grubbs, B., Marshal, T. J., and Schachter, J. (1988). Role in vivo for gamma interferon in control of pneumonia caused by Chlamydia trachomatis in mice. Infect. Immun. 56, 3004–3006. Williams, D. M., Grubbs, B., Darville, T., Kelly, K., and Rank, R. (1998). A role for interleukin 6 in host defense against Chlamydia trachomatis. Infect. Immun. 66, 4564–4567. Williams, D. M., Grubbs, B., Kelly, K. A., and Rank, R. G. (1997). Humoral and cellular immunity in secondary infection due to murine Chlamydia trachomatis. Infect. Immun. 65, 2876–2882. Williams, D. M., Grubbs, B., and Schachter, J. (1987). Primary murine Chlamydia trachomatis pneumonia in B-cell-deficient mice. Infection and Immunity 55, 2387–2390. Williams, D. M., Grubbs, B. G., Schachter, J., and Magee, D. M. (1993). Gamma interferon levels during Chlamydia trachomatis pneumonia in mice. Infection and Immunity 61, 3556–3558. Williams, D. M., Schachter, J., Coalson, J. J., and Grubbs, B. (1984). Cellular immunity to the mouse pneumonitis agent. J. Infect. Dis. 149, 630–639. Williams, D. M., Schachter, J., Drutz, D. J., and Sumaya, C. V. (1981). Pneumonia due to Chlamydia trachomatis in the immunocompromised (nude) mouse. J. Infect. Dis. 143, 238–241. Williams, D. M., Schachter, J., Grubbs, B., and Sumaya, C. V. (1982). The role of antibody in host defense against the agent of mouse pneumonitis. J. Infect. Dis. 145, 200–205.
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Williams, D. M., Schachter, J., Weiner, M. H., and Grubbs, B. (1984). Antibody in host defense against mouse pneumonitis agent (murine Chlamydia trachomatis). Infect. Immun. 45, 674–678. Wolner-Hanssen, P., Patton, D. L., Stamm, W. E., and Holmes, K. K. (1986). Severe salpingitis in pig-tailed macaques after repeated cervical infections followed by a single tubal inoculation with Chlamydia trachomatis. In Chlamydia infections, D. Oriel, G. Ridgway, J. Schachter, D. Taylor-Robinson, and M. Ward, eds., pp. 371–374. Cambridge University Press, New York. Wood, H., Fehlner-Gardner, C., Berry, J., et al. (2003). Regulation of tryptophan synthase gene expression in Chlamydia trachomatis. Mol. Microbiol. 49, 1347–1359. Yang, X., Hayglass, K. T., and Brunham, R. C. (1996). Genetically determined differences in IL-10 and IFN-γ responses correlate with clearance of Chlamydia trachomatis mouse pneumonitis infection. J. Immunol. 156, 4338–4344. Yang, Z., Kuo, C., and Grayston, J. T. (1995). Systemic dissemination of Chlamydia pneumoniae following intranasal inoculation in mice. J. Infect. Dis. 171, 736–738. Yang, Z.-P., Kuo, C.-C., and Grayston, J. T. (1993). A mouse model of Chlamydia pneumoniae strain TWAR pneumonitis. Infect. Immun. 61, 2037–2040. Zhang, J. P., and Stephens, R. S. (1992). Mechanism of C. trachomatis attachment to eukaryotic host cells. Cell 69, 861–869. Zhang, Y.-X., Fox, J. G., Ho, Y., Zhang, L., Stills, H. F., and Smith, T. F. (1993). Comparison of the major outer-membrane protein (MOMP) gene of mouse pneumonitis (MoPn) and hamster SFPD strains of Chlamydia trachomatis with other Chlamydia strains. Mol. Biol. Evol. 10, 1327–1342.
Chapter 14 Clostridial Species Kimberly S. Waggie
I. II.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clostridium piliforme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Bacterial Strains and Antigenic Relationships . . . . . . . . . . . . . . . . . . . . D. Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Clostridium perfringens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Bacterial Strains and Antigenic Relationships . . . . . . . . . . . . . . . . . . . . D. Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Control and prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Clostridium difficile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Bacterial Strains and Antigenic Relationships . . . . . . . . . . . . . . . . . . . . D. Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
350 350 350 350 350 351 351 351 353 353 353 355 355 355 355 356 356 356 356 356 356 357 357 357 357 357 357 358 358 358 358 358 359 359 359 359 360 360 360 Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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I.
INTRODUCTION
Over 100 bacterial species have been classified as members of the genus Clostridium. Morphologically, clostridia are straight to slightly curved, rod-shaped bacteria that range from 8–12 µm long and 0.4–1.2 µm in diameter. These bacteria form endospores. The shape, size, and location of the endospore within the mother cell can be used as an aid to species identification. The majority of the clostridia have peritrichous flagella and are motile. Most clostridia require enriched media for in vitro growth. Cultures are generally incubated at 37° C under anaerobic conditions. In biochemical tests they are fermentative and catalase and oxidase negative. Less than 20 species of clostridia have been associated with disease in animals. The pathogenic Clostridium species can be grouped into four categories based on the type of disease they produce. Clostridium botulinum and Clostridium tetani, the neurotoxic clostridia, release toxins that cause neuromuscular dysfunction but no visible tissue damage. Histotoxic clostridia, such as Clostridium chauvoei, the etiologic agent of blackleg, and Clostridium septicum, the etiologic agent of malignant edema, release toxins that produce relatively localized tissue damage and may subsequently produce toxemia. Members of the third category of clostridia, which includes Clostridium perfringens, release toxins that cause inflammation in the intestinal tract and enterotoxemia. Clostridia in the fourth category, such as Clostridium piliforme, Clostridium difficile, and Clostridium spiroforme, are associated with the production of sporadic disease, often in single animals (Merchant and Packer 1977; Quinn et al. 2002). Clostridial infections are uncommon in mice (Livingston and Riley 2003). Only two species of clostridium, Clostridium piliforme and Clostridium perfringens, have been reported in the literature to cause naturally occurring murine disease. A third species, Clostridium difficile, has been found in conventional mouse colonies and is reported to produce enterocolitis in experimentally infected mice.
II. CLOSTRIDIUM PILIFORME A.
History
Ernest Tyzzer first described Clostridium piliforme and the murine disease associated with this bacterium in 1917. Tyzzer lost his entire colony of Japanese waltzing mice (Mus bactrianus) and some of his Mus bactrianus × Mus musculus F1 hybrids following an outbreak of a disease characterized by either sudden death without premonitory signs or a short period of diarrhea followed by death. On microscopic examination of tissues from affected mice, Tyzzer observed long, rod-shaped
bacteria within the cytoplasm of hepatocytes and intestinal mucosal epithelial cells. Tyzzer named the organism Bacillus piliformis based on the bacterium’s morphology (Tyzzer 1917). Rights et al. (1947) named the disease associated with Bacillus piliformis “Tyzzer’s disease.” Wilson and Miles (1964) suggested that the bacterium be renamed Actinobacillus piliformis. This suggestion was not widely adopted and the bacterium continued to be called Bacillus piliformis. Bacillus piliformis was renamed Clostridium piliforme by Duncan et al. (1993), based on the sequence of its 16S ribosomal RNA (rRNA).
B.
Properties
Clostridium piliforme is an obligate intracellular organism that cannot be grown on artificial media. The bacterium is morphologically pleomorphic. Most forms of Clostridium piliforme are rod-shaped and 8–10 µm long by 0.5 µm wide. However, the organisms may range from 2–40 µm in length and can reach 1 µm in width. The longer forms may contain single, centrally located, fusiform swellings. Occasionally, thicker banded forms with tapered ends may be observed. Clostridium piliforme stains gram negative, PAS positive, and is non-acid fast (Fujiwara 1978; Ganaway et al. 1971a; Tyzzer 1917). Based on 16S rRNA sequencing, its closest relatives are Clostridium coccoides, Clostridium oroticum, Clostridium clostridiiforme, Clostridium symbiosum, and Streptococcus hansenii (Duncan et al. 1993).
C.
Bacterial Strains and Antigenic Relationships
Different strains of Clostridium piliforme have not been officially recognized. However, several lines of evidence suggest that multiple strains of the bacterium do exist. Some, but not all, isolates of Clostridium piliforme have been found to produce cytotoxins. The cytotoxins can be differentiated based on the ability of culture filtrates to produce cytopathic effects in BRL 3A and 3T3 cell lines. The cytotoxins are thermolabile, are partially digested by trypsin, and have a molecular weight of over 100 kDa (Livingston et al. 1996; Riley et al. 1992). In addition to differences in cytotoxin production, other lines of evidence that support the existence of multiple strains of Clostridium piliforme include differences in isolate stability in vitro (Fujiwara et al. 1973) and the ability of different isolates to produce disease in different species (Franklin et al. 1994; Fujiwara et al. 1973, 1974; Itoh and Kagiyama 1990; Takagaki et al. 1966; Waggie et al. 1987). In addition, protein and antigenic heterogeneity of different isolates are found by complement fixation, agar gel double diffusion, Coomassie blue-stained polyacrylamide gels, and Western blots (Fujiwara et al. 1971, 1985; Manning 1993; Riley et al. 1990). Differences in flagellar epitope expression have been identified by monoclonal antibodies (Boivin et al. 1993; Hook et al. 1995).
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D.
Growth In Vivo and In Vitro
There are a few reports of the growth of Clostridium piliforme in artificial media (Gard 1944; Kanazawa and Imai 1959; Simon 1977; Tyzzer 1917). However, numerous investigators have failed to repeat these findings (Craigie 1966; Fujiwara 1978; Ganaway et al. 1971b; Thunert 1984). Initial attempts at propagating Clostridium piliforme in vivo were not completely successful. The bacterium was frequently lost on repeated animal-to-animal passage. This problem was solved when Kaneko et al. (1960) discovered that concurrent cortisone administration potentiated the infection in experimentally infected mice, allowing repeated passage in this species. Craigie (1966) demonstrated that the bacterium could be isolated from infected livers by propagation in 7- to 10-day-old embryonated hen’s eggs, and that organisms propagated in hen’s eggs retained their virulence. The bacterium has also been isolated from fecal suspensions from naturally infected hamsters after passage in eggs (Zook, Huang, et al. 1977). Clostridium piliforme can be grown in primary chick and mouse embryo cell cultures. However, the organism loses its virulence upon passage into fresh primary cell cultures (Rights et al. 1947). Propagation and subculture without loss of virulence was accomplished by using primary mouse liver (Kawamura, Taguchi, Ishida, et al. 1983; Kawamura, Taguchi, and Fujiwara 1983) and primary chick embryo liver cultures (Ganaway et al. 1983). More recently, the bacterium has been successfully cultivated in a number of continuous cell lines (Riley et al. 1992; Spencer et al. 1990).
E. In Vivo 1.
Clinical Disease
Most murine Clostridium piliforme infections are inapparent (Fujiwara 1978; Ganaway 1982). Clinically apparent Tyzzer’s disease most often occurs in young animals, immunosuppressed animals, and animals stressed by transport, overcrowding, poor sanitation, inappropriate diet, or experimental procedures (Ganaway et al. 1971a; Tyzzer 1917). Tyzzer (1917) reported that the disease was found in the Mus bactrianus and some Mus bactrianus × Mus musculus F1 hybrids, but not in the Mus musculus in his colony. Differences in susceptibility among strains of Mus musculus have also been reported. In one study, CBA/N and C3.CBA/N mice, which are deficient in B cell function, were found to be more susceptible to experimental infection than immunologically competent mice and T cell–deficient nude (nu) mice (Waggie et al. 1981). However, it has been reported that homozygous nude mice were susceptible to clinical disease while heterozygous nude mice in the same colony were not (Livingston et al. 1996). Other strains of mice that have been reported to be more susceptible to Clostridium piliforme infection than other strains include ICR and DBA/2 (Fujiwara 1978; Van Andel et al. 1997). Clinical signs in mice may include watery to pasty diarrhea of several days’ duration followed by death, or death without prior clinical signs of illness. The most common lesions found at necropsy include reddening of the ileum and large intestine and variable sizes and numbers of yellow or grayish-white foci of necrosis in the liver (Fig. 14-1) (Tyzzer 1917). Variably sized,
Fig. 14-1 Multiple foci of hepatic necrosis in a C3H/HeN mouse experimentally infected with Clostridium piliforme.
352 pale grey foci of necrosis have also been reported to occur in the ventricles of the heart (Tsuchitani et al. 1983). Lesions may be present in any one or any combination of these organs. On microscopic examination of the intestine, characteristic clusters of Clostridium piliforme may be present within the cytoplasm of mucosal epithelial cells and, less frequently, in smooth muscle cells in the tunica muscularis and in neurons of Auerbach’s plexus (Fig. 14-2). Both vegetative and spore forms of the bacterium may be observed in tissues. Lesions, when present, may vary from foci of epithelial degeneration in the crypts (Tyzzer 1917) to patchy mucosal necrosis, transmural edema, and multifocal necrosis in the tunica muscularis. Inflammatory infiltrates in the wall of the intestine, if present, consist primarily of neutrophils and are generally mild in severity. The lymphatic vasculature and sinuses of mesenteric lymph nodes may be filled with cellular debris (Mullink 1968; Tsuchitani et al. 1983). Early lesions in the liver consist of neutrophil aggregates adjacent to Clostridium piliforme–infected hepatocytes. These lesions are often adjacent to portal veins; however, they may be found in all regions of the hepatic lobule. Older lesions consist of foci of coagulative necrosis or hepatocellular effacement. The latter foci contain only cellular debris and a fine reticular network of connective tissue. No or low numbers of neutrophils may be present, and a rim of hepatocytes containing characteristic intracytoplasmic bacteria may border the lesions. Healing may occur by hepatocellular regeneration or fibrosis. Healing lesions may have multinucleate giant cells of macrophage origin at the periphery, and healed lesions may contain sequestra of debris. Lesions of varying ages may be found in the same
K I M B E R LY S . WA G G I E
liver (Gard 1944; Hunter 1971; Meshorer 1974; Mullink 1968; Peace and Soave 1969; Porter 1952; Rights et al. 1947; Tsuchitani et al. 1983; Tyzzer 1917). Vegetative and spore forms of the bacterium are occasionally found in the cytoplasm of epithelial cells lining the bile duct and gall bladder (Tyzzer 1917). Microscopic lesions that have been described in the hearts of naturally infected mice include foci of myocyte degeneration characterized by vacuolization, hyalinization, and fragmentation. Occasional myocyte fibers may also be calcified. The areas of degeneration may also contain fibroblasts and infiltrates of polymorphonuclear cells and mononuclear cells. Clostridium piliforme may be found in apparently intact myocytes within or bordering the foci degeneration (Tsuchitani et al. 1983). Lesions have not been reported in the central nervous systems of mice naturally infected with Clostridium piliforme. However, multifocal necrosis and suppurative meningoencephalitis and meningomyelitis have been observed in mice following intracisternal, intranasal, or intraspinal injections of Clostridium piliforme-infected tissue suspensions. Clostridium piliforme has been observed within the cytoplasm of neurons, choroid plexus epithelial cells, and occasional glial cells adjacent to foci of necrosis in nervous tissue (Fujiwara et al. 1981; Okada et al. 1986; Onodera and Fujiwara 1970). Encephalitis has been reported in naturally occurring Tyzzer’s disease in gerbils (Veazey et al. 1992). These findings suggest that infection of the central nervous system may also occur in some outbreaks of Tyzzer’s disease in mice. Electron microscope images of Clostridium piliforme– infected cells show that the bacteria are not enclosed in vacuoles but either are in direct contact with the ground substance
Fig. 14-2 Cluster of Clostridium piliforme within the cytoplasm of an intestinal epithelial cell. Note the typical “pickup-sticks” arrangement. Warthin-Starry silver stain.
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of the cytoplasm or are surrounded by an electron lucent space. The majority of organisms are 300–500 nm in diameter, with occasional organisms reaching 1000 nm. The organism may have peritrichous flagella that are 7–20 nm in diameter and may contain terminally located spores. Vacuolation of endoplasmic reticulum and mitochondrial swelling may be present. There may be decreased numbers of cellular organelles in heavily infected cells (Fujiwara et al. 1963; Goto et al. 1974; Port et al. 1971). Shadow cast preparations of liver homogenates reveal 0.2–5 µm long by 16–20 nm diameter peritrichous flagella (Fujiwara et al. 1968). 2.
Pathogenesis
Electron microscopic studies of Caco-2 cell monolayers experimentally infected with Clostridium piliforme suggest that the mechanism of cell entry is by bacterially directed phagocytosis (Franklin et al. 1993). It has also been reported that invasion of primary cultures of mouse hepatocytes is enhanced by treating the cells with cytochalasin D, a microfilament disruptor. Treatment of mouse hepatocytes with vinblastine, a microtubule inhibitor, markedly suppressed entry into the cells by Clostridium piliforme (Kawamura et al. 1998). The host immune response to Clostridium piliforme is incompletely understood. Studies evaluating the cellular arm of the immune system have shown that depletion of either neutrophils or natural killer cells, but not macrophages, increases the severity of experimentally induced disease in both susceptible and resistant mouse strains (Van Andel et al. 1997). Experimental Clostridium piliforme infection has been shown to produce elevations in serum levels of the cytokines IL-6, IL-12, TNF-α, and interferon-γ (Van Andel et al. 1998, 2000a, 2000b). In addition, administration of neutralizing antibodies directed against IL-6 or IL-12 prior to experimental infection increased the severity of disease in both Clostridium piliforme susceptible and resistant mouse strains (Van Andel et al. 1998, 2000b). The humoral arm of the immune system may also play a role in host defense. Naturally acquired and experimentally administered anti-Clostridium piliforme antibodies have been demonstrated to be protective against challenge infection in mice (Fries 1979; Fujiwara et al. 1969).
F.
Epizootiology
Tyzzer’s disease has been described in a broad range of laboratory, domestic, and wild mammalian species throughout the world (Allen et al. 1965; Bonney and Schmidt 1975; Canfield and Hartley 1991; Carter et al. 1969; Chalmers et al. 1983; Errington 1946; Hum and Best 1988; Kovatch and Zebarth 1973; Kubokawa et al. 1973; Marler and Cook 1976; Niven 1968; Qureshi et al. 1976; Schmidt et al. 1984; Sparrow and Naylor 1978; Stanley et al. 1978; Swerczek et al. 1973; Takagaki et al. 1968; Takasaki et al. 1974; Webb et al. 1987; White and
Waldron 1969; Wojcinski and Barker 1986; Zook, Albert, et al. 1977). Two cases have been reported in avian species (Raymond et al. 2001; Saunders et al. 1993). The descriptions of the disease in all of the above species are similar to those described in Tyzzer’s waltzing mice. Clostridium piliforme infection has also been described in an immunocompromised human. The lesions in the human patient consisted of a group of crusted, verrucous skin lesions that were confined to a localized area on the chest. Morphologically typical bacteria were found within keratinocytes in the affected area. The bacteria were confirmed to be Clostridium piliforme by 16S ribosomal RNA sequence analysis (Smith et al. 1996). Additional evidence for the widespread presence of the bacterium is the demonstration of antibodies against Clostridium piliforme in sera from healthy members of numerous species, including humans (Fries 1980). The method of transmission of Clostridium piliforme is thought to be primarily by the ingestion of spores that have been shed into the environment by symptomatic individuals or inapparent carriers (Fujiwara 1978; Ganaway et al. 1971b). The vegetative phase of the bacterium is extremely unstable and rapidly loses infectivity when outside of host cells (Craigie 1966; Kawamura, Taguchi, and Fujiwara 1983). In contrast, Clostridium piliforme spores have been found to survive in infected bedding for at least 1 year (Tyzzer 1917) and in the natural environment for at least 5 years (Errington 1954). The spores can also survive multiple cycles of freezing and thawing (Craigie 1966; Ganaway et al. 1971b). There are no reports in mice of naturally occurring in utero transmission of Clostridium piliforme from dam to offspring. However, a presumed case of vertical transmission has been reported in guinea pigs (Boot and Walvoort 1984). In addition, the bacterium has been observed in the myometrium and endometrium of pregnant mice intravenously inoculated with the bacterium. Clostridium piliforme was also found within placental membranes and hepatocytes of the fetuses from these mice (Fries 1978; Kadowaki et al. 1980).
G.
Diagnosis
Tyzzer’s disease has classically been diagnosed on the basis of clinical signs, finding typical lesions on gross and microscopic examination of tissues, and observing typical “pickup-sticks” clusters of organisms in apparently viable cells bordering the lesions. The bacterium is often poorly demonstrated in H&E and Gram-stained tissue sections. Giemsa stains are useful for demonstrating Clostridium piliforme in hepatocytes and epithelial cells, but are not effective for organisms in smooth or cardiac muscle cells. Silver stains, such as the Warthin-Starry or Steiner’s, are the stains of choice for demonstrating the bacterium in tissue sections. Clusters of Clostridium piliforme may also be demonstrated in impression smears of infected tissues stained with Giemsa, methylene blue, or thionin (Fujiwara 1978; Ganaway et al. 1971a).
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Fig. 14-3 Liver from a mouse experimentally infected with Clostridium piliforme. Note the numerous clusters of organisms within the cytoplasm of hepatocytes. This liver sample was fixed in 10% neutral buffered formalin immediately after the death of the animal. Warthin-Starry silver stain.
Clostridium piliforme rapidly disappears from tissues following the death of the host (Figs. 14-3 and 14-4). Therefore, it is imperative to collect tissues as rapidly as possible. Immunofluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) tests have been developed to detect serum antibodies directed against Clostridium piliforme (Boivin et al. 1994; Fries 1977a; Motzel, Meyer, et al. 1991; Motzel and
Riley 1991; Toriumi et al. 1986). However, because of the antigenic diversity of Clostridium piliforme, IFA and ELISA tests may not detect antibodies directed against all strains of the bacterium. More recently, the polymerase chain reaction (PCR), based on highly conserved sequences in the bacterium’s 16S rRNA, has been used to identify Clostridium piliforme in tissues and feces (Furukawa et al. 2002; Goto and Itoh 1994, 1996; Wilson, 1994).
Fig. 14-4 Same liver as in Fig. 14-3. The liver sample was fixed in 10% neutral buffered formalin after the liver was held at room temperature for 24 hours. Very few Clostridium piliforme remain.
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Additional methods that may be used to detect Tyzzer’s disease in mouse colonies include the cortisone provocation test, inoculation of suspect material into embryonated hen’s eggs, tissue culture cell monolayers, other animals, and use of sentinel animals. The cortisone provocation test is used to attempt to activate inapparent infections. Test animals are given 100–200 mg/kg cortisone acetate subcutaneously or intramuscularly, then examined for the presence of lesions 7–10 days later (Fujiwara 1978). Sterilely harvested and processed tissue suspensions may be inoculated into the yolk sacs of embryonated hen’s eggs. If the suspension contains Clostridium piliforme, the embryos will usually die in 6–9 days (Fries 1977b; Ganaway et al. 1971b). Pieces of tissue may be directly inoculated onto tissue culture monolayers in an attempt to isolate the bacterium (Spencer et al. 1990). Tissue suspensions may also be inoculated into other mice or into a sensitive species such as the Mongolian gerbil (Meriones unguiculatus). The inoculated animals may be checked 5–10 days postinoculation for the presence of typical gross and microscopic lesions (Waggie et al. 1984, 1987). Mouseto-mouse passage is aided by the concurrent subcutaneous administration of 2.5 mg cortisone/mouse (Kaneko et al. 1960). Naive sentinel animals, either of the same species or a sensitive species, may be placed on soiled bedding and examined for lesions or tested for seroconversion. Bedding trials must be carefully controlled to insure that the sentinel animals were not already infected with Clostridium piliforme before being placed on suspect bedding (Gibson et al. 1987). If sentinels other than mice are used, it must be kept in mind that strains of Clostridium piliforme vary in their ability to infect different species of animals and may not infect a nonmurine species (Motzel and Riley 1992).
feces of infected animals. Therefore, effective methods of preventing the introduction and spread of the bacterium include housing mice in microisolator cages, avoiding cage overcrowding, sterilizing or disinfecting feed, bedding, cages, and cage accessories, and frequently changing bedding and cages. The spores of Clostridium piliforme are inactivated when heated to 80° C for 30 minutes. Chemical disinfectants that have been reported to inactivate spores include 0.4% peracetic acid, 0.3% sodium hypochlorite, 1% iodophor, 5% phenol, Alcide, and 0.37% formaldehyde (Ganaway 1980; Itoh et al. 1987). Cesarean derivation can potentially be used as a means to eliminate the organism from an infected colony. Naturally occurring vertical transmission of Clostridium piliforme has not been demonstrated in mice. However, the bacterium has been found in the myometrium and endometrium of the uterus, placental membranes, and in hepatocytes of fetuses of pregnant mice intravenously inoculated with the organism (Fries 1978; Kadowaki et al. 1980). Testing of Cesarean-derived animals for the presence of Clostridium piliforme prior to their reintroduction into an animal colony would help ensure that the infection had been eliminated. Commercial vaccines against Clostridium piliforme are not available. An experimental vaccine using formalized infected liver has been found at least partially effective against challenge infection. Vaccinated mice challenged with Clostridium piliforme did not exhibit clinical signs of disease; however, they did develop liver lesions (Fujiwara et al. 1965).
III. H.
CLOSTRIDIUM PERFRINGENS
Treatment A.
Because of the brief course of the disease, treatment of mice with clinical signs of Clostridium piliforme infection is probably futile. However, antibiotics may be useful in controlling outbreaks of Tyzzer’s disease. Tetracycline, administered at 14–19 mg/kg in drinking water containing sucrose, has been reported to abate epizootics of the disease in mouse colonies (Hunter 1971). Use of chloramphenicol, administered intramuscularly, has been reported to be partially effective in controlling outbreaks (Friedmann et al. 1969). In experimental studies, the following antibiotics have been found to be effective in the treatment of murine Tyzzer’s disease: cephaloridine (Yokoiyama and Fujiwara 1971), tetracycline (Takagaki et al. 1964; Thunert 1982; Yokoiyama and Fujiwara 1971), chlortetracycline (Kaneko et al. 1960), and oxytetracycline (Thunert 1982).
I.
Control and Prevention
The usual method of transmission of Clostridium piliforme is thought to be by ingestion of spores that have been shed in the
History
Clostridium perfringens was first described by Welch and Nuttall in 1892 after they had isolated the organism from a cadaver. The bacterium was first associated with a disease, gas gangrene in humans, in 1893 by Fraenkel, who called the organism Bacillus phlegmonis emphysematosae. The first probable isolation of the bacterium from a domestic species was in 1926 when Dalling isolated what he called Bacillus paludis from a sheep. Other names that have been used for Clostridium perfringens include Clostridium welchii, Bacillus welchii, Bacterium welchii, Bacillus aerogenes capsulatus, Bacillus enteridtidis sporogenes, Bacillus perfringens, Granulo bacillus saccharo butyricus immobilis, Bacillus anaerobicus cryptobutyricus, Bacillus cadaveris butyricus, and Bacillus emphysematis vaginae (Merchant and Packer 1977). Clostridium perfringens is associated with a number of diseases in domestic animals and humans. Clostridium perfringens type A causes gangrene in humans and other animals and food poisoning in humans. Types A–E are associated with a variety of enteric diseases in both mammals and birds (Quinn et al. 2002).
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B.
Properties
Clostridium perfringens is a rod-shaped, encapsulated, anaerobic bacterium measuring 4 µm by 8 µm in length and 0.8 µm to 1.5 µm in diameter. The bacterium forms subterminal spores; however, these may not be observed in direct smears from clinical samples or culture media. Unlike other clostridia, Clostridium perfringens is nonmotile (Merchant and Packer 1977; Murray et al. 2002; Swartz 1990). Colonies of Clostridium perfringens grown on blood agar are smooth, round, grayish in color, and are surrounded by a double zone of hemolysis. Clostridium perfringens is oxidase and catalase negative and forms acid and gas from glucose, fructose, galactose, maltose, lactose, and sucrose but not from mannitol, dulcitol, or salicin. The organism is lecithinase and gelatinase positive and indole negative. Inoculation of the bacterium into litmus milk produces coagulation and gas (Merchant and Packer 1977; Quinn et al. 2002; Swartz 1990). A positive CAMP test, resulting in the enhancement of the partial zone of hemolysis produced by Clostridium perfringens, occurs after incubation with Streptococcus agalactae (Quinn et al. 2002).
C.
Bacterial Strains and Antigenic Relationships
Clostridium perfringens is grouped into five types based on the production and secretion of four major toxins (Table 14-1). In addition to the major toxins, Clostridium perfringens produces a number of other virulence-enhancing toxins and hydrolytic enzymes. The most significant of these is probably enterotoxin. Unlike the other toxins and enzymes produced by Clostridium perfringens, which are secreted by the bacterium, enterotoxin is released with the bacterial spore after cell lysis. Enterotoxin is primarily produced by Clostridium perfringens type A (Murray et al. 2002; Petit et al. 1999).
D.
Growth In Vivo and In Vitro
The organism grows rapidly on media such as blood agar enriched with yeast extract, vitamin K, and hemin. To ensure the absence of oxygen, the media should be freshly prepared or pre-reduced. Cultures should be incubated under anaerobic
TABLE 14-1
MAJOR TOXINS PRODUCED BY DIFFERENT TYPES OF CLOSTRIDIUM PERFRINGENS AND THEIR SITES OF ACTION
conditions (e.g., anaerobe jars containing hydrogen supplemented with 5% to 10% carbon dioxide) at 37° C (Merchant and Packer 1977; Murray et al. 2002; Quinn et al. 2002; Swartz 1990). E. 1.
Types
Site of action
α β ε τ
A-E B, C B, D E
cell membrane cell membrane cell membrane intracellular
Clinical Disease
There are only a few reports in the literature describing clinical disease associated with Clostridium perfringens infection in mice (Clapp and Graham 1970; Matsushita and Matsumoto 1986; Rozengurt and Sanchez 1999). Disease has been observed in mice of both sexes, from 2 to 32 days old, and in female mice of breeding age. There is no apparent strain predilection. Clinical signs have included hunched posture, ruffled hair coat, enlarged painful abdomen, soft or impacted feces, hindquarter paralysis, and dyspnea. Sudden death without premonitory signs has also been reported. The toxin types of Clostridium perfringens isolated from these cases were reported to be non-type A (Matsushita and Matsumoto 1986), type B (Rozengurt and Sanchez 1999), and type D (Clapp and Graham 1970). The majority of changes observed at necropsy are found in the intestinal tract. The small and large intestines may be variably enlarged and filled with gas and liquid feces. The intestinal mucosa may be hyperemic, contain petecchial hemorrhages and ulcerations, and be covered by pseudomembranes. Rupture of the small intestine resulting in peritonitis has also been described in association with Clostridium perfringens infection. Mucosal necrosis in both the large and small intestine is a consistent finding on microscopic examination of tissues from mice with clinically apparent Clostridium perfringens infections. The lumen of the intestine may contain large numbers of rodshaped bacteria (Matsushita and Matsumoto 1986; Rozengurt and Sanchez 1999). Microscopic polypoid proliferations of mucosal epithelium have been found in the duodenum (Rozengurt and Sanchez 1999). Changes that have been described in other tissues include karyopyknosis and karyorrhexis of lymphocytes in the thymus, spleen, and mesenteric lymph nodes, vacuolation in renal tubular epithelial cells (Matsushita and Matsumoto 1986), mild arteritis in the lung, and thrombosis and inflammation in the left atrium of the heart (Rozengurt and Sanchez 1999). Clostridium perfringens was commonly isolated from the intestines of affected mice (Clapp and Graham 1970; Matsushita and Matsumoto 1986; Rozengurt and Sanchez 1999). The organism was also isolated from blood, liver (Clapp and Graham 1970), and atrial thrombi (Rozengurt and Sanchez 1999). 2.
Toxin
In Vivo
Pathogenesis
Clostridium perfringens is most likely acquired by the ingestion of spores that originated in the soil or in the intestinal tract of a carrier animal. The organism is often a member of the normal microbiota in man and domestic animals. Factors that have been associated with the proliferation of the organism in
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these species include poor husbandry and sudden dietary changes (Quinn et al. 2002). As noted previously, Clostridium perfringens produces a number of major and minor toxins. Different types of the bacterium produce different toxins and different disease syndromes. Three of the major toxins, α, β, and ε, act on the cell membrane to cause membrane disruption or pore formation. The fourth major toxin, τ toxin, acts inside the cell to cause depolymerization of actin filaments (Table 14-1). The final results of toxin secretion include tissue necrosis and increased vascular permeability. Enterotoxin, primarily produced by Clostridium perfringens type A, binds to receptors on the brush border of enterocytes, disrupting ion transport and altering membrane permeability (Murray et al. 2002; Petit et al. 1999). A number of minor toxins are also produced, and some may enhance virulence of the organism. These include δ and θ hemolysins, a collagenase (κ), and a hyaluronidase (µ) (Quinn et al. 2002).
F.
Epizootiology
Clostridium perfringens is a common soil inhabitant throughout the world. In addition, Clostridium perfringens type A is a constituent of the normal microbiota of the intestine of humans and other animal species. The major diseases associated with the various types of Clostridium perfringens include the following: (1) Type A—necrotic enteritis in chickens, necrotizing enterocolitis in piglets, and canine hemorrhagic gastroenteritis; (2) Type B—lamb dysentery and hemorrhagic enteritis in calves and foals; (3) Type C—“struck” in adult sheep, sudden death in goats and feedlot cattle, necrotic enteritis in chickens, and hemorrhagic enteritis in neonatal piglets; (4) Type D—pulpy kidney in sheep and enterotoxemia in calves and goats; and (5) Type E—hemorrhagic enteritis in calves and enteritis in rabbits (Quinn et al. 2002).
G.
Diagnosis
Because postmortem clostridial invaders may rapidly spread from the intestine to other tissues, specimens for bacterial culture should only be taken from live or recently dead animals. Samples for bacterial culture should be placed in anaerobic transfer medium for transport to a microbiology laboratory and should be promptly cultured after their arrival (Quinn et al. 2002). A presumptive diagnosis of Clostridium perfringens infection can be based on finding large gram-positive rods in fecal smears or in histologic sections of intestine (Quinn et al. 2002). Definitive diagnosis is based on toxin identification. The toxins of Clostridium perfringens classically have been identified by seroprotection tests (Merchant and Packer 1977). However, the neutralizing antibodies used in these tests are no longer commercially available. Immunoassays have been developed as an alternative method for toxin identification. However, because
clostridial exotoxins tend to be labile, inability to detect them in fecal contents does not exclude a diagnosis of clostridial disease (Quinn et al. 2002). DNA-based techniques, such as PCR and hybridization, have also been used to determine the genotype of the organism (Petit et al. 1999).
H.
Treatment
Chlortetracycline hydrochloride administered to mice for 2 weeks in drinking water at a level of 11 mg/L has been reported to eliminate disease due to Clostridium perfringens (Matsushita and Matsumoto 1986). Incorporation of penicillin G into the diet or changing the diet has also been thought to be effective in eliminating the disease. Antibiotics that have been noted to be efficacious against Clostridium perfringens in domestic species include ampicillin, amoxicillin-clavulanate, tylosin, clindamycin, metronidazole, and bacitracin (Greene 1998; Keir et al. 1999; McGorum et al. 1998). Bacterins are used in domestic species to control the disease (Quinn et al. 2002). However, the use of a commercially available bacterin in mice did not effectively control the disease (Clapp and Graham 1970).
I.
Control and Prevention
Methods to control and prevent Clostridium perfringens infections have not been evaluated in mice. Because the bacterium is most likely acquired by the ingestion of spores, it can probably be excluded from mouse colonies by maintaining good sanitation and sterilizing feed, bedding, cages, and cage accessories. Sudden dietary changes have also been associated with proliferation of the organism and should be avoided if possible (Quinn et al. 2002).
IV.
CLOSTRIDIUM DIFFICILE A.
History
Clostridium difficile (Bacillus difficilis) was first isolated from the feces of human neonates in 1935. The bacterium was given its species name because it was difficult to isolate and grew slowly in vitro. The original isolates of Clostridium difficile produced a toxin when grown in broth and were pathogenic for guinea pigs and rabbits. However, the bacterium’s ability to produce disease in humans was not defined (Hall and O’Toole 1935). Clostridium difficile attained new notoriety in the late 1970s when it was identified as the etiology of antimicrobial-associated pseudomembranous colitis in humans (George et al. 1978; Larson et al. 1978). Clostridium difficile has also been implicated in antibiotic-associated colitis in Syrian hamsters (Bartlett et al. 1977), guinea pigs (Lowe et al. 1980), rabbits
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(Thilsted et al. 1981), prairie dogs (Muller et al. 1987), and ostriches (Frazier et al. 1993). In domestic species, the bacterium has been associated with enteric disease in piglets (Waters et al. 1998), foals (Jones et al. 1987, 1988), and dogs (Berry and Levett 1986).
B.
Properties
Clostridium difficile is a strict anaerobe. The bacterium is rodshaped, 0.5 µm wide by 2–8 µm long, forms subterminal to terminal spores, and is motile (Hall and O’Toole 1935; George et al. 1979). The organism is generally Gram positive. However, bacteria from older colonies may exhibit variable gram staining. After 48 hours of growth on the selective medium cycloserinecefoxitin-fructose agar (CCFA), colonies of Clostridium difficile are circular in shape with a filamentous edge, 4–8 mm in diameter, spreading, yellowish to white, have a flat to low umbonate profile, and have a ground-glass appearance. Colonies grown on CCFA have a golden-yellow fluorescence under long-wave ultraviolet light (UV). When grown on blood agar, colonies of Clostridium difficile are nonhemolytic, gray, have a slightly raised umbonate profile, filamentous edge, and ground-glass appearance. Under UV light, colonies grown on blood agar have a chartreuse fluorescence. Colonies of Clostridium difficile also have a distinctive horse manure–like odor (George et al. 1979; Knoop et al. 1993). Clostridium difficile forms acid from glucose and fructose but yields negative reactions with lactose, maltose, and sucrose. It is lecithinase, indole, and gelatinase negative, and does not coagulate litmus milk (Swartz 1990).
C.
Bacterial Strains and Antigenic Relationships
Clostridium difficile is closely related to Clostridium bifermentans and Clostridium sordellii. These three species of clostridia are all members of the Johnson and Francis rRNA homology group II (Wilson et al. 1988). Numerous methods have been employed to group isolates of Clostridium difficile into strains. The grouping methods include serotyping, restriction endonuclease analysis (REA), polymerase chain reaction (PCR)-ribotyping, restriction fragment length polymorphism (RFLP), and toxinotyping (Brazier 1998; Rupnik et al. 2001). Depending on the method used, the number of distinct Clostridium difficile strains ranges from fewer than 10 to over 100. Two closely related exotoxins, Toxin A and Toxin B, are produced by Clostridium difficile. These toxins share considerable sequence homology with toxins produced by Clostridium sordelli and Clostridium novyi (Pothoulakis and Lamont 2001).
D.
Growth In Vivo and In Vitro
The vegetative phase of Clostridium difficile is extremely oxygen-sensitive. Because of this, culture media should be
freshly prepared or pre-reduced prior to use. Clostridium difficile can be grown on blood agar. However, blood agar is nonselective and clinical samples are easily overgrown with other bacteria. CCFA is a commonly used selective medium for Clostridium difficile. Cultures are incubated under anaerobic conditions at 35–37° C. Because the bacterium grows slowly, plates are generally incubated for 48 hours prior to examination (George et al. 1979; Murray et al. 2002). E. 1.
In Vivo
Clinical Disease
Naturally occurring asymptomatic Clostridium difficile infection has been documented in conventionally housed mice. In one study, 19.4% of mice tested from seven commercial and two academic colonies were Clostridium difficile positive. The percentage of positive animals in these colonies increased to 63.6% when the mice were intraperitoneally given 2 mg ampicillin for 4 days prior to culture. Mice from four commercial and two academic “specific pathogen free” colonies were negative for the bacterium (Itoh et al. 1986). Experimental inoculation of germfree mice with toxigenic strains of Clostridium difficile has resulted in disease production. Findings in experimental infections include diarrhea, cecitis, polymorphonuclear cell infiltration of the lamina propria of the intestinal mucosa, pseudomembrane formation, and death (Onderdonk et al. 1980; Corthier et al. 1986; Wilson et al. 1986). Antibiotic treatment of conventional mice prior to experimental Clostridium difficile infection also resulted in disease production. Germfree mice that had been colonized by normal hamster microbiota prior to infection with Clostridium difficile did not develop disease (Wilson 1986). Human patients with Clostridium difficile–associated disease usually experience variably severe diarrhea, abdominal cramping, and tenderness in the abdomen. Patients with severe colitis may present with fever, nausea, anorexia, malaise, and dehydration. The CBC may reveal polymorphonuclear leukocytosis. Increased numbers of leukocytes and occult blood may be present in the feces. The earliest microscopic lesions of the disease include patchy necrosis of the intestinal mucosal epithelium and exudates in the intestinal lumen that are comprised of fibrin and neutrophils. In later stages, the exudate appears to erupt from focal ulcerations in the mucosa (“volcano” lesions). Severe lesions are characterized by diffuse mucosal necrosis and ulceration overlaid by pseudomembranes formed of inflammatory cells, fibrin, mucin, and cellular debris (Kelly et al. 1994). Clostridium difficile–associated disease in hamsters is comparable to the disease in humans. Clinical signs in hamsters include anorexia, ruffled fur, dehydration, diarrhea, and soiled perianal fur. The characteristic gross lesion observed at necropsy of affected hamsters is hemorrhagic ileocolitis. Microscopic findings include severe congestion of the blood vasculature that is most prominent in the distal ileum, cecum, and colon. This is accompanied by an accumulation of acute
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inflammatory cells in the lamina propria of the mucosa. Tips of the villi are frequently distended by edema fluid or cellular exudates and there is focal erosion of the mucosal surface. Fluid and neutrophils are present in the intestinal lumen (Small 1987). 2.
Pathogenesis
Clostridium difficile is most likely acquired from the ingestion of spores that are present in the environment or in the feces of carrier animals. The spores pass through the stomach and then convert to the vegetative phase in the intestine. Many Clostridium difficile infections are asymptomatic. Asymptomatic infections may occur because the normal gut microbiota suppresses the potential pathogen or because the infecting strain does not produce toxins. Alteration of the normal microbiota by stressors such as antibiotics or sudden changes in diet can result in proliferation of the pathogen and production of enteric disease (Wilson and Freter 1986). Clostridium difficile is not tissue invasive, and only toxigenic strains are associated with disease. Once elaborated, Clostridium difficile Toxins A and B bind receptors located on the apical surface of enterocytes and are internalized by the cell. The toxins open tight junctions between cells, leading to increased vascular permeability. Both are cytotoxic and induce the production of pro-inflammatory mediators such as tumor necrosis factor-alpha. Toxin A is believed to play the more critical role in disease pathogenesis. Toxin A, but not Toxin B, produces intestinal damage and accumulation of fluid in the intestinal lumen of experimental animal models. Toxin B is thought to play a role only after the intestinal wall is damaged by Toxin A. However, Toxin A is probably not essential for virulence because Toxin A–negative/Toxin–B positive Clostridium difficile have been isolated from patients with clinical disease (Kelly et al. 1994; Pothoulakis and Lamont 2001; Poutanen and Simor 2004). Several defense mechanisms help the host to combat Clostridium difficile infection. The first is gastric acidity, which can inactivate ingested cytotoxins and decrease the number of infective spores that enter the small intestine. A second factor that can inhibit pathogen growth is the normal intestinal microbiota. Normal intestinal peristalsis also aids in host defense by speeding elimination of Clostridium difficile and its toxins (Lee 1985; Kelly and LaMont 1998). The humoral immune system also appears to be important in limiting Clostridium difficile–associated disease. Vaccination with a toxoid containing Clostridium difficile toxins A and B has been found to prevent fatal clindamycin-induced cecitis in hamsters (Libby et al. 1982; Kim et al. 1987). Antitoxoid antibody was demonstrated in the serum and cecal contents of vaccinated hamsters. Infant hamsters from vaccinated dams were also protected from disease. Toxin-neutralizing antibodies were demonstrated in the serum and cecal contents of the protected infants and in the serum and milk of their dams (Kim et al. 1987). Human patients with Clostridium difficile–associated disease and low anti–Clostridium difficile toxin A antibody
levels have clinical symptoms for a longer time and more frequent relapses than people with higher antibody levels (Warny et al. 1994).
F.
Epizootiology
Clostridium difficile can be found throughout the world and is a common inhabitant of soil and water. It exists in the environment as spores that can persist for months or years. A wide variety of animal species, including humans, can asymptomatically carry the organism in the intestinal tract (Brazier 1998).
G.
Diagnosis
Diagnosis of Clostridium difficile–associated disease is generally based on detection of cytotoxins. The “gold standard” for cytotoxin detection is the tissue culture cytotoxicity assay. This test has a specificity of 99%–100% and a sensitivity of 80%–90%. However, it is expensive, time consuming, and only detects Toxin B. Enzyme immunoassays have been developed for the detection of Toxin A alone and for both Toxins A and B. The enzyme immunoassays have a reduced sensitivity (65%–85%) but approximately equal specificity (95%–100%) as the tissue culture cytotoxicity assay (Poutanen and Simor 2004; Steiner 2004). Samples for immunoassay can be collected by vigorously rubbing cotton-tipped applicators over the mucosal surface of the colon. The applicator tips can be snapfrozen in liquid nitrogen and held in an ultra-low temperature freezer until analysis. Cultures are generally less useful for diagnosis of Clostridium difficile–associated disease because they cannot differentiate toxigenic from nontoxigenic strains of the bacterium. PCR assays for detection of both Clostridium difficile and its cytotoxins are in development (Poutanen and Simor 2004; Steiner 2004).
H.
Treatment
There are no published regimens specifically for the treatment of natural Clostridium difficile infections in mice. Two daily oral doses of 2 mg of vancomycin administered for 7 days to gnotobiotic mice experimentally infected with Clostridium difficile resulted in a 2- to 3-log decrease in vegetative bacterial cell count and no detectable cytotoxin. However, bacterial counts and cytotoxin levels returned to previous levels once treatment was stopped. Clindamycin treatment of experimentally infected gnotobiotic mice had no effect on Clostridium difficile numbers (Onderdonk et al. 1980). In humans with antibiotic-associated Clostridium difficile disease, the offending antibiotic is stopped. This is often all that is needed for the resolution of the disease. Metronidazole is the drug of choice for treatment of more severe disease in humans. Additional antibiotics that have been used in humans
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are vancomycin and bacitracin (Kelly and Lamont 1998; Craig 2004). Metronidazole, vancomycin, and bacitracin have been used to treat Clostridium difficile infections in horses (Staempfli et al. 1992; Jang et al. 1997).
I.
Control and Prevention
Specific methods to control and prevent Clostridium difficile infections in mouse colonies have not been evaluated. The method of transmission of this bacterium via ingestion of spores is similar to that of Clostridium piliforme and Clostridium perfringens. Therefore, all three of these clostridia can probably be excluded from mouse colonies by maintaining strict husbandry practices, good sanitation, and sterilizing feed, bedding, cages, and cage accessories. Sudden dietary changes and other stressors should be avoided when possible, and antibiotics should be used judiciously to minimize alteration of the animals’ normal gut microbiota.
ACKNOWLEDGMENTS The author would like to thank Lillian Maggio-Price, Cara Al-Hassan, and Ralph Bunte for their helpful advice and Molly Bernard for her excellent technical assistance.
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Rupnik, M., Brazier, J. S., Duerden, B. I., Grabner, M., and Stubbs, S. L. (2001). Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes. Microbiology 147, 439–447. Saunders, G. K., Sponenberg, D. P., and Marx, K. L. (1993). Tyzzer’s disease in a neonatal cockatiel. Avian Dis. 37, 891–894. Schmidt, R. E., Eisenbrandt, D. L., and Hubbard, G. B. (1984). Tyzzer’s disease in snow leopards. J. Comp. Pathol. 94, 165–167. Simon, P. C. (1977). Isolation of Bacillus piliformis from rabbits. Can. Vet. J. 18, 46–48. Small, J. D. (1987). Drugs used in hamsters with a review of antibiotic-associated colitis. In Laboratory hamsters, G. L. van Hoosier, Jr. and C. W. McPherson, eds., pp. 179–199. Academic Press, Orlando. Smith, K. J., Skelton, H. G., Hilyard, E. J., et al. (1996). Bacillus piliformis infection (Tyzzer’s disease) in a patient infected with HIV-1: confirmation with 16S ribosomal RNA sequence analysis. J. Am. Acad. Dermatol. 34, 343–348. Sparrow, S., and Naylor, P. (1978). Naturally occurring Tyzzer’s disease in guinea pigs. Vet. Rec. 102, 288. Spencer, T. H., Ganaway, J. R., and Waggie, K. S. (1990). Cultivation of Bacillus piliformis (Tyzzer) in mouse fibroblasts (3T3 cells). Vet. Microbiol. 22, 291–297. Staempfli, H. R., Prescott, J. F., Carman R. J., and McCutcheon, L. J. (1992). Use of bacitracin in the prevention and treatment of experimentally-induced idiopathic colitis in horses. Can. J. Vet. Res. 56, 233–236. Stanley, S. M., Flatt, R. E., and Daniels, G. N. (1978). Naturally occurring Tyzzer’s disease in the gray fox. J. Am. Vet. Med. Assoc. 173, 1173–1174. Steiner, T. (2004). Pseudomembranous colitis. In Cecil textbook of medicine, L. Goldman and D. Ausiello, eds., pp. 1836–1838. Saunders, Philadelphia. Swartz, M. N. (1990). Anaerobic spore-forming bacilli: the clostridia. In Microbiology, B. D. Davis, R. Dulbecco, H. N. Eisen, and H. S. Ginsberg, eds., pp. 633–646. J.B. Lippincott, Philadelphia. Swerczek, T. W., Crowe, M. W., Prickett, M. E., and Bryans, J. T. (1973). Focal bacterial hepatitis in foals: preliminary report. Mod. Vet. Pract. 54, 66–67. Takagaki, Y., Ito, M., Fujiwara, K., Maejima, M., Naiki, M., and Tajima, Y. (1964). [Effects of antibiotics and sulfonamides on Tyzzer’s disease in experimentally infected mice]. C. R. Soc. Biol. 158, 414–418. Takagaki, Y., Ito, M., and Naiki, M. (1966). Experimental Tyzzer’s disease in different species of laboratory animals. Jap. J. Exp. Med. 36, 519–534. Takagaki, Y., Tsuji, K., and Fujiwara, K. (1968). Tyzzer’s disease-like lesions observed in young rats. Jikken Dobutsu [Exp. Anim.] 17, 67–69. Takasaki, Y., Oghiso, Y., Sato, K., and Fujiwara, K. (1974). Tyzzer’s disease in hamsters. Jap. J. Exp. Med. 44, 267–270. Thilsted, J. P., Newton, M. W., Crandell, R. A., and Bevill, R. F. (1981). Fatal diarrhea in rabbits resulting from the feeding of antibiotic-contaminated feed. J. Am. Vet. Med. Assoc. 179, 360–361. Thunert, A. (1984). Is it possible to cultivate the agent of Tyzzer’s disease (Bacillus piliformis) in cellfree media? Z. Versuchstierkd. 26, 145–150. — — —. (1982). Therapy and prophylaxis of Tyzzer’s disease. (In vivo antibiogram of three strains of B. piliformis.) Z. Versuchstierkd. 24, 206–213. Toriumi, W., Kawamura, S., and Fujiwara, K. (1986). Application of enzymelinked immunosorbent assay (ELISA) to detection of antibodies against Tyzzer’s organism (Bacillus piliformis) in mice. Nippon Juigaku Zasshi (Jap. J. Vet. Sci.) 48, 1241–1244. Tsuchitani, M., Umemura, T., Narama, I., and Yanabe, M. (1983). Naturally occurring Tyzzer’s disease in a clean mouse colony: high mortality with coincidental cardiac lesions. J. Comp. Pathol. 93, 499–507. Tyzzer, E. E. (1917). A fatal disease of the Japanese waltzing mouse caused by a spore-bearing bacillus (Bacillus piliformis, N. sp.). J. Med. Res. 37, 307–338. Van Andel, R. A., Franklin, C. L., Besch-Williford, C. L., Hook, R. R., and Riley, L. K. (2000a). Prolonged perturbations of tumour necrosis factoralpha and interferon-gamma in mice inoculated with Clostridium piliforme. J. Med. Microbiol. 49, 557–563. — — —. (2000b). Role of interleukin-6 in determining the course of murine Tyzzer’s disease. J. Med. Microbiol. 49, 171–176.
14. CLOSTRIDIAL SPECIES
Van Andel, R. A., Hook, R. R., Jr., Franklin, C. L., Besch-Williford, C. L., and Riley, L. K. (1998). Interleukin-12 has a role in mediating resistance of murine strains to Tyzzer’s disease. Infect. Immun. 66, 4942–4946. Van Andel, R. A., Hook, R. R., Jr., Franklin, C. L., Besch-Williford, C. L., van Rooijen, N., and Riley, L. K. (1997). Effects of neutrophil, natural killer cell, and macrophage depletion on murine Clostridium piliforme infection. Infect. Immun. 65, 2725–2731. Veazey, R. S., 2nd, Paulsen, D. B., and Schaeffer, D. O. (1992). Encephalitis in gerbils due to naturally occurring infection with Bacillus piliformis (Tyzzer’s disease). Lab. Anim. Sci. 42, 516–518. Waggie, K. S., Ganaway, J. R., Wagner, J. E., and Spencer, T. H. (1984). Experimentally induced Tyzzer’s disease in Mongolian gerbils (Meriones unguiculatus). Lab. Anim. Sci. 34, 53–57. Waggie, K. S., Hansen, C. T., Ganaway, J. R., and Spencer, T. S. (1981). A study of mouse strain susceptibility to Bacillus piliformis (Tyzzer’s disease): the association of B-cell function and resistance. Lab. Anim. Sci. 31, 139–142. Waggie, K. S., Thornburg, L. P., Grove, K. J., and Wagner, J. E. (1987). Lesions of experimentally induced Tyzzer’s disease in Syrian hamsters, guinea pigs, mice and rats. Lab. Anim. 21, 155–160. Warny, M., Vaerman, J-P., Avesani, V., and Delmee, M. (1994). Human antibody response to Clostridium difficile toxin A in relation to clinical course of infection. Infect. Immun. 62, 384–389. Waters, E. H., Orr, J. P., Clark, E. G., and Schaufele, C. M. (1998). Typhlocolitis caused by Clostridium difficile in suckling piglets. J. Vet. Diagn. Invest. 10, 104–108.
363 Webb, D. M., Harrington, D. D., and Boehm, P. N. (1987). Bacillus piliformis infection (Tyzzer’s disease) in a calf. J. Am. Vet. Med. Assoc. 191, 431–434. White, D. J., and Waldron, M. M. (1969). Naturally occurring Tyzzer’s disease in the gerbil. Vet. Rec. 85, 111–114. Wilson, G. S., and Miles, A. A. (1964). Topley and Wilson’s principles of bacteriology and immunity. Williams and Wilkins, Baltimore. Wilson, K. H. (1994). Detection of culture-resistant bacterial pathogens by amplification and sequencing of ribosomal DNA. Clin. Infect. Dis. 18, 958–962. Wilson, K. H., Blitchington, R., Hindenach, B., and Greene, R. C. (1988). Species-specific oligonucleotide probes for rRNA of Clostridium difficile and related species. J. Clin. Microbiol. 26, 2484–2488. Wilson, K. H., and Freter, R. (1986). Interaction of Clostridium difficile and Escherichia coli with microfloras in continuous-flow cultures and gnotobiotic mice. Infect. Immun. 54, 354–358. Wilson, K. H., Sheagren, J. N., Freter, R., Weatherbee, L., and Lyerly, D. (1986). Gnotobiotic models for study of the microbial ecology of Clostridium difficile and Escherichia coli. J. Infect. Dis. 153, 547–551. Wojcinski, Z. W., and Barker, I. K. (1986). Tyzzer’s disease as a complication of canine distemper in a raccoon. J. Wildl. Dis. 22, 55–59. Yokoiyama, S., and Fujiwara, K. (1971). Effect of antibiotics on Tyzzer’s disease. Jap. J. Exp. Med. 41, 49–57. Zook, B. C., Huang, K., and Rhorer, R. G. (1977). Tyzzer’s disease in Syrian hamsters. J. Am. Vet. Med. Assoc. 171, 833–836. Zook, B. C., Albert, E. N., and Rhorer, R. G. (1977). Tyzzer’s disease in the Chinese hamster (Cricetulus griseus). Lab. Anim. Sci. 27, 1033–1035.
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Chapter 15 Enterobacteriaceae, Pseudomonas aeruginosa, and Streptobacillus moniliformis Hilda Holcombe and David B. Schauer
I.
II.
III.
IV.
V.
General Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Growth Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Virulence Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Pathogenicity Islands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Citrobacter rodentium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Treatment and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Clinical Disease and Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteus mirabilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
366 366 367 367 369 369 369 370 370 371 371 371 372 373 373 373 374 374 375 376 376 377 377 377 377 377 377 377 377 378 Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
365
366
H I L D A H O L C O M B E A N D D AV I D B . S C H A U E R
C. Clinical Disease and Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Treatment and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Klebsiella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Pseudomonas aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Clinical Signs and Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Streptobacillus moniliformis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Clinical Signs and Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Diagnosis and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
GENERAL OVERVIEW A.
Introduction
The family Enterobacteriaceae consists of 41 genera of gram-negative, facultatively anaerobic bacteria (Table 15-1). Many of the organisms are normal inhabitants of the gastrointestinal tract of animals; however, several have been identified as primary or opportunistic pathogens. Of these, only a few are documented to cause spontaneous disease in laboratory mice. Salmonella has historically been a concern in mouse facilities due in part to its zoonotic potential, but with the increased standards of sanitation and husbandry practiced in most laboratory animal facilities today, natural outbreaks of salmonellosis are rare. Escherichia, the type genus, includes strains capable of causing disease in humans and domestic animal species, yet mice are largely resistant to infection with pathogenic strains of Escherichia coli. However, immunodeficient mice are susceptible to infection and disease caused by certain lactose nonfermenting strains. A related organism, Citrobacter rodentium, is highly pathogenic, particularly in young mice, but like Salmonella, it has been excluded from most barrier facilities today. Other reports of spontaneous disease caused by Enterobacteriaceae in mice are predominantly sporadic cases involving opportunistic pathogens such as Klebsiella and Proteus, which, like E. coli, are primarily a concern in immunodeficient and immunocompromised animals.
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The mechanism responsible for the relative resistance of immunocompetent mice to infection with pathogenic enteric organisms is unclear, but would seem to involve more than simply appropriate innate and/or adaptive immune responses. It may be that laboratory mice lack suitable receptors for bacterial attachment, which are expressed on cells of host species that are susceptible to infection. Few receptors for specific bacterial attachment factors have been identified, but a comparable situation exists for at least one bacterial exotoxin. Certain strains of E. coli produce Shiga toxin (Stx), a potent exotoxin that has been implicated in hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans, particularly children. Attempts to develop a mouse model for HUS have been met with limited success. Even under conditions where laboratory mice can be successfully infected with Shiga toxigenic E. coli (STEC), the animals fail to develop glomerular lesions that are characteristic of HUS. Different outcomes with STEC infection between humans and mice appear to be due, at least in part, to differences in expression of globotriaosylceramide (Gb3), the cellular receptor for Stx (Rutjes et al. 2002). The presence of normal enteric microbiota is also important in resistance to infection with enteric pathogens (Hudault et al. 2001). Thus, one commonly used method to increase the susceptibility of mice to infection with virulent strains of E. coli and other enteric bacteria is to pre-treat the animals with antibiotics, such as streptomycin (Myhal et al. 1982). Possible mechanisms that have been proposed to account for the protection against infection, or colonization resistance,
15. PSEUDOMONAS AERUGINOSA, AND STREPTOBACILLUS MONILIFORMIS
TABLE 15-1
GENERA OF THE FAMILY ENTEROBACTERIACEAE a,b Alterococcus Arsenophonus Brenneria Buchnera Budvicia Buttiauxella Calymmatobacterium Cedecea Citrobacter Edwardsiella Enterobacter Erwinia Escherichia Ewingella Hafnia Klebsiella Kluyvera Leclercia Leminorella Moellerella Morganella Obesumbacterium Pantoea Pectobacterium Photorhabdus Plesiomonas Pragia Proteus Providencia Rahnella Saccharobacter Salmonella Serratia Shigella Sodalis Tatumella Trabulsiella Wigglesworthia Xenorhabdus Yersinia Yokenella from Garrity and Holt 2001. documented to cause spontaneous disease in laboratory mice are shown in bold. bGenera
conferred by normal microbiota include niche competition (i.e., attachment sites and nutrients) and production of soluble factors (i.e., antibacterial microcins, colicins, and short-chain fatty acids). The relative importance of each of these mechanisms, or of other as yet uncharacterized mechanisms, in colonization resistance remains to be determined.
B.
in length. Many species other than Klebsiella and Shigella are motile by peritrichous flagella. The majority grow well at 37°C on simple bacteriological media. Species are identified based on biochemical assays and growth on selective media. Biochemical properties of selected pathogens are shown in Table 15-2. Most are oxidase negative, catalase positive, reduce nitrate, and can utilize D-glucose as a sole source of carbon, resulting in the production of acid and often gas. Historically, the ability to ferment lactose has been used as a surrogate marker for virulence; many commensal strains ferment lactose, while some biochemically inactive strains of E. coli, as well as Salmonella and Shigella, do not. There are exceptions, however.
C.
aTaken
Growth Characteristics
Members of the family Enterobacteriaceae are gramnegative, rods, generally 0.5–1 µm in diameter, and 2–5 µm
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Virulence Factors
A number of features common to the members of the family Enterobacteriaceae are involved in infection and/or disease, and can thus be considered virulence determinants. Other, more specialized virulence determinants, such as adherence factors, exotoxins, and dedicated secretion systems, are limited to certain species or strains. Here, we briefly review some of the more common virulence determinants found in most, if not all, Enterobacteriaceae. Lipopolysaccharide (LPS), a key constituent of the gramnegative bacterial outer membrane, is also an important virulence factor. It is composed of 3 covalently linked parts: a hydrophobic lipid A portion, a core polysaccharide, and an O-linked sugar moiety. The O-linked polysaccharide chain is the most variable, and structures can differ between bacterial species as well as between strains of a given species. Recent evidence indicates that bacteria can modify their LPS in response to environmental conditions, which can in turn affect virulence (Ernst et al. 2001). Adaptive chemical modifications of LPS occur in both the lipid A portion as well as in the O side chain. Although the O side chain of LPS is more antigenic, it is the lipid A portion that contains endotoxin activity, a potent activator of innate immunity that in sufficient quantities can cause endotoxic shock and death. The mechanism of cellular activation by LPS is well characterized, and cell surface receptors that recognize LPS have been identified. For more details of LPS toxicity and signaling, the reader is referred to a review by Dobrovolskaia and Vogel (2002) (Volume 4, Chapter 2). Although the topic is beyond the scope of this chapter, it should be noted that C3H/HeJ and C57BL10/ScCr inbred strains of mice have mutations in Toll-like receptor 4 (TLR4), a component of the LPS receptor complex, rendering them hyporesponsive to LPS (Qureshi et al. 1999). In serotyping schemes, such as those developed by White and extended by Kauffmann for Salmonella in the 1930s and E. coli in the early 1940s, the somatic or O antigen corresponds to the heat-stable polysaccharide portion of LPS. The specificity
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TABLE 15-2
BIOCHEMICAL PROPERTIES OF SELECTED ENTEROBACTERIACEAEa Test Indole Methyl red Voges-Proskauer Citrate (Simmons) Hydrogen sulfide Urea hydrolysis Phenylalanine deaminase Lysine decarboxylase Arginine dihydrolase Ornithine decarboxylase Motility Gelatin hydrolysis, 22°C KCN, growth Malonate utilization D-Glucose, gas Acid produced from: D-Glucose Lactose Sucrose D-Mannitol Dulcitol Salicin D-Adonitol i-Inositol D-Sorbitol L-Arabinose Raffinose L-Rhamnose Maltose D-Xylose Trehalose Cellobiose alpha-CH3-d-glucoside Melibiose Glycerol D-Mannose Mucate Tartrate (Jordans) Acetate utilization Esculin hydrolysis Lipase Nitrate reduction Oxidase ONPGb
Salmonella
Citrobacter freundii
Citrobacter rodentium
E. coli
E. coli (inactive)
K. oxytoca
K. pneumoniae
Proteus mirabilis
– + – + + – –
– + – + [+] d –
– + – – – [+] –
+ + – – – – –
[+] + – – – – –
+ [–] + + – + –
– [–] + + – + –
– + d d + + +
+ D + + – – – +
– d [–] + – + [–] +
– – + – – – + +
+ [–] d + – – – +
d – [–] – – – – –
+ – – – – + + +
+ – – – – + + +
– – + + + + – +
+ – – + + – – d + + – + + + + – – + – + + + + – – + – –
+ d d + d – – – + + d + + + + d – d + + + + [+] – – + – +
+ + – + – – – – + + – + + + + + – – – + + + – – – + – +
+ + d + d d – – + + d +/– + + + – – [+] d + + + + d – + – +
+ [–] [–] + d – – – [+] [+] [–] d [+] d + – – d d + d [+] d – – + – d
+ + + + d + + + + + + + + + + + + + + + + + + + – + – +
+ + + + d + + + + + + + + + + + + + + + + + [+] + – + – +
+ – [–] – – – – – – – – – – + + – – – d – – [+] [–] – + + – –
aAll tests at 36°C (except gelatin), all tests read at 48 hours. +, >90% positive; [+], 76–89% positive; d, 26–75% positive; [–], 11–25% positive; −, ≤10% positive. Taken from Holt et al. 1994 and Schauer et al., 1995. bONPG, o-nitrophenyl-beta-D-galactopyranoside.
of antibodies raised against the O antigen results from variation in the chemical composition of the O-linked sugar side chains. Serotype analysis has been used for diagnostic purposes in the past, but today it is used primarily for epidemiology. The H antigen corresponds to the monomeric protein subunit of flagella, the organelle used for motility by members of the
family Enterobacteriaceae. This structural component of flagella is called flagellin. In contrast to the O antigen, the H antigen is heat- and alcohol-labile. The carboxyl and amino ends of flagellin are conserved, with an antigenically variable domain found in the surface-exposed central portion of the protein. Some pathogenic Enterobacteriaceae, such as
15. PSEUDOMONAS AERUGINOSA, AND STREPTOBACILLUS MONILIFORMIS
C. rodentium, are not motile, and do not appear to express flagella. However, analysis of isogenic mutants has demonstrated a clear role for flagella and motility in the pathogenesis of infection by other members of the Enterobacteriaceae family, such as Proteus mirabilis (Mobley et al. 1996; Zunino et al. 1994). K antigens, or capsular polysaccharides, are expressed by some genera of Enterobacteriaceae and have also been used for serotyping. Because the capsule is external to the outer membrane, presence of a K antigen can mask the presence of an O antigen and prevent agglutination by O-specific antisera. Klebsiella strains frequently produce abundant capsular polysaccharide. The K antigen has been implicated as a virulence determinant. It can prevent phagocytosis by polymorphonuclear leukocytes (PMNs) and macrophages as well as inhibit complement deposition on the surface of the bacterium (Cortes et al. 2002; Merino et al. 1992). Fimbriae or pili are proteinaceous surface structures, smaller than flagella, that are expressed by many Enterobacteriaceae. These surface appendages are not involved in motility, but rather promote attachment to other bacteria (where they are involved in conjugation) or to surfaces and/or host cells. 1.
Pathogenicity Islands
There is growing appreciation that genes encoding bacterial virulence factors can be physically linked, and can be found on plasmids, on lysogenic bacteriophage, or in discrete regions of the bacterial chromosome in what are called pathogenicity islands (PAI). PAI can include large clusters of genes, spanning hundreds of kilobases (kb) of sequence. Smaller regions have been called pathogenicity islets. Neither is unique to Enterobacteriaceae, but rather they have been found in many gram-positive and gram-negative bacterial pathogens (reviewed by Schmidt and Hensel 2004). PAI often contain distinct genes that confer related activities necessary to exploit a particular niche. They are dispensable and not required for survival of the bacterium. Nonetheless, the presence of a PAI may dramatically change the phenotype of a bacterium, and may be sufficient to bestow virulence. Several lines of evidence suggest that PAI are foreign DNA, in the sense that they have been acquired by the bacteria in which they reside, but did not originate there. During the evolution of virulent bacteria, PAI have been horizontally transferred between closely and distantly related bacteria. It seems likely that horizontal transfer of PAI (along with plasmids and bacteriophage) continues to generate diversity in Enterobacteriaceae, shuffling different combinations of virulence determinants, and possibly creating new categories of pathogens. Some PAI are unstable and can be lost during serial passage of a strain. A single bacterial strain can have multiple PAI. Some PAI encode fimbriae and exotoxins, while others encode complex, macromolecular, dedicated secretion systems that inject bacterial proteins directly into host cells. Many of the most well-studied dedicated secretion systems are related to flagellar
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secretion systems (for assembly of the organelle) or plasmid conjugation systems (for transferring protein-nucleic acid complexes between bacteria). These cognate type III secretion systems (T3SS) and type IV secretion systems (T4SS), respectively, inject distinct effector proteins to usurp normal host cell functions for the benefit of the pathogen. Effector proteins can be encoded by genes within the PAI or outside the PAI. There is also evidence that certain effector proteins can be substrates for multiple secretion systems in a single organism. While different dedicated T3SS and T4SS share common features of protein translocation, and injection of protein by these unrelated groups of secretion systems are analogous, specific effector proteins are individually tailored to each pathogen’s life style. Salmonella uses 2 different T3SS on 2 different PAI to induce its own phagocytosis by normally nonphagocytic enterocytes in order to gain entry to the host from the intestinal lumen and to modify the intracellular environment of the macrophage in order to survive within these professional phagocytes after being ingested. In contrast, Yersinia uses several different nonfimbrial adhesins to gain entry to the host from the intestinal lumen, including the outer membrane protein invasin, which binds to certain integrins, presumably allowing translocation through M cells located in the domed epithelium over Peyer’s patches. Yersinia then uses a plasmidencoded T3SS to inject proteins into macrophages to prevent phagocytosis, such that the bacteria can replicate in their preferred, extracellular niche. Distinct effector proteins, substrates for these cognate T3SS, confer specific virulence properties on these 2 different Enterobacteriaceae.
II. SALMONELLA A.
Introduction
There are approximately 2,500 serotypes, or serovars, in the genus Salmonella. DNA-DNA hybridization demonstrates that most of the serovars are related at the species level with a single exception, Salmonella bongori, which was shown to be a separate species (Reeves et al. 1989). Based on these findings, attempts have been made to simplify and standardize Salmonella nomenclature (Euzeby 1999; Le Minor and Popoff 1987). After an opinion on this proposed change was requested from the Judicial Commission of the International Committee on Systematics of Prokaryotes (ICSP), formerly the International Committee for Systematic Bacteriology, the proposed nomenclature gained wide acceptance among microbiologists. Unfortunately, the proposed change was ultimately rejected by the Judicial Commission, resulting in widespread use of an invalid nomenclature by not only researchers but also by the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC). Arguments for (Ezaki et al. 2000)
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and against (Yabuuchi and Ezaki 2000) adoption of the revised system of Salmonella nomenclature have been advocated, but the situation remains unresolved. Here we provide a brief overview of both the ICSP-approved nomenclature and the popular, but unofficial, system. The reader is cautioned that the basis for Salmonella taxonomy has changed considerably from the early days of clinical considerations, to serology and to our current standard of DNA relatedness; thus, trying to compare nomenclature in different reports can be confusing. For more information, the reader is referred to a review by Brenner et al. (2000) and to the CDC Salmonella Surveillance Summary (2002). Officially, there are 5 species of Salmonella recognized by the ICSP, with Salmonella cholerasuis being the type species (Ezaki et al. 2000; Yabuuchi and Ezaki 2000). The other species are Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, and S. bongori. S. typhi is the causative agent of typhoid fever in humans. S. enteritidis and S. typhimurium are the species most commonly associated with disease in mice, and S. typhimurium is commonly used in experimental infections as a mouse model of typhoid fever. The CDC, the WHO, and many published reports use nomenclature originally proposed by LeMinor and Popoff (1987). Because all serotypes other than S. bongori are genetically related, it was proposed that Salmonella be divided into 2 species, Salmonella enterica and S. bongori, and that S. enterica be further divided into 6 subspecies. Under this system, essentially all medically relevant isolates of Salmonella, including all of the named serovars, are included in S. enterica subsp. enterica. Thus, S. typhimurium and S. enteritidis are designated S. enterica subsp. enterica serovar Typhimurium and S. enterica subsp. enterica serovar Enteritidis, which can simply be referred to as S. Typhimurium and S. Enteritidis.
B.
History
S. typhi (originally Bacillus typhi) was first isolated from the spleen of a patient with typhoid fever by George Gaffky in 1884. S. typhimurium (originally Bacillus typhimurium) was determined to be the causative agent of murine typhoid in 1892 by Friedrich Loeffler (Santos et al. 2001). Bacterial distribution in the tissues of infected mice was comparable to that in human typhoid fever. Thus, murine typhoid was established as a model for studying human disease, and it remains a popular model today for studying both disease pathogenesis and vaccine efficacy. In addition, the mouse model of typhoid fever has been used to study herd immunity and the effects of nutrition and heredity upon resistance to disease. Infection of mice with S. enteritidis has also been used as a model for studying Salmonella gastroenteritis; however, other models, including experimental infection of calves, have been advocated as being more appropriate models of non-typhoid Salmonella diarrheal disease. Recent advances in understanding its molecular
pathogenesis have made Salmonella one of the most wellcharacterized enteric bacterial pathogens. As a result, a great deal is known about both host and bacterial factors that contribute to Salmonella infection and disease. Spontaneous outbreaks of salmonellosis in laboratory mouse colonies were a common occurrence through the middle of the 20th century. As the advantages of using specific pathogen-free animals became increasingly apparent, improved husbandry and biocontainment practices, including rederivation of mouse colonies, led to reduced incidence of outbreaks. However, Salmonella remains a potential cause of disease in conventional mouse colonies, as well as in barrier-maintained colonies when biocontainment practices are compromised.
C.
Properties
Salmonella are gram-negative, non-spore-forming, facultatively anaerobic rods that measure 0.7–1.5 µm in diameter and 2.0–5.0 µm in length. They are usually motile by peritrichous flagella. Salmonella reduce nitrate, produce acid from glucose, usually with the production of gas, and usually utilize citrate as a sole carbon source. Most strains produce hydrogen sulfide on triple sugar iron agar, except for S. typhi. Salmonella are indole and urease negative. Serotype analysis is typically determined by agglutination using a battery of O- and H-specific antisera. Salmonella can express 2 different flagellar antigens, although expression is typically coordinated so that only 1 antigen is expressed at a time (Fierer and Guiney 2001). Of the 2 antigenic types or phases, phase 1 flagellin is a more potent activator of the host immune system. Switching to phase 2 flagellin may facilitate immune evasion by the bacteria. As discussed in the general overview, Salmonella are facultative intracellular parasites that can induce their own uptake by normally nonphagocytic enterocytes, and can survive and replicate inside macrophages. The Salmonella pathogenicity island 1 (SPI-1) is a 40-kb chromosomal locus that encodes a T3SS that is required for bacterial entry, or invasion, of epithelial cells. SPI-1 effectors also cause caspase 1 dependent apoptotic death of macrophages and dendritic cells. Consequently, SPI-1 mutant strains are attenuated when administered orally but are virulent when given by intravenous or intraperitoneal routes of inoculation. The Salmonella pathogenicity island 2 (SPI-2) encodes a distinct T3SS that is required for survival and replication of Salmonella within macrophages. SPI-2 effectors alter various aspects of endocytic trafficking, permit avoidance of NADPH oxidase-dependent killing, and induce a delayed apoptosis-like host cell death in macrophages. Consequently, SPI-2 mutants are attenuated in virulence by at least 5 orders of magnitude, whether given orally or by a systemic route (Shea et al. 1996). The other Salmonella PAI are less well characterized. SPI-3 harbors 10 genes, but only 1 gene, mgtC, has been identified as a virulence factor and is
15. PSEUDOMONAS AERUGINOSA, AND STREPTOBACILLUS MONILIFORMIS
required for survival of Salmonella in macrophages and for growth in low-magnesium environments. SPI-4 contains 18 genes and is also believed to be involved in macrophage survival. SPI-5 encodes 6 genes, 4 of which have been shown to be involved in enteritis in calves.
D.
Epizootiology
Salmonella has a broad host range that probably includes most, if not all, vertebrate animal species. Potential reservoirs for exposure of mice and humans include many domestic and wild species of birds and mammals, as well as reptiles. Transmission occurs primarily through exposure to contaminated food, water, and bedding. Thus, feeding commercial mouse chow, providing disinfected or sterilized water and bedding, and preventing wild and feral rodents as well as wild birds from entering facilities in which laboratory mice are being housed are all effective in limiting contamination and helping to prevent Salmonella infection. It is worth mentioning that even in a closed or barrier-maintained colony where these measures are taken to eliminate likely sources, human carriers could serve as possible reservoirs.
E.
Pathogenesis
Many factors influence the outcome of natural or experimental exposure to Salmonella, including virulence of the bacterial strain, route of exposure, dose, age and genetic background of the host, nutritional status, concurrent diseases, and other experimental stresses that suppress immunity, to name but a few. Thus, the incubation period is variable, but usually lasts 3–6 days. Most of the challenge dose is excreted in the feces; only a few bacteria, possibly less than 1%, gain entry into the host by translocating across M cells and invading underlying Peyer’s patches in the ileum. The LD50 of virulent S. enteritidis in conventional outbred mice with normal gut microflora was shown to be 2.5 × 103 by the intraperitoneal route, 1 × 104 by the intravenous route, and 5 × 106 by the oral route. The fact that comparable LD50 values for germ-free mice by similar routes were 4 × 103, 2 × 103, and 3 to 5, respectively (Collins and Carter 1978), indicates the importance of resident intestinal microflora in preventing the establishment of Salmonella infection, particularly following oral exposure. Inbred strains of mice vary considerably in their susceptibility to Salmonella (reviewed by Lam-Yuk-Tseung and Gros 2003). Extremely susceptible strains include C57BL/6J, C3H/HeJ, and BALB/cJ; intermediate strains are A/J and CBA/N; and 129S6/SvEvTac are resistant. Wild-derived strains also vary in their susceptibility. CAST/Ei are very resistant, while MOLF/Ei and SPRET/Ei are more susceptible to Salmonella infection. Several different genetic factors contribute to resistance and susceptibility. One important locus is the natural
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resistance-associated macrophage protein 1 (Nramp1), which was originally referred to as the immunity to typhimurium (Ity) locus, and has more recently been designated solute carrier family11 member a1 (Slc11a1). This member of the Nramp family is a proton/divalent cation antiporter expressed in reticuloendothelial cells that apparently controls host resistance to certain pathogens by regulating intraphagosomal concentrations of Fe2+ and/or other cations. Studies with transgenic and targeted gene mutant (knockout) mice confirm that lack of expression of Nramp1 leads to fatal, uncontrolled replication of Salmonella in the spleen and liver 4–5 days after intravenous or subcutaneous inoculation. It is also clear that TLR4 plays an important role in susceptibility and resistance to Salmonella infection. TLRs are a growing family of proteins that recognize pathogen-associated molecular patterns (PAMPs). As of this writing, there are 11 individual TLRs (10 in humans), which together with other pattern recognition receptors (PRRs), allow cells to sense and respond to conserved microbial motifs (Janssens and Beyaert 2003; Zhang et al. 2004). Activation of TLR-signaling pathways results in transcriptional responses and secretion of pro-inflammatory cytokines by phagocytes and epithelial cells, as well as other cell types. Each TLR has a distinct set of PAMPs to which it responds; TLR4 recognizes LPS. Mouse strains with spontaneous null mutations in TLR4 include C3H/HeJ, which has a histadine to proline point mutation at position 712 in the signaling domain of TLR4, and C57BL/ 10ScCr, which has a deletion of the entire TLR4 locus (Qureshi et al. 1999). C3H/HeJ mice were shown to be extremely susceptible to S. typhimurium, with an LD50 of <2 organisms (Malo et al. 1994). TLR5 recognizes flagellin and is therefore also likely to be involved in determining susceptibility to Salmonella (Gewirtz et al. 2001; Hayashi et al. 2001); however, the role of TLR5 in vivo has not been definitively established. Additional genes involved in disease susceptibility have been described (Lam-Yuk-Tseung and Gros 2003). CBA/N inbred mice, which have a known X-linked defect in humoral immunity characterized by impaired maturation of B cells, diminished immunoglobulin production, and generally compromised T-independent immune responses, have an increased susceptibility to S. typhimurium. This defect can be corrected by passive transfer of immune serum containing anti-Salmonella antibodies. The CBA/N defect maps to the X-linked immunodeficiency (xid) locus, which is the murine equivalent of Brunton’s tyrosine kinase (btk) that is mutated in human patients with X-linked agammaglobulinemia (XLA), although the immunodeficiency in xid mice is less profound than in patients with XLA.
F.
Clinical Disease
Perhaps surprisingly, there are relatively few reports of spontaneous disease due to Salmonella infection in mice (Foster et al. 1982). In fact, it has been suggested that subclinical
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Salmonella infection in mice is a more frequent outcome than clinically apparent disease. When present, clinical signs are generally nonspecific and include ruffled fur, hunched posture, reduced activity, and weight loss (Casebolt and Schoeb 1988). Diarrhea and conjunctivitis may also be observed. Lesions found at necropsy are quite variable. In animals that die acutely with septicemia, there may be no obvious lesions, although hyperemia and congestion of viscera, particularly the ileum, may be present. In subacute cases, the intestine may appear normal, or the ileum may be thickened, and the cecum may be empty or filled with fluid. Multiple white to yellow foci in the liver, splenomegaly, enlarged mesenteric lymph nodes, with or without a scant fibrinous peritoneal exudate, are often present (Fig. 15-1). Chronic carriers may not have gross lesions at all. In experimentally infected, germ-free mice, all of the animals died within 10 days of a progressive systemic infection involving the lower intestine, liver, spleen, lymph nodes, and lung (Foster et al. 1982). Ileocecitis, mesenteric lymphadenitis, and multifocal inflammation in the liver and spleen are the predominant microscopic lesions caused by Salmonella infection. Edema in the submucosa and lamina propria, with sloughing of enterocytes, and leukocytic infiltration may be seen in the terminal ileum and cecum. Multifocal necrosis and venous thrombosis with leukocytic infiltration occur in the mesenteric lymph nodes, liver, and spleen (Fig. 15-2). Hepatic lesions are often granulomatous in nature.
G.
Fig. 15-1 Salmonellosis in a mouse, featuring disseminated, miliary foci or “typhoid nodules” in the liver (Courtesy of Dr. Cynthia Besch-Williford, University of Missouri–Columbia).
Diagnosis
Salmonellosis is diagnosed by culture and isolation of the organism. Isolation of Salmonella from the liver, spleen, mesenteric lymph nodes, or blood during the septicemic stage of disease is relatively straightforward. More challenging is
Fig. 15-2 Microscopic appearance of a “typhoid nodule” in the liver of a mouse with salmonellosis demonstrating focal mixed mononuclear and polymorphonuclear inflammation with central necrosis (200×).
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15. PSEUDOMONAS AERUGINOSA, AND STREPTOBACILLUS MONILIFORMIS
the isolation of Salmonella from feces or intestinal tissue, which usually necessitates enrichment with selenite F broth and/or isolation on selective, differential media in order to avoid overgrowth by commensal enteric microbiota. Descriptions of some of the most commonly used selective, differential media are presented in Table 15-3. Detection of chronic carriers is even more problematic. This can be accomplished by enrichment culture of feces and plating on selective, differential media, but given the expected low rate of carriage and the possibility intermittent shedding, false negatives are a real concern. Suspected positive colonies should be tested on triple sugar iron agar, and confirmed serologically.
III.
CITROBACTER RODENTIUM A.
Introduction
Citrobacter rodentium, formerly designated C. freundii biotype 4280, is the causative agent of transmissible murine colonic hyperplasia (TMCH). Other terms used in the literature to describe this disease include hyperplastic colitis, catarrhal enterocolitis, and colitis cystica. Although most Citrobacter are opportunistic pathogens capable of causing extraintestinal infections in animals and humans, C. rodentium is unique in that it
is a primary pathogen of mice. In an early study characterizing C. rodentium infection in mice, it was the only Citrobacter of 20 isolates from diverse sources tested that caused TMCH in outbred NIH Swiss mice (Barthold et al. 1977).
B.
History
In 1969, Muto et al. described an explosive outbreak of diarrhea accompanied by high mortality in a closed colony of suckling DDY mice at the National Institute of Health in Tokyo, Japan. A novel gram-negative rod was isolated from infected animals (Nakagawa et al. 1969). The pathogen was classified as an atypical E. coli based on biochemical characteristics, and was designated mouse-pathogenic E. coli (MPEC). More recently, genetic and biochemical analyses have demonstrated that MPEC is actually a misclassified C. rodentium strain, and should be referred to as such (Luperchio et al. 2000). Other outbreaks of C. rodentium, in addition to that at the NIH in Japan, have been reported. Brennan et al. described an outbreak of diarrheal disease at the Argonne National Laboratory caused by an atypical strain of Citrobacter in 1965. Ediger et al. described an outbreak of increased mortality associated with rectal prolapse, but not diarrhea, in outbred Swiss Webster mice at the National Cancer Institute-Frederick Cancer Research Center in 1974. Oral administration of the
TABLE 15-3
PROPERTIES OF SOME COMMONLY USED SELECTIVE, DIFFERENTIAL MEDIA FOR THE ISOLATION OF ENTEROBACTERIACEAEa Medium
Carbon source
Indicators
Inhibitors
Bacteria inhibited
Principle of use
Selenite F broth MacConkey lactose agar
Lactose Lactose
None Neutral red
Sodium selenite Bile salts, crystal violet
Coliforms Gram-positives
Brilliant green agar
Lactose, sucrose
Phenol red
Brilliant green
Gram-positives, coliforms, Shigella
Hektoen-enteric (HE) agar
Lactose, sucrose, salicin
Ferric ammonium citrate, acid fuchsin, and bromthymol blue
Bile salts
Gram-positives
Xylose lysine desoxycholate (XLD) agar
Xylose, lactose, sucrose
Phenol red, ferric ammonium citrate
Desoxycholate
Gram-positives
Enrichment for Salmonella. Gram-negative bacteria that utilize lactose lower the pH of the medium, causing the neutral red indicator to turn red. Lactose nonfermenters are colorless and translucent. Salmonella (except S. typhi); colonies range from reddish or pink to nearly white with a red zone. Lactose- and sucrose-fermenting bacteria appear as yellow-green colonies surrounded by a zone of yellow-green in the agar. Rapid lactose fermenters are moderately inhibited and produce yellow-orange to salmon pink colonies. Salmonella produce blue-green colonies with black centers. Proteus are somewhat inhibited and produce glistening colonies. Salmonella produce red or red-green colonies with black centers. Xylose-, lactose-, and sucrosenonfermenters produce red colonies. Xylosefermenting lysine-decarboxylating bacteria appear as red colonies. Xylose-fermenting lysinenondecarboxylating bacteria appear as opaque yellow colonies. Lactose- or sucrose-fermenting bacteria produce yellow colonies.
aTaken
from Atlas and Parks 1997.
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Citrobacter organism isolated from affected animals to pathogenfree mice reproduced the clinical and histopathological features of the disease. In 1976, Barthold et al. reported on a Citrobacter strain isolated from an outbreak that had occurred in 1972. Experimental inoculation studies confirmed the virulence of the organism, and it was designated C. freundii biotype 4280 on the basis of a battery of biochemical tests. It was in this report that the term TMCH was first proposed to describe the disease caused by C. rodentium (Barthold et al. 1976). The organism was formally renamed in 1995, when it was shown by DNA-DNA hybridization that it was a distinct species, and not an atypical C. freundii (Schauer et al. 1995).
C.
Properties
Members of the genus Citrobacter are gram-negative, facultatively anaerobic, nonmotile rods that measure approximately 1 µm in width by 2–6 µm in length. Several phenotypic and biochemical characteristics can be used to distinguish C. rodentium from other Citrobacter species. C. rodentium is negative for indole production, growth in KCN, arginine dihydrolase activity, and motility, but positive for ornithine decarboxylase (Schauer et al. 1995). Also helpful in distinguishing C. rodentium from other species is the fact that it is negative for hydrogen sulfide production and fermentation of sucrose, dulcitol, melibiose, and glycerol. Although most Citrobacter species utilize citrate as a sole carbon source, C. rodentium is negative or weakly positive for citrate utilization. It does, however, utilize malonate. C. rodentium, along with enteropathogenic E. coli (EPEC), some STEC, and a limited number of Escherichia strains
that were originally misidentified as Hafnia alvei, comprise a group of attaching and effacing (AE) pathogens. These diarrheagenic pathogens share a common ultrastructural feature of infection, namely the AE lesion (Fig. 15-3), which is characterized by dissolution of the apical brush border, cupping or pedestal formation by the plasma membrane, and cytoskeletal rearrangements in the underlying cytoplasm at the site of bacterial attachment to enterocytes (Johnson and Barthold 1979; Schauer and Falkow 1993). All of the bacterial genes that are required for AE lesion formation are contained in a PAI called the locus of enterocyte effacement (LEE). The LEE encodes a T3SS that injects bacterial effector proteins into the host cell, including a bacterially derived receptor for the outer membrane protein intimin called the translocated intimin receptor (Tir) (Deng et al. 2004; Luperchio and Schauer 2001). Intimin-Tir binding is sufficient to trigger focused actin rearrangement that forms pedestals or AE lesions. In addition to the conserved LEE-encoded effector proteins, Deng et al. (2004) have identified additional effector proteins that are encoded by genes outside the LEE. Interestingly, some of these putative effectors are also located in PAI, at least in STEC, for which the complete genome sequence has been determined. At the time of this writing, the C. rodentium genome is in the process of being sequenced and annotated (http://www.sanger.ac.uk/ Projects/C_rodentium/).
D.
Pathogenesis
C. rodentium forms AE lesions in the distal colon beginning 4 days after oral inoculation (Johnson and Barthold 1979).
Fig. 15-3 Transmission electron micrograph of Citrobacter rodentium bacteria (arrows) and attaching and effacing lesions in the descending colon of a mouse. Note microvillus effacement, destruction of the terminal web, and raised cups or pedestals of the apical cytoplasmic membrane of the colonic enterocyte.
15. PSEUDOMONAS AERUGINOSA, AND STREPTOBACILLUS MONILIFORMIS
Bacterial infection induces a T helper 1 (Th1) type immune response characterized by increased IL-12, IFN-γ, and TNF-α (Higgins et al. 1999). The Th1 response appears to be important in the clearance of infection, because experimental infection of knockout mice deficient in IL-12p40 or IFN-γ with C. rodentium resulted in increased bacterial loads, delayed bacterial clearance, and more severe colitis compared to wild-type mice (Simmons et al. 2002). TNF-α receptor (TNFRp55)–deficient mice also have higher bacterial loads and increased severity of colitis compared to wild-type mice following experimental infection with C. rodentium (Goncalves et al. 2001). Other studies have characterized the nature of the adaptive immune response in resistant strains of mice that leads to clearance of C. rodentium 3–4 weeks post-inoculation. Mice deficient in recombinase activating gene 1 (Rag1) that lack functional B and T cells fail to clear C. rodentium (Simmons et al. 2003; Vallance et al. 2002). Perhaps surprisingly, clearance of C. rodentium infection requires T cell–dependent humoral immunity, but not secretory IgA or IgM (Bry and Brenner 2004; Maaser et al. 2004). Passive transfer of immune sera is sufficient to provide protection against C. rodentium infection in CD4+ T cell deficient mice, indicating that serum Ig reaches the colonic mucosa, presumably through breaks in the epithelium that arise during the acute phase of infection (Bry and Brenner 2004). The mechanism by which C. rodentium causes epithelial cell hyperproliferation is not fully understood. The colonic hyperplasia that results from C. rodentium infection was found to promote the development of colonic adenomas after administration of the carcinogen 1, 2 dimethylhydrazine (DMH) (Barthold and Jonas 1977). Mice experimentally infected with C. rodentium and treated with DMH have a reduced latency for tumor development, compared to uninfected mice treated with DMH (1 month versus 3 months). C. rodentium infection also promotes colon tumorigenesis in Apc+/Min (Min) mice, which have a germ line mutation in one allele of the Apc tumor suppressor gene (Newman et al. 2001). A number of factors, including age, strain background, and diet, have been shown to influence the severity of lesions and clinical outcome of infection with C. rodentium. Colitis and mortality are typically greater in older suckling and weanling mice than in adults. In strain backgrounds where C. rodentium infection causes colonic hyperplasia with little inflammation, infected adults may exhibit only mild clinical signs of disease or may be subclinically infected. Mortality is considerably higher in susceptible strains, such as C3H/HeJ, which tend to develop more severe and extensive colitis than DBA/2J, BALB/c, or C57BL/6J, or outbred stocks such as NIH Swiss and Swiss Webster (Barthold and Jonas 1977; Vallance et al. 2003). The genetic loci controlling susceptibility to C. rodentium are not known, but TLR4 does not appear to be involved, because C3H/HeN and C3H/OuJ mice that express wild-type TLR4 had mortality rates that were comparable to the LPS nonresponder C3H/HeJ strain (Vallance et al. 2003). Although a
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comprehensive survey of the susceptibility of inbred strains to C. rodentium has not been reported, it appears that FVB and AKR mice develop high mortality as is seen with the various C3H substrains (unpublished observation, D. B. Schauer).
E.
Clinical Disease
Clinical signs of disease can be nonspecific and include ruffled fur, listlessness, weight loss, runting, and diarrhea. Rectal prolapse is common (Fig. 15-4). Signs are most pronounced during the second and third week of infection. Mice that recover from infection are resistant to reinfection, but protection appears to eventually wane (Barthold 1980). At necropsy, gross lesions are most commonly observed in the descending colon, which appears thickened and rigid and is often devoid of feces. Lesions may extend to the transverse and ascending colon and the cecum. Detailed descriptions of gross and microscopic lesions and ultrastructural changes that occur during the course of experimental C. rodentium infection in suckling and adult NIH Swiss mice have been reported by Barthold et al. (1978) and Johnson and Barthold (1979). Colonic thickening was initially observed approximately 1 week post-inoculation and peaked 2–3 weeks post-inoculation. No additional colonic thickening was observed after 4 weeks in adult mice, but the change persisted in suckling mice for at least 8 weeks. Hyperplasia was first detected microscopically in the descending colon 4 days after experimental infection and increased in severity up to the third week. The most severe lesions, observed between weeks 2–3, included over a 4-fold increase in the number of cells in mucosal crypts, absence of goblet cells, focally dysplastic surface epithelium, and
Fig. 15-4 Rectal prolapse in laboratory mice. Rectal prolapse is common in laboratory mice with colitis due to specific and nonspecific causes. Examples include Citrobacter rodentium infection, and enterohepatic Helicobacter species infection (see Chapter 17, this volume), particularly in genetically engineered mice or other mutant mice with immunodeficiency or altered immune functions.
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A
B
Fig. 15-5 Influence of strain or stock background on disease outcome in adult mice experimentally infected with Citrobacter rodentium. Transmissible murine colonic hyperplasia in outbred Swiss Webster mice (A) features gland elongation, goblet cell depletion, and limited inflammation. In contrast, C57BL/6 mice (B) develop a mixed necrotizing and proliferative colitis with less gland elongation and more inflammation. Both micrographs 100× (Courtesy of Dr. Arlin Rogers, Massachusetts Institute of Technology).
increased mitotic activity along the entire crypt column and surface mucosa (Fig. 15-5). Cecal mucosal hyperplasia occurred less frequently, and was more common in older mice. Inflammation was not consistently observed and did not correlate with the severity of hyperplasia. Suckling mice had more severe inflammation, associated with areas of mucosal necrosis and erosion, than adult mice. PMNs infiltrated the lamina propria and submucosa early in the infection. After 2–3 weeks, mononuclear cells were the predominant infiltrating cell type, and lymphocytes and plasma cells predominated during disease regression. Regression was complete in adult mice by 42 days post-inoculation, while some suckling mice still had hyperplastic lesions on day 56.
F.
Epizootiology
C. rodentium is probably globally distributed in laboratory mice. It is not a normal inhabitant of the mouse intestine. Transmission is presumed to be fecal-oral, either from direct contact with infected animals or from exposure to contaminated food or bedding. Some reports indicate that the type strain of C. rodentium (ATCC 51116T) was isolated from the ileum of a hamster. However, it seems likely that the records regarding the source of this strain are inaccurate and the strain was actually isolated from a mouse. In addition, oral inoculation of Syrian hamsters and inbred F344 rats with C. rodentium failed to result in intestinal infection (Barthold et al. 1977). Thus, C. rodentium appears to be extremely host adapted to the laboratory mouse. There is a single report of an outbreak of
C. rodentium in a colony of Mongolian gerbils that was associated with high mortality (de la Puente-Redondo et al. 1999).
G.
Diagnosis
Definitive diagnosis is made by culture and isolation of C. rodentium from the feces or the colon of affected animals. Since C. rodentium is cleared prior to the resolution of lesions, samples should be taken from multiple animals, preferably early in the course of infection. Plating on MacConkey lactose agar allows for presumptive identification of C. rodentium colonies based on their characteristic “fisheye” appearance. Because C. rodentium is a delayed lactose fermenter, these colonies will have a red center surrounded by a thin white or colorless margin. Over time, however, the entire colony will become red. Additional biochemical tests should be performed to confirm the identity of suspected colonies of C. rodentium. Serotype analysis may also have some utility, since C. rodentium LPS cross-reacts with E. coli O173 antisera. However, the type strain of C. rodentium appears to be rough, lacking an O-polysaccharide component of its LPS. A C. rodentium-specific PCR assay based on a LEE-encoded effector protein gene has also been reported (McKeel et al. 2002). PCR is perhaps slightly more sensitive than culture and isolation, but it too is limited by the fact that the organism is typically only present early in infection. In practice, routine diagnosis is made by demonstration of typical lesions in the colon of affected animals. Because lesions may be restricted to the most distal portion of the descending colon, appropriate samples should be collected at necropsy if a diagnosis of TMCH is suspected.
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H.
Treatment and Control
C. rodentium rarely occurs in barrier facilities where good husbandry and biocontainment practices are employed. Outbreaks continue to be a concern in conventional mouse facilities. It has been suggested that routine surveillance by fecal culture is likely to be unrewarding. Detection is probably more effectively accomplished by routine histopathologic evaluation of mice exhibiting morbidity combined with an aggressive surveillance program for other agents. During an outbreak, antimicrobial therapy, such as neomycin or tetracycline in the drinking water, may reduce mortality. Enrofloxacin added to acidified drinking water at a final concentration of 170 mg/L for 14 days was used to treat T cell receptor transgenic mice during an outbreak (Maggio-Price et al. 1998). The majority of the mice remained negative for C. rodentium, but treatment failures are possible and this method should probably be reserved for cases where rederivation by embryo transfer or hysterectomy or repopulation with clean stock is not practical.
IV.
ESCHERICHIA COLI A.
Introduction
Commensal E. coli strains are common inhabitants of the gastrointestinal tract of mice and are not typically pathogenic. On the other hand, there is a single report of an atypical E. coli strain causing colonic hyperplasia in immunodeficient mice (Waggie et al. 1988).
including defective bactericidal activity of granulocytes, severe deficiency of natural killer (NK) cells, and defective cytotoxic T cell responses. Other immunodeficient and immunocompetent stocks were also infected with the lactose nonfermenting E. coli strain, but only immunodeficient mice developed gross or microscopic lesions. C.
Clinical Disease and Pathogenesis
Affected N:NIH(S) III mice were listless and had diarrhea, along with a 5%–8% mortality rate among 7- to 8-week-old animals (Waggie et al. 1988). Other mice housed in the same room, including wild-type N:NIH(S) mice and mice homozygous or heterozygous for the nu mutation or the xid and/or bg mutations, were subclinically infected. At necropsy, the colon and occasionally the cecum were found to be thickened, and in some cases, the contents of the ileum and cecum were blood-tinged. Microscopically, there was segmental mucosal hyperplasia of the cecum and colon, with loss of goblet cells, and occasional mild PMN cell infiltrate in the lamina propria and submucosa. Clusters of bacteria were observed attached to the surface and within the cytoplasm of mucosal epithelial cells in the large and small intestine. C57BL/6N nu/nu mice experimentally inoculated with the lactose nonfermenting E. coli did not develop clinical signs of disease or gross lesions. All of the experimentally inoculated mice became infected, and some had multifocal areas of intracellular bacteria in the colon associated with epithelial hyperplasia. A similar condition has been observed in severe combined immunodeficient (scid) mice, in association with a lactose nonfermenting E. coli (personal communication, D. B. Schauer). D.
B.
Properties
In many animal host species and in humans, E. coli strains can cause a wide range of diseases, including diarrhea, dysentery, urinary tract infection, septicemia, pneumonia, and meningitis. In general, distinct strains are associated with each of these conditions, because acquisition of specific virulence determinants permits exploitation of a particular niche. A major diagnostic challenge, particularly for diarrhea-causing E. coli, is discrimination between pathogenic and commensal strains. Where direct detection of virulence determinants is not convenient or not possible, surrogate markers for virulence, such as serotype or biotype, have been used. Enteroinvasive E. coli (EIEC) are often less biochemically active than other E. coli strains. Waggie et al. (1988) reported on a lactose nonfermenting E. coli strain that was associated with colonic hyperplasia in outbred NIH Swiss triple immunodeficient (N:NIH(S) III) mice. These mice are homozygous for nude (nu), xid, and beige (bg) mutations; therefore, they lack mature T cells, have functional B cell defects, and have a variety of leukocyte abnormalities,
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Diagnosis
Observation of segmental hyperplastic lesions in the large intestine of immunodeficient mice along with isolation of lactose nonfermenting E. coli should be sufficient to establish a diagnosis. Preliminary studies suggest that the lactose nonfermenting E. coli are Congo red positive and possess ipaH loci, a multicopy family of genes encoding antigens, both features common to EIEC and Shigella (unpublished observations, D. B. Schauer). The true significance of this pathogen in immunodeficient mice remains to be determined. It does not appear to be clinically significant in immunocompetent mice.
V.
PROTEUS MIRABILIS
A.
Introduction and History
The genus Proteus was first described in 1885 by the German microbiologist Gustav Hauser. The bacterium has the ability to
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undergo dramatic morphological changes when grown on solid media versus in liquid, and was named after the Greek god of the sea Proteus, who was able to change form at will. A more complete description of the history, taxonomy, and clinical significance of the genus can be found in the review by O’Hara et al. (2000). The genus Proteus contains 5 named species—Proteus vulgaris, which is the type species, Proteus mirabilis, Proteus penneri, Proteus myxofaciens, and Proteus hauseri—as well as 3 unnamed species (O’Hara et al. 2000). These organisms are ubiquitous in nature and are commonly recovered from contaminated water, sewage, and soil. They are also found in the upper respiratory tract and feces of many animal species, including mice, and may be recovered from normal animals. P. mirabilis is the species most frequently recovered from mice. Proteus are opportunistic pathogens that are an important cause of urinary tract infections in humans, particularly in individuals with indwelling catheters or structural abnormalities of the urinary tract (Coker et al. 2000). Proteus have also been reported to cause significant morbidity and mortality in both immunocompetent and immunodeficient mice.
B.
Properties
Among the Enterobacteriaceae, the genus Proteus is most closely related to Providencia and Morganella, but can be differentiated from them by its lack of D-mannose fermentation, and by the fact that it is positive for gelatin liquifaction at 22°C, hydrogen sulfide production, and lipase activity (O’Hara et al. 2000). Proteus are motile by peritrichous flagella, which are overproduced when the organism differentiates from the infectious single-cell form (swimmer cell) to a multicell elongated form (swarmer cell). Proteus also possess a number of other virulence factors, including fimbrial adhesins, urease, hemolysin, an amino acid deaminase, and an IgA-degrading protease. These virulence determinants and their role in Proteus pathogenesis have been reviewed by Coker et al. (2000).
C.
Clinical Disease and Pathogenesis
There are relatively few reports of spontaneous P. mirabilis infection in mice. Taylor found P. mirabilis to be the predominant cause of acute pyelonephritis in male MM mice (Taylor 1988; Taylor et al. 1988). This strain of mouse is prone to develop diabetes mellitus, which apparently allows P. mirabilis, present as a latent opportunist, to cause pyelonephritis (Taylor et al. 1987). There are also two reports of P. mirabilis causing spontaneous nephritis in C3H/HeJ mice (Jones et al. 1972; Maronpot and Peterson 1981). Jones et al. found P. mirabilis– associated suppurative nephritis in 26 of 58 animals that died over a 28-month period. Maronpot et al. found the overall
incidence of P. mirabilis nephritis to be 1.2% among 2,836 control or treated (dermal carcinogenesis bioassays) male mice. The incidence was similar in control and treated mice, ranging from 2.5% to 32.5% among 73 different treatment or control groups. Clinical signs in mice with pyelonephritis are nonspecific, including listlessness and urine staining of the abdominal fur (Taylor et al. 1988). Grossly, focal discrete or coalescing, raised, tan areas are generally present on the surface and cut cortical sections of the kidney (Fig.15-6) and occasionally extend into the renal medulla (Maronpot and Peterson 1981). Histological lesions are most severe in the cortex and consist of suppurative nephritis with associated bacterial organisms (Maronpot and Peterson 1981). There is often a necrotizing papillitis and purulent pyelitis. Leukocytic infiltrates present in peri-ureteric and renal fat and in the ureter lumen consist of lymphoid and plasma cells (Taylor et al. 1988). The distribution of histologic lesions suggests a hematogenous route of dissemination (Jones et al. 1972; Taylor et al. 1988). In addition to pyelonephritis, suppurative cystitis, occasionally accompanied by submucosal edema, necrosis, and epithelial sloughing, has also been associated with P. mirabilis in mice. P. mirabilis has also been reported to cause systemic disease in scid and compound scid bg mutant mice (Percy and Barta 1993). Disease was most severe in adult females during the prenatal period. Clinical signs were nonspecific, consisting of wasting, ruffled hair coat, dehydration, and listlessness. At necropsy, animals tended to have decreased body fat, splenomegaly, and well-delineated, multifocal, irregular, pale tan areas on the liver. Acute fibrinopurulent peritonitis was observed in some cases. Microscopic lesions include interstitial pneumonia, fibrinopurulent peritonitis, focal meningitis, multifocal hepatic necrosis, and thrombophlebitis. Histological findings in the liver included multifocal areas of coagulation necrosis, in subcapsular regions and around central veins with infiltrating PMNs, and thrombi in many affected vessels. Proteus have also been sporadically isolated from the lungs or urogenital tract of mice. P. mirabilis was an infrequent opportunist isolated from the lungs of B6.129S6-Cybbtm1Din/J mice (Bingel 2002), which have a mutation in the gp91 subunit of the NADPH oxidase, resulting in compromised function of phagocytic cells. These mice are used as a model of chronic granulomatous disease. There is an early report of P. mirabilis being isolated from the genital tract of male C57BL/6N breeder mice following self-mutilation of the penis (Hong and Ediger 1978). Although P. mirabilis is the species most commonly reported in mouse infections, P. vulgaris has been isolated from utero-ovarian lesions of B6C3F1 breeding female mice (Davis et al. 1987).
D.
Epizootiology
P. mirabilis is an opportunistic pathogen of humans and laboratory animals. It has been reported that mice are highly
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A
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B
Fig. 15-6 Gross view of the capsular (A) and cut surface (B) of the kidney of a mouse with suppurative pyelonephritis. There is marked dilation of the renal pelvis and ureter with suppurative exudate that segmentally extends to the renal medulla, cortex, and capsular surface.
susceptible to experimental infection with the organism (Maronpot and Peterson 1981). Its ability to cause disease in immunocompromised mice is well established, and P. mirabilis should be considered when nonspecific signs of unthriftiness are observed. Disease has been reproduced in scid mice inoculated by oral gavage, indicating that acquisition of infection from contact with subclinical carriers or contaminated food or bedding can lead to morbidity and mortality (Percy and Barta 1993).
E.
F.
Treatment and Control
P. mirabilis, like other opportunists, is best controlled by strict adherence to good husbandry and sanitation procedures. In conventional facilities, decreasing the population density of animals per cage may reduce disease incidence. Antibiotic therapy is not generally used to treat P. mirabilis in mice. However, in one report, kanamycin sulfate was found to be effective for P. mirabilis, Morganella morganii, and Klebsiella sp. (Tsai et al. 1969). Penicillin G, streptomycin sulfate, and ampicillin were not effective in treating the mice in that study.
Diagnosis
P. mirabilis can be cultured from the feces of normal mice and perhaps from the respiratory tract. Isolation of the organism from other sites implicates the organism as a cause of disease. The organism grows readily on most solid media. It can be presumptively identified by its swarming growth on blood and chocolate agar; however, other swarming bacteria do exist, and confirmatory tests are needed for a definitive diagnosis. Proteus do not ferment lactose; thus, they produce white or colorless growth on MacConkey lactose agar. Because Proteus produce hydrogen sulfide, resulting in black colonies on HE and XLD agar, they must be differentiated from Salmonella.
VI. KLEBSIELLA A.
Introduction and History
Klebsiella are normal inhabitants of the gastrointestinal tract of humans and animals and are ubiquitous in water, sewage, and soil, as well as in agricultural and forest products. They are opportunistic pathogens that can cause urinary tract infections, pneumonia, meningitis, and/or septicemia in primates, including humans, and are a particularly important
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cause of nosocomial infection in immunocompromised patients. Nosocomial Klebsiella infections are caused mainly by Klebsiella pneumoniae, the medically most important species of the genus. To a much lesser degree, K. oxytoca has been isolated from human clinical specimens. Klebsiella species are rarely associated with disease in mice and are not considered to be significant pathogens.
B.
were positive for Mycoplasma arthritidis by ELISA, although no mycoplasma could be isolated. Aged female mice had suppurative endometritis, salpingitis, and perioophoritis and/or peritonitis. Experimental infection of mice with K. oxytoca by the intrauterine or intraperitoneal route produced only mild endometritis in a few cases, suggesting that additional factors may be involved in severe, life-limiting manifestations of disease.
Properties D.
Klebsiella are gram-negative, nonmotile, usually encapsulated bacilli, 0.3–1 µm in diameter by 0.6–6 µm in length. Different systems of taxonomy are in use, but Ørskov’s classification, which currently appears to be the most popular, features 5 species: K. pneumoniae, K. oxytoca, Klebsiella terrigena, Klebsiella planticola, and Klebsiella ornithinolytica. K. pneumoniae is further subdivided into 3 subspecies: subsp. pneumoniae, subsp. ozaenae, and subsp. rhinoscleromatis. Only a few virulence determinants of K. pneumoniae have been identified, including capsular polysaccharide, fimbrial adhesins, and siderophores. Capsules are essential for Klebsiella virulence. The capsular material forms thick bundles of fibrillous structures that cover the entire bacterial surface. This protects the organism from phagocytosis by professional phagocytes and confers resistance to bactericidal serum factors, probably by preventing the activation of complement components (reviewed by Podschun and Ullmann 1998). The capsular polysaccharide also provides a basis for the most widely used system of serotyping. There are 77 types in the standard serotyping scheme. K. pneumoniae K1 and K2 have been reported to be highly virulent for mice (Holt et al. 1994).
C.
Epizootiology
Klebsiella are ubiquitous in nature, and can be frequently isolated from surface water, sewage, soil, and plants. They are also frequently isolated from the feces of domestic and wild animal species and from humans.
E.
Diagnosis
Diagnosis is by culture and isolation of the organism. Klebsiella colonies typically have a mucoid appearance on solid media, due to their abundant capsular polysaccharide. Because they can be recovered from normal animals, isolation from the gastrointestinal tract, feces, or in low numbers from mucosal surfaces is not sufficient to demonstrate an association with disease. Klebsiella are typically identified and differentiated on the basis of biochemical reactions. They decarboxylate lysine but not ornithine, and are typically positive in the Voges-Proskauer test. When it is necessary to type individual strains for epidemiological purposes, K antigen serotyping or biotyping can be used.
Clinical Disease
Spontaneous disease in mice with K. pneumoniae and with K. oxytoca has been reported; however, neither organism appears to be a particularly important murine pathogen. There are two reports of infection with an untyped K. pneumoniae (Flamm 1957) and a K6 isolate (Schneemilch 1976). In these outbreaks, mice exhibited inappetance, hunched posture, rough hair coats, sneezing, and hyperpnea. Necropsy revealed enlargement of cervical lymph nodes, cervical abscesses, renal and hepatic abscesses, empyema, and pneumonia. In some mice, there was leukocytic infiltration and thrombosis in the ventricular endocardium and myocardium. Disease could be reproduced experimentally by intraperitoneal inoculation (Flamm 1957) or inoculation into the buccal mucosa (Schneemilch 1976). K. oxytoca has been reported to be associated with suppurative female reproductive tract lesions in a large population of aging B6C3F1 mice (Davis et al. 1987). The mice were also positive for K. pneumoniae, E. coli, and Enterobacter, and
VII.
PSEUDOMONAS AERUGINOSA A.
Introduction
Pseudomonas aeruginosa, the type species of the genus, is widespread in nature and particularly abundant in soil and water, and frequently can be isolated from a variety of clinical specimens. However, it only rarely causes disease in animals and humans and is classified as an opportunistic pathogen. It is not part of the indigenous microbiota of mice, but is commonly isolated from the oropharynx and feces, particularly in conventionally housed animals. Although it is only a sporadic cause of naturally occurring infections in mice, experimental inoculation of laboratory mice with P. aeruginosa is frequently used to model its role in human infections, including ulcerative keratitis and bacteremia in patients with severe burns (Markley and Smallman 1968; Stieritz and Holder 1975; Walker et al. 1964). Notably, P. aeruginosa is the
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most important pathogen in the cystic fibrosis (CF) lung, infecting approximately 60% of all patients and close to 80% of adolescents and adults with the condition (Lyczak et al. 2002). As a result, several mouse models have been developed to investigate the role of P. aeruginosa in CF, including transgenic lines and scid mouse-human chimeras (Davis et al. 1987; Schroeder et al. 2001; Starke et al. 1987; van Heeckeren and Schluchter 2002).
B.
History
P. aeruginosa was first isolated from blue-green pus in superficial wounds in 1882, leading to the original name of Bacillus (rod) pyo (pus) cyaneus (blue) (Villavicencio 1998). It was implicated in 2% of battlefield infections in World War II and the Korean War. During the Vietnam War, it was one of the three most common isolates from wounds. It remains an important opportunistic pathogen and cause of nosocomial infections today. Early interest in P. aeruginosa as a murine pathogen was stimulated by investigations of acute radiation injury (Hammond, Colling, et al. 1954; Hammond, Tompkins, et al. 1954; Hammond et al. 1955).
C.
Properties
In contrast to the organisms discussed so far in this chapter, P. aeruginosa is not a member of the family Enterobacteriaceae. It is a member of the family Pseudomonadaceae, a large and important group of gram-negative bacteria. P. aeruginosa is nonfermentative (it does not ferment glucose), motile by monotrichous flagella, non-spore-forming, and rod shaped (measuring 1.5 to 3.0 µm in length and 0.5 to 0.7 µ in diameter). It is oxidase positive, may produce a zone of β-hemolysis on blood agar, and produces a characteristic fruity odor. P. aeruginosa is capable of growth on simple minimal media by using a broad variety of low molecular weight organic compounds, and it will grow under aerobic or anaerobic conditions. When cultured on selective media such as King’s Medium B or Rosenthal’s Medium, many, but not all, P. aeruginosa strains produce characteristic fluorescent pigments as well as a blue pigment called pyocyanin, which is very important in species identification. As far as is known, this blue pigment of the phenazine family is absolutely diagnostic, since no other bacterial species has been found to produce it (Palleroni 1992). The fluorescent pigments fluoresce at a short wavelength of ultraviolet light (254 nm), in contrast to so-called fluorescent organisms belonging to other groups, which produce pigment fluorescing only at long-wavelength of ultraviolet (around 350 nm). P. aeruginosa is resistant to many antibiotics, which is due, at least in part, to multiple active efflux pumps (Poole 2000; Poole and Srikumar 2001; Schweizer 2003). Genomic analysis
381
demonstrates 6 functional, and an additional 6 potential, efflux systems in P. aeruginosa (Schweizer 2003). Another important factor in antibiotic resistance is the ability of P. aeruginosa to produce biofilms, in which exopolysaccharide-encased microcolonies on solid surfaces are more resistant than organisms in the planktonic state (reviewed by Mah and O’Toole 2001). A recent report indicates that production of periplasmic glucans by P. aeruginosa biofilms may play a role in antibiotic resistance by binding to and sequestering antibiotics (Mah et al. 2003). Biofilms can form on medical implants and on water sources such as taps and sipper tubes, serving as a continual source of infection in animal and laboratory facilities. P. aeruginosa strains produce a number of virulence factors that determine pathogenicity. Surface factors such as flagella and type IV pili are involved in dissemination throughout the host (Sato et al. 1988) and in biofilm production (O’Toole and Kolter 1998; Stickler 1999). Alginate, the polysaccharide encasing P. aeruginosa biofilms, is also important for bacterial adherence to host cells and evasion of phagocytosis (Lyczak et al. 2000; Meluleni et al. 1995). Pyocyanin is critical for lung infection in mice (Lau et al. 2004) and has multiple effects, including inhibition of ciliary function (Wilson et al. 1987), disruption of calcium homeostasis (Denning et al. 1998), and induction of neutrophil apoptosis (Usher et al. 2002). Several extracellular products (proteases, elastase) play a role in invasion and dissemination of P. aeruginosa. Most isolates produce exotoxin A (ExoA), which is induced under iron-limiting conditions that characterize many animal tissues. The target of ExoA is the same as that of diphtheria toxin, an elongation factor required for host cell protein synthesis. Strains incapable of producing ExoA are avirulent (Liu 1979; Nicas and Iglewski 1986).
D.
Pathogenesis
Disease due to P. aeruginosa generally occurs in animals that are immunocompromised, such as following irradiation (Gordon et al. 1955; Hammond et al. 1955; Miller et al. 1952), cyclophosphamide or cortisone administration (Nugent et al. 1984; Pierson et al. 1976; Urano and Maejima 1978), or as a consequence of burn stress (Pierson et al. 1976). Infection with viruses such as CMV that alter host immune responses may also enhance susceptibility (Hamilton and Overall 1978). Spontaneous disease has been described in immunodeficient scid mice co-infected with Enterococcus durans (Dietrich et al. 1996). Vestibular signs including circling and rolling due to P. aeruginosa infection have been described in outbred Swiss Webster (Ediger et al. 1971) and inbred C3H (Kohn and MacKenzie 1980) mice. Disease outbreaks in these instances were attributed to stress-induced immunosuppression following shipping. Antibiotic treatment can also lead to an increase in the number of P. aeruginosa organisms in mice, as evidenced by increased fecal shedding (Hentges et al. 1985;
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Hoag et al. 1965; Urano and Maejima 1978). As a result of immunosuppression, P. aeruginosa breaches mucosal surfaces, enters lymphatics and/or the circulatory systems, and produces septicemia within 3–14 days. Although natural infections generally occur in mice with neutropenia or lymphopenia, experimental methods have been developed to induce corneal (Fleiszig et al. 1994) or pulmonary (Starke et al. 1987) P. aeruginosa infections in immunocompetent mice. Results from these models underscore the complexity of disease pathogenesis and immune modulation of disease. For example, experimental infection studies demonstrate that certain mouse strains, including C57BL/6 and DBA/2, develop more severe pulmonary (Morissette et al. 1995; Stevenson et al. 1995; Tam et al. 1999) or corneal (Hazlett et al. 2000) lesions than do other strains, such as BALB/c. Milder lesions are associated with the recovery of fewer organisms from BALB/c mice than from C57BL/6 mice in both disease models. While this may appear to suggest that proinflammatory Th1 responses, typical of C57BL/6 mice, promote tissue damage and inhibit bacterial clearance while Th2 responses, typical of BALB/c mice, resolve the infection, the actual mechanism of disease progression is proving to be more complicated. Although a complete review of these studies is beyond the scope of this chapter, it appears that chemokines such as macrophage inflammatory protein-2 (MIP-2) and proinflammatory cytokines such as IFN-γ are required for bacterial elimination, but if their production persists, extensive damage can result. For a thorough review of the role of various immune mediators in P. aeruginosa-mediated keratitis see Hazlett 2004.
E.
Clinical Signs and Lesions
Due to the rapid course of bacteremia, often the only clinical signs observed in animals with P. aeruginosa disease are listlessness, anorexia, and death. Mice that were treated with cyclophosphamide in their drinking water developed bilateral conjunctivitis, serosanguinous nasal discharge, and generalized edema of the head, with death occurring within 4–5 days following treatment (Brownstein 1978). Vestibular signs including circling and rolling due to P. aeruginosa infection have been described in outbred Swiss Webster (Ediger et al. 1971) and inbred C3H (Kohn and MacKenzie 1980) mice. At necropsy, ulceration and epithelial necrosis may be observed at the site of bacterial entry, in the upper respiratory tract, and in gingival tissues (Percy and Barthold 2001). Focal hemorrhage and necrosis has been observed in multiple organs of mice with septicemia (Brownstein 1978). C3H mice with vestibular signs had purulent otitis media extending to the inner ear and meninges (Kohn and MacKenzie 1980). In Swiss Webster mice, lesions within the inner ear were limited to the cochlea and vestibular apparatus and consisted of chronic proliferation and inflammation (Olson and Ediger 1972).
Variable lesions included dissolution of the bone surrounding the inner ear and abscesses in the cerebellum and cerebrum.
F.
Epizootiology
P. aeruginosa has been isolated from a wide variety of sources, including soil, fresh or sea water, sewage, many types of clinical specimens and materials commonly handled in clinical laboratories, including distilled water, solutions, and even antiseptics, assorted foods and food industry wastes, flowers, fruits, and vegetables, as well as subclinically infected animals. Disease is generally latent in immunocompetent animals, but fatal septicemia may occur in immunocompromised individuals. The most common source of exposure of mice to P. aeruginosa is thought to be contaminated water (Beck 1963; Homberger et al. 1993).
G.
Diagnosis
A presumptive diagnosis of P. aeruginosa can be made based on clinical signs following a recent history of immunosuppression. Definitive diagnosis is based on culture and isolation of the organism, which can usually be accomplished from the blood, spleen, intestine, or lymph nodes of animals with septicemia. P. aeruginosa colonies are flat, grayish, and have irregular edges. Over time, they tend to spread on the surface of the agar. The cultures have a characteristic fruity odor and a metallic sheen; however, pigment production is not a reliable phenotypic characteristic (Palleroni 1992). An important test for distinguishing P. aeruginosa from members of the Enterobacteriaceae family is oxidase. P. aeruginosa and most other nonfermentative, gram-negative bacilli are oxidase positive, while all of the Enterobacteriaceae are oxidase negative.
H.
Control
Because of its ubiquitous occurrence, it is difficult to maintain mice free of P. aeruginosa unless they are maintained under gnotobiotic conditions. Decontamination of drinking water can reduce colonization of the nasopharynx and intestines of mice, but will not eliminate an established infection. To limit exposure of immunosuppressed or immunodeficient mice to P. aeruginosa, or during outbreaks, a number of techniques for decontamination of drinking water have been used, including reverse osmosis, deionization, microfiltration, reverse osmosis-deionization-ultrafiltration, autoclaving, hyperchlorination, and acidification. Chlorination (6–8 ppm recommended by Homberger et al. 1993, with ranges of 0.5–20 ppm reported in the literature) or acidification (pH 2.5 with hydrochloric acid) are probably the most straightforward methods. A further consideration for control measures is the
15. PSEUDOMONAS AERUGINOSA, AND STREPTOBACILLUS MONILIFORMIS
fact that transmission of P. aeruginosa from animal caretakers to mice, but not from animals to humans, has been documented (van der Waaij et al. 1963).
VIII.
STREPTOBACILLUS MONILIFORMIS A.
Introduction
Streptobacillus moniliformis is currently the sole species in the genus. Taxonomically, S. moniliformis has an uncertain affiliation, but has been suggested to be similar to some Mycoplasmatales (see Chapter 18, this volume), based on its low G+C DNA content (24%–26%), serum or blood requirements for growth, cholesterol incorporation into its cell membrane, and the occurrence of L-form variants (Greenwood and Harvey 1992). The International Committee on Systematics of Prokaryotes’ Subcommittee on the Taxonomy of GramNegative Anaerobic Rods is scheduled to consider S. moniliformis at its 2005 meeting, which may help clarify the organism’s standing. S. moniliformis is the cause of rat-bite fever and Haverhill fever in humans. It causes a septicemic disease in mice. The organism is a commensal of the upper respiratory tract of wild rats and occasionally laboratory rats. While it remains important as a zoonotic agent of human disease, S. moniliformis is no longer common in laboratory rats and, consequently, disease in laboratory mice has also become rare.
B.
History
The natural host of S. moniliformis is the rat, which usually harbors the infection without clinical signs. Before the widespread use of cesarean derivation and barrier maintenance of laboratory rodents, S. moniliformis was a common commensal of laboratory rats. In humans, two diseases are included under the general term of rat-bite fever: streptobacillary fever, caused by S. moniliformis, which is more common in the United States, and spirillary fever or Sodoku, caused by Spirillum minor, which is more common in Asia. Streptobacillary fever usually develops within 10 days of a rat bite that heals normally. There is often an abrupt onset of fever with chills, headache, and myalgia, followed by a macropapular palmar and plantar rash. One or more joints usually then become swollen, red, and painful. If untreated, bacterial endocarditis and focal abscesses can develop, and the case fatality rate is approximately 7%–10%. The organism is shed in oral, nasal, and ocular secretions of infected animals, and is most frequently transmitted by a bite or scratch. Infections from bites of squirrels and carnivores that prey on rats, such as weasels, cats, dogs, and pigs, have also been reported. Sporadic cases without a history of a bite have
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been documented, as have cases of people living in rat-infested buildings without a history of direct contact with the animals. A similar syndrome, called Haverhill fever, has been associated with outbreaks traced to ingestion of food contaminated with rat excrement. The first-well documented epidemic was described in 1926 in Haverhill, Massachusetts. Two large outbreaks have occurred worldwide, which were associated with contaminated milk and drinking water. Rat-bite fever is rare in the United States; between 1958 and 1983, Anderson et al. (1983) documented 13 cases, none of which were fatal. It has been estimated that half of the rat-bite fever cases in the United States involve laboratory personnel who handle rats, while the other half involve children. Because rat-bite fever is not a nationally reportable disease, the true incidence may be underestimated. In recent years, there have been reports of rapidly fatal cases of rat-bite fever in previously healthy adults (CDC 2005). In one case, a 52-year-old woman employed in a pet store was bitten on her right index finger by a rat. Two days later she developed headache, abdominal pain, diarrhea, lethargy, right axillary lymphadenopathy, and progressive myalgia. When she visited an emergency clinic 2 days later, she was admitted and treated aggressively for gram–negative sepsis with ciprofloxacin, metronidazole, and vancomycin; however, she died approximately 12 hours after admission. The woman also had regular contact with her own pet rats, cats, a dog, and an iguana, but no bites from these animals were reported. In a second case, a 19-year-old woman was pronounced dead on arrival at a hospital emergency clinic. This woman was employed as a dog groomer and lived in an apartment with 9 pet rats. One rat was recently started on doxycycline therapy for respiratory signs, but there was no history of animal bites during the 2 weeks preceding her death. None of the rats were tested for S. moniliformis. An acquaintance reported that this woman had experienced a 3-day history of fever, headache, myalgia, nausea, and profound weakness, without cough, diarrhea, or rash. Rapidly fatal pediatric cases of rat-bite fever had been reported previously, but these were the first reports of fulminant sepsis and death in previously healthy adults (within 12 hours of presentation). With little opportunity for assessment and treatment, prevention would appear to depend on greater awareness of risks and signs consistent with rat-bite fever in people with exposure to rats.
C.
Properties
S. moniliformis is a nonmotile, nonencapsulated, gramnegative rod that is pleomorphic, and can grow singly or as long filamentous strands. Chains of organisms with occasional thickenings that resemble a necklace (hence the Latin Monilifomis, meaning necklace-shaped) are common (Greenwood and Harvey 1992). The organisms measure 0.1–0.7 µm in width by 1–5 µm in length. S. moniliformis is nonhemolytic, and negative for catalase, oxidase, nitrate reduction, and indole production.
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D.
Clinical Signs and Lesions
Following oral inoculation, S. moniliformis can be isolated from submaxillary and cervical lymph nodes. Septicemia develops subsequently, and is associated with conjunctivitis, cyanosis, diarrhea, anemia, hemoglobinuria, emaciation, and high mortality. In mice that survive, septicemia resolves over the course of a few weeks, but infection persists in joints for approximately 6 months (Savage et al. 1981). During this chronic phase, animals may exhibit diffuse limb or tail swelling associated with arthritis, deformity and ankylosis that can progress to spinal involvement, posterior paralysis, kyphosis, and priapism. Lesions during the acute phase of disease are those associated with septicemia, particularly focal necrosis and inflammation in the liver, spleen, and lymph nodes, often with serosal petechial and ecchymotic hemorrhage. Suppurative embolic interstitial nephritis, dermatitis, and mastitis in an outbreak of streptobacillosis in breeding female mice have also been reported. In the chronic phase of disease, arthritis of varying stages and severity predominate. Spontaneous gangrenous amputation of limbs and tails has also been reported.
E.
Epizootiology
S. moniliformis is a commensal inhabitant of the nasopharynx of rats. In documented outbreaks of streptobacillosis in mice, carrier rats were maintained in the same room (Freundt 1956, 1959). Transmission is by rat bites, aerosols, and fomites. Mouseto-mouse transmission can also occur.
F.
Diagnosis and Control
Definitive diagnosis is made by culture and isolation of the organism. The organism is fastidious and slow growing. Growth media must be supplemented with blood or serum; 15% defibrinated rabbit blood or 20% horse serum have been used. The organism is a facultative anaerobe, and optimal growth conditions are between 35–37°C in a humid environment with increased CO2, such as in a candle jar or in a humidified CO2 incubator. Using a reduced partial pressure of oxygen may also be beneficial. L-phase variants may be present in clinical specimens, and these are more easily observed on media supplemented with serum, such that the media is translucent, rather than with blood that makes the media opaque. Bacterial colonies are smooth, convex, grayish, and 1–2 mm in diameter after 3 days of incubation, while L-phase colonies have a typical mycoplasma-like fried egg appearance and are considerably smaller (Greenwood and Harvey 1992). When performing blood cultures for isolation, thioglycolate broth can be used; however, broth containing sodium polyanethol sulfonate (SPS) as an anticoagulant should be avoided, since it has been shown to inhibit S. moniliformis growth even when present at
concentrations as low as 0.0125% (Rupp 1992). To confirm the identity of the organism after isolation, a fluorescent antibody test using an in-house polyclonal antibody that is not commercially available (Graves and Janda 2001) and a PCR-RFLP assay that amplifies a 296-bp fragment of the S. moniliformis 16S rRNA gene followed by BfaI restriction endonuclease digestion (Boot et al. 2002) have been described. The PCRRFLP assay may have utility for diagnosis using DNA isolated directly from clinical specimens, without bacterial culture and isolation. Control can be achieved by using mice and rats that have been cesarean derived, barrier maintained, and regularly monitored for pathogens by a comprehensive health surveillance program. Housing mice and unmonitored rats in the same room should be avoided.
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15. PSEUDOMONAS AERUGINOSA, AND STREPTOBACILLUS MONILIFORMIS
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Chapter 16 Aerobic Gram-Positive Organisms Cynthia Besch-Williford and Craig L. Franklin
I. II.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .390 Staphylococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .390 A. Bacterial Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .390 B. Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .390 C. Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .390 D. Clinical Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .391 E. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .394 F. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .394 G. Treatment and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .395 III. Streptococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .395 A. Bacterial Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .395 B. Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .396 C. Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .396 D. Clinical Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .396 E. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .398 F. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .398 G. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399 IV. Corynebacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399 A. Corynebacterium bovis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399 1. Bacterial Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399 2. Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399 3. Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399 4. Clinical Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400 5. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400 6. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .401 7. Treatment and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .401 B. Corynebacterium kutscheri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402 1. Bacterial Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402 2. Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402 3. Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402 4. Clinical Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402 5. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .403 6. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .403 7. Treatment and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .404 V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .404 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .404
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
389
390
CYNTHIA
I.
INTRODUCTION
Gram-positive bacterial infections in mice are among the most common causes of sporadic infections in research colonies, but the lack of recent reports of disease under-represents disease prevalence in contemporary research facilities. Clinical expression of infection is typical of pyogenic disease, with clinical signs that range from localized conjunctivitis and dermatitis to fulminate septicemia. Treatment of infections is often instituted to salvage valuable mutant mice until studies are concluded or until mice can be rederived. Many grampositive bacteria that cause disease in mice are commensals on the skin and mucous membranes of other laboratory animals and people. Housing and handling procedures must be implemented to minimize transmission by contact with colonized mice or contaminated fomites, including materials used in experimentation. In this chapter, we discuss gram-positive micrococci and corynebacteria as pathogens of concern to researchers who use mice.
II.
STAPHYLOCOCCUS
Staphylococci are hardy gram-positive, coccoid bacteria that commonly colonize the skin, mammary glands, mucous membranes, and gastrointestinal tract of man and animals, including laboratory mice (Bannerman 2003). Surveys of staphylococcal carriage revealed cutaneous colonization of about 90% of healthy people and approximately 75% of conventional laboratory mice (Nagase et al. 2002). The predominant isolate from man was Staphylococcus epidermidis, with S. warneri as a distant second. In contrast, Staphylococcus xylosus and S. sciuri were most often recovered from mice. In both man and mouse, fewer than 10% carried Staphylococcus aureus. While the distribution of staphylococcal species is quite different between man and mouse, one similarity is that the predominant staphylococcal skin commensals do not produce coagulase. Coagulase-negative isolates were once thought to be nonpathogenic. When coagulasenegative staphylococci were recovered from wounds, especially cutaneous wounds, it was difficult to determine if these bacteria were bystanders or pathogens. Isolation of coagulase-negative staphylococci from biofilms on indwelling medical devices and from pyogenic infections in immunosuppressed mice and people suggest these staphylococci are pathogenic, but have a different virulence factor repertoire than those of the well-studied coagulase-positive staphylococci such as S. aureus (Bannerman 2003). In general, staphylococci may be more accurately described as opportunistic pathogens, benignly colonizing host tissue but capable of proliferating and liberating virulence factors once the epithelial barrier is breached and bacteria contaminate wounded tissues.
BESCH-WILLIFORD
A.
AND
CRAIG
L.
FRANKLIN
Bacterial Properties
Staphylococci produce adhesins, a capsular polysaccharide, and various exoproteins or secreted proteins that help divert the host immune responses and that break down host tissue into readily available nutrients for rapid growth (Bannerman 2003). The exoprotein repertoire includes 4 hemolysins (α, β, γ, δ) and a variety of proteolytic enzymes, including coagulase, catalase, DNase, lipase, phosphatase, gelatinase, hyaluronidase, staphylokinase, and protein A. In addition, staphylococcal protein toxins, such as toxic shock syndrome toxin-1, dermatonecrotoxin, leucocidin, and enterotoxins, are potent virulence factors in staphylococcal diseases of man (Dinges et al. 2000), but have not yet been implicated in spontaneous diseases of mice. Staphylococcus aureus produces catalase, coagulase, and β hemolysin, which produces a clear zone of complete lysis around colonies grown on sheep or bovine blood agar. Neither Staphylococcus xylosus nor S. epidermidis produces coagulase, but both secrete catalase and α hemolysin, which produces a green zone of incomplete hemolysis around colonies on blood agar. Diagnostic laboratories providing microbiological services for rodent health monitoring programs screen nasopharyngeal cultures for S. aureus, but staphylococcal isolates that are negative for coagulase and not β-hemolytic may not be speciated unless the bacteria are recovered from a lesioned tissue. Speciation is accomplished by carbohydrate fermentation patterns and enzymatic activity (Bannerman 2003).
B.
Cultivation
Multiple artificial media will support growth of staphylococci, although blood-based media allow identification on the basis of colony morphology and type of hemolysis. Salt-supplemented media (7%–8% sodium chloride) such as mannitol salt agar is selective for staphylococci, which can grow in high salinity and ferment mannitol to produce yellow pigmented colonies. Chromogenic media have been developed for rapid identification of S. aureus. These media contain chromogenic substrates that yield insoluble pigments when cleaved by enzymes produced by S. aureus, such as α-glucosidase (S. aureus ID, bioMerieux, Durham, NC). Chromogenic media are ideal as primary isolation media in clinical microbiology laboratories since growth of coliforms is minimized and S. aureus colonies are quickly recognized on the basis of colony color (Perry et al. 2003). Confirmation is typically performed with a slide-based latex agglutination assay.
C.
Strains
Epidemiologic typing methods to classify pathogenic human strains have historically relied on susceptibilities of S. aureus to lytic bacteriophages (phage type). Application of phage typing
16. AEROBIC
GRAM-POSITIVE
ORGANISMS
to mouse isolates has yielded variable results. In a multiyear analysis of staphylococcal disease in research animal facilities, Markham and Markham (1966) found that staphylococci from conventionally housed mice were not all typable. They concluded that nontypable strains were likely “murine” strains of the bacteria, since phage types were based on human isolates. Interestingly, when S. aureus was recovered from mouse colonies previously free from staphylococcal infection, the staphylococcal isolates were typable strains. The typable strains recovered from these asymptomatic mice and from clinically ill mice were often identical to the phage types isolated from the animal care staff, documenting that mice can acquire S. aureus through human contact (Blakemore and Francis 1970; Lenz et al. 1978). Genetic typing analyses with multilocus sequence typing and enzymatic digestion with pulse-field gel electrophoresis methods have generally replaced phage typing for epidemiologic studies. Multilocus sequence typing involves sequencing of fragments of 7 housekeeping genes found in bacteria and performing a cluster analysis of similar sequence patterns. Pulse-field gel electrophoresis allows discrimination of DNA fragments cleaved from S. aureus by treatment with a restriction enzyme. Compared with phage typing, these assays offer higher discrimination of clonal bacterial populations, and provide more readily reproducible results between laboratories (Boerlin et al. 2003; Grundmann et al. 2002). Epidemiologic studies using these newer technologies have not yet been reported for characterizing staphylococcal isolates from mice.
D.
Clinical Manifestations
Several host factors, including age, physiologic state, and genotype, appear to increase the susceptibility of mice to staphylococcal infections. Staphylococcal infections occur more often in young and aged mice, and in mice under the physiologic stressors of experimentation. While all mouse strains are considered susceptible to colonization, those with an increased prevalence of clinical disease include BALB/c, DBA/2, C57BL/6, and C3H/He strains and their hybrids, and mice homozygous for the nude mutation (Percy and Barthold 2001). Staphylococcal infections are also diagnosed in genetically altered mice with known or unrecognized dysfunction of immune responses (Bingel 2002; Shapiro et al. 1997). Nonhost factors that predispose to clinical disease include staphylococcal contamination of the environment or materials used in mouse experimentation that exposes naive mice to infection. The most frequent manifestations of staphylococcal infections in mice are localized superficial to deep pyogenic infections of the skin or mucous membranes, particularly of the conjunctiva and genital mucosa. Cutaneous infections are believed to develop secondary to a traumatic break in the skin surface from normal social behaviors (grooming, scratching, or biting) or from experimental procedures. Staphylococcal
391 dermatitis occurs frequently on the face and neck and ranges from superficial infections such as pyoderma and ulcerative dermatitis to cellulitis and subcutaneous abscess formation. The event that causes the skin wound is often unrecognized, but grooming or scratching the damaged skin introduces staphylococci in the wound. The subsequent proliferation of bacteria and liberation of virulence factors exacerbates tissue damage, increases inflammatory cell infiltrates, and results in pruritis. Mice more vigorously groom and scratch wounded skin, resulting in a cycle of self-inflicted skin damage with inoculation and spread of staphylococci. Certain mouse strains are noted to be predisposed to development of this condition. The ulcerative dermatitis syndrome with opportunistic staphylococcal infection is frequently observed in mice of C57BL/6 lineage (Sundberg et al. 1994) (Figs. 16-1 and 16-2). The possible role of S. aureus as a primary skin pathogen was proposed in a report of an epizootic of ulcerative dermatitis in a colony of hairless DS-Nh mice (Hikita et al. 2002; Haraguchi et al. 1997). When housing conditions for the mice changed from a sterile isolator to a conventional environment, the cutaneous staphylococcal flora shifted from predominantly S. chinii in unaffected skin to pure cultures of S. aureus from lesioned skin (Haraguchi et al. 1997). The DS-Nh mouse is a model for allergic dermatitis and has been suggested to be especially susceptible to staphylococcal dermatitis. More commonly, multiple species of staphylococci or mixed colonies of staphylococci and gram-negative bacteria are isolated from ulcerative skin wounds. A similar pathogenesis of infection was described in reports of necrotizing staphylococcal dermatitis associated with coagulase-negative staphylococcal infections in immunodeficient mice (Bradfield et al. 1993; Won et al. 2002). In the report by Bradfield and associates, male nude mice developed flat, discolored, dry, erythematous skin lesions 1–24 hours prior to death (Fig. 16-3). Lesions were characterized by full-thickness
Fig. 16-1 Ulcerative dermatitis in a C57BL/6 mouse. Secondary colonization of the skin wound with staphylococci results in pruritis and scratching, causing extensive disfiguring skin lesions about the head, neck, and thorax.
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Fig. 16-2 Histopathologic appearance of the ulcerative skin wound in a C57BL/6 mouse. Coagulation necrosis of the epidermis and dermis is coincident with the presence of a multiple colonies of coccoid bacteria (arrows) in the seropurulent scab. The dermis is infiltrated with neutrophils. (H&E stain, bar = 100 microns)
epidermal coagulative necrosis with ulceration and dermal infiltration with neutrophils and macrophages. Colonies of gram-positive bacteria were present in necrotic wounds. The unusual component of the syndrome was the rapid decline with death in affected mice. Either Staphylococcus xylosus or S. epidermidis was most often isolated from affected mice and occasionally both staphylococci were recovered from wounds. To verify the causal role of these coagulase-negative staphylococci in the fatal disease syndrome, pure cultures of field isolates of each bacterium were inoculated subcutaneously in nude mice, and a silk suture soaked in the same bacterial inoculum was threaded through the skin at the inoculation site.
Fig. 16-3 Staphylococcus xylosus was isolated from this dry, nonulcerated necrotic skin lesion on the thorax of a male nude mouse. This mouse had become moribund a day after the skin lesion was first noticed.
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Another group was administered S. xylosus in an agarose suspension by subcutaneous inoculation. Dermatitis developed 3–4 weeks postinoculation, and affected mice became moribund in treatment groups that received S. xylosus only. From these findings, the authors suggested that an initial wound, perhaps from biting in group-housed male mice, provided the opportunity for secondary infection with resident staphylococcal flora. Since the wounds alone were not extensive enough to cause death, the possibility of exotoxin production by S. xylosus was proposed. Dermatitis from which S. xylosus was recovered was also described in mice deficient in nitric oxide synthase 2 (currently classified as B6.129P2-NOS2tm1Lau) (Won et al. 2002). Nitric oxide synthase 2 produces abundant and sustained nitric oxide in response to various inflammatory mediators. Nitric oxide production by macrophages and leukocytes has antimicrobial activities, mediated primarily through oxidative damage (Wray and Thiemermann 1999). Mice deficient in this enzyme have altered responses to experimental infections with a variety of bacterial agents and delayed wound healing (http://jaxmice. jax.org/strain/002609.html). The consequences of the enzyme deficiency and the predisposition of mice of C57BL/6 lineage to ulcerative dermatitis may have contributed to the development of staphylococcal dermatitis. Lesions were located about the head and ears, and were ulcerative with pustule formation. S. xylosus recovered from skin lesions was verified by biochemical patterns and nucleic acid genotyping. Immunocompetent mice are also susceptible to cutaneous infections with S. xylosus. Thornton and colleagues (2003) describe a disease syndrome in a colony of SJL/J mice that developed necrotic tail lesions of such severity that some mice sloughed most of their tails and had to be euthanized. To verify the role of S. xylosus in the tail lesion, naive SJL/J mice were inoculated with either high or low doses of the field strain of S. xylosus cultured from diseased tails. The inoculation was accomplished by passing a silk suture through the skin, scarifying epidermis adjacent to the suture site and applying the bacterial inoculum to the scarified sutured region on days 1 and 21. Tail lesions developed in most of the experimentally inoculated mice, regardless of bacterial dose. The experimental lesions were less severe than in the spontaneous disease, but S. xylosus was recovered from the wounds. The experimental disease recapitulated most of the clinicopathologic features of the spontaneous outbreak and highlights that coagulasenegative S. xylosus can contribute to skin wounds in mice with competent immune systems. Dermal and subcutaneous abscesses or furunculosis is a well-recognized manifestation of deep cutaneous infection with S. aureus. This condition occurs in mice of various genotypes, particularly in homozygous nudes and genetically altered mice and less often in outbred stocks and inbred strains (Percy and Barthold 2001; Shapiro et al. 1997). Staphylococcal abscesses develop as dermal and subcutaneous nodules on the face and muzzle and occasionally other sites of the
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Fig. 16-5 Histologic appearance of a cutaneous staphylococcal abscess. At the core are bacterial colonies surrounded by neutrophils and macrophages. Splendore-Hoeppli material is deposited at the interface between bacteria and the inflammatory cells. (H&E stain, bar = 50 microns)
Fig. 16-4 Furunculosis on the muzzle of a nude mouse. These cutaneous abscesses caused by Staphylococcus aureus are usually located on the head and muzzle.
body (Fig. 16-4). Nodules progressively enlarge, often with no or minimal ulceration of overlying skin. Occasionally, the inflammatory process causes destruction of underlying bony structures. Histologic appearance of these nodules is characteristic of a septic abscess, with a central core of necrotic neutrophils and colonies of coccoid gram-positive bacteria surrounded by pink amorphous to fibrillar material referred to as Splendore-Hoeppli material (Fig. 16-5). Hypersegmented neutrophils, macrophages, lymphocytes, and a fibrous capsule surround the necrotic core. In immune-deficient mice, the inflammatory cell component of the abscess wall may vary, based on the defect in the immune system. Abscesses often are multiple and coalesce, which suggests slow propagation of bacteria and the ineffectiveness of the host response to adequately wall off and eliminate the bacterial infection. Infections of tissue adjacent to bone can result in osteomyelitis and osteolysis. The histologic manifestations of S. aureus abscesses have been described as botryomycotic granulomas, with botryo meaning “grape-like” and mycotic incorrectly linking the presence of Splendore-Hoeppli material to the histologic appearance of granules formed in tissues infected with certain fungi. Staphylococcus aureus is usually isolated in pure culture from the lesion and can also be recovered from cultures of the nasopharynx and feces. Given the propensity of the lesions to occur on the face, one proposed pathogenesis is that bacteria
are introduced into the dermis through traumatic breaks in oral mucosa or the skin of the muzzle. Staphylococci can be isolated from the mucous membranes of clinically healthy mice. When these sites become inflamed, the presence of staphylococci is considered a complicating factor rather than the etiology of the condition. Conjunctivitis from which staphylococci are recovered may manifest as unilateral or bilateral epiphora or ocular exudation with photophobia (Fig. 16-6). Extension of infection can lead to keratitis and occasionally panophthalmitis. Generally multiple bacteria, including S. aureus, are recovered from these superficial ocular and periocular lesions. Retrobulbar cellulitis and abscesses may extend from these superficial infections. Mice with retrobulbar abscesses develop periocular swelling and
Fig. 16-6 Blepharospasm and exudation are signs of conjunctivitis. Staphylococcus aureus was among the bacteria isolated from the inflamed conjunctiva.
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Suppurative to pyogranulomatous adenitis with intralesional bacterial colonies are typical histologic features of this condition. Rupture of the gland and release of bacteria and sebaceous secretions in surrounding fascia leads to moderate to severe suppurative to pyogranulomatous cellulitis and occasionally abscess formation. S. aureus may be recovered in pure culture, but more often is recovered with a mixed bacterial population of enterococci and gram-negative enteric bacteria.
E.
Fig. 16-7 A severe case of bilateral retrobulbar abscesses in an ICR mouse. A pure culture of Staphylococcus aureus was recovered from the abscesses.
exophthalmus (Fig. 16-7). Pure cultures of S. aureus are usually isolated from deeper periocular infections. Staphylococcal colonization of the genital mucosa in male mice is speculated to be the source of bacteria in the development of preputial gland abscesses. Preputial gland abscesses manifest as subcutaneous nodules at the base of the genital (preputial) papilla (Fig. 16-8). Ulceration of the overlying skin can occur from self-mutilation or rupture of the abscessed gland(s).
Fig. 16-8 Unilateral preputial gland abscess in a young male breeder mouse. Staphylococcus aureus was recovered from this lesion, but other bacteria can be associated with infection of this gland.
Epizootiology
Epidemiologic surveys of staphylococcal colonization of mice are infrequently reported in the literature even though cultures for staphylococci, especially S. aureus, are often included in rodent health monitoring programs in both biomedical institutions and production companies. Interestingly, there is even less data on the nature of the staphylococcal flora of healthy people. Transmission of staphylococci among mice occurs by direct contact with infected mice, people, and fomites. The almost ubiquitous presence of staphylococci on the skin and mucous membranes of mice and people makes direct contact the most common form of staphylococcal transmission. There are several reports of staphylococcal spread in rodent facilities, in which transmission of a certain phage type of S. aureus from one mouse facility to another correlated with transfer of an animal care person carrying the same phage type (Blakemore and Francis 1970; Lenz et al. 1978). The survivability of S. aureus in dried exudates facilitates the spread of staphylococci through contact with contaminated cages, gloves, or shared equipment.
F.
Diagnosis
Diagnosis of staphylococcal infections is based on isolation of bacteria from pyogenic lesions. The demonstration of large clusters of gram-positive coccoid bacteria on direct smears of exudates or on gram-stained histologic section of affected tissues is also a diagnostic tool that can identify the causative or contributing bacterial component when culture is not possible. Staphylococcal isolates are characterized by growth in 24–48 hours in aerobic CO2-supplemented, 37°C environments on 5% sheep or bovine blood-based media. Staphylococci grow as round, glistening, opaque, white-to-yellow colonies with α or β hemolytic patterns. Selective media, such as mannitol-salt agar or commercially available chromogenic media, improve identification of staphylococci because of suppression of other bacteria, especially motile bacteria that can swarm over colonies on the agar and prevent further workup of suspect staphylococcal colonies. Hemolytic gram-positive colonies are tested for the ability to produce catalase and coagulase. Coagulase-negative staphylococci are speciated by inoculation on various carbohydrate media and enzyme substrates.
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Carbohydrate fermentation patterns and enzymatic cleavage of various substrates are compared to published data to determine staphylococcal species. Characterization of species is facilitated by the availability of several commercial test kits that contain key biochemical tests for species identification. Staphylococcus aureus can be confirmed by latex agglutination assay that assesses the presence of protein A. All staphylococcal species can be verified by analysis of bacterial genomic sequences from the 16S rRNA gene.
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Treatment and Control
Antibiotic treatment of staphylococcal infections may be performed if the infection is localized and accessible and the mouse is valuable enough to warrant the effort. Most strains of staphylococci, including Staphylococcus aureus, recovered from mice are susceptible to commonly used antibiotics, and susceptibility tests will identify appropriate antibiotics for therapy. In colonies of valuable genetically altered mice, treatments for staphylococcal infections range from topical treatment of conjunctivitis to surgical drainage of abscesses, combined with localized or systemic antibiotic regimens. Therapy can successfully treat early infections but is unlikely to eliminate carriage of the bacteria on mucous membranes (Thornton et al. 2003). Staphylococci are readily eliminated from inanimate surfaces with common disinfectants used in animal facilities, and cannot survive sterilization temperatures. The bacteria are relatively resistant to desiccation and can remain viable in dried exudates for weeks, so transmission is possible if uninfected mice contact contaminated materials such as dirty bedding or cages, gloves, laboratory coats, and treatment areas. Animal care individuals are considered biological and mechnical fomites, and the potential for zooanthroponotic transmission of staphylococci will be minimized by adopting management practices that include use of disposable gloves, masks, and protective clothes; handling mice with disinfected or heatsterilized forceps in a biosafety hood; and housing mice in cages with sterile interiors. Prevention of infection is also accomplished by screening replacement mice for staphylococcal colonization before introduction into a Staphylococcus-free colony. Since antibiotic treatment does not eliminate colonization of mucous membranes, methods proven to render mice free from staphylococcal colonization include embryo and caesarian rederivation and implementation of rigid husbandry practices described above.
III. STREPTOCOCCUS Streptococci are gram-positive coccoid (<2 µm in diameter) bacteria that are facultative anaerobes and form chains. Commensal or nonvirulent streptococci colonize the nasal
cavity, mouth and dental plaque, intestinal tract, and genital mucosa. Virulent streptococci also colonize throat, mucous membranes, and skin but are generally differentiated from commensal species by hemolysin production, colony appearance, and expression of polysaccharide surface antigens that can be classified by type-specific antisera into Lancefield’s groups (Ruoff et al. 2003). Classic taxonomic nomenclature for streptococci has been modified in recent years by inclusion of information from bacterial genetic analyses, resulting in reassignment as subspecies or even new genera (Facklam 2002). For example, Lancefield’s group D streptococci are now in the genus Enterococcus. Streptococci reported to cause clinical disease in mice are β-hemolytic and include strains from Lancefield’s group A (Hook et al. 1960), group B (Geistfeld et al. 1998; Schenkman et al. 1994), group C (Greenstein et al. 1994), and group G (Stewart et al. 1975). There is one report of an epizootic infection of mice attributed to Enterococcus (Gledhill and Rees 1951). Group A streptococci, for which S. pyogenes is the prototype species, are the most common cause of pyogenic infections in man, ranging from pharyngitis or “strep throat” to deep invasive infections. However, Lancefield’s group B streptococci are the more frequently reported cause of invasive disease in mice, followed by streptococci from Lancefield’s group C, which include S. dysgalactia subsp. equisimilis and S. equi subsp. zooepidemicus.
A.
Bacterial Properties
Streptococci pathogenic to mice are part of the “pyogenic” division, which includes β-hemolytic strains that express a Lancefield’s group antigen and have the ability to grow under various conditions, including environmental temperatures of 10°C and 45°C and in broths with 6% NaCl or high pH (reviewed by Facklam 2002). These streptococci have a repertoire of surface and extracellular proteins that facilitate adherence to host cells, interfere with the innate and adaptive immunologic responses, and cause cellular destruction for release of nutrients and to permit spread of bacteria in infected tissues. Most virulent streptococci of man and animals produce a hyaluronate capsule in vivo that protects the bacterium from proteases (Cunningham 2000; Doran et al. 2004; Timoney 2004). The capsule helps resist phagocytosis and complementmediated opsonization and activation. The capsule also preserves the three-dimensional structure and function of hydrophobic surface-anchored proteins called M or M-like proteins, which are encoded by the emm or emm-related genes of the M gene superfamily. Functionally, M proteins bind to fibronectin and laminin on host cells in the process of bacterial adherence. M proteins also bind to fibrinogen and immunoglobulins, with subsequent impairment of complement activation. Thus, capsule and M proteins are key virulence factors in bacterial persistence in the host.
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Streptococci produce various secreted proteins that have known pathogenic actions on the host. The characteristic hemolytic action of virulent streptococci is from production of a cytotoxin that is generally identified as a streptolysin or hemolysin/cytotoxin among the various groups of streptococci. Functionally, these toxins produce transmembrane pores in host cells, which results in cell lysis in vivo and production of pericolonial zones of hemolysis when cultivated on blood agar plates. Streptococci produce streptokinase and streptokinaselike proteins that bind plasminogen to the surface of bacteria, which may help conceal bacteria from the immune cells and also may enhance bacterial invasion (Cunningham 2000; Humar et al. 2002; Doran et al. 2004; Timoney 2004). Other streptococcal enzymatic exoproteins include peptidases, proteases, collagenases, and hyaluronate lyase that cause tissue damage but are not as well studied as other virulence factors. Streptococci also produce a wide assortment of exotoxins that are known for pyrogenicity and their immunomodulatory effects as superantigens. Superantigens are exotoxins that are mitogenic for certain subsets of T lymphocytes by simultaneously binding Vβ-chain of a set of T cell receptors and Class II MHC molecules on macrophages, B lymphocytes, and dendritic cells. The outcome is nonspecific proliferation of T cells and release of abundant proinflammatory cytokines that cause fever, hypotension, neutrophilia, and fibrinogenemia—the classic clinical features of toxic shock syndrome.
B.
Cultivation
Complex nonselective media containing blood (usually 5% sheep blood) are often used by clinical laboratories to isolate streptococci and recognize hemolytic patterns surrounding bacterial colonies. A culture environment of 35°C to 37°C with 5% CO2 allows improved growth of some virulent streptococcal strains. The lower oxygen tension also enhances production of streptolysin and hemolysin/cytotoxin, which results in red blood cell hemolysis. A clear zone of β-hemolysis surrounding bacterial colonies is a diagnostic feature of many pathogenic streptococci. Group A, C, and G strains produce broad β-hemolytic bands around small (0.5 mm in diameter) grey to white colonies. Group B streptococci have slightly larger colonies with less distinct zones of β-hemolysis. Streptococci are catalase-negative and appear as gram-positive chains of oval to round cocci when stained by the Gram method (Ruoff et al. 2003). For many clinical microbiology laboratories, β-hemolytic streptococci are classified by Lancefield’s group antigens, but may not be further classified unless there is more than one species in that group or the treatment regimen is tailored for a particular streptococcal species. In addition, some streptococcal isolates can express multiple Lancefield’s group antigens; for these isolates, additional biochemical testing would be required for speciation (Facklam 2002). Several key biochemical reactions for speciation include
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bacitracin susceptibility, CAMP reaction (increased β-hemolysis of an S. aureus indicator strain by a factor produced by streptococci) and Voges-Proskauer reaction (acetoin production from glucose degradation), pyrrolidonylarylamidase production, hippurate hydrolysis, and carbohydrate fermentation patterns. These tests are often included in commercially available kits.
C.
Strains
Use of type-specific antisera to classify polysaccharide surface antigens into Lancefield’s groups continues to be the first step in identification of streptococci. Virulence cannot be predicted by Lancefield’s grouping, so other typing systems have been developed to help predict pathogenicity of isolates and also to track the spread of streptococcal strains. The variable amino-terminal end of the M protein has been used for subtyping of group A streptococci by serologic-based precipitant assays (Facklam 2002). There has been good correlation between M-protein serotyping and molecular-based emm genotyping, and M-protein subtypes have been used as an epidemiologic tool to track group A streptococcal infections in man and prepare M-protein-based vaccines. Similarly, M-like proteins from Lancefield’s group C streptococci are used for evaluation of host specificities (Timoney 2004). There is one species assigned to group B streptococci, S. agalactia, and capsular serotyping is used to categorize group B streptococci into 9 subgroups. Aside from serotype, there is phenotypic diversity in group B streptococci isolated from different animal hosts. Group G streptococcal isolates share many virulence factors with group A streptococci (S. pyogenes) (Humar et al. 2002), and group G isolates can be differentiated by phenotypic and genotypic features. For example, a large colony type with broad zones of β-hemolysis has been correlated to pathogenicity (Ruoff et al. 2003), and emm genotyping has been used to establish group G strains. Identification by phenotype and genotype can be verified at the CDC Streptococcus Laboratory (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm).
D.
Clinical Manifestations
Infections of mice and other domestic animals with group A streptococci are uncommon. In the classic report of mouse infection, streptococci were isolated from brain and other tissues from Swiss mice that died during experiments in which they received parenteral sublethal doses of bacterial endotoxin (Hook et al. 1960). The inocula containing endotoxin were bacteriologically sterile, but group A streptococci were cultured from the pharynges from other mice shipped from the same source that had not been used in studies. Infected mice displayed cervical lymphadenitis, lethargy, and depression before death. Pure cultures of group A streptococci were recovered
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from abscessed lymph nodes and from thoracic and abdominal viscera that suggested the mice were bacteremic. Group B streptococcal infections of mice have been reported as isolated events or outbreaks of invasive disease. In one report, weanling nu/nu mice from a colony used for human brain cancer xenograft studies developed group B streptococcal meningoencephalitis (Schenkman et al. 1994). About a third of affected mice carried subcutaneous xenografts and other affected mice were breeder mice or mice not yet included in the study. Clinical signs included rapid loss of body condition and development of neurologic signs. Histological exam revealed suppurative meningoencephalitis, often with ependymitis and periventriculitis, and colonies of gram-positive cocci in purulent exudate. In several mice, suppurative inflammation extended through the cribriform plate to include the olfactory epithelium of the nasal turbinates. Nonhemolytic group B streptococcus serotype Ib were isolated from brain and blood cultures and from nasopharyngeal cultures from asymptomatic nu/+ females from the colony. Disease was suggested to have progressed from rhinitis to meningitis (or possibly vise versa), with transmission of bacteria between mice from direct contact. Another reported group B streptococcal epizootic occurred in adult immunocompetent mice in a barrier production facility (Geistfeld et al. 1998). Adult female and male DBA/2 mice developed depression and unkempt hair coats. Necropsy findings included multiple pale foci in heart, kidneys, spleen, and liver. The most common lesion observed microscopically was chronic severe pyelonephritis. Other lesions included multifocal necrotizing myocarditis, metritis, hepatitis, and pneumonia with suppurative inflammation. Gram-positive coccoid bacteria were present in most organs (Fig. 16-9). A β-hemolytic streptococcus was isolated from organs and oral swabs from affected mice, and from oral and fecal swabs from asymptomatic DBA/2 and hybrid B6D2F1 and CD2F1 mice in the barrier. Group B streptococci were not cultured from NOD, C3H, or C57BL/6 mouse strains in the same barrier, suggesting that DBA/2 mice were uniquely susceptible to infection and disease. The isolate was identified as group B streptococcus serotype V and was biochemically biotyped as S. agalactiae. Although the animal care staff were not screened for carriage of group B streptococci, the authors theorized that zooanthroponosis was likely. Depopulation of the barrier, sanitization with a chlorine dioxide disinfectant, and sterilization of equipment eliminated environmental bacterial contamination such that replacement colonies remained free from group B streptococcal colonization. In the single report of group C streptococcal disease, Greenstein and associates (1994) described a asymptomatic infection in the affected mouse colony. ICR mice that failed to respond appropriately in an immunologic study were found to have hepatic abscesses. A β-hemolytic group C streptococcus was recovered and identified as Streptococcus equisimilis (currently classified as S. dysgalactiae subsp. equisimilis).
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Fig. 16-9 Bacterial embolus (arrow) in a renal glomerular capillary in a mouse with group B streptococcal bacteremia. (H&E stain, bar = 50 microns)
Sentinel mice that had come from the same shipment as the study mice were removed for bacteriologic examinations. Abscesses were observed in the subcutis, liver, and abdomen in 30% of the mice (Fig. 16-10). To assess whether the mice contracted group C streptococcus at the research facility, additional mice were ordered from the supplier for a bacteriologic screen. Two of six retired breeder female mice from the same barrier colony that produced the study mice had hepatic and abdominal abscesses. Streptococcus dysgalactiae subsp. equisimilis was isolated from the abscesses as well as from nasopharyngeal
Fig. 16-10 Abscess of a mesenteric lymph node (arrow) from which a group C streptococcus was isolated.
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and fecal cultures. Mice from different production barriers were not colonized with the bacterium. The authors concluded that affected mice were colonized at the supplier’s barrier facility by contact with contaminated feces, direct contact, or perhaps by the umbilical route in neonates. The lack of isolation of S. dysgalactiae subsp. equisimilis from mice on other studies in the research facility suggested that the bacterium was not readily spread by fomite transmission. The only reported superficial infection caused by streptococci was an epizootic of necrotizing dermatitis from which a group G streptococcus was isolated (Stewart et al. 1975). Dry, necrotic skin lesions were first noticed on the dorsal thoracic or lumbar region and progressively spread to cover the entire shoulder and pelvic areas. Underlying musculature was not involved, even though mice with extensive lesions developed paresis or paralysis. The lesion was characterized histologically as an ulcerative dermatitis with vasculitis and occasional vascular thrombosis. Gram-positive cocci were identified on the margins of epidermal ulcers, in underlying dermal collagen, and occasionally in subcutaneous fascia distant to ulcerated lesions. The skin disease was reproduced when naive mice were inoculated with the field strain of group G streptococci by either subcutaneous injection or tiny cutaneous puncture wounds.
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to internal organs in healthy animals (Wells et al. 1991; Dahlinger et al. 1997; Von Bultzingslowen et al. 2003) and in animals exposed to various manipulations, including antibiotic treatments (Krueger et al. 2004), irradiation (Brook et al. 2004), transportation (Swildens et al. 2004), and surgery (Oliveros Hernandez et al. 2004). Prevalence of colonization of people with group A streptococci ranges from 1%–16% (Hoe et al. 2003) and with group B streptococci ranges from 18%–35% (Bliss et al. 2002; Facklam 2002). Zooanthroponosis has not been documented from man to mice, but there are a few reports comparing isolates from man and animals that suggest interspecies transmission may be possible. For example, group B streptococci from dogs, cats, and man were more similar than cattle isolates in serotype subgroup, carbohydrate fermentation patterns, pigmentation, and genotype of several surface proteins (Yildrim et al. 2002). The authors suggested these phenotypic and genotypic similarities indicate an epidemiologic relationship among isolates from man, dogs, and cats. Use of biochemical and phenotypic features along with genotypic analyses such as emm genotyping, pulse-field gel electrophoresis, and multilocus sequence typing system can be applied to animal and human isolates to establish epidemiologic patterns of bacterial transmission and virulence (Nicholson et al. 2000; Facklam 2002; Jones et al. 2003; Ruoff et al. 2003).
Epizootiology F.
From a five-year review of bacteriologic results archived at the Research Animal Diagnostic Laboratory (Columbia, MO), β-hemolytic streptococci isolated from nasopharynges of asymptomatic, virus-antibody-free mice were mostly group B streptococci. Group B streptococci were also the most likely βhemolytic streptococcus to be recovered from suppurative processes in normally sterile sites, including liver, kidney, and mammary gland. Many mouse strains and stocks are susceptible to experimental infection with β-hemolytic streptococci and are the principal animal models for evaluation of host response to infection. In the few reports of spontaneous outbreaks, host factors involved with susceptibility to infection are not easily delegated to distinct categories with respect to age, immune competence, or sex. Young and old mice and immune deficient and immune competent mice have been affected with clinical disease. The exception is the enhanced susceptibility of DBA/2 mice to group B streptococcal colonization and infection as compared to NOD, C3H, or C57BL/6 mouse strains in the same barrier (Geistfeld et al. 1998). Physiologic demands resulting from transportation and experimental treatments also appear to predispose to clinical expression of disease in colonized mice (Hook et al. 1960; Stewart et al. 1975; Dunigan and Percy 1992). Experimental studies in various animal models that investigate the events that lead to invasive disease have documented streptococcal bacterial translocation from oral and intestinal mucosa
Diagnosis
Diagnosis of streptococcal infections is made by isolation of β-hemolytic bacteria from skin wounds or from inflamed tissues, including blood, kidney, liver, or brain. Clinical laboratories recommend inoculation of samples on agar containing sheep or bovine blood, as the hemolytic patterns can vary when blood from other species is used. Inoculated blood media should be incubated in 5% CO2 conditions, since hemolysin production is facilitated in low-oxygen conditions. Virulent streptococci can be nonhemolytic. The recovery of heavy loads of nonhemolytic cocci from normally sterile sites should drive further testing for bacterial identification. Speciation of isolates can be performed by demonstration of Lancefield’s group, biochemical reactions, and serosubtyping with use of commercially available kits. Direct antigen detection methods that employ agglutination or immunoassay techniques are available for group A and group B streptococci (Rouff et al. 2003). These methods are specific and rapid, but suffer from low sensitivity and could provide false negative results if low numbers of streptococci are present in the sample. Bacterial genotyping of signature genomic regions such as the 16S rRNA genes are also used to establish bacterial species and strain information. Since streptococci are commonly found in high numbers in purulent exudates, a presumptive diagnosis of streptococcal infection may also be achieved by demonstration of chains or pairs of gram-positive
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cocci on impression smears of exudates or in histologic sections of affected tissues.
G. Control and Prevention There are no published reports on treatment of streptococcal infections in mice. Given the propensity of superficial infections to become systemic diseases, there likely would be little success in attempting antibiotic therapy once the mice were moribund. However, antibiotic treatment of carriers of streptococci might be valuable prior to rederivation procedures to minimize bacterial loads in the dam prior to embryo or fetal harvest. Control of disease has been primarily accomplished through elimination of affected mice and their cohorts. Elimination of β-hemolytic streptococcal colonization is possible in mice that have been rederived through embryo transfer or caesarean section. People are likely involved as biological reservoirs or as fomites in the introduction of virulent streptococci to mice. Use of masks, gloves, and air barriers between mice and the human handlers should prevent inadvertent exposure of mice. Streptococci are susceptible to disinfectants used in animal facilities, such that routine disinfection of transfer forceps, bench tops, and shipping crates should effectively decontaminate these surfaces.
IV. CORYNEBACTERIUM Corynebacteria are gram-positive, non-spore-forming, partially acid-fast, nonmotile, small rods. The stem “coryne” comes from the Greek word koryne, which means club. The morphologic appearance of corynebacteria is a slightly curved rod with one end wider than the other that stains unevenly with the Gram method. From broth cultures, corynebacteria are often arranged in pairs or haphazardly arranged clusters, figuratively called Chinese letter formations (Funke and Bernard 2003). Corynebacteria are considered part of the normal flora of the skin and mucous membranes of man and animals. There are two pathogenic corynebacteria of mice, Corynebacterium bovis and C. kutscheri. Corynebacterium bovis, which causes hyperkeratotic dermatitis in nude mice, is a common inhabitant of bovine mammary gland and has been reported as a rare human pathogen, principally isolated from blood cultures from ill patients (Bernard et al. 2002, Funke et al. 1997). Corynebacterium kutscheri is primarily a rodent pathogen, responsible for abscess formation in colonized mice and rats, and has rarely been associated with disease conditions in other animals or people (Funke and Bernard 2003). Corynebacteria have also been isolated from mice with conjunctivitis; however, the etiologic role for these bacteria has not been demonstrated (Sundberg et al. 1991).
The clinical disease manifestations and husbandry measures required to control bacterial transmission are quite different between the two corynebacterial pathogens of mice. To clearly distinguish between these diseases, the microbiological characteristics and disease features of C. bovis will be discussed separately from those of C. kutscheri.
A. 1.
Corynebacterium bovis
Bacterial Properties
Little is known of the virulence factors of C. bovis, but as with most all coryneform bacteria, the mycolic acid composition of the cell wall is known to play a role in immunomodulation of the host immune system. No corynebacteriophage with an exotoxin gene typical for the C. diphtheriae group has been identified in C. bovis (Funke et al. 1997). 2.
Cultivation
Corynebacterium bovis is a lipophilic coryneform bacterium that exhibits enhanced growth in media supplemented with 0.1–1% Tween 80 or several drops of serum. Colonies are small and slower growing than nonlipophilic coryneforms. Colonies may be apparent within 24 hours of incubation in a microaerophilic environment, but are readily visible with extended incubation for 48–72 hours. Colonies are typically small (less than 1 mm), punctiform, nonhemolytic, and white to light grey. Corynebacterium bovis isolates are catalasepositive, reduce nitrate, and produce β-galactosidase. Carbohydrate fermentation and biochemical patterns available in a kit format are commonly used for bacterial speciation. Since the databases for the commercial kits are not comprehensive for the heterogeneous genera of Corynebacterium, biochemical identification alone may give false identification (Funke et al. 1997). Other methods for species identification include sequence analysis of the 16S rRNA gene (Bernard et al. 2002; Duga et al. 1999) and cell wall fatty acid analysis (Funke and Bernard 2003). 3.
Strains
The earliest published report of hyperkeratotic skin disease in nude mice was associated with Corynebacterium sp. that was biochemically distinct from C. bovis (Nomura 1984). Characteristics of infection were similar to those attributed to C. bovis in later reports, but the organism could not be assigned to a species from biochemical patterns, and was not analyzed genetically. It is uncertain if this isolate was a unique Corynebacterium sp., or a novel strain of C. bovis. Changes in cultivation and genomic identification strategies since that early report have improved the ability to assign corynebacteria into distinct species.
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Clinical Manifestations
Clinical manifestations of C. bovis infection almost exclusively involve the skin of affected mice even though the bacterium has also been recovered from oral mucous membranes and feces. A dramatic hyperkeratosis syndrome was first recognized in nude mice (Clifford et al. 1995). Yellow-white flakes of keratin that adhere to skin give the appearance of scales, and the condition is often referred to as “scaly skin” disease (Fig. 16-11). Hyperkeratosis is most frequently observed on the dorsum and, in severe cases, can extend from the head to rump and can include the face and lateral portions of the trunk. The skin condition can be transient, with spontaneous resolution, although recurrence has been reported. A pattern of resolution and recrudescence and the observation that affected skin is commonly associated with vestigial hair suggest that susceptibility to disease expression may be linked to the wave-like growth cycle of hair (Clifford et al. 1995). Other manifestations of clinical disease from C. bovis infection include pruritis, weight loss, and neonatal mortality (Clifford et al. 1995; Scanziani et al. 1997). Mice that develop this disease during the course of a study often are removed prematurely because of the addition of an unwanted experimental variable. Histologic examination of affected skin reveals epidermal hyperplasia and hyperkeratosis without ulceration. Acanthosis and a hyperplasia of the stratum corneum are accompanied by layers of orthokeratotic hyperkeratosis (Fig. 16-12). Keratin horn cysts, which can be seen in sections of normal nude mouse skin, are more frequent in affected skin. Squamous metaplasia
Fig. 16-12 Histologic appearance of hyperkeratotic dermatitis from a nude mouse with Corynebacterium bovis infection. Epidermal hyperplasia and orthokeratotic hyperkeratosis are hallmarks of the disease. (H&E stain, bar = 50 microns)
of adnexal components has also been reported. The dermis is generally infiltrated with macrophages and neutrophils. Tiny coccobacillary to diphtheroid rods clustered in the layers of keratin and occasionally in keratin-filled hair follicles are best visualized in tissue sections stained with the Gram method (Fig. 16-13). 5.
Fig. 16-11 Nude mouse with cutaneous hyperkeratosis. Corynebacterium bovis infection was confirmed by PCR testing of a skin swab that contained keratin scales.
Epizootiology
Mouse strains other than thymic-deficient nude mice have been examined for their susceptibility to infection and expression of clinical disease. Hirsute immunocompetent mice of several strains and stocks, including C57BL/6, heterozygous nude, BALB/c, DBA/2, C3H/HeN, and Swiss mice, were found to develop low-level, often transient, skin colonization when conventionally housed in the same room as clinically infected nude mice (Gobbi et al. 1999). Transmission from affected nude mice was likely by contact with fomites. No clinical or histologic abnormalities were observed in those mice with cutaneous colonization, and the authors concluded that hirsute immunocompetent mice are unlikely reservoirs of bacteria. Hirsute SCID mice were also susceptible to infection when
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Fig. 16-13 Colonies of tiny gram-positive rod-shaped coryneform bacteria in the keratin scale. (Modification of the Brown & Hopps Gram method, bar = 50 microns)
housed with affected nude mice, and on occasion developed alopecia with scaly dermatitis (Scanziani et al. 1998). Hairless immunocompetent mice (SKH-1) were determined to be susceptible to experimentally induced infection and hyperkeratotic disease when inoculated with a field isolate of C. bovis (Clifford et al. 1995). Taken together, these results suggest that a lack of hair and contact with bacterial reservoirs are the most significant predisposing factors for expression of clinical disease. The immune competence seems to be associated with duration and severity of colonization. Nude mice are persistently colonized even though they may not express clinical disease, while immunocompetent mice tend to have transient colonization (Gobbi et al. 1999). 6.
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Diagnosis
Definitive diagnosis is accomplished by detection of C. bovis on skin samples by culture or PCR assays. The presence of hyperkeratosis and the observation of gram-positive coccobacillary bacterial colonies in the keratin scale by histopathological examination can be used to make a presumptive diagnosis if culture or PCR is unavailable. Since the coryneform bacteria colonize the keratin layers of skin, collection of samples for culture or PCR are directed toward sampling the superficial keratin layer. If collecting samples from multiple mice at a time, care must be exercised to minimize contamination of the work area, gloves, and instruments with keratin scales so that carryover between mice does not occur. For isolation of bacteria by culture, the skin is swabbed with isopropyl alcohol prior to sample collection to disinfect the surface of commensal bacteria that could overwhelm the slow-growing colonies of corynebacteria on enriched isolation medium of 5% sheep blood agar. Dry, sterile cotton-tipped
swabs are vigorously rubbed on the skin to remove the superficial keratin layers, and then swabbed on the blood agar plates. Sites with hyperkeratotic lesions should be chosen for culture. In hirsute mice, mild lesions can be obscured by hair, so examination of the pelage with aid of a dissecting microscope may be warranted to aid in the identification of subtle lesions. In the absence of hyperkeratotic lesions, skin of the lateral thorax or abdomen has been swabbed (Clifford et al. 1995). While C. bovis has been recovered from other sites, including oral cavity and feces, the skin was up to 1.5 times more likely to yield Corynebacterium from infected animals than these other sites (Clifford et al. 1995). Blood agar plates are incubated at 37°C with 5% CO2 for 48 to 72 hours and pinpoint white to tan colonies are then picked for Gram staining. Diphtheroid grampositive bacteria are then speciated by inoculation of various biochemical media, conveniently available in kit format, or by fatty acid analysis. Confirmation of C. bovis can be made by sequence analysis of the 16S rRNA gene. PCR also can be used as a primary or confirmatory diagnostic test. One advantage of PCR over culture is the ability to identify C. bovis in the presence of other skin bacteria, such as Proteus, which can swarm the surface of the isolation medium and prevent the recovery of pure cultures of C. bovis needed for inoculation of the biochemical test media. Primer sets that anneal to regions of the 16S rRNA gene specific for C. bovis are used in reaction mixtures with DNA recovered from skin samples. Skin samples can be collected in various ways. Rubbing the skin with dry, sterile cotton swabs (as is done for culture) or scraping the skin surface with sterile scalpel blades both provide bacteria-laden samples. Activities designed to minimize contamination of work surfaces, gloves, or instruments with keratin scales are even more critical when collecting samples for PCR testing because a few contaminating bacteria could yield a positive test result. Precautionary processes include soaking instruments in 10% bleach prior to use, changing gloves between animals, using capped sterile tubes that can be closed after addition of the skin swabs or scrapes, and wiping down the work area with 10% bleach or 70% ethanol. PCR results can be obtained in several days, so that decisions about the disposition of infected mice can be made expediently. 7.
Treatment and Control
Evaluation of the antibiograms of C. bovis revealed that the bacterium is sensitive to many antibiotics, including tetracycline, enrofloxacin, and ampicillin. In the report from Clifford and associates (1995), C. bovis was resistant to trimethoprim sulfamethoxazole, an antibiotic commonly used to suppress growth of Pneumocystis in immunecompromised mice. Therefore, use of this antibiotic would not interfere with the ability to detect C. bovis infection in susceptible mice. Antibiotic use may decrease clinical signs of infection, but recrudescence of disease generally occurs
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when antibiotic use is discontinued. Replacement of infected mice and rederivation by caesarean section or embryo transfer have been the most reliable practices to obtain Corynebacterium-free mice. Control methods are designed to eliminate environmental contamination with corynebacteria. In the outbreak of disease reported by Clifford and colleagues (1995), cage-to-cage transmission was best prevented when susceptible mice were housed in semi-rigid isolators. Naive mice developed disease when housed in rooms that previously contained C. bovis-infected mice, suggesting the environment was contaminated enough for indirect transmission of bacteria to newly introduced mice. Infections have also been diagnosed by examination by PCR testing of samples wiped from the interior walls of the cages. The positive results from environmental sampling suggests that keratin flakes bearing bacteria are readily dispersed from infected mice and can coat the cage interior. These findings indicate that inadvertent contact with the wirebar lid, water bottles, or even the inner surfaces of microisolator lids during cage changing can contaminate the gloves and shirtsleeves of the handler and the interior of an air displacement work station. In those situations in which affected mice have not yet been or cannot be removed, several practices could be employed to minimize environmental contamination. Antibiotics delivered in water can ameliorate the scaly clinical disease, which would markedly reduce the potential for dispersal of contaminated keratin flakes. Changing gloves between cages or rotational use of several chlorine dioxide-soaked forceps that remain in contact with the disinfectant for approximately 5 minutes prior to use will diminish contamination of the animal handler. Cage-changing stations that do not allow horizontal displacement of air into the animal room will decrease dispersal of bedding, dust, or flakes into the room. Spraying the interior of the cage-changing station with disinfectant prior to introduction of cages will also minimize contamination of the station during use. The lipophilic nature of C. bovis and its keratin niche may also account for difficulties in adequate disinfection of environmental surfaces in animal facilities. Decontamination of the environment has involved autoclaving of equipment coupled with either gluteraldehyde fumigation after quaternary ammonium disinfection or chlorine dioxide disinfection alone (Scanziani et al. 1997). The success of these efforts was measured by lack of disease development when uninfected nude mice were housed in rooms that previously contained infected mice. Other options for determining successful removal of bacterial fomites include swiping environmental surfaces and evaluating swipe samples for corynebacteria by PCR. Specific sites that benefit from this type of monitoring include those items that cannot be autoclaved and have more frequent contact with mice or the mouse handlers such as interior of cage-changing stations, platforms or tubes used with imaging equipment, ultrasound probes, and other specialized equipment.
BESCH-WILLIFORD
B. 1.
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Corynebacterium kutscheri
Bacterial Properties
Corynebacterium kutscheri is a member of the C. diphtheriae group of fermentive, nonlipophilic corynebacteria. C. kutscheri is genetically related to C. diphtheriae, as determined by 16S rRNA gene analysis, but is biochemically dissimilar. Virulence factors, aside from the mycolic-rich cell wall, have not been described for C. kutscheri. 2.
Cultivation
Corynebacterium kutscheri grows as punctate white to grey colonies when incubated on a blood-based medium for 48–72 hours at 37°C in a microaerophilic environment. A selective medium, FCN or furazolidone-colimycin-nalidixic acid agar, permits primary isolation of C. kutscheri by inhibiting growth of gram-negative rods, including Proteus, and gram-positive cocci (Amao et al. 1995). Aside from the catalase production and nitrate reduction typical for corynebacteria, carbohydrate fermentation patterns are used to distinguish C. kutscheri from C. bovis. Type-specific antiserum produced with an ATCC isolate of C. kutscheri from a rat has been used for a rapid slide microagglutination assay to verify C. kutscheri isolates from both mice and rats (Amao et al. 1995).
3.
Strains
There is minimal information about strains of Corynebacterium kutscheri recovered from diseased mice. What is available for review is primarily serologic cross-reactivity of C. kutscheri isolates from mice and rats. In a report from Boot and associates (1995), multiple isolates of C. kutscheri from mice and rats were collected from laboratories in Europe and Japan. Serologic cross-reactivity among the 7 strains was demonstrated by evaluating isolate-specific antisera binding with homologous and heterologous antigens in an ELISA format. Interestingly, isolates from Japan were serologically distinguishable from those collected in Europe.
4.
Clinical Manifestations
Infection of mice with C. kutscheri is usually asymptomatic. Bacteria colonize the gastrointestinal tract, with highest frequency of isolation from oral cavity and large bowel in naturally infected mice (Amao et al. 1995). Pyogenic infections characterized as arthritis or abscesses in the subcutis or viscera are hypothesized to result from bacteremia when the mouse immune defenses are compromised from experimentation (Schechmeister et al. 1953; Zucker et al. 1954; Fauve et al. 1964) or concurrent microbial infections (Weisbroth et al. 1968), or genotype (Amao et al. 1993, 1995). Disease expression may manifest as acute outbreaks associated with mortality, or as
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intermittent morbidity, especially in colonies of mice carrying C. kutscheri as part of their intestinal flora. Affected mice exhibit a variety of clinical signs depending on the organ system involved. Rough hair coat, lethargy, anorexia with weight loss, oculonasal discharge and tachypnea, ulcerative dermatitis and subcutaneous nodules, and swollen joints of the lower aspect of the limbs have been described. The distribution of lesions in affected mice is typical of a bacteremia. Bacteria embolize in vessels of the skin, lung, liver, kidney, and joint synovium, leading to formation of abscesses. Abscesses are generally small (1–2 mm), grey, firm nodules that may occur in the subcutis or in multiple viscera, including lung, liver, spleen, and kidney (Fig. 16-14). Lymphadenopathy of cervical and mesenteric lymph nodes is also observed. Transection of abscesses reveals casseous material, similar to that of other corynebacterial visceral infections in man and domestic animals. Subcutaneous abscesses are often recognized as cutaneous ulcers after rupture. Joint disease typically involves the carpal/metacarpal and tarsal/metatarsal joints, which are swollen, and the overlying skin is erythematous (Fig. 16-15). Subsequent ischemic necrosis of digits with autoamputation can be confused with one of the clinical signs of ectromelia virus infection. Histologic features of C. kutscheri abscesses in soft tissues include central core of necrotic debris surrounded by neutrophils. Clusters of gram-positive small bacilli within the necrotic core can be demonstrated with the tissue Gram method. Arthritic lesions are characterized by neutrophilic infiltration and edema of joint capsule and synovium, and neutrophilic effusion in joint spaces. More chronic lesions include periosteal fibroplasia, and cartilage erosion and ulceration.
Fig. 16-14 Splenomegaly, unilateral renomegaly, renal abscess (arrow) and multifocal pneumonia in a mouse with spontaneous Corynebacterium kutscheri infection.
5.
Epizootiology
Age, sex, and strain susceptibilities to experimental C. kutscheri infection have been reported (Amao et al. 1993; Komukai et al. 1999). Four- and 12-week-old ICR mice of both sexes developed subclinical infections when experimentally inoculated orally with either a million or a billion colony-forming units of a field strain of C. kutscheri (Amao et al. 1993). Four weeks postinoculation, bacteriologic surveys using the selective FCN medium revealed that all mice inoculated as weanlings were colonized with C. kutscheri, whereas only the adult mice in the higher dose group developed infection. Regardless of age group, male mice were about 1.5–2 times more frequently colonized and carried higher bacterial loads in the cecum as compared to female mice. Susceptibility of mouse strains to disease was examined in 9 inbred strains and 1 outbred stock inoculated orally with a field strain of C. kutscheri (Komukai et al. 1999). The course of disease was monitored by expression of clinical signs, documentation of gross and histologic lesions and recovery of the organism from lesions, and gut. Susceptible mouse strains, as determined by colonization rate and disease expression, included BALB/c nude, A/J, CBA/N, MPS, and BALB/cCr. Mice of intermediate susceptibility were C3H/He, and mice most resistant to colonization were C57BL/6Cr, B10.BR/SgSn, ddY, and ICR. 6.
Diagnosis
A presumptive diagnosis can be made from the identification of small gram-positive rods in visceral abscesses by histology or via impression smears. Corynebacterium kutscheri can be readily cultivated from inflamed tissues, so a definitive diagnosis is made by culture. Use of the selective FCN medium for swabs from mucosal sites will enhance primary isolation of the slow-growing C. kutscheri by inhibiting growth of gramnegative rods (Amao et al. 1995).
Fig. 16-15 Septic arthritis of the tarsus and metatarsus in a mouse with naturally occurring Corynebacterium kutscheri infection.
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An agglutination assay is available commercially for detection of serum antibodies to C. kutscheri (Denka Seiken Co., Ltd. Tokyo, Japan). The agglutination assay is relatively insensitive, as it does not yield positive results from serum with low-level antibody, typical in asymptomatic naturally infected or experimentally inoculated mice (Amao et al. 1995; Komukai et al. 1999). However, the agglutination assay is very specific, and did not yield false positive results when antibodies to other corynebacteria were used in the test (Ackerman et al. 1984). ELISA-based serodiagnostic tests have been developed over the years to identify anti-C. kutscheri antibody (Ackerman et al. 1984; Boot et al. 1995), but are not routinely employed in rodent diagnostic laboratories. ELISA assays are inherently more sensitive than agglutination tests but typically are less specific. Ackerman and associates (1984) reported that antisera to C. ulcerans and Rhodococcus (previously Corynebacterium) equi did bind to C. kutscheri antigen in the ELISA but not in the agglutination test. The ELISA developed by Boot and colleagues (1995) was more specific, yielding no significant cross-reactivity to antisera produced by experimental vaccination of rats to C. bovis, Rhodococcus equi, C. murisepticum, C. pseudodiphtheriticum, C. pseudotuberculosis, Arcanobacterium (formally Corynebacterium) pyogenes, C. renale, and C. xerosis. Differences in specificity of the ELISA in these two reports may have been due to the particular C. kutscheri isolate chosen as the antigen source and the methodology used to prepare it for use in the ELISA. Aside from issues of specificity, the more significant drawback to serologic monitoring of naturally infected rat and mouse colonies for C. kutscheri infection has been the inconsistency in identifying asymptomatically infected rodents. Two explanations have been proposed for this observation. One is the sensitivity of the serologic test, with the ELISA outperforming the agglutination assay for detection of low levels of circulating antibody. To date, reports on seropositivity in subclinically infected mice used the agglutination assay. The ELISA has principally been used to survey colonized rats (Boot et al. 1995; Amao et al. 1995). Another explanation is that circulating antibody gradually increases as the animal ages. Boot and associates (1995) reported a low (<50%) prevalence of positive ELISA test results in 5- to 6-week-old rats as compared to high (92%) prevalence of detectable antibody in young adult (12- to 16-week-old) rats. As maternal antibody wanes, circulating antibody is produced, but not at levels sufficient to result in a positive serologic test. By 12–16 weeks of age, however, colonized rats produce enough antibodies to result in positive ELISA tests. There is no comparable report of surveying infected mice by ELISA. Komukai and colleagues (1999) used the agglutination assay to survey for antibody production in young (4-week-old) and adult (14-week-old) experimentally infected ICR mice of both sexes. Regardless of age or sex, the agglutination assay identified antibody in no more than 30% of colonized mice, confirming the poor sensitivity of this testing platform.
BESCH-WILLIFORD
7.
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L.
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Treatment and Control
Treatment of infected or carrier mice has not been reported. Reports recovering C. kutscheri-free mice through rederivation techniques also have not been published, but these techniques would likely have a good success rate in mice without active disease. To keep mice free from C. kutscheri, husbandry practices such as use of microisolator housing can be used to prevent fomite transmission of the bacteria. Shared equipment or common use areas should be thoroughly disinfected, since rats are also carriers of C. kutscheri.
V.
SUMMARY
In summary, gram-positive bacterial infections are common causes of sporadic pyogenic disease in research mice. Clinical expression of disease is typically pyogenic with micrococcal infections and varies from cutaneous hyperkeratosis to septicemia with corynebacterial infections. Treatment of infections may reduce overt disease in affected mice, but elimination of colonization requires rederivation. To raise and maintain mice free from pathogenic gram-positive bacteria, research and resource staff must employ husbandry and handling procedures that minimize contact with contaminated fomites.
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405 Humar, D., Datta, V., Bast, J.D., et al. (2002). Streptolysin S and necrotising infections produced by group G streptococcus. Lancet 359, 124–129. Jones, N., Bohnsack, J.F., Takahashi, S., et al. (2003). Multilocus sequence typing system for group B streptococcus. J. Clin. Microbiol. 41, 2530–2536. Komukai, Y., Amao, H., Goto, N., et al. (1999). Sex differences in susceptibility of ICR mice to oral infection with Corynebacterium kutscheri. Exp. Anim. 48, 37–42. Krueger, W.A., Krueger-Rameck, S., Koch, S., et al. (2004). Assessment of the role of antibiotics and enterococcal virulence factors in a mouse model of extraintestinal translocation. Crit. Care Med. 32, 467–471. Lenz, W., Thunert, A., Brandis, H. (1978). [Epidemiologic investigation of staphylococcal infections of stocks of SPF-animals (author’s transl)]. Zentralbl. Bakteriol.[Orig. A] 240, 447–465. Markam, N.P., and Markham, J. (1966). Strains of staphylococci in man and animals. J. Comp. Path. 76, 49–56. Nagase, N., Sasaki, A., Yamashita, K., et al. (2002). Isolation and species distribution of staphylococci from animal and human skin. J. Vet Med. Sci. 64, 245–250. Nicholson, M.L., Ferdinand, L., Sampson, J.S., et al. (2000). Analysis of immunoreactivity to a Streptococcus equi subsp. zooepidemicus M-like protein to confirm an outbreak of post-streptococcal glomerulonephritis, and sequences of M-like proteins from isolates obtained from different host species. J. Clin. Microbiol. 38, 4126–4130. Nomura, T. (1984). Genetic and microbiological control in immunodeficient laboratory animals. In Immune-deficient animals, 4th International Workshop on Immune-deficient Animals in Experimental Research, Chexbres, 1982, B. Sordat, ed., pp. 160–171. S. Karger, Basel, Switzerland. Percy, D.H., and Barthold, S.W. (2001). Pathology of laboratory rodents and rabbits, pp. 46–70. Iowa State University Press, Ames. Perry, J.D., Rennison, C., Butterworth, L.A., et al. (2003). Evaluation of S. aureus ID, a new chromogenic agar medium for detection of Staphylococcus aureus. J. Clin. Microbiol. 41, 5695–5698. Ruoff, K.L., Whiley, R.A., Beighton, D. (2003). Streptococcus. In Manual of clinical microbiology, P.R. Murray, E.J. Baron, J.H. Jorgensen, M.A. Pfaller, R.H. Yolken, eds., pp. 405–421. American Society of Microbiology Press, Washington, DC. Scanziani, E., Gobbi, A., Crippa, L., et al. (1998). Hyperkeratosis-associated coryneform infection in severe combined immunodeficient mice. Lab. Anim. 32, 330–336. — — —. (1997). Outbreaks of hyperkeratotic dermatitis of athymic nude mice in northern Italy. Lab. Anim. 31, 206–211. Schechmeister, I.L., and Adler, F.L. (1953). Activation of pseudotuberculosis in mice exposed to sublethal total body radiation. J. Infect. Dis. 92, 599–600. Schenkman, D.I., Rahija, R.J., Klingenberger, K.L., et al. (1994). Outbreak of group B streptococcal meningoencephalitis in athymic mice. Lab. Anim. Sci. 44, 639–41. Shapiro, R.L., Duquette, J.G., Nunes, I., et al. (1997). Urokinase-type plasminogen activator-deficient mice are predisposed to staphylococcal botryomycosis, pleuritis, and effacement of lymphoid nodules. Am. J. Pathol. 150, 359–369. Shulman, S.T., Tanz, R.R., Kabat, W., et al. (2004). US Streptococcal Pharyngitis Surveillance Group. Group A streptococcal pharyngitis serotype surveillance in North America, 2000–2002. Clin. Infect. Dis. 39, 325–332. Stewart, D.D., Buck, G.E., McConnell, E.E., et al. (1975). An epizootic of necrotic dermatitis in laboratory mice caused by Lancefield group G streptococci. Lab. Anim. Sci. 25, 296–302. Sundberg, J.P., Brown, K.S., Bates, R., et al. (1991). Suppurative conjunctivitis and ulcerative blepharitis in 129/J mice. Lab. Anim. Sci. 41, 516–518. Sundberg, J.P., Brown, K.S., McMahon, W.M. (1994). Chronic ulcerative dermatitis in black mice. In Handbook of mouse mutations with skin and hair abnormalities, J.P. Sundberg, ed., pp. 485–492. CRC Press, Boca Raton, FL. Swildens, B., Stockhofe-Zurwieden, N., van der Meulen, J., et al. (2004). Intestinal translocation of Streptococcus suis type 2 EF+ in pigs. Vet. Microbiol. 103, 29–33.
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Thornton, V.B., Davis, J.A., St. Claire, M.B., et al. (2003). Inoculation of Staphylococcus xylosus in SJL/J mice to determine pathogenicity. Contemp. Top. Lab. Anim. Sci. 42, 49–52. Timoney, J.F. (2004). The pathogenic equine streptococci. Vet. Res. 35, 397–409. Von Bultzingslowen, I., Adlerberth, I., Wold, A.E., et al. (2003). Oral and intestinal microflora in 5-fluorouracil treated rats, translocation to cervical and mesenteric lymph nodes and effects of probiotic bacteria. Oral Microbiol. Immunol. 18, 278–284. Weisbroth, S.G., and Scher, S. (1968). Corynebacterium kutscheri infection in the mouse. I. Report of an outbreak, bacteriology, and pathology of spontaneous infections. Lab. Anim. Care 18, 451–458.
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Wells, C.L., and Erlandsen, S.L. (1991). Localization of translocating Escherichia coli, Proteus mirabilis, and Enterococcus faecalis within cecal and colonic tissues of monoassociated mice. Infect. Immun. 59, 4693–4697. Won, Y.S., Kwon, H.J., Oh, G.T., et al. (2002). Identification of Staphylococcus xylosus isolated from C57BL/6J-Nos2(tm1Lau) mice with dermatitis. Microbiol. Immunol. 46, 629–632. Wray, G., and Thiemermann, C. (1999). Nitric oxide in sepsis. In Nitric oxide and infection, F.C. Fang, ed., pp. 265–280. Kluwer Academic/Plenum Publishers, New York. Yildirim, A.O., Lammler, C. Weiss, R., et al. (2002). Pheno- and genotypic properties of streptococci of serological group B of canine and feline origin. FEMS Microbiol. Lett. 212, 187–192.
Chapter 17 Helicobacter Infections in Mice James G. Fox and Mark T. Whary
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Biochemical, Phenotypic, and Molecular Classification . . . . . . . . . . . . . . . IV. Species of Helicobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Helicobacter hepaticus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Host Susceptibility to H. hepaticus-Induced Hepatitis . . . . . . . . . . 3. Possible Mechanisms of Tumor Induction or Promotion . . . . . . . . . 4. Biomarkers of Oxidative Stress and Cytotoxicity in H. hepaticus-Associated Hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Effects of H. hepaticus on Gene Expression in Liver and Ceca of Infected Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Helicobacter bilis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Helicobacter rodentium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Helicobacter ganmani . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Helicobacter typhlonius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Helicobacter muridarum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Helicobacter muricola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Helicobacter mastomyrinus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Helicobacter rappini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Experimental Helicobacter Infections in Mice . . . . . . . . . . . . . . . . . . . . . . . A. Mouse Models of Helicobacter-Associated Hepatitis and Hepatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mouse Models of Helicobacter-Associated IBD-like Disease . . . . . . . 1. Experimental Infection Using H. trogontum . . . . . . . . . . . . . . . . . . . C. Gastritis Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Helicobacter felis Gastritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Helicobacter pylori Gastritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Helicobacter-Associated Gastric Cancer . . . . . . . . . . . . . . . . . . . . . . . . . VI. Interference with Research Attributable to Helicobacter Infections of Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Diagnostic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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D. Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Genomic Analysis and Diversity of H. hepaticus . . . . . . . . . . . . . . . . . IX. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Antibiotic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Colony Management of Helicobacter-Free Mice . . . . . . . . . . . . . . . . . . . . . A. Principles of Helicobacter Eradication in Mice . . . . . . . . . . . . . . . . . . 1. Restocking with Helicobacter-Free Mice . . . . . . . . . . . . . . . . . . . . . 2. Embryo Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cross Fostering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Colony Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Use of Sentinel Mice to Monitor for Helicobacter spp. . . . . . . . . . . . . XI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
Helicobacter pylori was first identified by Marshall and Warren in 1982 (Marshall and Warren 1983) as an infectious cause of chronic, active gastritis. H. pylori is now known to cause peptic ulcers, gastric adenocarcinoma, and MALT lymphoma in humans and has been classified as a class I carcinogen by the World Health Organization (Marshall 1995). A new group, named enterohepatic helicobacter, was subsequently identified by Fox and Ward in the early 1990s (Fox et al. 1994; Ward et al. 1994b). The Helicobacter genus now includes at least 26 formally named species (Fig. 17-1), many of which colonize the intestine of rodents, including mice (Table 17-1). Beyond the impact of H. pylori on human health (Blaser and Atherton 2004), considerable research has focused on other Helicobacter spp. isolated
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from humans and animals and the associated natural diseases these organisms produce in the gastrointestinal tract and liver. Identification and characterization of novel Helicobacter spp. associated with clinical disease in their natural host has allowed investigations of idiopathic disease syndromes through experimental Helicobacter infections of animal models, most notably in mice. Natural and experimental Helicobacter infections in mice reproducibly recapitulate important features of human disease. These models are providing new information on human diseases, including gastritis and its progression to gastric atrophy and cancer, infectious hepatitis, cholecystitis, hepatocellular carcinoma, bacteremia in immunodeficient humans, and inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis (Rogers and Fox 2004). H. hepaticus and the gastric Helicobacter (H. felis, H. pylori) that experimentally
Fig. 17-1 Dendogram demonstrating the genetic relationships among Helicobacter species.
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colonize mice are examples of Helicobacter-associated disease models that are exploring the link between chronic infections, inflammation, and cancer within the liver and gastrointestinal tract (Table 17-3). A large amount of the data presented in this chapter has been distilled and reformatted from reviews and articles written by the authors (Fox 2002; Fox et al. 2000b; Fox and Lee 1997; Fox et al. 2002b; Whary and Fox 2004b). Because H. hepaticus is the prototype enterohepatic Helicobacter and is a commonly occurring pathogen in mice and has been studied extensively, the chapter will highlight the epidemiology, treatment, and pathogenesis of this helicobacter.
II.
HISTORY
Helicobacter hepaticus, an enterohepatic Helicobacter in mice, was the first murine Helicobacter discovered in the 1990s as a confounding factor in long-term carcinogenesis studies (Fox et al. 1994; Ward et al. 1994b) and is known to cause typhlocolitis, colon cancer (Table 17-2) hepatocellular adenomas, carcinomas, and hemangiosarcomas in susceptible mouse strains (Table 17-3). H. bilis was isolated soon thereafter from the livers of aged inbred strains of mice (Fox et al. 1995). Prior to the isolation of H. hepaticus, H. muridarum and “H. rappini” were isolated from intestines of mice but were not associated with disease, nor were they formally named within the genus Helicobacter until a later date (Dewhirst et al. 2000a; Lee et al. 1992; Phillips and Lee 1983; Schauer et al. 1993). It is now known that several distinct Helicobacter spp. colonize the cecum and colon of mice and other rodents and have been associated with gastrointestinal and liver disease in select strains of mice and rats. Naturally acquired infections are persistent, and the organism is chronically shed in the feces. Endemic Helicobacter infections are common unless specific steps are taken
to prevent introduction and dissemination. Helicobacter-associated disease is dependent on interaction between host factors including age, sex, genetics, and immune competence and bacterial virulence factors that either are known or are suspected to influence tissue tropism and host immune responses. In addition to mice, naturally acquired helicobacter infections have been identified in other commonly used laboratory animal rodent species (rats, gerbils, hamsters) as well as in wild rodents, such as woodchucks (Fox et al. 2002a). Helicobacter spp. have coevolved with their hosts and other commensal microbiota of the gastrointestinal tract to reach a homeostatic equilibrium that minimizes the risk of clinical disease in the vast majority of their natural hosts. Dependent on the host range and tissue tropism of individual Helicobacter sp., clinical disease develops not only in immunocompromised humans and animals but also in a subset of immunocompetent individuals. Risk factors for overt disease in otherwise clinically normal hosts presumably include environmental factors and host-pathogen genetic polymorphisms (El-Omar et al. 2000; El-Omar et al. 2001). The ability of these organisms to persist in the host reflects in part the adaptation of different Helicobacter spp. to their natural niche. The importance of genetic and environmental predisposition in predicting disease outcome is illustrated by the number of genetically engineered mouse models that have diseases attributed to natural Helicobacter infection.
III.
BIOCHEMICAL, PHENOTYPIC, AND MOLECULAR CLASSIFICATION
The International Committee of Systematic Bacteriology Subcommittee on the Taxonomy of Campylobacter and
TABLE 17-1
HOST RANGE AND TISSUE TROPISM OF RODENT HELICOBACTER SPECIES Helicobacter taxon
Source(s)
Primary site
H. aurati H. bilis H. cholecystus H. cineadi H. ganmani H. hepaticus H. marmotae H. mastomyrinus H. mesocricetorum H. muridarum ‘H. rappini’a
Hamster Mouse, dog, rat, cat, human Hamster Human, hamster, macaque, dog Mouse Mouse, gerbil Woodchuck, cat Mastomys, mouse Hamster Mouse, rat Human, cat, dog, mouse, sheep, swine
Stomach, intestine Intestine Gallbladder Intestine Intestine Intestine Intestine Intestine Intestine Intestine Intestine
H. rodentium H. trogontum H. typhlonius
Mouse, rat Rat Mouse, rat
Intestine Intestine Intestine
aFormerly
regarded as ‘Flexispira rappini’; now subgrouped into 10 taxa.
Secondary site
Liver (mouse, rat) Gallbladder, liver (human) Blood, brain, joint (human) Liver (mouse), fetus (mouse) Liver (woodchuck) Liver (mastomys) Stomach (mouse) Placenta/fetus (sheep) Blood (humans)
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TABLE 17-2
HELICOBACTER-ASSOCIATED TYPHLOCOLITIS AND COLON CANCER IN MICE Genetic status of mice
Type of defect
Pathology
CD45RBhighreconstituted scids TCR α−/−, β−/− defined flora Scid ICR defined flora IL-10−/− RAG2−/−
Reconstitution with naïve CD4+ T cells Abnormal T cell receptors Lack T and B cells
Typhlocolitis
Lack IL-10 Lack T and B cells Lack IL-7, T and B cells Normal Normal
Typhlocolitis, colon cancer Typhlocolitis, colon cancer (129 background) None
Lack NF-κB Lack P-glycoprotein Lack TGFβ
Typhlocolitis Typhlocolitis Typhlocolitis, colon cancer
IL-7−/−/RAG2−/− A/JCr Swiss Webster gnotobiotic NF-κB (p50−/−, p65+/−) Mdr1a−/− TGFβ−/−
Typhlocolitis Typhlocolitis
availability of advanced molecular techniques, comparison of near-complete 16S ribosomal DNA sequences does not always provide conclusive evidence for species-level identification and can be misleading (Vandamme et al. 2000). Additional identifying information can be gained from 23S rDNA analysis, fatty acid analysis, restriction fragment length polymorphism analysis (RFLP), random amplified polymorphic DNA fingerprinting (RAPD) analysis, and fluorophore-enhanced repetitive element PCR (FERP or repPCR) analysis (Danon et al. 1998; Kiehlbauch et al. 1995; On 1996; Patterson et al. 2000; Shen et al. 2005).
IV.
Typhlitis Enterocolitis
SPECIES OF HELICOBACTER A. Helicobacter hepaticus
Related Bacteria has established minimum requirements for the formal description of new species of the genus Helicobacter (Dewhirst et al. 2000b). Formal naming of a new species or subspecies is based on phenotypic and genotypic examination of at least five strains to characterize the range of potential variation between independent isolates. Helicobacter spp. are gram-negative bacteria that are speciated by morphology (curved to spiral in shape, variation in flagella, and presence or absence of periplasmic fibers), growth requirements (microaerobic and/or anaerobic, optimal growth at 37°C and/or 42°C), biochemical profiles (such as oxidase, catalase, and urease production), antibiotic sensitivity and sequencing of conserved 16S rRNA as well as whole-cell protein electrophoresis and DNA-DNA hybridization (Table 17-4). Despite the
To date, nine species of Helicobacter have been identified in mice (see Table 17-1). H. hepaticus has a spiral shape and bipolar, single-sheathed flagella and was first isolated from the livers of A/JCr mice with active, chronic hepatitis and liver cancer in 1994 (Fox et al. 1994; Ward et al. 1994b). The bacteria also colonize the cecal and colonic crypts of mice (Fig. 17-2). The bacterium grows at 37°C under microaerobic and anaerobic conditions, rapidly hydrolyzes urea, is catalase and oxidase positive, reduces nitrate to nitrite, and is resistant to cephalothin and metronidazole (Fox et al. 1994). 1.
Pathogenesis
The A/JCr mouse, an immunocompetent strain, and SCID/ NCr, an immunodeficient strain, appear to be among the most hepatitis-prone strains identified to date with liver lesions that become progressively more severe with age (Fox et al. 1996b; Fox et al. 1996c; Li et al. 1998). H. hepaticus-associated
TABLE 17-3
SUMMARY OF KEY MOUSE MODELS OF HELICOBACTER GASTROINTESTINAL AND LIVER CANCERa Strain
Infectious agent/Transgene
Tumor
Comment
C57BL/6
H. felis
Gastric adenocarcinoma
INS-GAS FVB
H. felis and H. pylori
Gastric adenocarcinoma
BALB/c Genetically engineered mice: IL10-/-, especially on 129Sv background Lymphocyte-deficient mice: Rag-/-; especially on 129Sv background A/JCr, B6C3F1, AB6F1, AXB recombinant inbred
Several Helicobacter spp. “Endogenous microbiota” or H. hepaticus
Gastric MALT lymphoma Lower bowel carcinoma (cecum ± colon)
H. hepaticus
Lower bowel carcinoma (cecum ± colon)
H. hepaticus
Hepatocellular carcinoma (HCC) hemangiosarcoma
Natural feline pathogen, but lacks vacA and cag pathogenicity island Constitutive hypergastrinemia promotes tumorigenesis Usually requires 18 to 24 months Bacteria in endogenous microbiota models not well defined; H. hepaticus reliably induces disease Often used for adoptive transfer studies; H. hepaticus induces tumors in untreated Rag2-/- mice Natural murine pathogen induces chronic active hepatitis, HCC, and hemangiosarcoma
aTable
adapted from Rogers and Fox (2004).
Growth
Helicobacter taxon *H. bilis +H. ganmani H. hepaticus H. mastomyrinus ‘H. muricola’ H. muridarum *’H. rappini’ *H. rodentium H. typhlonius
Resistance to
Catalase production
Nitrate reduction
Alkaline phosphatase
Urease
Indoxyl acetate hydrolysis
γ-Glutamyl transferase
At 42°C
With 1% glycine
Nalidixic acid
Cephalothin
Flagella
Flagella sheathed
+ + + + + + +/− + +
+ + + − + − − + +/−
− − − − − + − − −
+ − + + + + + − −
− − + − − + − − −
+ ND − ND ND + + − −
+ − − + − − + + +
+ − + + − − − + +
R R R R S R R R S
R S R R R R R R R
Bipolar Bipolar Bipolar Bipolar Bipolar Bipolar Bipolar Bipolar Bipolar
+ − + + + + + − +
17. HELICOBACTER INFECTIONS IN MICE
TABLE 17-4
BIOCHEMICAL PROFILE, GROWTH CHARACTERISTICS, AND FLAGELLAR MORPHOLOGY OF HELICOBACTER SPECIES ISOLATED FROM MICE
ND = not determined, R = resistant, S = susceptible *periplasmic fibers +anaerobic growth only
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A
B
C Fig. 17-2 Electron micrograph of H. hepaticus. Notice the spiral morphology as well as the single, bipolar flagellum (A) in the colon crypt (B) and within the biliary canaliculi of the liver (C). Bars = 0.5 µm. Reproduced with permission from Dr. Schauer and Dr. Solnick (Solnick and Schauer, 2001).
hepatitis and progression to hepatocellular carcinoma in A/JCr mice is most prevalent after 18 months postinfection, and males are predisposed to more severe lesions for unknown reasons (Fox et al. 1996b). SCID/NCr mice naturally infected with H. hepaticus developed hepatitis, proliferative typhlitis, and colitis (Li et al. 1998). Lesions in the liver of SCID mice consisted of Kupffer, Ito, and oval cell hyperplasia, along with multifocal to coalescing coagulative hepatocyte necrosis. Numerous Warthin-Starry-positive bacteria were observed in the parenchyma, and there were minimal to mild accumulations of monocytic cells and neutrophils. Proliferative typhlitis was characterized by moderate to marked mucosal epithelial cell hyperplasia with mild monocytic and neutrophilic infiltration. In contrast, infected immunocompetent A/JCr mice, which develop a significant immune response to H. hepaticus associated with prominent multifocal mononuclear cell infiltrates in the liver (Whary et al. 1998), typically have low numbers of
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H. hepaticus observable at the periphery of inflammatory foci and colonizing the biliary canaliculi (Fig. 17-3). Therefore, the success rate for recovery of H. hepaticus in culture from infected A/JCr mice is high when the cecum and colon are sampled but much lower from liver samples. Natural H. hepaticus infection in A/JCr mice has been characterized on a longitudinal basis through 18 months of age (Fox et al. 1996b). H. hepaticus colonization persisted in the large bowel and liver and was associated with a chronic proliferative hepatitis and liver cancer and in some mice, a chronic typhlitis (Fox et al. 1996b). Infected A/JCr mice developed sustained serum IgG antibody responses to H. hepaticus and elevated serum enzymes indicative of hepatocellular injury. To fulfill Koch’s postulates, germ-free mice were colonized with H. hepaticus and went on to develop chronic hepatitis and, in select mice, an enterocolitis (Fox et al. 1996c). A clinical syndrome of typhlocolitis, with and without rectal prolapse or concomitant hepatitis, has been described in a variety of inbred strains of immunocompromised mice infected with H. hepaticus (Table 17-3) (Fig. 17-4) (Foltz et al. 1998; Shomer et al. 2001; Ward et al. 1996). In immune dysregulated mice, the cecum and colon develop mononuclear inflammatory cell infiltrates in the mucosa and submucosa. Epithelial cell hyperplasia can be significant with frequent villous to papillomatous folds extending into the lumen and a high mitotic index in the crypts. Goblet cells are variably decreased in number in the proliferative mucosa. H. hepaticus infection has been associated with development of lower bowel adenocarcinoma in RAG2−/− mice on a 129/SvEv background (Fig. 17-5) (Erdman et al. 2003a; Erdman et al. 2003b) (see Mouse Models of IBD-like Disease later in this chapter). 2.
Host Susceptibility to H. hepaticus-Induced Hepatitis
Why some mouse strains appear more susceptible to development of liver lesions is unknown, but our initial studies indicated that susceptibility was related to host genotype. We and others determined that A/JCr, BALB/cANCr, SCID/NCR, C3H/HeNCr, and SJL/NCr mice were susceptible to infection, whereas C57BL mice were not (Fox et al. 1996c; Ward et al. 1994a). Given that B6C3F1 mice are produced by interbreeding susceptible C3H and resistant C57BL strains of mice, the finding of severe liver disease and tumor induction in B6C3F1 infected with H. hepaticus infers that genetic susceptibility to H. hepaticusinduced neoplasia has a dominant pattern of inheritance (Hailey et al. 1998). This dominant trait of liver tumor susceptibility has also been recently demonstrated in ABF1 mice (García et al. 2004). This experimental approach is similar to a recent study by Sutton et al. which showed that by interbreeding two inbred strains of mice, one resistant and the other susceptible to Helicobacter-induced severe gastric inflammation, the gastric inflammation phenotype was not inherited as a dominant trait (Sutton et al. 1999). Furthermore, it is well known that genetic background is often one of the determinants in conferring
17. HELICOBACTER INFECTIONS IN MICE
413
Fig. 17-3 Photomicrograph of a section of the liver from an A/J mouse infected with H. hepaticus. Notice characteristic lesions associated with chronic active hepatitis, including cholangiohepatitis (upper left white arrow), interface hepatitis (lower right black arrow), hepatocellular dysplasia (black arrowhead), and lobular inflammation (central upper white arrow). H&E stain; magnification, 20× (B) Inset depicts Warthin-Starry-positive organisms with morphology consistent with H. hepaticus in the liver. Magnification, 100 ×. Reproduced with permission from Dr. Whary and Dr. Fox (Whary and Fox 2004a).
resistance or susceptibility to a number of infectious agents as well as carcinogens (Malkinson et al. 1985; Malo et al. 1993; Skamene et al. 1982). Recombinant inbred (RI) strains of mice are an important and useful tool for analyzing genetic traits in mice. RI sets are derived by crossbreeding two parental inbred strains of mice, then developing a set of inbred lines from the F2 generation of that cross. As a consequence, the genetic composition of each strain in the RI set is a unique combination of genetic material from the parental strains, representing approximately 50% of each parental genome. Therefore, a genetic marker recognized in either of the parental strains will have a characteristic strain distribution pattern (SDP) among the RI strains. If a single gene is responsible for a trait with differing phenotypes in the parental strains, the gene can be mapped to a chromosomal region using linkage analysis to compare the SDP of a particular phenotype with the SDPs of all the known genetic markers. The AXB RI set utilized to study H. hepaticus genetic susceptibility was derived from parental A/J and C57BL/6J inbred strains (Ihrig et al. 1999). It is one of the best characterized of the RI panels, typed for over 400 genetic markers spanning the entire genome, and has been used extensively to study host-related mechanisms of immunity to infectious agents (Dindzans et al. 1986; Kongshavn 1986; Manly et al. 1997).
The phenotypes exhibited in response to chronic H. hepaticus infection among nine of the AXB recombinant inbred strains have been characterized (Ihrig et al. 1999). Not surprisingly, the most discriminating of the phenotypes were the extent and severity of hepatic inflammation scored morphometrically (Fig. 17-5). Five males and five females from each of the nine AXB RI strains were inoculated by gavage with H. hepaticus at 10 weeks of age and euthanized at 14 months postinoculation (Ihrig et al. 1999). Microscopic evaluation of hematoxylin and eosin-stained liver sections revealed lesions consisting of inflammation and necrosis of the hepatic parenchyma, intralobular veins, and portal areas that varied in severity for different strains. Morphometric scoring indicated that AXB strains 1 and 8 had the most severe changes, while strains 4 and 13 were the least affected. The variation in hepatic inflammation between strains was statistically significant, and the continuous, rather than bimodal, distribution of inflammation scores among the strains was indicative of a multigenic trait (Ihrig et al. 1999). Linkage analysis suggested that foci on chromosome 19 (D19Mit34 and D19Mit36) may contribute, in part, to Helicobacter-induced disease in AXB RI mice. Interestingly, a number of immunologically important genes are located on chromosome 19, including CD5, which has been shown to be a phenotypic marker of activated B lymphocytes in mice (Ye et al. 1998).
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Hepatic Inflammation Scores
Area of Inflammation(log)
4.000 3.800 3.600 3.400 3.200 3.000 2.800 2.600 4
13
5
6
2 10 Strain
12
1
8
Fig. 17-5 Hepatic inflammation scores in A × B recombinant inbred strains infected with H. hepaticus (Ihrig et al. 1999).
Fig. 17-4 Tac:ICR:HascidfRF mouse infected with a novel Helicobacter. Note enlarged distal colon with evidence of fecal staining at base of the the tail.
Bile ductule and oval cell hyperplasia were present in some severely affected portal areas, and occasionally, dysplasia of biliary epithelium was present. Proliferative lesions ranging from nodular hyperplasia to hepatocellular adenomas and carcinomas were present in several cases with severe, chronic inflammation (Ihrig et al. 1999). Typically, oval cell proliferation has been linked to chronic, ongoing hepatic damage, usually from carcinogens (Steinberg et al. 1994). More recently, however, proinflammatory cytokines produced by leukocytes have been recognized as important mitogens for oval cells (Isfort et al. 1998). In H. hepaticus-induced hepatitis, both direct cell damage and inflammatory cell-derived cytokines probably contribute to oval cell proliferation. This constellation of lesions is consistent with lesions observed in A/JCr mice that were similarly infected with H. hepaticus. In contrast, uninfected A/JCr mice had minimal or no lesions, and, as expected, all C57BL/6J mice, whether or not infected with H. hepaticus, had minimal or no lesions (Ihrig et al. 1999). Notably, two mice from AXB RI strain 12 developed lymphosarcoma that involved the liver, mesenteric lymph nodes,
and large intestine, and both neoplasms were of B-cell origin, as are H. pylori-associated MALT lymphomas in humans (Spencer and Wotherspoon 1997; Yumoto et al. 1998). Helicobacter hepaticus causes hepatitis in susceptible mouse strains including A/J, BALB/c, C3H, B6C3F1, and SJL as well as hepatocellular carcinoma in A/J mice but minimal or no disease in resistant C57BL mice, despite persistent colonization with the organism. To dissect the basis of differing genetic predisposition to hepatic disease associated with H. hepaticus infection, hybrid B6AF1 mice were used to ascertain which phenotype was dominantly inherited (Garcia et al. 2003). Sixty mice, male and female, were divided equally into control and H. hepaticus-infected groups and necropsied 18 months postinfection. Lymphohistocytic lobular and lymphoplasmacytic portal hepatitis and tigroid cells were observed in B6AF1 mice at 18 months indicating a dominant inheritance of susceptibility to H. hepaticus-induced hepatitis. Tigroid hepatocytes, characterized ultrastructurally by orderly stacks of dilated endoplasmic reticulum cisternae richly decorated with ribosomes, were first noted by Rogers et al. in H. hepaticus-infected A/J mice (Rogers et al. 2004). These cells were originally described in rats used in chemical carcinogenicity studies (Bannasch et al. 1985). Their physiometabolic origin is uncertain, but they belong to the lineage of basophilic hepatocytes in rats that appear at high risk for malignant transformation. Morphologic consistency with rat tigroid cells was independently verified (Bannasch, personal communication). It is possible that small numbers of tigroid cells may have been overlooked in other mouse studies. Alternatively, the combination of bacterial strain, inoculation protocol, and environment may have uniquely predisposed these mice to tigroid cell formation in our setting. Further investigation will be required to determine whether tigroid cells in mice behave similarly to those in rats.
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There was a positive correlation between severity of inflammation and development of hepatocellular atypia and tumors in B6AF1 hybrid mice infected with H. hepaticus. Hepatocellular carcinomas and foci of altered hepatocytes displaying variable phenotypic patterns, including clear cell (glycogen) type, vacuolated (fatty) type, and tigroid cell type, were noted in 3 of 12 H. hepaticus-infected male mice but not in female infected mice or in control animals (García et al. 2004). All of these cell types have been noted previously (Rogers et al. 2004). A hemangiosarcoma was also noted in a H. hepaticus-infected male mouse. We have not observed hemangiosarcomas in A/JNCr mice in related studies, but this tumor type is statistically increased in H. hepaticus-infected B6C3F1 mice (Hailey et al. 1998). Similarly, the data in B6AF1 mice support earlier studies in B6C3F1 mice in which susceptibility to H. hepaticusinduced liver disease was dominantly inherited. 3.
Possible Mechanisms of Tumor Induction or Promotion
Cancer development in the liver is incompletely understood. The major pathway in the development of hepatocellular carcinoma involves altered hepatic foci and hepatic nodules. Foci of altered hepatocytes can be recognized histologically; the foci show increased eosinophilia, and they may appear vacuolated due to the accumulation of glycogen. Growth of these foci leads to hepatic nodules. Nodules are distinguished from foci by size, although they show tinctorial properties that are similar to foci. As in other organ systems, the mechanism of tumor induction in the liver is considered to be a two-step process involving initiation followed by promotion. The initiation phase is thought to result from an irreversible effect of genotoxic agents on DNA, while promotion occurs as a result of proliferation of the initiated cells. Agents that work through the stage of promotion are often called epigenetic or nongenotoxic carcinogens. There is no requirement for mice to be exposed to genotoxins for neoplasia to result; animals appear to have within the liver a population of spontaneously initiated cells. As animals age, an increasing number of altered foci are seen. It has been suggested that these altered foci indicate that initiation occurs spontaneously, as a consequence either of low-level exposure to genotoxins or inherent metabolic processes leading to free radical formation (Schulte-Hermann et al. 1983). Following exposure to genotoxic agents, the initial biochemical lesions may be considered premutagenic. The functions of the ensuing mutations are poorly understood. Tumors that develop in C3H and B6C3F1 mice show a high incidence of mutation at codon 61 of the H-ras oncogene, although there is some question as to whether such mutations are a cause of transformation or a result of it. Genetic analysis of recombinant inbred strains of mice derived from C57BL and C3H mice has indicated that susceptibility to neoplastic development may be controlled by two genetic loci, one of which plays a predominant (85%) role. This difference relates to the promotional stage, and susceptible strains have a more rapid
growth of altered foci (Drinkwater 1994; Drinkwater and Ginsler 1986). PCR and single-stranded conformation polymorphism (SSCP) have been used to examine DNA from H. hepaticusassociated hepatocellular tumors for the presence of H-, N-, and K-ras mutations, as well as mutations in the tumor suppressor gene p53 (Sipowicz et al. 1997b). In this study, mutations were not observed. These findings support the hypothesis that H. hepaticus infection causes liver tumors through a promotion-like mechanism (Table 17-5). The inbred A/J strain normally has a low incidence and multiplicity of liver tumors. Thus, their susceptibility to H. hepaticus-induced hepatitis and development of hepatocellular tumors has provided a unique opportunity to dissect helicobacter-associated tumorigenesis. Our laboratory, and subsequently others, have demonstrated that marked hepatocyte proliferation, strongly linked to tumor promotion and presumably the result of H. hepaticus-induced chronic inflammation, is in part responsible for the increased rates of hepatocellular tumors in male A/J mice (Fox et al. 1996b; Ihrig et al. 1999; Nyska et al. 1997). However, these histological features are expressed only in certain inbred strains of mice and not in other inbred strains (e.g., C57BL/6). These strain differences in susceptibility to H. hepaticus-associated disease also suggest a mechanism involving tumor promotion, because of evidence that tumor promotion in the mouse liver is influenced strongly by host genetics (Diwan et al. 1997; Weghorst et al. 1989). In addition, a tumor promotion mechanism is supported by a lack of mutagenic response in the Ames assay as well as lack of demonstrated mutations in H-, K-, and N-ras and p53 tumor suppresser genes in liver tumors of A/J mice infected with H. hepaticus (Canella et al. 1996; Diwan et al. 1997; Sipowicz et al. 1997b). Furthermore, the presence of a long-term chronic inflammatory response that precedes tumor development in H. hepaticus-infected mice supports this hypothesis (Fox et al. 1996b, 1998b). Helicobacter mustelae, a natural gastric pathogen in ferrets, significantly promotes carcinogenesis initiated by the gastric carcinogen, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (Fox et al. 1993). Others have demonstrated that liver fluke infection in hamsters also promoted nitrosodimethylamine (NDMA)-nitiated bile duct cancer (Flavell and Lucas 1983). It is interesting to speculate as to whether these hamsters were
TABLE 17-5
CHARACTERISTICS THAT CLASSIFY H. HEPATICUS AS A TUMOR PROMOTER Mouse strain susceptibility Long-term inflammatory state with increased reactive oxygen species prior to tumor formation Increased hepatocyte proliferation Lack of mutations in ras and p53 genes Negative Ames assay H. hepaticus promotion of tumors initiated by hepatic carcinogens
416 also infected with H. cholecystus, a novel Helicobacter known to colonize the bile of hamsters (Franklin et al. 1996). Male A/J infant mice infected with H. hepaticus were given a single IP dose of NDMA and developed a statistically higher incidence of hepatocellular adenomas at two time points (31–36 weeks and 51–64 weeks p.i.) than in uninfected mice similarly treated with the carcinogen (Diwan et al. 1997). There was also a fourfold increase in multiplicity of adenomas at 31–36 weeks p.i. and a fivefold increase in both incidence and multiplicity of carcinomas after 50 weeks. These data indicate that H. hepaticus not only stimulates growth of tumors from initiated cells, but also enhances progression to malignancy (Diwan et al. 1997). Helicobacter hepaticus may predispose the liver to develop cancer through the induction of apoptosis and proliferation. Several studies have shown that H. pylori infection leads to an increased proliferative index and that the effect on proliferation may be one of the earliest identifiable abnormalities in the progression to gastric cancer (Cahill et al. 1996). The factors that lead to an increased proliferation rate have not been clearly delineated, although the possibilities studied include direct bacterial factors (e.g., adherence) and chronic inflammation with release of cytokines and growth factors. Apoptosis (programmed cell death) is the primary mechanism for cell loss in the gastric mucosa (Hall et al. 1994), and the rate of apoptosis is normally closely aligned with the proliferative rate in the gastric mucosa. In the setting of H. pylori-gastritis, apoptotic rates are markedly increased. For example, the mean apoptotic rate rose from 2.9% in uninfected patients to 16.8% in H. pyloriinfected patients (Moss et al. 1996). Changes in the apoptotic rate are normally matched by changes in the rate of proliferation, and thus the induction of apoptosis by H. pylori (and possibly H. hepaticus) could be the stimulus for the increases in proliferation. Preliminary studies performed on A/JCr and B6C3F1 naturally infected with H. hepaticus also indicated that apoptosis and proliferation characteristic of hepatitis in mice may be linked to progression to tumors (Fox et al. 1996b; Hailey et al. 1998). This observation suggested that growthpromoting factors that increase the proliferative rate might also lead to increases in the rate of apoptosis. The induction of apoptosis appears to be mediated by direct effects of H. hepaticus, with significant contributions by cytokines. Several groups have now shown that infection of gastric cancer cell lines with H. pylori leads to dose-related increases in the apoptotic rate, which is increased in a synergistic manner through co-incubation with interferon-gamma (IFN-γ) or tumor necrosis factor-alpha (TNF-α) (Fan et al. 1998; Wagner et al. 1997). Interestingly, increased levels of IFN-γ are also noted in A/J mice infected with H. hepaticus (Myles et al. 2003; Whary et al. 1998). Thus, previous work has suggested that both direct bacterial effects and immune responses may induce apoptosis, which may be a possible stimulus for increased epithelial proliferation. The combination appears to lead, through unknown mechanisms, to altered differentiation and progression to cancer.
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4. Biomarkers of Oxidative Stress and Cytotoxicity in H. hepaticus-Associated Hepatitis
In addition to alterations in cell kinetics in the liver of mice infected with H. hepaticus, there is now strong evidence for the presence of elevated levels of oxidative stress in H. hepaticusassociated hepatitis. In H. pylori-associated gastritis, it is well recognized that the chronic-active inflammation is characterized by elevated levels of proinflammatory cytokines, including IL-1, IL-6, IL-8, TNF-α, and interferon IFN-γ (Crabtree et al. 1991; Karttunen et al. 1995; Noach et al. 1994). The inflammatory responses engendered by this cytokine milieu, however, do not confer protective immunity; instead, the cytokines promote chronic inflammation and elevated levels of reactive oxygen species (ROS) in the gastric mucosa (Davies et al. 1994). The elevated level of ROS is coincident with increased nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) (Mannick et al. 1996). NO reacts with ROS to form additional reactive species. A major fate of NO is a reaction with superoxide anion to yield peroxynitrite, which is even more reactive than NO itself. Importantly, the formation of NO and superoxide occurs simultaneously in activated inflammatory cells (Assreuy et al. 1994), and nitrotyrosine, a specific marker for peroxynitrite formation, is produced in H. pylori-associated gastritis (Mannick et al. 1996). In addition to nitrotyrosine, the oxidation and nitration products of DNA, 8-oxo-dG and 8-nitrodeoxyguanosine (8-nitro-dG), are considered useful biomarkers for monitoring promutagenic DNA damage. The role of 8-oxo-dG in mutagenesis and carcinogenesis has been widely investigated, and several studies have shown a correlation between the formation of 8-oxo-dG and increased cancer risk (Floyd 1990). There is a time-dependent increase in 8-oxo-dG in the liver of H. hepaticus-infected A/JCr mice compared to uninfected, agematched controls (Sipowicz et al. 1997a). In this study, the source of the ROS was determined to be the hepatocytes, as evidenced by formazan deposition following perfusion of 4-nitro-blue tetrazolium chloride (NBT). Immunohistochemistry revealed an increased number of cells expressing P450 (CYP) isoforms IA2 and 2AI, and co-localization of formazan and the 2A5 isoform of the enzyme (CYP2A5), suggesting a possible mechanism for the production of ROS (Sipowicz et al. 1997a). In addition, immunohistochemistry for glutathione S-transferase indicated that the hepatocytes produced increased amounts of reduced glutathione (GSH). GSH is involved in protecting cells from killing by NO and by ROS, and both de novo synthesis of GSH and reduction of oxidized glutathione (GSSG) are important responses to increased oxidative stress (Luperchio et al. 1996). TNF-α, IFN-γ, iNOS, nitrotyrosine, 8-oxo-dG, and GSH may serve as useful biomarkers for oxidative stress and cytotoxicity in H. hepaticusassociated hepatitis. It must be cautioned, however, that the use of 8-oxo-dG as a biomarker is questionable in light of the fact that it is ~1000-fold more susceptible to oxidation than dG itself (Burney et al. 1999; Uppu et al. 1996) and its analysis is subject to numerous artifacts (Cadet et al. 1997).
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A study performed by Shuker and coworkers revealed a lack of correlation between infection with H. hepaticus and the level of M1G, the pyrimidopurinone adduct of dG proposed to arise in reactions with malondialdehyde, a lipid peroxidation product (Singh et al. 2001). Of particular importance with regard to M1G is the possibility that it is not a product of lipid peroxidation but instead arises in reactions with base propenals generated by deoxyribose oxidation in DNA (Dedon et al. 1998). Interestingly, Shuker and coworkers did observe age-related lobe-specific increases in M1G levels that were independent of H. hepaticus infection (Singh et al. 2001). Additional biomarkers representative of the chemistry occurring at sites of H. hepaticus-induced inflammation are clearly needed. 5. Effects of H. hepaticus on Gene Expression in Liver and Ceca of Infected Mice
Helicobacter hepaticus-associated cancer in A/JCr mice is a well-studied model of infectious liver cancer. H. hepaticusinfected male A/JCr mice are more prone to severe disease than female mice, but display a bimodal pattern of susceptibility. Hepatic global gene expression was profiled in male H. hepaticusinfected and control A/JCr mice at 3 months, 6 months, and 1 year using an Affymetrix-based oligonucleotide microarray platform (Boutin et al. 2004). The authors compared livers from infected male mice with severe preneoplastic lesions to uninfected mice and also to infected mice without any lesions/ disease. Quantitative real-time RT-PCR confirmed microarray results on selected genes. Model-based expression index comparisons generated by dChip (Li and Wong 2001) yielded consistent profiles of the most highly transcriptionally upregulated and downregulated genes for H. hepaticus-infected male mice with progressive disease versus uninfected control mice within each age group. The number of transcriptionally upregulated and downregulated genes temporally increased for all groups. Linear discriminant analysis and principal component analysis clearly allowed segregation of mice based on combined age and lesion status, or age alone. Transcriptionally downregulated genes in mice with liver lesions included those related to peroxisome proliferator pathways and the enzyme responsible for dihydrotestosterone catabolism, 3β-hydroxysteroid dehydrogenase V (Boutin et al. 2004). Genes associated with cholelithiasis were also transcriptionally altered in Helicobacterinfected C57L mice on a lithogenic diet, suggesting that different Helicobacter species may predispose to gallstone formation (Maurer et al. 2004). A/J mice may develop mild to moderate typhlitis after chronic infection with H. hepaticus (Fox et al. 1996b). To correlate histologic changes with proinflammatory influences from the infection, cecal tissues from A/JCr mice infected with H. hepaticus were compared for gene expression profiles to determine potential differences prior to the onset of inflammation (1 month) and after chronic inflammation was established for 3 months (Myles et al. 2003). One month postinfection, 25 genes
were upregulated and 3 were downregulated in contrast to upregulation of 31 and downregulation of 2 genes at the 3-month time point. A subset of proinflammatory genes, including IFN-γ, IP-10, MIP 1α, and serum amyloid A1, were among the upregulated genes (Myles et al. 2003). Helicobacter hepaticus-infected A/JCr female mice had higher inflammation scores in their ceca compared to infected male mice 3 months after experimental infection. This corresponded to higher levels of Th1 cytokines in ceca of female versus male A/JCr mice (Livingston et al. 2004).
B. Helicobacter bilis Helicobacter bilis was first isolated from the livers, bile, and intestines of aged, inbred mice in 1995 (Fox et al. 1995). H. bilis was identified by Warthin-Starry silver stain in livers with lobular and periportal chronic inflammation (Fig. 17-6, 17-7). H. bilis is a fusiform bacterium with periplasmic fibers and has 3 to 14 multiple bipolar-sheathed flagella (Fig. 17-8). H. bilis grows at 37°C and 42°C under microaerobic conditions and is urease, catalase, and oxidase positive. H. bilis has since been isolated from dogs, rats, and gerbils. As additional evidence that the wide host range of H. bilis potentially includes humans, H. bilis was detected by PCR and identified by 16S rRNA analysis in gallbladder and bile samples from Chileans with chronic cholecystitis (Fox et al. 1998a). More recently, it was associated with biliary tract and gallbladder cancers in two high-risk populations, Japanese and Thai (Matsukura et al. 2002). Like H. hepaticus, H. bilis infection of mice is associated with moderate to severe proliferative typhlitis and chronic active hepatitis in immunocompromised mice (Franklin et al. 1998). Coinfection with H. bilis and H. rodentium was associated with diarrhea in a breeding colony of SCID/Trp53−/− mice (Shomer et al. 1998). A third of the colony developed mucoid, watery, or severe hemorrhagic diarrhea with mortality. H. bilis and H. rodentium were isolated from microaerobic culture of feces or cecal specimens from affected mice and confirmed by PCR. Lesions consisted of rectal prolapse and proliferative typhlocolitis with focal ulcers in the cecum, colon, and rectum. Sentinel Swiss mice exposed to bedding from cages containing affected SCID/Trp53−/− mice acquired H. bilis and H. rodentium infection but did not develop clinical signs (Shomer et al. 1998). Helicobacter bilis infects mice worldwide and recently has been associated with hepatitis in outbred Swiss Webster (SW) mice (Fox et al. 2004). SW mice were cultured for the presence of H. bilis in the intestine and liver and analyzed as to whether infection status was associated with H. bilis seroconversion, and/or hepatitis. H. bilis was cultured from the colon of all mice, but only from the liver of one 12- to 13- month-old female mouse. Livers were H. bilis PCR positive, particularly those of older mice. PCR for H. bilis was negative, and H. bilis was not cultured from control mice. Histopathologic lesions of the liver in H. bilis-infected outbred mice ranged in severity from minimal
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Fig. 17-6 High-power view of a focus of inflammation in a mouse H. bilis-infected liver depicting the lymphocyte and lipid- and pigment-laden macrophage composition of these foci. The stain used was hematoxylin and eosin. Magnification, 300×. Reproduced with permission from Dr. Fox et al. (Fox et al. 1995).
to moderately severe for lobular and periportal chronic inflammation. H. bilis infection was statistically associated with increased portal inflammation mice in comparison to age-matched Helicobacter-free mice. A comparison of potential sex predisposition to liver disease indicated that H. bilis-infected female mice developed statistically more severe portal inflammation
than H. bilis-infected male mice. The authors concluded that H. bilis infection was associated with hepatitis in SW mice and therefore natural infection can confound experimental results (Fox et al. 2004). More recently, C57L mice infected with H. bilis and fed a lithogenic diet developed gallstones at a high frequency (Maurer et al. 2005).
Fig. 17-7 An example of H. bilis-like organisms (arrows) observed in mouse liver. The stain used was Warthin-Starry. Magnification, 750×. Reproduced with permission from Dr. Fox et al. (Fox et al. 1995).
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different animal facilities (Robertson et al. 2001). Isolates were unusual in that they only grew anaerobically at 37°C and were incapable of growth under microaerobic conditions. Like H. rodentium, isolates possessed single, bipolar, unsheathed flagella and were urease negative. H. ganmani is positive for oxidase and reduces nitrate to nitrite but does not hydrolyze hippurate or indoxyl acetate, and can be cultured on charcoal agar with resistance to cephalothin. Analysis of 16S rDNA sequence indicates that H. rodentium is the most closely related species with 98.2% similarity. Analysis of whole-cell proteins by SDS-PAGE for nine isolates differentiated H. ganmani from H. rodentium and other Helicobacter spp. (Robertson et al. 2001).
E. Helicobacter typhlonius
Fig. 17-8 Transmission electron micrograph of H. bilis (Hb1) illustrating different cell morphologies. Note that flagella are sheathed. Bar, 0.5 µm. Reproduced with permission from Dr. Fox et al. (Fox et al. 1995).
C.
Helicobacter rodentium
Helicobacter rodentium was the first urease-negative Helicobacter species isolated from laboratory mice (Shen et al. 1997). H. rodentium is spiral-shaped with bipolar, single, nonsheathed flagella. H. rodentium grows at 37°C and 42°C under microaerobic and anaerobic conditions, is urease negative, and only weakly positive for catalase and oxidase. In immunocompetent mice, H. rodentium appears nonpathogenic as part of commensal intestinal flora, but the pathogenic potential of H. rodentium has not been systematically studied. Coinfection with H. rodentium and H. bilis was implicated in an outbreak of diarrhea and typhlocolitis in a colony of SCID mice (Shomer et al. 1998). Experimentally, H. rodentium alone does not induce disease in A/JCr and SCID mice, whereas in combination with other murine Helicobacter it can exacerbate the disease (Myles et al. 2004). More recently, C57L mice coinfected with H. hepaticus and H. rodentium but not H. pylori, developed gallstones when fed a lithogenic diet, whereas uninfected mice fed the same diet had a very low incidence of gallstones (Maurer et al., 2005, 2006). D.
Helicobacter ganmani
Helicobacter ganmani sp. nov., a urease-negative anaerobe, was isolated from the intestines of laboratory mice at four
Helicobacter typhlonius, a urease-negative Helicobacter, was isolated from colonies of laboratory mice independently by two laboratories (Fox et al. 1999b; Franklin et al. 2001; Franklin et al. 1999) and has been detected in rat feces by PCR (Livingston and Riley 2003). Typhlocolitis in C.B-17 SCID mice was observed, but in contrast to mice infected with H. hepaticus (Li et al. 1998) or H. bilis (Franklin et al. 1998), lesions of chronic active hepatitis in C.B-17 SCID mice were not detected in mice inoculated with H. typhlonius (Franklin et al. 1999). IL-10−/− mice naturally infected with a helicobacter later identified as H. typhlonius developed diarrhea, perianal ulceration, intestinal bleeding, and rectal prolapse (Fox et al. 1999b). Helicobacter-free IL-10−/− mice were experimentally challenged with H. typhlonius, and typhlocolitis was reproduced (Fox et al. 1999b). H. typhlonius is closely related to H. hepaticus and has similar morphology with spiral shape and bipolar sheathed flagella. A large intervening sequence in its 16S rRNA gene and biochemical differences, including being urease negative, distinguishes H. typhlonius from H. hepaticus. H. typhlonius infection of research mice appears to be less common than H. hepaticus and H. rodentium and similar to the lower prevalence of H. bilis in colonies of mice in academic institutions (Whary et al. 2000b).
F.
Helicobacter muridarum
Helicobacter muridarum was first isolated from the intestinal mucosa of rats and mice (Lee et al. 1992; Phillips and Lee 1983). Natural H. muridarum infection of immunocompetent rodents has not been associated with clinical disease. Although the natural niche for H. muridarum is the lower bowel, H. muridarum, which is urease positive, has been associated with gastritis in mice, some of which had developed gastric atrophy that presumably promoted colonization of H. muridarum in the less acidic stomach (Queiroz et al. 1992). In a T cell transfer model of IBD in C.B-17 SCID mice, monoassociation with H. muridarum provoked typhlocolitis upon receipt of CD45RBhi CD4+ T cells from conventionally reared congenic BALB/c mice
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A
B
C Fig. 17-9 (A and B) Transmission electron micrographs of Helicobacter sp. flexispira (Lockard type 1) taken from a pure culture (A) and from an intestinal crypt in a mouse (B). Bipolar flagella and periplasmic fibers are apparent. (C) Scanning electron micrograph of Helicobacter sp. flexispira shows the organisms among intestinal microvilli in a mouse. Bars, 1 µm. Reprinted from (Schauer et al., 1993) with permission from the publisher (Solnick and Schauer 2001).
(Jiang et al. 2002). After cell transfer, C.B-17 SCID mice monoassociated with H. muridarum developed typhlocolitis within 5 to 6 weeks compared to 8 to 12 weeks in conventionally housed cohorts.
G.
rRNA gene sequence analysis. ‘H. muricola’ is a microaerophilic Helicobacter with a pair of nonsheathed bipolar flagella and is urease, catalase, and oxidase positive, reduces nitrate to nitrite, does not hydrolyze hippurate, is susceptible to nalidixic acid, and is resistant to cephalodin. Its pathogenic potential is unknown.
‘Helicobacter muricola’ H.
A novel enteric helicobacter was isolated from the cecum and feces of Korean wild mice (Mus musculus molossinus) (Won et al. 2002). This isolate was provisionally named ‘H. muricola’ based on phenotypic characteristics and 16S
‘Helicobacter mastomyrinus’
A spiral-shaped bacterium, with bipolar single-sheathed flagella, was isolated from the liver and cecum of clinically normal mastomys (the African rodent, Mastomys natalenis);
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A
from feces and ceca of normal mice, and also from the cecum of a p53−/− C57BL mouse with proctitis. The bacterium grew at 37°C under microaerobic conditions, rapidly hydrolyzed urea, and was catalase and oxidase positive. It did not reduce nitrate to nitrite, and was resistant to cephalothin and nalidixic acid. Like many other enterohepatic Helicobacter spp., this organism expresses cytolethal distending toxin and causes cell distention in vitro. On the basis of 16S rRNA gene sequence analysis, RFLP, and rep-PCR analysis, the organism was classified as a novel Helicobacter sp. Although H. mastomyrinus, like H. hepaticus and H. bilis, colonizes the inflamed liver of rodents, the pathogenic potential of this novel Helicobacter is unknown (Shen et al. 2005).
I.
B
‘Helicobacter rappini’
A novel bacterium with fusiform morphology, periplasmic fibers, and bipolar tufts of sheathed flagella was identified in the intestinal mucosae of laboratory mice (Schauer et al. 1993) (Fig. 17-9). The isolate grew under microaerobic conditions and was urease positive. On the basis of 16S rRNA gene sequence analysis, the organism was shown to be ‘Flexispira rappini,’ which is now referred to as ‘H. rappini.’ This Helicobacter is a member of closely related taxa (Dewhirst et al. 2000a).
V.
EXPERIMENTAL HELICOBACTER INFECTIONS IN MICE
A.
Mouse Models of Helicobacter-Associated Hepatitis and Hepatic Cancer
In experimental studies of H. hepaticus infection in A/JCr mice, the development of Helicobacter-associated lesions, particularly hepatitis, has been associated with the concomitant development of a significant serum IgG and Th1 cell-mediated immune response to H. hepaticus antigens (Whary et al. 1998), which are consistent with the robust response of IL-10−/− mice to H. hepaticus infection (Kullberg et al. 1998). Significant C
D
Fig. 17-10 Regulatory cells lacking IL-10 did not suppress inflammation or dysplasia when transferred either before or after H. hepaticus infection. (A) Infected untreated mice developed moderate to severe inflammation, hyperplasia, and dysplasia in the cecum and colon at 4 months after infection. (B) Inflammation and dysplasia were significantly suppressed after transfer of wild-type regulatory cells. (C) Regulatory cells lacking IL-10 were unable to suppress inflammation or dysplasia. Mice receiving IL-10−/− regulatory cells had an increased frequency of mucinous cancer. (D) Dense inflammatory infiltrate in the mucinous tumor (inset of C) comprised mainly of neutrophils (arrowheads) and a few macrophages. A–C: bar = 250 µm; D: bar = 25 µm. Reproduced with permission from Dr. Erdman et al. (Erdman et al. 2003b).
422 levels of mucosal IgA, systemic IgG, and T cell responses develop early postinfection but are ineffective in preventing chronic infection or development of lesions. The importance of host genetics is demonstrated by strain susceptibility to liver disease in mice. The predisposition of A/JCr to develop H. hepaticus-associated hepatitis contrasts with mild or no disease in C57BL/6 mice (Whary et al. 2001b). In this study, colonization of H. hepaticus in the cecum of experimentally infected A/JCr and C57BL/6 mice was quantified by use of real-time PCR analysis with primers for the H. hepaticus cdtB gene and mouse 18S rRNA. Colonization and lesions were evaluated after 6 months of H. hepaticus infection. Quantitative PCR analysis of H. hepaticus in cecal specimens indicated that C57BL/6 mice were colonized to a greater extent than were A/JCr mice. Consistent with prior reports, A/JCr mice developed more severe parenchymal necrosis, portal inflammation, and phlebitis in the liver compared with mild disease observed in infected C57BL/6 mice. Thus, hepatitis in A/JCr mice caused by H. hepaticus infection is associated with significantly lower colonization levels of H. hepaticus in the cecum, compared with those of hepatitisresistant C57BL/6 mice. Host responses of A/JCr mice that limit cecal colonization with H. hepaticus may have important roles in the pathogenesis of hepatic lesions. Both H. hepaticus and H. bilis are members of the enterohepatic group of murine helicobacters and are urease positive, a purported virulence factor in common with H. pylori that depends on urease activity to survive in the acidic environment of the stomach (Clyne et al. 1995). Urease was shown to be critical for colonization of the ferret stomach with H. mustelae (Andrutis et al. 1995), and in vivo complementation of ureB restores the ability of isogenic H. pylori urease mutants to colonize mice (Eaton et al. 2002). H. pylori urease has also been shown to stimulate significant increases in macrophage iNOS expression and nitric oxide (NO) production, implicating it in NO-dependent mucosal damage and carcinogenesis (Gobert et al. 2002). In contrast to gastric helicobacters that are all urease positive, the functional role of urease in enterohepatic helicobacters is not clear because the natural niche of the cecum and colon is not acidic and many members of the enterohepatic group of helicobacters are urease negative. The urease gene of H. hepaticus has been cloned and shown to contain a homolog of each gene in the H. pylori urease cluster (Beckwith et al. 2001). Isogenic urease mutants of H. hepaticus were compared to the wildtype parental strain for viability in a low pH environment in vitro and for ability to colonize outbred Swiss Webster mice (Ge et al., unpublished data). The urease mutants, despite being susceptible to acid killing, were still able to infect outbred Swiss Webster mice by oral gavage. These results indicate that urease activity is not essential for intestinal infection by H. hepaticus. Early exposure of A/JCr mice to H. hepaticus infection induced the most severe hepatitis and preneoplastic changes in the liver, particularly in male mice (Rogers et al. 2004).
J A M E S G . F O X A N D M A R K T. W H A RY
Mouse pups were exposed to H. hepaticus by oral infection of the pregnant female, and/or weanlings born to infected and uninfected dams were experimentally re-dosed with H. hepaticus at 3 or 12 weeks of age. Effects of gestational exposure to H. hepaticus were not significant, but male A/J offspring infected by 3 weeks of age developed the most severe hepatitis while those first infected at 12 weeks of age were hepatitisresistant. Interestingly, males were most prone to severe disease compared to females, and males in this study displayed a bimodal pattern of susceptibility as previously reported (Fox et al. 1996b). The most severe lesions in males included lobular necrogranulomatous and interface (chronic active) hepatitis, while females were more prone to develop portal (chronic persistent) hepatitis. Hepatic bacterial load and precursor lesions of hepatocellular carcinoma, including clear and tigroid cell foci of altered hepatocytes, were strongly associated with lobular hepatitis severity (Rogers et al. 2004). A urease-negative Helicobacter sp. isolated from a wild mouse induced proliferative typhlocolitis in defined flora SCID mice (Tac:ICR:HascidfRFscid) accompanied by severe hemorrhagic diarrhea, weight loss, phlebothrombosis, and hepatitis (Shomer et al. 2001). Infected A/J mice did not develop clinical signs but had mild to moderate proliferative typhlocolitis and moderate to marked cholangiohepatitis at 7 and 24 weeks postinfection. This same novel helicobacter isolate was used to examine the ontogeny of a characteristic lesion of H. hepaticus-induced hepatitis in the A/J mouse (Shomer et al. 2003). H. hepaticusassociated hepatitis is characterized by random distribution of multifocal accumulation of mononuclear inflammatory cells around small intralobular hepatic venules. These foci of inflammatory infiltrates resemble tertiary lymphoid development that has been observed in other models of chronic autoimmunity or inflammation and are thought to arise by a process termed lymphoid organ neogenesis. The small high endothelial venules in inflammatory lesions in A/J mice infected with the novel helicobacter were positive on immunohistochemistry for features typical of lymphoid organ neogenesis that included peripheral node addressin and the mucosal addressin cell adhesion molecule. The chemokines SLC (CCL 21) and BLC (CXCL13) were also present, as were B220+ B cells and the naïve T cell subset that expresses CD45loCD62Lhi. These findings suggest that lymphoid aggregates observed in lesions of helicobacterinduced chronic active hepatitis arise through the process of lymphoid organ neogenesis (Shomer et al. 2003). B.
Mouse Models of HelicobacterAssociated IBD-like Disease
The roles of the immune response in either limiting colonization or in contributing to the intensity of the inflammatory response to helicobacter infections that can progress to cancer in mouse models have been widely investigated (Rogers and Fox 2004). Soon after the discovery of H. hepaticus in 1991,
17. HELICOBACTER INFECTIONS IN MICE
case reports associated H. hepaticus infection with inflammatory bowel syndromes in nude and SCID mice as well as other immune dysregulated mice, manifested as rectal prolapse accompanied by chronic proliferative typhlitis, colitis, and proctitis (Foltz et al. 1998; Ward et al. 1996). Infected A/JCr mice produce predominantly IgG2a serum antibodies to H. hepaticus, which is consistent with a Th1 immune response, as reported for humans infected with H. pylori and mice with H. felis. Spleen mononuclear cells from infected A/JCr mice proliferate in vitro to H. hepaticus antigens and produce more IFN-γ than IL-4 or IL-5, also characteristic of a Th1 immune response (Whary et al. 1998). The Th1 immune response observed in the ceca of H. hepaticus-infected A/JCr mice is also supportive of the proinflammatory response noted previously in H. hepaticus-infected A/JCr mice (Livingston et al. 2004). Murine models such as IL-10−/− mice, which develop Th1mediated typhlocolitis, mimic some features of the pathogenesis associated with human IBD. Epithelial hyperplasia and progression to dysplasia with neoplastic sequelae have been associated with dysregulated responses to enteric flora, including H. hepaticus, in certain strains of mice (Berg et al. 1996; Erdman et al. 2003a; Whary et al. 2001a). The most susceptible strains of IL-10−/− mice develop severe spontaneous typhlocolitis when maintained under conventional housing conditions. The importance of genetic background has been highlighted by evidence that severity of typhlocolitis in response to enteric flora varies with mouse strain. Most severe disease developed in IL-10−/− 129/SvEv and IL-10−/− BALB/c strains, intermediate severity was noted in the IL-10−/− 129 x C57BL/6J outbred crosses, and least severe disease was observed in IL-10−/− C57BL/6J mice (Berg et al. 1996). The associated chronic inflammation and epithelial dysplasia progresses to colorectal adenocarcinoma by 6 months of age in a high percentage (60%) of IL-10−/− 129/SvEv mice (Berg et al. 1996). It is presumed but not stated in the paper that these mice were infected with Helicobacter spp. IL-10−/− mice experimentally infected with H. typhlonius (Fox et al. 1999b) and most notably H. hepaticus (Kullberg et al. 1998) develop IBD-like clinical signs, including diarrhea, perianal ulceration, intestinal bleeding, and rectal prolapse (Berg et al. 1996; Fox et al. 1999b; Kuhn et al. 1993). The cecal-colic junction and distal colon are most severely affected with mucosal ulceration and focal transmural inflammation characterized by extensive infiltration of the lamina propria with lymphocytes, plasma cells, macrophages, and scattered neutrophils. IL-10−/− C57BL/10J mice coinfected with the murine nematode Heligmosomoides polygyrus, which induces a Th2 host response, and H. hepaticus, which induces a Th1 response, developed less severe typhlocolitis and epithelial hyperplasia than IL-10−/− mice infected with H. hepaticus alone (Whary et al. 2001a). The reduction in mucosal damage in the H. polygyrus / H. hepaticus coinfected IL-10−/− mice was associated with suppressed Th1 responses to H. hepaticus, despite increased colonization of H. hepaticus in the cecum, typical of a Th2 bias toward helicobacter infection. The ameliorating effect of parasitism on the
423 mucosal damage associated with H. hepaticus in the IL-10−/− mouse IBD model supports prior findings that parasitism can reduce helicobacter-associated gastric tissue damage (Fox et al. 2000a). Efforts to define the relevant contributions of innate immunity versus adaptive immunity to IBD have taken advantage of the ability of H. hepaticus to reproducibly cause IBD-like disease in immunodeficient mice such as the SCID C.B-17 strain (Cahill et al. 1997; Maloy et al. 2003), T cell receptor knockouts (Burich et al. 2001; Chin et al. 2000), NF-kappa B-deficient mice (Erdman et al. 2001), RAG2−/− mice (Erdman et al. 2003b), and IL-10−/− mice (Kullberg et al. 2002; Kullberg et al. 1998). SCID mice develop typhlocolitis upon adoptive transfer of CD4+ T cells expressing high levels of CD45RB, a phenotype of naïve T cells. Germfree SCID mice do not develop inflammation upon cell transfer, indicating that a response to the intestinal flora is key to the model. When SCID mice were experimentally infected with H. hepaticus, the development of typhlocolitis was accelerated upon reconstitution with CD45RBhighCD4+ T-cells (Cahill et al. 1997). Transfer of CD45RBlowCD4+ T cells that contain a regulatory T cell subset expressing the CD25 IL2 receptor did not cause inflammation and suppressed the inflammatory response mediated by the CD45RBhighCD4+ T cells when co-transferred. The mechanism(s) for immune suppression by CD4+CD25+ T regulatory cells was further studied in H. hepaticus-infected RAG−/− mice that lack functional B and T cells. In this model, H. hepaticus infection elicited both T cell-mediated and T cell-independent intestinal inflammation, both of which were inhibited by adoptively transferred CD4+ CD25+ regulatory T cells. T cell-independent pathology was accompanied by activation of the innate immune system that was also inhibited by CD4+CD25+ cells. Suppression of innate immune pathology was dependent on T cell-derived IL-10 and also on the production of TGF-β (Maloy et al. 2003). Lacking functional B and T cells, the inflammatory response of RAG2−/− mice to H. hepaticus is mediated by the innate immune system. RAG2−/− mice naturally infected with H. hepaticus developed severe colitis, but IL-7−/− RAG2−/− mice colonized by the same flora did not develop spontaneous colitis, suggesting that IL-7 plays a critical role in exacerbating a non-T cell/non-B cell-mediated chronic inflammatory response (von Freeden-Jeffry et al. 1998). Administration of rIL-10 was able to prevent the occurrence of colitis in susceptible mice, suggesting a pivotal role for macrophages (von Freeden-Jeffry et al. 1998). H. hepaticus-infected RAG2−/− mice on the 129/SvEv background were reported to rapidly develop colitis and large bowel carcinoma (Erdman et al. 2003a). Adoptive transfer with CD4+CD45RBlowCD25+ regulatory T cells into these mice significantly inhibited H. hepaticus-induced inflammation and colon cancer. Regulatory cells lacking anti-inflammatory cytokine IL-10 were unable to inhibit inflammation, dysplasia, or cancer, indicating that IL-10 was required for the protective effects of lymphocytes in these experiments and that the regulatory T cells expressing IL-10
424 had an anti-inflammatory effect on the innate immune system (Figure 17.10) (Erdman et al. 2003b). IBD syndromes in humans such as Crohn’s disease and ulcerative colitis are idiopathic, but data from germfree studies have clearly demonstrated the role of intestinal flora in stimulating the host response to bacterial antigens. In the IBD model of H. hepaticus infection in IL-10−/− mice, the aberrant, Th1-driven inflammatory response (Kullberg et al. 1998) was demonstrated to be T cell-specific for helicobacter antigens (Kullberg et al. 2003). Regulatory T cells also develop in response to specific helicobacter antigens as these cells transferred from H. hepaticusinfected, but not uninfected, wild-type mice prevented H. hepaticus-induced typhlocolitis in IL-10−/− recipient mice (Kullberg et al. 2002). Thus, H. hepaticus infection of donor wildtype mice was required for induction of regulatory function. Interestingly, in this model the disease-protective CD4+ T cells were contained within the CD45RBlow fraction (as others have demonstrated) but also were variable in expression of CD25, considered a hallmark phenotype of T regulatory cells in many studies. T regulatory cell effects were even more potent in the CD25− subpopulation of cells. The suppressive role of IL-10 was shown by production of IL-10 by H. hepaticus-stimulated CD4+CD45RBlow cells from infected wild-type mice in vitro with concurrent suppression of IFN-γ production by IL-10−/− CD4+ cells. In addition, IL-10-mediated regulatory cell effects were demonstrated by treatment in vivo with anti-IL-10R antibody, which blocked the ability of transferred T regulatory cells to prevent colitis in H. hepaticus-infected IL-10−/− mice. The attribution of T regulatory cell function to the CD25− subset of CD4+CD45RBlow cells illustrates that protective effects are likely impacted by timing of cell transfer in relationship to onset of helicobacter infection in donor as well as recipient mice and established dose effects related to the number and purity of transferred cell subsets. NF-kappaB is a transcription factor activated during the inflammatory response. Thus, it was unexpected that spontaneous colitis would develop in mice lacking the p50 subunit and having only one allele of the p65 subunit of NF-kappaB (p50−/− p65+/−) (Erdman et al. 2001). These mice were rederived into a helicobacter-free health status, and the phenotype of spontaneous colitis decreased dramatically. Experimental infection of NF-kappaB p50−/−p65+/− mice with H. hepaticus caused severe colitis to develop within 6 weeks, was milder in control genotypes such as p50−/−p65+/+, p50+/+ and p65+/− and absent in wildtype mice. This suggests that the p50 and p65 subunits of NFkappaB have a role in inhibiting the development of colitis, potentially through control of proinflammatory IL-12. It was further demonstrated that NF-kappaB activity is required within the innate immune system to inhibit H. hepaticus-induced colitis and that NF-kappaB regulatory activity is mediated, at least in part, through inhibition of the expression of the p40 subunit of IL-12 (Tomczak et al. 2003). Together, these studies and others highlight the intriguing potential to treat human IBD with manipulation of regulatory
J A M E S G . F O X A N D M A R K T. W H A RY
T cell subpopulations and indicate the value of mouse models in identifying mechanisms for rational design of novel therapies. 1.
Experimental Infection Using H. trogontum
NIH outbred germfree mice experimentally infected with H. trogontum were persistently colonized at different levels of the gastrointestinal tract when necropsied at 6 weeks postinoculation (Moura et al. 1999). All infected mice developed histological inflammation in at least one region of the bowel, including gastritis. The predominant histological change observed was a moderate diffuse inflammatory infiltrate of mononuclear cells in the lamina propria, often accompanied by a mild infiltration of polymorphonuclear cells. Two animals presented focal infiltration of inflammatory cells in the liver, although no bacteria could be demonstrated. Chronic H. trogontum infection of NIH outbred germfree mice that were subsequently housed in open cages over 18 months resulted in focal infiltration of inflammatory cells in the liver, although no bacteria could be recovered by culture (Moura et al. 2003). In addition, no significant bowel lesions were noted.
C.
1.
Gastritis Models
Helicobacter felis Gastritis
Isolation of H. felis from the cat stomach made it possible to model helicobacter gastritis in mice after it was shown that H. felis would colonize germfree mice (Lee et al. 1990) and gnotobiotic rats (Fox et al. 1991) and produce gastritis. The pattern and intensity of helicobacter-associated gastritis range from minimal in BALB/c mice to moderate in C3H to severe in C57BL/6 mice (Fox et al. 1996a; Mohammadi et al. 1996b). The robust inflammatory response of C57BL/6 mice to H. felis was opportune for further model development of helicobacter gastritis because the C57BL/6 background is commonly used for producing transgenic and knockout mice. H. felis infection in mouse models has been used to investigate gastric atrophy and cancer (Correa, 2003; Fox et al. 2003a; Fox et al. 2003b; Wang et al. 2000), the roles of both innate (Ismail et al. 2003a; Ismail et al. 2003b) and adaptive immune responses (Mohammadi et al. 1996a; Roth et al. 1999), colonization levels, and inflammatory responses of mice to H. felis. Vaccine strategies to prevent infection with H. felis (Jiang et al. 2003) or therapeutically ameliorate gastritis (Corthesy-Theulaz et al. 1995) are of considerable interest for application of vaccine development in humans. 2.
Helicobacter pylori Gastritis
The ability to genetically engineer the host response in mice is a powerful advantage in studying the basic mechanisms of
17. HELICOBACTER INFECTIONS IN MICE
H. pylori gastritis and its sequelae. Initial attempts to colonize mice with H. pylori were unsuccessful until the Sydney strain of H. pylori known as SS1 was isolated by dosing mice with a variety of fresh clinical isolates and evaluating long-term colonization (Lee et al. 1997). High levels of colonization were achieved in C57BL/6, BALB/c, DBA/2, and C3H/He mice, and persistent colonization of mice led to chronic active gastritis with progression to gastric atrophy. The capability of SS1 to colonize mice and cause gastritis has had significant impact on H. pylori research. SS1 is cag+ and vacA+ (cytotoxin associate gene and vacuolating cytotoxin positive, respectively). It should be noted that the functional status of cag+ in SS1 has been debated because several laboratories have reported that SS1 does not induce IL-8 secretion from human gastric epithelial cells in vitro (Crabtree et al. 2002). Recently, cag+ SS2000 H. pylori strain has been reported to cause significant gastritis in mice (Thompson et al. 2004). Investigators interested in cagA as a virulence factor and in particular, the significance of IL-8 as a proinflammatory mediator, must be aware that mice do not produce IL-8.
D. Helicobacter-Associated Gastric Cancer Epidemiological evidence in humans has linked H. pylori to the development of gastric cancer. Indeed, the World Health Organization has classified H. pylori as a Class I carcinogen (WHO 1994). H. felis infection in C57BL/6 mice leads to alterations in gastrin and somatostatin levels as the gastritis progresses to atrophy, characterized by loss of parietal and chief cells and impairment of gastric acid secretion (Dial et al. 2000). C57BL/6 mice fed a high-salt diet colonize to a greater level with H. pylori and develop more severe gastritis (Fox et al. 1999a). H. felis infection in insulin-gastrin (INS-GAS) transgenic mice that develop hypergastrinemia accelerates the development of gastric carcinoma (Wang et al. 2000). In the INS-GAS mouse model, male gastric tissue responded more rapidly and aggressively to H. pylori infection, a high-salt diet, and the combination when compared with females; this finding appears consistent with the greater incidence of gastric carcinoma in men. Only male INS-GAS mice infected with H. pylori developed in situ and intramucosal carcinoma (Fox et al. 2003a). These results showed that H. pylori can accelerate the development of gastric cancer in the INS-GAS mouse model and that salt has less of a procarcinogenic effect in the setting of endogenous hypergastrinemia. This same model of INS-GAS mice infected with H. pylori, or an isogenic H. pylori cagE mutant of the cag pathogenicity island, developed atrophy, intestinal metaplasia, and dysplasia by 6 weeks and carcinoma by 24 weeks (Fox et al. 2003b). Inactivation of cagE delayed the progression to carcinoma, but neoplasia ultimately developed in all males infected with the H. pylori mutant. These studies highlight the importance of using both sexes to investigate the pathogenesis of H. pylori.
425 H. pylori infection and adenomatous polyposis coli (Apc) gene mutations have been linked to gastric cancer in humans. C57BL/6 and Apc1638 heterozygous mice infected with H. felis were studied for 7.5 months (Fox et al. 1997). The infected Apc1638 mice had less epithelial proliferation and gastritis, lower serum IgG responses, and higher bacteria and urease scores than infected C57BL/6 mice. The Apc1638 truncating mutation led to gastric dysplasia and polyposis of the antrum and pyloric junction, but H. felis infection did not increase the risk for gastric neoplasia. This study suggested that this Apc mutation is associated with decreased immune, inflammatory, and gastric hyperplastic responses to H. felis infection, indicating the possibility of a novel role for this tumor suppressor gene in the immune and local tissue responses to gastric bacterial infection. More recently, investigators have reported that Apc min+/− mice develop a progressive loss of immature and mature thymocytes from 80 days of age with complete regression of the thymus in these mice by 120 days of age (Coletta et al. 2004). There is also a partial depletion of splenic natural killer cells and mature B cells and progenitor cells in bone marrow due to complete loss of IL-7 dependent B cell progenitors. The lymphocyte depletion noted in Apc min+/− mice may in part be responsible for the reduced gastritis and hyperplasia seen in H. felis-infected mice. Heterozygous p53 mice infected with H. felis for up to 15 months also had a lower risk for helicobacter-associated cancer (Fox et al. 2002b). Antral inflammation and submucosal invasive foci were observed in wild-type C57BL/6 mice accompanied by invasion of adjacent submucosal blood vessels by glandular epithelia in a subset of infected C57BL/6 mice. None of these lesions were observed in p53+/− mice, infected or not, and p53+/− mice had significantly higher helicobacter colonization consistent with an anti-inflammatory Th2 response. Proinflammatory Th1 responses were significantly higher in C57BL/6 mice compared to p53+/− mice. These results suggested that germ-line deletion of one p53 allele resulted in a downregulated Th1 response to gastric helicobacter infection, possibly related to T cell senescence, which indirectly would be protective against the development of gastric cancer and other epithelial-derived neoplasms associated with chronic inflammation. In contrast, infection with H. felis in TSG-p53/Big Blue mice (p53+/−) resulted in enhanced gastritis and a threefold increase in mutations after 7 months compared to wild-type mice (Jenks et al. 2003). TSG-p53/Big Blue mice (p53+/-) infected with H. felis or H. pylori for 6 months developed severe gastritis that was associated with increased iNOS expression and a 4-fold and 1.7-fold higher rate, respectively, of mutation compared to wild-type mice (Touati et al. 2003). After 12 months of infection, gastric hyperplasia was prominent, but the mutagenic effects and iNOS expression decreased in H. pylori- and H. felis-infected mice. These data suggest a synergistic action between infection with gastric helicobacters and p53 deficiency in the accumulation of mutations within gastric tissue. Our laboratory reported Th1-promoted gastric atrophy secondary to H. felis infection in mice was ameliorated by the Th2
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host response to the murine helminth, Heligmosomoides polygyrus (Fox et al. 2000a). These results suggest that the low incidence of gastric cancer in African populations with a high incidence of H. pylori infection is attributable, in part, to a high prevalence of concurrent parasitic infection in Africans that tempers the host response to H. pylori. More recently, using BALB/c infected with H. felis and Toxoplasma gondii, enhanced tachyzoite replication resulted in severe organ-specific tissue damage, whereas BALB/c mice mono-infected with T. gondii developed no significant disease (Stoicov et al. 2004). In addition, coinfection with T. gondii altered the H. felis-specific immune response converting a previously resistant, BALB/c mouse (i.e., Th2 response), to a susceptible Th1 phenotype with elevated proinflammatory gastric cytokines and severe gastric inflammation (Stoicov et al. 2004). BALB/c mice infected with H. felis for up to 22 months developed B cell lymphoid follicles in the gastric wall that were reminiscent of MALT lymphoma of humans infected with H. pylori (Enno et al. 1995). Others have also shown that BALB/c mice infected with H. pylori cag– strain SS2000 for 15 months developed aggregates of gastric lymphoid tissue compared to mice infected with the cag+ H. pylori SS1 strain (Thompson et al. 2004).
VI.
INTERFERENCE WITH RESEARCH
ATTRIBUTABLE TO HELICOBACTER INFECTIONS OF RODENTS The discovery of H. hepaticus was the result of an investigation into an abnormally high incidence of hepatic tumors and hepatitis in control A/JCr mice that were part of a long-term carcinogenesis study (Ward et al. 1994b). Subsequent to the isolation and characterization of H. hepaticus, a review of the impact of H. hepaticus infection on 12 National Toxicology Program two-year carcinogenesis studies was published in 1998 (Hailey et al. 1998). Male and female B6C3F1 mice from all 12 studies were naturally infected with H. hepaticus with associated hepatitis in many of the male mice from 9 of these studies. There were increased incidences of hepatitis and hepatocellular and hemangiosarcoma of the liver in H. hepaticus-infected control male B6C3F1 mice compared with uninfected control males. Typical of the chronic hepatitis attributable to H. hepaticus, the livers from infected males had increased hyperplasia and apoptosis. Interestingly, H-ras codon 61 CAA to AAA mutations were less common in liver neoplasms from infected males than historical and study control samples. In inhalation toxicity and carcinogenicity studies of cobalt sulfate, male B6C3F1 mice had liver lesions consistent with a H. hepaticus infection and an increased incidence of hepatic hemangiosarcomas. Because of the confounding infection with H. hepaticus, no conclusion could be reached concerning an association between liver hemangiosarcomas and cobalt sulfate (Bucher et al. 1999).
Helicobacter hepaticus contamination of nonfrozen transplantable human tumors was infectious to SCID mice via subcutaneous injection of infected tissue (Goto et al. 2001). Contaminated but cryopreserved samples were not infectious. This is the first report suggesting that H. hepaticus has the ability to spread via biomaterials and that freeze-thawing is able to reduce the numbers of organisms to levels insufficient for inadvertent infection of immunodeficient mice. H. bilis infection was demonstrated to accelerate, and H. hepaticus infection to delay, the development of (otherwise) expected spontaneous colitis that develops with age in multiple drug resistancedeficient (mdr1a−/−) mice (Maggio-Price et al. 2002). The cause of spontaneous colitis has been attributed to a lack of P-glycoprotein that contributes to a presumed epithelial cell barrier defect in mdr1a−/− mice. Experimental H. bilis infection induced diarrhea, weight loss, and typhlocolitis in mdr1a−/− mice within 6 to 17 weeks postinoculation and before the expected onset of spontaneous typhlocolitis. In contrast, H. hepaticus infection delayed the onset of disease, and spontaneous colitis was also less severe. Similar effects were reported in IFN-γ −/− mice naturally coinfected with H. hepaticus and mouse hepatitis virus (MHV). Clinical disease developed that included pleuritis, peritonitis, hepatitis, pneumonia, and meningitis. Experimental infection with H. hepaticus alone did not produce clinical signs, but when mixed with MHV challenge, H. hepaticus infection appeared to reduce the severity of MHV-induced lesions during the acute phase of infection, and exacerbated hepatitis and meningitis at a later point in time (Compton et al. 2003). Importantly, the subclinical effects of helicobacter infection may confound research. For example, as described in the section Mouse Models of Helicobacter-Associated IBD-like Disease, altered gene expression in cecal tissues prior to the development of any features of pathology attributable to H. hepaticus infection was reported in A/JCr mice (Myles et al. 2003).
VII.
EPIZOOTIOLOGY
Recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barriermaintained mouse colonies (Fox et al. 1998b, 2004; Nilsson et al. 2004; Shames et al. 1995). Furthermore, H. hepaticus and H. bilis (and probably other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and are readily detected in feces. These results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies. In addition, H. hepaticus has been recovered from the fetuses of pregnant SCID mice, suggesting that in utero transmission of the organism is possible (Li et al. 1998). Limited epidemiology studies in research colonies of mice have indicated that the most prevalent enterohepatic species of helicobacter in mice include H. hepaticus and H. rodentium, with fewer mice colonized with H. typhlonius or H. bilis
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(Livingston and Riley 2003; Whary et al. 2000b). The prevalence of more recently named and provisionally named novel helicobacter species in research or wild mice is unknown (Table 17-1). The significance of enterohepatic helicobacter infections in humans is actively being investigated, but to date, it has been substantiated to only a limited extent. Documented infections of humans with helicobacters that also naturally colonize rodents, such as H. cineadi, have implicated human contact with hamsters but not mice (Whary and Fox 2004a). Thus, the zoonotic potential of natural murine helicobacter infections appears limited. H. bilis appears to have the widest host range, and molecular evidence of H. bilis infection has been demonstrated in humans screened because of their high risk for gallbladder and liver disease and associated cancers (Fox et al. 1998a; Matsukura et al. 2002; Murata et al. 2004).
VIII.
DIAGNOSTIC METHODS A.
Culture
Diagnostic samples (feces, cecal scrapings, tissues) should be stored at –70°C in brain–heart infusion or Brucella broth containing 30% glycerol. For culture from the intestinal tract, samples are homogenized in 1 ml of PBS and filtered through a 0.45 µm filter to eliminate competitive commensals. For larger helicobacters such as H. bilis or H. trogontum, a 0.65 µm filter is used to aid in isolation. Filtrate from GI samples is cultured on blood agar supplemented with 1% trimethoprim, vancomycin, and polymyxin, and samples from liver can be plated directly onto blood agar. Plates are incubated at 37°C or 42°C under microaerobic conditions in vented jars (90% N2, 5% H2, and 5% CO2; alternatively, a 80:10:10 mixture of the same gases) for 3 to 7 days. Recently, however, H. ganmani was grown only under anaerobic conditions (Robertson et al. 2001). Plates should be held for 21 days before concluding there is no growth. Growth of murine helicobacters occurs as a mucoid film or lawn on plates and not in distinct colonies. Bacterial numbers can be expanded by inoculation into Brucella broth containing 5% fetal bovine serum and incubated for 24–48 hours on a rotary shaker. Many helicobacter species require moist agar for efficient growth.
B.
PCR
PCR methods for rodent helicobacters have been previously described (Beckwith et al. 1997; Riley et al. 1996; Shames et al. 1995). Samples for PCR should be collected aseptically and stored at –20°C prior to DNA extraction. PCR amplification of bacterial DNA extracted from feces can be performed using the QIAamp Tissue Kit (Qiagen Inc., Valencia, California) following the kit protocol for the isolation of nucleic acids
from blood. PCR assay for detection of helicobacter DNA in feces permits serial monitoring of individual or groups of laboratory rodents. Cecal scrapings obtained at necropsy are extracted for PCR using the Boehringer-Mannheim High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Indianapolis, Indiana) following the kit protocol for the isolation of nucleic acids from tissue. Primer sequences specific for H. hepaticus have been published; 5′-GCA TTT GAA ACT GTT ACT CTG–3′ (C68) and 5′-CTG TTT TCA AGC TCC CC–3′ (C69) yield an amplification product of 414bp. H. hepaticus has three urease structural genes (ureA, ureB, and ureC) that are highly conserved and are homologous to those of the gastric Helicobacter species. A RFLP assay using the nucleotide sequence of the ureAB gene sequence can be used in a PCR and RFLP assay for diagnosis of H. hepaticus (Shen et al. 1998). RFLP analysis has also been used to differentiate between multiple enterohepatic helicobacters (Riley et al. 1996; Shen et al. 2000). A fluorogenic nuclease PCR assay that eliminates post-PCR processing provided sensitive, specific, and high-throughput capacity for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in fecal DNA samples from rodents (Drazenovich et al. 2002). Because H. hepaticus does not grow in discrete colonies in vitro, standard limiting dilution methods for bacterial quantification of H. hepaticus are difficult to interpret. A real-time PCR assay has been developed to quantify H. hepaticus recovered from feces or cecal scrapings using primers for the H. hepaticus cdtB gene and mouse 18S rRNA (Ge et al. 2001c).
C.
Serology
Commercial serology for helicobacter infection of rodents is not available. Experimentally, helicobacter-specific serum IgG or mucosal IgA antibody responses by ELISA can be measured using a bacterial sonicate (Fox et al. 1996c), membrane digest preparations (Livingston et al. 1997; Whary et al. 1998), or recombinant antigen (Feng et al. 2002; Kendall et al. 2004; Livingston et al. 1999). The presence of infection is readily supported by positive ELISA serology to helicobacter antigens and has been shown to be an assay with sensitivity exceeding 90%, though with variable specificity, particularly if mice are infected concurrently with multiple Helicobacter spp. (Whary et al. 2000b). PCR analysis has been more sensitive than culture or serology in determining H. bilis infection status in experimental studies, particularly during the first month postinoculation because seroconversion can take several months or be undetectable (Hodzic et al. 2001; Shomer et al. 1997; Whary et al. 2000b). For example, Eight of eleven, 6- to 8- month-old mice infected with H. bilis seroconverted to H. bilis outer membrane antigen using ELISA (Fox et al. 2004). Efforts to improve serologic sensitivity and specificity have included identifying an immunodominant H. bilis-specific antigen by testing sera from infected
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mice against a H. bilis genomic DNA expression library (Feng et al. 2002). Immunoreactive clones contained sequences that encoded a predicted 167-kDa protein that identified H. bilis but not H. hepaticus-infected mice by ELISA. Western blot reactivity of sera from H. bilis-infected mice against select bands in H. bilis lysates was high in comparison to no reaction on immunoblots prepared from H. hepaticus, H. muridarum, or unrelated bacterial lysates. However, primers for sequences within the coding region of the 167-kDa protein amplified DNA from H. hepaticus and H. muridarum, indicating these species express homologous antigens. Recombinant protein fragments of the P167 protein were compared to H. bilis membrane extracts in an ELISA protocol to determine the seroconversion status of 76 mice naturally infected with H. bilis or H. hepaticus alone or with an unspeciated Helicobacter sp. (Kendall et al. 2004). Assay sensitivity was highest for the membrane extracts, but specificity of the assay was greatly improved using the recombinant P167 fragment proteins. Characterization of nine immunogenic polypeptides proteins in outer membrane preparations of H. bilis demonstrated that isolates from a variety of sources (mouse, dog, and rat) had similar protein profiles that were distinct from those of H. pylori (Ge et al. 2001a). Immunoblot cross-reactivity was limited to their flagellins. N-terminal sequences of the two membrane proteins had no homology with protein sequences available in public databases, indicating that H. bilis has a conserved, unique outer membrane protein profile that is distinct from that of H. pylori.
D.
Histology
Warthin-Starry or Steiner silver stains are used to localize H. hepaticus and H. bilis in the liver parenchyma and biliary system or gastric helicobacters in the stomach. Silver staining of the lower bowel is unrewarding because many other enteric organisms will also be visualized with a silver stain. In the liver of infected mice, bacteria appear localized between hepatocytes. Severity of the hepatic lesions appears to be associated with the relative abundance of bacteria in SCID mice; however, the opposite is true in immunocompetent mice. For example, in infected A/J mice, the organisms can be difficult to find in the liver. Transmission electron microscopy has demonstrated spiral bacteria with morphology consistent with H. hepaticus within bile canaliculi (Fig. 17-2). H. hepaticus in tissues can also be localized by immunofluorescence using polyclonal rabbit antiH. hepaticus sera (Fox et al. 1998b).
E.
Genomic Analysis and Diversity of H. hepaticus
Pulsed-field gel electrophoresis (PFGE) was used to establish genomic diversity in 11 strains of H. hepaticus obtained from geographically distant locations in the United States and Europe (Saunders et al. 1997). Isolates from three of four independent
U. S. sources had very similar PFGE patterns, suggesting relative genomic conservation as well as diversity. The balance of seven isolates from different areas in Europe differed significantly in PFGE patterns from those of the U. S. isolates and from one another. DNA fingerprinting could be useful in epidemiological studies of H. hepaticus and highlights the potential strain diversity within H. hepaticus. The genetic relationship between H. hepaticus, C. jejuni, and H. pylori was initially examined by comparative sequence analysis of an ordered cosmid library (Ge et al. 2001b). The complete sequence of H. hepaticus has since been published (Suerbaum et al. 2003). The analysis focused on the comparison between H. hepaticus, H. pylori, and C. jejuni, and provided insights into the diverse mechanisms of tissue tropism and pathogenesis of these three pathogens. H. hepaticus has a genome of 1,799,146 bp, predicted to encode 1875 proteins; approximately 50% have homologs in H. pylori. Importantly, H. hepaticus lacks almost all H. pylori-specific colonization and virulence factors, including adhesins, vacuolating cytotoxin A (vacA), and the cytotoxin-associated gene pathogenicity island (cagPAI). H. hepaticus has orthologs of C. jejuni adhesin PEB1 and the cytolethal distending toxin (CDT). Experimental infection with an isogenic stain of H. hepaticus lacking CDT did not promote typhlocolitis in IL-10−/− mice to the extent caused by wild-type H. hepaticus, suggesting a role for CDT in inflammation (Young et al. 2004). Importantly, H. hepaticus has a novel genomic island and putative pathogenicity island encoding the basic components of a type IV secretion system as well as other virulence protein homologs. Whole genome microarray analyses showed that this island is lacking in many isolates of H. hepaticus (Suerbaum et al. 2003). H. hepaticus predictably induces colitis, hepatitis, and hepatocellular carcinoma in susceptible strains of mice. Some investigators have reported lack of expected morbidity and lesion development in H. hepaticus infection studies (Dieleman et al. 2000). These differences in outcome may be related to the genetic diversity illustrated by these genomic studies.
IX. A.
TREATMENT
Antibiotic Therapy
No long-term, large-scale study has reported success in using antibiotics to eradicate helicobacters from a mouse colony. Several oral antimicrobial formulations have been evaluated for eradication of H. hepaticus (Foltz et al. 1995; Foltz et al. 1996). Amoxicillin- or tetracycline-based triple therapy (amoxicillin or tetracycline in combination with metronidazole and bismuth) given by oral gavage three times daily for weeks eradicated H. hepaticus in 8- to 10-week-old A/JCr mice. In a second study (Foltz et al. 1995; Foltz et al. 1996), A/JCr male mice received amoxicillin-based triple therapy in drinking water or by oral gavage, or received tetracycline-based triple
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therapy in the drinking water. DBA/2J female mice received amoxicillin-based triple therapy in a specially formulated dietary wafer or by oral gavage, or received enrofloxacin in drinking water. All treatments were given for a 2-week period. One month after treatment, H. hepaticus was recovered from the liver, cecum, or colon in mice receiving amoxicillin- or tetracycline-based triple therapy in drinking water but not in mice receiving amoxicillin-based triple therapy by oral gavage. H. hepaticus was not recovered from DBA/2J mice receiving amoxicillin-based triple therapy in dietary wafers or by oral gavage but was recovered from mice receiving enrofloxacin in water. Although these results indicate that amoxicillin-based triple therapy administered in the diet or by oral gavage could be effective in eradicating H. hepaticus, eradication of H. hepaticus was not successful in a breeding colony of immunodeficient RAG1−/− mice treated with amoxicillin-based triple therapy in dietary wafers or in mice of various genetic backgrounds that had received medicated wafers 4 months after manufacture. In SCID/Trp53−/− mice coinfected with H. bilis and H. rodentium, antibiotic treatment for 2 weeks with food wafers containing 1.5 mg of amoxicillin, 0.69 mg of metronidazole, and 0.185 mg of bismuth/mouse per day, previously shown to eradicate H. hepaticus in immunocompetent mice, controlled the diarrhea but did not eliminate H. bilis or H. rodentium infection (Shomer et al. 1998). Thus, immunocompetence of the mice and stability of antibiotic activity in wafers are necessary considerations before undertaking antibiotic treatment modalities (Foltz et al. 1996). Amoxicillin-based therapy was also shown to have efficacy in preventing hepatitis and typhlitis in SCID mice infected with H. hepaticus, but eradication data were not reported (Russell et al. 1995).
X.
COLONY MANAGEMENT OF HELICOBACTERFREE MICE A.
Principles of Helicobacter Eradication in Mice
Before the impact of helicobacter infection on the colony health of research mice was appreciated, the diagnostic laboratory at the MIT Division of Comparative Medicine frequently identified helicobacter-infected mice from both commercial and academic sources (Shames et al. 1995). Currently, mice received at MIT from noncommercial sources continue to be infected with Helicobacter spp. (Taylor et al. 2004). The majority of commercial vendors can now supply mice that are free of H. hepaticus and H. bilis, and by the same production methods (i.e., cesarean section, embryo transfer) mice should also be free of other Helicobacter spp. Natural infection with several murine Helicobacter spp. remains common in conventional mouse colonies because of horizontal transmission through fecal-oral contact (Livingston and Riley 2003; Whary et al. 2000b).
Because of the endemic nature of the infection, successful eradication strategies consist of restocking with helicobacter-free mice, closely adhering to husbandry practices demonstrated to prevent introduction and dissemination of helicobacter, and monitoring colony and sentinel mice to maintain the helicobacterfree health status. 1.
Restocking with Helicobacter-Free Mice
Commercial vendors should be selected based on the availability of helicobacter-free colonies and quality assurance practices that afford a reasonable guarantee that the helicobacter-free health status will be maintained and adequately monitored. A percentage of newly received animals should be tested by PCR of cecal scrapings or feces using genus-specific primer sets. The veterinary staff should develop a working relationship with the vendor to emphasize the importance of helicobacter-free mice and to encourage rapid communication should a break in health status be detected. “Gift mice” received from noncommercial sources can be assumed to be infected with one or more Helicobacter spp. until proven otherwise (Taylor et al. 2004). The best methods for maintaining a helicobacter-free barrier is to use only helicobacterfree vendors and ensure that gift mice be rederived into the facility via embryo transfer as standard policy. Although PCR screening is highly sensitive and has reasonable specificity, it requires skill to perform and is subject to false negatives. Thus, reliance on diagnostic screening of gift animals is subject to error. 2.
Embryo Transfer
Embryo transfer is the most efficient method of rederiving mice into a helicobacter-free (as well as other bacterial, viral, and parasitic agents) health status. It requires significant investment in technical skills and necessitates support colonies (helicobacterfree recipient females and vasectomized males) that must be monitored closely for changes in health status. For large institutions that import significant numbers of gift mice, an embryo transfer program in a separate quarantine facility can be very cost-efficient and yields the greatest assurance of maintaining helicobacter-free mice. 3.
Cross Fostering
Cesarean section with fostering onto helicobacter-free dams is less ideal than embryo transfer but has been successfully used in obtaining helicobacter-free offspring (Fox et al. 1999b; Singletary et al. 2003). Neonatal mice appear to acquire helicobacter infections soon after birth, and thus immediate transfer (within 24 hours) of pups to clean mothers is imperative. This method is labor intensive and inherently subject to failure from inadvertent contamination. H. hepaticus was cultured from fetal viscera of 2 of 11 pups sampled late in gestation from infected SCID/NCr females (Li et al. 1998), suggesting that transplacental infection of H. hepaticus is possible in immunodeficient
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mice. Cross fostering or cesarean section to derive helicobacterfree mice should be reserved for immunocompetent strains of mice. 4.
Colony Management
Fecal-oral spread is the route of natural acquisition of infection with helicobacters in rodents, and H. hepaticus (and others) can be transferred on soiled bedding (Livingston et al. 1998; Whary et al. 2000b). Thus, biocontainment necessary to prevent horizontal transmission of H. hepaticus within a mouse colony must prevent fecal-oral contact. Use of microisolator caging, transfer of mice with disinfected forceps, and a change order of uninfected mice before known infected mice or mice of unknown helicobacter status provide barriers to horizontal transmission (Whary et al. 2000a).
B.
Use of Sentinel Mice to Monitor for Helicobacter spp.
Outbred Tac: (SW)fBR sentinel mice have been used to monitor colonies by PCR and serology for infection acquired from colony mice shedding H. hepaticus, H. rodentium, and H. bilis (Whary et al. 2000b). The experimental design utilized an ongoing colony health surveillance program that required systematic transfer of dirty bedding from cages housing colony mice to cages housing sentinel mice. Screening colony mice by speciesspecific PCR of cecal scrapings indicated that H. hepaticus and H. rodentium were most prevalent, whereas a low incidence of H. bilis was noted. Concurrence of helicobacter infection detected by a PCR-based assay between colony mice and sentinel mice exposed via contact with dirty bedding was 82% for H. hepaticus, 88% for H. rodentium, and 94% for H. bilis. The antihelicobacter serum IgG ELISA demonstrated high sensitivity but low specificity in detecting helicobacter infection as early as one month postexposure to dirty bedding. Low sensitivity may be related to the high antigenic challenge that sentinel mice experience when chronically exposed to dirty bedding from a large number of colony mice. Thus, the high sensitivity of the serum ELISA predicts helicobacter infection, but the specificity of the assay was too low for reliable identification of helicobacter species, necessitating the use of culture and PCR for confirmation. Transmission of H. hepaticus infection to sentinel C57BL/6Ncr mice was detected by PCR as early as 2 weeks postexposure to dirty bedding twice a week (Livingston et al. 1998). By 4 weeks postexposure, all sentinels were PCR positive and by 6 weeks, all sentinel mice had seroconverted to H. hepaticus as measured by an IgG ELISA. Both of the published protocols for sentinel mice exposure (Livingston et al. 1998; Whary et al. 2000b) relied on well-defined conditions consisting of controlled amounts and frequency of dirty bedding exposure as well as which cages of colony mice were sampled. As for other infectious agents, management programs that depend on sentinel exposure to dirty bedding for reliable surveillance of the helicobacter infection
status of rodent colonies must standardize their methods and potentially validate them with controlled studies. Increased exposure to dirty bedding or testing an increased number of sentinel mice per time point may improve the sensitivity of surveillance programs that depend on similar protocols.
XI.
CONCLUSION
The genus Helicobacter has rapidly expanded owing to the isolation of new species from a wide range of animals. More novel helicobacters isolated from mice can be anticipated in the future. The genus now includes 26 formally named species as well as numerous other novel helicobacters currently being characterized. Continued study of the genus Helicobacter is yielding new information for varied scientific disciplines. The zoonotic potential of Helicobacter spp. has been established but is likely underreported. Advanced molecular techniques have given investigators the ability to study these fastidious organisms when culture has been difficult or appeared unachievable. Technological advances allowing evaluation of the host and bacteria using genetic and proteonomic analysis continue to evolve. These approaches will be applicable to the study of the pathogenesis between Helicobacter spp. and mice of various genetic backgrounds. Genomic studies of Helicobacter spp. and their interactions with murine hosts will undoubtedly shed light on critical genes and associated molecular events that could be targets for preventing or treating infectious diseases in the future. As examples, new insights are emerging concerning the dysregulated host response to enteric flora thought to be the basis for inflammatory bowel diseases of humans. Particularly noteworthy, mouse models of helicobacter-associated disease are strengthening the link between chronic infections, the associated tissue damage from chronic inflammation, and the progression of dysplastic lesions to cancer within the liver and gastrointestinal tract. As more is learned about the potential negative impact that Helicobacter spp. infection has on animals being used in biomedical research, the more imperative the maintenance of helicobacter-free research colonies will become.
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Chapter 18 Mycoplasma pulmonis, Other Murine Mycoplasmas, and Cilia-Associated Respiratory Bacillus Trenton R. Schoeb
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. M. pulmonis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties of the Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Bacterial Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Gross and Microscopic Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Host Range and Geographic Distribution . . . . . . . . . . . . . . . . . . . . . 2. Prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Immunofluorescence and Immunohistochemistry . . . . . . . . . . . . . . 6. Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. DNA Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Polymerase Chain Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Other Mycoplasmas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mycoplasma arthritidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mycoplasma neurolyticum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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D. Mycoplasma collis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Mycoplasma muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. “Grey Lung” Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. CAR Bacillus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Properties of the Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Bacterial Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Host Range and Geographic Distribution . . . . . . . . . . . . . . . . . . . . . 2. Prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Immunofluorescence and Immunohistochemistry . . . . . . . . . . . . . . 6. Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Polymerase Chain Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
Although once common, clinically evident murine respiratory mycoplasmosis (MRM), the respiratory disease caused by Mycoplasma pulmonis, essentially has been eliminated from most rodent colonies as a result of increased use of specific pathogen-free (SPF) mice and rats, barrier maintenance facilities and procedures, and generally improved husbandry. Nonetheless, M. pulmonis remains potentially important as a mouse pathogen. A recent survey showed that non-SPF mouse and rat colonies still are common among user institutions (Jacoby and Lindsey 1998). Evidence of mycoplasma infection was found in a significant proportion of such colonies and even in some barrier-maintained colonies. As a result, 25% of responding user institutions had infected colonies representing a risk of contamination to other colonies. The degree of risk is unknown but probably significant, because of the large and growing numbers of genetically engineered mice now maintained by, and shipped between, biomedical research institutions, and because many of the mice at risk have mutations that could alter resistance to infectious agents. Several other mycoplasmas also are known to infect mice, including Mycoplasma arthritidis, Mycoplasma neurolyticum, Mycoplasma collis, Mycoplasma muris, and the “grey lung” agent. These are not known to cause natural disease in mice. Infection with cilia-associated respiratory (CAR) bacillus appears to be rare in laboratory mice, and disease has been reported only in conventional mice. CAR bacillus respiratory disease resembles MRM closely in several respects and presents similar challenges in diagnosis and control.
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II. M. PULMONIS A.
History
Disease having the characteristics of MRM in laboratory rats was first reported by Hektoen (1915–1916). Nelson (1937a) first described the natural disease in mice. He called the disease “infectious catarrh” and attributed it to “coccobacilliform bodies” he identified in the tissues of affected mice (Nelson 1937b; Nelson 1937c). M. pulmonis also was isolated at about this time by others (Klieneberger and Steabben 1937; Klieneberger and Steabben 1940; Sabin 1941). Because pneumonia was not a consistent feature of the disease, Nelson believed the pneumonia, which he called “endemic pneumonia” or “enzootic bronchiectasis,” had a separate, viral etiology, and he referred to “infectious catarrh” and pneumonia together as “chronic respiratory disease” (Nelson 1955, 1963, 1967). Other investigators used other terms, so that eventually numerous different terms had been used (Cassell et al. 1973, 1979). However, in the 1960s and 1970s, several reports described reproduction of both upper respiratory tract disease and bronchopneumonia in SPF mice and rats inoculated with M. pulmonis (Atobe and Ogata 1974; Howard et al. 1978; Jersey et al. 1973; Kohn and Kirk 1969; Lindsey et al. 1971; Lindsey and Cassell 1973; Lutsky and Organick 1966; Organick et al. 1966; Taylor et al. 1977; Whittlestone et al. 1972). These studies established M. pulmonis as the etiologic agent, making MRM an etiologically specific disease name (Lindsey et al. 1983). Results of other studies showed that the variable expression of
1 8 . M Y C O P L A S M A S P P. A N D C A R B A C I L L U S
M. pulmonis disease is due, at least in part, to various organism, host, and environmental factors (discussed in Section II.E.3.a).
B.
Properties of the Agent
MYCOPLASMAS (Mycoplasma spp.) are among the smallest prokaryotes. Based on the lack of a cell wall and other characteristics, mycoplasmas and related organisms of the genera Ureaplasma, Entomoplasma, Mesoplasma, Spiroplasma, Acholeplasma, Anaeroplasma, and Asteroleplasma are placed in the class Mollicutes, from the Latin mollis (soft) and cutis (skin) (Razin et al. 1998). Mycoplasmas share the family Mycoplasmataceae, order Mycoplasmatales, with ureaplasmas (Ureaplasma spp.). Based on analyses of sequences of 16S rRNA, 5S rRNA, tRNA, and the complete genomes of several species, the Mollicutes are believed to have evolved from the ancestor of contemporary gram-positive bacteria having low G + C DNA, which include the genera Clostridium, Bacillus, Lactobacillus, Staphylococcus, Streptococcus, and Enterococcus (Chambaud et al. 2001; Dybvig and Voelker 1996; Glass et al. 2000; Himmelreich et al. 1996; Papazisi et al. 2003; Razin et al. 1998; Sasaki et al. 2002). This relationship is further supported by homologies among the predicted peptide sequences of proteins encoded by various mycoplasma genes and those of gram-positive bacteria. Major aspects of mycoplasma biology pertinent to M. pulmonis are summarized below. Further information is available in Razin et al. (1998), Dybvig and Voelker (1996), and Maniloff et al. (1992). M. pulmonis is classified as a member of the Mycoplasma fermentans group on the basis of its 16S rRNA sequence (Maniloff 1992). The organism has had several synonyms, including Murimyces pulmonis (Sabin 1941), Musculomyces histotropicus (Sabin 1941), Asterococcus pulmonis (Prévot 1961; Sabin 1941), Mycoplasma histotropicus (Sabin 1941; Tully 1965), and Mycoplasma mergenhagen (Euzéby 2004; Grace et al. 1965; Skerman et al. 1980). Members of the Mollicutes in general have small genomes, and those of mycoplasmas are particularly so, ranging from approximately 600 kbp to 1400 kbp as determined by pulsedfield gel electrophoresis (Herrmann 1992). The smallest known is that of Mycoplasma genitalium, at 580,070 bp as determined by sequencing (Fraser et al. 1995), whereas the genome of M. pulmonis is 963,879 bp (Chambaud et al. 2001). Thus, mycoplasma genomes are about one-third to one-tenth the size of those of common bacteria such as Escherichia coli and Pseudomonas aeruginosa. Mycoplasma genomes also typically have low G + C content, below 30% in most cases (Herrmann 1992), and utilize TGA to encode tryptophan rather than as a stop codon (Dybvig and Voelker 1996). In keeping with their small genomes, the metabolic capabilities of mycoplasmas are limited (Pollack 1992; Razin et al. 1998). Lacking a tricarboxylic acid cycle, quinones, and cytochromes, they cannot produce ATP by oxidative phosphorylation.
439 Many mycoplasmas, including M. pulmonis, produce ATP primarily by glycolysis. Growth of glycolytic or fermentative mycoplasmas in artificial medium results in acidification, which allows growth detection by use of a color pH indicator such as phenol red. Most nonglycolytic mycoplasmas, as well as a few glycolytic species, can produce ATP by arginine hydrolysis, although whether this actually is a primary means of ATP production is unclear. A few mycoplasmas do not metabolize either arginine or glucose and instead oxidize organic acids such as pyruvate and lactate. Mycoplasmas have purine and pyrimidine salvage pathways, but cannot synthesize purines and pyrimidines, and also are deficient in synthesis of amino acids, cofactors, fatty acids, and sterol (Razin et al. 1998). These organisms are thus obligate parasites and must obtain complex nutrients from the host in vivo. It is likely that mechanisms for doing so also contribute to mycoplasmal disease pathogenesis. In vitro, the organisms’ growth requirements must be met by complex culture media. A few mycoplasmas are reported to be capable of gliding motility, but the mechanism is unknown (Razin et al. 1998). This phenomenon was first recognized by Andrewes and Welch (1946) in a mouse isolate of M. pulmonis. Gliding motility of M. pulmonis also was described by Nelson and Lyons (1965), who reported observing it only in fresh isolates, and by Bredt and Radestock (1977). Motile M. pulmonis cells are reported to have a protruding, flexible “stalk” that can be somewhat thickened at the end and that imparts a flask-like shape to the cells (Kirchhoff et al. 1984). One of the most intriguing aspects of the biology of mycoplasmas is their ample capacity for the genetic variation necessary to adapt to their environment and resist host defenses, despite their small genomes (Dybvig and Voelker 1996). A major mechanism by which this is accomplished is by chromosomal gene rearrangements that enable coding for large numbers of related proteins, which are associated with antigenic and other changes that are likely to be related to host colonization and disease pathogenesis. For example, M. pulmonis undergoes high-frequency changes in expression of surface lipoproteins of the Vsa (variable surface antigen) family that are associated with variations in phenotype, including growth characteristics on agar media (Dybvig et al. 1989), hemadsorption and adherence to plastic surfaces (Simmons and Dybvig 2003; Watson et al. 1990b), and susceptibility to the mycoplasma virus P1 (Dybvig et al. 1988). The functions of Vsa proteins are not yet fully understood, but one function clearly is resistance to complement (Simmons and Dybvig 2003). Vsa protein expression varies both in vivo and in vitro, suggesting that Vsa proteins are related to adaptation to different environments. Vsa phenotype also has been linked to disease expression (Talkington et al. 1989; Watson et al. 1990a). Bacterial surface proteins containing variable repeat units often mediate such virulence factors as adherence to host cells, a likely function of Vsa proteins in M. pulmonis, inasmuch as the organism lacks a specialized attachment organelle or identified adhesins such as those of
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Mycoplasma pneumoniae and a few other mycoplasmas. The potential for variation among Vsa proteins could allow extensive diversity in surface antigen expression (Bhugra et al. 1995); thus, another function of Vsa proteins may be to provide a means of thwarting host immune responses. Vsa proteins are encoded by the vsa locus, and their expression is regulated by a unique site-specific DNA inversion system (Bhugra et al. 1995; Shen et al. 2000; Simmons et al. 1996). Variants occur at such high frequency that the M. pulmonis genome is one of the most variable known. Vsa variants always can be obtained from cultures of subclones. For example, minor expression of vsaB can be detected in subclones of vsaA-expressing parent clones and vice versa, indicating that M. pulmonis populations are always heterogeneous with respect to Vsa phenotype (Bhugra et al. 1995). Thus, variant subpopulations of the organism continuously arise, within the limits of the vsa gene repertoire of a particular strain. Vsa variation occurs in vivo in mice and seems to be associated with disease severity (Talkington et al. 1989). Vsa proteins or other surface molecules probably also are involved in virulence in the reproductive tract. After 50 in vitro passages, a virulent, hemadsorbing strain became nonhemadsorbing and failed to establish infection in most mice after intravaginal inoculation. In those mice in which it did establish infection, the isolates that recovered had regained hemadsorptivity (Taylor-Robinson and Furr 1985). M. pulmonis also expresses a family of restriction and modification (R-M) enzymes encoded by hsd (host specificity determinant) loci (Dybvig et al. 1998). Expression of R-M activity is regulated by rearrangement of an invertible element in the manner of the vsa locus, and hsd rearrangements frequently occur in association with vsa rearrangements (Bhugra et al. 1995). Another unusual feature of M. pulmonis is that inversions in both hsd and vsa loci are catalyzed by a single recombinase, HsvR. HsvR has dual specificity for both vsa and hsd recombination sites, which are not homologous (Sitaraman et al. 2002). The function of the variant enzymes induced by hsd rearrangements is not clear, but such rearrangements do occur in vivo (Gumulak-Smith et al. 2001).
et al. 1986), and UAB CT was derived from UAB T by passage in mice (Davidson et al. 1988c). Negroni was cultivated from medium from contaminated tissue cultures, and the origins of Ogata T, 66, and Peter C strains are unknown (Davidson et al. 1988c). M. pulmonis strains differ in infectivity and virulence for both mice and rats. Davidson et al. (1988c) compared pathogenicity of 18 strains for mice. In C3H/HeN mice, PG34(Ash), Ogata T, WRAIR, Nelson A, 7MC-A-1, JB, Kon, and PG22 were not infective. UAB 5782C, UAB 6510, UAB 8145D, UAB T, UAB CT, Ml, 66, Peter C, Nelson C, and Negroni established infections, but the median infectious dose varied over a range of 103 CFU to > 107 CFU. Only UAB CT and M1 produced grossly evident pneumonia. Strains UAB CT, UAB T, M1, UAB 5782C, and PG34(Ash) were further evaluated for pathogenicity in both C57BL/6N and C3H/HeN mice. Lesions varied both among mycoplasma strains and between mouse strains. UAB CT was the most virulent strain, with a dose of 107 CFU causing acute fibrinohemorrhagic pneumonia, and a dose of only 103 CFU consistently producing microscopic lesions. In contrast, UAB 5782C and UAB 6510, which are rat isolates, colonized the respiratory tracts of inoculated mice but caused minimal or no disease. Results of other studies also indicate that rat isolates such as UAB 5782C and UAB 6510 are less virulent for mice than rats, whereas UAB CT, a mouse isolate, is highly pathogenic for mice (Cartner et al. 1995, 1998; Davidson et al. 1988c; Davis and Cassell 1982; Davis et al. 1985b; Lindsey et al. 1971). Differences in virulence, including host preference, loss of virulence after repeated in vitro passage, and restoration of virulence upon in vivo passage, appear to be associated with Vsa protein expression, but the underlying mechanisms are not clearly understood. Antigenic relationships among M. pulmonis strains have not been extensively studied. However, most proteins are conserved among the strains that have been examined (Watson et al. 1987b), with the major variant proteins being those of the Vsa family (Bhugra et al. 1995).
D. C.
Bacterial Strains
Many strains of M. pulmonis have been reported. The type strain is PG34(Ash) (ATCC 19612), originally isolated and designated L3 by Klieneberger-Nobel (1937). Among the best studied strains are UAB 5782C, UAB 8145D, UAB T, UAB CT, Ogata T, Peter C, Ml, WRAIR, Nelson C, 7MC-A-1, Negroni, JB, Kon, and PG-22 (Davidson et al. 1988a, 1988b, 1988c; Watson et al. 1988). UAB 5782C, PG34 (Ash), WRAIR, JB, and Kon were isolated from rats, and UAB 8145D, UAB T, M1, Nelson C, 7MC-A-1, and PG22 were cultured from mice (Davidson et al. 1988c). UAB 6510 and UAB X1048 were isolated from rats experimentally inoculated with UAB 5782C (Brown and Steiner 1996; Cassell and Davis 1978; Williamson
Growth In Vivo and In Vitro
In vivo, M. pulmonis occurs primarily in the respiratory tract, where it colonizes the epithelium of the nasal passages, auditory (eustachian) tubes, and tympanic bullae preferentially, with extension to the larynx, trachea, bronchi, bronchioles, and lungs in more severe or advanced disease (Cassell et al. 1979; Cassell et al. 1973; Lindsey et al. 1983; Schoeb et al. 1996). The numbers of CFU attained in the lungs can reach 106 to 107 per lung in mice inoculated with a virulent strain such as UAB CT, whereas considerably smaller numbers, in some cases only a few colonies, may be recovered from the lungs of mice inoculated with avirulent or weakly virulent strains (Cartner et al. 1995, 1998; Davidson et al. 1988c). In rats, numbers of CFU of virulent M. pulmonis strains can reach 109 CFU per lung (Schoeb et al. 1982, 1985; Schoeb and Lindsey 1987).
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M. pulmonis also colonizes the female reproductive tract of mice (Banerjee et al. 1985; Casillo and Blackmore 1972; Hill 1974; Nelson 1954; Saito et al. 1976). In immunodeficient mice or mice of susceptible genotype, experimental inoculation via the nasal passages can lead to dissemination to the joints, spleen, and other organs (Berglof et al. 1997; Cartner et al. 1998; Evengard et al. 1994). The organism also has been isolated from the brain of naturally infected mice (Banerjee et al. 1985; Hill 1974; Saito et al. 1976), but the significance of this is unknown. Because of its metabolic limitations and nutrient requirements, cultivation of M. pulmonis in vitro requires use of specially formulated mycoplasma media such as Hayflick’s (Freundt 1983), SP-4 (Whitcomb 1983), or modified versions of these (Shen et al. 2000). Davidson et al. (1994) recommend Medium A for cultivation of murine mycoplasmas and provide protocols for broth and agar plate versions. M. pulmonis ferments glucose and thus acidifies culture media; therefore, media intended for growth of M. pulmonis and other fermentative mycoplasmas usually include a pH indicator such as phenol red. M. pulmonis does not grow in media intended for arginine-utilizing mycoplasmas unless such media also contain glucose. On agar, M. pulmonis colonies are pinpoint to a few millimeter in diameter and finely granular (Fig. 18-1). Color and opacity vary from translucent and nearly colorless to yellow-tan and almost opaque.
1.
Clinical Signs
Clinical signs are variable and may be absent. However, affected mice usually can be identified by careful observation for chattering, Nelson’s term for the characteristic sound thought to result from the accumulation of exudate in the nasal passages and from the thickening of the nasal mucosa. In Nelson’s outbreak, severely affected mice had weight loss, lethargy, rough coat, and dyspnea. Morbidity and mortality were high, with the loss of 95% of the mice over several months. Severe outbreaks observed by others resulted from the combined effects of M. pulmonis and Sendai virus (Lindsey et al. 1983). 2.
Gross and Microscopic Lesions
Signs and lesions of M. pulmonis disease have been reviewed previously (Cassell et al. 1973, 1979; Lindsey et al. 1983, 1991a; Lindsey and Cassell 1973; Schoeb et al. 1996; Simecka et al. 1992).
A. RESPIRATORY DISEASE Lesions in mice are generally similar to those in rats, although there are a few differences, as noted later. Development of the disease in mice appears to follow patterns similar to those in rats. Rhinitis is the primary lesion early in the course of the disease, and it is frequently accompanied by unilateral or bilateral otitis media. The disease progresses distally, leading to tracheitis and bronchopneumonia; however, this occurs inconsistently, so that pneumonia is less common than rhinitis and otitis media. Although the inflammation can later decrease in severity, the disease is chronic and persists for weeks to months in mice in which it is not fatal. Rhinitis frequently is bilateral but can be unilateral. Both olfactory and nonolfactory mucosae are affected, with differentiated epithelium becoming hyperplastic or being replaced by less differentiated “squamoid” cells in more severe lesions (Figs. 18-2 and 18-3). Also commonly observed, especially in acute disease, are epithelial syncytia (Fig. 18-2), which are not seen in rats. The lumina contain neutrophilic exudate.
Fig. 18-1 M. pulmonis strain UAB CT colonies on agar medium viewed by transmitted light. Main panel: Typical translucent colony morphology. Bar = 1 mm. Inset: Colony with dark sector representing appearance of a variant with increased opacity. Bar = 0.5 mm. (Adapted from images provided by W. L. Simmons)
Fig. 18-2 Ethmoid turbinate (above) and nasal septum (below) of a BALB/c mouse with M. pulmonis rhinitis. The lumina contain numerous neutrophils (N), and lymphocytes and plasma cells (L) have accumulated in the mucosa. The olfactory mucosa (O) on one side of the septum is extensively altered. Several syncytia are present in the epithelium (arrows). HE. Bar = 66 µm.
E.
In vivo
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Fig. 18-3 Lateral nasal mucosa of a B6D2F1 mouse with M. pulmonis rhinitis. The epithelium has lost its normal differentiation, and the cells have become flattened (arrow). In the underlying mucosa, the glands have been lost, and large numbers of plasma cells (P) have accumulated. HE. Bar = 33 µm.
TRENTON R. SCHOEB
Fig. 18-5 Left lung lobe of a BALB/c mouse with bronchopneumonia caused by M. pulmonis. The inflammatory process affects the mainstem bronchus (B) and its branches. Adjacent to affected airways are patches of suppurative pneumonia (P). HE. Bar = 1300 µm.
Lymphoid cells accumulate in the submucosa, and submucosal glands can be hyperplastic or lost if the inflammation is severe. Similar changes occur in the tympanic bullae, eustachian tubes, larynx, and trachea. The lumina of affected tympanic bullae typically are filled with neutrophilic exudate (Fig. 18-4). Extension to otitis interna and meningitis occurs in a few cases. Extension of the disease to the lung results in bronchopneumonia (Figs. 18-5–18-9). The distribution is variable; any or all lobes can be affected. Gross lesions, which are indistinguishable from those of CAR bacillus disease, are welldemarcated gray-purple areas of atelectasis, pneumonia, or both. Such areas may show elevated yellow-tan nodules representing
exudate-filled airways. In sections, neutrophilic exudate is evident in airway lumina. Epithelial changes are similar to those in the proximal respiratory tract, and syncytia occur occasionally. Although mice lack the prominent bronchus-associated lymphoid tissue (BALT) found in rats, mice develop extensive peribronchial and peribronchiolar accumulations of lymphoid cells similar to those in rats (Figs. 18-6 and 18-7). However, in mice, the cells are predominantly plasma cells rather than lymphocytes as in rats. Alveolitis can develop around affected airways and in some cases can involve entire lobes, with the alveolar lumina containing foamy macrophages, neutrophils, or
Fig. 18-4 M. pulmonis-induced otitis media in a BALB/c mouse. The tympanic bulla is filled with neutrophilic exudate (E). The mucosa (M) contains lymphocytes and plasma cells, and the epithelium is hyperplastic. The inflammation extends into the auditory tube (A). HE. Bar = 330 µm.
Fig. 18-6 Chronic bronchiolitis and bronchiectasis in a BALB/c mouse with M. pulmonis disease. The lumina (L) are dilated with neutrophils, and there is heavy peribronchiolar accumulation of plasma cells and lymphocytes (P). HE. Bar = 330 µm.
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Fig. 18-7 M. pulmonis-induced chronic bronchitis in a BALB/c mouse. There are neutrophils (N) in the lumen (N) and extensive peribronchial accumulation of plasma cells (P). The mucosa (M) is heavily infiltrated with lymphocytes, plasma cells, and neutrophils, and the epithelium has lost its normal differentiation. HE. Bar = 33 µm.
Fig. 18-9 Bronchopneumonia in a B6D2F1 mouse with concurrent infection with M. pulmonis and Sendai virus. The alveolar epithelium surrounding the affected airway has undergone hyperplasia and metaplasia (“adenomatoid” change). HE. Bar =130 µm.
both (Fig. 18-8). Severely affected airways can progress to bronchiectasis or bronchiolectasis (Fig. 18-6) and, occasionally, bronchial abscesses. In cases of concurrent Sendai virus infection, changes attributable to the virus can be present, such as the alveolar epithelial hyperplasia and metaplasia characteristic of sendai virus disease in DBA mice (Brownstein et al. 1981) (Fig. 18-9).
of naturally occurring oophoritis or perioophoritis caused by M. pulmonis in mice (Banerjee et al. 1985). However, others report isolating the organism from the vagina and uterus of naturally infected mice or mice infected by contact with experimentally inoculated mice (Hill 1974; Saito et al. 1976), and experimental reproductive tract or respiratory infection in mice can lead to oophoritis or perioophoritis (Cartner et al. 1998; Goeth and Appel 1974; Nelson 1954). In rats, natural infection can be associated with decreased fertility (Cassell et al. 1979), and experimental genital infection results in fetal infection, death, and resorption; smaller litter sizes; and decreased birth weight (Brown and Steiner 1996; Steiner and Brown 1993; Steiner et al. 1993). The potential for M. pulmonis to affect fertility in mice is unclear. Decreased fertility has not been reported in naturally infected mice. In most experimental studies, effects on fertility were not assessed, and in studies in which effects on fertility were evaluated, the organism was inoculated intraperitoneally (Goeth and Appel 1974) or intravenously (Taylor-Robinson et al. 1975). The risk of reproductive tract disease may be somewhat greater for immunodeficient mice, in which M. pulmonis tends to disseminate from the respiratory tract to other organs (Berglof et al. 1997; Bhugra et al. 1995; Cartner et al. 1998; Evengard et al. 1994). M. pulmonis could complicate procedures involving in vitro fertilization or manipulation of ova, inasmuch as mouse spermatozoa incubated with M. pulmonis had reduced ability to fertilize ova in vitro (Fraser and Taylor-Robinson 1977; Swenson 1982). M. pulmonis adheres to mouse ova and resists removal by washing (Hill and Stalley 1991).
GENITAL DISEASE Genital tract infection and resultant chronic suppurative oophoritis, perioophoritis, salpingitis, and endometritis are significant features of naturally occurring M. pulmonis disease in rats (Cassell et al. 1979), but there is only one report
Fig. 18-8 Pneumonia in a BALB/c mouse with M. pulmonis disease. Alveoli on the left contain primarily foamy macrophages, whereas those on the right are filled with neutrophils. HE. Bar = 66 µm.
POLYARTHRITIS There are numerous reports of experimental induction of polyarthritis by intravenous or intraperitoneal
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Fig. 18-10 Carpal arthritis in an experimentally inoculated C3H/HeN SCID mouse. The joint space contains fibrinopurulent exudate (E). The synovial cells have been destroyed, and the inflammatory process extends into the capsule and periarticular tissue (P). HE. Bar =130 µm. (From a histologic slide provided by S. C. Cartner)
inoculation of M. pulmonis, but naturally occurring arthritis has not been reported. However, the organism can disseminate to joints from the respiratory tract in experimentally inoculated B cell-deficient mice (Berglof et al. 1997) and mice with severe combined immunodeficiency (SCID) (Evengard et al. 1994). This also can occur in experimental infections in susceptible C3H/HeN mice (Cartner et al. 1998) (Fig. 18-10). C57LB/6 mice are resistant, but, when they are Fas or FasL deficient, they develop severe chronic arthritis (Hsu et al. 2001). M. pulmonisinduced arthritis is characterized initially by suppurative inflammation affecting primarily the carpal and tarsal joints (Harwick et al. 1976; Kono et al. 1980). Tendon sheaths also can be affected. As the process becomes chronic, lymphoid cell accumulation and synovial proliferation take place. The organism and accompanying inflammation can persist for weeks or months. Immunodeficient mice with chronic arthritis can have repeated recurrences of acute inflammation (Kono et al. 1980). 3.
Pathogenesis
FACTORS AFFECTING DISEASE EXPRESSION Several factors other than immunodeficiency are known to influence development of M. pulmonis disease. In rats, severity of M. pulmonis disease is affected by host genotype (Davis et al. 1982; McIntosh et al. 1992; Reyes et al. 2000), M. pulmonis strain (Schoeb et al. 1993b), exposure to ammonia (Broderson et al. 1976; Schoeb et al. 1982), concurrent infection with Sendai virus or sialodacryoadenitis virus (Schoeb et al. 1985; Schoeb and Lindsey 1987), advancing age, and experimental vitamin deficiencies (Tvedten et al. 1973). Severity in mice also is influenced by host genotype (Cartner et al. 1995, 1996; Lai et al. 1993, 1996; Tanaka et al. 1998). Davis et al. (1985b)
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showed that C57BL/6 and C3H/HeN mice inoculated with M. pulmonis strain UAB CT 14 days previously did not differ in severity of upper respiratory tract disease, but C3H/HeN mice were much more susceptible to pulmonary disease, having median lethal and lesion-inducing doses nearly 100-fold less than the doses for C57BL/6 mice. C3H/HeN mice had more severe changes in airway epithelium, more extensive alveolitis, and more severe accumulation of lymphoid cells than C57BL/6 mice. In another study, in which C3H/HeN and C57BL/6 mice were examined at weekly intervals after inoculation with M. pulmonis UAB CT, C3H/HeN mice had 102 to 105 more M. pulmonis CFU in their lungs than C57BL/6 mice, as well as more severe lung lesions (Cartner et al. 1995). C3H mice also are more susceptible to experimental M. pulmonis arthritis than C57BL/10 mice (Keystone et al. 1982). As in rats, disease severity in mice is affected by M. pulmonis strain (Cartner et al. 1995, 1996; Davis et al. 1985b; Lai et al. 1993), exposure to ammonia (Saito et al. 1982), and concurrent infection with Sendai virus (Howard et al. 1978; Saito et al. 1981). In addition, Yancey et al. report that in C3H/HeN mice inoculated with M. pulmonis UAB CT, males had significantly more severe alveolitis than females (Yancey et al. 2001). Development of experimental respiratory disease in mice, unlike rats, is highly dependent on the number of CFU inoculated (Lindsey and Cassell 1973). Inocula of less than 105 CFU result in mild upper respiratory tract disease, whereas higher doses induce severe fibrinopurulent and hemorrhagic pneumonia, which is fatal in many mice. Chronic bronchopneumonia, bronchiectasis, and bronchial abscesses develop in mice that survive the acute disease. Experimental infection of mice with Sendai virus prior to inoculation with M. pulmonis resulted in increased severity of pulmonary disease and increased numbers of M. pulmonis organisms in the lungs (Howard et al. 1978). Sendai virus infection also exacerbated existing M. pulmonis disease, a more likely scenario in natural infections. Outbreaks of disease observed by Lindsey et al. (1983) in which there was significant mortality were due to concurrent infection with M. pulmonis and Sendai virus. Affected mice had severe and extensive bronchopneumonia. Alveolar epithelial proliferation and secretory metaplasia were prominent features (Fig. 18-9). Affected alveoli contained large numbers of neutrophils, and there were globular masses of DNA from disintegrating inflammatory cells in airways, with massive accumulation of neutrophils. Brennan et al. (1969) reported that experimental combined infection with M. pulmonis and Pasteurella pneumotropica caused more severe disease than M. pulmonis alone. However, whether P. pneumotropica is a potential contributing factor in natural MRM in mice is unclear. Neither experimental results nor findings in natural outbreaks supporting such a role have been reported. The mechanisms by which the above factors alter disease expression remain poorly understood. The high susceptibility of LEW rats to M. pulmonis respiratory disease has been linked
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to more extensive proliferation of lymphoid cells in the lungs (Davis et al. 1982), a larger percentage of T helper cells in lymphoid organs (Davis et al. 1985a), higher responsiveness of lymphoid cells to nonspecific activation (Davis et al. 1985a; Naot et al. 1984; Williamson et al. 1986), and differences in subclass distribution of anti-M. pulmonis antibodies (Simecka et al. 1989a) as compared with resistant F344 rats. From comparisons of disease expression in C57BL/6, BALB/c, and IL-2-treated ICR mice, Tanaka et al. (1998) suggested that differences in the pattern of pulmonary disease could be related to predominance of Th1 or Th2 responses. The possible role of Vsa surface proteins as virulence determinants is discussed in Section II.B. Viral infections of the respiratory mucosa may be expected to alter innate mucosal defenses, such as mucociliary clearance, or specific immune responses, but none of these has been shown specifically to be involved. In rats, exposure to up to 100 ppm ammonia did not alter pulmonary bacterial clearance, and its effects were confined to the nasal passages (Schoeb et al. 1982). The effect of ammonia may be mediated by direct effects on upper respiratory epithelium, as exposure to ammonia enhances growth of M. pulmonis in tracheal organ cultures (Pinson et al. 1988). Enhanced adherence of M. pulmonis to respiratory epithelium could be a factor; however, such an effect was not consistently observed in a study of adherence of M. pulmonis to rat tracheal mucosa in vitro (Schoeb et al. 1993b). The number of adherent organisms was increased in tracheas from rats with sialodacryoadenitis virus infection, but not in tracheas from rats with Sendai virus infection. Vitamin A deficiency, which is reported to increase MRM severity (Tvedten et al. 1973), also increased the number of adherent organisms, whereas ammonia exposure and host age (40 weeks versus 8 weeks) did not. Pulmonary clearance of M. pulmonis in rats was not affected by Sendai virus or sialodacryoadenitis virus infection (Nichols et al. 1992). The only available information regarding increased susceptibility with advancing age is from one study in which susceptibility could not be related to changes in avidity of anti-M. pulmonis antibody responses (Steffen and Ebersole 1995). Production of antibodies to M. pulmonis does not appear to be responsible for differences in susceptibility among mouse strains. C57BL/10 mice were more resistant to experimentally induced arthritis and had higher serum concentrations of complement-fixing antibody than C3H mice (Keystone et al. 1982), but in studies of experimental respiratory disease, average serum antibody concentrations in resistant C57BL/6 and susceptible C3H/HeN mice were not significantly different, and serum antibody concentrations were highest in mice of each strain with the most severe disease (Cartner et al. 1995; Davis et al. 1985b). There were differences in subclass distribution, with C3H/HeN producing more antibodies of the IgG1 and IgG2a subclasses than C57BL/6 mice (Cartner et al. 1995), but the functional significance of such differences has not been determined. The relative resistance of C57BL/6 mice is more likely related to innate immunity. The numbers of M. pulmonis CFU in the
445 lungs of C57BL/6 mice decreased by 83% over the first 72 hours after inoculation, whereas in the same period the numbers of CFU in the lungs of C3H/HeN increased 180-fold (Davis et al. 1985b). This innate resistance is related to pulmonary alveolar macrophages, because mycoplasmacidal activity is detectable in the lungs of C57BL/6 mice within 4 hours after inoculation and prior to influx of other inflammatory cells (Cartner et al. 1995; Davis et al. 1985b, 1992; Parker et al. 1987), and because this early resistance is reduced or eliminated by nitrogen dioxide-induced pulmonary macrophage injury (Davis et al. 1992) or depletion of pulmonary macrophages with toxic liposomes (Hickman-Davis et al. 1997). Also, pulmonary macrophages collected from C57BL/6 mice 5 days after inoculation were more inhibitory to M. pulmonis growth in vitro than macrophages from C3H mice (Lai et al. 1993). C3H and C57BL/6 mice also differ in changes in mucosal vascular remodeling in response to inflammation induced by M. pulmonis (Thurston et al. 1998). In the tracheas of C3H mice, the total number of vessels did not change, but the proportion of capillaries decreased and the proportion of venules increased. The average diameter of mucosal vessels also increased, as did the total number of endothelial cells. In contrast, the total numbers of both capillaries and venules increased in C57BL/6 mice. One study of the genetic basis of differences in susceptibility among mouse strains linked susceptibility to H-2 haplotype (Davis et al. 1985b). C3H/HeN and CBA mice (H-2k) were most susceptible, C57BL/6 mice (H-2b) were most resistant, and H-2d 310.D2 mice and H-2b/k C3B6 Fl hybrid mice were intermediate in susceptibility. However, others did not find an association between susceptibility and H-2 haplotype (Lai et al. 1993). In that study, C3H/HeJ (H-2k), C3H/Bi (H-2k), A.By (H-2b), and BALB.B (H-2b) mice were susceptible, and C57BL/6 (H-2b) and B10.BR (H-2k) mice were resistant. Results of typing B6C3F2 mice for H-2 haplotype and disease severity indicated that resistance was not H-2-linked and that resistance was due to a single dominant gene. Subsequently, these investigators used C57BL/6 × C3H and BALB/c × C57BL/6 recombinant inbred mice to map the resistance gene to chromosome 4. In addition, C57BL/6 mice congenic for the H18 (histocompatibility 18) allele of BALB/c mice were more susceptible than C57BL/6 mice but less susceptible than BALB/c mice, indicating that other loci modify susceptibility. Cartner et al. (1996) evaluated susceptibility to M. pulmonis UAB CT in mice of 17 inbred strains and concluded that susceptibility is a complex trait. C57BR/cdJ, C57BL/6NCr, C57BL/10ScNCr, and C57BL/6J mice were resistant, whereas C57L/J, SJL/NCr, BALB/cAnNCr, A/JCr, C3H/HeJ, SWR/J, AKR/NCr, CBA/NCr, C58/J, DBA/2NCr, C3H/HeNCr, C3HeB/FeJ, and C3H/HeJCr were variably susceptible. Differences in susceptibility were not associated with H-2 loci or with the Slc11a1 (Bcg) host resistance locus. MECHANISMS OF HOST INJURY The pathogenesis of M. pulmonis disease is not clearly understood. Ability to adhere to
446 host cells probably is an important virulence factor, but how M. pulmonis induces host cell injury is unknown. Because mycoplasmas cannot synthesize purines, pyrimidines, sterol, and other essential substances, they must obtain them from the host. How this is accomplished is not clear, but mycoplasmas produce nucleases, proteases, and phospholipases that could function in this role. Hydrogen peroxide production has been linked to M. pulmonis virulence (Kinbara et al. 1992), but its importance remains unknown. Whatever the mechanisms, M. pulmonis has substantial capacity to directly damage respiratory epithelial cells (Pinson et al. 1988; Stadtlander et al. 1991) and to induce vigorous inflammatory and immune responses. HOST RESPONSES Macrophages constitute a critical host defense against M. pulmonis pulmonary disease. M. pulmonis is rapidly killed in the lungs of resistant C57BL/6N mice in the first 72 hours after inoculation (Parker et al. 1987). Killing by lung lavage cells is demonstrable in vitro when M. pulmonis is inoculated into mice, then recovered by tracheobronchial lavage and incubated with the cells in the lavage sample (Davis et al. 1992). Most of the recovered organisms are associated with macrophages, and both killing and macrophage viability are sharply reduced in mice exposed to NO2, which damages pulmonary macrophages, indicating that macrophages are responsible for M. pulmonis killing. Direct evidence for this is provided by a study in which liposomes containing dichloromethylene bisphosphonate were administered intratracheally to deplete pulmonary macrophages in resistant C57BL/6 mice and susceptible C3H/HeN mice (Hickman-Davis et al. 1997). Intrapulmonary killing of M. pulmonis in treated C57BL/6 mice and C3H/HeN mice was comparable to that in untreated C3H/HeN mice, showing that macrophages are the primary effectors of killing, and indicating that differences in macrophage function are at least partially responsible for the difference in susceptibility between the two mouse strains. Subsequently, killing of M. pulmonis by C57BL/6 pulmonary macrophages in vitro was shown to require surfactant protein A (SP-A), inducible nitric oxide synthase (iNOS) activity, and generation of peroxynitrite (Hickman-Davis et al. 1998, 1999, 2001, 2004). In vivo, SP-A or iNOS deficiency increased disease severity in C57BL/6 mice, and in the case of SP-A deficiency, rendered them as susceptible as C3H/HeN mice. In rats, pulmonary macrophages efficiently clear M. pulmonis from the alveoli in vivo (Cassell et al. 1973), and unstimulated rat pulmonary macrophages have antimycoplasmal activity in vitro (Davis et al. 1980). Thus, vigorous activity of pulmonary macrophages against M. pulmonis may at least partially account for the observation that rats are not susceptible to induction of acute pneumonia by inoculation of M. pulmonis, as are mice (Cassell et al. 1973). Resistance to phagocytosis or extracellular killing by macrophages could be one mechanism by which the organism resists host defenses and causes chronic disease
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(Razin et al. 1998), and may account in part for differences in virulence among M. pulmonis strains (Howard and Taylor 1979). Phagocytosis of M. pulmonis by mouse lung macrophages is induced by anti-M. pulmonis antibodies of IgG1, IgG2a and IgG2b subclasses, and clearance of the organism from the respiratory tract is enhanced by pre-inoculation treatment of M. pulmonis with IgG1, IgG2a, IgG2b, or IgA antibodies (Taylor and Howard 1981). Results of in vitro studies indicate that M. pulmonis is resistant to phagocytosis by pulmonary or peritoneal macrophages unless opsonized with antibody or complement (Marshall et al. 1995), although Davis et al. (1980) could demonstrate antibody-mediated phagocytosis of M. pulmonis by mouse pulmonary macrophages only with rabbit, not mouse, antiserum. M. pulmonis is more easily killed by macrophages when the organisms are cultured in vitro than when recovered from mice with chronic disease (Taylor and Howard 1980b), which could be related to altered Vsa lipoprotein expression. Whether these observations are applicable to in vivo disease pathogenesis is unclear. Specific antibody is not required for intrapulmonary killing in mice, at least in the early stages of infection after experimental inoculation (Davis et al. 1992; Parker et al. 1987). M. pulmonis stimulates enzyme release by cultured mouse peritoneal macrophages (Taylor-Robinson et al. 1978) and induces expression of class I and class II MHC antigens on macrophages, B cells, and airway dendritic cells and epithelial cells (Ross et al. 1990; Stuart et al. 1989; Umemoto et al. 2002). The roles of such effects in M. pulmonis disease pathogenesis have not been evaluated. Killing of M. pulmonis by neutrophils in vitro can be induced by opsonization with specific antibody, but neutrophils seem to be ineffectual against M. pulmonis in vivo (Davis et al. 1992). In contrast with resistant C57BL/6 mice, C3H/HeN mice have much greater accumulation of neutrophils in their lungs following experimental inoculation, but poor intrapulmonary killing of M. pulmonis (Davis et al. 1992). The role of NK cells in M. pulmonis disease is uncertain, and results of existing studies are somewhat conflicting. Inoculation of mice with M. pulmonis can enhance NK activity of lung and spleen cells (Kamiyama et al. 1991; Lai et al. 1990b). NK cells from the lungs and spleen of recently inoculated C57BL/6 mice can inhibit growth of M. pulmonis in vitro, and clearance of the organism from the lungs was more rapid in acutely infected SCID mice than uninoculated mice, suggesting that activation of NK cells could contribute to host resistance during early infection (Lai et al. 1990b). However, Swing et al. (1995) studied the effects of virulent and avirulent M. pulmonis strains on NK cell activity in resistant C57BL/6 and susceptible C3H/HeN mice, and found no relationship between activation of NK cells in vitro and either host or mycoplasma strain. The temporal courses of intrapulmonary killing and NK cell activity also are inconsistent with an important role of NK cells in acute infection, inasmuch as intrapulmonary killing is most pronounced in the first 24 hours after inoculation, whereas the greatest NK cell activity occurs three days after inoculation (Lai et al. 1990b).
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Complement-deficient mice are reported to have increased susceptibility to chronic arthritis induced by intraperitoneal inoculation of M. pulmonis (Keystone et al. 1978). However, in vitro, complement does not promote attachment to, or ingestion by, mouse peritoneal macrophages, although the organism is killed by purified C3a and by mouse IgG1, IgG2a, and IgG2b antibodies and guinea pig complement. Recently, Simmons and Dybvig (2003) showed that M. pulmonis strain UAB CT variants that had one of two Vsa proteins with short tandem repeats were more susceptible to killing by guinea pig complement than organisms expressing a Vsa protein with long tandem repeats, indicating that complement resistance is mediated by Vsa proteins. As would be expected, M. pulmonis-induced pneumonia is accompanied by production of inflammatory cytokines (Faulkner et al. 1995b; Romero-Rojas et al. 2001a). Faulkner et al. (1995b) assessed expression of TNFα, IL-1α, IL-1β, IL-6, and IFNγ mRNA in the lungs of C57BL/6 and C3H/HeN mice inoculated with M. pulmonis strain UAB CT and measured the corresponding proteins in lung lavage fluid and in serum. TNFα was produced first, and mostly, by lavage cells. Concentrations of TNFα and IL-6 were higher in lavage fluid samples from susceptible C3H/HeN mice than those from resistant C57BL/6 mice, and increased concentrations persisted for longer times after inoculation. The serum concentration of IL-6 also was higher in C3H/HeN mice. Mice inoculated with avirulent M. pulmonis strain UAB T also had increased concentrations of TNFα in lung lavage fluid shortly after inoculation, but concentrations declined more quickly than in samples from mice given virulent M. pulmonis strain UAB CT. Similar results were reported by Nishimoto et al. (1994). Tanaka et al. (1996) found that administration of IL-2 to experimentally infected ICR mice increased cuffing of lymphocytes around airways and vessels and accumulation of macrophages in bronchioles. Indomethacin treatment of experimentally infected rats resulted in decreased concentrations of PGE and T×A2 in lung lavages and increased growth of M. pulmonis in the lung, suggesting a contribution of prostaglandins to pulmonary defenses against the organism (Reinhard and Chandler 1990). Acute severe pneumonia in C3H/HeN mice is accompanied by coagulopathy, with increased serum concentrations of fibrin degradation products (Faulkner et al. 1995a). This observation, and the finding that intravenous injection of M. fermentans cells or their membranes induced lethal shock in mice (Gabridge et al. 1972), suggests that mycoplasma components are capable of activating cytokine and coagulation cascades in a manner similar to that of endotoxin. Expression of mRNAs of the β-chemokines macrophage chemoattractant factor 1 and macrophage inflammatory peptides 1α and 1β is increased in the lungs of experimentally infected mice, indicating that these chemokines probably contribute to accumulation of inflammatory cells in M. pulmonis respiratory disease (Simecka 1999). M. pulmonis induces generation of noncomplement factors from rat serum that are
447 chemotactic for macrophages and neutrophils in vitro (Ross et al. 1992), but the identity of these factors and whether they have a role in M. pulmonis disease pathogenesis are unknown. M. pulmonis is mitogenic for mouse B, T, and NK cells and for rat B and T cells, both in vitro and in vivo (Ginsburg and Nicolet 1973; Lai et al. 1990b; Lapidot et al. 1995; Simecka et al. 1993; Swing et al. 1995). These effects appear to be mediated by proteins other than those of the Vsa family (Lapidot et al. 1995; Watson et al. 1989). The role of nonspecific lymphocyte activation in M. pulmonis disease is unclear. Lymphocytes of susceptible LEW rats respond more strongly than those of resistant F344 rats (Davis et al. 1985a; Simecka et al. 1987). LEW rats also have more pronounced inflammatory responses in viral diseases (Liang et al. 1995) and various experimental inflammatory conditions (Simecka et al. 1992). M. pulmonis is about equally stimulatory for lymphocytes of arthritis-resistant C57BL/10 mice and those of arthritis-susceptible C3H mice (Keystone et al. 1982). Stimulation of mouse spleen cells by M. pulmonis has been attributed to superantigen-like molecules (Romero-Rojas et al. 2001b). The relative roles of adaptive and innate immunity have been investigated in studies using immunodeficient mice. Athymic nude, neonatally thymectomized, X-linked immunodeficient, and SCID mice develop less severe respiratory tract lesions in response to experimental infection with M. pulmonis than immunocompetent mice (Cartner et al. 1998; Sandstedt et al. 1997). However, unlike immunocompetent mice, such mice are susceptible to dissemination of the organism from the respiratory tract and development of polyarthritis. In studies of experimental arthritis induced by intravenous or intraperitoneal injection of M. pulmonis, immunosuppressed mice and mice with B cell, T cell, or complement deficiency develop more severe arthritis and more lesions in other organs, with larger numbers of organisms in lesions, than immunocompetent mice (Berglof et al. 1997; Cartner et al. 1998; Evengard et al. 1994; Sandstedt et al. 1997). Cartner et al. (1998) inoculated C3H/HeSnJ and C57BL/6 SCID and control mice intranasally with virulent M. pulmonis UAB CT, examined respiratory and other organs histologically, and determined the numbers of M. pulmonis organisms in lungs and spleens in groups of mice sacrificed at 14 or 21 days after inoculation. They found that the numbers of M. pulmonis CFU in the lungs did not differ according to ability to mount adaptive immune responses, but that C3H mice had larger numbers of CFU in the lungs than did C57BL/6 mice. Although colonization of the spleen occurred in all groups of mice, as did lesions in organs other than those of the respiratory tract, spleens were colonized more frequently, and with larger numbers of CFU, in SCID mice of both strains. SCID mice of both strains also had less severe lung disease, but they had more frequent and more severe suppurative arthritis and tenosynovitis than their immunocompetent counterparts. At both 14 and 21 days after inoculation, C3H SCID mice had a greater frequency of such lesions as suppurative splenitis, pericarditis,
448 myocarditis, atrioventricular valvulitis, and perioophoritis than C57BL/6 SCID mice. Transfer of immune serum from infected immunocompetent mice to C3H SCID mice did not alter the severity of pulmonary disease, but it inhibited the development of arthritis, whereas C3H SCID mice engrafted with immunocompetent naive spleen cells developed lung lesions similar to those of immunocompetent C3H mice. Immunized and naturally or experimentally infected mice and rats produce vigorous antibody responses to M. pulmonis, both systemically and locally in the respiratory tract (Brown and Reyes 1991; Cartner et al. 1995; Cassell et al. 1974; Horowitz and Cassell 1978; Simecka and Cassell 1987; Simecka et al. 1989a, 1989b; Simecka et al. 1991; Steffen and Ebersole 1992a; Steffen and Ebersole 1992b; Steffen and Ebersole 1995; Taylor 1979; Taylor and Howard 1980a). In experimentally infected rats, antibodies are first produced in the local lymph nodes of the upper respiratory tract, with antibody-producing cells subsequently appearing in the nasal mucosa, lungs, the lymph nodes of the lungs, and spleen, but the upper tract nodes remain the major sources of antibody production. In both mice and rats, IgM antibodies usually are detected first. In mice, IgG antibodies appear next in serum, but in rats, the appearance of IgA antibodies precedes that of IgG antibodies. IgM, IgG1, IgG2, and IgA antibodies are produced in the respiratory tract of mice as well as in local lymph nodes. Mice produce large amounts of antibodies of the IgG1 subclass, whereas F344, but not LEW, rats immunized with M. pulmonis produce antibodies primarily of the IgG2b subclass. The role of antibodies in M. pulmonis respiratory disease is not clear. Antibody responses do not lead to resolution of disease, but results of adoptive transfer experiments indicate that transfer of serum or fractionated antibodies of different classes and subclasses from immunized mice confers partial resistance to respiratory disease, whereas spleen cells are ineffective (Cassell et al. 1973; Taylor and Howard 1981; Taylor and Taylor-Robinson 1976). In rats, however, transfer of immune serum does confer resistance (Lai et al. 1991b). Few studies have been done on the role of T cell subsets in M. pulmonis disease pathogenesis. Jones et al. (2002) characterized changes in populations of CD4+ and CD8+ T cells in M. pulmonis respiratory disease in C3H mice inoculated with M. pulmonis UAB CT, using assays for IFN-γ and IL-4 production to assess Th1 and Th2 activity. At 7 days after inoculation, infected mice had increased numbers of CD4+ cells, but not CD8+ T cells or B220+ B cells, in the regional lymph nodes, with no detectable changes in populations of these cells in the lungs. At 14 days after inoculation, numbers of T cells, and, to a lesser extent, B cells, were increased in the lungs. Numbers of CD4+ and CD8+ T cells both were increased, but the increase in numbers of CD4+ cells was more pronounced than that of CD8+ cells. CD4+ cells also were most numerous in the regional lymph nodes. In in vitro assays of mycoplasma-specific responses, both Th1 and CD8+ cell responses were detected among lung lymphocytes, with Th1 responses predominating,
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whereas the major responses in regional lymph nodes were Th2. Depletion of CD4+ cells reduced the severity of lung lesions, without affecting the numbers of M. pulmonis CFU recovered, whereas CD8+ cell depletion increased lesion severity. These results indicate that Th1 and CD8+ T cells have important roles in the pathogenesis of respiratory disease induced by M. pulmonis. In summary, innate immune mechanisms appear to be of major importance in pulmonary defenses against M. pulmonis, at least in the early phases of infection. Adaptive immune responses are important in limiting dissemination of the organism from the respiratory tract to other organs, but they do not result in elimination of the infection and resolution of the disease. Rather, adaptive responses appear to contribute to the severity of pulmonary lesions, leading some to characterize M. pulmonis respiratory disease as an immunopathologic process (Jones et al. 2002; Tanaka et al. 1996).
F. 1.
Epizootiology
Host Range and Geographic Distribution
In addition to laboratory mice and rats, M. pulmonis has been isolated from cotton rats (Andrewes and Niven 1950), Syrian hamsters (Hill 1974), guinea pigs (Cassell and Hill 1979), rabbits (Deeb and Kenny 1967), and horses (Allam and Lemcke 1975). Occurrence in laboratory mice and rats or in wild Norwegian rats and other Rattus species is reported from Europe, Japan, and Brazil as well as the United States, and probably is worldwide (Busch and Naglic 1995; Hill 1974; Juhr et al. 1970; Kagiyama et al. 1986; Koshimizu et al. 1993; par Tram et al. 1970; Saito et al. 1978; Sparrow 1976; Timenetsky and De Luca 1998).
2.
Prevalence
Information regarding the prevalence of M. pulmonis among laboratory rodents has been reported only sporadically, and comprehensive current data are not available. Reports of surveys in the 1970s showed that the organism was prevalent among laboratory mice in the United Kingdom (Sparrow 1976) and Japan (Saito et al. 1978). According to results of testing by enzyme-linked immunoabsorbent assay (ELISA) and culture in the 1980s, M. pulmonis was common among conventional mice in the United States and also was present in some barrier-maintained colonies, with up to 91% of mouse populations and up to 78% of rat populations testing positive (Casebolt et al. 1988; Cassell et al. 1981b). The most recent data are from the survey by Jacoby and Lindsey (1998). Among the 72 responding institutions, 30% of mouse colonies and 40% of rat colonies were not SPF. Evidence of M. pulmonis infection was found in SPF mice at two institutions (2.8%) and in non-SPF mice at 12 (16%). Infection in SPF rats was not reported, but the organism’s presence in non-SPF rats was reported by 26 respondents (36%).
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Thus, it is likely that M. pulmonis remains common among conventional mice and rats, and may occasionally be encountered in SPF colonies. Information regarding prevalence within infected colonies is sparse. Prevalence can be high among rats maintained in unfiltered cages (Lindsey et al. 1985), but no data are available regarding prevalence within colonies of mice or rats maintained in filtered cages.
3.
Transmission
Observations of natural infections are consistent with transmission from mother to suckling pups by direct contact or aerosol (Cassell et al. 1979; Lindsey et al. 1985). Transmission among cagemates and among cages occurs among older animals (Hill 1972; Jersey et al. 1973; Lindsey et al. 1971). Although the frequency of occurrence in natural infection is unknown, transplacental transmission has been documented in experimental genital infections in rats (Steiner et al. 1993), and is the most likely route of infection in hysterectomy-derived animals thought to have been germ-free (Cox et al. 1988; Ganaway et al. 1973; Juhr et al. 1970; par Tram et al. 1970). M. pulmonis is an occasional cell culture contaminant (Bolske 1988; Nicklas et al. 1993), and such contamination is a possible source of infection for mice receiving injections of cells or cell culture products. The role of genital transmission in the epizootiology of M. pulmonis is unknown. Rats with experimental genital infections frequently also develop respiratory tract infection (Steiner and Brown 1993), but whether this represents extension to another organ system in the same individual, transmission between individuals, or both, is unknown. The risk of transmission by other means, such as by fomites, also is unknown. Little information concerning environmental resistance of M. pulmonis is available, and that which exists is contradictory.
G. 1.
Diagnosis
General Considerations
Methods of diagnosis of M. pulmonis infection are reviewed by Lindsey et al. (1983), Cassell et al. (1986), Simecka et al. (1992), and Davidson et al. (1994). Diagnosis of clinically evident M. pulmonis disease is straightforward in most cases. However, detection of inapparent or cryptic infection in clinically healthy populations for health-monitoring purposes imposes somewhat different requirements on diagnostic tests. In that case, the objective is not to identify an etiologic agent where it probably exists in large numbers but to provide a high degree of confidence that specific agents are not present. To do this, the user needs to be able to assess the reliability of negative test results under the husbandry and management conditions of the population being monitored. Such information rarely is available. Another consideration is the lack of standardization of test
449 methods among laboratories. In the absence of such standardization, differences in procedures and quality control inevitably result in tests that do not provide precisely the same results from the same sample. Whether these differences are of practical significance in a particular case is difficult to determine. As a consequence of modern husbandry and the widespread use of filtered cages to reduce the risk of introduction and spread of infectious agents, the difficulty of detecting an unwanted agent is greatly increased, because the proportion of cages in which it is present is likely to be limited, especially in the case of agents of low transmissibility, such as M. pulmonis. The efficiency of sentinel systems in detecting such agents has not been thoroughly investigated but probably is not high. Another issue is the large number of mice having mutations that do not result in an overtly immunodeficient phenotype, but nevertheless have various immunological abnormalities. The relative performance of serologic tests for M. pulmonis infection in such mice is unknown. For optimal detection of M. pulmonis, no single test serves well in all situations. Davidson et al. (1994) recommend using a combination of serology, culture, and histopathology to increase the probability of detecting inapparent infections. Investigation of respiratory disease in mice should take into account differential diagnoses and the possibility that more than one potential etiologic agent could be present. Accordingly, it should be standard practice to employ a combination of appropriate serologic, bacteriologic, histopathologic, and other techniques as necessary to identify not only the primary etiologic agent, but also any secondary or exacerbating agents. Other than M. pulmonis, possible causes of clinically apparent respiratory disease in immunocompetent mice include CAR bacillus and Sendai virus. CAR bacillus is discussed in Section IV. Sendai virus disease is distinguished by its clinical presentation, which typically is that of an acute epizootic, and by its characteristic lesions, which in immunocompetent mice are acute and necrotizing (Brownstein 1996) rather than chronic and suppurative, as are lesions caused by M. pulmonis or CAR bacillus. Sendai virus infection can be concurrent with M. pulmonis infection and can exacerbate MRM in mice (Howard et al. 1978; Lindsey et al. 1983; Saito et al. 1981). Pasteurella pneumotropica is not regarded as a significant respiratory pathogen of mice (Percy and Barthold, 2001), but it has been reported to occur concurrently with M. pulmonis (Brennan et al. 1969). Although concurrent infection with both M. pulmonis and CAR bacillus has not been reported in mice, it is possible, inasmuch as M. pulmonis often is present in rats with CAR bacillus infection (Schoeb et al. 1996; Schoeb and Lindsey 1996). In the case of immunocompromised or immunodeficient mice, diagnosis of respiratory disease having a clinical presentation and gross lesions suggesting MRM or CAR bacillus disease requires consideration of additional differential diagnoses. Nude and SCID mice are susceptible to wasting with chronic pneumonitis induced by Sendai virus, pneumonia virus of mice (PVM), or Pneumocystis carinii (Iwai et al. 1979; Richter et al. 1988;
450 Roths et al. 1990; Shultz et al. 1989; Ueda et al. 1977; Ward et al. 1976; Weir et al. 1988). PVM is reported to exacerbate pneumocystosis in SCID mice (Bray et al. 1993), and severe pulmonary disease is reported in SCID mice with concurrent P. pneumotropica and P. carinii infections (Macy et al. 2000). Recombinase activating gene (Rag1 or Rag2) knockout mice, which have a severe combined immunodeficiency phenotype, are susceptible to induction of lethal P. carinii pneumonia when engrafted with effector CD25− CD4+ T cells in the absence of CD25+ CD4+ regulatory T cells (Hori et al. 2002). 2.
Serology
A variety of methods have been used to detect antibodies to M. pulmonis (Cassell et al. 1986; Davidson et al. 1994; Lindsey et al. 1983; Simecka et al. 1992; Taylor 1979). Complement fixation, hemagglutination, hemagglutination inhibition, and metabolic inhibition methods rarely are used since development of a successful ELISA (Horowitz and Cassell 1978). ELISA is at least as sensitive and reproducible as culture, immunofluorescence, and histopathology (Cassell et al. 1981b; Davidson et al. 1981; Horowitz and Cassell 1978). It also is economical, provides results more rapidly than culture, and is commercially available from several laboratories. For these reasons, serologic testing by ELISA is the most widely used screening method for M. pulmonis infection. In cases of overt disease, the diagnosis should be confirmed by direct demonstration of the organism by culture, immunofluorescence, immunohistochemistry, or polymerase chain reaction (PCR) methods. Other than the necessity for rigorous quality control, the chief difficulties in use of ELISA for screening purposes are cross reaction with other murine mycoplasmas, primarily Mycoplasma arthritidis (Kobayasi et al. 1989; Minion et al. 1984; Watson et al. 1987a), and delayed antibody responses in young animals infected with small numbers of organisms (Davidson et al. 1981). Detection in the latter situation can be somewhat improved by use of an IgM ELISA. Cross reactions are most troublesome in the case of SPF colonies, in which it may become necessary to distinguish responses to M. pulmonis from those to other, relatively innocuous murine mycoplasmas. Cross reactions can be resolved by the immunoblotting of serum against proteins of M. pulmonis and those of other species (Minion et al. 1984; Watson et al. 1987a). In general, specific responses will be indicated by the presence of numerous bands; patterns of 10 or more bands usually allow a specific diagnosis. However, in a large proportion of individual animals, only a few bands may be present with proteins of any mycoplasma species. This probably indicates early infection, in which responses initially are indicated by four or fewer bands, with reactivity to additional bands appearing over a period of months (Cox et al. 1988). 3.
Culture
Methods of isolation and identification of murine mycoplasmas are described by Cassell et al. (1983) and Davidson et al. (1994).
TRENTON R. SCHOEB
M. pulmonis can be cultivated with Hayflick’s, SP-4, modified versions of these, and other media, and recovery of M. pulmonis from clinically affected animals is not difficult in most cases. Davidson et al. (1994) recommend Medium A and provide detailed protocols for its preparation and for sample collection. Media for growth of M. pulmonis from specimens from animals usually include antibiotics, thallium acetate, or both, to prevent overgrowth by other bacteria. Cefoperazone is preferable to other antibiotics and to thallium, which is highly toxic. Blood and tissues can contain inhibitory substances, which can be countered by the addition of ammonium reinechate or lysophospholipase to culture media (Davidson et al. 1994). Growth inhibition by tissue components is of most concern when the sample is a homogenate, such as an organ or a tissue being quantitatively cultured for experimental purposes. In general, addition of anti-inhibitors is not necessary as a routine measure with samples collected by lavage, probably because of the dilution of the sample that occurs in sample collection and inoculation of broth and agar media. An atmosphere containing 5% or more CO2 is sometimes recommended for mycoplasma culture. This is unnecessary for M. pulmonis, but at least 90% humidity should be maintained for incubation of agar plates. Optimal incubation temperature is 37°C. In most cases, growth is evident within one week, but M. pulmonis strains occasionally are encountered that are difficult to grow on primary isolation. For this reason, all broth tubes and agar plates should be incubated for three weeks before being discarded. In a few cases, serial blind passages may be required to obtain visible growth. Quality control of media is important for isolation and cultivation of M. pulmonis and other murine mycoplasmas. Occasionally, batches of medium components are encountered that are inhibitory or do not support growth well. For example, Davidson et al. (1994) recommend purified agarose because some agar preparations are inhibitory. Plasticware should be of cell culture quality, and if nondisposable glassware is used, it should be washed and rinsed as if for cell culture. When medium components are purchased, small test quantities should be obtained and used to prepare trial batches of broth and agar media, which are then tested for ability to support growth, preferably with a relatively fastidious strain. Davidson et al. (1994) recommend the M. pulmonis type strain, PG34(Ash), which is available from the American Type Culture Collection. They also recommend assessing growth in trial batches of media by quantitative culture of serial dilutions of stock culture, observing the time required for growth to become evident and evaluating colony size and morphology. After the quality of the medium components has been established, larger quantities should be purchased from the same lot number. Subsequent batches of medium should be tested by inoculation with stock culture before use to culture samples from mice. The primary problems with use of culture for screening are that it is expensive and not highly sensitive. Although rates of recovery of M. pulmonis are improved by sampling multiple organs, the organism was not isolated from 30% of rats inoculated
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with 106 CFU and sacrificed 28 days later (Davidson et al. 1981). The organism tends to be most consistently present in the nasal passages and tympanic bullae, but it can inhabit any part of the respiratory tract, and its distribution is not necessarily consistent within an animal or among different individual animals. Samples should be collected from the nasal passages, tympanic bullae, and lungs, either with small swabs or by lavage. Lentsch et al. (1979) report the highest rate of success with cultures of the tympanic bullae. Lavage of each of these sites with a 23 ga needle attached to a 1 ml syringe containing buffered saline works well. The syringe can then be used to inoculate broth tubes and agar plates. If swabs are used, they must be prevented from drying, preferably by placing them directly into liquid culture medium. Reproductive tract samples also can be collected either with swabs or by lavage with a syringe. If there is evidence of arthritis or other reason to suspect dissemination of the organism, joints can be lavaged with a syringe and a fine needle, or they can be aseptically opened and swabbed. M. pulmonis has been isolated from many other organs, such as brain, spleen, and liver, but culture of these is unnecessary for routine diagnostic purposes. Mycoplasma isolates that utilize glucose and adsorb guinea pig erythrocytes to colonies on agar are likely to be M. pulmonis, although M. collis shares these characteristics, and some M. pulmonis strains do not hemadsorb (Davidson et al. 1994). Definitive identification requires use of specific antisera in growth inhibition assays (Clyde 1964, 1983) or immunofluorescence (Del Giudice et al. 1967; Gardella et al. 1983; Lehmkuhl and Frey 1974) or immunoperoxidase (Hill 1978; Polak-Vogelzang et al. 1978) techniques, or PCR (Harasawa et al. 1990; Kunita et al. 1990; Takahashi-Omoe et al. 2004; van Kuppeveld et al. 1992). 4.
Kohn and Chinookoswong 1989; Kraft et al. 1982; Lutsky et al. 1986; Organick and Lutsky 1968; Polak-Vogelzang et al. 1978). The chief advantages of such methods are that they can provide results more rapidly than culture and can be used in cases in which samples or expertise for mycoplasma culture are not available. However, immunohistochemical and immunofluorescence methods require training and experience to obtain consistent results and interpret them, and frequently require special fixation or processing of tissues. For immunofluorescence, frozen sections or sections fixed in cold 95% ethanol and specially processed for paraffin sectioning according to the method of Sainte-Marie (1962) can be used. The most significant problem with immunofluorescence is that it lacks the sensitivity to detect small numbers of organisms (Davidson et al. 1981). Immunohistochemical methods have the advantage of not requiring a specially equipped fluorescence microscope, but the relative sensitivity of different immunofluorescence and immunohistochemical techniques has not been investigated.
6.
Electron Microscopy
Electron microscopy can be useful in investigation of outbreaks of respiratory disease. M. pulmonis is characteristically found on the surface of respiratory epithelial cells in transmission electron micrographs (Fig. 18-11). The organism can be expected to be numerous in clinically ill animals, but negative results in a given sample or limited number of samples should be interpreted with caution, because the organisms may not be uniformly present in all organs or in all animals in an outbreak.
Histopathology
In cases of overt disease, the histopathologic pattern is characteristic, and CAR bacillus is the only other primary etiologic agent causing similar lesions. For monitoring of apparently healthy animals, histologic examination of the upper respiratory mucosa and tympanic bullae can be a useful adjunct to serologic testing and culture, although the same difficulties of insensitivity and possible lack of findings in young animals infected with small numbers of organisms apply. Among contemporary SPF mice, suppurative rhinitis and otitis media are much more likely to be caused by opportunistic bacteria than M. pulmonis. In either case, additional techniques are required for specific identification of the organism. Because of its lack of a cell wall, routine histochemical techniques for bacteria do not stain M. pulmonis. 5.
Immunofluorescence and Immunohistochemistry
Various immunohistochemical and immunofluorescence methods can be used to identify M. pulmonis in tissue sections (Brunnert et al. 1994; Davidson et al. 1981, 1994; Hill 1978;
Fig. 18-11 M. pulmonis cells (arrows) among cilia of a respiratory epithelial cell. Inset: M. pulmonis cells adherent to the surface of a non-ciliated epithelial cell. Uranyl acetate and lead citrate. Bar = 500 µm.
452 7.
DNA Probes
Methods have been developed for specific identification of M. pulmonis by DNA hybridization using radioactively and nonradioactively labeled probes (Ferebee et al. 1992; Kunita et al. 1989; Matsuzaki et al. 1989). One such method is reported to detect as little as 150 pg of M. pulmonis DNA in dot blots (Ferebee et al. 1992). These methods would not be practical for routine screening, and, with the availability of PCR-based methods, there has been little reason for their use. However, they could serve as a means of specific identification of mycoplasmas isolated from mice. 8.
Polymerase Chain Reaction
Several PCR methods for detection of M. pulmonis have been described (Goto et al. 1994; Harasawa et al. 1990; Kunita et al. 1990; Sanchez et al. 1994; Schoeb et al. 1997a; Takahashi-Omoe et al. 2004; van Kuppeveld et al. 1992). In general, such methods are at least somewhat more sensitive than culture (Goto et al. 1994; Sanchez et al. 1994; Schoeb et al. 1997a; van Kuppeveld et al. 1993) or immunohistochemistry (Brunnert et al. 1994). PCR also can provide results more rapidly than culture, and it does not require the presence of live organisms, allowing detection of the organism in degraded samples or paraffin embedded tissues (Brunnert et al. 1994; Schoeb et al. 1997a). Inasmuch as samples such as nasal or oropharyngeal swabs can be analyzed, PCR also allows animals to be tested directly without sacrificing them. Conventional PCR methods for M. pulmonis are reported to have limits of detection of 0.5 pg to 1 pg of M. pulmonis DNA, or as few as 10 CFU (Harasawa et al. 1990; Kunita et al. 1990). Reverse transcriptase PCR to amplify 16S rRNA sequences can reportedly detect the equivalent of a single mycoplasma cell (van Kuppeveld et al. 1992). However, the potential sensitivity of PCR methods is sometimes overemphasized. One pg of M. pulmonis DNA is the equivalent of about 1000 mycoplasma cells, a limit of sensitivity approximately that of well-optimized culture techniques. Expression of detection limits in terms of CFU is likely to be misleading because the organisms tend to clump and because cultures can contain dead organisms whose DNA still is detectable. Efficiency can be increased considerably by use of nested PCR, blotting and probing of PCR products, and reverse transcriptase PCR to amplify ribosomal RNA sequences (Schoeb et al. 1997a; van Kuppeveld et al. 1992, 1993). However, the efficiency of amplification of purified nucleic acids is not necessarily attained with actual clinical samples, because it is subject to variables related to sample type and preparation and potential PCR inhibitors. Use of internal controls in PCR tests to detect mycoplasmas has shown that many samples can be inhibitory (Moalic et al. 1998; Schoeb et al. 1993a, 1997b; Ursi et al. 1992). Thus, proper controls must be used to ensure accuracy and reduce the risk of false negative results.
TRENTON R. SCHOEB
Properly designed PCR testing can reduce the risk of false negative results inherent in serologic and cultural methods in cases of early infections, colonization with small numbers of organisms, or infection of immunodeficient mice. However, conventional PCR methods are too labor-intensive and expensive for routine use in screening mouse populations. Real-time PCR for detection of M. pulmonis, as described for Mycoplasma pneumoniae (Hardegger et al. 2000; Templeton et al. 2003; Ursi et al. 2003), could be more efficient, especially if combined with automated sample preparation, but it still could be a challenge to make cost competitive.
H.
Treatment
In general, mycoplasmas are inhibited by various antibiotics that inhibit DNA function or protein synthesis, but, as would be expected, they are not susceptible to the bacteriocidal effects of antibiotics that interfere with cell wall synthesis, such as penicillins and cephalosporins (Taylor-Robinson and Bebear 1997). However, treatment of infected mice is unlikely to be completely effective and rarely is indicated. Antibiotics have been administered to mice or rats with natural or experimental M. pulmonis infection (Bowden et al. 1994; Chambaud et al. 2001; Dolowy et al. 1960; Ganaway and Allen 1969; Haberman et al. 1963; Lane-Petter et al. 1970; Tanaka et al. 1994; Taylor-Robinson and Furr 2000; Vasenius and Tiainen 1966). Administration of tylosin in drinking water can achieve concentrations in the serum and lung tissue above in vitro minimal inhibitory concentrations (Carter et al. 1987). Tetracycline administered in drinking water not only is poorly absorbed but also reduces water consumption (Porter et al. 1985), and injected oxytetracycline, unless in a long-acting form, has a short serum half-life (Curl et al. 1988). Oxytetracycline also is reported to reduce disease severity without eliminating the infection (Dolowy et al. 1960). In the author’s experience, tylosin or oxytetracycline administered in drinking water decreases the numbers of M. pulmonis CFU in the respiratory tract of experimentally infected rats, but neither antibiotic completely eliminates the organism, even with prolonged treatment, and both reduce water consumption. Taylor-Robinson and Furr (2000) report elimination of M. pulmonis from the vagina, but not oropharynx, of a large proportion of experimentally infected mice by oxytetracycline or lymecycline given by subcutaneous injection. Antibiotic treatment could, however, be justified under certain circumstances. Inasmuch as M. pulmonis is probably still present among populations of mice or rats at some institutions, it is possible that the organism could be identified in mice in a non-SPF facility or in mice quarantined for health assessment before being introduced into an SPF facility. Mutant mice that are difficult or impossible to replace could be salvaged by embryo transfer or by hysterectomy and fostering, in which case antibiotic treatment could reduce the probability of
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in utero transmission. Banerjee et al. (1987) report eliminating M. pulmonis from rats by such a method. If infected mice were clinically ill, antibiotic treatment could be used to attempt to maintain them until they could be bred and derivation accomplished. Mice derived by hysterectomy or embryo transfer to eliminate M. pulmonis infection must be rigorously monitored because of the risk of transplacental transmission (Cox et al. 1988; Ganaway et al. 1973; Juhr et al. 1970; par Tram et al. 1970; Steiner et al. 1993) and because of the potential difficulties of detecting M. pulmonis infection if only small numbers of organisms are present (Cox et al. 1988; Davidson et al. 1981, 1994).
that M. pulmonis can be eliminated by serologic testing and removing seropositive animals from the colony (Cox et al. 1988; Lindsey et al. 1983). However, there is little reason to consider either approach, inasmuch as M. pulmonis-free mice are widely available from modern vendors and derivation by hysterectomy or embryo transfer is available commercially or can be accomplished in-house.
III.
OTHER MYCOPLASMAS A.
I.
Control and Prevention
Control of M. pulmonis is accomplished by establishing breeding colonies from hysterectomy-derived, M. pulmonisfree stock and maintaining them according to barrier protocols. Control and prevention of M. pulmonis infection in mouse populations in research institutions should take into account several potential problems related to efficiency of detection of the organism. First, use of filtered cages in concert with absence of disease-promoting environmental conditions or concurrent infections makes it unlikely that M. pulmonis contamination would be signaled by epizootic clinical disease. If M. pulmonis were introduced into mice under these conditions, it would likely remain confined to a small proportion of cages, creating a problem of how best to sample the populations at risk. Second, there is clear evidence from studies in rats that the organism can be present in small numbers that can be difficult to detect by culture and that do not induce diagnostic serologic responses for weeks or months. Third, many institutions have large numbers of mice with mutations that do not result in overt immunodeficiency but potentially compromise the ability to mount antibody responses, and it is unknown whether such mutations would further compromise the ability of serologic testing to detect M. pulmonis infection. Fourth, widespread exchange of large numbers of mutant mice within and among institutions poses risks for the introduction of M. pulmonis from infected user colonies. Fifth, it is unclear that sentinel systems are effective for detecting of M. pulmonis. Finally, M. pulmonis occasionally contaminates cultured cells and thus could be transmitted to mice inoculated with contaminated cells or cell culture products. Procedures for vaccinating mice or rats against M. pulmonis have been reported (Atobe and Ogata 1977; Barry et al. 1995; Cassell and Davis 1978; Cassell et al. 1981a; Lai et al. 1990a, 1991a, 1991b, 1994, 1995, 1997; Taylor et al. 1977; Taylor and Taylor-Robinson 1976), but none has been shown to provide more than partial protection. That is, immunization can reduce severity of disease, but it does not prevent infection, leaving the possibility that immunized mice could become asymptomatic carriers (Simecka et al. 1992). There is some evidence
Introduction
Other mycoplasmas of laboratory mice are of much less concern than M. pulmonis, and, in general, little is known about their biology, interactions with the host, and epizootiology. Most probably are simply commensals. Available information about these agents, reviewed by Davidson et al. (1994) and Lindsey et al. (1991; 1986b), is summarized briefly below. Methods of isolation and identification are described by Davidson et al. (1994) and Cassell et al. (1983), and sensitive detection of M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis by PCR is described by van Kuppeveld et al. (1992). However, efforts to detect these organisms in animals rarely are made in settings other than research laboratories specifically concerned with rodent mycoplasmas. To the recognized mycoplasma species discussed in the following subsections probably will soon be added the agent currently known as Eperythrozoon coccoides, an obligate parasite of erythrocytes in mice (Lindsey et al. 1991b). Several similar organisms, including Haemobartonella canis, Haemobartonella felis, Haemobartonella muris, Eperythrozoon suis, Eperythrozoon wenyonii, and others, have been shown by genetic analysis to comprise a new group of mycoplasmas (Harasawa et al. 2002; Messick 2003; Messick et al. 2002; Neimark et al. 2001, 2002a, 2002b; Neimark and Kocan 1997; Rikihisa et al. 1997). B.
Mycoplasma arthritidis
M. arthritidis was isolated by Klieneberger and reported as a cause of polyarthritis in wild and laboratory rats in the late 1930s (Collier 1938; Findlay et al. 1939; Klieneberger 1938). There are a few other reports of polyarthritis, and on rare occasions M. arthritidis has caused abscesses or polyarthritis as a result of contamination of transplanted tumors. However, most publications about M. arthritidis concern its superantigen (Cole et al. 2000) or its use to induce experimental arthritis. Infection in mice was reported by Davidson et al. (1983). M. arthritidis requires arginine, is facultatively anaerobic, and grows in broth or on solid media containing yeast extract and horse serum. The type strain is PG6 (ATCC 19611).
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M. arthritidis varies within and among strains in antigenic and protein composition in a manner similar to that of M. pulmonis (Droesse et al. 1995; Stadtlander and Watson 1992). Virulence mechanisms of M. arthritidis are not well understood, but its superantigen probably contributes (Cole et al. 2000). Components of the organism have chemotactic and stimulatory activities for neutrophils, are toxic for rats and mice, induce hemolysis, and share antigenic epitopes with rat chondrocytes (Kirchhoff et al. 1989). The MAV1 virus of M. arthritidis is a proven virulence factor in both mice and rats (Tu et al. 2002). The organism has been isolated from the respiratory tract, tympanic bullae, and salivary glands of rats with inapparent infections, and from the conjunctiva, respiratory tract, and reproductive tract of mice. Only small numbers of organisms were recovered from the tissues of infected mice (Davidson et al. 1983). From the limited information available, most natural M. arthritidis infections are inapparent. Mice with M. arthritidis infection had no evidence of disease. They were housed in the same room with rats and had positive results in IgM ELISAs for serum antibodies to M. pulmonis. Lesions of experimental arthritis are similar to those caused by M. pulmonis (Fig. 18-10), and are reviewed by Lindsey et al. (1986a; 1978). Experimentally induced arthritis is reported to be more severe in nude athymic rats than in normal littermates (Binder et al. 1993). Rats, and probably mice, are the natural hosts. The organism has been isolated from wild Rattus spp. in Japan (Koshimizu et al. 1993), from pigs (Binder et al. 1990), and from human patients (Jansson et al. 1983), but the significance of such findings is not known. Geographic distribution, transmission, and prevalence are unknown, although results of serologic testing suggest that infection may be common (Cassell et al. 1986; Lindsey 1986; Lindsey et al. 1986a). Infection has been found in hysterectomy-derived, barrier-maintained rats and mice (Davidson et al. 1983; Thirkill and Gregerson 1982) and in isolator-housed rats thought to be germ-free (Cox et al. 1988).
C.
Mycoplasma neurolyticum
M. neurolyticum has been known since 1938 and was reported to be associated with conjunctivitis in mice by Nelson (1950). However, conjunctival, intranasal, or intravenous inoculation did not result in conjunctivitis or other disease, and M. neurolyticum is considered a nonpathogenic commensal. It has been isolated from the nasopharynx, brain, conjunctiva and Harderian gland of laboratory mice, and from the conjunctiva of wild mice and laboratory rats. M. neurolyticum ferments glucose and grows on solid media or in broth containing horse serum and yeast extract. For unknown reasons, growth is inhibited by penicillin (Hottle and Wright 1966). The organism produces a toxin, which, when injected intravenously into mice or rats, causes cerebral edema with
side-to-side rolling, but “rolling disease” does not occur naturally. The organism produces a mitogen (Katz et al. 1983; Naot et al. 1977), but its significance in vivo is unknown.
D. Mycoplasma collis M. collis ferments glucose and is cultivable with media containing horse serum and yeast extract. It has been isolated from the conjunctiva, nasopharynx, and Harderian gland of rats and the conjunctiva of mice from a few colonies in the United Kingdom (Hill 1983). It was associated with conjunctivitis in some infected rats, but experimental inoculation did not produce disease.
E.
Mycoplasma muris
M. muris utilizes arginine and is a strict anaerobe that grows only on SP-4 medium. It was isolated from the vagina of mice, which did not have evidence of associated disease (McGarrity et al. 1983).
F.
“Grey Lung” Agent
The “grey lung” agent originally was reported as a virus that caused pneumonia in mice and rats (Andrewes and Glover 1946). Subsequent studies showed that the agent was transmissible, that it caused respiratory disease similar to that caused by M. pulmonis, and that it had the ultrastructural characteristics of a mycoplasma (Gay 1967; Gay 1969; Gay and Attridge 1967; Gay et al. 1972). The organism never has been cultured, but 16S rRNA sequence analysis shows it to be another, previously unrecognized mycoplasma (Neimark et al. 1998). It is possible that it was the etiologic agent of pneumonia in mice described by Ebbesen (1968a, 1968b). In that study, disease was transmissible to other mice by inoculation of suspensions of affected lung, but M. pulmonis could not be isolated, and serologic responses to M. pulmonis were not demonstrable. The risk of infection for contemporary mice is unknown but probably low.
IV.
CAR BACILLUS A.
History
Colonization of the respiratory mucosa of rats and rabbits with filamentous bacilli among and parallel to the cilia is evident in electron micrographs published prior to reports of such organisms in association with respiratory disease, but these earlier publications were primarily concerned with ciliary ultrastructure, and the authors either did not mention the bacilli or did not
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consider them to be significant (Afzelius 1979; Burian and Stockinger 1963; Hilding and Hilding 1966). Respiratory disease associated with such organisms was first reported by van Zwieten et al. (1980), who described the bacteria in association with an outbreak of respiratory disease in laboratory rats in the Netherlands. Shortly thereafter, MacKenzie et al. (1981) reported similar findings in wild rats, a laboratory mouse, and laboratory rabbits. That report also cites a personal communication that the infection had been observed in African white-tailed hamsters (Mystromys albicaudatus) with “chronic respiratory disease.” The organisms could not be cultured on bacteriological media and were identified by morphologic and staining characteristics. In 1985, Ganaway et al. (1985) introduced the term cilia-associated respiratory bacillus and reported cultivation of the organism in embryonated chicken eggs, induction of respiratory disease in SPF rats and mice inoculated with egg-passaged CAR bacilli, and recovery of the organism from inoculated rats. Spontaneous CAR bacillusassociated respiratory disease in laboratory mice and isolation of the organism from mice were reported by Griffith et al. (1988) and Shoji et al. (1988b).
B.
Properties of the Agent
Most of the information about the properties of CAR bacilli of rodents comes from studies of organisms of rats. CAR bacilli of rats are gram negative, with a typical trilaminar cell wall; 3.5 µm to 9 µm in length and 0.12 µm to 0.35 µm in diameter; slightly fusiform with slightly bulbous ends; and without flagella, pili, or filaments (Cundiff et al. 1994a; Ganaway et al. 1985; MacKenzie et al. 1981; Matsushita and Joshima 1989; Schoeb et al. 1993a). The organisms have flexing and gliding motility in culture (Ganaway et al. 1985; Schoeb et al. 1993a). CAR bacilli of mice resemble those of rats by transmission electron microscopy (Griffith et al. 1988). Analyses of 16S rRNA gene sequences indicate that CAR bacilli of rats and mice belong to the flavobacteria subdivision of the flavobacteria-bacteroid eubacterial phylum and are most closely related to Flavobacterium ferrugineum and Flexibacter sancti (Cundiff et al. 1994b; Schoeb et al. 1993a; Wei et al. 1995), but the organisms remain unclassified and are known only as inhabitants of ciliated respiratory mucosa. CAR bacilli from different host species may represent different strains or even species, as discussed in Section IV.F.1.
SMR (Matsushita and Joshima 1989). Strains NIH and StL were derived from Ganaway’s original isolate; X1247C, X1331B, and X1428D were isolated from naturally infected rats; and X2006C was cultured from a laboratory rabbit (Schoeb et al. 1993a). CBM is a mouse isolate, and CBR and SMR were recovered from rats (Matsushita and Joshima 1989; Shoji et al. 1988a). These have not been characterized extensively. Antigenic differences (Hook et al. 1998) and minor differences in 16S rRNA sequences (Cundiff et al. 1994b; Schoeb et al. 1993a; Wei et al. 1995) among mouse and rat isolates are reported. Isolates from rats differ in growth characteristics and motility in culture and in virulence in experimentally infected rats (Schoeb et al. 1993a, 1997c).
D.
Growth In Vivo and In Vitro
CAR bacilli colonize ciliated epithelium throughout the respiratory tract (Ganaway et al. 1985; Griffith et al. 1988; Itoh et al. 1987; MacKenzie et al. 1981; Matsushita 1986; Matsushita 1991; Matsushita and Joshima 1989; Shoji et al. 1988b; van Zwieten et al. 1980). No other organ system is reported to be affected. Attempts to cultivate CAR bacilli with conventional microbiological media have been unsuccessful (Ganaway et al. 1985; MacKenzie et al. 1981; Schoeb et al. 1993a; van Zwieten et al. 1980), and embryonated eggs or cell cultures were used to obtain most reported isolates. However, CAR bacilli can be grown in various cell culture media. Shoji et al. (1992) reported that a rat isolate and a mouse isolate grew in Eagle’s minimum essential medium supplemented with 10% fetal calf serum and 20% conditioned medium from hamster tracheal organ cultures, and that the organisms retained virulence after in vitro passage. Rat and rabbit CAR bacilli can be isolated from animals and grown in several cell culture media supplemented with 10% fetal calf serum (Schoeb et al. 1993a). A 50:50 mixture of Dulbecco’s minimal essential medium and Coon’s modified F12 medium with fetal calf serum supports optimal growth (Schoeb and Lindsey 1996). In such cultures, the organisms adhere lengthwise to the flask surface and grow singly or in interlacing fascicles (Fig. 18-12), or they can form pincushion- or brush-like aggregates with one end adherent to the flask. Rapid flexing movement often is evident in such clusters. Mixtures of these patterns also occur. Some isolates grow more rapidly than others and attain higher densities, or are more vigorously motile.
E. C.
In Vivo
Bacterial Strains
Several laboratories have obtained isolates of CAR bacillus (Cundiff et al. 1994a; Ganaway et al. 1985; Matsushita and Joshima 1989; Nietfeld et al. 1999; Schoeb et al. 1993a; Shoji et al. 1988a). Some are given strain designations, including NIH, StL, X1247C, X1331B, X1428D, and X2006C (Schoeb et al. 1993a); CBM and CBR (Itoh et al. 1987; Shoji et al. 1988a); and
Signs and lesions of CAR bacillus disease are closely similar to those of MRM (Schoeb et al. 1996; Schoeb and Lindsey 1996). Naturally occurring CAR bacillus disease in mice is described by Griffith et al. (1988). The authors report identifying CAR bacillus infection in 72 of 202 C57BL/6J obese mice and wild-type littermates. Gross lesions, when present, consisted of firm, discolored, well-demarcated areas in the cranioventral
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Fig. 18-12 CAR bacillus strain StL growing in cell culture medium. Phase contrast. Bar = 28 µm.
aspects of the lungs (Fig. 18-13). Affected airways had pronounced accumulation of lymphocytes and plasma cells, luminal neutrophilic exudate, and epithelial hyperplasia (Figs. 18-14 and 18-15). Lesions were bilateral in most mice. Colonization of the epithelial surface was evident in sections stained with either hematoxylin and eosin (HE) or Warthin-Starry silver impregnation (Fig. 18-16). Morphology of the bacteria in transmission electron micrographs was similar to that of rat CAR bacilli, with the typical trilaminar cell wall of gram-negative bacteria and location among and parallel to the cilia of respiratory epithelial cells. M. pulmonis was not isolated from any of the mice that were cultured, and results of tests of 33 mice for antibodies to M. pulmonis all were negative. However, there was evidence of concurrent viral infection in 89 of the mice, which had multifocal necrotizing bronchitis, bronchiolitis, and alveolitis,
Fig. 18-13 CAR bacillus-induced bronchopneumonia in a C57BL/6J mouse, showing areas of consolidation (arrows) in the right apical lobe, azygous lobe, and the anterior part of the left lobe. Gross lesions are indistinguishable from those of M. pulmonis disease. Bar = 1 cm. From Griffith et al. (1988), Fig. 1, p. 74, with permission.
TRENTON R. SCHOEB
Fig. 18-14 Bronchiolitis in a C57BL/6J mouse with CAR bacillus disease. The lumen is filled with neutrophils (N), and there is extensive peribronchiolar accumulation of lymphocytes and plasma cells (L). The epithelium (E) is hyperplastic and is densely colonized with CAR bacilli (arrow). HE. Bar = 30 µm. From Griffith et al. (1988), Fig. 2, p. 74, with permission.
with syncytia, eosinophilic cytoplasmic inclusions, and alveolar epithelial metaplasia. Sendai virus antigen was identified in the lesions by immunohistochemistry. Thus, concurrent Sendai virus infection may have potentiated development of CAR bacillus disease. Obese mice have decreased resistance to bacterial disease and are partially immunodeficient (Busso et al. 2002; Loffreda et al. 1998; Mancuso et al. 2002), but it does not seem likely that these defects contributed to the disease, inasmuch as a smaller proportion of obese mice were affected than nonobese littermates. The pathogenesis of CAR bacillus disease has not been extensively investigated. Kendall et al. (2000) studied antibody and
Fig. 18-15 CAR bacillus-induced bronchiolitis in a C57BL/6J mouse. Neutrophils (N) fill the lumen. The epithelium (E) is hyperplastic and densely colonized with CAR bacilli (arrow). Bar = 20 µm. From Griffith et al. (1988), Fig. 3, p. 74, with permission.
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Fig. 18-16 Bronchiolitis in a C57BL/6J mouse with CAR bacillus disease. Silver impregnation staining shows colonization of epithelial surface with CAR bacilli (arrow). Warthin-Starry stain. Bar = 30 µm. From Griffith et al. (1988), Fig. 4, p. 74, with permission.
cytokine responses to experimental infection with a mouse isolate of CAR bacillus in BALB/c and C57BL/6 mice. In BALB/c mice, disease developed rapidly, progressively increased in severity, and was accompanied by production of IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA serum antibodies and TNF-α, IFN-γ, and IL-4 in the lungs. C57BL/6 mice had less severe lesions and less vigorous antibody responses, with modest increases in IgM, IgG1, IgG2b, and IgG3, and no detectable increases in cytokines. The results were interpreted to indicate that adaptive immune responses are ineffective in eliminating infection and may actually contribute to disease severity. In addition, adaptive responses are not essential host defenses against CAR bacillus, suggesting that innate immune mechanisms are more effective. These findings are thus reminiscent of those of studies of M. pulmonis disease pathogenesis. Studies of susceptibility of immunodeficient mice to CAR bacillus infection have not been reported. C.B-17 SCID mice inoculated with a CAR bacillus of rat origin had abundant colonization of respiratory epithelium (T.R. Schoeb, M.K. Davidson, and J.K. Davis, unpublished) but lesions generally less severe than those reported by Griffith et al. (1988). As would be expected, the inflammatory response was primarily neutrophilic, and accumulation of lympoid cells was absent (Figs. 18-17–18-19). F. 1.
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Fig. 18-17 Rhinitis in a C.B-17 SCID mouse inoculated with CAR bacillus strain NIH. There is loss of mucosal mucous glands, the lumen contains neutrophils (N), and the epithelium is colonized with CAR bacilli (arrows). HE. Bar = 33 µm.
in wild rats (Brogden et al. 1993), African white-tailed hamsters (MacKenzie et al. 1981), rabbits (Oryctolagus cuniculus) (Caniatti et al. 1998; Hilding and Hilding 1966; Kurisu et al. 1990; MacKenzie et al. 1981; Oros et al. 1997c; Schoeb et al. 1993a), cattle (Hastie et al. 1993; Nietfeld et al. 1999), pigs (Hafner and Latimer 1998; Nietfeld et al. 1995, 1999), goats (Fernandez et al. 1996; Oros et al. 1997a, 1997b), and cats (Ramos-Vara et al. 2002). The host range of CAR bacillus isolates is restricted according to the host of origin. Isolates originating from mice and rats are infective and pathogenic for both species (Cundiff et al. 1994a,
Epizootiology
Host Range and Geographic Distribution
CAR bacillus infection in mice has been reported from the United States and Japan, and in rats from the United States, Japan, Europe, and Australia (France 1994). In addition to laboratory rats and mice, CAR bacillus infection has been identified
Fig. 18-18 Tracheitis in a C.B-17 SCID mouse given CAR bacillus strain NIH. The epithelium (E) is distorted and colonized with CAR bacilli (arrows). Neutrophils (N) infiltrate the mucosa and are within the epithelium. HE. Bar = 33 µm.
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Lindsey (1998), evidence of CAR bacillus infection was found in non-SPF mice at one of 72 responding institutions (1.4%), in SPF rats at 5 (6.9%), and non-SPF rats at 17 (24%). Experimental infections in mice can be inapparent, that is, without lesions, detectable organisms in Warthin-Starry-stained sections, or seroconversion for up to 8 weeks after inoculation, but with CAR bacillus recovered by culture from inoculated mice (Schoeb and Lindsey 1996). It is not known whether such infections occur naturally. 3.
Fig. 18-19 Bronchiolitis in a C.B-17 SCID mouse inoculated with CAR bacillus strain NIH. The bronchiolar lumina are filled with exudate (E) containing neutrophils and macrophages. HE. Bar = 130 µm.
1995a; Matsushita and Joshima 1989; Matsushita and Suzuki 1995). A mouse isolate induced mild rhinitis, tracheitis, and bronchitis in Syrian hamsters (Shoji-Darkye et al. 1991), and a rat isolate was pathogenic in Mongolian gerbils (St. Clair et al. 1999). However, natural CAR bacillus infection has not been reported in hamsters or gerbils. In contrast, rabbits and guinea pigs inoculated with CAR bacillus strains from mice or rats had no histologic evidence of infection or disease, although they responded serologically, and CAR bacillus antigen could be detected in the respiratory tract (Matsushita et al. 1989; ShojiDarkye et al. 1991). CAR bacilli from rabbits are pathogenic in that species, but are not infective in mice and rats, or induce antibody responses but not disease (Cundiff et al. 1994a, 1995a). Isolates from pigs also are not infective for mice or rats (Nietfeld et al. 1999). Whether such host preferences are the result of host adaptation of closely related strains or whether CAR bacilli from different hosts represent distinct species is unclear. Rat and mouse isolates appear to be closely related (Cundiff et al. 1994b; Schoeb et al. 1993a; Wei et al. 1995). Rabbit CAR bacilli are reported to be related to Helicobacter spp. rather than flavobacteria, based on 16S rRNA sequence analysis (Cundiff et al. 1995a). However, preliminary analysis of the 16S rRNA sequence of one rabbit isolate indicates that it is related to flavobacteria and to rat CAR bacilli rather than Helicobacter spp. (K.F. Dybvig and T.R. Schoeb, unpublished). Further studies of these organisms clearly are needed.
2.
Transmission
Transmission of CAR bacillus probably is similar to that of M. pulmonis. In breeding populations, sucklings acquire the organism from the mother, often within a week after birth (Ganaway 1986). In experimentally infected mice, transmission occurs slowly among cagemates and can require several weeks to become evident, whereas transmission between mice in separate cages did not occur (Matsushita et al. 1989). Thus, transmission among adults probably occurs primarily by direct contact. G. 1.
Diagnosis
General Considerations
Most information regarding diagnosis of CAR bacillus infection comes from studies of rats, but also should be applicable to mice. General considerations for diagnosis of M. pulmonis infection discussed above in Section II.G.1 are equally appropriate for CAR bacillus. Although CAR bacillus can cause respiratory disease in rats in the absence of M. pulmonis (Medina et al. 1994; Schoeb et al. 1997c), the two agents frequently occur concurrently in rats, so techniques to detect M. pulmonis and other exacerbating or secondary agents also should be employed. 2.
Serology
Antibodies to CAR bacillus can be detected by immunofluorescence (Matsushita et al. 1987) or ELISA (Shoji et al. 1988a). ELISA testing is commercially available. 3.
Culture
As described in Section IV.D, CAR bacilli can be cultured in embryonated eggs, cell culture, and cell culture media without cells, and these methods can be used to isolate the organisms from infected animals. However, this is expensive, time-consuming, and inefficient, and is thus most useful for research purposes.
Prevalence
CAR bacillus disease has been reported to occur only in conventional, not hysterectomy-derived and barrier-maintained, laboratory mice and rats. In the survey by Jacoby and
4.
Histopathology
Identification of CAR bacillus colonization of the respiratory mucosa by silver impregnation histochemical staining is the usual
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method for diagnosis of CAR bacillus infection. A microwave modification of Steiner’s method is reported to give more consistent results and less background staining than the commonly used Warthin-Starry stain (Medina et al. 1996). In the author’s experience, the Warthin-Starry method provides good results when solutions are freshly prepared with ultrapure water, all glassware is scrupulously clean, and the stain is developed over a water bath at 43°C. 5.
Immunofluorescence and Immunohistochemistry
CAR bacilli can be demonstrated in tissues by immunoperoxidase (Griffith et al. 1988; Nietfeld et al. 1995; Oros et al. 1996, 1997c) and immunofluorescence (Matsushita and Joshima 1989; Shoji-Darkye et al. 1991) methods. It is possible that immunoperoxidase methods may be somewhat more sensitive than histochemical staining, but no comparisons of different methods have been reported. 6.
Electron Microscopy
Transmission electron microscopy is useful for demonstration of CAR bacilli, which are readily identified by their characteristic ultrastructure and location among cilia at the surface of respiratory epithelium (Fig. 18-20). 7.
swab samples. PCR testing of oral swab samples was considerably more sensitive than indirect immunofluorescence (Goto et al. 1995).
H.
Treatment
There is only one report of treatment of CAR bacillus infection in laboratory rodents (Matsushita and Suzuki 1995). In that study, sulfamerazine, ampicillin, or chlortetracycline at 500 mg/l in were administered via drinking water to experimentally infected mice. Colonization of the respiratory mucosa was not evident in mice treated with sulfamerazine and ampicillin, whereas chlortetracycline was ineffective. However, treatment rarely, if ever, would be indicated.
I.
Control and Prevention
Little information specifically regarding control and prevention is available, but procedures used for M. pulmonis should be equally applicable to CAR bacillus. The risk to well-maintained SPF populations is probably low. However, Cundiff et al. (1995b) report failure of a soiled bedding sentinel system to detect CAR bacillus infection. Such potential problems should be considered in designing monitoring protocols.
Polymerase Chain Reaction
CAR bacillus infection also can be detected by PCR (Cundiff et al. 1994b; Franklin et al. 1999; Goto et al. 1995; Nietfeld et al. 1995). Cundiff et al. (1994b) report a method capable of detecting CAR bacillus in either frozen or formalin-fixed, paraffinembedded tissues of naturally and experimentally infected mice and rats. Goto et al. (1995) and Franklin et al. (1999) describe detection of the organism in nasal, oral, and tracheal
Fig. 18-20 CAR bacilli (arrows) among the cilia of a respiratory epithelial cell. Uranyl acetate and lead citrate. Bar = 650 nm.
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Williamson, J. S., Davis, J. K., and Cassell, G. H. (1986). Polyclonal activation of rat splenic lymphocytes after in vivo administration of Mycoplasma pulmonis and its relation to in vitro response. Infect. Immun. 52, 594–599. Yancey, A. L., Watson, H. L., Cartner, S. C., and Simecka, J. W. (2001). Gender is a major factor in determining the severity of mycoplasma respiratory disease in mice. Infect. Immun. 69, 2865–2871.
Chapter 19 Pasteurellaceae Werner Nicklas
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Family Pasteurellaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Genus Pasteurella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Genus Actinobacillus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Genus Haemophilus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Properties of the Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Family Pasteurellaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Sensitivity to Environmental Conditions . . . . . . . . . . . . . . . . . . . . . . 3. Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phenotypic Characteristics of Pasteurellaceae Infecting Mice . . . . . . . . 1. Pasteurella pneumotropica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Actinobacillus muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Haemophilus influenzaemurium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Growth Factor-Dependent Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . C. Molecular Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Growth In Vivo and In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Growth In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Suitable Culture Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Growth In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Locations, Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. In Vivo — Clinical Disease, Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . A. Morbidity and Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Clinical Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Primary Pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Co-pathogens, Association with Additional Pathogens, Synergistic Effects, Opportunism . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Additional Factors Responsible for Increased Pathogenicity . . . . . . 4. Latency or Latent Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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5. Organ Systems and Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Interference with Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Epizootiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Host Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Importance for Humans, Zoonotic Potential . . . . . . . . . . . . . . . . . . . . . C. Prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Prevalence in Populations of Laboratory Mice . . . . . . . . . . . . . . . . . 2. Carrier Rate in Infected Colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Geographical Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Mode(s) of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Isolation of the Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Phenotypic Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Chemotaxonomic Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Molecular Methods (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Control and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Eradication of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Gnotobiotic Rederivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Other Eradication Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General Hygiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Laboratory Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Wild Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Biological Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Cryopreserved Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
All Pasteurellaceae species are commensal parasites living on mucous membranes of vertebrates, particularly mammals and birds. At present, no free-living species are known. During their phylogenetic adaptation to parasitic life, the whole group of organisms has lost much of the genome information of their free-living ancestors, resulting in defects in their biosynthetic capabilities. For these reasons, all species are more or less fastidious organisms and have high nutritional requirements. Many species have some pathogenic potential that may become manifest under conditions such as stress or immunodeficiency and are, therefore, opportunistic pathogens. Pasteurellaceae were among the first agents identified as pathogens of laboratory mice. It is thought that the vast majority of wild mice are carriers of these organisms. The same is very likely the case in laboratory mouse populations if special precautions to avoid contamination are not undertaken. Together with other bacterial agents such as various Helicobacter species, they are among the most frequently found agents in contemporary laboratory mouse colonies, and it might be very difficult to find an experimental facility without at least a few populations that are carriers of Pasteurellaceae. However, it seems that many
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agents, among them Pasteurellaceae, are gaining importance owing to the development of thousands of new lines of genetically modified mice, many of which are not fully immunocompetent or are “immunovague.” There is general agreement that at least members of the Pasteurella (P.) pneumotropica complex, which are the most well-known bacteria of this family found in mice, may play a role as pathogens or as factors that may influence the outcome of animal experiments. These agents are therefore commonly included in health monitoring programs worldwide, and information on their presence or absence in a population is usually given in health reports. In general, Pasteurellaceae species infecting mice have low pathogenic potential. They are therefore often considered part of the “normal” flora and are frequently tolerated in populations of laboratory mice. For these reasons they have never been of major research interest. Hard scientific data on these bacteria are difficult to find in the literature, and knowledge of this group of bacteria is very limited. Although culture and subsequent identification are possible without specific equipment, insufficient information has been published on their phenotypic characteristics, resulting in frequent misidentification and perhaps false assumptions regarding their host range and other biological properties.
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19. PASTEURELLACEAE
Few molecular studies have been undertaken, and many strains and species infecting mice have not yet been sufficiently characterized. Although Pasteurellaceae in mice have low importance compared to some viral infections, they have a high degree of host specificity. Their presence in a population that is intended to be free of unwanted agents may therefore indicate a management failure or a breach in the system. The correct taxonomic position of these agents has not yet been defined. Sometimes, different organisms that were not clearly characterized were used in experimental studies or found as causative agents of disease. It seems therefore appropriate to describe all members of the family Pasteurellaceae most commonly found in mice using their traditional names.
II. A.
HISTORY
Family Pasteurellaceae
The family Pasteurellaceae was conceived to accommodate a large group of gram-negative facultative anaerobic and fermentative bacteria and originally consisted of the genera Actinobacillus, Haemophilus, and Pasteurella (Mannheim 1984). However, various studies (Mannheim 1981; Dewhirst et al. 1992) showed that the assignments into these three genera do not reflect their true genetic relatedness and that members of the genera are widely distributed among the family. The phenotypic classification of Pasteurellaceae and especially the differentiation among genera has been difficult, if not impossible, on the basis of the original concepts. Criteria used for classification were frequently arbitrarily chosen. For example, colonial adherence and the absence of indole production were long thought to be the features most consistently distinguishing Actinobacillus from Pasteurella. Other criteria were growth on MacConkey agar and urease, hemolysis, or fermentation of trehalose. Lentsch and Wagner (1980) suggested that a combination of features could be used to separate the two genera. None of these attempts, however, satisfactorily solved the problem of phenotypic classification. Increasing knowledge of this group of bacteria and reclassification according to genomic relatedness instead of phenotypic characteristics led to the transfer of species from one genus to another. Some species were split into several new species, and some disappeared by amalgamation into one new species. Several species previously classified as Haemophilus, Pasteurella, or Actinobacillus have been eliminated from the family Pasteurellaceae and assigned elsewhere. As a consequence, traditional names had to be changed repeatedly. At present, traditional terminology and corrected names coexist and frequently lead to confusion. The history of the family has been reviewed in more detail by Zinnemann (1981) and Mutters et al. (1989) and most recently by Olsen et al. (2004).
1.
Genus Pasteurella
The first bacteria of the genus Pasteurella were isolated in the nineteenth century. The type species of this genus, P. multocida, has had a variety of names over the years and was first isolated from outbreaks of disease in fowl (for details see Carter 1981; Frederiksen 1989a). This best-known species infects different hosts but does not play a role as a natural pathogen of mice (Manning et al. 1989a). However, because of the high susceptibility of mice to experimental infection by P. multocida, mice have been used to recover the agent from contaminated samples (Carter 1981). A. PASTEURELLA PNEUMOTROPICA The first report of Pasteurella isolation from mice might have been published by Andrewes et al. (1934), who cultured small bipolar gramnegative bacteria from the lungs of mice during their studies on the susceptibility of mice to influenza viruses. However, the bacteria were not further characterized because they were not regarded as etiologically significant. P. pneumotropica was first described and studied by Jawetz (1948, 1950). This agent was latent in normal mice, but similar bacteria were also reported in the lungs of normal rats and guinea pigs. Serial passages in mice by intranasal instillation resulted in necrotizing pneumonia and death within two to seven days. Ten times higher doses failed to produce lesions or death if administered intravenously, intraperitoneally, or subcutaneously to young mice. All attempts to produce disease or lesions in laboratory animals other than mice (e.g., rats, cotton rats, hamsters, guinea pigs, rabbits, chicken) after exposure by various routes failed. In view of this apparent predilection for the lung, the name P. pneumotropica was proposed. During an extensive study of Pasteurella cultures derived from the human respiratory tract, Henriksen and Jyssum (1961) observed two isolates among a number of different strains that were considered special biotypes of P. multocida. In an extension of his first communication, Henriksen (1962) classified these cultures as P. pneumotropica. These bacteria were later considered as P. ureae (Mutters et al. 1984a, Carter 1984) and were subsequently reclassified as Actinobacillus (A.) ureae (Mutters et al. 1986). Heyl (1963) examined 52 cultures of Pasteurella isolated from mice, the majority of which came from cases of pneumonia. Colonies on blood agar had a yellowish pigmentation. In contrast to mouse isolates described by Jawetz (1950), the bacteria were xylose-positive and inositol-negative. He suggested the name P. pneumotropica var. xylophila for strains comparable with his cultures and defined the strains described by Jawetz as P. pneumotropica var. pneumotropica. Frederiksen (1973) divided P. pneumotropica into three biotypes that he named after those who first described strains belonging to these biotypes. He studied 32 strains belonging to these three biotypes. All had in common positive reactions for indole and urease, and he separated the biotypes on the basis of a number of biochemical reactions (see Table 19-1). Some years
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TABLE 19-1
CLASSIFICATION OF PASTEURELLA PNEUMOTROPICA BIOTYPES ACCORDING TO FREDERIKSEN (1983)
P. pneumotropica type Heyl P. pneumotropica type Jawetz P. pneumotropica type Henriksen
Indole
Urease
ONPGa
LDCb
ODCc
Arabinose
Xylose
Melibiose
+ + +
+ + +
+ + −
+ − −
+ + −
+ − −
+ + −
+ − −
β-galactosidase lysine decarboxylase cODC: ornithine decarboxylase aONPG: bLDC:
later he found out that biotypes Heyl and Jawetz came from rodents, whereas the Henriksen biotype was mainly associated with dogs and cats and was identical with bacteria provisionally called Pasteurella “Gas” (Frederiksen 1981). It was also shown by DNA:DNA hybridization studies (Pohl 1981) that the three biotypes of P. pneumotropica were not members of one natural group and that type Henriksen does not show any remarkable binding to other P. pneumotropica biotypes. DNA:DNA hybridization studies conducted by Mutters et al. (1984b, 1985) with all recognized species of the genus led to the reclassification of the genus. Some of the formerly recognized species were excluded from the genus on the basis of these studies, among them the biotypes Jawetz and Heyl of P. pneumotropica. Even today the taxonomic position of this group of organisms has not been defined. In the most recent edition of Bergey’s Manual of Systematic Bacteriology, P. pneumotropica is listed as a species incertae sedis in the Pasteurella chapter (Mutters et al. 2004). B. ADDITIONAL PASTEURELLA SPECIES Several reports exist of isolation of additional Pasteurellaceae species from mice. However, many reports in the literature of unusual species infecting mice are confusing and should be regarded with caution. As mentioned elsewhere, there is a high risk of misidentification owing to inappropriate or insufficient numbers of criteria tested and therefore of false conclusions on the host spectrum. It is in many cases very unlikely that the results of phenotypic classification would be confirmed by genetic tests. Most species within the family Pasteurellaceae show a high degree of host specificity, which in the past has not been considered by many authors. Kunstyr and Hartmann (1983) described the isolation of eight Pasteurellaceae species, including P. aerogenes and P. ureae, in addition to three biotypes of P. pneumotropica from mice, rats, hamsters, guinea pigs, rabbits, and cats without specifying the host species. It would be very useful to have information on the species from which the agents were cultured. Also, they did not present details on phenotypic characteristics of their bacteria so that it is not possible to reevaluate their identification results. Hooper and Sebesteny (1974) found a variant of P. pneumotropica that differed from “typical” strains in several biochemical reactions (e.g., indolenegative, dulcitol-positive). Dickie et al. (1996) reported isolation
of P. gallinarum from mice. An “endogenous” mannitol-positive Pasteurella sp. was detected in bronchopulmonary tissues after infection with a reovirus (Phillips et al. 1970). Also, the isolation of P. multocida has occasionally been reported from mice (Rehbinder and Tschäppät 1974), but the criteria leading to this diagnosis are not mentioned. However, more convincing reports exist that P. multocida has been cultured from rats, especially from wild rats (Curtis et al. 1980; Roberts and Gregory 1980; Kirschnek 2000). It is frequently mentioned in health reports that P. haemolytica (which has been reclassified and is now Mannheimia sp.), P. multocida, or P. aerogenes were detected in mouse colonies, but convincing publications or scientific evidence based on appropriate classification methods do not exist. Isolation of further Pasteurellaceae species (e.g., P. dagmatis) from small rodents has been published (e.g., Boot and Bisgaard 1995; Champlin et al. 2002). These species do not cause natural infections in mice, and diagnoses are likely to be misidentifications and should be questioned. Such isolates have frequently been retested by specialized laboratories using appropriate tests (including 16S rDNA sequencing, DNA:DNA hybridization) and are usually classified as P. pneumotropica type Heyl or Jawetz (author’s laboratory, unpublished data). 2.
Genus Actinobacillus
The firsts species of the genus Actinobacillus was described in 1902 as the causative agent of infections in cattle causing lesions resembling those of Actinomycosis. As with the genus Pasteurella, DNA:DNA hybridization studies led to the reclassification of the genus, and additional species were added to those listed in Bergey’s Manual of Systematic Bacteriology (Phillips 1984). Ackerman and Fox (1981) reported isolation of P. ureae from genital tracts of mice that had received antibiotics to treat metritis combined with infertility, stillbirth, and abortion due to P. pneumotropica. Isolation of P. ureae from mice was also reported by Mraz et al. (1980), Gialamas (1981), Kunstyr and Hartmann (1983), and Hagele and Bielanski (1987). Isolation of this organism from an animal source, most likely from a mouse, was also reported by Wang et al. (1996). Mutters et al.
19. PASTEURELLACEAE
(1984a) reinvestigated rodent strains isolated by Mraz et al. (1980) and Ackerman and Fox (1981) by conventional phenotypic characterization and DNA:DNA hybridization and demonstrated that rodent isolates were not related to P. ureae. They identified the isolate described by Mraz et al. (1980) as an indole-negative variant of P. pneumotropica, whereas the remaining murine isolate (Ackerman and Fox 1981) was unrelated to P. ureae and P. pneumotropica and was considered to represent an unrecognized species. This strain had the same phenotypic characteristics as strains belonging to Bisgaard’s taxon 12, which had been isolated from the oral mucosa of laboratory mice and was found to represent a new species, named A. muris (Bisgaard 1986). Mutters et al. (1984a) concluded that members of P. ureae do not colonize rodents. This species seems to be limited to humans. Reports of its occurrence in rodents and other animals are highly questionable. It was later shown to be unrelated to the genus Pasteurella and was renamed A. ureae (Mutters et al. 1986). Isolation of Actinobacillus spp. has also been reported by Lentsch and Wagner (1980) and Simpson and Simmons (1980). Colony morphology and biochemical characteristics were described to be similar to those of P. pneumotropica, but the information given in both papers does not allow reliable identification. Kunstyr and Hartmann (1983) reported the isolation of A. lignieresii, A. capsulatus, and Actinobacillus spp. from laboratory animals, but they did not give clear information on the animal species from which these isolates were obtained or on the criteria used for classification. It must be noted that the term Actinobacillus muris was historically used also for Streptobacillus moniliformis, which still occasionally leads to confusion. 3.
Genus Haemophilus
The genus Haemophilus is one of the classical genera in medical microbiology and was established by the end of the nineteenth century (Nicolet 1981) when Haemophilus (H.) influenzae was first cultured and believed to be the causative agent of influenza. Bacteria classified as Haemophilus are frequently found on mucous membranes of humans and several animal species (Kilian and Biberstein 1984). The term Haemophilus has classically been used for Pasteurellaceae (small coccobacilli or rods) that are dependent on preformed growth factors and do not grow on blood agar. It has been known for more than a decade that the taxonomic significance of growth factor requirements was overestimated in the past, and we know from various studies that these phenotypic criteria do not reflect real relatedness. For example, the rat isolates designated as Taxon B (Kilian 1976) have been excluded from the genus Haemophilus and have been adjoined to P. pneumotropica biotype Jawetz (Ryll et al. 1991). Genera belonging to the family Pasteurellaceae may comprise both growth factor-requiring and growth factor-nonrequiring species.
473 Also, strains may be found which depend on growth factors (although this is not a typical characteristic of the species), as well as growth factor-independent strains of Pasteurellaceae commonly requiring growth factors (see Section III.B.4). It has even been demonstrated by different approaches that two growth factor-dependent species, H. parainfluenzae and H. influenzae, are phylogenetically only distantly related to each other (Dewhirst et al. 1993; Busse et al. 1997). A. HAEMOPHILUS INFLUENZAEMURIUM The bacterium H. influenzaemurium was one of the first pathogens and the first Pasteurellaceae species described in laboratory rodents. This organism, suspected to be the causative agent of an epizootic respiratory infection, was isolated from mice in 1935 and was named Bacterium influenzae murium (Kairies and Schwartzer 1936). Bacteria grew best on chocolate agar, and it was reported later (Ivanovics and Ivanovics 1937) that these organisms require hemin as a growth factor. The only reference strains existing from the first reports were destroyed during World War II (Zinnemann 1980). The organism was listed in the eighth edition of Bergey’s Manual of Determinative Bacteriology (Zinnemann and Biberstein 1974) but was later omitted from the Approved List of Bacterial Names because no representative cultures existed and classification was not possible. It is therefore not included neither in Bergey’s Manual of Systematic Bacteriology (Kilian and Biberstein 1984) nor in the ninth edition of Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994). However, this organism was reisolated in Hungary in 1973 by Csukas (1976), and one single reference strain is available from this study. The type strain (H. influenzaemurium, NCTC 11146) was subject to further studies by Bisgaard and Mutters (1986), who showed that growth of this organism is independent of hemin. It was excluded from the genus Haemophilus on the basis of its DNA relatedness (Mutters et al. 1989). This organism is presently considered to be a member of the rodent group within the family Pasteurellaceae (Christensen et al. 2003b). B. ADDITIONAL HAEMOPHILUS SPECIES Bacteria that fulfill the classically used criteria of growth factor dependency for the genus Haemophilus have been cultured from mice and other rodents. Kunstyr and Hartmann (1983) described isolation of H. influenzae, H. parainfluenzae, and H. paragallinarum from laboratory animals, including mice, but they did not mention that growth factor dependency was tested. Growth factorrequiring Pasteurellaceae were reported to occur frequently in rats (Nicklas 1989) but have also been cultured from mice (Nicklas et al. 1993) at a low prevalence rate of 2.4%. Also, strains designated as Haemophilus taxon B (Kilian 1976) have rarely been found in mice (Nicklas, unpublished). After a few passages of these NAD-dependent bacteria and extended incubation on blood agar, colonies occasionally grow on blood agar after several days. This property is stable and can be maintained for more than 20 passages. In fact, these bacteria have been shown by Ryll et al. (1991) to be closely related to P. pneumotropica type Jawetz.
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III.
PROPERTIES OF THE AGENTS A. Family Pasteurellaceae
1. General Characteristics
Pasteurellaceae are gram-negative, non–spore-forming coccobacilli or rods, usually 0.3 × 0.3-2 µm in size. The mol % G+C of the DNA is 37-47, and the genome size is 1.2–2.2 × 109 Daltons (Mannheim et al. 1980; Mannheim 1984; Olsen et al. 2004). They may show some pleomorphism, and sometimes long bacillary forms are prevalent after a number of subcultures. Some species (e.g., H. influenzaemurium) may form long filaments, but it is not possible to distinguish species on the basis of gram-stained smears. They require organic nitrogen sources and sometimes also vitamins, amino acids, and other factors for growth. They are nonmotile, facultatively anaerobic, and fermentative (Table 19-2). Most species do not produce gas from carbohydrates. Nitrate is reduced to nitrite, and oxidase and catalase tests are usually positive. The oxidase reaction is usually weak and may not be detected with methods that are not sufficiently sensitive. Typical for Pasteurellaceae is the small amount of acidity from sugar fermentation compared to Enterobacteriaceae. Many fermentation tests may therefore fail to detect acid with some of the fastidious members of the family (e.g., H. influenzaemurium). Citrate cannot be used as a carbon source, and arginine dihydrolase is not produced. Pasteurellaceae of homoeothermic vertebrates grow best at 35–37°C. They will also grow at several degrees above 37°C, but growth is markedly reduced as the temperature is lowered toward 25°C (Carter 1981). Members of the Pasteurellaceae are usually isolated from mucous membranes of the respiratory, alimentary, and genital tracts. They may also be recovered from other tissues (e.g., from the conjunctivae) or from secretions. Most species infect nonhuman animals and do not play an important role in humans. Bacteria belonging to the genera Pasteurella and Actinobacillus
are typical animal pathogens and are only occasionally transmitted to humans by animal contact. Most members of the family show a high degree of host specificity. With few exceptions, each single species seems to be associated with one or very few specific hosts. This is also the case for species infecting mice, although it has repeatedly been reported in the literature that P. pneumotropica, the most frequently mentioned organism occurring in mice, may infect a broad spectrum of different hosts. However, it seems that this assertion may in many cases be a result of misidentification of bacterial isolates. Pasteurella, Actinobacillus, and Haemophilus are historical terms. The genera are very similar in their mole percentages of G+C and in their chromosomal DNA. There is no single phenotypic feature that clearly distinguishes the genera. It has been stated by Mannheim et al. (1980) that the classification into these three genera requires reconsideration. Molecular studies demonstrate that certain species currently assigned to a genus or even to a species may belong to separate genera according to their 16S rDNA sequences and other molecular criteria. This is especially the case for Pasteurellaceae infecting mice and other rodents because sufficiently detailed taxonomic studies have not been conducted. The information available on properties of these agents is therefore incomplete and frequently misleading. Even today the state of taxonomy of the family Pasteurellaceae is not comparable with that of well-studied groups of bacteria with similar importance (e.g., the Enterobacteriaceae). During the last decade, additional genera have been created within the family Pasteurellaceae by reclassification of existing species [e.g., the genera Lonepinella (Osawa et al. 1995), Mannheimia (Angen et al. 1999), Phocoenobacter (Foster et al. 2000), Gallibacterium (Christensen et al., 2003a), Histophilus (Angen et al. 2003), and Volucribacter (Christensen et al. 2004)]. It will be necessary in the future for rodent Pasteurellaceae to be reclassified because the presently used historical names do not reflect their true relatedness and taxonomic position. 2.
TABLE 19-2
PHENOTYPIC CHARACTERISTICS OF PASTEURELLACEAE ●
● ● ● ● ● ● ● ● ● ●
Gram-negative short rods or coccobacilli; some may form filaments or morse-code forms (rods interspersed with coccal elements) Optimum growth temperature: 35–37°C Facultative anaerobic Fermentative with weak production of acid from various sugars Nonmotile Oxidase usually positive (frequently weak) No utilization of citrate as a carbon source Arginine dihydrolase negative Reduction of nitrate to nitrite Mol% G+C of the DNA: 38–47 Genome size: 1.2–2.2 × 109 daltons
Sensitivity to Environmental Conditions
Pasteurellaceae have a limited survival time outside an appropriate host. They should be subcultured after at least 4 to 6 days, and some strains do not survive on blood agar in the incubator over the weekend. Strains of A. muris are similarly sensitive and should be subcultured after no more than three days. The survival time in the refrigerator (4°C) may be similarly short. After drying on various surfaces (laboratory coat, cardboard, plastic), P. pneumotropica usually survives for less than two hours, with the longest survival time of 2 hours on mouse hair (Scharmann and Heller 2001). When stored in phosphate buffered saline (PBS), the number of viable Pasteurella constantly decreased at various temperatures (+37, +24, +4, –20°C), while the number of other viable bacteria remained constant during the observation period or decreased much more slowly (Shimoda et al. 1991). Survival times can be extended by the addition of 10% glycerol
475
19. PASTEURELLACEAE
or 3% skimmed milk with 5% glucose. Owing to the high sensitivity to environmental conditions, samples to be cultured for Pasteurellaceae (e.g., swabs) are best shipped in transportation media (e.g., Amies, Stuart). Shipping of bacterial isolates (e.g., to reference laboratories) on agar plates is possible. However, only young overnight cultures should be used. Transportation over the weekend must be avoided because many strains will not survive. Viability is increased when shipped in transportation media, and transportation on dry ice is also possible in a liquid medium containing a cryoprotectant. Both approaches are recommended, especially for transportation over long distances. 3.
Storage
Like other bacteria, Pasteurellaceae can easily be maintained by freezing without significant loss of viability when cryoprotectants are used (e.g., 15% glycerol in broth). In general, storage at temperatures above −30°C results in poor viability. The most common temperatures used are −80°C or −196°C in liquid nitrogen. Freezing on glass beads using a glycerol-containing medium has many advantages over freezing and storage in larger volumes, (e.g., in 1 ml aliquots). Lyophilization is another method that can successfully be used for long-term storage of Pasteurellaceae and other bacteria. We use calf serum containing 6% glucose, but other suspension media may also be used successfully. Various methods for the maintenance of bacteria and other microorganisms have been described in detail by Kirsop and Doyle (1991). They report storage and successful preservation of Actinobacillus, Haemophilus, and Pasteurella on glass beads or freeze dried for 20 years or more. B.
Phenotypic Characteristics of Pasteurellaceae Infecting Mice
Reports in the literature on the biochemical characteristics of rodent Pasteurellaceae are very inconsistent and contain conflicting information. Information from older articles is especially confusing, which may be due to differences in media and incubation times used in different laboratories. Frequently, only small numbers of isolates were studied, and most likely isolates belonged to different phenotypic groups or even species. For example, the ability of P. pneumotropica to ferment mannitol has repeatedly been reported (Hoag et al. 1962; Phillips et al. 1970; Hooper and Sebesteny 1974; Ward et al. 1978). This reaction is consistently negative in contemporary literature, and most likely isolates belonging to A. muris were tested. Many reports are based on simplified diagnostic keys that do not yield unambiguous identification of the species and cause confusion regarding the species delineation and epidemiology. The lack of reliable commercial identification systems adds to diagnostic problems and difficulties in phenotypic separation and identification of genera and species and is often a reason for false conclusions (e.g., on the host spectrum). Insufficiently
characterized or even misidentified strains may also have been used for molecular studies and are discussed as potential causes for confusing data (Liu et al. 1999). The information given here focuses on Pasteurellaceae species that are more or less common in laboratory or wild mice. Additional Pasteurellaceae species or phenotypic variants infecting mice exist, and it has to be expected that bacterial species commonly infecting related hosts may occasionally be cultured also from laboratory mice. Exceptions may therefore occur, and such strains should be submitted to reference laboratories for further characterization. Pasteurellaceae commonly infecting mice show great variability in their biochemical characteristics (Schulz et al. 1977). Differences also exist in the hemagglutinating properties (Boot et al. 1993) of various Pasteurelleacae from rodents and in their serological reactivity (Boot et al. 1994). Rodent Pasteurellaceae are positive for sucrose, mannose, fructose, and maltose, and produce catalase (Table 19-3). They do not split dulcitol and sorbitol. Key characteristics for the phenotypic differentiation of Pasteurellaceae infecting mice are alkaline phosphatase, urease, ornithine decarboxylase, β-galactosidase, and production of indole. Fermentation of sugars and alcohols such as mannitol, arabinose, xylose, and melibiose are similarly important for the separation of species and biotypes. 1.
Pasteurella pneumotropica
Colonies of P. pneumotropica in general are greyish-white, smooth, and nonhemolytic (Fig. 19-1). Sometimes, strains belonging to biotype Heyl have a typically yellow color, but many strains have the same color as strains belonging to biotype Jawetz and are indistinguishable. The colony color is therefore not an appropriate criterion to separate these biotypes with certainty. Bacteria belonging to this group show some variation in biochemical characteristics. Numerous attempts have been made to classify different types (e.g., Simmons and Simpson 1977). P. pneumotropica is usually urease- and ornithine-positive and does not ferment mannitol, cellobiose, and salicin. The organism does not produce gas from glucose. It is positive for alkaline phosphatase and β-galactosidase (Table 19-4). Most strains produce indole, but indole-negative strains occur (Blackmore 1972; Casillo and Blackmore 1972; Hooper and Sebesteny 1974; Schulz et al. 1977; Simmons and Simpson 1977; Ward et al. 1978; Nicklas et al. 1995). Typical for P. pneumotropica is a strong odor resembling that of H. influenzae and
TABLE 19-3
PHENOTYPIC CHARACTERISTICS OF PASTEURELLACEAE INFECTING MICE ● ● ●
Sucrose, maltose, galactose (sometimes weak), fructose, mannose positive Dulcitol and sorbitol negative Catalase positive
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WERNER NICKLAS
Fig. 19-1 Mixed culture of Pasteurella pneumotropica biotype Jawetz and Actinobacillus muris together with various Streptococci and Lactobacilli from the lungs of a mouse (48 hours incubation).
other Pasteurellaceae. The smell is typically described as resembling that of ink or sperm. Historically, urease- and indole-positive Pasteurellaceae were classified as P. pneumotropica. However, it has been shown that other species share these characteristics (e.g., P. dagmatis, which was formerly designated as P. pneumotropica biotype Henriksen). Urease-negative strains of P. pneumotropica are extremely unusual, but strains that do not produce indole occur more frequently. However, it must be considered that at least some of the reported weak, delayed positive or negative reactions (Hooper and Sebesteny 1974) might be due to the use of media that do not sufficiently support growth of these bacteria, and these might therefore be false-negative reactions (see Section VII.A.2). A. PASTEURELLA PNEUMOTROPICA BIOTYPES There have been several opinions about the separation of the two P. pneumotropica biotypes. Heyl (1963) separated the biotypes based on their ability to split xylose and inositol. Frederiksen (1973) uses differences in the ability to ferment arabinose and melibiose to
TABLE 19-4
PHENOTYPIC CHARACTERISTICS OF PASTEURELLA PNEUMOTROPICA ● ● ● ● ● ● ● ●
●
Positive for alkaline phosphatase and β-galactosidase Ribose and trehalose positive Urease positive No fermentation of mannitol, dulcitol, cellobiose, salicin, esculin No gas production from glucose Most strains produce ornithine decarboxylase (negative strains occur) Variable reactions for lactose, inositol Negative reactions for β-glucosidase, α-fucosidase, β-glucuronidase, β-xylosidase Positive reaction for α-glucosidase (except trehalose-negative rat isolates)
distinguish between biotypes. According to Mannheim et al. (1989), fermentation of mannose should be used. Mutters et al. (1989) suggest fermentation reactions of arabinose, inositol, and melibiose. Ryll et al. (1991) found that lysine decarboxylase (LDC) is the best criterion to differ between biotypes Heyl and Jawetz. A common feature of all these studies is that limited numbers of isolates (usually <20) were studied. Based on our phenotypic studies (Nicklas et al. 1995) for which more than 2000 isolates from numerous populations of rodents were tested and which were later confirmed by genetic studies (Nicklas and Kirschnek 1996), both biotypes can best be separated from each other by tests for arabinose and melibiose fermentation and a few other criteria (e.g., melibiose-positive strains also ferment raffinose). Kodjo et al. (1999) also observed that arabinose, melibiose, and raffinose fermentation are useful to separate both biotypes and could confirm the results by using phenotypic criteria and PCR methods. Biotype Heyl is positive for arabinose and also ferments melibiose and raffinose (Table 19-5). Typical strains belonging to biotype Jawetz are negative for arabinose and melibiose; however, a variant exists of this biotype, which ferments melibiose and is consistently negative for indole. Exceptionally, strains occur that are positive for arabinose and negative for melibiose or vice versa, and these cannot be unequivocally classified on the basis of phenotypic criteria. A definite classification in these rare cases is possible only using molecular methods. Positive reactions for LDC are found only among strains of biotype Heyl, but the majority of isolates are negative. The Heyl biotype seems to represent a homogeneous species that can be found in mice and rats and occasionally also in other rodent species (e.g., Syrian hamsters, Mastomys). Biotype Jawetz is more heterogeneous and consists of at least two different
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19. PASTEURELLACEAE
TABLE 19-5
2.
PHENOTYPIC CHARACTERISTICS OF PASTEURELLA PNEUMOTROPICA BIOTYPES JAWETZ AND HEYL Biotype Jawetz 1a 1aa − − − + − + − − + −
Arabinose Melibiose Raffinose LDC Indole
Biotype Heyl + + + d (+)
a These designations are used in our laboratory for the identification of different phenotypes of Pasteurella pneumotropica type Jawetz.
species (Ryll et al. 1991). Strains with characteristics of the type strain isolated by Jawetz (arabinose- and melibiose-negative) are usually found in mice and are less common in rats. The second phenotype, characterized by negative reactions for indole and arabinose fermentation, infects both mice and rats. Both are closely related to each other on the species level (Table 19-5). Another group exists which, in contrast to both phenotypes of the type Jawetz already mentioned, does not ferment trehalose (Table 19-6). These bacteria are almost exclusively found in rats and only exceptionally in mice and are not related to trehalose-positive strains on the species level (Ryll et al. 1991; Kirschnek 2000). These strains may also be negative for xylose, while the vast majority of strains fermenting trehalose show positive reactions.
TABLE 19-6
PHENOTYPIC CHARACTERISTICS OF BIOTYPES OF PASTEURELLA PNEUMOTROPICA (MINIMAL CHARACTERISTICS TO SEPARATE BETWEEN BIOTYPES.)
Actinobacillus muris
Colonies on blood agar are small after incubation at 37°C for 24 hours but are convex and mucoid with a diameter of 4–5 mm after 48 hours of incubation (Fig. 19-1). Upon subculture, colonies grown on blood agar in a normal atmosphere are heterogeneous in size after overnight incubation and may mimic a mixed culture while colonies are uniform and larger when grown in 5 to 10% CO2. After incubation for two days, colonies may almost reach the size of Enterobacteriaceae colonies; however, they can easily be distinguished by their failure to grow on MacConkey agar. Typical hemolysis is not observed, but sometimes a weak hemolytic zone is visible after removal of colonies. These hemolytic zones are intensified in the vicinity of Staphylococcus (Staph.) aureus. The cultures do not have the specific odor characteristic of P. pneumotropica. Bacteria are highly pleomorphic gram-negative rods on gram stain. Cell sizes range from short rods to filaments (up to 10 µm). The most typical characteristic to distinguish A. muris from P. pneumotropica is the negative reaction for alkaline phosphatase, with reactions for ornithine and β-galactosidase also negative (Table 19-7). The strains originally found by Ackerman and Fox (1981) and Bisgaard (1986), which were also studied in detail by Mutters et al. (1984a), are positive for mannitol, cellobiose, salicin, trehalose, raffinose, esculin, urease, β-glucosidase and β-glucuronidase, and negative for arabinose, xylose, sorbitol, and indole. This agent shows even more variation than P. pneumotropica. Various phenotypes exist, with differences in growth characteristics and culture morphology. They may also deviate from the biochemical characteristics published by Bisgaard (1986) in several reactions (Table 19-8). Variable reactions can be found, for example, for mannitol, indole, urease, cellobiose, salicin, and esculin.
Pasteurella pneumotropica Jawetz Reaction
Mannitol Urease Phosphatase Indole Arabinose Xylose Trehalose Melibiose Raffinose Esculin Host species Mouse Rat
3.
Jawetz-like (rat)
Heyl
Pp1aa
Pp1a
Pp4a
Pp5a
Pp6a
− (+) + (+) + + + + + −
− + + − − + + + + −
− + + + − + + − − −
− + + (+) − + − − − −
− + + + − − − − − −
− + + (+) + + − − − −
x x
x x
x (x)
x
(x) x
a These terms are used in our laboratory to describe phenotypes of Pasteurella pneumotropica.
Very little information has been published on this organism. Kairies and Schwartzer (1936) reported that they grow best on chocolate agar. We studied a number of isolates from both wild and laboratory mice, and the information given here is
TABLE 19-7
PHENOTYPIC CHARACTERISTICS OF ACTINOBACILLUS MURIS ●
●
x
Haemophilus influenzaemurium
● ●
●
Negative for alkaline phosphatase, β-galactosidase, α-glucosidase, and ornithine decarboxylase Ribose, trehalose, raffinose positive No fermentation of lactose, arabinose, and xylose Most strains produce urease and ferment melibiose (negative, weakly, or delayed positive strains may occur) Variable reactions for inositol
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WERNER NICKLAS
TABLE 19-8
PHENOTYPIC CHARACTERISTICS OF BIOTYPES OF ACTINOBACILLUS MURIS AND OTHER GROWTH FACTOR-INDEPENDENT PASTEURELLACEAE INFECTING MICE (MINIMAL CHARACTERISTICS TO SEPARATE BETWEEN BIOTYPES) .
Actinobacillus muris
Reaction
Mannitol Urease Phosphatase Indol Arabinose Xylose Trehalose Melibiose Raffinose Ribose Esculin Cellobiose Salicin β-glucosidase α-fucosidase β-glucuronidase
Past. pneum.a
A. murisb
Actino 11c
Actino 12c
Actino 21c
Actino 22c
Actino HWc
H. infl.d
− + +
+ + − − − − + d + + + + + + +
+ + − + − − + + + + + − + + +
+ + − + − − (+) + + + − − − − −
− (+) − − − − + + + + − − − − −
− + − + − − + + + + + (−) (+) (+) −
− − − − − − + + + + + + + + −
− − − − − − (+) − − + − − − − − −
a
Pasteurella pneumotropica. Biochemical profile as described by Bisgaard (1986). c These terms are used in our laboratory to describe phenotypes of Actinobacillus muris. d Haemophilus influenzaemurium. b
primarily based on these studies (Kirschnek et al. 1997; Nicklas et al. 1998). This organism is difficult to isolate from clinical material because the bacteria grow very slowly during primary isolation. It can best be cultured on blood agar, but it will also grow on chocolate agar. Growth is enhanced by 5 to 10% CO2 but is not improved by growth factors used to culture “true” Haemophilus, nor do they show satellite growth adjacent to Staph. aureus. Colonies are frequently mucoid after incubation for 2 to 3 days and may be yellowish. Dependence on X factor has been reported (Ivanovics and Ivanovics 1937; Csukas 1976). We could not confirm their dependency on X factor (hemin) and showed that they possess the enzymes of the hemin biosynthetic pathway and are therefore able to synthesize porphyrins from small precursor molecules. This is in agreement with data from Bisgaard and Mutters (1986), Mannheim et al. (1989), and Mutters et al. (1989), whereas a recently published textbook (Hansen 2000) mentions dependency of this species on hemin. Identification by conventional methods is difficult due to the lack of sufficient information available from the literature. In addition, high nutritional and growth requirements are responsible for weak reactions in biochemical tests and further facilitate its identification. Commercial test kits are not able to identify this organism or to discriminate it from other Pasteurellaceae of rodent origin. Only a few reactions (NO2, PNG, oxidase) are
positive in the API 20 NE kit, and the API 50 CH kit usually yields very weak reactions that are not always reproducible due to poor growth. Results of “classical” phenotypic tests are more reproducible. Sugar fermentation (e.g., galactose, trehalose, ribose) is often weak and requires a minimum of 48 hours of incubation. Identification of H. influenzaemurium and discrimination from other Pasteurellaceae-infecting rodents can easily be achieved by phenotypic methods (Tables 19-8, 19-9). In contrast to P. pneumotropica, this organism lacks alkaline phosphatase and is negative for indole, urease, and ornithine decarboxylase. It can be separated from A. muris and other organisms found in mice by negative reactions for urease, mannitol, melibiose, raffinose, indole, and esculin, and a positive reaction for β-galactosidase. Biochemical characteristics are very similar to those of a Pasteurellaceae species reported from rats (Schulz et al. 1977). Separation from this hitherto unidentified species TABLE 19-9
PHENOTYPIC CHARACTERISTICS OF HAEMOPHILUS INFLUENZAEMURIUM ●
●
●
5 to 10% CO2 advantageous, sometimes necessary for primary isolation and subculture Negative reactions for urease, indole, alkaline phosphatase, mannitol, inositol, melibiose, raffinose, esculin Positive reaction for ribose, β-galactosidase
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19. PASTEURELLACEAE
is more difficult, but it is possible due to different reactions for β-galactosidase and ribose fermentation. Lack of growth on media that do not contain blood or serum (e.g., Mueller-Hinton agar) agar seems to be characteristic for H. influenzaemurium, whereas other growth factor-independent Pasteurellaceae strains tested grow easily. 4.
Growth Factor-Dependent Bacteria
The term Haemophilus has classically been used for Pasteurellaceae that are dependent on growth factors and do not grow on blood agar. Growth depends on either X factor (protoporphyrin, hemin) or V factor. Various compounds such as nicotinamide adenin-dinucleotide (NAD) and nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) can serve as V factor. These growth factors are released from erythrocytes by heat (80°C) and are therefore available in chocolate agar but not in blood agar. They are also produced in excess by other bacteria, which therefore support growth of Haemophilus adjacent to their colonies (“satellite phenomenon”) (Fig. 19-2). It has been shown that dependency on growth factors does not justify definition of a genus based on these criteria and also that strains may be found which depend on growth factors, although this is not a typical characteristic of the species. Krause et al. (1987) showed by DNA:DNA hybridization that V factordependent bacteria isolated from swine lungs and exhibiting a pattern of biochemical reactions fitting with that of P. multocida subsp. multocida were really members of this species. Vice versa, for many species of Pasteurellaceae defined as V factor-dependent, V factor-independent variants have been identified. These include strains of A. pleuropneumoniae (Pohl et al. 1983), H. paragallinarum (Mouahid et al. 1992;
Bragg et al. 1993; Miflin et al. 1995), H. influenzae (Munson et al. 2004), and H. parainfluenzae (Gromkova and Koornhof 1990). The gene encoding V factor-independence has been shown to be present on a plasmid (Windsor et al. 1991; Martin et al. 2001). It has even been concluded by Niven and O’Reilly (1990) that all members of the family Pasteurellaceae require pyridine nucleotides or precursors for growth and that this “growth factor dependency” is an important familial criterion for the Pasteurellaceae. While members of the P. pneumotropica-complex and the A. muris group are frequently found in mice, growth factordependent bacteria seem to play a minor role. They are common in rats from experimental colonies as well as from commercial breeders but are only occasionally found in mice. This group of bacteria most frequently colonizes the deep respiratory tract (lungs and the trachea), and 80 to 90% of all strains were isolated from these sites (Nicklas and Benner 1994; Bootz et al. 1998). Growth factor-dependent bacteria colonizing laboratory rodents have not been classified satisfactorily. Most strains found in laboratory rodents are members of the H. parainfluenzae complex, which typically infect humans, but they are also found in several species of laboratory animals such as rats, guinea pigs, and occasionally also in mice. There is considerable variation in metabolic activity within this group of organisms. They are usually dependent on V factor (NAD), and some strains may produce small amounts of gas from glucose. They show positive reactions for sucrose, mannose, and fructose and do not ferment mannitol, arabinose, trehalose, and melibiose. They do not produce indole. Reactions for ribose are usually negative, and most strains are catalase-positive (Nicklas et al. 1993). In addition to the members of the H. parainfluenzae complex, additional strains
Fig. 19-2 Satellite growth of Haemophilus parainfluenzae adjacent to Staphylococcus aureus.
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WERNER NICKLAS
and species can be found. They are, however, extremely uncommon in mice.
C.
of the gene encoding the β-subunit of the RNA polymerase (rpoB) resulted in a similar relationship within the family Pasteurellaceae, and only a few discrepancies between the trees were observed (Korczak et al. 2004).
Molecular Classification
In the last edition of Bergey’s Manual of Systematic Bacteriology, P. pneumotropica was listed as one of six species of the genus (Carter 1984). However, it was already stated in an addendum that reference strains of P. pneumotropica biotype Heyl and Jawetz remain outside the genotypic cluster Pasteurella sensu strictu (Mutters and Mannheim 1984). Major advances in understanding the phylogeny of the members of the family Pasteurellaceae came from DNA:DNA hybridization studies by Pohl (1981) and Mutters et al. (1984b, 1985). These studies defined all recognized species belonging to the sensu strictu definitions of the three traditional genera. They revealed that biotypes Jawetz and Heyl of P. pneumotropica were more closely related to the Actinobacillus group. Biotype Henriksen, however, remained in the genus and was reclassified as P. dagmatis (Mutters et al. 1985). They also showed that the phylogenetic structure of the Pasteurellaceae is complex and that more than three genera are required to accommodate the species in this group. Studies by Ryll et al. (1991) on the P. pneumotropica complex have shown that biotype Jawetz represents a genus-like cluster containing several species, including the growth (V factor) factordependent “Taxon B.” Their studies showed further that biotype Heyl remains as a species without further relationship to other known genera, and it is also not related to biotype Jawetz on the genus level. An important additional step in molecular classification of Pasteurellaceae was done by the comparison of DNA sequences. Dewhirst et al. (1992) determined 16S rDNA sequences for 70 representative strains of species in the family Pasteurellaceae. The majority of strains fell within four large clusters with several subclusters; however, the type strain of P. pneumotropica (type Jawetz) branched alone to form a fifth group. Additional strains were included in subsequent studies (Dewhirst et al. 1993) showing that A. muris was related to P. pneumotropica in cluster 5. Meanwhile, it is generally accepted that based on 16S rDNA phylogenetic analysis as well as on DNA:DNA hybridizations, rodent species of the family Pasteurellaceae form a separate group and do not belong to the genus Actinobacillus. Consequently, rodent Pasteurellaceae have not been assigned to one of the traditional genera in a review by Bisgaard (1993) but were instead described under taxa of uncertain affiliation. Even today the taxonomic position of P. pneumotropica and other species infecting mice has not been clearly defined. A full phylogenetic tree based on 16S rDNA sequences has been published by Christensen et al. (2003b) and Christensen and Bisgaard (2004), which demonstrates that P. pneumotropica, A. muris, and H. influenzaemurium form a separate subcluster in the family Pasteurellaceae. A phylogenetic tree obtained with sequences
IV.
GROWTH IN VIVO AND IN VITRO
A. 1.
Growth in vitro
Suitable Culture Conditions
The optimum growth temperature for rodent Pasteurellaceae is 35–37°C. Under appropriate conditions, colonies are usually visible after incubation for 18–24 hours, and overnight incubation is considered to be sufficient by many laboratories. However, many strains will not be detected and are easily visible only after incubation for 48 hours or more. Most strains grow within 24 to 48 hours in a normal atmosphere, but an atmosphere enriched with 5 to 10% CO2 usually improves growth. Only the rarely found H. influenzaemurium grows very slowly or not at all in a normal atmosphere and is best detected after incubation in an atmosphere enriched with CO2 and an extended incubation time.
2.
Media
Pasteurellaceae of mouse origin are easily cultured on chocolate agar, but most species also grow on blood agar and on other enriched media. Occasional reports indicate that the growth rate can be increased by addition of serum to liquid and solid media (Hoag et al. 1962). In general, Pasteurellaceae grow relatively slowly and may easily be overgrown in mixed cultures. Growth is even suppressed if they have to compete with large numbers of other slowly growing bacteria or bacteria that form small colonies such as streptococci or lactobacilli, (e.g., in cultures from the intestinal tract). Therefore, for the purpose of surveying the incidence of these bacteria, a selective medium that increases the sensitivity of detection is advisable. These media facilitate the isolation of P. pneumotropica especially from the intestinal tract (e.g., cecum) or from feces but also from other organs and mucous membranes. Various media designed for the selective isolation of Pasteurellaceae have been listed by Kilian and Frederiksen (1981a). Improved success has been reported with chocolate agar or blood agar with addition of bacitracin, clindamycin, or other antibiotics which inhibit the gram-positive flora (Garlinghouse et al. 1981; Hansen 2000). Selective media containing potassium tellurite, kanamycin, and bacitracin were developed by Knight et al. (1983) and Mikazuki et al. (1987, 1994). Growth factor-requiring species or strains are traditionally cultured on chocolate agar but also grow on other enriched agar
481
19. PASTEURELLACEAE
media supplemented with 5 to 7% blood or serum and crossinoculated with a Staph. aureus strain to provide V-factor. P. pneumotropica and other Pasteurellaceae species infecting mice usually do not grow on selective media for Enterobacteriaceae (e.g., MacConkey agar). However, P. pneumotropica occasionally grows on MacConkey agar from clinical samples on primary isolation, but growth is usually not observed on MacConkey agar upon subculture attempts.
B. 1.
Growth in vivo
Locations, Organs
Pasteurellaceae infecting mice were first detected in the respiratory tract (Kairies and Schwartzer 1936; Jawetz 1948, 1950). Infections are usually clinically silent, but they may be associated with various clinical signs such as conjunctivitis, abscesses, metritis, mastitis, pneumonia, and other clinical diseases. In latently infected mice, Pasteurellaceae can be detected on all mucous membranes, and they are sometimes almost ubiquitous on the mucous membranes of their host. Main colonization sites are the respiratory and the genital tracts, and they are very frequently found also in the intestinal tract and in feces. Other important sites are mucous membranes of the genital tract (preferably prepuce and vagina). Pasteurellaceae are among the most frequently occurring bacterial species found in the female genital tracts of mice and rats (Larsen et al. 1976a; Yamada et al. 1983). Vaginal bacterial counts vary with the estrus cycle stage, and the highest bacterial counts are consistently observed during the estrus phase of the cycle (Larsen et al. 1976b; Yamada et al. 1983). Peak bacterial counts reach levels approximately 100 times average peak levels seen in ovariectomized animals. Also, the microflora is more diverse in females with a normal estrus cycle, and various agents such as anaerobes and also P. pneumotropica are only found in rats with a normal cycle (Larsen et al. 1977). Administration of estrogen to ovariectomized rats causes an increase in the number of P. pneumotropica nearly equal to that at estrus in intact animals (Yamada et al. 1986). These findings explain why Pasteurellaceae are found in varying numbers in the vagina and may also explain the high frequency of bacterial transmission during birth. Pasteurellaceae are also frequently isolated from the uteri of mice, but they are less frequently found in fetuses or in the uteri of pregnant mice. They are found in a high percentage of uteri after experimental intravaginal infection indicating ascending infection. Mixing infected males with females produces a similarly high incidence of uterine infection. The bacteria are also found in the uteri at a lower incidence after intravenous or intranasal inoculation (Blackmore and Casillo 1972). It is interesting that colonization of the uterus is transitory. Growth factor-dependent species can also be found on mucous membranes and in various organ systems including the intestinal tract.
However, they are most frequently found in the lungs and the trachea and less frequently in other organs.
V. IN VIVO — CLINICAL DISEASE, PATHOGENESIS While some species or “biotypes” of Pasteurellaceae infecting mice or rats seem to be host-specific, some variants of P. pneumotropica are found in several host species. Rats and mice and sometimes also hamsters are often housed in the same facility, and the agents may be transmitted among these species. Therefore, some information is also given for Pasteurellaceae infections in other species in addition to mice when necessary.
A.
Morbidity and Mortality
While Pasteurellaceae are prevalent in many rodent populations, reports of natural disease outbreaks are relatively rare. With few exceptions, clinical disease seems to be usually caused by P. pneumotropica, and only very few reports exist on disease caused by other Pasteurellaceae species. The morbidity rate induced by P. pneumotropica is variable but in general low. Most reports dealing with clinical disease caused by P. pneumotropica were published before 1990. Comparable to infections with other Pasteurellaceae species (e.g., P. multocida) and other bacterial and viral pathogens, additional factors are necessary to induce disease. Contemporary laboratory mice and rats are now frequently housed under optimal environmental conditions (e.g., temperature, humidity, nutrition) and are free of most other opportunistic agents (viruses, parasites, other bacterial agents such as Mycoplasma, Bordetella) that might compromise the host’s immune system. There is no mortality associated with clinical disease in adult immunocompetent mice. Disease expression is primarily dependent on host and environmental factors. It has been shown repeatedly that the severity and prevalence rate of clinical signs associated with Pasteurellaceae infections are very much dependent on the host genotype (Kunstyr et al. 1980; Roberts and Gregory 1980; McGinn et al. 1992). Immunodeficiency or immunosuppression are important predisposing factors for clinical disease (Artwohl et al. 2000), but also pathological states (e.g., renal failure) or experimental procedures (e.g., nephrectomy, anesthesia) result in the reduction of bactericidal activity of the host’s defense mechanisms (Goldstein and Green 1967). Although the organisms show some variability in biochemical reactions, correlations between specific biotypes and pathogenicity have not been firmly established, nor have specific criteria been developed for evaluating pathogenicity. During recent years it has repeatedly been reported that P. pneumotropica may cause severe problems in genetically modified animals (Dickie et al. 1996; Artwohl et al. 2000; Enzler et al. 2003) and may lead to high morbidity and mortality (Macy et al. 2000).
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Mice deficient in MHC class II antigens are more susceptible to infection than wild-type mice (Chapes et al. 2001). Infection by P. pneumotropica may even impede phenotypic characterization of such mice due to high mortality during the first days after birth (Erdman et al. 1999). Much information on the pathogenicity and other biological characteristics of P. pneumotropica is based on experimental infections conducted 30 or more years ago. The agents used for infection experiments were frequently not sufficiently defined, which may explain contradictory results. For example, Jawetz (1950) reported that mice were highly susceptible to intranasal infection, while studies from Goldstein and Green (1967), Jakab (1974), and Jakab and Dick (1973) revealed that viable organisms rapidly declined in the lungs until, at 24 hours p.i., less than 1% of the initial dose remained. Viable organisms were completely eliminated from the lungs within 72 hours. Also, animals used for infection studies were frequently undefined microbiologically or insufficiently defined (e.g., by serological tests for viral infections). Several authors (Sellers et al. 1961; Phillips et al. 1970; Jakab and Dick 1973; Jakab 1974) report endogenous Pasteurella sp., which had not been detected previously during their infection studies in mice. It is in many cases difficult to ascertain whether a pathogenic effect was due to increased virulence of the agent of interest, for example, by serial animal passages (Jawetz and Baker 1950), or if other undetected agents played a synergistic role or were even passaged as well. It seems that the importance of P. pneumotropica as a primary pathogen of the respiratory tract might have been overestimated in several reports. Not even reports of deaths resulting from infection with pure cultures of P. pneumotropica [while uninfected animals did not show any changes or death (e.g., Laubach et al. 1978)] conclusively exclude the possibility that undetected silent infection by other agents might have contributed to clinical signs.
B.
Pathogenesis
The pathogenesis of Pasteurellaeceae infections in mice has received little attention. Usually, the agents peacefully coexist with their animal hosts, and all of the species have the capacity to persist in their host, thus resisting phagocytosis and lysis. Clinically evident infections usually depend on an incapacitation of host defenses by various factors (e.g., immunosuppression, immaturity, concurrent infections, physical injuries, or challenges by different types of stress). The agents have a predilection for tissues that have been injured or damaged by various means, including infections by viruses and mycoplasmas. Colonization mechanisms of Pasteurellaceae have been discussed by Biberstein (1990). Pili and outer membrane proteins not identified with pili as well as capsules have been related to attachment. Some species have been shown to produce IgA endopeptidases for which a role in persistence is assumed. Capsules are important for proliferation and dissemination because they inhibit phagocytosis and complement-mediated killing.
Lipopolysaccharides (LPS) have been shown to be important contributors to the disease process and to manifestations of infections causing fever, organ damage, vascular derangements, or abortion. Host resistance to pulmonary infection depends on various genes, and it was shown that mice lacking functional alleles of certain genes are more susceptible to natural and experimental infection with P. pneumotropica than mice carrying the functional alleles (Chapes et al. 2001). Studies by Hart et al. (2003) showed that Toll-like receptor 4 (TLR-4) is the primary recognition molecule for lipopolysaccharides of gram-negative bacteria. Mice lacking this receptor on macrophages are susceptible to infection by P. pneumotropica, while reconstitution with these macrophages can provide host resistance to the agent. Lung lysophospholipase activity may be increased and associated with inflammatory responses after both helminthic and bacterial lung infections. However, it was not increased in studies by Laubach et al. (1978) after P. pneumotropica infection. Pore-forming cytolytic protein toxins (RTX toxins) are produced by a broad range of pathogenic gram-negative bacteria and are particularly widespread among species of Pasteurellaceae. This family of toxins is composed of several proteins (Kuhnert et al. 1997). They have been detected in various species of Pasteurella and Actinobacillus (Frey and Kuhnert 2002; Christensen and Bisgaard 2004). A positive CAMP reaction has been found related to the secretion of these toxins. A. muris exhibits a weak CAMP reaction, but no attempt has been made to detect such toxins in this agent or in other Pasteurellaceae species infecting mice. A lipoprotein that is exposed at the outer membrane surface where it functions to anchor capsular material to the cell surface and that was therefore considered to represent a virulence factor was described for P. multocida and other Pasteurellaceae species, among them P. pneumotropica (Champlin et al. 2002). However, the P. pneumotropica strain tested originated from a ferret, and it is not clear if the strain was identified properly. Iron-binding membrane proteins have been discussed as virulence factors. Iron uptake systems are indirectly related to virulence since the iron concentration is limited in the host and iron is essential for the function of metabolic electron transport chains of the pathogen. These transferrin-binding proteins have been detected in several Pasteurellaceae species (Morton and Williams 1990; Gerlach et al. 1992; Baltes et al. 2002; Bahrami et al. 2003), but studies have not been undertaken to demonstrate such proteins in species causing natural infection in rodents.
C. 1.
Clinical Disease
Primary Pathogenicity
Many reports of clinical signs of disease were published between 1960 and 1980. In many cases, predisposing conditions and confounding variables such as concurrent infections or
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environmentally associated problems were not considered. Well-defined animals free of latent or concurrent infections with other organisms were frequently not available. Many published reports should therefore be regarded with caution because additional factors that might have contributed to the incidence or severity of lesions cannot be excluded. There is, however, no doubt that in specific cases P. pneumotropica has been identified as a primary pathogen in clinical disease of mice (e.g., McGinn et al. 1992). These bacteria may be involved in suppurative infections of various organs from which they are usually isolated in pure culture. With the increased use of immunocompromised and genetically modified mice that occasionally have undetected immunodeficiencies, the incidence of clinical disease caused by Pasteurellaceae is increasing. 2. Co-Pathogens, Association with Additional Pathogens, Synergistic Effects, Opportunism
P. pneumotropica may be incriminated as the primary etiologic agent of purulent inflammatory lesions, but is more frequently regarded as an opportunist or a secondary invader, acting as one of several agents that contribute to disease. A number of reports suggest that P. pneumotropica plays a synergistic role with viruses, mycoplasmas, or other bacteria in the production of disease (Lindsey et al. 1982). In rats, P. pneumotropica has been associated with disease induced by Kilham rat virus (Carthew and Gannon 1981), Sendai virus (Carthew and Aldred 1988), sialodacryoadenitis virus (Hajjar et al. 1991), Bordetella bronchiseptica (Burek et al. 1972), and species of Staphylococcus, Corynebacterium, and Mycoplasma (Eamens 1984). Jawetz (1950) stated that mice used in his study had a natural Chlamydia trachomatis infection and spontaneous pulmonary consolidation possibly related to Sendai virus, Mycoplasma (M.) pulmonis, or other agents. In evaluating the relative importance of various etiologic agents of murine pneumonia, Brennan et al. (1969b) concluded that P. pneumotropica and M. pulmonis were the most common causes of murine pneumonia and that both are frequently responsible for fulminating pneumonia and death if present in the same animal. Brennan et al. (1969a) showed synergism or even additive effects in the extent and the severity of lesions when P. pneumotropica was combined with M. pulmonis in both conventional and axenic mice. Coinfection by both agents was also reported by Casillo and Blackmore (1972) and Young and Hill (1974). Further substantiation for synergistic effects was provided by Saito et al. (1978) who approached the same conclusions through epidemiologic methods. They surveyed 3270 mice from 22 breeding colonies for the presence of various agents such as P. pneumotropica, M. pulmonis, Corynebacterium kutscheri, and Sendai virus showing a correlation between the presence of these agents and the presence of lung lesions. The studies conducted by Jakab and Dick (1973) and Jakab (1974; 1981) demonstrated a correlation between the combined presence of Sendai virus and P. pneumotropica and the extent of lung lesions, compared with the extent when only a single
agent was present. While mice eliminated P. pneumotropica within 72 hours after infection by aerosol, the elimination was greatly delayed when mice had been infected with Sendai virus a few days earlier. A synergistic effect was also demonstrated by enhanced mortality as compared with that resulting from virus or bacteria alone. Previous immunization with Sendai virus prevented virus infection and thus the synergistic effect (Jakab and Dick 1973). Synergistic effects between Pasteurellaceae and influenza virus became obvious in studies from Sellers et al. (1961), who infected mice with an influenza virus and observed purulent bronchopneumonia that frequently progressed to abscess formation from which an endogenous Pasteurellaceae species of murine origin were cultured. Similar observations were made in studies by Phillips et al. (1970). Spontaneous severe pneumonia has been documented in immunodeficient mice co-infected with Pneumocystis (Pn.) carinii (Gordon et al. 1992; Marcotte et al. 1996; Macy et al. 2000). However, synergistic effects between P. pneumotropica and other pneumotropic agents do not always occur. Laubach and Kocan (1983) observed a mortality rate between 46 and 62% of rats after intranasal infection with P. pneumotropica while the mortality was significantly reduced when rats had received larvae of the pneumotropic helminth Angiostrongylus cantonensis. 3.
Additional Factors Responsible for Increased Pathogenicity
In addition to synergistic effects of infectious agents, properties of the host may also be responsible for an increased likelihood of clinical disease caused by Pasteurellaceae. McGinn et al. (1992) reported a high incidence of spontaneous otitis media caused by P. pneumotropica in CBA/J mice, while it was absent in CBA/Ca mice housed in the same room. The importance of the host genotype for the severity and prevalence rate of conjunctivitis in rats was also shown by Kunstyr et al. (1980). Induced or spontaneous mutations may also lead to an increased genetic predisposition to pasteurellosis (Kent et al. 1976). Immunosuppression and immunodeficiency increase susceptibility to clinical disease. P. pneumotropica should therefore not be tolerated in immunodeficient mutants or in animals with manipulations of the genome resulting in altered immune functions as well as in experiments requiring immunosuppression. Host defenses to P. pneumotropica have been reported to be lowered after nephrectomy (Goldstein and Green 1967), X-irradiation (McKenna et al. 1970), or exposure to nitrogen dioxide (Jakab 1987), and even placing mice in “cold wet air,” had a similar effect (Kairies and Schwartzer 1936). It has also been observed repeatedly that mice or rats did not recover from inhalation anesthesia when infected with P. pneumotropica, while the same concentration of the anesthetic is well tolerated by animals uninfected with Pasteurellaceae (Hansen 2000). Genetically modified mice may unexpectedly be more susceptible to disease than their wild-type counterparts. It was hypothesized that decreased survival time in mutant mice with a defect in the red blood cell membrane was caused by a severe
484 anemia (Grossmann et al. 1993), but the deaths were related to the development of pneumonia, pulmonary fibrosis, infarction, and bacteriemia by P. pneumotropica. 4.
Latency or Latent Infections
Various surveys revealed a high incidence of asymptomatic infection with Pasteurellaceae (Hoag et al. 1962; Sparrow 1976; Saito et al. 1978). This group of agents is therefore, in many cases, only detected if animals are carefully monitored. Pasteurellaceae were common in colonies maintained by commercial breeders (Flynn et al. 1965) and are occasionally found in colonies of commercial breeders even today. Growth factordependent Pasteurellaceae (usually classified as Haemophilus) are especially prone to colonize mucous membranes primarily of the respiratory tract of rats and less frequently of mice and often remain undetected because the culture conditions usually applied do not allow their detection (Nicklas et al. 1993). Repeatedly, “endogenous” Pasteurellaceae have been found in infection studies after tissue damage or immunosuppression by experimental infections (Sellers et al. 1961; Phillips et al. 1970; Jakab 1974), thus complicating and modifying infection studies conducted with defined agents. 5.
Organ Systems and Diseases
A. LUNG INFECTION, RESPIRATORY TRACT Early infection studies conducted by Jawetz and Baker (1950) indicating pneumotropism of P. pneumotropica were conducted with mice that were infected by additional agents. High pneumotropism of P. pneumotropica has also been described by Blackmore and Casillo (1972), who reported deaths associated with bacteriemia and pneumonia after experimental intranasal inoculation of P. pneumotropica, whereas intravenous and intravaginal inoculation did not lead to clinical signs of disease. Wheater (1967) readily transmitted P. pneumotropica to mice or rats by instilling a broth culture into the nostrils or by aerosol, but no infection followed intravenous infection, and the organism was not recovered from the animals after more than 24 hours. A high incidence of pneumonia in sick animals with isolation of P. pneumotropica was also found by Brennan et al. (1965). The same authors (Brennan et al. 1969a) produced lung lesions with areas of consolidation and perivascular and peribronchial lymphocyte infiltration by experimental infection with P. pneumotropica, but lesions were more severe when animals were co-infected with M. pulmonis. Spontaneous interstitial pneumonia by dual infection with Pn. carinii and P. pneumotropica was observed in B cell-deficient mice (Marcotte et al. 1996; Macy et al. 2000). Pasteurella-induced bronchopneumonia, lobar pneumonia, or pleuropneumonia were found in clinically affected mice, and in the same population P. pneumotropica was also recovered from mice with suppurative necrosis of muscle, subcutis, uterus, and from middle ears. Although mice were infected
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by two agents, the authors conclude that their findings suggest that clinical disease and mortality were associated with Pasteurella-induced pneumonia. B. OCULAR INFECTIONS, CONJUNCTIVITIS Among the lesions associated with P. pneumotropica, those of the eye and ocular adnexa are probably the most frequently documented. In mice, conjunctivitis, panophthalmitis, and dacryoadenitis (Gray and Campbell 1953; Brennan et al. 1965; Wagner et al. 1969; Weisbroth et al. 1969; Needham and Cooper 1975) have been reported, but the agent has also been cultured from the conjunctivae of healthy mice (Rehbinder and Tschäppät 1974) in almost 50% of animals tested. The incidence of clinical signs in an infected population may be quite variable. Young and Hill (1974) found clinical conjunctivitis in 60% of rats examined. P. pneumotropica was present in the conjunctival sack of all animals, but Streptobacillus moniliformis and Mycoplasma were also cultured. Wagner et al. (1969) and Weisbroth et al. (1969) reported P. pneumotropica-associated eye lesions in 5 to 10% of weanlings or adult breeding mice, resulting in a substantial economic loss in the colonies. In both incidents as well as in another case report (Black 1975), Harderian glands and other orbital structures were involved. Needham and Cooper (1975) reported an incidence of 45 affected animals (yearly turnover of about 72,000 mice) over a period of 25 months from which P. pneumotropica was cultured in most cases. Brennan et al. (1965) and Wagner et al. (1969) found a high incidence of conjunctivitis in weanling mice that also developed orbital abscesses and grossly visible conjunctivitis. It is reported in both publications that lesions were no longer detectable by three weeks after weaning, and lesions did not appear in mice after weaning. In contrast, Weisbroth et al. (1969) did not observe orbital abscesses in mice younger than 6 months of age. The incidence of eye infections may be high, with serious consequences among immunodeficient mice. Nude mice and rats frequently develop large abscesses in the orbita (Moore and Aldred 1978; Moore 1979) (see Fig. 19-3), but abscesses are also found in immunocompetent mice (Vallee et al. 1967). Abscesses develop when ports of entry for bacteria arise from small lesions that are created by bedding particles that penetrate into the conjunctiva due to the absence of eyelashes in nude mice, although the infection could also ascend the tear ducts (Dickie et al. 1996). These abscesses are the most characteristic signs of infection by P. pneumotropica in nude mice and may result in the loss of eyes and/or the absence of recognizable Harderian glands. However, similar abscesses are sometimes also caused by other opportunistic agents such as Klebsiella (McGarry et al. 1976). Lesions similar to those in nude mice may be found in genetically modified animals as a consequence of immunodeficiency, which may predispose specific lines to infections, whereas cohabiting, nontransgenic littermates may not be affected (Artwohl et al. 2000). Dickie et al. (1996) observed weight loss, muscle wasting, and premature death at 6 months of age in transgenic lines containing Moloney murine leukemia virus (MLV) and human
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Fig. 19-3 Multiple abscesses (head, orbita) in a nude mouse caused by Pasteurella pneumotropica (courtesy of Dr. I. Kunstyr).
immunodeficiency virus type 1 (HIV-1) proviruses and expressing viral RNA in various tissues. The first signs were observed at the time of eye opening with lacrimal secretions and progression to inflammation of Harderian glands and periocular abscess formation. C. OTITIS P. pneumotropica is occasionally cultured from the external ear of mice with lesions of the external ear duct. McGinn et al. (1992) reported purulent otitis media with a high incidence (up to 60% of the population) in CBA/J mice used in hearing research, and P. pneumotropica was isolated from infected bullae. The authors state that no evidence of other known pathogens of the mouse respiratory tract [e.g., Sendai virus, pneumonia virus of mice (PVM), mouse hepatitis virus (MHV), Reovirus type 3, mice minute virus (MMV), Chlamydia, M. pulmonis, CAR bacillus] was found in the affected colony. The incidence was highest in old animals (>1 year of age). Similar findings were also reported by Harkness and Wagner (1975) in outbred mice. They reported violent scratching of the external ears and adjacent tissues of the head and neck and severe self-mutilation (Fig. 19-4). All mice with external lesions had otitis media from which P. pneumotropica was isolated as the primary etiological agent, but other bacterial species were also found. Eamens (1984) isolated P. pneumotropica in middle ears in most cases of otitis media together with other bacterial species. D. ABSCESSES Abscesses caused by P. pneumotropica have been reported in both rats and mice (Moore and Aldred 1978; van der Schaaf et al. 1970; Weisbroth et al. 1969). They are most frequently seen in the orbita (see above) but may also be found in bulbourethral glands (Sebesteny 1973), in muscles such as the masseter muscle (Wilson 1976), and at various other locations. The regional lymph nodes are usually enlarged. Healing of the abscesses may occur spontaneously (Wagner et al. 1969)
or after opening the skin and discharging the pus. In general, the incidence was quite low or not defined in published reports. Experimental studies have shown that P. pneumotropica can cause abscesses in mice and rats when pure cultures are injected subcutaneously (van der Schaaf et al. 1970; Sebesteny 1973). The bacteria are also found in suppurative and other skin lesions (Sundberg et al. 1994). E. MASTITIS P. pneumotropica has been reported to cause mastitis in mice and rats (Wheater 1967; van der Schaaf et al. 1970). Hong and Ediger (1978) isolated this agent from rats with chronic necrotizing mastitis, usually in pure culture, but sometimes also in combination with other agents such as Pseudomonas aeruginosa, Escherichia coli, and Staph. aureus. Histologically, the affected glands are characterized by massive necrosis of the mammary parenchyma. A thick mass of pyogenic granulation tissue separated the viable tissue from the necrotic tissue. Gialamas (1981) reported spontaneous mastitis and abscess formation in the mammary glands in 10% of females in a breeding colony from which P. pneumotropica was isolated as the only pathogen. Usually, the organism was also isolated from other locations of infected animals. It must be noted that infection of the mammary glands affects not only the health of the dam but also of the weaning rats (Wheater 1967). F. INFECTIONS OF THE UROGENITAL TRACT (UTERINE INFECTIONS, METRITIS, ACCESSORY SEX, AND PREPUTIAL GLAND INFECTIONS) The occurrence of P. pneumotropica in the genital tract has been reported frequently. The agent is present in many mice without causing disease and can easily be isolated from mucous membranes of the vagina, uterus, and the prepuce. Colonization is usually asymptomatic, but this agent may produce uterine infections and accessory sex and preputial gland abscesses. P. pneumotropica has also been found to be associated with
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Fig. 19-4 Self-mutilation of the ear pinna caused by scratching as a consequence of an infection of the external ear canal by Pasteurella pneumotropica.
chronic spontaneous pyelonephritis in diabetic mice (Taylor 1988). Experimental uterine infections could easily be produced by intravenous, intranasal, and intravaginal inoculation, or by mixing susceptible mice with animals carrying the organism, but these infections may be temporary in nature (Blackmore and Casillo 1972). Some animals even died in these infection experiments. The incidence of uterine infections in mice has been reported to be between 3.6% (Casillo and Blackmore 1972) and more than 10% (Hoag et al. 1962; Brennan et al. 1965; Flynn et al. 1965; Juhr and Hiller 1973). Flynn et al. (1968) found infected uteri in 20% of virgin female mice, and P. pneumotropica was the organism usually recovered. Uterus infections were not eliminated by pregnancy and parturition. Ward et al. (1978) describe an epizootic of abortion in one mouse strain. Necrotizing and suppurative metritis was found among aborting females, and P. pneumotropica was isolated from uteri and fetuses of aborting females but also from other organs as well as the genital tracts of male mice (e.g., testes, seminal vesicles). Although detailed profiles of the isolated bacteria were not given, the biochemical reactions of some isolates reported to be positive for esculin and mannitol indicate that other species, most likely A. muris, might have been involved. This agent was also reported by Ackerman and Fox (1981), who found P. pneumotropica
associated with infertility, abortion, metritis, and stillbirth in addition to A. muris (identified as A. ureae).
D.
Pathology
In most cases, no or very mild lesions are noted (Burek et al. 1972; Jakab 1974) even after experimental infection. In animals with suppurative lesions, patterns may vary. Purulent conjunctivitis, periorbital and subcutaneous abscesses, abscessation of lymph nodes, rhinitis, bronchopneumonia, and metritis are possible manifestations of infection (Percy and Barthold 2001). Jawetz and Baker (1950) described in detail the pathological changes observed in experimental P. pneumotropica infections, but mice were coinfected by additional pathogens that are likely to have contributed to the lesions found. Lesions were found only following intranasal inoculation of P. pneumotropica and a bronchopneumonia developed which progressed to necrosis and the formation of abscesses. Immune suppression facilitates establishment of the pathogen in the lungs, with lesion development characterized by diffuse, suppurative interstitial pneumonia (Goelz et al. 1996). Lesions developing at a mucocutaneous junction are often purulent, with a pronounced neutrophilic component (Dickie et al. 1996).
19. PASTEURELLACEAE
In studies by Ackerman and Fox (1981), histopathological evaluation revealed acute suppurative metritis and dead fetuses in utero. Animals sacrificed months after treatment with antibiotics were still positive by culture but did not show any pathological lesions. Giddens et al. (1971) and Brennan et al. (1969a) have described the lesions of chronic murine pneumonia complicated with P. pneumotropica. While conventional mice experimentally infected with P. pneumotropica and M. pulmonis were obviously latently infected with both agents in studies published by Brennan et al. (1969a), experimental infection in germ-free mice led to consolidation of lungs with hemorrhages and abscesses. Microscopically, they found a severe polymorphonuclear response and extensive lymphocyte infiltration. In combination with Sendai virus, histopathologic changes may include occlusion of bronchi with a purulent exudate, peribronchiolar inflammation, and areas of consolidation in the pulmonary parenchyma (Jakab 1974).
E.
Interference with Research
Compared to most viral infections, extremely little information is available on the impact of overt or silent Pasteurellaceae infections on research. Bacteria colonizing animals are still in most cases tolerated if they are not pathogenic or are only weakly so, whereas viruses and parasites are usually considered unacceptable even when their pathogenicity is similarly low. Detection of Pasteurellaceae in a mouse population does not usually result in termination of a population. As a consequence of tolerating these agents, many experimental colonies are infected, and the agents may have more frequent impact on results of animal experiments than agents for which more information is available and which are for this reason not tolerated. Although usually mild, the inflammation of mucosa and tissues induced by P. pneumotropica would interfere with studies of those systems. Natural infection of laboratory rodents could compromise research involving the dermal, enterohepatic, reproductive, and respiratory systems, and could confound other studies if the general health of the animals was compromised. Experimental nephrectomy increases the susceptibility of the lung to infection by P. pneumotropica (Goldstein and Green 1967). It is known that various members of the family Pasteurellaceae share the general property of being resistant to cellular defense mechanisms. This occurs through a variety of means, including the presence of an antiphagocytic capsule, active insult to phagocytes, and alteration of leukocyte and macrophage function (Czuprynski and Sample 1990). Therefore, it is possible that P. pneumotropica and other members of the family may also interfere with studies involving the lymphoreticular system. Colonization of the uterus sometimes results in reduced fertility. Animals occasionally do not respond to hormonally induced superovulation or do not get pregnant after transfer of fertilized eggs or blastocysts into the oviduct or the uterus.
487 Failure of an in vitro culture system to support development of mouse embryos collected at the four-cell stage and older was linked to Pasteurella infection endemic in a mouse colony used as the source of embryos. Pasteurella was cultured from the uteri and oviducts of donors and from seminal vesicles and testes of donor males. Only 15% of embryos collected from an infected population developed, while 78% developed normally in the same culture system when taken from noninfected mice (Hagele and Bielanski 1987). Simpson et al. (1980) observed spontaneous regression of a transplanted tumor that was contaminated with P. pneumotropica. After treatment with antibiotics, the cells grew normally when injected into rats, whereas untreated cells from the same batch either failed to form tumors or the tumors regressed spontaneously. McGinn et al. (1992) reported otitis media in CBA/J mice used in hearing research at an incidence of 90% in older animals (>400 days of age), with P. pneumotropica being the only potential pathogen isolated. Royston et al. (1983) showed that P. pneumotropica causes some alteration of alveolar-capillary barrier permeability in the respiratory tract by comparing rats from two colonies, one naturally infected with P. pneumotropica and one without. Despite latent infection, P. pneumotropica may render the lungs more susceptible to other pathogens by altered permeability of capillaries in the pulmonary alveoli. Certain genetically modified mouse strains may show increased susceptibility to infection by P. pneumotropica, resulting in suppurative infection of the respiratory tract and high mortality during the first days after birth, thus impeding phenotypic characterization (Erdman et al. 1999). Also, infection of adult immunocompromised mice by P. pneumotropica frequently leads to clinical disease (e.g., abscesses) and reduced life expectancy of a population. Thus, long-term experiments (e.g., studies with slowly growing tumors) or experiments involving surgery may not be possible. Repeatedly, increased mortality during experimentation has been reported for animals infected by Pasteurellaceae as compared to noninfected animals. The incidence of spontaneous deaths during inhalation or injection anesthesia in rodents might be increased in P. pneumotropica-infected animals (Hansen 2000). Premature deaths in mutant mice with a defect in the red blood cell membrane were unexpectedly caused by pneumonitis with thrombosis and infarction, and P. pneumotropica was found in blood cultures (Grossmann et al. 1993). In long-term drug toxicity experiments, gross lesions containing P. pneumotropica developed in the lungs, and up to 50% of the animals in certain groups died (Wheater 1967). P. pneumotropica infections may complicate natural and experimental infections with other agents such as Kilham rat virus (Carthew and Gannon 1981), Sendai virus (Jakab and Dick 1973; Jakab 1974, 1981; Carthew and Aldred 1988), and Mycobacterium tuberculosis (Gray and Campbell 1953), and several publications describe the interaction of natural Pasteurella infection with experimental infections. Synergistic effects between Pasteurellaceae and other agents may lead to
488 an increased morbidity or mortality in natural infections with other agents. Experimental infections may be influenced by undetected or silent natural infections (Nicklas et al. 1999). It may in many cases be difficult to decide if observations are caused by the agent used for experimental infection or by the naturally occurring agent. Synergisms between agents may not only lead to an increased pathogenicity of latent Pasteurellaceae infections, but undetected latent infections with Pasteurellaceae most likely have also influenced the outcomes of experiments with the agents of interest in numerous experimental studies and may therefore be responsible for false conclusions. Repeatedly, “endogenous” Pasteurella infections were observed to emerge and proliferate in infection experiments conducted with various agents. Phillips et al. (1970) infected mice with an avian reovirus and observed that in the presence of endogenously derived P. pneumotropica the infection with the virus of interest was greatly increased in severity and had a fatal outcome in a high percentage of mice, while the virus alone caused only transient disease. Similarly, Sellers et al. (1961) observed purulent pneumonia and spontaneous appearance of infections with murine Pasteurellaceae in pulmonary tissues of 40 to 50% of mice previously infected with influenza virus. Appearance of these agents in the lungs exerted considerable influence on the pathological evolution of the influenza infection and led to purulent bronchopneumonia, progressing to formation of lung abscesses. The absence of purulent complications in animals that were protected from endogenous infection by the administration of antimicrobial agents supports the hypothesis that the purulent bronchopneumonia was a consequence of spontaneous secondary infection with these Pasteurellaceae. Due to low pathogenicity and the comparatively sparse evidence of modification of research results, the presence of Pasteurellaceae is frequently not regarded as cause for destruction or rederivation of colonies or for classification of animals as inappropriate for research. However, there is general agreement that these agents should be included in monitoring programs and that positive findings should be mentioned in health reports (Kunstyr 1988; Hansen 2000; Waggie et al. 1994; Yamamoto et al. 2001). While most laboratories or institutions report the presence or absence of P. pneumotropica, the Federation of European Laboratory Animal Science Associations (FELASA) has suggested that monitoring should not be restricted to P. pneumotropica and recommends monitoring for and reporting of Pasteurella spp. (Kraft et al. 1994). Subsequent recommendations published by FELASA suggest that all Pasteurellaceae should be included in monitoring programs with further identification as far as possible to avoid misunderstandings due to different identification criteria (Rehbinder et al. 1996; Nicklas et al. 2002). Monitoring for other Pasteurellaceae species (in addition to P. pneumotropica) does not significantly increase the costs of monitoring because other species (with a few exceptions such as growth factor-dependent strains and species) will grow on the same media under the same conditions. Although action may in many cases not be necessary and will frequently not be
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taken, it will allow the reader of a health report or the person responsible for the microbiological quality to decide whether such animals are acceptable under the conditions given.
VI.
EPIZOOTIOLOGY A.
Host Range
One of the most important characteristics of the family Pasteurellaceae is the marked degree of specificity they show for a host species. With few exceptions, a species of the group seems to be exclusively associated with one or very few specific hosts (Kilian and Frederiksen 1981a). The only Pasteurellaceae species with a broad host spectrum, P. multocida, is usually not found in mice, and convincing reports on spontaneous infections of mice by P. multocida do not exist in the literature (Manning et al. 1989a). Most strains belonging to this species are moderately to highly pathogenic for mice after experimental infection. The pathogenicity of this agent for mice has been used for decades to recover the agent from heavily contaminated material or to confirm a bacteriological diagnosis (Carter 1981; Muhairwa et al. 2001). When inoculated with P. multocida intraperitoneally or subcutaneously, mice will usually die within 18 to 96 hours, and the organisms can be readily recovered from heart, blood, liver, and spleen. Mice are therefore unlikely to be silent carriers of this agent, and it is considered of no importance as a natural pathogen of mice. P. pneumotropica has been reported to colonize a wide variety of mammals including humans. Species include laboratory animals such as rats, mice, hamsters, guinea pigs, and rabbits (Biberstein 1981; National Research Council 1991; Bisgaard 1993; Besch-Williford and Boivin 1994), and poultry (Bisgaard 1982). The majority of these reports (if not all) should be questioned because there is evidence that P. pneumotropica has a narrow host spectrum. Shepherd et al. (1982) reported isolation of P. pneumotropica with variable biochemical profiles from various species of wild rodents in South Africa. Sparrow (1976) reported P. pneumotropica as the most common organism in rats and mice, but he reports isolation also from hamsters, guinea pigs, and rabbits. However, he does not mention identification criteria or biochemical data for the bacteria. Kirchner et al. (1983) described isolation of P. pneumotropica from a Pasteurella-free colony of rabbits. Although very few criteria were tested, the biochemical reactions reported are not characteristic for P. pneumotropica and their diagnosis is likely to be wrong. McKenna et al. (1970) isolated P. pneumotropica from abscesses in irradiated golden hamsters, and Lesher et al. (1985) described these bacteria as causative agents of enteritis in hamsters (the correct species name was not given). Kunstyr et al. (1976, 1987) reported isolation of P. pneumotropica from purulent processes of European hamsters, but subsequent DNA:DNA hybridization studies revealed that these bacteria were not identical to
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P. pneumotropica and represented at least two new species (Krause et al. 1989). Ryll et al. (1991) found high DNA:DNA hybridization rates between an avian isolate and P. pneumotropica Jawetz, but Christensen et al. (2003b) stated that a final conclusion on this avian isolate has to await analysis of more strains. There are also reports from many other host species. Van Dorssen et al. (1964) reported the isolation of P. pneumotropica from cats and van der Schaaf et al. (1970) from the oral cavities of dogs. Their identification was based on very few reactions, and they most likely cultured strains that would now be identified as P. dagmatis. The same is likely to be the case for additional isolates from dogs (Brennan et al. 1965), but the authors also report bacteria with identical profiles from hamsters and kangaroo rats. Ward et al. (1998) identified 8 among 73 isolates from horses (11%) as P. pneumotropica. The authors stated that most isolates would have been presumptively identified as P. pneumotropica on the basis of the reactions in the API 20 NE strips. Similarly, the identification of P. pneumotropica isolated from flagellates (Lu et al. 2000) was based solely on the API identification system. Also, the proper identification of a bacterial strain that originated from a ferret and was identified as P. pneumotropica should be questioned (Champlin et al. 2002). Evaluating the identification results from these reports is unfortunately not possible in most cases because only a few characteristics were tested or insufficient information was given regarding diagnostic criteria. Some of this information is probably a consequence of misidentifications, and it is very unlikely that P. pneumotropica really has such a broad host spectrum. This organism apparently does not play a significant role in the majority of these host species and is very unlikely to be transmitted to rodents from all the species mentioned. Considering the difficulties in delineation of species within this family, some of the published data should be used only with great care since many isolates have been classified according to simplified diagnostic keys that do not permit proper identification of species within the family Pasteurellaceae. Based on present knowledge, there is no doubt that both biotypes of P. pneumotropica are typical rodent organisms. The term P. pneumotropica describes a group of bacteria typically infecting mice (Mus musculus) and rats (Rattus norvegicus) and closely related muridae. According to 16S rDNA sequences, P. pneumotropica and other species infecting mice and rats (A. muris, H. influenzaemurium) together form a separate cluster in the phylogenetic tree (“rodent group”). Evidence exists of a strict host specificity, at least for some biotypes. Infection studies with various isolates from several host species showed that hamster and gerbil strains colonize mice and rats to a lower extent than strains isolated from mice and rats (Boot et al. 1994). Nakagawa and Saito (1984) demonstrated antigenic differences between mouse and rat strains by agglutination tests, although common antigens were also shared. Also, Boot et al. (1994–1996) reported differences between mouse and rat strains in agglutination assays supporting evidence for host specificity of Pasteurellaceae in mice and rats. However, all
these studies have in common that strains used were not sufficiently characterized, and it is unclear which agents were used for the experiments. Several reports indicate that P. pneumotropica of rat origin, at least some specific biotypes, are not easily transmissible to mice. Van der Schaaf et al. (1970) could not induce any changes after experimental infection of mice with a rat isolate of P. pneumotropica, while abscesses were induced in rats by the same bacteria. Nakagawa et al. (1981) reported that mouse isolates could be transmitted to mice and rats, whereas rat strains were easily transmissible to rats but were less infective for mice. Typical strains of biotype Heyl seem to have a broader host spectrum and have been isolated from mice and rats as well as from other rodent species (e.g., multimammate mice, Syrian hamsters). Also, the indole-negative and melibiose-positive variant of biotype Jawetz (see Section III.B.1.a) is frequently found in both mice and rats, whereas the “normal” variant having phenotypic properties of the type strain (Table 19-6) is primarily cultured from mice and is rarely found in rats. Additional phenotypes exist which are isolated primarily from rats and are only exceptionally cultured from mice. Typical for these bacteria is their negative reaction for trehalose. Actinobacillus muris has been reported only from mice, but comprehensive published information is not available. It seems that mice are the major host for this agent, although very similar strains have, as rare exceptions, also been cultured from rats. Similarly, little information is available on H. influenzaemurium which, however, also seems to infect only mice. Growth factor-dependent strains are frequently cultured from rats, but bacteria with biochemical properties identical to rat isolates are also occasionally found in mice and in guinea pigs. The majority of these bacteria have biochemical properties identical to human isolates belonging to the H. parainfluenzaecomplex. Indeed 16S rDNA sequences also indicate that these bacteria are very closely related or identical to H. parainfluenzae and might be transmitted to laboratory rodents from humans. V factor-dependent strains belonging to Taxon B (Kilian 1976; Nicklas et al. 1993) and their growth factor-independent counterparts primarily infect rats and are only occasionally found in mice and in other species (e.g., guinea pigs). Insufficient information has been published on the host specificity of these agents and their transmissibility among host species. There is no question that additional species of Pasteurellaceae that have not yet been classified exist in laboratory and wild mice (Mus musculus). Also, closely related rodents (e.g., Apodemus, Microtus) seem to have their own Pasteurellaceae species, which may occasionally also be found in laboratory mice when these species are housed together.
B.
Importance for Humans, Zoonotic Potential
With the exception of infections caused by typical human pathogens like, for example, H. influenzae, Pasteurellaceae
490 infections are uncommon in humans. However, infections with Pasteurellaceae of animal origin occur in humans, usually after animal bites (Bailie et al. 1978) or after other contact with animals. The agent most frequently implicated in zoonotic infections is P. multocida (Frederiksen 1989b; Holst et al. 1992; Escande and Lion 1993). This bacterium is capable of producing a wide variety of disease states in humans, ranging from local infection (bite wounds) to respiratory disease to serious systemic illness that may lead to death. Isolation of other Pasteurellaceae species from humans like P. stomatis, P. canis, P. dagmatis, P. trehalosi, P. caballi, P. aerogenes, P. bettyae, A. equuli, A. lignieresii, or A. suis are less frequently reported in the literature (Starkebaum and Plorde 1977; Bisgaard et al. 1991; Fajfar-Whetstone et al. 1995; Ejlertsen et al. 1996; Moritz et al. 1996; Escande et al. 1997). Sometimes, more than one species can be isolated from the same specimen (Zbinden et al. 1988). Several reports exist of isolation of P. pneumotropica from humans. Most cases are related to dog or cat bites (Miller 1966; Olson and Meadows 1969; Medley 1977; Gadberry et al. 1984; Ashdown and Mottarelly 1990; Minton 1990) or close contact (Frebourg et al. 2002) with these animal species, but a report exists of isolation of P. pneumotropica from a horse bite (Dibb and Digranes 1981). Also, infection caused by P. pneumotropica in a dialysis patient was reported after a hamster bite (Campos et al. 2000) and endocarditis after a guinea pig bite. One case of a skin infection in a child was reported after a rat bite (Olive et al. 1994). Bacteria identified as P. pneumotropica were found in an AIDS patient (Cuadrado-Gomez et al. 1995), but also in immunocompetent humans. Such organisms were cultured from the human respiratory tract (Henriksen and Jyssum 1961; Henriksen 1962; Sakazaki et al. 1984; Cuadrado-Gomez et al. 1995) as well as from a wide variety of infections of different organ systems including skin wounds (Winton and Mair 1969; Medley 1977; Peloux et al. 1979; Olive et al. 1994), bone and joint infections (Gadberry et al. 1984; Sammarco and Leist 1986), septicemia (Rogers et al. 1973; Mansoor et al. 1992; Nimri et al. 2001; Frebourg et al. 2002), endocarditis (Cornaert et al. 1987), meningitis (Cooper et al. 1973; Minton 1990), and peritonitis (Campos et al. 2000). One report described isolation of a X factor-requiring Haemophilus species from a perianal abscess of a woman (Ryan 1968). The isolate was considered to be a variety of H. influenzaemurium. However, the diagnosis was unlikely to be correct owing to the colony morphology, growth characteristics, and biochemical properties reported. In most reports the diagnosis was based on commercial test kits intended for the identification of human pathogens (Ashdown and Mottarelly 1990; Minton 1990) or on few phenotypic criteria (Olson and Meadows 1969), sometimes with questionable significance (Weaver 1992). Various authors agree that commercially available systems for speciation frequently fail to identify Pasteurellaceae properly (Sakazaki et al. 1984; Fajfar-Whetstone et al. 1995; Elsaghier et al. 1998). The relevant species in question are not always included in the databases used for identification. Sometimes, different diagnoses were obtained
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when other or additional criteria were used to identifiy such bacteria (Eckert et al. 1991), indicating that the results of bacterial identification are not always correct. Some authors therefore suggest that identification of unusual pathogens by commercial identification systems should be confirmed by other methods that take into account the basic characteristics of the organism (Lester et al. 1992; Elsaghier et al. 1998). Diagnoses of human infections with P. pneumotropica were frequently based on positive reactions for indole and urease (Medley 1977). It was in some cases stated that isolates differed from typical P. pneumotropica or from reference strains in their reactions for ornithine, ONPG, or xylose fermentation (Winton and Mair 1969; Dibb and Digranes 1981; Sakazaki et al. 1984), or gas production (Rogers et al. 1973), thus showing typical reactions for P. dagmatis which — prior to its definite classification — was designated as P. pneumotropica-type Henriksen. A list of published cases of P. pneumotropica infections in humans given by Frederiksen and Kilian (1981) demonstrates that in most cases biotype Henriksen was isolated. Many isolates identified as P. pneumotropica were transmitted from dogs and cats and had typical biochemical characteristics of P. dagmatis (Sakazaki et al. 1984) and are likely to have been misidentified. For those publications containing sufficient details, it appears that the isolate would in most cases now be identified as P. dagmatis. Sakazaki et al. (1984) studied 96 strains isolated from humans and identified one of them as P. pneumotropica. However, this strain, too, had characteristics typical of P. dagmatis. Frederiksen (1989b; 1993) has also stated that there should be very few reports of P. pneumotropica in humans if the proper nomenclature is used. Only in very few cases were more sophisticated and convincing methods, such as 16S rDNA sequencing, used for identification, and in very few cases is there sufficient evidence that human infections might really have been caused by P. pneumotropica. Frebourg et al. (2002) obtained a bacterial isolate from septicemia in an immunocompetent patient living in the company of dogs and cats and by 16S rDNA sequencing identified it as P. pneumotropica. However, it must be considered that not even a high sequence similarity of the 16S rRNA gene warrants proper classification on the species level (Stackebrandt and Goebel 1994). Weaver et al. (1985) have also stated that P. pneumotropica is of little importance as a cause of human disease, although they mentioned that a few cultures were also isolated from human sources. Pickett et al. (1991) concluded that members of this species were less common than other species of Pasteurellaceae as agents of human disease. It becomes obvious from the literature that the majority of human infections attributed to P. pneumotropica are likely to have been caused by P. dagmatis, which has previously been labeled Pasteurella sp. new species 1, Pasteurella “gas,” and P. pneumotropica biotype Henriksen and has already been reclassified by Mutters et al. (1985). It is not considered impossible for humans to become infected by true P. pneumotropica, although such cases seem to be extremely rare. However, it is very unlikely that humans are
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carriers of P. pneumotropica and serve as sources of infection for other animals, including laboratory rodents.
C. 1.
Prevalence
Prevalence in Populations of Laboratory Mice
Historically, the number of rodent colonies infected with P. pneumotropica has been highest for conventional colonies, perhaps close to 100%, but the agent has also been common in many barrier-bred colonies including commercial breeders (Flynn et al. 1965; Sparrow 1976; Nakagawa et al. 1984). A high prevalence rate in laboratory rodent populations also became obvious from compilations given by Lindsey (1986) and the National Research Council (1991). In a more recent retrospective study, more than 40% of mouse and rat colonies were reported to be positive for P. pneumotropica in France (Zenner and Regnault 1999/2000) with a declining tendency over time. However, only a very low percentage of mice monitored by Livingston and Riley (2003) were found to be positive for P. pneumotropica. The actual prevalence of Pasteurellaceae in colonies of laboratory rodents is unknown but appears to be very high. It has to be expected that even today P. pneumotropica and other Pasteurellaceae are endemic in numerous populations of laboratory mice. No detailed information is available for A. muris because it is identified by only a few laboratories. Although it is seldom reported to be cultured or identified, it is prevalent in a high percentage of conventional and barrier-housed mice and is even found in mice from commercial breeders (Nicklas, unpublished). Growth factor-dependent Pasteurellaceae (Haemophilus) are frequently found in the majority of rat populations and are common even in commercial breeding colonies of rats but seem to occur less frequently in mice. Nicklas et al. (1993) cultured growth factor-dependent Pasteurellaceae from 2.4% of 2800 mice and in more than 20% of rats. Even today, Pasteurellaceae are frequently tolerated as “normal flora,” and eradication steps are not taken, which is one reason for the high prevalence rate in experimental colonies. However, it seems that at least P. pneumotropica is becoming less frequently accepted in contemporary commercial breeding colonies.
2.
Carrier Rate in Infected Colonies
Screening of conventionally housed and clinically healthy mice by culture methods revealed a prevalence of colonization in the nasopharyngeal region or in the respiratory tract ranging from 48 to almost 100% (Brennan et al. 1965; Saito et al. 1978; Sparrow 1976). In contrast, Wang et al. (1996) found P. pneumotropica in 1.4% of mice by direct culture and in 4.1% after enrichment, while 21.9% of samples were positive by PCR. Depending on the mouse strain, Rehbinder and Tschäppät (1974) cultured P. pneumotropica from 16 to 48% of mice in an infected population.
Uterine colonization was reported to occur in 4 to 36% of breeder and virgin mice raised in conventional facilities (Casillo and Blackmore 1972; Ward et al. 1978; Brennan et al. 1965). It is obvious that the recovery rate and the number of carriers detected in a population depend on various factors. The detection rate by bacterial culture is dependent on the type and number of organs tested, the culture conditions and media used, and other variables. Very importantly, experience has a great influence on the isolation rate. Higher rates of animals testing positive may be expected if serological tests are used and if animals are exposed to the agent for a sufficiently long period of time. PCR may also yield higher rates of positive findings compared to culture, but the selected primer sets also have an impact on the specificity of the tests and on the spectrum of agents and thus on the detection rate (see Section VII.B). The carrier rate in a colony also depends on the agent or the biotype. Obviously, certain biotypes are less infectious and are found in one population less frequently than others. 3.
Geographical Distribution
Pasteurellaceae infections of mice have a worldwide distribution. Most published reports mention only P. pneumotropica, but it has to be expected that all species are likely to be found in laboratory rodents in all parts of the world due to the extensive exchange of laboratory mice between research institutes. P. pneumotropica has been isolated from wild house mice and rats (van der Schaaf et al. 1970; Shepherd et al. 1982; Boot et al. 1986) but also from conventional and barrier colonies of rodents in all parts of the world. Also, other species such as A. muris and all variants of V factor-dependent bacteria are found in rodents worldwide but systematic surveys have not been reported.
D.
Mode(s) of Transmission
P. pneumotropica and other species infecting mice are primarily cultured from mucous membranes of spontaneously infected animals. They are shed by excretions from the upper respiratory tract (saliva, nasal, or lacrimal secretions), but also by feces. Direct contact seems to be the most important mode of transmission between animals. An important route of entry is via the nasopharynx. The agent is not transmitted readily between cages by inhalation of infectious droplets or attached to dust particles or fomites. Spreading of the agent within a unit is facilitated by an increased population density. The vagina is very often colonized by large numbers of organisms, and the bacteria can frequently also be found in the uterus. The highest bacterial counts are consistently observed during the estrus phase of the cycle (Larsen et al. 1976b). Intrauterine infection may reach prevalences as high as 60 to 70% (Casillo and Blackmore 1972), and it has been demonstrated that mice may harbor P. pneumotropica and other Pasteurellaceae species (e.g., A. muris, Haemophilus sp.) in
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both gravid and nongravid uteri (Flynn et al. 1968; Ackerman and Fox 1981). Jawetz (1948) and Jawetz and Baker (1950) reported that the organism seems to be acquired early in life and concluded that it is obviously transmitted from the mother to the progeny because it was frequently recovered 24 to 48 hours after birth in the respiratory tract of newborn mice. It was demonstrated by others (Mikazuki et al. 1994) that transmission frequently occurs during birth or in the suckling phase by direct contact between the dam and offspring, and the infection is thus perpetuated in the colony. Fetuses may also be infected in the uterus during pregnancy, and the bacteria may therefore be transmitted during cesarean section. Indeed, P. pneumotropica is the agent that is most frequently responsible for failures of rederivation by cesarean section. Indirect contact between mice is less efficient in transmitting Pasteurellaceae (Wheater 1967). Compared to direct contact between animals, transmission occurred later and at a lower extent when uninfected mice were exposed to contaminated bedding (Myers et al. 2003). No transmission of P. pneumotropica was observed by handling uninfected animals immediately after infected animals. Transmission also did not occur when animals were housed in individually ventilated cages at negative pressure even in a densely populated room. Scharmann and Heller (2001) did not observe transmission by dirty bedding at all. They concluded that transmission of P. pneumotropica by contaminated materials is very unlikely owing to the short survival time on various materials such as cardboard, plastic, or bedding. Pasteurellaceae frequently colonize reproductive organs in mice, and this may enhance the risk of embryo contamination during processing. It has even been suspected that embryos stored in liquid nitrogen may become contaminated with pathogens (Rall 2003), and the author also makes suggestions about how to reduce the likelihood of cross-contamination as a result of storage at low temperatures. However, Kyuwa et al. (2003) cryopreserved two-cell embryos in cryotubes in the same nitrogen tank together with cryotubes containing mouse hepatitis virus (MHV) or P. pneumotropica. Implantation of the embryos did not result in infection of the foster mothers or their progeny, and the authors conclude that it is unlikely that such a crosscontamination between cryotubes occurs in liquid nitrogen tanks.
very similar to that of other members of the Pasteurellaceae (Barrow and Feltham 1993). In fact, the phenotypic classification is still based on information that relies on a very small number of isolates identified by a few biochemical tests more than 30 years ago (see Table 19-1). The key to diagnosis of P. pneumotropica and other species of the family is isolation and discrimination of the organisms from phenotypically similar species to avoid confusion and false conclusions by misidentifications. False conclusions in the case of clinical disease may also be possible if clinically ill animals are monitored only for a limited range of agents and if synergistic effects between different agents are not taken into consideration. It is therefore important to completely characterize the bacteriologic, parasitologic, and viral status of animals for which the diagnosis is considered. Commonly, readers of a publication accept a bacteriological diagnosis given, even if details are not stated. However, authors should be encouraged to give full bacterial descriptions when an uncommon organism is isolated and when an organism is isolated under unusual circumstances. Reference laboratories should be involved much more frequently, or well-characterized reference strains should be included in comparative studies. Although the pathogenicity of Pasteurellaceae for mice is low, there is no doubt that they should be considered in health monitoring programs and should be mentioned in health reports. Information in the literature pertains almost exclusively to P. pneumotropica, and there is general agreement that this group of agents has some importance for laboratory mice and closely related host species. Presence or absence of P. pneumotropica in mouse colonies is mentioned in health reports from commercial breeders worldwide, but other members of the family are detected or mentioned only exceptionally due to insufficient information on their biological characteristics and on identification criteria. FELASA, however, recommends that all Pasteurellaceae should be listed in health reports (Nicklas et al. 2002) to avoid discrepant information or confusion owing to the fact that uniform identification criteria are not used, even for P. pneumotropica.
A. 1.
VII.
DIAGNOSIS
More than many other bacterial pathogens, isolation and correct characterization of Pasteurellaceae challenge the microbiologist. Specifically, the classification of P. pneumotropica and its various biotypes and other Pasteurellaceae species infecting mice has not evolved significantly since the classification into biotypes as suggested by Frederiksen (1973). Precise definition of species, biotypes, and their host range is difficult, particularly as their biochemical profile may be variable and is sometimes
Culture
Isolation of the Agents
A. GROWTH CONDITIONS Diagnosis of Pasteurellaceae infections is usually based on bacterial culture and subsequent phenotypic characterization. The minimum incubation period for primary isolation of Pasteurellaceae isolation from animal organs or clinical samples should be 48 hours, but extended incubation for an additional 1 to 2 days may in some cases increase the isolation rate. The majority of strains grow well on blood agar (most commonly, Columbia agar with 5 to 7% sheep blood). Growth is sometimes improved on chocolate agar, and some growth factor-requiring strains grow only on chocolate agar.
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Growth of these bacteria is also possible on blood agar if a bacterial strain producing growth factor (NAD) in excess, usually Staph. aureus, is cross-inoculated. Isolation of Pasteurellaceae may be facilitated by the use of selective media in addition to nonselective media. This will increase the isolation rate and thus the sensitivity of isolation by culture. Various selective media (see Section IV.A.2) have been described, and some are commercially available. It must, however, be noted that growth of some Pasteurellaceae species may also be inhibited. For example, use of bacitracin in chocolate agar may inhibit growth of not only gram-positive flora but also A. muris, while both biotypes of P. pneumotropica grow very well and can easily be isolated from various sites, including the intestinal tract. Selective media should be tested carefully prior to routine use to be sure that growth of all agents of interest is sufficiently supported. B. ORGANS Mouse organs to be cultured should be collected aseptically during necropsy. In contrast to organs from larger animals, mouse organs (due to their small size) should not be flamed on the outside to remove contaminants because the heat might damage bacteria and prevent them from growing. Enrichment in broth is possible, but Pasteurellaceae are frequently not detected owing to overgrowth by other faster growing bacteria. A sectioned organ surface, swabs (e.g., from the vagina), intestinal contents, or feces are usually streaked over the culture plate surface to assure discrete colonies. Inocula can also be taken from incised tissues (e.g., lungs) with a swab or an inoculating loop. Sites to culture should include the upper respiratory tract (nasopharynx, trachea) and the genital tract (prepuce, vagina). In addition, the organisms can frequently be cultured from the conjunctivae. Often, large numbers of Pasteurellaceae can also be cultured from the lungs. Isolation from the intestinal tract is possible, but Pasteurellaceae are usually difficult to detect due to overgrowth not only by Enterobacteriaceae but also by more slowly growing organisms. They are frequently detected accidentally, but the likelihood of isolation can be increased by the use of selective media. Saito et al. (1981) and Mikazuki et al. (1994) reported the highest isolation rate from the pharynx of mice, but other main sites were the lower intestinal tract and the vagina. Other authors have reported that the vagina in particular is colonized by P. pneumotropica at a high frequency (Larsen et al. 1976a; Yamada et al. 1983, 1986), and this site is therefore important for the isolation of these bacteria. The likelihood of isolation and thus the sensitivity of culture techniques is enhanced by culturing multiple sites (e.g., nasal cavity, lungs, trachea, and uterus in addition to nasopharynx and vagina). The intestinal tract is an important site of isolation if appropriate selective media are used (Mikazuki et al. 1987) and is therefore also recommended for routine health monitoring. Careful testing for Pasteurellaceae is advisable after hysterectomy because these bacteria may be transmitted to the offspring at hysterectomy. If testing gnotobiotic mice, the gut and especially the caecum may be the preferred location for isolation
(Moore et al. 1973). In neonates, the organisms can be isolated within 24 hours after birth from the respiratory and intestinal tracts. C. PECULIARITIES OF DIFFERENT SPECIES FOUND IN MICE Isolation of P. pneumotropica is easy for the experienced microbiologist if the appropriate organs are streaked on blood agar or on other media supporting growth. Isolation is sometimes also possible on MacConkey agar where these agents occasionally grow when material (e.g., intestinal contents) is streaked directly on the culture medium. Growth is usually not observed on MacConkey agar upon subculture. Colonies of A. muris on blood or chocolate agar are usually small after overnight incubation and are typical after incubation for an additional day (Section III.B.2.). H. influenzaemurium is best detected on blood or chocolate agar after incubation at an increased CO2 concentration for 48 to 72 hours or more. Growth of NAD-dependent organisms (Haemophilus) is usually also improved in an atmosphere with increased CO2 levels. They are indistinguishable from other Pasteurellaceae such as P. pneumotropica on chocolate agar. Typical for this group of bacteria is satellite growth on blood agar adjacent to V factorproviding bacteria (Staph. aureus, Acinetobacter) (Fig. 19-2) and normal growth on chocolate agar. 2.
Phenotypic Identification
Small to moderate sized dewdrop-like colonies are usually evident after 18 to 24 hours of incubation. Colonies are round, grayish or yellow-white, and nonhemolytic. To the uninitiated eye, colonies do not look too unlike species of Enterobacteriaceae; however, with sufficient practice one can usually distinguish the colonies of Pasteurellaceae species from other bacteriae. The characteristic odor of most cultures is suggestive. The colonial morphology will vary considerably with strains and with culture conditions (Fig. 19-1). Smears may be made from colonies and Gram stained. All of the Pasteurellaceae species are gram-negative, non–sporeforming coccobacilli or rods. As a second step, some basic tests may be performed to characterize an isolate as a member of the Pasteurellaceae (e.g., oxidase, OF-test, motility, nitrate reduction, utilization of citrate, splitting of arginine). When a decision is made that the culture belongs to the Pasteurellaceae, additional tests are conducted to further identify them, if possible, to the species level. Detailed information on phenotypic tests that are useful for the identification of Pasteurellaceae may be found in several publications (Kilian and Frederiksen 1981b; Kilian 1976, Mannheim et al. 1980; Barrow and Feltham 1993, Holt et al. 1994). Criteria that are useful for the identification of Pasteurellaceae infecting mice are given in Tables 19-2–19-5, 19-7 and 19-9. For mice, relatively few species have to be separated from each other. These distinctions are possible when appropriate identification criteria and methods are used. Biotypes of
494 P. pneumotropica can usually be identified by the biochemical reactions given in Table 19-6. Various phenotypic variants of A. muris can be separated from each other by criteria given in Table 19-8. Pasteurellaceae sometimes do not grow sufficiently in certain media that were developed for the identification of less fastidious organisms such as Enterobacteriaceae. It is important that media are used which sufficiently support growth of this fastidious group of bacteria to avoid false-negative reactions. Some sugar fermentation media (e.g., with brom thymol blue) do not always support growth sufficiently while growth is much better in others (e.g., phenol red broth) leading to more reliable results. To avoid false-negative reactions and potential misidentification, nonproliferative tests should be favored whenever possible because they may be more sensitive than differentiation media requiring growth. These tests have been developed for the phenotypic identification of Haemophilus species (Kilian 1976; Kilian and Biberstein 1984). For example, strains of P. pneumotropica or other species infecting mice are found that are negative for urease or indole in commonly used differentiation media due to reduced metabolic activity, while positive results are obtained in nonproliferative media indicating that the enzymes for the reactions are synthesized. These tests should at least be used to confirm unexpected results from key reactions (e.g., indole, urease, ornithine decarboxylase). Also, results of testing for oxidase may be very much dependent on the test used. The oxidase activity is usually low, and the more sensitive tetramethyl reagent should be used instead of the dimethyl reagent (Bisgaard 1982; Holmes et al. 1995). Misidentification occurs frequently for various reasons of which some have been mentioned above. Sometimes criteria are used which do not allow species identification. Different results may therefore be obtained if the same strains are tested in different laboratories. Wagner et al. (1969) have reported considerable discrepancy between different laboratories studying the same isolates. Hooper and Sebesteny (1974) studied 28 strains of rodent Pasteurellaceae in two different laboratories, and only 76.5% of the fermentation tests correlated while the remaining tests gave uncertain or even contradictory results. Application of additional criteria or use of more recently published identification tables may lead to reclassification which, however, does not necessarily mean that even these results are correct. Compared to Enterobacteriaceae, extended incubation periods are advisable for biochemical testing. As a rule, differentiation media requiring growth and metabolism of the bacteria should be incubated for a minimum of 48 hours as P. pneumotropica and related species, in addition to having high nutritional requirements, are rather slow fermenters. Results from nonproliferative media can usually be read after incubation for 4 to 24 hours. For testing of biochemical characteristics of growth factordependent bacteria, growth factors (usually NAD) must be added to differentiation media, or nonproliferative media should be used. In many cases it is necessary to incubate differentiation media for 3 to 5 days. Details are given by Kilian (1976) and Holt et al. (1994).
WERNER NICKLAS
A. IDENTIFICATION BY COMMERCIAL KITS Most commercially available identification kits are not useful for the proper identification of Pasteurellaceae species and frequently fail to detect these organisms (Sakazaki et al. 1984; Eckert et al. 1991; Frederiksen 1993; Fajfar-Whetstone et al. 1995; Elsaghier et al. 1998). Usually only very few Pasteurellaceae species are included in databases, or only a limited number of relevant reactions are tested. Proper identification of some mouse organisms such as A. muris is very unlikely or not possible because the agent is not included in databases of most commercial identification kits. It is therefore in many cases advisable to use appropriate “conventional” methods for identification or for confirmation of unexpected results obtained by commercial test kits or to make sure that a commercial test kit is appropriate for the agents that are expected to be found. Commercial identification kits do not always identify Pasteurellaceae correctly even when they do yield a diagnosis. This has been demonstrated for bacteria of human origin (Lester et al. 1992; Weaver 1992; Elsaghier et al. 1998; Hamilton-Miller 2003) but is the case also for strains isolated from mice and other animals (Ward et al. 1998). For example, API 20 NE, which is a frequently used test kit, has only four Pasteurellaceae species included in the database [P. multocida, P. pneumotropica, P. aerogenes, and “P. haemolytica,” the last cited renamed a few years ago (Angen et al. 1999) and consists of several species]. Even fewer Pasteurellaceae species (e.g., P. aerogenes, P. haemolytica, P. multocida) are included in others. For this reason, isolations of P. haemolytica or P. aerogenes (e.g., Champlin et al. 2002) are frequently reported from rodents. Both agents are found primarily in farm animals (sheep, swine), and more careful examination usually identifies these as P. pneumotropica. Also, identification of mouse isolates as P. multocida may happen and should, like all unexpected identification results, be handled with care and verified by appropriate methods or by reference laboratories.
3.
Chemotaxonomic Criteria
The cellular fatty acid patterns seem to be uniform within the family with minor variations (Jantzen et al. 1981, Guettler et al. 1999). These criteria indicate the relationship of species within the family and are not suited for identifiying species within the family, but separation from other groups (e.g., from Neisseriaceae and Moraxella) is possible. Caution is necessary because the growth medium may have a significant effect on the cellular acid composition and thus on the reproducibility of data (Boot et al. 1999). The lipoquinone contents are more useful for the discrimination of groups within the family but do not necessarily reflect the degree of genomic relatedness (Mutters et al. 1993). Also, analysis of polyamines was found promising in discrimination of Pasteurellaceae (Busse et al. 1997). These studies also included four strains of P. pneumotropica, and the polyamine patterns confirmed that these organisms should be classified separately from the genus Pasteurella sensu strictu.
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Additional criteria have been discussed in more detail by Olsen et al. (2004).
B.
Molecular Methods (PCR)
Several PCR tests have been described for the identification of cultured mouse Pasteurellacae or, more importantly, for their detection in clinical samples. Target sequences for primers are usually located on the 16S rRNA gene which has been sequenced for numerous bacterial species. PCR tests have been reported to be more sensitive than bacterial culture (Wang et al. 1996; Nozu et al. 1999; Bootz et al. 1998). However, detection by PCR requires isolation of DNA from one or several locations, and conducting specific PCR tests will lead to increased expenses. The test described by Wang et al. (1996) is based on 16S rRNA sequences that were at that time available from GenBank and detects P. pneumotropica Jawetz but not the Heyl biotype. However, Weigler et al. (1998) showed that A. muris is also detected by this test, and the resulting amplicon cannot be distinguished from P. pneumotropica specimens, even after restriction endonuclease digestion. The PCR method developed by Wang et al. (1996) was modified by Hasegawa et al. (2003) who collected swab specimens from the laryngopharyngeal regions of anesthetized mice and subsequently cultured them in a liquid enrichment culture medium. Bacteria grown after incubation were washed and used for DNA extraction and subsequent PCR testing. A PCR test described by Nozu et al. (1999) is based on the sequence of P. pneumotropica biotype Jawetz from GenBank and an additional isolate that has obviously not been identified in detail. A number of reference organisms and field strains were used to evaluate the PCR. Differences were shown between their PCR results and those obtained with primers described by Wang et al. (1996), but neither the sensitivity nor the specificity can be evaluated on the basis of the data given because P. pneumotropica and non-P. pneumotropica had been identified by API-20 NE, which is definitely not suited for this purpose. The PCR described by Kodjo et al. (1999) is based on sequences of P. pneumotropica types Jawetz and Heyl and detects both biotypes, which can be separated from each other by different sizes of the PCR product; specificity can be achieved by restriction endonuclease digestion. Bootz et al. (1998) aimed at developing a PCR that detects all Pasteurellaceae infecting rodents, as suggested by the FELASA recommendations (Rehbinder et al. 1996; Nicklas et al. 2002). As a result, A. muris and H. influenzaemurium as well as H. parainfluenzae are detected. This often leads to confusion because positive results are obtained in populations that are negative for P. pneumotropica by culture. This PCR detects all members of the family Pasteurellacae infecting mice and rats. If an animal or a colony tests negative, further workup is not required. If an animal tests positive, one may choose to attempt to identify the strain. This may be done by traditional methods such as culture and biochemical characterization.
More straightforward options are sequencing the PCR product and comparing it with published sequences of other strains or restriction endonuclease digestion. PCR can be used not only for detecting Pasteurellaceae from clinical materials but also for identifiying them. Kodjo et al. (1999) developed their PCR primarily for the identification and differentiation of P. pneumotropica into both biotypes. They also used random amplified polymorphic DNA analysis (RAPD) or arbitrarily primed PCR (AP-PCR) to demonstrate further genetic diversity. These methods can therefore be used for the identification of strains to the subspecies level. They are also useful to identify genetic differences between strains and can contribute to epidemiological studies (Weigler et al. 1996).
C.
Serology
Rodents colonized with Pasteurellaceae produce antibodies, and serologic testing may enhance identification of carriers (Manning et al. 1989b; Wullenweber et al. 1988). Agglutination and complement fixation techniques are not sensitive enough to detect antibodies to Pasteurellaceae in subclinically infected animals (Hoag et al. 1962; Weisbroth et al. 1969). Most commonly, ELISA or immunofluorescence tests (IFA) are used. Use of ELISA and IFA (Nicklas 1989) prepared with whole-cell antigens allow detection of antibodies in spontaneously infected mice, but antibodies may cross-react with other members of the Pasteurellaceae, and serology may therefore fail to differentiate between species (Wullenweber-Schmidt et al. 1988; Manning et al. 1989b; Boot et al. 1995). Compared to whole-cell antigens which are shared among Pasteurellaceae, the specificity may be improved by use of lipooligosaccharides as antigens (Manning et al. 1989b, 1991). However, these tests may be too specific and sometimes do not detect all types (Manning et al. 1994). Also, Pasteurellaceae live on mucous membranes of their host, and this may result in delayed formation of antibodies of the IgG class. Commonly used tests for the detection of antibodies to Pasteurellaceae might therefore be positive only if the agent has invaded host tissues (Weisbroth et al. 1969; Percy and Barthold 2001) or several weeks postinfection. Antibodies (IgG) to P. pneumotropica antigens were detected initially by ELISA at 28 days after infection when whole-cell lysates were used, whereas antibodies to lipooligosaccharides were detectable by 7 weeks after infection (Manning et al. 1989b). Wullenweber et al. (1988) did not detect an immune response after experimental infection of mice with a low infectious dose. Slightly increased serological reactions were found 3 weeks after experimental infection with higher doses, and high levels of antibodies were detectable after 70 days. Although serology is cheaper and faster than bacterial culture and does not require killing and necropsy of the animal, it is used far less commonly for the detection of bacterial infections in mice. Serologic tests are routinely used for the detection of Pasteurellaceae infections only by a small number of laboratories.
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This is because bacteria have a complex antigenic structure resulting in an increased risk of nonspecific reactions or crossreactivity with other bacteria. Serologic tests for Pasteurellaceae can easily be used in combination with other methods of detection (culture, PCR) but should not be used as stand-alone techniques. They are useful in routine screening programs, but definite diagnosis is best done by culture or PCR.
VIII.
TREATMENT
In general, administration of therapeutics may influence the outcome of animal experiments and cannot be considered as a means to replace improvement of hygienic standards. Antibiotic treatment may induce resistance in bacteria especially when used on a large scale or may result in overgrowth of other bacterial species owing to an imbalance in intestinal flora. However, antibiotic treatment of bacterial infections may be of help or may even be necessary during quarantine and in the case of spontaneously infected animals to provide a better chance for a successful rederivation, especially in immunodeficient animals (Macy et al. 2000). Use of antibiotics can be necessary to suppress clinical disease and to extend the survival time of valuable animals or after experimental immunosuppression (e.g., by sublethal irradiation). Pasteurellaceae are usually sensitive to a wide variety of antibiotics, which can be given orally by addition to the drinking water or food, or by injection. Nevertheless, injection of individual animals for several days is practicable only for small numbers or if animals are highly valuable. Various authors (Moore and Aldred 1978; Hansen and Velschow 2000) tested the antibiotic sensitivity of P. pneumotropica and demonstrate that the bacteria have a good in vitro sensitivity to a number of substances. However, antibiotics should only be used according to results of sensitivity testing because some strains are resistant to specific drugs. Harkness and Wagner (1975) have pointed out that many isolates of P. pneumotropica are resistant to tetracycline and streptomycin. Reports also exist that tetracyclines may be less efficient when used in vivo (McGinn et al. 1992; Goelz et al. 1996), but there are also reports of regression of clinical signs (Moore and Aldred 1978). Hansen (1995) reported that treatment with four different antibiotics or antibiotic mixtures suppressed P. pneumotropica, but the agent reappeared after the medication was stopped. Klebsiella pneumoniae appeared in addition after the treatment, although it had never been observed prior to the use of the antibiotics. Also, human isolates identified as P. pneumotropica have been subjected to sensitivity testing (Olson and Meadows 1969; Rogers et al. 1973), but these isolates were most likely members of other species, and the data should be considered with care. Several reports have demonstrated the positive effects of antibiotics on Pasteurella infections of several organ systems (see Table 19-10). Antibiotic regimens of ampicillin, chloramphenicol,
and tetracyclines (oxytetracycline) reduced the prevalence of the organism in an infected population and produced regression of clinical signs (Gray and Campbell 1953; Moore and Aldred 1978). A number of antibiotics were also used by Wheater (1967), but the bacteria could be cultured again shortly after the end of the treatment. Conjunctivitis with mildly purulent lacrimal discharge in immunodeficient animals was alleviated by daily washing of the eyes with warm water, and severely affected animals received daily injections of ampicillin for 5 days resulting in regression of abscess formation and conjunctivitis (Moore 1979). Ackerman and Fox (1981) placed mice with P. pneumotropica uterine infections on tetracycline in the drinking water, which minimized the incidence of abortion and metritis within the colony, but the agents were isolated again some months after the therapy had been terminated. Oral or intramuscular administration of chloramphenicol to mice with otitis media associated with self-mutilation of the external ear resulted in the cessation of scratching and subsequent wound healing (Harkness and Wagner 1975). Moore and Aldred (1978) reported on antibiotic treatment of nude mice with abscesses in the orbital or preputial regions. While treatment by injection and, to a lesser extent by administration in the drinking water, caused regression of the abscesses, P. pneumotropica was in most cases isolated from the nasopharynx even after a month under treatment. More recently, the elimination of P. pneumotropica from asymptomatic mice has been reported after oral or parenteral administration of enrofloxacin at concentrations of 25.5 (170 mg/l drinking water) and 85 mg/kg body weight (570 mg/l drinking water) for 14 days (Goelz et al. 1996; Macy et al. 2000). Ueno et al. (2002) also reported eradication of P. pneumotropica from experimentally and naturally infected mice by enrofloxacin treatment (170 mg/l in the drinking water) for two weeks. Although treatment was repeated at intervals of two weeks, the agent was found again in one animal 45 weeks after final treatment. Treatment with antibiotics to which the organisms were sensitive in vitro failed to clear the bacteria in vivo (e.g., Kent et al. 1976; Moore and Aldred 1978; Ueno et al. 2002).
IX.
CONTROL AND PREVENTION
Relatively few recent surveys exist, but it is obvious that the incidence in most research facilities (Zenner and Regnault 1999/2000; Baker 2003) and thus the risk of introduction of Pasteurellaceae from outside or from infected populations on the campus is extremely high. Very general rules help to prevent introduction of agent into a unit, but frequently P. pneumotropica is among the first agent detected if hygiene measures are not strict enough (Cooper et al. 1977; Carthew and Aldred 1988). However, total exclusion from commercial breeding and from research colonies is possible if appropriate measures are taken.
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19. PASTEURELLACEAE
A. 1.
Eradication of Infection
Gnotobiotic Rederivation
A. HYSTERECTOMY The traditionally applied technique for the elimination of unwanted agents has been hysterectomy and subsequent maintenance using aseptic techniques and gnotobiotic technology (flexible film isolators). These are necessary together with careful testing to demonstrate the success of rederivation. Pasteurellaceae are, in fact, the agents that are most frequently responsible for failures of hysterectomy. Several studies have demonstrated that vertical transmission of Pasteurellaceae during rederivation may result from uterine colonization with the organism (Hoag et al. 1962; Brennan et al. 1965; Ward et al. 1978). P. pneumotropica and other species of this family are frequently isolated from the genital tract. Since an infected animal may harbor Pasteurellaceae in the uterus especially at the time of cesarean derivation, this method may result in introduction or reintroduction of the organism into isolator or barrier colonies (Casillo and Blackmore 1972; Blackmore and Casillo 1972). When gravid uteri are introduced into a unit, only the outer surface is disinfected. Hysterectomy derivation of animals should therefore always be followed by a quarantine period during which animals are carefully monitored to ensure successful elimination of the organism. It is best if uteri are transferred into plastic film isolators and if hysterectomy-derived fetuses are crossfostered by germ-free foster mothers. The placenta should immediately be examined microbiologically, and samples from weanling mice housed in the isolator should be submitted for laboratory examination before the young are transferred into a new unit. Studies conducted by Flynn et al. (1965) revealed that in most cases when an organism was cultured from the uterus, it was also cultured from fetuses and fetal membranes, thus demonstrating that cesarean-derived mice are not always free of Pasteurellaceae infections. Another source of failure may be animals intended to be used as foster mothers with undetected colonization by Pasteurellaceae. Especially NAD-dependent bacteria may not be detected during routine bacteriological monitoring if appropriate media are not used. Moore et al. (1973) reported isolation of P. pneumotropica from rats and mice that had been hysterectomy-derived and maintained under barrier conditions. The organisms were also found in feces of animals in isolators considered either germfree or gnotobiotic. A combination of antibiotic administration and cesarean derivation aimed at eliminating P. pneumotropica may also be successful if embryo transfer technology fails or is not available (Macy et al., 2000). B. EMBRYO TRANSFER Embryo transfer is a more reliable method for the eradication of agents (Reetz et al. 1988; Erdman et al. 1999; van Keuren and Saunders, 2004). If properly conducted, the risk of transmission of Pasteurellaceae is negligible. It has the advantage of avoiding postimplantational vertical transmission of infection, which frequently occurs in the case of Pasteurellaceae. It has been shown by Reetz et al. (1988)
that P. pneumotropica can be removed from eight-cell stage embryos by a few washing steps after in vitro contamination with high numbers of bacteria. In vitro fertilization followed by embryo transfer has been successfully used to rederive mice infected with P. pneumotropica (Suzuki et al. 1996). This procedure has the advantage that many eggs can be fertilized from a single male, thus reducing the risk of agent transmission as compared to natural matings. 2.
Antibiotics
First attempts to eliminate P. pneumotropica from mouse colonies, to prevent transmission from pregnant mice to their offspring, or for control of the organism in the course of intranasal passage of virus-containing material were reported already by Jawetz (1950), Jawetz and Baker (1950), and Gray and Campbell (1953), but the long-term effect of the treatment was not studied. Elimination of Pasteurellaceae from mice has repeatedly been reported by use of several antibiotics (see Section VIII. Treatment), but this approach will in many cases only lead to a significant reduction of the number of bacteria. Antibiotic therapy generally does not eliminate Pasteurellaceae or other bacterial organisms from a population or improve the microbiological quality of the animals, although it may improve the breeding performance or eliminate clinical signs (see also Table 19-10). 3.
Other Eradication Methods
Attempts have been made to immunize animals by both live and dead vaccines injected either intravenously or into the footpad. These led to antibody production but not to protection from subsequent challenge by nasal instillation (Wheater 1967). Early weaning and separation of pups from the dam, which may be used to eradicate other bacterial agents, are not successful in the elimination of Pasteurellaceae because the bacteria may be transmitted to the offspring in utero prior to birth, intravaginally at parturition, and by oronasal infection through maternal saliva and feces immediately after birth.
B. 1.
Prevention
General Hygiene
Modes of agent introduction into a unit are usually difficult to detect. Aerogenic transmission of Pasteurellaceae over long distances is very unlikely because the bacteria survive desiccation only for short periods. The chance of introducing these bacteria via the ventilation system is therefore very low. Also, transmission of these bacteria by materials is less likely due to their low resistance to environmental conditions (Scharmann and Heller 2001). Transmission with inanimate materials (clothes, animal house equipment, food, drinking water) can easily be avoided if appropriate methods (e.g., disinfection and
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WERNER NICKLAS
TABLE 19-10
EFFICIENCY OF ANTIBIOTIC TREATMENT OF MICE INFECTED BY P. PNEUMOTROPICA Antibiotic
Route
Dose
Treatment period
Result
Reference
Ampicillin
s.c.
9 mg/mouse/day
5 days
Moore and Aldred (1978)
Oxytetracycline
i.m.
3.5 mg/mouse/day
5 days
Tylosin base
i.m.
5 mg /mouse/day
5 days
Ampicilline
oral
1 g/l drinking water
30 days
Oxytetracycline
oral
2.5 g/l drinking water
30 days
Tylosin tartrate
oral
0.2 g/l drinking water
30 days
Tetracycline
oral
0.5 g/l drinking water
two months
Tetracycline
oral
0.5 g/l drinking water
four months
Enrofloxacin
s.c.
14 days
Enrofloxacin
s.c.
Enrofloxacin
oral
Enrofloxacin
oral
Tetracycline hydrochloride
oral
Enrofloxacin
oral
Enrofloxacin
oral
25.5 mg/kg body weight every 12 hours 85 mg/kg body weight every 12 hours* 25.5 mg/kg body weight ≈ 170 mg/l drinking water 85 mg/kg body weight ≈ 570 mg/l drinking water 60 mg/kg body weight ≈ 400 mg/l drinking water 40-50 mg/kg body weight 25.5 mg/kg body weight ≈ 170 mg/l drinking water
Complete regression of abscesses, but culture positive Complete regression of abscesses, but culture positive Complete regression of abscesses, but culture positive Complete regression of abscesses, but culture positive Complete regression of abscesses, but culture positive No regression of abscesses, culture positive Incidence of otitis media reduced from 90% to 25% Incidence of otitis media reduced from 90% to 8% P. pneumotropica not recovered 30 days after treatment P. pneumotropica not recovered 30 days after treatment P. pneumotropica not recovered 30 days after treatment
14 days 14 days
Moore and Aldred (1978) Moore and Aldred (1978) Moore and Aldred (1978) Moore and Aldred (1978) Moore and Aldred (1978) McGinn et al. (1992) McGinn et al. (1992) Goelz et al. (1996) Goelz et al. (1996) Goelz et al. (1996)
14 days
P. pneumotropica not recovered 30 days after treatment
Goelz et al. (1996)
14 days
Culture positive for P. pneumotropica at treatment termination
Goelz et al. (1996)
14 days in drinking water 14 days, 2-3x with intervals of 2 weeks
Decreased mortality, improved breeding performance P. pneumotropica not detectable in 3 out of 4 rooms 45 weeks after treatment termination, no eradication in the fourth room
Macy et al. (2000) Ueno et al. (2002)
*Skin ulcerations at the injection site.
sterilization procedures) are used for the introduction of materials. Transmission by staff is possible if they are given access to colonies with lower hygiene quality or in cases of other inappropriate management rules, thoughtlessness, or insufficient discipline. It is therefore important to select staff who are sufficiently motivated and trained to work in a facility governed by principles of microbiology. A barrier unit should be designed in a way so that it is difficult for staff to ignore or forget rules. Staff with special knowledge of microbiology must be involved in any change of policy. Typical rodent Pasteurellaceae do not infect humans, and it is therefore unlikely that infection is introduced by active carriage of the organism by staff. Exceptions are strains belonging to the H. parainfluenzae-complex, which are frequently found in humans and are rarely isolated from mice. Implementation of appropriate preventative measures, such as protective clothing and masks for personnel coming into contact
with animals and controlled access to animal rooms to maintain the Pasteurella-free status, are important prophylactic measures. Gloves and careful hand disinfection especially help to avoid passive transfer by personnel. Spreading of Pasteurellaceae infections between populations within a facility can be avoided by husbandry procedures that are common in well-managed mouse colonies (e.g., the use of chlorinated or acidified water in automatic watering systems). Like other agents, Pasteurellaceae can easily spread in populations housed in open cages, but inter-cage transmission can be avoided by the use of individually ventilated cages if proper cage-handling practices are undertaken (Hasegawa et al. 2003). 2.
Laboratory Rodents
The most important sources of infection are latent carriers of the same or a closely related species. Pasteurellaceae are endemic
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19. PASTEURELLACEAE
in numerous rodent colonies, even in populations housed behind barriers. Colonization by Pasteurellaceae is frequently not detected during routine health monitoring. Introduction of animals with unknown or undetected colonization by Pasteurellaceae is likely to be the most important mode of agent introduction. It is therefore of crucial importance that, despite a negative declaration in a health report, animals are rechecked prior to introduction into a Pasteurella-free population if this status is to be maintained. 3.
Wild Rodents
Important sources of infection are also vermin, especially wild rodents, gaining access to an animal housing unit. Wild house mice (Mus musculus) are usually infected with P. pneumotropica, A. muris, and other members of the Pasteurellaceae and represent a highly important risk factor (Shepherd et al. 1982; Boot et al. 1986). Wild rats may also transmit Pasteurella pneumotropica and other Pasteurellaceae (van der Schaaf et al. 1970) to laboratory rodents. Other animal species such as pigs and dogs are at risk if they are also infected with P. multocida (Curtis et al. 1980). Therefore, the area surrounding a building in which animals free of Pasteurellaceae are housed should not contain material such as food, clean or used bedding, or waste likely to attract rodents. 4.
Biological Materials
It is important that biological materials are monitored not only for viruses but also for bacteria and parasites (e.g., Encephalitozoon). Contamination of transplantable murine tumors by P. pneumotropica after animal-to-animal passages has been reported in the literature (Simpson et al. 1980; Nakai et al. 2000) and has also been found in our laboratory. The agent can therefore be transmitted by inoculation of contaminated samples or by animal-to-animal passages and introduced into a facility or a unit if tumors or other biological materials are introduced without prior testing. It is important that such materials can be stored frozen without loss of infectivity and may be a source of infection even after storage for decades. 5.
Cryopreserved Samples
There is some evidence that liquid nitrogen may be a potential source of cross contamination if some samples are contaminated with pathogens. To reduce the likelihood of cross contamination as a result of cryopreservation of gametes, embryos, sperm, and other biological samples, Rall (2003) has suggested that only aseptic procedures should be used to collect, prepare, and transfer gamete, embryo, and cell suspensions. In addition, decontamination of all external surfaces prior to cryopreservation and after thawing is strongly recommended. It is also important that the container is hermetically sealed to avoid nitrogen coming in direct contact with the cell suspensions. Freezing in vapor phase has the same effect.
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Chapter 20 Fungal Diseases in Laboratory Mice Virginia L. Godfrey
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Spontaneous/Natural Fungal Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pneumocystis murina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. History/Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Epidemiology/Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pathology/Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Treatment/Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Dermatophytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. History/Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Epidemiology/Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pathology/Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Treatment/Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Systemic and Opportunistic Infections . . . . . . . . . . . . . . . . . . . . . . . . . 1. History/Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pathology/Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Treatment/Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Animal Models of Fungal Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pneumocystis murina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Systemic and Localized Fungal Infections . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
Laboratory mice are widely used as animal models of fungal infections. In contrast, recent reports of spontaneous fungal diseases in laboratory mice are rare. This has largely been due to the generation of specific pathogen-free (SPF) mice by commercial vendors and repositories, the widespread use of barrier housing methods and sanitation for disease control, and the THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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increased microbiologic surveillance of experimental rodent colonies. The dramatic increase in the use of genetically altered (transgenic and knockout) mouse models has opened new avenues for the investigation of fungal diseases. Genetically altered mice with both intended, and sometimes unexpected, immune deficits have elucidated many of the biological responses to fungal agents. However, these immune-compromised animals can also serve as reservoirs for pathogens such as Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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Pneumocystis murina, and they may be highly susceptible to opportunistic infections by environmental fungi. For these reasons, fungal diseases in mice will continue to be of interest to the laboratory animal community.
II. SPONTANEOUS/NATURAL FUNGAL INFECTIONS A. 1.
Pneumocystis murina
History/Taxonomy
Pneumocystis organisms were first described by Chagas in 1909, although he mistook them for a form of Trypanosoma cruzi (Chagas 1909). Within a few years, further studies established that the organism was not a trypanosome but a new species that was named Pneumocystis carinii (Delanoe and Delanoe 1912). As recently as the 1980s, Pneumocystis was widely thought to be a protozoan. This view was based on morphologic features more similar to protozoa than fungi, the ineffectiveness of antifungal drugs in treating Pneumocystis carinii pneumonia (PcP), and the effectiveness of drugs generally used to treat protozoan infections. Some investigators had pointed out that Pneumocystis organisms exhibit morphologic similarities to fungi (Vavra and Kucera 1970). Nevertheless, the protozoan hypothesis remained predominant until 1988, when analysis of ribosomal RNA (16S-like rRNA) from P. carinii and Saccharomyces cerevisiae suggested an evolutionary relationship between Pneumocystis and fungi (Edman et al. 1988; Stringer 1993). Additional RNA data showed that Pneumocystis organisms in different mammals are quite distinct (Weinberg and Durant 1994; Cushion 1998), which led to an interim trinomial naming system (The Pneumocystis Workshop 1994; Stringer et al. 1997). In this system, the organism naturally infecting mice was named Pneumocystis carinii formae speciales muris (Pneumocystis carinii f. sp. muris). Because the trinomial system was clumsy, and genetic analysis showed more differences among the organisms than implied by subspecies nomenclature, a classic binomial system was subsequently proposed. Pneumocystis carinii now refers to a species infecting only rats, Pneumocystis jiroveci to the species infecting humans, and Pneumocystis murina to the species infecting mice. However, the acronym PcP is so well established as a medical term that it has been retained and now refers to Pneumocystis Pneumonia. The history of Pneumocystis and developments in its nomenclature are nicely described in recent reviews (Stringer et al. 2002; Wazir and Ansari 2004). 2.
Epidemiology/Morphology
Relatively little is known about the life cycle of P. murina, in part due to the extreme difficulty of propagating these organisms in vitro. Although phylogenetically classified as an ascomycete,
Pneumocystis sp. have features atypical of these yeasts (Edman et al. 1988). Generally, Pneumocystis sp. have four morphologic forms: trophozoites, cysts, precysts, and intracystic bodies (sporozoites) (Wazir and Ansari 2004). Trophozoites are pleomorphic unicellular structures measuring 2 to 4 µm with thin walls and 1 to 2 nuclei. Pneumocystis trophozoites divide either by binary fission or, after a fusion event, transform into early cysts. The cyst form measures 4 to 8 µm and appears spherical, cup-shaped, or crescent-shaped under light microscopy. In silverstained preparations, the cysts may appear empty or contain dark dots, which are focal thickenings of the cyst wall. Electron microscopy reveals an irregular pore in the thickened area of cell wall, which may be used to release sporozoites (Wazir 1993). As the cyst matures, meiosis and mitosis occur, producing eight intracystic bodies, similar to an ascus with ascospores. These intracystic bodies were formerly called sporozoites when it was thought that Pneumocystis was a protozoan, and occasionally this term is still used. The eight intracystic bodies are released to grow as haploid trophozoites. Slight ultrastructural differences have been noted in Pneumocystis cysts and trophozoites derived from different host species (Nielsen et al. 1998). The acquisition and transmission of Pneumocystis infection is still not clearly understood. The cyst would seem to be the transmissible form, but trophozoites are also infective to experimental animals. Each species of Pneumocystis appears to be specific for the mammal, in which it is found. The species that infects humans, Pneumocystis jiroveci (P. jiroveci), has not been found in any other mammal and the species of Pneumocystis found in other mammals have not been seen in humans (Stringer 2002). Animal models have clearly demonstrated that the infection can be transmitted from one animal to another via the airborne route (Dumoulin et al. 2000; Myers et al. 2003), although experimental infections across species lines have failed (Durand-Joly et al. 2002). It was thought for many years that Pneumocystis pneumonia resulted from reactivation of latent infections acquired as a neonate. This was based on the prevalence of anti-Pneumocystis antibodies in children, the presence of Pneumocystis organisms in immune-competent hosts without evidence of pneumonia, and the special forms of Pneumocystis associated with each mammalian host (Morris et al. 2002). However, studies from mice (Chen et al. 1993), rats (Vargas et al. 1995), and humans (Keely et al. 1995; Keely et al. 1996; Beard et al. 2000) all suggest that Pneumocystis pneumonia is an actively acquired infection. Chen et al. infected severe combined immunodeficiency (SCID) mice with Pneumocystis, establishing a clinical pneumocystosis, then reconstituted the SCID immune systems with transplanted normal spleen cells. Following immune reconstitution, no Pneumocystis organisms could be detected by cytology or PCR. Subsequent immune suppression of these animals did not induce pneumocystosis, indicating that the organisms had been cleared. In addition, studies of recurrent episodes of pneumocystosis in AIDS patients were done using mitochondrial RNA markers to categorize the organism at each occurrence (Keely et al. 1996).
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Genotype switching indicative of reinfection was found in four of five patients studied. Finally, genetic variation of Pneumocystis jiroveci found in different geographic regions is consistent with acquisition from a common source (humans or the environment) but not with reactivation of latent infections (Keely et al. 1996). Taken together, these data imply that murine Pneumocystis is actively acquired from other mice and not from reactivation of latent infections. However, an environmental reservoir cannot be ruled out since Pneumocystis DNA has been amplified from filtered air samples (Wakefield 1996; Olsson et al. 1998). Neonatal mice may serve as a reservoir for Pneumocystis murina in that their lung microenvironment is less capable of clearing the infection. Using an intranasal inoculation of Pneumocystis, Garvy and Qureshi (2000) showed that neonatal mice developed a subclinical infection that took 6 weeks to resolve, whereas adult mice resolved a comparable challenge within 3 weeks. The clearance delay in neonates corresponded to delayed kinetics of expression of lung cytokines TNF-alpha and IFN-gamma mRNA and chemokines lymphotactin, RANTES, and macrophage inflammatory protein-1ss mRNA. Along with deficits in antigen presentation, these differences in chemical mediators led to an inefficient T cell response in the lungs of the neonatal mice.
the host cells (Limper and Martin 1990). Diagnosis of Pneumocystis pneumonia is usually done by necropsy of clinically affected mice with demonstration of the organism in the lungs. Clinical signs of pneumocystosis are usually seen in immunesuppressed or immune deficient animals. These signs may include wasting, ruffled fur, dyspnea, cyanosis, and death. At necropsy, the lungs are enlarged, rubbery, and dark red to grayish-brown. Portions may fail to collapse upon opening the chest. Histologic changes include alveolar septal thickening with mononuclear inflammatory cells and alveolar filling with macrophages and a finely vacuolated eosinophilic material (Fig. 20-1). This alveolar exudate consists of organisms, sloughed alveolar cells, edema fluid, and pulmonary surfactant (Baker 1998). The Pneumocystis organisms can be demonstrated in bronchoalveolar lavage (BAL) fluids or lung touch imprints using modified Giemsa stain (Diff-Quik; Baxter American Scientific Products, Chicago) and in histologic sections by silver stains such as Grocott-Gomori methenamine silver (GMS). The trophic forms and intracystic bodies are stained by Diff-Quik and the cyst walls (spore cases) are stained by GMS (Fig. 20-2). Pneumocystis organisms may also be demonstrated by immunostaining with specific antibodies and by PCR analysis of lung tissues (Myers et al. 2003; Vestereng et al. 2004).
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4.
Pathology/Diagnosis
Pneumocystis sp. have a unique tropism for the lung, where they attach tightly to the alveolar epithelium without invading
Treatment/Control
Standard treatment for spontaneous pneumocystosis in laboratory mice is oral trimethoprim (50 mg/kg/day) plus
Fig. 20-1 Pneumocystis pneumonia in a C3H-scid/scid mouse. Alveolar septae are thickened with mononuclear inflammatory cells, and alveolar spaces are filled with a characteristic finely vacuolated eosinophilic exudate (*). Hematoxylin and eosin stain. Bar equals 20 µm.
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Fig. 20-2 Histiocytic pneumonia due to Pneumocystis murina. Pneumocysts are visible as dark oval structures (arrow). Grocott’s methenamine silver stain. Bar equals 20 µm.
sulfamethoxazole (250 mg/kg/day) in drinking water. The efficacy of this treatment is thought to reside entirely with the sulfamethoxazole component (Kunz et al. 1995). A number of other drugs such as sulfamethoxypyridazine, dapsone, and pentamidine have shown efficacy in experimental Pneumocytis infections in mice (Bartlett et al. 1998). Although treatment of immune-deficient mice can reduce morbidity, eradication of Pneumocystis infection in immune-deficient strains requires rederivation via Cesarean section or embryo transfer (Ito et al. 1991). Coinfection with other pulmonary pathogens can influence the treatment of immune competent strains (Roths et al. 1993; Macy et al. 2000) and may dictate the need for rederivation.
B. 1.
with a high mortality rate. The eminent microbiologist, Raimond Sabouraud, classified the dermatophytes into four genera: Achorion, Epidermophyton, Microsporum, and Trichophyton based on cultural and microscopic features of the organisms (Sabouraud 1910). The etiologic agent of human ringworm was named Achorion schoenleinii, and the mouse agent was termed Achorion quinckneum. In 1934, Emmons modernized the taxonomic classification of dermatophytes based on the morphology of spores and accessory organs. This led to the elimination of the genus Achorion and the renaming of the mouse dermatophyte as Trichophyton quinckneum (Emmons 1934). Tests have since revealed that T. quinckneum is not a distinct species, and it is now classified as a variant of T. mentagrophytes (Ajello et al. 1968).
Dermatophytosis
History/Taxonomy
The origins of medical mycology trace back to the discovery of the fungal etiology of favus by European physicians in the mid-nineteenth century (Weiztman and Summerbell 1995). Favus (from the Latin word for “honeycomb”) describes a distinctive type of tinea capitis characterized by formation of yellow, cupshaped crusts composed of dense mats of mycelia and epithelial debris that enlarge to form prominent honeycomb-like masses. Murine ringworm was first reported by Bennett in England in 1850 when he noted vegetative lesions on the face of a house mouse that were similar to those seen in human favus (cited in Buchanan 1919). This fulminant skin disease of mice was referred to as favus herpetiformis or favus muris and was often associated
2.
Epidemiology/Morphology
Classical murine ringworm, caused by Trichophyton mentagrophytes var. quinckneum, is usually restricted to feral and wild rodents. Buchanan (1919) cited numerous nineteenthcentury reports from Europe concerning favus in mice with natural and experimental transmission of the disease to humans and to other animals such as cats and dogs. Zoonotic transmission of murine ringworm has been well documented in persons occupationally exposed to rodents such as agricultural workers (Chmel et al. 1975) and laboratory technicians (Booth 1952). Prior to the widespread use of barrier housing methods and the creation of SPF populations by commercial vendors, various surveys found instances of dermatophytosis in laboratory mice
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that ranged from asymptomatic infestation to severe clinical disease. Dolan et al. (1958) reported 43% of healthy mice from various breeders in the United States were infected with T. mentagrophytes, though only 2% displayed clinical lesions. Mackenzie also isolated T. mentagrophytes from the coats of laboratory white mice, with 2% exhibiting clinical lesions (Mackenzie 1961). A similar survey in England showed a prevalence of T. metagrophytes in laboratory mice as high as 80 to 90%, with less than 1% showing clinical signs (Davies and Shewell, 1964). Çetin et al. (1965) documented a higher incidence of clinical disease that affected 60 of 400 white mice. Reports of spontaneous dermatophyte infections in laboratory mice are rare in the recent literature, with the disease more often associated with pet rodents. Lopez-Martinez et al. (1984) isolated T. mentagrophytes from 8% of 50 clinically normal mice, while Feuerman et al. (1975) found T. mentagrophytes (10%) and Microsporum gypseum (0.05%) in a similar survey. Papini et al. (1997) described dermatophytosis in laboratory animals housed under conventional conditions. In this report, Microsporum canis was isolated from a mouse exhibiting dorsal alopecia. The source of this murine infection was undetermined, but contamination of workers’ clothing, either by pets or M. canis-infected cats in an adjacent facility, was considered.
the detection of dermatophytes in skin scrapings and hair from infected companion animals (Kano et al. 2003). A similar system might be used for rapid detection of suspected dermatophytes in laboratory mice.
4.
Treatment/Control
Eradication of the disease can be accomplished by depopulation of infected animals and/or rederivation of breeding stock by Cesarean section or embryo transfer. Treatment of unique or exceptionally valuable mice might be attempted in order to generate breeding stock for rederivation. Systemic therapy should be used with caution because of the danger of overdose and adverse effects (Donnelly et al. 2000). Russell et al. (1981) suggest an oral dosage of 25 mg/100 g body weight of griseofulvin every 10 days (Hawk and Leary 1999). Pollock (2003) lists a griseofulvin dose of 15 to 25 mg/kg once daily (SID) for 3 to 5 weeks and cautions against the use of griseofulvin in pregnant rodents. Itraconazole has been shown to be effective against experimental T. mentagrophytes infection in mice at a dosage of 5 mg/kg/day for 5 days (Odds et al. 2004).
C. 3.
Systemic and Opportunistic Infections
Pathology/Diagnosis
Spontaneous dermatophytosis in laboratory mice can be asymptomatic or cause variable alopecia and scaling, usually on the head and back. Dolan (1958) noted cutaneous lesions in 2% of a newly arrived shipment of male albino (Webster strain) mice. These lesions consisted of scaly patches of alopecia measuring 0.5 to 2 cm. in diameter. Çetin et al. (1965) reported an epizootic of Trichophyton metagrophytes var. interdigitale in white laboratory mice. Gross cutaneous lesions in affected mice included generalized alopecia and brittleness of hair, large plaques with depressed centers and chalky crusts, and smaller hairless plaques without crusts. Histopathologic analyses revealed thick mats of mycelia covering the epithelium in the crater-like plaques with a mild chronic inflammation of the dermis. The hairless plaques consisted of papillary proliferation of the epidermis with hyperkeratosis, parakeratosis, and dermal fibrosis. Hair follicles were atrophic and sometimes dilated and filled with spores. Some mice displayed systemic signs of emaciation, depression, and epistaxis, which usually culminated in death. At necropsy, Çeftin found pancreatitis, liver necrosis, and hemorrhagic changes in the lungs and kidneys, which he attributed to a toxic effect since no fungal elements were found in the viscera. However, concomitant infection(s) with other pathogens were not considered and may have caused these systemic lesions. The diagnosis of dermatophytosis can be made by standard laboratory methods such as microscopic examination of plucked hair in 10% KOH preps and cultures in appropriate media such as Sabouraud or Mycobiotic agar (Fischman et al. 1976; Papini et al. 1997). An experimental PCR assay has been reported for
1.
History/Epidemiology
The older literature contains several reports of spontaneous systemic mycoses in laboratory mice. Sacquet et al. (1959) isolated Cryptococcus neoformans from an Af/A strain mouse displaying multifocal renal necrosis and a 1-mm diameter splenic abscess. Goetz and Taylor (1967) reported an outbreak of Candida tropicalis infection in Swiss-Webster mice. Clinical signs of inappetence, decreased lactation, weight loss, hunched posture, and death were noted in female mice transferred to an adjacent facility. Necropsy of affected mice revealed suppurative nephritis with intralesional yeast-like organisms. The cause of the infection was thought to be contamination of a customized soft diet combined with cold stress from a malfunctioning thermostat in the animal room. Austwick et al. (1974) isolated Candida tropicalis from a single 9-week-old mouse with suppurative nephritis. Lesions consisted of yeast cells and/or pseudohyphae surrounded by necrotic debris and polymorphonuclear leukocytes. Actinomycosis was the suspected etiology of a retroperitoneal mass in an adult NZW mouse (Mullink 1968). Although this mass contained multiple abscesses with fungal hyphae, cultures were negative, and the diagnosis of actinomycosis was based on the histopathologic lesions. More recently, Bingel (2002) described an array of spontaneous fungal infections in a colony of Cybbtm1 knockout mice. These mice lack the β subunit of NADPH oxidase and represent a murine model of X-linked chronic granulomatous disease of humans. They are reported to be highly susceptible to experimental infection with Aspergillus fumigatus.
512 During a 5-year period, Bingel (2002) documented 16 cases of suppurative and necrotizing fungal pneumonia caused by Paecilomyces sp. (11 of the 16 mice), A. fumigatus (3 mice), Rhizopus sp. (1 mouse), or Candida guilliermondii (1 mouse). Lacy et al. (2003) described 5 cases of spontaneous trichosporonosis in p47 (phox) knockout mice. These mice had pyogranulomatous inflammation in multiple organs, including lung, liver, lymph nodes, salivary gland, and skin. Fungal elements were identified in many of the lesions using special histochemical stains, and Trichosporon beigelii was cultured from affected sites. In 2001, the author investigated several cases of spontaneous pneumonia in p47phox −/− mice with a concomitant mutation in the gamma interferon gene. The animals had been housed in microisolator cages but were placed on nonautoclaved corncob bedding. Portions of the lungs in these mice were swollen, pale, and dry to rubbery on cut surfaces (Fig. 20-3). Histologic sections showed extensive necrosis and pyogranulomatous inflammation with intralesional hyphae with Periodic Acid Schiff (PAS) stains. Aspergillus terreus was cultured from affected lungs.
VIRGINIA L. GODFREY
Immune-deficient mice can also harbor asymptomatic infections that nonetheless interfere with experimental results. Ishihara noted an acceleration of the elimination of transfused human red blood cells (huRBC) in C.B-17-scid/scid (SCID) mice (Ishihara et al. 1998). Yeast-like organisms were isolated from their drinking water in pure culture, and the two isolates were identified as Candida guilliermondii. Inoculation of these isolates into SCID and BALB/c mice produced asymptomatic infection with oral and fecal shedding, seroconversion in the BALB/c mice, and recapitulation of the accelerated huRBC clearance in the SCIDs. 2. Pathology/Diagnosis
Clinical signs of systemic or invasive mycoses in laboratory mice include inappetence, weight loss, hunched posture, ruffled fur, cessation of lactation, and death. The clinical course, gross appearance, and microscopic pathology are dependent on the immune capabilities of the infected animals. Affected organs may
Fig. 20-3 Gross appearance of pyogranulomatous pneumonia due to spontaneous Aspergillus terreus infection in a p47phox –/– mouse. Lungs are pale, dry, leathery, and fail to collapse when the thorax is opened.
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be enlarged and often have multifocal to confluent pale nodules while the spleen and lymph nodes may show reactive enlargement. Extensive necrosis, hyphal invasion of the vasculature, multifocal infarction, and pyogranulomatous inflammation with intralesional hyphae can be demonstrated histopathologically. Fungal elements may be visualized with appropriate special stains such as Periodic Acid Schiff (PAS) and Grocott-Gomorri methenamine silver (GMS). 3.
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Treatment/Control
Eradication of the disease is best accomplished by depopulation of infected animals and/or rederivation of breeding stock by Cesarean section or embryo transfer. Systemic therapy may be attempted to salvage unique or exceptionally valuable mice; however, caution should be used because of the danger of overdose and adverse effects, especially in debilitated animals (Donnelly et al. 2000). Fluconazole (Diflucan®) is used to treat experimental deep mycoses in mice at a wide range of dosages (MacCallum and Odds 2004), although 10 to 20 mg/kg IP once daily (SID) has been recommended clinically (Pollock 2003). Itraconazole (Sporonax®) at a dosage of 5 to 10 mg/kg daily is another possible treatment (Pollock 2003). Amphotericin B (Fungizone®) at 1 mg/kg IP daily is a commonly used treatment regimen for experimental mycoses (MacCallum and Odds 2004). Identification of the source of the fungal pathogen is critical to prevent reinfection of rederived or reintroduced populations. Immune-deficient mice must be maintained in barrier housing environments with sterilized food, water, and bedding and handled only with appropriate sterile techniques under controlled isolation conditions such as HEPA-filtered laminar flow hoods or biosafety cabinets. It is critical to educate investigators to the dangers of cross breeding immune-deficient mice to immunecompetent mice reared in less stringent conditions. Such clinically normal breeder mice are often the source of opportunistic infections when introduced to the immune deficient population.
III.
ANIMAL MODELS OF FUNGAL INFECTIONS A.
Pneumocystis murina
Progress in continuous culturing of Pneumocystis sp. has been reported recently (Merali et al. 1999), but the complexity and cost involved in maintaining this system could limit its usage. Although several short-term culture methods have been previously described, they only achieve limited replication of the organism (Sloand et al. 1993; Atzori et al. 1998) and are used primarily for preliminary screening of potential therapeutic agents (Gangjee et al. 2003). As continuous culture systems to isolate and propagate Pneumocystis sp. are not readily available, animal models are critically important in Pneumocystis research.
Initial mouse models were based on immunosuppression of normal mice by administration of corticosteroids (Walzer et al. 1979; Walzer et al. 1983) or antilymphocyte antibodies (Bartlett et al. 1994) to initiate reactivation of supposedly latent infections. A marked variability of parasite levels among corticosteroid-treated animals and the fact that the origin of the parasite strain remained uncertain were important drawbacks of the corticosteroid-treated reactivation models (Dei-Cas et al. 1998). For these reasons, inoculated animal models of Pneumocystis pneumonia (PcP) were developed. Intratracheal or intranasal inoculation of lung homogenates containing viable organisms to immune-suppressed or immune-deficient mice results in extensive, reproducible Pneumocystis infections.
B.
Systemic and Localized Fungal Infections
Laboratory mice are used extensively to study pathogenic mechanisms in systemic and localized fungal infections. These models often provide the first in vivo screens for potential therapeutic compounds. Initiation of invasive infections usually requires immune suppression of the host by such mechanisms as radiation, corticosteroids, chemotherapeutic drugs, or, more recently, specific genetic alterations that induce immune deficiency. However, some agents such as Candida albicans and Cryptococcus neoformans can infect common inbred strains of mice such as DBA, BALB/c, A/JCr, or C57BL/6 without the need for immune suppression. Infections at specific sites may require unique manipulations such as antibiotic-induced alteration of microflora in gastrointestinal candidiasis (Mellado et al. 2000) or estrogen treatments for establishment of vaginal Candida infections (Hamad et al. 2002). Even minor modifications in the route of administration may alter the utility of model systems. For example, mouse models of invasive aspergillosis have utilized a pulmonary route for delivery of conidia, largely through intranasal instillation. However, radio-labeled particle studies have shown that aerosol delivery is preferable to intranasal instillation to create a more homogeneous delivery to the lungs. Steinbach et al. (2004) developed an inhalational model of Aspergillus fumigatus infection using a Hinners-type inhalation chamber and demonstrated by quantitative polymerase chain reaction that this creates a more homogeneous murine pneumonia. While Candida sp. and Aspergillus sp. have provided the most commonly used models of invasive mycoses, mice are also used to test antifungal compounds against human pathogens like Cryptococcus neoformans (Clemons et al. 1996) and Coccidioides immitis (Lutz et al. 1997). Cryptococcus neoformans intranasal inoculation in mice mimics well the progression of the human disease, starting with pulmonary infection, then systemic spread, and eventually cryptococcal meningitis (Cox et al. 2003). With increasing numbers of humans living with immune deficiency or immunosuppressive therapy, the study of atypical and opportunistic fungal infections has gained importance. Laboratory mice provide animal models for the
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study of such diverse agents as zygomycetes (Kamei 2001), ascomycetes (Cermeno-Vivas et al. 1998), and saprophytic molds like Scedosporium sp. (Capilla et al. 2003). The use of murine models, especially genetically altered mice, should continue to provide new insights into the pathogenesis and treatment of fungal diseases in animals and humans.
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Chapter 21 Protozoa Katherine Wasson
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Flagellates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Giardia muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle and Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Prevention, Treatment, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Spironucleus muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle and Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Prevention, Treatment, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Tritrichomonas muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle and Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Treatment, Prevention, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Other Intestinal Flagellates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Trypanosoma musculi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle and Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Treatment, Control, Research Implications . . . . . . . . . . . . . . . . . . . III. Amoebae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Entamoeba muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle and Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Treatment, Prevention, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Apicomplexans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Eimeria spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Treatment, Prevention, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Sarcocystis muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Prevention, Treatment, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Klossiella muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Prevention, Treatment, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Toxoplasma gondii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Treatment, Prevention, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Cryptosporidium muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Prevention, Treatment, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Microsporidia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Encephalitozoon cuniculi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Disease and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Prevention, Treatment, Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Research Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .....................................................
I.
INTRODUCTION
The Kingdom Protozoa contains a diverse collection of single-celled eukaryotic organisms that in many ways bridge the gap between the plant and animal worlds. Protozoa may be free living or parasitic, may be capable of photosynthesis, or may be thriving in a microaerophilic environment. Others can be considered extremophiles, living in ice, hydrothermal vents, or sulfur-emitting fumaroles. A relatively small number
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of these organisms are parasitic for mammals. Although there is no universal agreement on the correct terminology for these organisms, the parasitic protozoa of mammals fall into one of four phyla: the amoebae (Sarcodina), the flagellates (Mastigophora), the apicomplexans (Sporozoa, or the coccidia), and the ciliates (Ciliophora). Of these, mice can be naturally infected with members from the first three phyla; there are no known reports of murine-specific ciliates. A fifth phylum, the Microspora (which includes Encephalitozoon cuniculi), is now considered a member of the Kingdom Fungi (see Table 21-1 for
21.
519
PROTOZOA
TABLE 21-1
COMPARISON OF MURINE PROTOZOANSa Phylum
Genus and species
Diagnostic stage(s)
Diagnostic test(s)
Size (µm)
Description
Amoeba
Entamoeba muris
Cyst
Fecal float, smear
9–19 diameter
Trophozoite
Fecal smear
12–30 diameter
Cryptosporidium spp.
Oocyst
Fecal float
5×7
Eimeria falciformis
Oocyst
Fecal float
14–27 × 11–24
E. ferrisi
Oocyst
Fecal float
12–22 × 11–18
E. papillata
Oocyst
Fecal float
18–26 × 16–24
E. vermiformis
Oocyst
Fecal float
18–26 × 15–21
Klossiella muris
Sporocyst
Urinary centrifugation
16 × 13
Sarcocystis muris Toxoplasma gondii
Bradyzoite-filled cyst Cyst (intermediate host) Oocyst (Cat)
Histology Histology
4–6 × 14–16 Variable
Fecal float, smear
11–14 × 9–11
Tachyzoite
Histology
2–3 × 6–7
Bradyzoite
Histology
Giardia muris
Cyst Trophozoite
Fecal float, smear Fecal smear
15 × 17 7–13 × 5–10
Spironucleus muris
Cyst
Fecal float
7×4
Trophozoite
Fecal smear
10–15 × 3–4
Tritrichomonas muris
Trophozoite
Fecal smear
16–19 × 7–9
Trypanosoma musculi Encephalitozoon cuniculi
Trypomastigote Spore
Blood smear Histology
2–3 × 16–34 1.5 × 2.5
Round; 8 nuclei; lumen of cecum and ascending colon Pleomorphic, 1 nucleus; lumen of cecum and ascending colon Round to ellipsoid; gastrointestinal mucosa; intracellular but extracytoplasmic Round to ellipsoid; smooth; clear to light brown; crypt epithelium of cecum and colon Round; smooth; clear to light brown; villus epithelium of cecum and colon Round to ellipsoid; papillated; yellowish brown; villus epithelium of distal small intestine Ellipsoidal; pitted; yellowish brown; crypt epithelium of distal small intestine Round; glomerular endothelium and renal tubule epithelium Banana-shaped; myocytes Bradyzoites within cysts 5-8 × 1-2; prominent cyst wall; CNS, muscle, other tissues Round to subspherical; small intestinal epithelium Crescent shaped; within variety of nucleated cells Fusiform shaped; found within tissue cysts in variety of tissues Ellipsoid; 4 nuclei; lumen of small intestine Bilaterally symmetrical, pear-shaped; two nuclei; 4 pairs of flagella; closely associated with small intestinal villus brush border; “falling leaf” motility Ellipsoid; 2 nuclei; “Easter egg”; lumen of small intestinal Ellipsoid, tapered; found in mucus layer of small intestinal villus and crypts; “zig-zag” motility Pear-shaped; lumen of cecum and colon; “rolling” motility Elongated, vermiform shaped Rod-shaped; free or within parasitophorous vacuoles in brain, renal tubules
Apicomplexans
Flagellates
Microsporidia
aSee
text for references.
a summary). Because of E. cuniculi’s historical classification as a protozoan, and because of its importance as a pathogen in laboratory mice, this organism will be included in this discussion of murine protozoa. Not all of these organisms are pathogenic for mice. Indeed, the flagellate Tritrichomonas muris and the amoeba Entamoeba muris can be considered commensals of the murine large intestine. Others, for example, the flagellate Giardia muris and the apicomplexan Cryptosporidium muris, tend to cause disease in immunologically naïve, neonatal mice. Susceptibility to these organisms tends to have a mouse
strain and gender predisposition, and it is expected that genetically engineered mice will vary in their susceptibility to disease as well. Lastly, several of the parasitic protozoa of mammals were first recognized and described in mice, and mice continue to be instrumental in understanding the pathology, immune response to, clearance, and treatment of these infections. Although parasitic infections are rare in well-managed facilities utilizing rederived and barrier-maintained mice, surveillance for and familiarization with these organisms in mouse colonies are still warranted.
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K AT H E R I N E WA S S O N
II. A. 1.
FLAGELLATES
G. lamblia, or unspeciated Giardia (Franjola et al. 1995; Sogayar and Yoshida 1995).
Giardia muris 2.
Introduction
Life Cycle and Morphology
Genus
Species
Primary host
Giardia
muris agilis ardeae lamblia (duodenalis, intestinalis) microti psittaci
Rodents Amphibians Herons Mammals including humans
G. muris, as with all Giardia species, has a simple and direct life cycle. Infection is acquired through the ingestion of environmentally resistant cysts. Experimental infections indicate that the minimal infectious dose of G. muris in outbred Swiss or athymic nude mice is 10 spores (range, 5 to 20), and the prepatent period prior to fecal shedding is 8 days (range, 5–14; Roberts-Thomas et al. 1976; Stachan and Kunstyr 1983). Cysts undergo excystation upon exposure to gastric pH and secretions from the upper gastrointestinal tract. Upon excystation, two trophozoites are released into the small intestine. Trophozoites are bilaterally symmetrical and pear-shaped, possess four pairs of flagella, and measure 7–13 × 5–10 µm. Two nuclei and the median body often give trophozoites a “ghost” face appearance under light microscopy. Trophozoites are found in the upper small intestine where they attach to intestinal epithelial cells by means of a ventral “adhesive” disk. Trophozoites replicate by binary fission to produce two identical daughter trophozoites. As trophozoites are sloughed into the intestinal lumen toward the large bowel, a certain percentage will encyst and be excreted as cysts into the environment. The length of time that cysts are shed in the feces is dependent on the dose of the original inoculum, host immune status, mouse strain, and possibly gender (Belosevic and Faubert 1983; Belosevic et al. 1984; Daniels and Belosevic 1995; Heyworth 1988). Cysts are ellipsoid in shape, possess four nuclei (two trophozoites), and measure 15 × 17 µm. To provide organisms for in vitro studies, G. muris trophozoites can be collected from the duodenum and jejunum of infected mice (or other infected hosts) and isolated in axenic culture media (Tillotson et al. 1991). Trophozoites can also be generated by harvesting cysts from fecal pellets and inducing them to excyst in the presence of trypsin and low pH (Schaefer et al. 1984).
Voles, muskrats Parrots
3.
muris andersoni baileyi canis felis galli hominis meleagridis molnari parvum serpentis saurophilium wrairi
Rodents Cattle Chickens, turkeys Dogs Cats Finches, chickens Primates including humans Turkeys Fish Cattle, sheep, goats Snakes, lizards Lizards Guinea pigs
Mice are natural hosts to Giardia muris and can be experimentally infected with G. lamblia. G. lamblia (also referred to as G. intestinalis, or G. duodenalis; Thompson et al. 2000) is a waterborne pathogen and an important cause of human diarrhea on a global basis (Adam 2001). Although the classification and nomenclature of Giardia is still evolving, it is generally agreed that six distinct species exist and that G. lamblia can be further subdivided into two major genotypes (Table 21-2; Adam 2001). The virulence and zoonotic potential between species and genotypes varies. Among the flagellates found in the intestines of mice, only G. muris is considered a primary pathogen of mice (Sebesteny 1969). Giardiasis in laboratory-reared mice should be nonexistent under proper rederivation, husbandry, and management standards. Colonies of G. muris–infected mice still exist, however. G. muris was diagnosed in 6.9% of “white” laboratory mice, and co-infection with other murine intestinal protists was noted (Jalili et al. 1995). Giardia infection is also common in wild rodent populations, which can serve as potential sources of infection for laboratory mice. Recent field studies of wild rodents (including Mus musculus and Rattus rattus) indicated that 14.3 to 100% of the various populations harbored G. muris,
TABLE 21-2
VALIDATED GIARDIA AND CRYPTOSPORIDIUM SPECIESb
Cryptosporidium
bAdapted
from Adam (2001) and Xiao et al (2004).
Cell Biology
Giardia spp. possess two membrane-bound nuclei, a complex cytoskeleton, and a Golgi-like membrane system. However, they lack several features found in other eukaryotes, including nucleoli, mitochondria, enzymes involved in oxidative phosphorylation, and most enzymes involved in amino acid and nucleoside synthesis. As a result, Giardia are anaerobic and must forage for amino acids, purines, and pyrimidines from the intestinal milieu. The nuclei of Giardia are unique in that both are transcriptionally active and replicate at the same time. A second unique feature of Giardia is the ventral disk. This cytoskeletal structure allows the trophozoite to attach to intestinal epithelial cells of the duodenum and jejunum, preventing premature exit from the host. It is composed of
21.
several cytoskeletal proteins, including β–tubulins (Parsons 1995). The median body, located on the midline, is another unique component of the trophozoite’s cytoskeleton system. This structure is thought to be the assembly site for microtubule bundles to be incorporated into the ventral disk (Meng et al. 1996). The morphology of the median body can be used to distinguish between some of the Giardia species: G. lamblia has one or two transverse median bodies that are shaped like hammer claws; G. muris has one small, rounded median body (Adam 2001). Giardia lack mitochondria and the ability to generate energy through aerobic metabolism. However, identification of mitochondria-like heat-shock protein genes and mitochondrial remnant organelles (“mitosomes”) suggests that Giardia may have lost this organelle during the course of adapting to a parasitic lifestyle (Roger et al. 1998; Tovar et al. 2003). Lastly, Giardia trophozoites are capable of antigenic variation by expressing a family of cysteine-rich, immunodominant proteins across their surfaces known as variable surface proteins (Aggarwal et al. 1988). The ability to undergo antigenic variation is likely a means of evading the host immune response and may give the organisms a survival advantage in differing intestinal microenvironments (Adam 2001). 4.
521
PROTOZOA
Disease and Diagnosis
G. muris infections generally do not cause clinical signs in immunocompetent mice. Immunocompromised mice may exhibit weight loss and failure to thrive. These clinical signs are also observed in mice infected as weanlings, presumably due to a less mature immune system at this age (Buret et al. 1990). Unlike giardial infection in other mammals, diarrhea is not a clinical feature of disease in mice (Eckmann and Gillin 2001). Giardia clearance, as well as acquired immunity to the parasite, is primarily dependent on IgA production (Langford et al. 2002). Cellular immune responses and innate immunity factors such as defensins and host microflora also contribute to disease resistance and clearance (Singer and Nash 1999, 2000). G. muris can be diagnosed by identifying the characteristic trophozoites or cysts in feces by light microscopy. A “falling leaf” motility may be observed in fresh, unstained wet-mount samples taken from the duodenum or jejunum. Wright or Giemsa stains can be used to enhance the “ghost” face appearance of trophozoites. Cyst stages can be identified on fecal flotation or smears, and differentiated from other cysts or oocysts by their size (Table 21-1). Four nuclei may be seen on trichrome- or iodine-stained preparations of cyst forms. Owing to irregular shedding of cysts, several fecal examinations may need to be performed prior to ruling out an antemortem diagnosis of giardiasis. Histologically, trophozoites are found attached to the brush border of epithelial cells of the duodenum and jejunum. Trophozoites are lightly eosinophilic, crescentshaped on longitudinal view, and noninvasive. Careful examination under oil immersion may reveal the presence of
the ventral disk (Fig. 21-1A). There may be mild reduction in the villus:crypt ratio, and mild to moderate lymphocyte infiltration in the underlying mucosa and lamina propria (MacDonald and Ferguson 1978). Organisms need to be differentiated from Spironucleus muris, which tend to localize in the distal small intestine and are often found packed in intestinal crypts (Brett and Cox 1982, Owen et al. 1979). Enzyme-linked immunosorbent assays, flourescence antibody assays, and molecular-based methods are also available for diagnosis of giardiasis (Garcia et al. 1992; Nash et al. 1987; Sedinova et al. 2003). These tests are used for the detection of human giardial infection, and several are available in kit format. Use of these or comparable diagnostic kits for screening rodent colonies has not been reported. 5.
Prevention, Treatment, Control
G. muris is readily transmitted between mice and other rodent species (Belosevic et al. 1986a; Kunstyr et al. 1992; Saxe 1954). Therefore, rederivation is the treatment of choice for eliminating Giardia spp. from rodent colonies. Metronidazole and albendazole are the drugs of choice for treating humans infected with G. lamblia (Gardner and Hill 2001). The efficacy of these compounds may not be complete in mice (Bemrick 1963; Oxberry et al. 1994). Treatment with oral metronidazole resulted in only a 58.3% cure rate in mice naturally infected with G. muris, based on histologic examination of the small intestine for the presence of trophozoites (Cruz et al. 1997). Cyst forms shed into the environment are resistant to chlorine and ozone exposure, but can be inactivated by autoclaving of bedding and caging material (Lane and Lloyd 2002). 6.
Research Implications
Subtle alterations in intestinal epithelial cell kinetics, brush border enzyme composition, and cytokine production have been documented in immunocompetent mice with G. muris infection. Although villus height remained the same, a higher turnover rate of intestinal epithelial cells was observed in mice naturally co-infected with G. muris and Spironucleus muris when compared to control mice (MacDonald and Ferguson 1978). Significant decreases in the brush border enzymes lactase, sucrase, trehalase, and maltase were documented in C57BL/6 mice by day 10 of infection with G. muris (Daniels and Belosevic 1992). Reduced production of the epithelial cytokine IL-6 was reported in CD-1 mice (Scott et al. 2000). In addition, an increased epithelial lymphocyte infiltration of the small intestine has been observed with resolving Giardia infection (Brett and Cox 1982). All of these factors make endemically infected mice unsuitable for research involving intestinal physiology. A decreased T cell response has also been reported in mice experimentally infected with G. muris (Brett 1983). This was documented by a decreased ability of infected mice to mount
522
K AT H E R I N E WA S S O N
A
B
C
D
Fig. 21-1 Intestinal flagellates of mice. (A) Two Giardia spp. trophozoites in the upper small intestine of a mouse. Note the pear-shaped trophozoite with two nuclei and posterior flagella, and its close association to the intestinal brush border; the second trophozoite is oriented longitudinally, demonstrating the cup shape of the ventral adhesive disk. (B) Spironucleus muris trophozoites in the distal small intestine of a mouse; trophozoites are small and tapered, and associated with the mucus layer. (C) Tritrichomonas muris and (D) T. minuta in the ceca of mice; note the foamy cytoplasm and larger size of T. muris compared with T. minuta. H&E; A through D, bar = 50 µm.
21.
523
PROTOZOA
an immune response to sheep red blood cells. T cell responses returned to normal with resolution of infection (Brett 1983). Not surprisingly, exogenous administration of the immunosuppressive drugs cortisone and cyclosporin A resulted in increased cyst shedding and prolonged time to clearance of infection (Nair et al. 1981; Belosevic et al. 1986b). In the case of cortisone, recrudescence of subclinical infection could also be induced (Nair et al. 1981). These results suggest that administration of immunosuppressive agents to mice on experiment may exacerbate occult G. muris infection. A murine model of giardiasis was first described in 1976, and although it does not replicate all the features of human giardiasis, mice have been valuable in understanding the pathophysiology of this disease (Roberts-Thomas et al. 1976). Choice of mouse to use for infection studies should take into account strain, major histocompatibility complex haplotype, and perhaps gender. Early work demonstrated that C57BL/6, C57BL/10, and DBA/2 strains have a longer prepatent period, lower fecal cyst output, and faster resolution of experimental G. muris infection than A/J, BALB/c, or C3H/He strains of mice (Belosevic et al. 1984; Daniels and Belosevic 1992). The differences in the time course of infection were hypothesized to be due to differences in production of IgG2a and gamma interferon (γ-INF) inherent in the different mouse strains. A more virulent giardial infection could be induced in C57BL/10 mice by the administration of anti-γ–INF antibodies (Venkatesan et al. 1996). No gender differences were observed in the number of cysts shed in feces in BALB/c mice during acute infection with G. muris (Heyworth 1988); however, female C57BL/6 mice cleared G. muris infection by 18 days post-inoculation, while male mice of the same strain continued to shed cysts in the feces beyond 60 days (Daniels and Belosevic 1995). Murine haplotype also plays a role in clearing infection. BALB mice of the H-2b haplotype shed G. muris cysts for a longer period of time than BALB mice of the H-2d or H-2k haplotypes (Venkatesan et al. 1993). The commercial source of mice should also be considered. Isogenic mice from two different vendors were found to have different susceptibilities to experimental G. lamblia infection (Singer and Nash 1999). The inherent resistance in mice from one vendor could be overcome when the intestinal flora was altered with the antibiotic neomycin. Resistance in these mice could also be overcome when housed with susceptible mice from the second vendor (Singer and Nash 1999). These results suggest that differences in the endogenous microflora and fauna can affect both experimental and natural Giardia infection.
B. 1.
Spironucleus muris
Introduction
Originally called Hexamita muris, Spironucleus muris has been renamed to differentiate the exclusively parasitic (“Spironucleus”)
from the usually free-living (“Hexamita”) members of this genus (Brugerolle et al. 1980). Recent surveys of laboratory and wild mouse populations suggest that the prevalence of S. muris ranges from 4.1 to 38.6% (Franjola et al. 1995; Jalili et al. 1995). Spironucleus muris is probably represented by subspecies with differing host preferences and infectivity. In several separate transfaunation studies, isolates of S. muris from mice and golden Syrian hamsters were infectious for the reciprocal host; isolates obtained from a rat and European hamster were not infectious for mice; and in a third study, isolates from a mouse and rat were infectious for hamsters and rats (Saxe 1954; Schagemann et al. 1990; Sunstyr et al. 1993). Regardless of host specificity, these organisms are likely commensals in their respective rodent hosts (Baker et al. 1998; Sebesteny 1969). Several clinical reports from the 1970s describe “outbreaks” of spironucleosis in laboratory mice, with mortality rates ranging from 20 to 50%. Clinical signs included chronic wasting in athymic nude and thymectomized mice (Boorman et al. 1973); or depression, distended abdomens, “sticky stools,” and nonbloody, catarrhal enteritis in weanling age mice (Flatt et al. 1978; Wagner et al. 1974). Large numbers of Spironucleus muris trophozoites, and occasionally G. muris cysts, were observed at necropsy. Thirty years later, descriptions of these “outbreaks” are reminiscent of disease seen with enteric mouse hepatitis virus (MHV), transmissible murine colonic hyperplasia (Citrobacter rodentium), Tyzzer’s disease (Clostridium piliforme), and/or salmonellosis rather than primary infection with S. muris. Today, large numbers of S. muris organisms are frequently observed secondary to underlying disease, often MHV infection (Percy and Barthold 2001). A diagnosis of spironucleosis should prompt the clinician to search mouse colonies for an underlying infectious agent or disease condition. 2.
Life Cycle and Morphology
S. muris has a simple and direct life cycle. Infection is initiated with the ingestion of cysts. A single trophozoite is released from S. muris upon excystation in the upper gastrointestinal tract (Schagemann et al. 1990). Trophozoites replicate by longitudinal binary fission. Trophozoites have a slender, tapered body measuring 10–15 × 3–4 µm. They possess four pairs of flagella and two nuclei, but lack the specialized cytoskeletal features seen in Giardia spp. (Brugerolle et al. 1980). Trophozoites encyst as they move down the intestinal tract. Cysts measure 7 × 4 µm and resemble “Easter eggs” due to the presence of flagella under the cyst membrane, giving the cysts a banded appearance (Kunstyr 1977; Kunstyr et al. 1977). The minimal infective dose is one cyst, and the prepatent period has been reported between 2 and 8 days for mice (Kunstyr 1977; Stachan and Kunstyr 1983). In vitro culture and manipulation of S. muris have not been reported; organisms are passaged in flagellate-free mice for experimental manipulation (Schagemann et al. 1990).
524 3.
Cell Biology
Relatively little is known regarding the cellular and molecular biology of S. muris. Like Giardia, they are early eukaryotic organisms, possessing a membrane-bound nucleus, but lacking mitochondria and, presumably, the enzymes involved in aerobic metabolism (Brugerolle et al. 1980). S. muris lacks the ventral disk and median body observed in Giardia (Januschka et al. 1988). Trophozoites feed on luminal flora, as evidenced by the presence of bacteria in digestive vacuoles seen on electron microscopy (Brugerolle et al. 1980). An inverse ratio of Giardia and Spironucleus trophozoites has been observed in naturally infected mice, suggesting that these organisms compete for similar resources in the host (Sebesteny 1969). 4.
Disease and Diagnosis
Early descriptions of disease due to spironucleosis must be interpreted with caution, as S. muris is likely a “facultative pathogen” in mice infected with other disease agents (Kunstyr et al. 1977). In contemporary mouse colonies, heavy S. muris burdens are associated with enteric mouse hepatitis virus infection (Percy and Barthold 2001). Spironucleosis has also been observed in otherwise pathogen-free, genetically engineered mice with underlying immune alterations. Experimental S. muris infection of athymic nude mice resulted in abdominal distention, failure to thrive, and death after 2 to 3 months of infection (Kunstyr et al. 1977). A more recent experimental report noted a lack of clinical disease and intestinal pathology in a variety of inbred strains of mice orally inoculated with S. muris cysts (Baker et al. 1998). Diagnosis of S. muris depends on observing trophozoites or cysts in fecal smears or histopathology. Trophozoites have a fast, zigzag movement on fresh wet-mount samples taken from the distal small intestine and colon (Sebesteny 1969). The nuclei are best visualized with Giemsa or hematoxylin stain (Sebesteny 1969). Cysts are found in the large intestine and feces, and can be differentiated from Giardia cysts by their smaller size and “Easter egg” appearance (Kunstyr et al. 1977). On histologic sections, organisms are located in the distal small intestine and are usually found in the mucus layer or packed within intestinal crypts (Wagner et al. 1974; Brugerolle et al. 1980). Invasion of the intestinal mucosa and lamina propria by organisms has also been reported (Flatt et al. 1978). S. muris trophozoites appear as smudgy, lightly eosinophilic structures with hematoxylin-eosin stain (Fig. 21-1B). Under 100X oil immersion, they appear as plump banana-shaped organisms, with a single mid-body nucleus visible. Diagnostic immunologic or molecular techniques have not been reported for S. muris. 5.
K AT H E R I N E WA S S O N
dimetronidazole, metronidazole, or tinidazole was ineffective in eliminating cyst shedding in mice (Kunstyr et al. 1977; Sebesteny 1969). Albendazole, a microtubule inhibitor with some efficacy against Giardia infection, had minimal effect on the ultrastructure of S. muris (Oxberry et al. 1994). Cysts are susceptible to several common disinfectants, fixatives (70% ethanol, household bleach, aldehyde-based compounds), and temperatures above 45°C, suggesting that standard laboratory animal husbandry and management procedures will control cysts in the environment (Kunstyr and Ammerpohl 1978). 6.
Research Implications
S. muris infection complicates research with experimental intestinal flagellate infection (Owen et al. 1979). In that report, mice colonized with S. muris were less susceptible to experimental G. muris infection. Alterations in intestinal epithelial cell kinetics were observed in mice co-infected with S. muris and G. muris (MacDonald and Ferguson 1978). A decreased villus to crypt ratio, and decreased T cell–dependent immune response to sheep red blood cells, have been observed in S. muris–infected mice (Brett 1983; Brett and Cox 1982). C. 1.
Tritrichomonas muris
Introduction
Tritrichomonas muris is a nonpathogenic flagellate of mice, rats, hamsters, and other rodents. Early literature refers to this organism as Trichomonas muris, Trichomonas cricetus, or Tritrichomonas cricetus. However, these were determined to be synonyms for morphologically identical organisms found in a variety of laboratory and wild rodents (Daniel et al. 1971; Honigberg 1963). An exhaustive transfaunation experiment demonstrated the ease with which several trichomonads (including T. muris and Pentatrichomonas hominis) could be established in and transmitted between mice, rats, and hamsters (Saxe 1954). T. muris is related to the more pathogenic Trichomonas vaginalis and Tritrichomonas foetus of humans and cattle, respectively. These latter organisms are sexually transmitted protists that live in the genitourinary tracts of their hosts, causing reproductive disorders (BonDurant 1997; Petrin et al. 1998). T. muris resides in the cecum and colon of rodents, where it is considered a component of the normal fauna (Sebesteny 1969). Recent surveys of laboratory and wild colonies of mice demonstrate a 29.6 to 47.4% prevalence of the organism in the large intestines (Franjola et al. 1995; Stachan and Kunstyr 1983). Although little is known of T. muris, much of its life cycle and cell biology can be inferred from work done on more pathogenic family members.
Prevention, Treatment, Control
S. muris is readily transmitted between rodents (Saxe 1954). Rederivation and fostering on flagellate-free dams will eliminate S. muris from mouse colonies. Chemotherapy using
2.
Life Cycle and Morphology
T. muris has a simple and direct life cycle, and exists as motile trophozoites within the host. Pseudocyst forms of
21.
T. vaginalis and other trichomonads have been observed and are thought to represent degenerating trophozoites responding to unfavorable environmental conditions (Honigberg 1963; Petrin et al. 1998). The minimal infectious dose of T. muris “pseudocysts” for mice is 5, and the prepatent period is 10 days (Stachan and Kunstyr 1983). Trophozoites are pear- or teardropshaped, measuring 16–19 × 7–9 µm (Selukaite 1977; Fig. 21-1C). Trophozoites replicate by binary fission. Trophozoites reside in the cecum and colon but have been reported in the stomach and small intestines, likely as a result of recent ingestion (Koyama et al. 1987; Selukaite 1977). Newborn hamsters, and probably other rodent pups as well, are colonized by T. muris within a week after birth (Mattern and Daniel 1908). Cell-free cultivation of T. muris has not been reported; trophozoites have been harvested from mono-infected rodents for experimental manipulation (Saxe 1954). 3.
Cell Biology
All trichomonads possess a single membrane-bound nucleus, three anterior flagella, and a fourth posterior flagellum that forms the “backbone” of the undulating membrane. The undulating membrane runs partway down the length of T. muris and can be visualized in less motile trophozoites under light microscopy (Osada 1962). The axostyle is the cytoskeletal structure that appears to give the organism a “backbone.” It originates at the nucleus and tapers to a tail-like appendage at the distal end of the parasite (Daniel et al. 1971). In T. vaginalis, the axostyle is thought to be an attachment organ on vaginal epithelial cells (Petrin et al. 1998). On electron microscopy, a comb-like structure called the costa can be seen anchoring the base of the undulating membrane (Daniel et al. 1971). The parabasal body is a collection of flattened cisterns similar in structure to the Golgi complex in higher eukaryotes. Trichomonads lack mitosomes but possess double-membrane bound granules that metabolize carbohydrates and produce ATP and hydrogen, and are referred to as hydrogenosomes (Petrin et al. 1998). 4.
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Disease and Diagnosis
No disease has been attributed to T. muris in rodents. Impressive numbers of trophozoites can be seen in the cecal lumen of some mice in the absence of disease (Fig. 21-1C). Diagnosis can be made by examining fresh wet mounts from the cecum and colon by light microscopy. Organisms can be identified by their “rolling” or “quivering” movement (Petrin et al. 1998). Staining wet mounts with Giemsa or iodine enhances the appearance of the nucleus and undulating membrane.
indicator of breach in barrier maintenance of otherwise pathogen-free mice. Chemotherapeutic elimination of T. muris has not been reported. The lack of T. muris cyst forms implies that standard husbandry and management practices will eliminate organisms from the environment. 6.
Interference with research results due to the presence of T. muris in mice has not been reported. Due to the health significance of T. vaginalis in the human population, there has been interest in using the laboratory mouse as a model for this sexually transmitted disease. Patent, long-term infection requires pretreatment of female mice with estrogen and intravaginal doses of Lactobacillus spp. prior to the introduction of T. vaginalis organisms (McGrory and Garber 1992). Using this model, investigators have demonstrated that IgA antibodies protect against severe infection with T. vaginalis and that T-lymphocytes are important in parasite clearance in infected women (Paintlia et al. 2002).
D.
Treatment, Prevention, Control
Rederived and barrier-maintained mice are free of T. muris. The ease with which T. muris can be transmitted between mice and other rodents suggests that trichomonads can be used as an
Other Intestinal Flagellates
In addition to the above-mentioned protists, several other nonpathogenic flagellates have been identified in the intestines of the laboratory mouse. Most of these can be identified according to their morphology and size. Trichomonas minuta (4–9 × 2–5 µm) and Trichomonas wenyoni (4–16 × 2.5–6 µm) are smaller in size and have a less vacuolated or “foamy” cytoplasm compared to T. muris (Levine, 1973b). T. minuta has a more prominent and eosinophilic axostyle than T. muris (Fig. 21-1D). Chilomastix bettencourti is a cyst-forming flagellate found in the cecum of mice, rats, and hamsters. Trophozoites are asymmetrically piriform and measure 8–20 × 7–8 µm; cysts are lemon-shaped and measure 6.5–9 × 5.5–7 µm (Nie 1948). They possess three anterior flagella and a short, posteriorly directed flagellum. A distinctive, pouch-like cytostome can be seen in both forms, and is thought to be a feeding organelle (Levine 1973a). Octomitus pulcher is a bilaterally symmetrical flagellate that morphologically resembles Giardia spp. They measure 6–10 × 3–7 µm and possess six anterior and two posterior flagella, as well as two anteriorly located nuclei. O. pulcher is found in the cecum of mice, rats, hamsters, and other wild rodents (Gabel 1954; Levine 1973a).
E. Trypanosoma musculi 1.
5.
Research Implications
Introduction
Trypanosoma musculi is a nonpathogenic hemoflagellate parasite of wild mice living in the Mediterranean basin, West Africa, and Central America. T. musculi is host-specific for Mus musculus (Krampitz 1969). First described in 1909, this
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organism is also referred to as T. duttoni in the older literature (Kendall 1906). A relatively recent survey identified T. musculi in 3.8% of wild house mice in the Arabian peninsula (Molan and Hussein 1988). There are no published reports of natural infection with this parasite in laboratory-raised mice.
recta of the kidneys, and suggested that mice can be persistently infected for life (Viens et al. 1972). Subsequent work has shown that capillaries of the vasa recta provide nutrients and an “immunologically” privileged site for T. musculi trypomastigotes (Monroy and Dusanic 1997).
2.
3.
Life Cycle and Morphology
The life cycle and morphology of T. musculi is similar to that of other African trypanosomes. T. musculi is transmitted by fleas, including the Oriental rat flea, the Northern rat flea, and the mouse flea (Xenopsylla cheopis, Nosopsyllus spp., and Leptosylla segnis, respectively). Mice become infected by ingesting infected fleas or flea feces containing trypanosomes. Mice become parasitemic, during which time trypomastigotes replicate by multiple fission, and can be identified in peripheral blood smears. Trypomastigotes have an elongated, vermiform shape and measure 2 to 3 µm in width and 10 to 34 µm in length (Taliaferro and Pavlinova 1936; Fig. 21-2). Parasitemia lasts 2 to 3 weeks, after which time organisms are difficult to identify in blood smears. The life cycle is perpetuated when fleas consume a blood meal from parasitemic mice. The peak parasite burden varies considerably in immunocompetent strains of mice (Derothe et al. 1999). Mice develop immunity and are resistant to reinfection with T. musculi. However, Viens et al. demonstrated that a small percentage of parasites persisted (and continued replicating) in the vasa
Trypanosomes possess a membrane-bound nucleus and a single flagellum. Trypanosomes also possess a single mitochondrion referred to as a kinetoplast. In addition to performing metabolic functions as in other eukaryotic organisms, this organelle has a unique mitochondrial genome organization and function (McFadden 2003). Indeed, several unique RNA processing mechanisms were first described in the kinetoplast of trypanosomes (Gott and Nilsen 2003). These include transsplicing and RNA editing. The DNA in the kinetoplast is organized in 20 to 50 “maxicircle” DNA segments and 5000 to 10,000 small circular DNA segments. The large content of DNA in this organelle contributes to its intense staining. Instead of the conventional linear method of transferring genomic information from DNA to RNA (transcription), and then to protein (translation), various transcripts of RNA from different areas of the kinetoplast genome can be ligated together prior to being translated (trans-splicing). The ability to perform trans-splicing is the mechanism behind the phenomenon of antigenic variation in African trypanosomes, a major means by which parasites evade the host immune system. In addition to modifying RNA by ligating pieces together, sections of transcribed RNA can be altered by the insertion or deletion of uridine residues in order to create messenger RNA that will code for functional proteins (RNA editing). Once thought to be unique to the kinetoplastid parasites, trans-splicing has since been identified in parasitic and free-living nematodes, and RNA-editing has been described in plant mitochondria and chloroplasts. 4.
Fig. 21-2 Trypanosoma sp. in a peripheral blood smear from a cow. Note the kinetoplast, undulating membrane, and single flagellum. Similar features would be seen in T. musculi from mice. H&E; bar = 50 µm (Image courtesy of H. Gelberg).
Cell Biology
Disease and Diagnosis
T. musculi is relatively nonpathogenic for immunocompetent mice. Mice develop mild anemia, splenomegaly, hepatomegaly, and lymph node hyperplasia that resolve after one month (Hirokawa et al. 1981). Mice experimentally infected between the fourth and fifteenth day of gestation, however, develop fatal parasitemia, with large numbers of replicating trypomastigotes present in the maternal vessels of the placenta (Krampitz 1969). Athymic nude mice develop persistent infection, while splenectomized mice develop fatal parasitemia, when experimentally inoculated with T. musculi trypomastigotes (Rank et al. 1977; Taliaferro and Pavlinova 1936). The trypomastigote forms of T. musculi are morphologically similar to those of other mammalian trypanosomes. When stained with Giemsa, they have a large, red, centrally placed nucleus and a smaller, posteriorly located kinetoplast. The cytoplasm
21.
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stains blue. An undulating membrane can be seen by light microscopy running the length of the trypomastigote. The membrane continues past the main body of the parasite as a free, anteriorly located flagellum. Infection is best diagnosed on peripheral blood smears. Trypomastigotes may also be observed in histologic sections of spleen, liver, and kidneys (Hirokawa et al. 1981). 5.
Treatment, Control, Research Implications
Because of the self-limiting nature of T. musculi infection, treatment of infected mice has not been reported. Experimental infection in laboratory mice has been proposed as an in vivo model for screening chemotherapeutics against Chagas’ disease in humans (Jennings and Gray 1982). However, laboratory mice can be experimentally infected with the etiologic agents of both human American and African trypanosomiasis (T. cruzi and T. brucei spp., respectively). Infection in mice with these organisms mimics many of the pathologic and immunologic features of chronic human infection (Kennedy 1999; Marinho et al. 2004). Consequently, T. musculi infection in mice is rarely reported as a model for human trypanosomiasis. In areas where T. musculi is endemic in wild mouse populations, prevention of infection in the laboratory setting involves appropriate vermin and insect control. There are no published reports of natural T. musculi infection confounding experimental data obtained from mice.
III. A. 1.
AMOEBAE
Entamoeba muris
Introduction
The genus Entamoeba is composed of a diverse group of parasitic and free-living, single-celled organisms with an amoeboid mode of locomotion (Silberman et al. 1999). Most Entamoeba are nonpathogenic. Entamoeba muris is the only amoeba identified in laboratory mice and is considered a commensal inhabitant of the cecum. The older literature refers to this organism as Amoeba muris, Entamoeba muris decumani, Councilmania muris, Councilmania decumani, Endamoeba ratti, or Entamoeba coli var ratti. Based on morphology, these terms are now considered synonyms for E. muris (Neal 1950). Surveys of laboratory and wild mouse populations demonstrate a prevalence of E. muris between 5 and 55% (Franjola et al. 1995; Jalili et al. 1995; Livingston 2004; Pruss 1960). Organisms morphologically identical to E. muris are also identified in laboratory and wild rats, and hamsters (Pruss 1960). E. muris is related to the more pathogenic E. histolytica and E. invadens of humans and reptiles, respectively.
2.
Life Cycle and Morphology
Entamoeba spp. have a simple and direct life cycle. Trophozoites are found in the cecum and anterior colon, where they feed on bacteria, protozoa, and other luminal material (Lin 1971). They are pleomorphic on wet mounts, round or ovoid in histologic sections, and possess a single nucleus (Neal 1950). The pseudopod, an ectoplasmic extension distinct from the endoplasm (cytoplasm), and a trailing uropod may be observed in wet-mount preparations and account for the amoeboid motion of these organisms. They lack cilia, flagella, or other organized cytoskeletal structures. The cytoplasm contains vacuoles of varying size. Trophozoites replicate by binary fission. Encystation occurs in the cecum, with mature cysts possessing eight nuclei and measuring 9 to 20 µm in diameter. Immature or “precyst” stages may possess four nuclei. Amoebae that pass from the cecum without encysting do not survive outside the host (Lin 1971). Cysts are excreted in the feces and are available for ingestion and infection in the next host. The signals involved in excystation of ingested cysts in the cecum are not known. The environmental stability of E. muris is unknown. Cell-free cultivation of E. muris has not been reported. 3.
Cell Biology
The genus Entamoeba has classically been divided into groups based on the number of nuclei in the mature cyst forms (Neal 1966). In general, nonpathogenic Entamoeba have one nucleus (E. chattoni from nonhuman primates; E. polecki from pigs and humans) or eight (E. coli from humans; E. muris from mice) nuclei per mature cyst. The pathogenic E. histolytica and nonpathogenic E. dispar of humans possess four nuclei per mature cyst. In addition, E. invadens of reptiles possess four nuclei in the mature cyst. This classification has held up to phylogenetic analysis of ribosomal DNA and protein sequences of Entamoeba (Silberman et al. 1999). This morphologic detail is important when diagnosing amoeba infection in other species. The presence of octonucleate cysts in the feces of a reptile suggests that these are E. muris that were transiently acquired from feeder mice, while quadranucleate cysts suggest parasitism with E. invadens. Like the flagellates, Entamoeba lack mitochondria, peroxisomes, and a Golgi complex. A mitochondrial-like organelle, variably termed crypton, cryptome, or mitosome, has been identified in E. histolytica (Mai et al. 1999; McFadden 2003; Tovar et al. 1999). Its function has yet to be determined. Entamoeba spp. generate energy through glycolysis. Elongated, rhomboidshaped cytoplasmic inclusions called chromatoid bodies can be seen by light microscopy in cyst stages of Entamoeba spp. These bodies are aggregates of ribosomes, and their function is unknown (Kusamrarn et al. 1975; Neal 1966). 4.
Disease and Diagnosis
Disease due to the presence of E. muris in the cecum and colon of mice has not been reported. Although molecular and
528 cellular analysis of E. muris has not been reported, it is likely that this species lacks the virulence factors that are well characterized in E. histolytica. These factors include lectin-binding molecules, channel-forming amoebapores, and cysteine proteases responsible for tissue destruction in cases of invasive human amoebiasis (Espinosa-Cantellano and Martinez-Palomo 2000). E. muris can be diagnosed by examining fresh wet mounts from the cecum and colon by light microscopy. If slides are kept at 37°C, amoeboid movement may be observed. When cooled, organisms tend to round up and are more difficult to identify. Visualization of octonucleate cysts is enhanced by the addition of iodine to wet mounts. In histologic sections, trophozoites are round to oval in shape and vary from 8 to 30 µm in diameter (Fig. 21-3). They live in colonies of varying number within the mucus layer of the cecum, and rarely, anterior colon. Trophozoites stain eosinophilic with a granular and often highly vacuolated cytoplasm. Close examination of the vacuoles may reveal ingested bacteria or other protists. Cyst forms are smaller and possess a thin cell wall. Refractile chromatoidal bodies may be present in the cytoplasm. Due to plane of sectioning, mature cysts often appear to contain less than eight nuclei. 5.
Treatment, Prevention, Control
As with the intestinal flagellates, rederived and barriermaintained mice are free of E. muris. Chemotherapeutic elimination of E. muris from infected mice has not been reported,
Fig. 21-3 Entamoeba muris cysts from the cecum of a mouse. Note variably sized intracytoplasmic vacuoles. H&E; bar = 50 µm.
K AT H E R I N E WA S S O N
although luminal amoebicides (iodoquinol, diloxanide furoate, paromomycin) or imidiazoles (metronidazole) used as therapy for human amoebiasis may be effective (Upcroft and Upcroft 2001). The environmental stability of E. muris cysts has not been reported. 6.
Research Implications
Interference with experimental design or reproducibility has not been reported from mice harboring E. muris. There has been tremendous interest in creating a murine model of human amoebic dysentery and invasive amoebiasis. Unfortunately, E. histolytica is not very pathogenic for mice (Ghadirian et al. 1987; Gold and Kagan 1978; Stern et al. 1984). Early reports of mouse mortality due to intracecal inoculation with E. histolytica were complicated by the fact that the organisms were co-cultured with a cocktail of potentially pathogenic bacteria (including E. coli and clostridial species) in order to generate organisms for mouse experiments (Owen 1985, 1990). Since then, methods for cultivating E. histolytica in monoand axenic media have been developed, and more mousevirulent strains isolated (Clark and Diamond 2002). Still, patent intestinal E. histolytica infection in mice depends on intracecal inoculation of xenically passaged organisms (Thompson et al. 2000). Infection is also dependent on mouse strain: 60% of cecally inoculated C3H mice developed typhlitis and remained persistently infected 18 months post-inoculation, compared with no infection or histologic lesions in BALB/c, C57BL/6, or INF-γ-, IL12-, or iNOS-knockout mice (Cieslak et al. 1992). An interesting variation on the mouse model of amoebiasis is the SCID (severe combined inmmunodeficient) mousehuman intestinal xenograft model (Seydel et al. 1997). In this model, human fetal intestinal sections are transplanted under the skin of C.B-17-Prkdcscid (SCID) mice and allowed to develop for several weeks prior to direct inoculation into the intestinal lumen with E. histolytica trophozoites. With this model, researchers have characterized the proinflammatory cytokines released by human intestinal epithelial cells in response to E. histolytica infection and have examined the virulence of genetically manipulated E. histolytica trophozoites (Zhang et al. 2003, 2004). Models of invasive amoebiasis, the most common form in humans being hepatic abscess, can be recreated by intrahepatic inoculation of trophozoites into SCID mice (Cieslak et al. 1992).
IV.
APICOMPLEXANS
A.
General Introduction
The phylum Apicomplexa is a large and diverse group of spore-forming, sexually reproducing protozoans that share
21.
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PROTOZOA
an anteriorly located, apical complex important for host-cell invasion. This phylum includes the coccidia (Cryptosporidium, Eimeria, Isospora, Klossiella, Sarcocystis, Toxoplasma gondii), piroplasms (Babesia), and hemoapicomplexans (Plasmodia). Apicomplexans of importance in the laboratory mouse include Eimeria spp., Sarcocystis muris, Klossiella muris, Cryptosporidium spp., and Toxoplasma gondii. Although apicomplexans infect a variety of cell types and cause distinctly different diseases in their respective hosts, they share a common basic life cycle (Levine and Ivens 1990). This life cycle starts with the oocyst (or spore form), which must undergo sporulation (meiosis) before becoming infective for the host. Once sporulated, the oocyst is composed of sporoblasts, each of which contains a specific number of invasive sporozoites, depending on the species. After the sporulated oocyst is ingested by a susceptible host, the sporozoites are released and invade the appropriate host cells. These forms replicate by multiple fission resulting in daughter merozoites (also referred to as schizozoites or trophozoites). Merozoites are released by rupture of the host cell and invade neighboring cells. This cycle of asexual replication, rupture, and re-invasion (referred to as merogony or schizogony) is repeated for one or more generations, depending on the parasite species. After asexual replication, the parasites cease replicating and differentiate into microgametes or macrogametes within the host cells. Macrogametes are fertilized by motile microgametes and develop into oocysts, which mature and rupture from the host cell to start the parasite life cycle again. Variations in this basic apicomplexan life cycle include length of time and location where sporulation occurs, number and arrangement of sporoblasts and sporozoites within the sporulated oocyst, tissue and cell preference of the sporozoites, number of generations of merogony, presence or absence of intermediate hosts, parasite host specificity, and production of cyst or pseudocyst forms within hosts. These variations will be dealt with when discussing the specific protozoans. Another common feature shared by these parasites is the apical complex, the defining criterion for inclusion in the phylum Apicomplexa. The apical complex refers to a group of organelles that are present in one or more stages of the protozoan’s life cycle. These organelles—visible by electron microscopy—include the polar ring, conoid, micronemes, and rhoptries (Morrissette and Sibley 2002). The polar ring is a cogwheel-like, microtubule-organizing center from which microtubules radiate out of and down the length of the sporozoite. Engagement of these structures is thought to play a role in parasite gliding motility. The conoid is a retractable protuberance at the apical end of the parasite. It is composed of tightly coiled microtubules and is thought to be responsible for mechanical penetration of epithelial cell membranes by the invading sporozoite. Micronemes and rhoptries are secretory organelles containing proteins required for parasite motility, and adhesion to and invasion into host cells. Unlike the flagellates and amoebae, most apicomplexans possess
mitochondria and a Golgi complex. Most apicomplexans also possess an apicoplast, a chloroplast-like organelle homologous to the plastid of algae (Kohler et al. 1997; Roos et al. 1999). This organelle contains its own genomic material and is thought to have an endosymbiotic origin in apicomplexans similar to that of mitochondria in eukaryotic cells. Although its function is not completely understood, its presence is essential for parasite survival (Roos et al. 1999). In addition, because the genes on this episomal DNA appear prokaryotic in nature, it is an attractive target for drug development against the apicomplexans (McFadden 2003). B. 1.
Eimeria spp.
Introduction
There is a surprisingly large body of literature regarding Eimeria infection in mice, especially when compared with other protozoal agents of rodents. Much of the early literature deals with species identification and life-cycle elucidation. Recent work focuses on Eimeria infection in mice as a model for investigating the immunopathogenesis of coccidiosis, a disease that accounts for large economic losses in the livestock industries. In general, members of the genus Eimeria are homoxenous (completing their life cycle in one host) and are host-specific (Fernando 1990; Levine and Ivens 1988). Most, but not all, are intestinal pathogens (Fernando 1990). Eighteen species of Eimeria have been described in Mus musculus, of which four of these (E. falciformis, E. vermiformis, E. papillata, and E. ferrisi) are considered pathogenic (Levine and Ivens 1990). Infection with Eimeria is rare in well-managed, laboratory mouse colonies. A recent study indicated a 26.3% infection rate with Eimeria spp. in a variety of wild rodents including Mus musculus (Franjola et al. 1995). 2.
Life Cycle
The life cycle of Eimeria spp. in mice is simple and direct, and follows the basic apicomplexan life-cycle scheme described above. Oocysts are shed in the feces and require 3 to 6 days in the environment to undergo sporulation (Levine and Ivens 1990). Sporulated oocysts contain four sporoblasts, each of which contains two infective sporozoites. After ingestion, sporozoites are released and invade the intestinal epithelium. As with other members in the genus Eimeria, those infecting mice have preferred host-cell and intestinal tract niches: E. falciformis infects the crypt epithelium of the cecum and colon (Fig. 21-4A); E. vermiformis, the crypt epithelium of the distal small intestine (Fig. 21-4B); E. papillata, the villus epithelium of the distal small intestine; and E. ferrisi, the villus epithelium of the cecum and colon (Table 21-1; Schito et al. 1996). E. falciformis undergoes four generations of asexual replication in the host, while E. vermiformis and E. ferrisi undergo three (Levine and Ivens 1990). The number
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A
B
C
D
E
F
Fig. 21-4 Eimeria spp. from the intestinal tract of mice. (A) Eimeria falciformis oocysts in the crypts of the proximal colon. (B) E. vermiformis oocysts in the crypts of the distal small intestine. (C) and (D) Mature meronts of E. falciformis and E. vermiformis, respectively. (E) E. falciformis microgametes (bottom of figure), and macrogametes (top of figure). A single macrogamete being fertilized by microgametes is present in the middle of the figure. (F) A cluster of E. vermiformis macrogametes. H&E; A and B, bar = 100 µm; C through F, bar = 50 µm.
21.
of rounds of asexual replication in E. papillata has not been reported. Sexual replication begins with differentiation of the parasites into microgametes and macrogametes. After fertilization, mature oocysts are released from host cells and shed in the feces to begin the cycle again. The infective dose for naïve mice is 103 oocysts, and the prepatent period ranges from 4 to 8 days (Schito et al. 1996). Resistance to reinfection with Eimeria is dependent on the host mouse strain, immunocompetency, and initial parasite dose and species (Mahrt and Shi 1988; Mesfin and Bellamy 1979; Schito et al. 1996). 3.
Cell Biology
Eimeria species possess an apical complex, Golgi complex, mitochondria, and apicoplast (see Section IV.A; Adams and Todd 1983; Cai et al. 2003; Chobotar et al. 1975). Although the functions of the apicoplast genes are unknown, portions of its genome have been used to investigate phylogenetic relationships within the rodent lineages of Eimeria (Zhao and Duszynski 2001). 4.
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Disease and Diagnosis
Four Eimeria species are considered pathogenic for mice: E. falciformis, E. vermiformis, E. ferrisi, and E. papillata. All result in similar clinical disease and histologic lesions. Concurrent infection with multiple species has been observed in mice (Allen and Fetterer 2002). Virulence factors have not been identified in Eimeria—disease is due to the damage sustained by the intestinal epithelium during multiple rounds of parasite replication and rupture from the host cells. Clinical signs are dose-related and include soft, mucus-covered feces with perianal staining, depression, dehydration, and anorexia (Allen and Fetterer 2002; Blagburn and Todd 1984; Mesfin et al. 1978). In experimentally infected mice, mortality rates increase with doses of 103 and higher for E. falciformis and E. vermiformis oocysts (Blagburn and Todd 1984; Mesfin et al. 1978). Histologic lesions vary by intestinal location and include multifocal mucosal erosions of the intestinal epithelium with a pyogranulomatous cellular infiltrate and hemorrhage, submucosal congestion, and edema (Blagburn and Todd 1984; Levine and Ivens 1990; Mesfin et al. 1978). Intralesional parasites, at various stages of replication within intestinal epithelial cells, may be observed during acute infection. As infection resolves, a reduction in the intestinal villus to crypt ratio, villus shortening or crypt hyperplasia, and a nonspecific mononuclear cell infiltrate may be all that is observed (Blagburn and Todd 1984). Focal granulomas with oocysts in the lamina propria of the colon have been described in resolving E. falciformis infections (Mesfin et al. 1978). Hyperplasia of the mesenteric lymph nodes and splenic follicles may also be seen histologically (Smith and Hayday 2000a). Eimeria spp. are readily observed in hematoxylin-eosin stained histologic sections. Meronts (asexually replicating forms) are
found within intracellular, parasitophorous vacuoles. Meronts vary in number and size depending on their stage of development. Immature forms are round to indistinct in shape, while mature forms are crescent or banana-shaped (Fig. 21-4C, D). Most are uninucleate and lightly basophilic. Microgametes stain intensely basophilic, are comma-shaped, and possess two to three flagella (Fig. 21-4E). Macrogametes have a prominent nucleus and nucleolus. Refractile eosinophilic material or periodic acid-Schiff (PAS) positive staining material may be observed within the cytoplasm of the macrogamete (Fig. 21-4E, F). Oocysts can be distinguished from macrogametes by the presence of one or two refractile cell walls. Speciation of Eimeria parasites can be tentatively assigned based on the tissue and cell location of parasite infection (see Section IVB2). Eimeria spp. infection can also be diagnosed by fecal flotation, although speciation is difficult. In fresh fecal pellets, oocysts are round to ellipsoidal and vary considerably in size both within and between species. Most range in size between 18–26 × 11–24 µm (see Table 21-4; Levine and Ivens 1990). Sporulated oocysts are smaller (ranging in size between 8–14 × 5–10 µm), and contain four sporoblasts with two sporozoites each. Oocyst walls of E. falciformis and E. ferrisi are smooth and clear to light brown in color; those of E. vermiformis and E. papillata are yellowish brown and pitted or papillated (Levine and Ivens 1990). 5.
Treatment, Prevention, Control
A variety of anticoccidial drugs and coccidiostats are used in the livestock industry to control Eimeria infection (Allen and Fetterer 2002). The efficacy of some of these compounds has been examined in naturally infected mice, with varying results (Haberkorn et al. 1983). Toltrazuril (Baycox®) was effective in eradicating a mixed Eimeria infection in C57BL and CFW1 mice, while amprolium, several ionophores, and sulfa-based drugs were only partially effective (Haberkorn et al. 1983). Efficacy was enhanced when some of these less effective drugs where given simultaneously (Harder and Haberkorn 1989). Autoclaving is sufficient to inactivate Eimeria oocysts. E. falciformis oocysts were found to be nonviable after 7 days at 40°C (summarized in Schneider et al. 1972). However, due to the large reproductive potential of Eimeria spp., oocyst numbers build up quickly in the environment and can be difficult to eliminate from some types of laboratory animal housing (Haberkorn et al. 1983; Wilkinson et al. 2001). Rederived and barrier-maintained mice housed in clean environments are free of coccidia. 6.
Research Implications
Outbred mice previously infected with E. falciformis were noted to be resistant to experimental infection with Salmonella abortus-ovis (Lantier et al. 1981). Survival time was found to be greater in mice previously infected with E. falciformis and
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experimentally inoculated with Toxoplasma gondii (Chinchilla et al. 1986). The authors of these reports suggest that Eimeria infection induces a nonspecific inflammatory response that provides protection against other pathogens, but state that the mechanisms involved are unknown. Due to immunologic stimulation and potential for severe intestinal pathology, mice infected with Eimeria spp. are unsuitable for most research involving the immune system and gastrointestinal tract. Experimental Eimeria spp. infection in mice, however, has served as a marvelous tool for investigating gut immune responses to a natural murine pathogen. The stimulus for this research is the hope of developing vaccines against these pathogens in poultry and cattle (Rose et al. 1997). Primary infection with Eimeria spp. in immunocompetent mice results in cellular and humoral immune responses and clearance of infection in approximately 3 weeks (Nash and Speer 1988; Smith and Hayday 2000a). Mice are resistant to reinfection with the same species. Experiments with genetically modified mice have allowed these responses to Eimeria spp. to be immunologically “dissected.” The inability of athymic nude mice to develop immunity to Eimeria falciformis suggested that T cell responses were important in clearance of infection (Mesfin and Bellamy 1979). Using several knockout mouse models, it was later shown that major histocompatibility (MHC) II-restricted CD4+ α/β T cells and the production of interferon gamma were important for effective primary immune responses against Eimeria spp. (Roberts et al. 1996; Rose et al. 1989, 1992). Gamma delta (γ/δ) T cells, noncirculating lymphocytes found in the intestinal epithelium of mice, were found to have an immunomodulatory effect on Eimeria spp. infection. Mice deficient in γ/δ T cell receptors have more severe intestinal pathology than mice deficient in γ/δ and α/β T cell receptors when infected with E. vermiformis (Roberts et al. 1996). The ability to mount an effective secondary immune response was not altered by the lack of γ/δ T cells, however. Work done with severe combined immunodeficient-beige, perforin-knockout mice, and mice treated with monoclonal antibodies to deplete CD4+ or CD8+ T cells demonstrated that cytotoxins produced by natural killer cells and cytotoxic (CD8+) T cells play a role in immunity to secondary infection with E. papillata or E. vermiformis (Rose et al. 1992; Schito and Barta 1997). Lastly, infection of TAP1, interleukin-4, Fas ligand, and inducible nitric oxide knockout mice with E. vermiformis has demonstrated that MHC class I-restricted T cells, B cell activation, and respiratory burst activity of macrophages are not important for clearance of primary Eimeria spp. infections (Smith and Hayday 2000b). C. 1.
Sarcocystis muris
Introduction
Sarcocystis muris is a coccidial parasite of murine skeletal muscle (Miescher 1843). In the early part of the twentieth
century, the prevalence of Sarcocystis infection in laboratory mice was reported to be between 14 and 60% (Smith 1901; Twort and Twort 1932). Although modern husbandry and management practices such as separation of species, vermin control, rederivation, and barrier housing have decreased the incidence of S. muris, this parasite is still occasionally observed in laboratory-bred mice (Tillmann et al. 1999). 2.
Life Cycle
The source of Sarcocystis muris infection in mice remained obscure until 1976, when cats were identified as the definitive host (Ruiz and Frenkel 1976). Limited cross-transmission studies demonstrate that Mus musculus is the only intermediate host for S. muris (Ruiz and Frenkel, 1976). The life cycle follows the basic scheme as described above with a few exceptions: Oocysts sporulate within the carnivore’s intestinal tract and are immediately infective when released into the environment; sporulated oocysts contain two sporocysts, each of which contains four sporozoites; after ingestion of sporocysts by mice, asexual replication first occurs in the liver, then in the skeletal muscle; in the muscle, replication of parasites results in the formation of large cystic structures (sarcocysts) filled with thousands of organisms (bradyzoites). When infected skeletal muscle is consumed by the carnivore, bradyzoites are released, invade the host intestinal epithelium, and undergo sexual replication. This heteroxenous life cycle begins again with the release of sporocysts into the environment. Recent data suggest that S. muris can also be sustained within a mouse colony due to cannibalism of infected individuals (Koudela and Modry 2000). 3.
Cell Biology
Sarcocystis muris possess the standard complement of organelles found in apicomplexans, including an apical complex, Golgi complex, and mitochondria. An apicoplast organelle has been identified in S. muris by electron microscopy (Hackstein et al. 1995). Partial sequencing of this apicoplast genome revealed an herbicide-binding region, suggesting that S. muris may be susceptible to triazine chemotherapeutics such as toltrazuril (Hackstein et al. 1995). 4.
Disease and Diagnosis
Difficulty moving is the only clinical symptom associated with sarcocystosis in mice (Ruiz and Frenkel 1976). Mice of that report were experimentally inoculated, and approximately 80% of muscle fibers were infected with S. muris by 100 days post-inoculation (Ruiz and Frenkel 1976). A dose of 50 sporocysts is reported to result in widespread and severe muscle infection with S. muris in NMRI and AKR/N mice (Rommel et al. 1981). Natural infections in mice are usually not that severe (Tillmann et al. 1999; Twort and Twort 1932).
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Diagnosis of S. muris is made by histologic examination of skeletal muscle; organisms are occasionally observed in the myocardium (Tillmann et al. 1999; Twort and Twort 1932). Within muscle, spherical or cylindrical cysts—often several millimeters long—can be seen within myocytes. Hundreds of banana-shaped, uninucleate bradyzoites are present within the cysts. Often there is no inflammatory reaction to these structures. Bradyzoites are visible with routine hematoxylineosin and periodic acid-Schiff staining, and measure 4–6 × 14–16 µm (Ruiz and Frenkel 1976). Sporocysts are shed in the feces of infected cats and can be diagnosed by fecal floatation. Sporocysts measure 7.5–9 × 8.7–11.7 µm, contain four sporozoites, and have a smooth and colorless wall (Cawthorn and Speer 1990; Ruiz and Frenkel 1976). 5.
Prevention, Treatment, Control
There is a single report documenting elimination of S. muris from the livers of experimentally infected mice with sulfaquinoxaline and pyrimethamine (Rommel et al. 1981). It is not clear if this drug combination was effective against bradyzoite forms found in muscle. Other anticoccidial drugs were not effective (Rommel et al. 1981). Sporocysts are environmentally resistant and remain infectious for at least 119 days at 21°C in fecal flotation solutions (Smith and Frenkel 1978). Although Sarcocystis spp. have an obligatory heteroxenous life cycle (requiring definitive and intermediate hosts), direct contact with cats or cat feces is not needed to transmit S. muris to mice. Cockroaches exposed to S. muris–infected cat feces transmitted infection to naïve mice for 20 days (Smith and Frenkel 1978). Cannibalism—although less efficient—will sustain S. muris in a mouse population (Koudela and Modry 2000). For these reasons, rederivation and maintenance in a clean barrier is recommended to eliminate S. muris from mouse colonies. 6.
Research Implications
Experimental infection with S. muris was found to suppress humoral and cell-mediated immune responses to vaccination with unrelated proteins, and to induce splenomegaly in mice (Gill et al. 1988a, 1988b). These nonspecific immune aberrations make infected mice unsuitable for research involving the immune system. D. 1.
Klossiella muris
Introduction
Klossiella muris is the least characterized apicomplexan parasite of mice. Infection in the kidneys of mice was first reported in 1889 and further described in 1902 (Smith and Johnson 1902). In a 1932 histologic survey of mice used in carcinogenesis studies, renal infection with K. muris was estimated to be 60% (Twort and Twort 1932). Infection in
wild-caught mice has been reported as high as 93% (Rosenmann and Morrison 1975). Under current laboratory animal housing conditions, infection with K. muris is seldom reported. 2.
Life Cycle
Infection in mice begins with ingestion of sporocysts and uptake into the portal vascular system (Yang and Grice 1964; Smith and Johnson 1902). Sporozoites are released, circulate through the vascular system, and preferentially invade the endothelium of the glomeruli, where they undergo several rounds of asexual replication (Fig. 21-5A). Parasites have also been observed in capillaries and arterioles of the lungs, liver, spleen, and thymus (Twort and Twort 1932). At some point, asexually replicating forms rupture from the glomeruli into Bowman’s space and pass to the renal tubules. Parasites invade the tubular epithelium and differentiate into microand macrogametes. Fertilization results in the formation of sporonts, each of which contains as many as 30 sporoblasts and a residual body (Fig. 21-5B; Levine and Ivens 1990; Smith and Johnson 1902; Yang and Grice 1964). Sporoblasts mature into sporocysts, each of which in turn contains 25 to 35 crescent-shaped sporozoites. The life cycle is completed when infective sporocysts are released from ruptured host cells, pass into the urinary bladder, and are shed into the environment. A homoxenous life cycle has been inferred due to the large number of infected mice diagnosed histologically after remaining caged together for several months (Smith and Johnson 1902). 3.
Cell Biology
Ultrastructural examination of Klossiella life-cycle stages and organelles has not been reported. They presumably possess components of the apical complex found in other apicomplexans. The means by which they identify and invade host cells, generate energy, and acquire nutrients for replication have not been investigated. 4.
Disease and Diagnosis
Clinical disease has not been reported with K. muris infection in mice. Diagnosis is usually made histologically and is dependent on identifying replicating forms within the glomerular endothelium or renal tubular epithelium (Smith and Johnson 1902; Yang and Grice 1964). Asexually replicating forms (merozoites) often cause distention of endothelial cells and may appear as multiple minute (< 0.5 µm) bodies surrounded by clear halos within a parasitophorous vacuole in the cell (Yang and Grice 1964). Sporocysts are subspherical, measuring 16 × 13 µm, and are located in the renal tubule epithelium (Levine and Ivens 1990; Yang and Grice 1964). Multiple banana-shaped sporozoites may be seen budding within each sporocyst (Levine and Ivens 1990; Yang and Grice 1964). Heavily parasitized kidneys show evidence of nonsuppurative
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A
B
C
D
Fig. 21-5 Klossiella muris from the kidneys of a mouse. (A) Merozoites replicating in endothelial cells of a glomerulus. (B) Sporont within renal tubular epithelial cell; note budding sporoblasts and residual body. (C) High magnification of mature sporocysts in the renal tubular epithelium. (D) Low magnification of K. muris infection in the renal medulla of a mouse. H&E; A through D, final magnification unknown (Images courtesy of S. W. Barthold).
interstitial nephritis with tubular degeneration. Often, there is no inflammatory response associated with intracellular stages (Yang and Grice 1964). Organisms are periodic acid-Schiff negative but readily identifiable by routine hematoxylin-eosin staining (Fig. 21-5C, D; Yang and Grice 1964). Clinical diagnosis of Klossiella equi by examination of urine has been described and should be possible for diagnosis in mice (Reppas and Collins 1995). However, the authors of that report note that sporocysts were destroyed by fecal flotation solutions and that organisms were apparent only after examining the pellet of centrifuged urine samples (Reppas and Collins 1995).
6.
5.
1.
Prevention, Treatment, Control
Treatment of endemic K. muris infection with coccidiostats reduced the histologic incidence of infection in mice from 93 to 23% (Rosenmann and Morrison 1975). Unfortunately, that report does not specify the compound, dose, or route used. Infection does not occur in rederived, barrier-maintained mouse colonies.
Research Implications
A single report documents the effects of K. muris infection in mice on research results (Rosenmann and Morrison 1975). Endemically infected mice exhibited decreased oxygen consumption and endurance when compared with uninfected controls. The authors suggest that under certain environmental conditions, K. muris infection impairs the metabolic capabilities of infected mice.
E.
Toxoplasma gondii
Introduction
Toxoplasma gondii was first described in 1908, in Ctenodactylus gundii (or “gundi”), a guinea pig–like rodent found in North Africa (Nicolle and Manceaux 1908). Although T. gondii was subsequently shown to cause disease in a variety of animals around the world, it took 50 years to identify the cat as the definitive host (Hutchison 1965). Hutchison correctly
21.
surmised that cats excreted infective forms in their stools. However, concurrent Isospora spp. infection in his experimental cats obscured identification of T. gondii oocysts on fecal examination. In addition, the presence of Toxocara cati ova in the feces led him to conclude that T. gondii was transmitted in conjunction with this common nematode of cats (Hutchison 1965). This was quickly rectified once parasite-free cats were used for transmission experiments (Sheffield and Melton 1969). Mice of the genus Mus are but one of several hundred mammals (including humans), birds, and reptiles since identified as intermediate hosts in the complex life cycle and biology of this protozoal parasite. Natural infection of most animals (including mice) with T. gondii is subclinical. Because of the pervasiveness of this parasite and its role in human and animal disease on a global basis, there is a tremendous amount of published research on various aspects of toxoplasmosis. Mice have played an integral role in understanding the virulence and pathogenesis of this organism. 2.
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Life Cycle
The life cycle of T. gondii is perhaps the most complicated of all the apicomplexans of mice. Oocysts are shed in the feces of infected cats, and sporulate in the environment after several days. Sporulated oocysts contain two sporocysts with four sporozoites each and can be differentiated on fecal floatation from Isospora spp. by size: T. gondii oocysts measure 12 × 10 µm, while those of Isospora spp. are larger: I. rivolta measure 22 × 17 and I. felis 42 × 35 µm (Foyet 2001). After ingestion, sporozoites are released in the small intestine of intermediate hosts. In experimentally infected mice, sporozoites can be identified in cells of the ileal lamina propria as early as 2 hours post-inoculation (PI; Dubey, Speer et al. 1997). After invading a suitable host cell, sporozoites differentiate into tachyzoites and replicate asexually for an infinite number of generations. These rapidly replicating (“tachy-” = fast) asexual forms are responsible for “acute” toxoplasmosis. From the small intestine, parasites invade additional tissues after being transported by blood or lymph. Unlike other apicomplexans, T. gondii can infect virtually any nucleated cell in the host. In experimentally infected mice, tachyzoites can be identified in the mesenteric lymph nodes 8 hours PI (Dubey et al. 1997). By day 3 PI, parasites are present in the lungs, spleen, and kidneys; by day 4, in the heart and liver; by day 6 in the pancreas and brain; and by day 7, in the skeletal muscle (Dubey et al. 1997). If the host survives acute infection, most parasites in these peripheral locations are cleared by the immune response. This immune pressure is thought to stimulate tachyzoites to differentiate into bradyzoites within a protective tissue cyst wall. These cyst forms are typically found in the central nervous system, or skeletal or cardiac muscle (Dubey et al. 1997). In experimentally infected mice, tissue cysts are observed by day 15 PI (Dubey et al. 1997). These slowly replicating (“brady-” = slow) asexual forms are responsible for “chronic” toxoplasmosis in
intermediate hosts. Ingestion of tissue cysts by felids results in release of bradyzoites, invasion into intestinal epithelial cells, and differentiation into the sexually replicating forms of the parasite. As with the other coccidians, fertilization of macrogametes by microgametes results in the formation of oocysts and completion of the life cycle. Although the life cycle of T. gondii is considered heteroxenous, mice can sustain T. gondii infection through cannibalism or congenital transmission, in addition to ingestion of sporulated oocysts. Stage conversion between tachyzoites, bradyzoites, and oocysts can be demonstrated in vitro (Dubey et al. 1998). However, in vivo generation and collection of oocyst or tissue cysts from cats or mice, respectively, are usually required to generate sufficient numbers of organisms for experimental work. 3.
Cell Biology
T. gondii possesses an apical complex, mitochondrion, Golgi complex, and apicoplast similar to the other apicomplexans (see Section IV. A). The large complement of secretory organelles (micronemes and rhoptries of the apical complex, dense granules) and their functions are best—though not completely—understood in T. gondii. The evolution of this extensive network of secretory organelles is thought to be an adaptation to an obligate intracellular lifestyle. Micronemes release proteins involved in the early stages of tachyzoite attachment and invasion into host cells (Carruthers et al. 1999). Depletion of microneme proteins results in transient loss of tachyzoite infectivity. Rhoptries are distinctive, club-shaped organelles numbering 8 to 16 per tachyzoite. Rhoptry proteins are secreted later in the host-cell invasion process; some are also incorporated into the parasitophorous vacuole that encloses tachyzoites once within the host cell (Carruthers and Sibley 1997). Dense-granule proteins are secreted late in the invasion process. These proteins insert in the parasitophorous membrane and are thought to be involved with nutrient acquisition and importation from the host cytoplasm to the replicating tachyzoites (Carruthers 1999). Formation of these organelles, and the biogenesis, trafficking, processing, storage, and secretion of their respective protein cargos suggest that an intricate and complex network of secretory pathways are present in apicomplexans. These pathways must be executed in a precise and sequential manner for parasite survival. A second set of unique organelles found in T. gondii are the acidocalcisomes (Moreno and Zhong 1996). First identified in Trypanosoma cruzi, these structures have since been identified in other trypanosomatids, apicomplexans, algae, slime molds, and the bacterium Agrobacterium tumifaciens (Docampo et al. 1995; Docampo and Moreno 2001; Seufferheld et al. 2003). As the name implies, these organelles contain acidified stores of calcium and other elemental minerals. Although the exact function of acidocalcisomes is not known—and may vary between species—several functions have been proposed. These include as an energy source, an intracellular calcium store for
536 parasite signaling pathways, a mechanism for intracellular pH homeostasis, and for parasite osmoregulation (Docampo and Moreno 1999). In addition, since acidocalcisomes are not present in mammalian cells, they represent an attractive target for antiprotozoal therapies.
4.
Disease and Diagnosis
T. gondii infection in mice is subclinical with minimal gross necropsy lesions (Perrin 1942). Virulence of experimental infection is dependent on mouse strain, T. gondii–type strain, stage of parasite (sporulated oocysts being more pathogenic than bradyzoites, which in turn are more pathogenic than tachyzoites), and parasite dose (Araujo et al. 1976; Suzuki et al. 1995). Swiss Webster mice inoculated with the mildly pathogenic type III strain of sporulated oocysts remained clinically normal until 3 weeks PI, at which time they appeared unthrifty, lost weight, and developed paralysis (Dubey et al. 1997). Mice euthanized at early time points had enlarged and edematous mesenteric lymph nodes. The ileum was congested and edematous, with small white pinpoint foci visible through the serosa. Necrosis was the prominent histologic lesion, with the organs involved dependent on the time point of infection at which mice were euthanized. Necrosis and infarction of the ileal lamina propria and mesenteric lymph nodes were seen in the first days of infection. Focal hepatitis and myocarditis with mixed leukocytic infiltration were observed during the first week of infection. Interstitial pneumonia with intralesional parasites developed during the second week of infection. Intra- and extracellular tachyzoites were seen in brain parenchyma as early as 9 days PI. By 87 days after inoculation, bradyzoite-filled tissue cysts with little associated inflammation were present in most mouse brains (Dubey et al. 1997). Diagnosis of toxoplasmosis in mice is primarily by histology. Individual tachy- and bradyzoites are difficult to identify in histologic sections, but tissue cysts may be observed in the CNS, myocardium, or skeletal muscle (Fig. 21-6A). Cysts are spherical and of variable size, with a thin wall that is argyrophilic and faintly periodic acid-Schiff (PAS) positive (Frenkel 1956). Cysts are packed with PAS positive, fusiform bradyzoites. Unstained impression smears of brain material can also be screened for the presence of T. gondii tissue cysts (Dubey et al. 1998). In intermediate hosts with patent parasitemia, individual tachyzoites may be identified in the lamina propria of the small intestine, endothelium of the small intestine or lungs, or within leukocytes on peripheral blood smears. Pre-cyst intracellular clusters of tachyzoites may also be observed in heart and skeletal muscles (Fig. 21-6B). Tachyzoites have a centrally located nucleus and measure 2–3 × 6–7 µm (Moller 1968). Tachyzoites exhibit a gliding motility that is usually only appreciated in tissue culture systems. T. gondii infection in mice can also be diagnosed by PCR and serology (James et al. 1994).
K AT H E R I N E WA S S O N
5.
Treatment, Prevention, Control
Because of a short shedding period and subclinical signs, cats with T. gondii infection usually do not receive treatment (Dubey 1994). Acute toxoplasmosis in intermediate hosts is amenable to chemotherapeutic intervention, usually a combination of pyrimethamine and sulfa drugs (Wilson et al. 2003). Tissue cyst forms are resistant to treatment, however. Experimentally infected mice treated with sulfonamides for 15 days had a lower mortality rate than those treated with chlortetracycline or left untreated (Frenkel 1956). In general, treatment of T. gondii infection in intermediate hosts is not warranted. Prevention of infection in rodent colonies relies on separation of species and elimination of potential transport vectors from the environment (Chinchilla et al. 1994). Because T. gondii can also be sustained through cannibalism and congenital infection, rederivation and barrier maintenance are recommended for naturally infected mouse colonies (Beverley 1959). As with other apicomplexans, the oocysts of T. gondii are remarkably hardy. Oocysts retain infectivity when stored at 4°C for 54 months or 0°C for 13 months; oocysts lose infectivity at temperatures above 40°C (Dubey 1998). Oocysts also survive exposure to 5.25% sodium hypochlorite (bleach) solution (Dubey et al. 1997). This method is used to “sterilize” oocysts harvested from cat feces prior to tissue culture or mouse inoculation. Tachyzoites and bradyzoites are less environmentally stable, although tissue cysts (filled with bradyzoites) retain infectivity when stored at 4°C for several months (Dubey 1997). Autoclaving or heat treatment at 70°C for 10 minutes will inactivate T. gondii oocysts from bedding, caging, and other equipment (Dubey 1994). 6.
Research Implications
Interference with research due to endemic toxoplasmosis has not been reported for mice. Several studies examining the behavioral effects of experimental T. gondii infection in mice have been published, however, and suggest that subclinical infection would interfere with behavioral phenotyping of mice. Experimentally and congenitally infected mice exhibited decreased motor coordination, spent less time grooming and exploring novel areas, and spent more time running on a home-cage wheel than uninfected control mice (Hay et al. 1983, 1984, 1985). The authors attributed these behavioral abnormalities to subclinical T. gondii–induced encephalitis in the otherwise clinically healthy mice, and suggested that this behavior increased their chance of predation (and therefore perpetuation of toxoplasmosis) by cats (Hay et al. 1985). More recent experimental mouse work has focused on three areas of importance in human toxoplasmosis: ocular, encephalitic, and congenital toxoplasmosis. Ocular toxoplasmosis in humans is thought to result from reactivation of congenital T. gondii infection, and occurs in both immunocompetent and
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A
B
C
D
Fig. 21-6 Toxoplasma gondii tissue cyst in the cerebellum (A) and tachyzoites in the heart muscle (B) of a wallaby. Note the difference in shape and size of bradyzoites within the mature cyst compared to the intracellular tachyzoites. (C) Cryptosporidium parvum in the small intestine of a mouse. (D) Higher magnification of C. parvum. Note the variation in size of the merozoites and their intracellular but extracytoplasmic location in the enterocytes. H&E; A, B and D, bar = 50 µm; C, bar = 100 µm (Images A, B courtesy of S. Tunev).
538 immunosuppressed individuals. Ocular toxoplasmosis causes a recurrent and progressive, necrotizing retinochoroiditis that eventually results in blindness of the affected eye (Gutierrez 2000). The disease can be experimentally reproduced in mice by intraperitoneal injection of tissue cysts or by intraocular injection of tachyzoites (Hu et al. 1999; Lyons et al. 2001). Using the mouse model, researchers have shown that T. gondii infection results in an upregulation of both inflammatory and anti-inflammatory mediators of the immune system and in apoptotic pathways in ocular tissues (Hu et al. 1999; Lyons et al. 2001). These responses may serve as a mechanism to minimize inflammation in the otherwise immune-privileged eye by eliminating parasite-infected cells while preserving uninfected tissues. As with ocular disease, toxoplasmic encephalitis results from reactivation of a congenital or subclinical infection. Toxoplasmic encephalitis tends to occur in profoundly immunosuppressed individuals and generally carries a poor prognosis (Suzuki 2002). A murine model of reactivated toxoplasmosis can be created by inoculating virulent strains of T. gondii into sulfadiazine-treated mice. Antibiotic treatment suppresses—but does not eradicate—parasite infection. This prevents development of acute toxoplasmosis while allowing the infection to establish itself in brain and muscle tissues. “Reactivation” of infection (renewed proliferation of tachyzoites) and development of fatal encephalitis occur when antibiotics are discontinued. Using resistant, susceptible, and genetically modified mouse strains, researchers have demonstrated that the source of gamma interferon (INF-γ), an immune mediator critical for preventing toxoplasmic encephalitis, is produced by T cells and an as-of-yet unidentified population of non–T cells within the brain (Kang and Suzuki 2001). This non–T cell source of INF-γ appears critical for host resistance against T. gondii encephalitis. Mice have also been key to determining the genetics of resistance to toxoplasmic encephalitis. Mice with the d haplotype (e.g., BALB/c mice) in the D region of the major histocompatibility complex (MHC) are resistant, while those with the b or k haplotype (C57BL/6 mice) are susceptible (Suzuki et al. 1994). This correlates with what is seen in human infection. Humans with the HLA-DQ3 haplotype of the MHC are more susceptible to toxoplasmic encephalitis than those with the HLA-DQ1 haplotype. Mice engineered to express human HLA-DQ1 had a greater degree of protection against toxoplasmic encephalitis compared with those expressing human HLA-DQ3 (Mack et al. 1999). Lastly, mice have been used as a model to investigate aspects of congenital transmission of toxoplasmosis. BALB/c mice infected with tissue cysts on day 12 of gestation—but not 8 weeks prior to breeding, or at 8 weeks prior to and on day 12 of gestation—gave birth to T. gondii–infected pups (Roberts and Alexander 1992). Pups had histologic evidence of toxoplasmic encephalitis, and some developed ocular disease at 2 months of age. Using this model, researchers have demonstrated the importance of natural killer cells—an important
K AT H E R I N E WA S S O N
component of the innate immune response—in protecting the fetus from congenital transmission of T. gondii (Abou-Bacar et al. 2004). This model has also been useful for evaluating the efficacy of candidate vaccines against toxoplasmosis (Roberts et al. 1994). This naïve pregnant mouse model replicates several of the epidemiologic and clinical features of human congenital toxoplasmosis. These include infection rates that are highest in fetuses born to mothers exposed to T. gondii for the first time during pregnancy; protection of fetuses against infection if mothers have been previously exposed to T. gondii; and a high incidence of ophthalmic and neurologic disorders in congenitally infected infants (Gutierrez 2000).
F. 1.
Cryptosporidium muris
Introduction
Cryptosporidium muris is the type species for a genus of parasites that were considered biomedical curiosities until the advent of HIV and AIDS (Current and Garcia 1991). Shortly after he identified C. muris in the gastric glands of mice, Ernest Tyzzer went on to identify and describe Cryptosporidium parvum, a second species that resided in the small intestine of mice (Tyzzer 1910, 1912). It wasn’t until 70 years after Tyzzer’s first descriptions that Cryptosporidium spp. were recognized as a cause of self-limiting diarrhea in immunocompetent humans and of severe life-threatening diarrhea in immunocompromised patients (Peterson 1992). There are now 13 validated Cryptosporidium species described, and most of these infect a wide range of hosts (Table 21-2; Xiao et al. 2004). Humans are the primary host for C. hominis but can be infected with various isolates of C. parvum and C. muris (Palmer et al. 2003; Xiao et al. 2004). Cryptosporidial infection of contemporary mouse colonies is seldom reported; natural infection with C. muris and/or C. parvum is occasionally observed in wild rodent populations (Bajer et al. 2003; Klesius et al. 1986; Torres et al. 2000).
2.
Life Cycle
Tyzzer’s 1910 light microscopic description of the life cycle and morphology of C. muris is remarkably accurate (Tyzzer 1910). Despite the parasite’s small size, he determined that the parasite had asexual and sexual stages, formed a unique organ of attachment to the host cell, produced two types of oocysts, and was likely related to coccidia (Tyzzer 1910). Seventy-five years later, electron microscopy confirmed Tyzzer’s findings, with few exceptions (Current and Reese 1986). Like Eimeria, Cryptosporidium spp. are homoxenous and follow the general apicomplexan life cycle described in Section IV. A. Sporulated oocysts are shed in feces. Once consumed, four sporozoites excyst and invade the gastrointestinal epithelium. These develop within a parasitophorous vacuole in the host cell
21.
into type I merozoites. Type I merozoites replicate asexually, rupture from the host cell, and re-invade neighboring epithelium, developing into either type I or type II merozoites. Type I merozoites continue the asexual replication cycle, while type II merozoites develop into micro- or macrogametes. After fertilization by microgametes, macrogametes develop either into thick-walled or thin-walled oocysts. Oocysts undergo sporulation and contain four “naked” sporozoites prior to rupture from the host cell (there are no sporocysts). Thick-walled oocysts are shed as fully infective parasites into the environment; thin-walled oocysts excyst while still in the host, infect new gastrointestinal epithelial cells, and initiate the asexual replication cycle again (Tyzzer 1910). The autoinfective ability of type II merozoites and thin-walled oocysts can result in an overwhelming parasitism despite a relatively small, initial inoculum, particularly in the immunocompromised host (Current and Garcia 1991). C. muris infects and replicates in the gastric mucosa, while C. parvum infects small intestinal epithelium (Current and Reese 1986; Tyzzer 1910). The complete life cycle of Cryptosporidium spp. has yet to be replicated in vitro. Oocysts for in vitro experimentation are usually purified from the feces of experimentally inoculated neonatal ruminants or suckling mice (Arrowood 2002; Current 1990). Oocysts can be induced to excyst upon exposure to sodium hypochlorite (bleach) or trypsin and bile salts, and the released sporozoites will infect a number of different cell culture lines and replicate asexually (Arrowood 2002). However, production of significant numbers of mature oocysts in vitro has not been reported (Arrowood 2002). 3.
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Cell Biology
Cryptosporidium spp. possess an apical complex, complete with preconoid rings, conoid, rhoptries, and micronemes, which warrants their inclusion in the phylum Sporozoa (Current and Reese 1986). However, they lack a Golgi complex, mitochondrion, and the apicoplast present in other coccidia (Abrahamsen et al. 2004; Current and Reese 1986; Zhu et al. 2000). In addition, they inhabit a unique intracellular, extracytoplasmic location in their host cells (Current and Reese 1986; Goebel and Brandler 1982). At the interface between the parasite and host-cell cytoplasm, a unique “feeding organelle” can be seen by electron microscopy (Current and Reese 1986). This organelle is present during asexual and sexual parasite replication, and is composed of a portion of the parasitophorous vacuole membrane and a microfilamentous sheet (Current and Reese 1986). As the name implies, this organelle is thought to be important in nutrient transport from the host cytoplasm to the developing parasite (Current and Reese 1986). Recent work demonstrates that this organelle is composed, in part, of a membrane transporter protein that belongs in the ATP-binding cassette protein superfamily. This superfamily of proteins regulates the flow of diverse compounds across cell membranes (Perkins, Riojas et al. 1999). Recent publication of the
C. parvum genome reveals that these parasites also possess several “plant-like” and bacterial-type enzymes that are either absent or divergent from those found in mammals (Abrahamsen et al. 2004). These proteins are likely to be good candidates in the search for drug targets against human and animal cryptosporidiosis. 4.
Disease and Diagnosis
Suckling mice infected with C. muris or C. parvum are reported to grow more slowly and are less active than their uninfected littermates (Current and Reese 1986; Tyzzer 1910). Neonatal mice are susceptible to experimental infection with C. parvum up until 14 days of age, at which time they clear and remain resistant to infection (Novak and Sterling 1991). Clinical signs of C. muris infection in immunocompetent adult mice are not reported, and it is not clear if an age-related clearance of infection occurs for this species (McDonald et al. 1996; Tyzzer 1910). Experimental infection of immunosuppressed mice results in persistent infection with clinical signs of weight loss and sticky stools (Perryman and Bjorneby 1991; Ungar et al. 1990). Diagnosis is made by histologic examination of gastrointestinal tract tissue. Dilation of gastric glands and minimal lymphoid infiltrate are seen in mice infected with C. muris (Tyzzer 1910). In heavy C. parvum infection, blunting and fusion of intestinal villi, crypt hyperplasia, and moderate lymphocytic-plasmacytic infiltration of the underlying lamina propria are seen (Current and Reese 1986). In athymic nude mice, C. parvum can also be seen infecting the hepatobiliary tree and pancreatic ducts (Ungar et al. 1990). Small, round, basophilic-staining parasites may be observed by routine hemoxylin-eosin staining of histologic sections of the gastrointestinal mucosa. Life-cycle stages are difficult to differentiate by light microscopy due to their small size. Meronts and oocysts of C. muris measure 5 × 7 µm and are located on the luminal surface of gastric epithelium (Tyzzer 1910). Similar stages of C. parvum measure 5 µm or less in diameter and are found “decorating” the brush border of ileal enterocytes (Fig. 21-6C, D; Current and Reese 1986). These differences in size and tissue location can be used to differentiate between the two species in mice (Perryman 1990). Diagnosis can also be made by fecal flotation. Oocysts are spherical or ellipsoidal, with a smooth colorless wall and a faint suture line running down one-half the oocyst length. C. muris oocysts measure 7–8 × 5–6.5 µm; those of C. parvum are smaller, measuring 4.5–5 × 4–5 µm (Levine and Ivens 1990). 5.
Prevention, Treatment, Control
In immunocompetent animals and humans, infection with Cryptosporidium spp. is self-limiting. Although treatment of laboratory mice for natural infection with C. muris or C. parvum has not been reported, suckling and immunosuppressed mice have been a useful in vivo model to test anticryptosporidial
540 compounds (see Blagburn and Soave 1997 for a summary of literature). Fifteen common anticoccidial compounds (including amprolium and sulfaquinoxaline) were found to be ineffective in protecting neonatal mice against infection with C. parvum (Angus et al. 1994). Lack of the apicoplast organelle in Cryptosporidium spp. has been hypothesized as one reason why these parasites are not susceptible to chemotherapeutics effective in other apicomplexans (Abrahamsen et al. 2004; Zhu et al. 2000). In addition to a lack of effective drug treatment, Cryptosporidium oocysts remain infective in chlorinated water (Carpenter et al. 1999; Korich et al. 1990). Oocysts are also remarkably resistant to common disinfectants. In an experiment examining the infectivity of oocysts after 18 hours incubation in various agents, only those treated with 10% formalin or 5% ammonia were noninfective for the suckling mouse model (Campbell et al. 1982). Steam sterilization, pasteurization, and ethylene oxide exposure for 24 hours will inactivate oocysts (Fayer et al. 1996; Harp et al. 1996). However, oocysts retain their infectivity when stored in 2% potassium dichromate solution at 4°C for greater than 6 months (Arrowood 2002). Because of a lack of treatment, resistance to common disinfectants, the environmental stability of oocysts, and demonstrated zoonotic potential of Cryptosporidium spp., endemically infected laboratory mouse colonies should be rederived and barrier maintained. 6.
Research Implications
Interference with research results due to natural infection in mice with C. muris has not been reported. In addition, C. parvum is thought to be primarily a pathogen of ruminants and humans, and is not likely to be identified in barrier-maintained mouse colonies (Klesius et al. 1986; Xiao et al. 2004). On the other hand, suckling mice have been useful for generating small amounts of pure isolates of Cryptosporidium parvum oocysts for in vitro work (Current 1990). Although Cryptosporidium spp. are not pathogenic for mice, work done in immunocompromised mouse models has helped elucidate the immune mechanisms responsible for clearance of infection. Similar to infection with Eimeria spp., clearance of C. muris and C. parvum infection was found to be dependent on production of interferon gamma and presence of CD4+ intraepithelial T cells (Chen et al. 1993; Culshaw et al. 1997; McDonald et al. 1996). The course of infection in mice with C. muris is also dependent on major histocompatibility complex (MHC) types. BALB/c mice (with the H-2d MHC type) were shown to produce fewer oocysts and to recover from infection with C. muris faster than BALB/B (H-2b) or BALB/K (H-2k) mice (Davami et al. 1997). This was attributed to a difference in interleukin-4 secretion by splenocytes between these strains of mice (Davami et al. 1997). Lastly, patent infection with Cryptosporidium spp. appears to depend on the host intestinal microflora. Axenic C.B-17/IcrTac-Prkdcscid mice
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shed oocysts sooner when infected with C. parvum than their cohorts bearing intestinal flora (Harp et al. 1992). The authors speculate that resistance to initial infection is due to competition by endogenous microflora for preferred intestinal niches.
V. A. 1.
MICROSPORIDIA
Encephalitozoon cuniculi
Introduction
Mice can be naturally and experimentally infected with Encephalitozoon cuniculi, the type species for a burgeoning collection of parasitic organisms that are more closely related to fungi than protozoans. Although frequently associated with rabbits and mice, E. cuniculi is a much more promiscuous organism than originally thought and has been identified as a pathogen in hamsters and guinea pigs, squirrel monkeys and cotton-top tamarins, carnivores (including fox and mink), domestic dogs and cats, goats, sheep, pigs, horses, muskrats, and shrews; and is a suspected cause of equine abortion (PattersonKane et al. 2003; Reetz et al. 2004; Wasson and Peper 2000; Wasson and Zbka 2003). It is also a pathogen of immunocompromised and immunocompetent humans (De Groote et al. 1995a; Mertens et al. 1997; van Gool et al. 2004). E. cuniculi infection was first described in rabbits in 1922 and in mice two years later (Cowdry and Nicholson 1924; Wright and Craighead 1922). E. cuniculi was reported in laboratory mice frequently thereafter, often in conjunction with additional parasite infestations including fleas, lice, pinworms, cysticerci, K. muris, S. muris, and T. gondii, and subclinical bacterial and—in all likelihood—viral infections (Innes et al. 1962; Lackey et al. 1953; Morris et al. 1956; Perrin 1942; Perrin 1943; Twort and Twort 1932). In several of these reports, granulomatous hepatitis or meningoencephalitis associated with endemic E. cuniculi confounded attempts to develop murine models of human viral hepatitis or encephalitis, respectively. It was estimated that E. cuniculi was prevalent in 15 to 50% of laboratory mouse colonies at this time (Innes et al. 1962; Twort and Twort 1932). This older literature variably refers to this organism as “haplosporidia,” “mouse ascitic agent,” Nosema muris, E. muris, or E. rabii (Lainson et al. 1964; Morris et al. 1956; Twort and Twort 1932). By 1964, the name Nosema cuniculi was proposed, to reflect its morphologic similarity by light microscopy to the familiar Nosema spp. parasites of insects (Weiser 1964). However, ultrastructural comparison of E. cuniculi with Nosema spp. confirmed the former’s status as a separate genus and reaffirmed its original name as given by Levaditi et al. 1923 (Levaditi et al. 1923; Pakes et al. 1975; Sprague and Vernick 1971). Until 1985, E. cuniculi was considered the only microsporidial parasite of mammals. With the arrival of HIV and AIDS, six genera—representing approximately
21.
12 species—have since been identified as human pathogens (Franzen and Muller 1999). Mice appear to be natural hosts for E. cuniculi only. However, they can be experimentally infected with several of these newly described species. And although rare, E. cuniculi infection is still reported in contemporary mouse colonies (El Nass et al. 1998). 2.
Life Cycle
E. cuniculi is an obligate intracellular parasite with a simple and direct life cycle, and exists outside the host as an environmentally resistant spore (Wasson and Peper 2000). The initial site of E. cuniculi infection in mice is not known but is thought to be the intestinal epithelium. Once ingested, E. cuniculi infects susceptible host cells through the deployment of a preformed polar tube (discussed below). This polar tube is extruded from the spore, penetrates a susceptible host-cell membrane, and deposits the spore’s infectious sporoplasm in the host-cell cytoplasm. The sporoplasm proliferates, resulting in the production of numerous plasmodial cells (also referred to as meronts) within a parasitophorous vacuole. These cells are amorphous with an indistinct nucleus and a simple cytoplasm when examined by transmission electron microscopy. Plasmodial cells develop into sporonts as the cell-limiting membrane (plasmalemma) becomes electron dense and the nucleus more distinct. Sporonts undergo binary fission to produce sporoblasts, which are no longer capable of dividing. Sporoblasts begin depositing organelles, including the polar tube and its associated polaroplast membrane, endoplasmic reticulum, the posterior vacuole, ribosomes, and electron dense bodies. Mature spores can be distinguished from sporoblasts by the presence of a thick spore wall, distinct cross sections through the coiled polar tube, and increased density of the spore cytoplasm. As nascent spores mature and increase in number, the host cell becomes distended and ruptures. Spores are released into the surrounding tissues to infect neighboring cells. E. cuniculi is capable of infecting a variety of host organs, although the mechanisms by which dissemination to these sites occur are not clear. In naturally infected, immunocompetent mice, parasites and associated inflammation are usually observed in the CNS and kidneys. Spores are shed in the environment through urine to complete the life cycle. E. cuniculi is also easily propagated in tissue culture, resulting in release of fully infective spores from a variety of mammalian cell lines and generating sufficient numbers of parasites for in vitro work (Visvesvara 2002). 3.
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Cell Biology
Microsporidia contain a curious mix of eukaryotic, prokaryotelike, and novel organelles. Like all eukaryotic organisms, they possess a membrane-bound nucleus, endoplasmic reticulum, Golgi apparatus, and membrane-bound vesicles. Although they lack mitochondria, a mitochondrial remnant—termed
mitosome—has been identified (Williams et al. 2002). The function of the mitosome is not clear, as microsporidia lack the mitochondrial enzymes associated with aerobic metabolism. All life stages of microsporidia are replete with ribosomes. These ribosomes are smaller than typical eukaryotic ribosomes, with a sedimentation rate and molecular organization similar to those found in prokaryotes. The defining criterion for inclusion into the phylum Microspora is possession of the polar tube and its associated structures. Also referred to as a polar filament, this unique apparatus is the means by which parasites invade and infect susceptible host cells. The polar tube is preformed and coiled around the sporoplasm within mature microsporidial spores. This tube is attached at the anterior end of the spore by the means of an anchoring disk and is surrounded by lamellar and vesicular membranes (also referred to as the polaroplast). At the opposite end of the spore is a membrane-bound posterior vacuole. After activation by some yet-to-be-determined signal (or signals), the polar tube is forcefully ejected from the spore, uncoiling and penetrating a susceptible host cell in the process. Once fully ejected, the spore’s sporoplasm (containing the nucleus and abundant ribosomes) travels through the tube and is deposited within the host-cell cytoplasm. The function of the polaroplast is not clear, but it may contribute to the limiting membrane of the discharged sporoplasm (Weidner et al. 1984). It is also not clear what role the posterior vacuole plays. However, this vacuole is seen to swell by electron microscopy during the course of polar tube extrusion and may provide additional osmotic force to propel the sporoplasm through the polar tube and into the host cytoplasm (Lom and Vavra 1963). Although the mechanisms behind this sequence of events are still being worked out, it appears that calcium signaling and water flux across the spore wall play key roles in the activation of this novel parasite invasion apparatus (Frixione et al. 1997; Weidner and Byrd 1982). 4.
Disease and Diagnosis
E. cuniculi does not appear to be transmitted vertically in mice, nor are neonates more susceptible to infection than adults (Liu et al. 1988). Infection in immunocompetent mice is subclinical, although susceptibility to infection will vary by strain. C57BL/6, DBA, and 129/J mice had a higher parasite burden and depressed antibody responses to intraperitoneal inoculation with E. cuniculi when compared with BALB/c mice (Niederkorn et al. 1981). In immunologically impaired or immunosuppressed mice, however, overt disease may be seen. Naturally infected, immunocompetent mice treated twice weekly with corticosteroids developed abdominal distention and ascites. Microscopic examination of ascites fluid, peripheral blood smears, and brain, liver, spleen, and kidneys tissues revealed clusters of intracellular E. cuniculi organisms after 5 weeks of this immunosuppressive regime (Bismanis 1969). Experimentally infected C57BL/6-Hfh11nu mice develop ascites,
542 or a chronic wasting syndrome characterized by lethargy, anorexia, dehydration, and death within 2 to 4 weeks of parasite inoculation (Didier et al. 1994). Diagnosis of encephalitozoonosis is made by serology, histopathologic examination of tissues, and PCR. Serologic screening is a routine and useful method for monitoring E. cuniculi in immunocompetent mouse colonies. Histologically, parasites may be observed in a variety of tissues, but lesions are most frequently identified in the brain and kidneys. In contrast to the lesions seen in the CNS of endemically E. cuniculi–infected rabbits, those present in mice are nongranulomatous. In infected immunocompetent mice, a diffuse, nonsuppurative meningoencephalitis, and multifocal aggregates of activated microglia, lymphocytes, and macrophages, often with a perivascular distribution, are seen in the CNS. In addition, diffuse lymphocytic interstitial nephritis is observed. In these mice, parasites are scarce and may be difficult to identify. In immunosuppressed mice, E. cuniculi infection tends to be extensive and disseminated. Multiple necrotic foci with abundant intralesional and extralesional parasites have been described in the brain, heart, lungs, liver, spleen, adrenals, kidneys, pancreas, intestine, and the visceral and parietal peritoneal tissues of mice (Didier et al. 1994). Parasites are best observed in tissue sections with special stains (Fig. 21-7A, B). E. cuniculi spores stain positive with a Brown and Brenn’s (B&B) tissue Gram stain, and bright red with PAS stain (Wasson and Peper 2000). Mature spore forms pick up stain the best—proliferative forms of the parasite are
A
K AT H E R I N E WA S S O N
more difficult to identify. Spores are rod-shaped and measure 1.5 × 2.5 µm. With B&B, spores often appear to have a clear vacuole at one end. With PAS, this vacuole appears as a deeply red staining granule. Other internal organelles are not visible by light microscopy. On occasion, it may be necessary to discriminate E. cuniculi from T. gondii infection, particularly in mice with CNS lesions. While T. gondii stains PAS positive, it does not stain with B&B. In addition, intracellular clusters of E. cuniculi spores can be distinguished from bradyzoitefilled T. gondii cysts by the presence of a cyst wall in the latter. Cyst walls stain faintly PAS positive and are argyrophilic (Frenkel 1956). Urinalysis for detection of E. cuniculi spores is theoretically possible but is more often used to monitor parasite shedding in experimentally infected mice. Molecular-based methods are available for diagnosis of encephalitozoonosis and may be useful when dealing with immunosuppressed mice and/or inconclusive histopathologic results (Didier et al. 1995). 5.
Prevention, Treatment, Control
Because of its subclinical nature and insidious effect on animal-based biomedical research, E. cuniculi-infected colonies should be depopulated and repopulated with clean stock. Alternatively, rederivation can be performed. Although E. cuniculi is not as prevalent in contemporary mouse colonies as in the earlier part of the twentieth century, investigators, mouse biologists, and diagnosticians should remain vigilant.
B
Fig. 21-7 Encephalitozoon cuniculi in the kidney of a mouse. Clusters of spores are present within intracytoplasmic parasitophorous vacuoles. (A) H&E. (B) B&B Gram stain. A and B, bar = 50 µm.
21.
E. cuniculi infects a variety of cell types and animal species, and may inadvertently be transmitted through the use of contaminated biologics in mice. Despite the handful of reports suggesting clinical improvement with the use of albendazole in humans with E. cuniculi infection, therapeutic efficacy has not been demonstrated in mice (De Groote et al. 1995b; Fournier et al. 2000; Koudela et al. 1994). Although E. cuniculi infection in humans has been documented, direct zoonotic transmission from animals to humans has not been demonstrated (Glaser et al. 1994). Human infections are likely obtained from water sources or the environment. In laboratory environments, Encephalitozoon spp. have been shown to be inactivated by 2% Lysol, 10% formalin, 70% ethanol, 10% bleach, quaternary ammonium compounds, and by boiling (Santillana-Hayat et al. 2002; Shadduck and Polley 1978). For this reason, steam sterilization of equipment and contaminated animal bedding, as well as routine disinfection of surfaces, should prevent environmental buildup of E. cuniculi spores in animal facilities. 6.
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Research Implications
As alluded to in the introduction, interference with research results due to subclinical E. cuniculi infection is well documented in rodents. The inflammatory lesions commonly observed in the CNS of infected mice were often misinterpreted as lesions due to experimental inoculation with rabies virus, herpes simplex, cercopithecine herpesvirus 1, poliomyelitis virus, hepatitis B virus, syphilis, psittacosis, scrub typhus, and toxoplasmosis (reviewed in Wasson and Peper 2000). In addition to infectious disease work, E. cuniculi also interfered with early cancer research. Rats were found to be resistant to tumor development when inoculated with E. cuniculi–infected rat sarcoma cells (Petri 1965). E. cuniculi–infected mice were shown to have significantly reduced tumor growth and prolonged survival rates compared in uninfected controls when inoculated with different strains of solid or ascites-producing murine tumor cell lines (Arison et al. 1966; Niederkorn 1985). Nonspecific resistance to tumor growth in these mice may in part be due to enhanced natural killer cell activity—and associated increase in gamma interferon (INF-γ) production—secondary to E. cuniculi infection (Niederkorn et al. 1983). Experimentally, E. cuniculi–infected C57BL/6 mice cleared lymphoma tumor cells from the lungs faster when compared to natural killer cell–deficient beige mice and infected, cyclophosphamide-treated C57BL/6 mice (both of which have reduced levels of INF-γ). In addition, infected C57BL/6 mice were significantly more resistant to pulmonary tumor formation with B16F10 melanoma cells when compared with uninfected, age-matched controls (Niederkorn 1985). The immune response to E. cuniculi infection in mice is now being elucidated from these early observations of enhanced tumor resistance. The importance of INF-γ was reaffirmed when INF-γ depleted and INF-γ knockout mice were shown to be highly susceptible to E. cuniculi
infection when compared to unmanipulated or wild-type controls (Khan and Moretto 1999). INF-γ production by natural killer cells and gamma delta T lymphocytes is thought to activate CD8+ cytotoxic T lymphocytes, which in turn results in parasite killing. Evidence for this was shown in CD8 knockout mice, which were more susceptible to E. cuniculi infection compared to CD4 knockout mice and wild-type controls (Khan et al. 1999; Moretto et al. 2000).
ACKNOWLEDGMENTS This work was supported in part by NIH grant AI49735. The author wishes to thank Drs. Stephen Barthold, Nicole Baumgarth, Howard Gelberg, Stefan Tunev, and Mr. Robert Munn, for translation of articles from German and Russian, and for assistance with histopathology antd figures.
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Chapter 22 Helminth Parasites of Laboratory Mice Kathleen R. Pritchett
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Helminths of Major Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Oxyurids: Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Syphacia obvelata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Aspiculuris tetraptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Effects on Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Helminths of Minor Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Syphacia muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Trichuris muris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Heligmosomoides polygyrus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cestodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Rodentolepis (=Hymenolepis) nana . . . . . . . . . . . . . . . . . . . . . . . . . 2. Hymenolepis diminuta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Rodentolepis (=Hymenolepis) microstoma . . . . . . . . . . . . . . . . . . . 4. Taenia taeniaeformis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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I.
INTRODUCTION
Since the last publication of this volume, significant strides have been made in eradicating the infectious diseases of laboratory mice. Mice are, without a doubt, freer from disease than they have ever been. However, animals that reside in today’s rodent facilities still frequently test positive for the presence of helminth parasites. In the prior edition of this chapter (Wescott 1982), the importance of the parasites addressed was based on their prevalence in the laboratory rodent population, a categorization that continues in the current version. The oxyurids, or pinworms, of mice remain important parasites in terms of the numbers of institutions with mice infected with these parasites and their potential impact on biomedical research (Boivin et al. 1996; Huerkamp 1990; Huerkamp et al. 2000; Jacoby and Lindsey 1997, 1998; Klement et al. 1996; Le Blanc et al. 1993; Lipman et al. 1994; Murphy-Hackley and Blum 1990; Pinto et al. 1994; Shibihara 1999; Zenner and Regnault 2000). This is likely due to the persistence of oxyurid eggs in the environment, their means of transmission, and their lack of overt symptomology. Of 68,110 samples submitted from outside sources to a large animal diagnostic laboratory for parastitological examination from February 2002 to November 2004, 206 tested positive for oxyurid parasites (Cosentino 2004). These samples represent biotech firms, universities, hospitals, pharmaceutical companies, and government institutions, with colleges and universities having the highest number of positives. This number probably underestimates the number of institutions harboring oxyurids, for many infections are discovered on routine health monitoring exams conducted in-house, treated, and cleared. Hymenolepis (=Rodentolepis) nana was previously treated as a parasite of major importance in laboratory mice. Although it remains important in terms of zoonotic potential, a survey of approximately 68,000 samples submitted to a major diagnostic laboratory over three years (2001–2004) failed to show a single case (Cosentino 2004). Since H. nana has a more complex life cycle than the oxyurid parasites, this would suggest that modern “mousekeeping” methods have had an effect on the prevalence of this parasite. Many other helminths may parasitize mice, especially wild mice, but they are not found in a laboratory setting, unless these parasites are serving as a model of host–parasite interaction. For a more detailed treatment of less common parasites of laboratory or wild mice, the reader is directed to Flynn’s Parasites of Laboratory Animals (Flynn 1973c). A second edition of this volume is currently in press.
II.
HELMINTHS OF MAJOR IMPORTANCE A.
Oxyurids: Overview
Oxyurids, the family to which the pinworms of mice, Syphacia obvelata and Aspiculuris tetraptera, belong, are cosmopolitan
monoxenous parasites that are transmitted through ingestion of embryonated eggs. Pinworms are routinely found in animals from modern animal facilities, even in facilities free of viral and bacterial diseases of mice (Jacoby and Lindsey 1998; Zenner and Regnault 2000). Oxyurids are also a common parasite of wild mice (Behnke et al. 1999; Derothe et al. 1997; Pisanu et al. 2001; Singleton et al. 1993). Mice may be infected with both species of pinworms concurrently (Agersborg et al. 2001; Bazzano et al. 2002; Eaton 1972; Goncalves et al. 1998; Hoag 1961; Jacobson and Reed 1974; Macarthur and Wood 1978; Nicklas et al. 1984; Pinto et al. 2001; Scott and Gibbs 1986; Taffs 1975, 1976b; Zenner 1998). The common finding of two species of pinworms in an infection can be explained by the species predilections for slightly different portions of the gastrointestinal tract. Since they do not compete directly for resources, they are able to maintain simultaneous infections. In concurrent infections, A. tetraptera may have higher worm numbers because its longer lifespan may allow it to accumulate in the host (Scott and Gibbs 1986). The prevalence of pinworms in an infected rodent population depends on many factors, including gender, age, strain, immune status, and the concentration of parasite ova in the environment. Male animals tend to have higher parasite burdens than female animals (Behnke 1975a; Derothe et al. 1997; Eaton 1972; Mathies 1959a, b). Studies suggest that this is not entirely due to the tendency of male mice to exhibit more exploratory behavior, and thus become infected at a higher rate through greater exposure to eggs. Rather, it may be attributed to some innate resistance in female mice resulting in a greater rate of parasite expulsion (Behnke 1975b). Young animals tend to have higher oxyurid burdens than older animals (Behnke 1976; Eaton 1972; Mathies 1959b; Panter 1969), a fact that is important to consider when designing sentinel programs to detect pinworm infection. In wild populations endemically infected with A. tetraptera, susceptibility to infection peaks in animals between 10 and 17 grams (or approximately 4 to 7 weeks of age) and subsides as animals age and apparently become increasingly immune to reinfection (Behnke 1976). In the laboratory setting, animals were shown to develop resistance to infection with S. obvelata, regardless of previous infection status, between 4 and 9 weeks of age (Panter 1969). Laboratory mice are more resistant to experimentally induced infection than wild mice (Derothe et al. 1997) and hybrids of two populations of wild mice are more susceptible to infection than either of the parent species (Sage et al. 1986). The “Columbia” and CF1 strains of mice differed in their susceptibility to pinworm infection (Chan 1952). AKR/ LwNIcr, DBA/2J, DBA/2An, and C3H/Cum mice were shown to be more susceptible to pinworm infection than other inbred strains of laboratory mice (Eaton 1972; King and Cosgrove 1963). Athymic mice have an increased susceptibility to infection (Clarke and Perdue 2004; Jacobson and Reed 1974), but the susceptibility of other immunocompromised animals has not been examined. Rodent pinworms are not a significant zoonotic hazard, with only S. obvelata reported to infect humans, and then only rarely
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(Flynn 1973a). A more recent source states that rodent pinworms are not transmissible to humans and vice versa (Marx 1991). The two common pinworms of mice, Syphacia obvelata and Aspiculuris tetraptera, are described next. A summary of morphologic and reproductive data may be found in Table 22-1. A comparison of the appearance and size of the ova of S. obvelata, A. tetraptera, and S. muris may be found in Fig. 22-1. Syphacia muris, the pinworm of rats, is occasionally found in mice, but it will not be discussed in detail. 2.
Syphacia obvelata
A. MORPHOLOGY Syphacia obvelata was first described in 1801 (Rudolphi 1801), but it would be several years until it was distinguished from Aspiculuris tetraptera and placed in the genus Syphacia (Seurat 1916). In the genus Syphacia, adult parasites have three fleshy lips, a round esophageal bulb, and small
TABLE 22-1
DIFFERENTIATION OF SYPHACIA OBVELATA AND ASPICULURIS TETRAPTERA S. obvelata Physical Characteristics Cervical alae Shape of tail of female Location of vulva Mamelons in male Spicule Ova size Life Cycle Location in host Prepatent period Location of ova Time to infectivity of ova
A. tetraptera
Subtle Long and pointed Anterior of body Present Present 134 x 36 µm, one side flattened
Prominent Conical Middle of body Absent Absent 86 µm x 37 µm, ovoid, symmetrical
Cecum and colon 11–15 days Perianal skin 5–20 hours
Colon and cecum 21–25 days Fecal pellet 5–8 days
140
120
100
80
60
40
20
0 Syphacia obvelata
Aspiculuris tetraptera
Syphacia muris
Fig. 22-1 Appearance and relative size of the ova of the oxyurid parasites of mice, Syphacia obvelata, Aspiculuris tetraptera, and Syphacia muris.
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cervical alae, or clear, cuticular flanges found on the anterior lateral margins of the body. Female S. obvelata are 3.4 to 5.8 mm long and 240 to 400 µm wide, with a 530 to 675 µm tail. The vulva is found in the anterior part of the body, behind the excretory pore. S. obvelata exhibit marked sexual dimorphism, and the males measure only 1.1 to 1.5 mm long with a 130 µm long tail. Males have a prominent spicule that measures 68 to 90 µm and two to three rounded protuberances, or mamelons, on their caudal ventral surface. These mamelons allow the male to grasp the female during copulation (Fig. 22-2). The ova of S. obvelata are pointed ovals that are flattened on one side and measure approximately 75 x 29 µm. Frequently, the eggs have embryonated before leaving the female, and larva may be seen in newly laid eggs. B. LIFE CYCLE S. obvelata has a direct life cycle and a prepatent period of 11 to 15 days (Levine 1968). S. obvelata eggs are infective once embryonated, which often occurs 5 to 20 hours
after release from the female (Chan 1952; Taffs 1976a). When the infective eggs are ingested by a suitable host, the eggs hatch and the larvae migrate to the cecum over a 24-hour period (Chan 1952). Syphacia spp. reside in the cecum or anterior colon, where they feed on bacteria present in the lumen. Two-thirds of the females are fertilized by 6 days after hatching (the usual lifespan of the male), and the females remain in the cecum for another 10 to 11 days while they produce ova. Gravid females migrate to the anus to lay their eggs on the perianal area of the host. S. obvelata females release an average of 350 eggs per female, after which they die (Chan 1952). Animals are usually infected through contact with surfaces or substances contaminated with embryonated eggs. Due to the short time necessary for eggs to embryonate and become infective, it is theoretically possible for S. obvelata ova to embryonate on the host and retroinfect the animal by migrating back into the body (Prince 1950), although this is considered an uncommon route of infection (Chan 1952).
Syphacia obvelata Lips Alae Esophageal bulb 100 µm
Mamelons
100 µm Vulvar pore
Spicule 1 mm
Fig. 22-2 Distinguishing features of Syphacia obvelata. Note the sexual dimorphism and the mamelons and spicule present in the male. In the female, the characteristic round esophageal bulb is illustrated, as are the small cervical alae and the fleshy lips. The vulvar pore is seen in the illustration of the head of the female.
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C. DIAGNOSIS Since the eggs of S. obvelata may be found on the perianal skin and hair, the usual method of diagnosing infection is the perianal cellophane tape test and variations thereof (Eguiluz et al. 2001). In this test, a strip of clear cellophane tape is pressed firmly to the perianal region of the mouse, then mounted on a glass microscope slide. The slide is examined for the presence of pinworm ova using a microscope. This test relies on the presence of oxyurid eggs on the perineum. For that to occur, the infection must have reached a point where gravid females are present and releasing ova. Mice are frequent, assiduous groomers and may remove evidence of eggs before they are found, or the mouse may be infected with a very light worm burden and very few eggs may be present. The “tape test” is therefore less sensitive than a test in which the animal’s gut contents are directly examined for the presence of parasites. S. obvelata ova may also be found using standard fecal flotation techniques, but this is much less common, since the animals release their eggs at the anus. An anal swabbing technique has been described in live mice and has been shown to be effective in diagnosing infection with S. obvelata (Goncalves et al. 1998). While the “tape test” and the anal swab technique are excellent tests for evaluation of treatment success
when the mouse must remain alive, examination of animals at necropsy should include both the evaluation of cecal and colonic contents for adult worms using a dissecting scope and flotation of the cecal and colonic contents (Klement et al. 1996; West et al. 1992). Although the ova of S. obvelata are present mainly on the perineal skin, maceration of worms present in the contents of the large intestine may yield positive results. 2.
Aspiculuris tetraptera
A. MORPHOLOGY Aspiculuris tetraptera was first described by Schulz in 1812. This pinworm of mice may be differentiated from S. obvelata by its broad cervical alae, oval esophageal bulb, and striated cuticle. Female A. tetraptera are 3 to 4 mm long and 215 to 275 µm wide, with a tail that is 445 to 605 µm long. The vulva of the female A. tetraptera is found anterior to the middle of the body, but the vulva is more posterior than in S. obvelata. The ova of A. tetraptera are symmetrical, oval, and approximately 86 x 37 µm. The eggs are at the morula stage at the time of release and the inner cell mass does not fill the shell. Males are 2 to 4 mm long and 120 to 190 µm wide, with a 117 to 169 µm tail. The male A. tetraptera has neither spicules nor mamelons (Fig. 22-3).
Lips
Aspiculuris tetraptera Alae Esophageal bulb
1 mm
Fig. 22-3 Distinguishing features of Aspiculuris tetraptera. There is less sexual dimorphism in this species, and the male lacks spicules and mamelons. The illustration of the female head does not show the vulvar pore since it is slightly lower on the body than in S. obvelata. The broad cervical alae and oval esophageal bulb of A. tetraptera are highlighted.
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B. LIFE CYCLE A. tetraptera has a 21- to 25-day prepatent period (Behnke 1974). After hatching in the cecum, A. tetraptera larvae may be found in the crypts of Lieberkühn in the proximal colon, where they remain for 3 to 5 days (Anya 1966a). Adult A. tetraptera reside in the colon and migrate from the proximal to distal colon to deposit eggs (Chan 1955). Each A. tetraptera female releases an average of 17 eggs/day, usually at night (Phillipson 1974). The eggs are excreted in the mucus layer of the feces, and are not infective for 5 to 8 days. Unlike S. obvelata, females live another 21 to 24 days after their first egg release, for a total lifespan of 45 to 50 days (Hsieh 1952). Due to the location of egg release in A. tetraptera and the extended time necessary for the eggs to reach infectivity, retroinfection is not thought to be a means of reinfection in A. tetraptera infections. C. DIAGNOSIS The eggs of A. tetraptera may be found on the perianal skin and hair, but it is not a common occurrence. The perianal tape test is not an effective diagnostic tool when dealing with a single-species infection with A. tetraptera. The anal swabbing technique described by Goncalves has been shown to be effective in diagnosing infection with A. tetraptera (Goncalves et al. 1998). Evaluation at necropsy of cecal and colonic contents for the presence of adult worms is the most certain way to diagnose A. tetraptera (Klement et al. 1996; West et al. 1992). Ova may also be found on fecal flotation. Table 22-2 describes a fecal concentration and centrifugation technique effective in isolating these ova.
3.
Effects on Research
When compared to the devastating effects of infection with certain murine viruses still in circulation, such as mouse hepatitis virus, mouse parvovirus, or ectromelia, oxyurid infection is more a nuisance than a life-threatening situation. Infections with pinworms are considered to be clinically silent in animals with normal immune systems (Harkness and Wagner 1995; Levine 1968; Taffs 1976a). Pinworms only rarely penetrate the mucosa of the gut, unlike other helminths, and reside mainly in the lumen of the intestines, where they feed on bacteria. A single report describes small (1 to 2 mm) granulomas in two mice (of an unrecorded number examined) caused by the penetration of
TABLE 22-2
FECAL CONCENTRATION AND CENTRIFUGATION TECHNIQUE 1. Soften up to 5 cm3 of feces in a 15-ml conical tube. A small amount of water may be used, but using the flotation solution is better. 2. Fill the tube with the flotation solution (a zinc sulfate solution at 1.18 sp gravity). 3. Put the filled tube into a centrifuge and add more flotation solution to the tube until there is a small positive meniscus. 4. Place a coverslip on each tube, ensuring that contact is made with the entire lip of the tube. 5. Spin at 616–760 RCF for 10 minutes. 6. Place coverslip on glass slide and evaluate under a microscope at 100x.
the colonic wall by an adult A. tetraptera (Mullink 1970). Undoubtedly, this is a rare occurrence. A variety of nonspecific signs have been attributed to heavy oxyurid infections: poor condition, rough hair coats, reduced growth rate, and rectal prolapse (Eaton 1972; Harwell and Boyd 1968; Hoag 1961; Taffs 1976a). The reports that describe these signs fail to exclude agents such as Citrobacter and Helicobacter, both of which may cause similar signs in susceptible mice (Foltz et al. 1998; Maggio-Price et al. 1998; Vallance et al. 2003; Ward et al. 1996). Despite the lack of clinical signs usually associated with infection, pinworms may interfere with research in a number of ways. One of the most important of these is by modification of the immune system. Host–parasite interaction is a complicated system that involves both T and B cell-mediated immunity. Infections with S. obvelata or A. tetraptera have been shown to increase the host humoral response to nonparasitic antigenic stimuli in AKR/J mice (Sato et al. 1995). Infection with S. obvelata was associated in B6AF1/J neonates with termination of the tolerance state and induction of a Th2-associated eosinophilic autoimmune oophoritis (Agersborg et al. 2001). In athymic mice, pinworm infection may induce proliferation of T and B lymphocytes in the spleen (Beattie et al. 1981) and cause the development of a lymphoproliferative disorder that eventually leads to lymphoma (Baird et al. 1982; Beattie et al. 1980). Pinworm infections also result in the inhibition of diabetes formation in the nonobese diabetic (NOD) mouse (Gale 2002), presumably through antigenic stimulation of the immune system. The prevalence and consistency of these effects are difficult to evaluate as few researchers are able to pinpoint pinworms as the cause and then publish work that directly explains why oxyurid infection produced unexpected experimental results. In addition to effects on the immune system, pinworms may affect other systems. Infection with Syphacia has been shown to accelerate the development of the hepatic monooxygenase system in young C57BL/6N and WHW/HOM mice (Mohn and Philipp 1981). S. obvelata, but not A. tetraptera, infections have been shown to inhibit exploratory behavior in C57BL/6NHsd mice (McNair and Timmons 1977). Retarded growth in a colony of C57BL/6N and HOM mice was attributed to heavy pinworm infection, but no further information on the health status of this colony was given (Mohn and Philipp 1981). A final consideration when evaluating the effect of pinworms on research programs is financial. Research programs and animal care facilities incur increased costs associated with treatment and environmental decontamination. Pinworm infection may also preclude the movement of animals between facilities or between portions of one facility, thereby delaying experiments. 4.
Treatment
Any treatment regimen considered for pinworms must take into account the conditions under which the animals are housed and the potential for environmental persistence of the pinworm ova.
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Heroic sanitation measures are often employed to rid animal rooms of contamination; however, few reports directly document the presence of oxyurid eggs in the environment (Boivin et al. 1996; Le Blanc et al. 1993; Macy 2000). In conventionally housed mouse colonies infected with both A. tetraptera and S. obvelata, Hoag described eggs in dust and air intake filters and on equipment, but did not say from which species the eggs originated (Hoag 1961). Cellophane tape impressions from the interior surfaces of filter-top cages housing S. obvelata-infected mice failed to reveal eggs (Lipman et al. 1994). In a colony of A. tetrapterainfected mice housed in individually ventilated caging, tape tests of the environment were negative for eggs (Boivin et al. 1996). The addition of a simple bonnet-type filter top to colonies of outbred mice was able to substantially reduce transmission between cages (Wescott et al. 1976). With many modern rodent facilities using individually ventilated cages and laminar flow changing stations to effectively completely isolate the mouse from the environment, the distribution of eggs around an animal room may be minimal. Treatment failure in current rodent housing conditions may be due less to environmental contamination than to failure to rid mice of immature worms, resulting in small pockets of infected mice, mice avoiding treatment through normal movement for breeding or experimental purposes, or persistent infection in permissive hosts, such as immunocompromised genetically modified animals. Recent work on the susceptibility of Syphacia ova to common disinfectants has focused on the ova of the rat pinworm, Syphacia muris. S. muris eggs may be less fastidious than S. obvelata ova as they were found to embryonate in tap water and saline while the eggs of S. obvelata did not (Stahl 1961; van Der Gulden and van Aspert-van Erp 1976). S. obvelata eggs do not embryonate in water, saline, moistened activated charcoal, agar, or glycerin (Chan 1952; Grice and Prociv 1993; Philpot 1924). Eggs harvested from gravid S. obvelata females and kept on a moistened slide at room temperature began to degenerate after only 24 hours (Grice and Prociv 1993). Although humidity was not measured in either Chan’s or Grice’s studies, eggs allowed to become wet either prematurely opened their opercula and/or ruptured, while eggs that dried out collapsed and were not viable (Chan 1952; Grice and Prociv 1993). Despite these differences, and the apparent delicacy and fastidiousness of S. obvelata ova, the results of disinfectant studies on the ova of S. muris should probably be extrapolated to the ova of S. obvelata until studies show otherwise. Miyaji et al. showed that S. muris eggs were embryonated after exposure to several common disinfectants, but that exposure to 80°C for 30 minutes killed all ova (Miyaji et al. 1988). Work by Dix et al. showed that either 100°C heat for 30 minutes or ethylene oxide exposure produced a 100% kill rate in S. muris eggs. Formaldehyde gas was 94% effective, and chlorine dioxide was 96% effective under the conditions described by Dix et al., which included both a technical failure in the formaldehyde trial that allowed for growth of the bacterial indicator used and a relatively brief (10-minute) exposure
557 period to chlorine dioxide (Dix et al. 2004). Both formaldehyde gas and chlorine dioxide may have higher ova kill rates when applied in a different fashion. Dix et al. also stated that 41% of the ova left exposed to room air for 4 weeks hatched when exposed to suitable conditions (Dix et al. 2004). This resistance of S. muris eggs to many common cleaning and disinfection chemicals emphasizes the importance of including rigorous environmental decontamination as part of the treatment plan for a pinworm-infected area, despite the fact that the eggs may not have left the immediate cage area. Little, if any, work has been done on the environmental persistence of A. tetraptera eggs, probably because they are excreted in the feces, and removal of feces should suffice to remove the eggs from the environment. However, removal of fecal matter from the environment may be difficult in situations such as open-topped caging environments or the dirty side of a shared cage wash. In 1952, Hsieh described hatching A. tetraptera eggs in distilled water at 27°C, which may indicate that if allowed to persist in the environment, A. tetraptera ova are not as fastidious as S. obvelata ova (Hsieh 1952). Anya described a similar experiment in which the ideal hatching temperature appeared to be 30°C (Anya 1966b). Regardless of which species of oxyurid infects the animals in a particular facility, environmental decontamination as part of a pinworm eradication program is controversial (Gaertner 2000). One cycle of decontamination and removal of potentially infective materials and fomites, preferably after the first week of treatment is completed, is probably sufficient. While potentially both expensive and time- consuming, environmental decontamination is an important part of any parasite eradication effort, if for no other reason than the perception of making a “clean sweep.” However, at least one author has shown success in ridding a colony of rats of S. muris through treatment without environmental decontamination (Barlow et al., 2005). The second part of a treatment plan for oxyurids should include the administration of anthelminthics to the infected mice. Mice have been treated with a variety of chemicals over the years in the quest to produce helminth-free research subjects. These have included gentian violet, crystal violet, sodium fluoride, hexylresorcinol, phenothiazine, terramycin, aureomycin, and bacitracin (Taffs 1976a). In addition, organophosphates such as dichlorvos and uredofos have been used in pinworm eradication programs (Tetzlaff and Weir 1978; Wagner 1970). Another family of agents occasionally used is the nicotinic agonist family, which includes levamisole and pyrantel (Brody and Elward 1971; Comley 1980; Scott 1988). These compounds are neither as safe nor as effective as the GABA-agonistic piperazine compounds, widely used and recommended throughout the 1960s and into the present, especially in combination with ivermectin or a benzimidazole (Lipman et al. 1994; Martin 1997; Owen and Turton 1979; Reiss et al. 1987; Taffs 1976a; Zenner 1998). The most common agents in use today for pinworm eradication are avermectins and benzimidazoles. The avermectins are macrocyclic lactones produced by the actinomycete Streptomyces avermitilis. Avermectins act by increasing muscle
558 Cl- permeability through a glutamate-gated ion channel that paralyzes parasites (Martin 1997). In the laboratory animal literature, they are represented mainly by ivermectin. Ivermectin at an oral dose of 2 mg/kg/day was shown to be effective against S. obvelata in mice by removing 100% of gravid females, 94% of males, and 97% of immature worms (Ostlind et al. 1985). Early studies of the use of ivermectin in mice reported administration by gavage or subcutaneous injection (Flynn et al. 1989; Huerkamp 1990; Murphy-Hackley and Blum 1990; Ostlind et al. 1985). Treatments were either single or paired, given 7, 9, or 10 days apart. Animals remained parasite-free for 2 to 6 months (the length of published follow-up) after treatment (Flynn et al. 1989; Huerkamp 1990, 1993). Ivermectin applied between the scapulae of mice, using a micropipettor, at 2 mg/kg and administered 10 days apart has also been reported to be effective in treating pinworm infection in mice, with animals remaining free of parasites for 6 months (West et al. 1992). Ivermectin has also been administered topically through the use of a spray bottle and found to be effective in the treatment of pinworms, with animals remaining parasite-free for 6 months (Le Blanc et al. 1993). These methods of ivermectin administration involve direct handling of the affected mice and are relatively time-consuming and difficult for personnel, especially when dealing with large numbers of rodents. Ivermectin has also been administered to mice in drinking water (Hasslinger and Wiethe 1987; Klement et al. 1996). Effective dosages were calculated to be 2 mg/kg/day, although, due to differences in water consumption, actual doses ranged from 1.7 to 4.8 mg/kg/day (Klement et al. 1996). The ivermectin formulation used by Klement was the liquid anthelminthic formulated for horses, Eqvalan® (Merial, Athens, Georgia), mixed in water (Klement et al. 1996). Klement examined several different treatment regimens, each of which consisted of 4 consecutive days of ivermectin treatment, spaced 3 days apart, but differed in total number of treatments (Klement et al. 1996). Animals were followed for 29 to 32 weeks, and pinworms were eradicated by the use of four or five treatment regimens, but no fewer (Klement et al. 1996). The combination of piperazine and ivermectin, both in drinking water, has also been shown to be effective in the elimination of oxyurid infections in mice (Lipman et al. 1994; Zenner 1998). Unintended deleterious effects may result from ivermectin treatment, especially in animals with compromised blood-brain barriers (Didier and Loor 1995; Paul et al. 1987; Roder and Stair 1998). This effect has been demonstrated in a subpopulation of the outbred mouse stock, CF-1 (Jackson et al. 1998; Lankas et al. 1997), which is deficient in P-glycoprotein, a protein that functions as a drug transport pump across the blood-brain barrier. In addition, mdr1a (Abcb4) and mdr1b (Abcb1) knockout mice, which are also deficient in P-glycoprotein, are exquisitely sensitive to ivermectin (Schinkel et al. 1994; Schinkel et al. 1997). Toxicity has also been reported in young C57BL mice
K AT H L E E N R . P R I T C H E T T
(Skopets et al. 1996). Young mice are more susceptible to ivermectin toxicosis due to postnatal blood-brain barrier closure and potential overdosing through receiving the drug via multiple routes, especially in milk, where concentrations are three to four times plasma concentrations (Lankas et al. 1989). Mice are more sensitive to the adverse effects of ivermectin than rats, and male mice are more sensitive than females (JEFCA 1991; Woodward 1993). An inadvertent overdose of ivermectin administered subcutaneously to BALB/cSim mice was shown to produce lesions in the liver and kidneys (Hamlen et al. 1994). Those lesions included mild to moderate diffuse microsvesicular fatty change of the liver and acute, diffuse tubular necrosis. Ivermectin may also affect some behaviors in rats and mice. In 129/SvEvTac, AKR/J, and C57BL/6J mice, ivermectin was not shown to affect swimming behavior or spatial learning, but had effects on more subtle behaviors, such as exploration of a novel open field (Davis et al. 1999). In Crl:CD1(SW) mice, ivermectin may also have immunomodulatory effects through the stimulation of helper T lymphocytes (Blakley and Rousseaux 1991). New avermectins, especially selamectin, which is administered topically, have been shown to be safe and effective in cats, cattle, and dogs, including Collies, a subpopulation of which are sensitive to ivermectin toxicosis (Bishop et al. 2000; Jacobs 2000; Krautmann et al. 2000; McTier et al. 2000). Doramectin, one of the new avermectins, has been shown to be efficacious against S. muris in rats (Öge et al. 2000). A limited study demonstrating the efficacy of selamectin in the treatment of both S. obvelata and A. tetraptera in Crl:CD1(SW) mice has been performed, and the compound was both safe and efficacious (Winchester et al. 2004). Before initiating the widespread use of new avermectins in a potentially drug-sensitive colony of genetically manipulated rodents, pilot applications on an age range of animals from the colony of interest may prevent deleterious side effects. The benzimidazole class of anthelminthics binds to nematode β-tubulin and inhibits microtubule formation. Microtubule binding results in a drug that is adulticidal, larvicidal, and ovicidal, since microtubules are necessary for cell division (Kirsch 1978; Lacey et al., 1987). Fenbendazole has been used since at least 1981 as a feed formulation to treat pinworm infection (Mohn and Philipp 1981). Fenbendazole is a relatively benign drug with no known teratogenicity and an acute oral LD50 of more than 10 g/kg in mice and rats. Toxicity occurred when rats were fed doses of 500 mg/kg/day (60 times the approximate dose of 8 mg/kg/day achieved by a 150 ppm feed level) for 14 days or longer. These changes included renal tubular hyperemia or hemorrhage, increased serum creatinine, and hepatocellular granular degeneration (Xu et al. 1992). In rats, fenbendazole appeared to promote liver tumor formation, but those changes were seen at doses 60 or more times the therapeutic dose (Shoda et al. 1999). In rats, at therapeutic dosages, over extremely long treatment periods (greater than 70 consecutive days, including pre- and postnatal exposure), fenbendazole was
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found to have minimal behavioral effects and was deemed safe (Barron et al. 2000). Fenbendazole did not affect the hepatic monooxygenase system in C57BL/6N mice (Mohn and Philipp 1981), nor did it affect the immune response in BALB/cByJ mice (Reiss et al. 1987). Fenbendazole administered to mice at doses of 100 to 300 mg/kg was found to have no effect on pain perception, no influence on hexobarbitone anesthesia sleep times, and no effect on maximal electroshock convulsions (Keller 1991). In mice, fenbendazole given at a dose of 150 mg/kg of feed for 3 alternating weeks of treatment and combined with environmental decontamination proved efficacious against A. tetraptera for one year (Boivin et al. 1996). A standard protocol in use at many institutions in the United States is the administration of feed containing 150 ppm (or mg/kg) of fenbendazole for at least three 7-day periods over at least 5 weeks. Fenbendazole is recommended over ivermectin due to its lack of documented interference with research, its large margin of safety, and its ovicidal, larvicidal, and adulticidal effects. The effectiveness of this treatment should be evaluated by necropsy of colony and susceptible sentinel animals and by both gross and microscopic evaluation (via fecal flotation) of the gastrointestinal contents. The treatment of an infection with A. tetraptera would seem to be substantially easier than the treatment of S. obvelata infections due to the difference in their life cycles. The egg of A. tetraptera is excreted in the feces and takes 5 to 8 days to reach an infective stage. Retroinfection through hatching on the perianal skin and migration through the anus is impossible. A. tetraptera infections would seem to be able to be controlled through the simple expedient of giving one treatment with an anthelminthic such as fenbendazole, cleaning the environment, and keeping treated animals from having access to the feces of infected animals. However, since many infections with pinworms are infections of more than one species, following the more rigorous treatment recommendations designed to remove S. obvelata from the environment is a wise choice. If facilities harbor wild-caught mice for research projects, these animals should be prophylactically treated for parasites to reduce the risk of zoonotic disease and to prevent contaminating other animals at the institution.
are smaller, measuring approximately 75 x 29 µm, and more symmetrical. Mixed infections of S. obvelata and S. muris in mice are uncommon. Treatment of S. muris would be as recommended above for the oxyurids of mice, plus the cessation of exposure to infected rats. The effects on research of S. muris infection in mice are probably similar to those seen with S. obvelata. 2.
Trichuris muris
Trichuris muris is the whipworm of mice. Infection is common in wild mice but vanishingly rare in laboratory mice, unless the animals are deliberately infected with T. muris as a model of host/parasite interaction. Eggs are not infective until 30 days after they are laid, so good housekeeping practices should preclude the spread of infection in laboratory mice (Fahmy 1954). The relatively large (16 to 25 mm) worm may be found in the cecum. Immunity to T. muris is strain-dependent (Else and deSchoolmeester 2003). Effects on research may include a modulation of the immune system or, with heavy infections, typical whipworm pathologies, such as anemia. Treatment may be accomplished by a single administration of oxantel at 12.5 mg/kg or two doses of mebendazole at 50 mg/kg (Rajasekariah et al. 1991). 3.
Heligmosomoides polygyrus
Heligmosomoides polygyrus is a trichostrongyloid nematode of mice with a strictly enteric life cycle. H. polygyrus is common in wild rodents and absent in laboratories unless animals are used for parasitology research. The worm resides in the anterior duodenum, where it penetrates tissues and feeds on tissue components (Bansemir and Sukhdeo 1994). Effects on research of infection with H. polygyrus are mainly related to the immune system (Barnard et al. 1998; Bashir et al. 2002). H. polygyrus appears to have some innate tolerance to ivermectin, requiring a dose of at least 1.7 mg/kg to remove fourth stage larvae from mice (Njoroge et al. 1997).
B.
III.
HELMINTHS OF MINOR IMPORTANCE A.
1.
Nematodes
Syphacia muris
Syphacia muris is the pinworm of the rat. Mice can be infected with S. muris (Hussey 1957; Ross et al. 1980). Adult S. muris are slightly smaller than adult S. obvelata but the easiest way to differentiate the two Syphacia species is to examine the ova. The ova of S. muris resemble S. obvelata eggs but
Cestodes
The characteristics of the three most common mouse cestodes are addressed in Table 22-3. A comparison of the relative size and appearance of their eggs may be found in Fig. 22-4. Infection with cestodes is extremely rare in modern mouse facilities, unless the animals are being used to study host/parasite interactions. 1.
Rodentolepis (=Hymenolepis) nana
Mice are the definitive host of Rodentolepis nana, also known as the “dwarf tapeworm” and the most common cestode parasite of mice. The parasite attaches to the intestinal villi
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TABLE 22-3
DIFFERENTIATION OF RODENTOLEPIS (=HYMENOLEPIS) NANA, HYMENOLEPIS DIMINUTA, AND RODENTOLEPIS MICROSTOMA R. nana
H. diminuta
R. microstoma
Physical Characteristics Length
25–40 mm
20–60 cm
Width Armed rostellum Ova size
0.25–0.5 mm Yes ~40 x 50 µm
4 mm No ~70 µm
8–50 mm (up to 120 mm) 0.5–4 mm Yes ~85 µm
Life Cycle Requires intermediate host Location in host Prepatent period
No Small intestine 14–16 d
Yes Small intestine 19–20 d
Yes Bile duct 16–17 d
using an armed rostellum and subsists on the host’s interstitial fluids. Alone among cestodes, R. nana can reproduce in immunocompetent hosts using either a direct or indirect life cycle (Flynn 1973b). In the indirect life cycle, invertebrates such as the flour beetle, Tribolium confusum, ingest eggs, which hatch and develop into cysticercoids in the intestines. Mice consume these invertebrates and are infected with the cysticercoids. Mice may also become infected by directly ingesting R. nana eggs, as may humans. Cysticercoids will form in the villi of the small intestine and then hatch in 5 to 6 days to become active infections. The parasites excyst in the duodenum, but most
parasites are found in the lower ileum of the mouse (the last 80 mm of small intestine), after the fourth day of infection (Henderson and Hanna 1987). As described by Henderson, the maximum mean worm length of R. nana is 51.5 mm but the worm is usually 25 to 40 mm long and 1 mm wide (Henderson and Hanna 1987). The eggs of R. nana are infective for 11 days outside of the host (Baskerville et al. 1988). Although R. nana is considered a zoonotic parasite, the human and rodent strains may be different and not cross-infective (al-Baldawi et al. 1989). Successful treatment for R. nana has been accomplished using benzimidazole compounds, but since zoonotic potential does exist, euthanasia or rederivation via hysterectomy or embryo transfer is recommended if a colony becomes infected (Baskerville et al. 1988; Taffs 1975, 1976b). Since the parasites attach to the mucosa and feed on host interstitial secretions, heavy infection of R. nana in mice may result in weight loss and retardation of growth (Flynn 1973b). This may have a negative effect on research projects, as may the antigenic stimulation inherent in parasitism. 2.
Hymenolepis diminuta
Despite its name, Hymenolepis diminuta is not the smallest of the cestodes infecting laboratory mice, with an average length of 20 to 60 cm and a width of 3 to 4 mm (Flynn 1973b). This cestode has an indirect life cycle, in which arthropods such as flour beetles, fleas, or moths are the intermediate hosts. H. diminuta adults reside in the small intestines of mice,
100
80
60
40
20
0
Hymenolepis diminuta
Rodentolepis nana
Rodentolepis microstoma
Fig. 22-4 Appearance and relative size of the ova of Hymenolepis diminuta, Rodentolepis nana, and Rodentolepis microstoma.
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where they feed on the interstitial fluid of the host. Zoonotic infections have been described, but this requires that a human ingest the intermediate host (Flynn 1973b). This tapeworm is often described as a tapeworm of rats, and infection may be difficult to establish in laboratory mice (Flynn 1973b; Read and Voge 1954). The treatments effective against R. nana would also be effective for H. diminuta (McCracken et al. 1992). 3.
Rodentolepis (=Hymenolepis) microstoma
Rodentolepis microstoma is found in the bile duct of its definitive hosts, which include mice. This cestode, which is similar in size to R. nana, has an indirect life cycle, in which arthropods such as flour beetles, fleas, or moths serve as intermediate hosts. R. microstoma may, however, exhibit a direct life cycle in immunocompromised hosts (Andreassen et al. 2004). After ingestion of the intermediate host, the parasites excyst in the duodenum and migrate to the bile duct in 5 to 7 days (Macnish et al. 2003). Mature proglottids are found at 15 to 16 days postinfection (De Rycke 1966). Treatment of mice with either mebendazole or albendazole at a dose of 50 mg/kg did not clear infection (McCracken et al. 1992), perhaps because of the parasite’s protected location in the bile duct. Cholangitis is associated with infection with R. microstoma (Percy and Barthold 2001). This parasite was recently described as a zoonotic agent (Macnish et al. 2003). 4.
Taenia taeniaeformis
The mouse is the intermediate host of this feline tapeworm. Approximately 30 days after ingesting eggs shed by an infected cat, tapeworm larvae, or strobilocerci, begin to form in an infected mouse’s muscle tissue or liver. This tissue phase of T. taeniaeformis may be found in older sources such as Cysticercus fasciolaris. The strobilocerci are white or clear and approximately 4 to 10 mm in diameter. The strobilocercus contains a scolex and a segmented strobila that appears exactly as an adult tapeworm, but there is a bladder on the end. There are usually only 1 to 2 per host (Owen 1992). Different strains of mice are more susceptible to infection than others (Conchedda and Ferretti 1984). If this parasite is found in animals in a facility, a thorough investigation as to how the mice are gaining access to and ingesting cat feces should be conducted (Balk and Jones 1970). Effects on research may be minimal, other than antigenic stimulation occurring with the development of the parasite, but the discovery of strobilocerci in the liver or muscle of affected mice is alarming and indicates a breakdown in sanitation procedures. ACKNOWLEDGMENTS The author wishes to acknowledge the illustrator, Sarah Williams, for production of the figures. In addition, the author would like to acknowledge the able editorial assistance of Dr. William White.
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Chapter 23 Arthropods David G. Baker
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Parasitic Lice of Laboratory Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Polyplax serrata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Parasitic Mites of Laboratory Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Demodex musculi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Myobia musculi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Myocoptes musculinus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Psorergates simplex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Radfordia sp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Trichoecius romboutsi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
Arthropod parasites are important pathogens of laboratory mice. Arthropod infestation can alter host behavior and physiology, thereby introducing unwanted variability into research data. Fortunately, however, they are also uncommon in wellmanaged animal facilities. Improvements in laboratory animal husbandry practices, pathogen surveillance, and treatment options have dramatically decreased the incidence of arthropod infestation. In particular, improvements in animal husbandry practices have interrupted the life cycles of many arthropods, especially those that spend portions of their life cycle off the host. Recently, an extensive survey was conducted of over 14,000 mice submitted to a commercial diagnostic laboratory.
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
565 566 566 568 568 569 572 574 575 576 577
Arthropod infestation was rarely diagnosed (Livingston and Riley 2003). Laboratory mice are susceptible to infestation with a wide range of arthropod parasites. Many of these parasites are of primarily historical importance (see Tables 23-1 and 23-2). Very little new information has been published on many of these ectoparasites for several years. The relatively few parasitic arthropods likely to be found in modern animal facilities constitute the primary focus of this chapter. It should be noted, however, that changes in the genetic makeup of laboratory mice may expand the scope of parasite infestations. Many genetically modified mice are immunedeficient and may be more permissive of infestation by arthropod parasites. An example is the recent finding of Demodex musculi in immunodeficient transgenic
Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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TABLE 23-1
INSECT PARASITES OF LABORATORY MICE Order
Genus
Species
Common name
References
Hemiptera Siphonaptera
Cimex Xenopsylla Nosopsyllus Nosopsyllus Leptosylla Polyplax Hoplopleura Hoplopleura
lectularis cheopis fasciatus londiniensis segnis serrata acanthopus captiosa
Bedbug Oriental rat flea Northern rat flea
Flynn (1973); Yunker (1964) Bakr et al. (1996); Flynn (1973); Yunker (1964) Flynn (1973); Yunker (1964) Flynn (1973) Flynn (1973); Murray (1961); Yunker (1964) Flynn (1973); Nelson et al. (1972); Sparrow (1976) Flynn (1973) Beaucournu (1968); Flynn (1973)
Anoplura
Mouse flea Mouse louse
mice (Hill et al. 1999). Where did that parasite come from? How long had it been there? Clearly, uncommon arthropod parasites still threaten colonies of laboratory mice. There is still a need for continued vigilance for arthropod parasites.
II.
PARASITIC LICE OF LABORATORY MICE A.
1.
Polyplax serrata
Description and Life Cycle
The genus Polyplax is in the family Polyplacidae, one of the families in the order Phthiraptera, suborder Anoplura. Members of this order are known as sucking lice. Adult female P. serrata are slender and about 1.5 mm long, whereas the males are thicker and shorter (1.0 mm) (Fig. 23-1). Both sexes are large enough to be seen with the unaided eye. Polyplax serrata may be differentiated from P. spinulosa (the spined rat louse) on the basis of both the sternal plate, which is triangular in P. serrata and pentagonal in P. spinulosa, and the fourth lateral abdominal
(paratergal) plate, which has setae of unequal length in P. serrata (the dorsal seta is longer than the ventral), whereas these setae are of equal length in P. spinulosa (Flynn 1973). The five stages recognized in the life cycle of P. serrata are the egg, three nymphal stages, and the adult (Murray 1961). The eggs are attached near the base of hair shafts. The young hatch by lifting the operculated cap in the dorsum of the egg. A wide distribution of first-stage nymphs may be found over the body surface; however, more mature stages generally favor the anterior dorsum of the host (Murray 1961). Nymphal stages may be differentiated by setal arrangements, as described by Murray (1961). Eggs of P. serrata hatch in 5 to 6 days, and nymphs develop into adults in 7 days, giving an average minimum life cycle of 13 days (Murray 1961). Louse populations are constrained by the development of a host immune response and by grooming behavior (Bell and Clifford 1964; Bell et al. 1962, 1966; Clifford et al. 1967; Murray 1961; Ratzlaff and Wikel 1990). Transmission from host to host is by direct contact. Using olfactory cues, female mice can discriminate between P. serrata-infested and uninfested male mice in an oxytocin-dependent manner, and exhibit a preference for the odor of uninfested males (Kavaliers et al. 2003).
TABLE 23-2
ECTOPARASITES OF LABORATORY MICE OF THE ORDER ACARINA Suborder
Genus
Species
Common name
References
Mesostigmata
Ornithonyssus Ornithonyssus Liponyssoides Haemogamasus Eulaelaps Laelaps Haemolaelaps Myobia Radfordia Psorergates Notoedres Demodex Myocoptes Trichoecius
bacoti sylviarum sanguineus pontiger stabularis echidninus glasgowi musculi affinis simplex musculi musculi musculinus romboutsi
Tropical rat mite Northern fowl mite House mouse mite
Fox (1982); French (1987); Keefe et al. (1964) Flynn (1973); Miller and Price (1977) Flynn (1973); Levine and Lage (1984) Morsy et al. (1994) Uchikawa and Rack (1979) Flynn (1973); Owen (1956) Zumpt and Till (1956) Flynn (1973); Friedman and Weisbroth (1977) Ewing (1938); Flynn (1955) Beresford-Jones (1965); Flynn (1955) Fain (1965) Hill et al. (1999); Hirst (1917) Flynn (1973); Gambles (1952); Watson (1960) Van Eyndhoven (1946); Fain et al. (1970)
Prostigmata
Astigmata
Spiny rat mite Fur mite Fur mite Hair follicle mite
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A
B
Fig. 23-1 (A) Polyplax serrata, male. (B) Polyplax serrata, female. From: Flynn, R. J. 1955. Ectoparasites of mice. Proc. Anim. Care Panel 6, 75–91. Reprinted with permission from AALAS.
2.
Susceptible Hosts
P. serrata has been reported from both wild and laboratory mice (Sparrow 1976; Sosnina et al. 1981). Infestation was not detected in a recent survey of over 14,000 mice submitted to a commercial diagnostic laboratory (Livingston and Riley 2003). 3.
Pathobiology
The Anoplura are blood-sucking lice, and the effects on the mouse host are related mainly to this feeding strategy. Heavy infestation with P. serrata may cause anemia and debilitation (Flynn 1973). Histopathologic findings include inflammatory changes compatible with the development of Type I hypersensitivity. Later stages of infestation also include epithelial hyperplasia (Nelson et al. 1972). Following a theme common to other host-ectoparasite relationships, these changes are largely immune mediated, involve both immediate
and delayed responses, and facilitate constraint of louse populations (Bell et al. 1982; Nelson et al. 1983; Ratzlaff and Wikel 1990). Arthropod-borne infections transmitted by P. serrata are known to include Eperythrozoon coccoides (Berkenkamp and Wescott 1988) and Francisella tularensis (Shaughnessy 1963). 4.
Clinical Symptoms
Heavy infestations with P. serrata cause localized reddening of the skin, pruritus, self-trauma, loss of condition, and, occasionally, death (Flynn 1973; Nelson et al. 1972; Oldham 1967).
5.
Diagnosis
The most effective method of detecting infestation with ectoparasites, including lice, is by direct examination of the pelage of dead or anesthetized mice under a bright light and
568 dissecting microscope. The pelage should be examined under low power, with the principal focus on the epidermal surface. The hair over the dorsum, nuchal crest area of the skull, the cervical dorsum, and the area between the scapulae generally yield the greatest number of parasites. The hair in these areas should be parted and examined for moving or stationary lice and for viable or hatched egg cases. Flynn (1963) presents an alternative method for detecting lice and non–follicle-inhabiting mites. Briefly, this involves placing the euthanatized mouse on a sheet of black paper surrounded by clear adhesive tape, sticky side up. After an overnight wait, the carcass is discarded, and the paper is examined under a dissecting microscope. 6.
Treatment
There are essentially two approaches to eradicating arthropod infestations in an animal colony: rederivation and chemical treatment. Rederivation procedures are labor intensive, may put valuable stock at risk, and may slow or halt research efforts. However, it is the most effective means of eradicating all ectoparasites, especially those that spend a portion of their life cycles off the host. Because lice spend their entire life cycle on the host, chemical treatments are a more viable option than for other ectoparasite infestations. Historically, several compounds have been used to treat for lice. These have been applied as dusts, insecticidal powders, or dips. These compounds are labor intensive and are often less than 100% effective, leaving a remnant louse population to reestablish the infestation. The more successful treatments include organophosphates such as malathione, diazinon, and methoxychlor; pyrethroids (Wall and Shearer 2001); and chlordane. Unfortunately, many of these compounds are known to induce physiologic changes and may be toxic to the host as well as to animal care personnel (Skopets et al. 1996). Among those listed, the pyrethroids, such as permethrin, are effective and generally safe. Pyrethroids may be applied as dips or sprays; they should be applied twice, 14 days apart (Wall and Shearer 2001). Pyrethroids may be fatal to mice if unintentionally given as an overdose (Constantin 1972). The insecticide fipronil has been shown to be effective in eliminating biting lice from dogs (Pollmeier et al. 2002) and humans (Downs et al. 2000). Fipronil has also been found effective for eliminating lice from laboratory mice when applied to the mouse’s entire body, with treatment repeated in 10 days (Wall and Shearer 2001). In one report, fipronil was clinically nontoxic after accidental ingestion by a person (Fung et al. 2003). However, it has been reported to increase plasma progesterone and decrease plasma estrogen levels, and to lengthen the estrous cycle of female Wistar rats (Ohi et al. 2004). Lastly, ivermectin administered at 200–400 µg/kg is also effective in controlling lice on mice (Wall and Shearer 2001). It should be noted, however, that the avermectins, a class of compounds that includes ivermectin, may affect host physiology and has been associated with toxicity in a number of inbred or
D AV I D G . B A K E R
genetically modified mouse strains (Toth et al. 2000). For example, ivermectin has anticonvulsant properties (Dawson et al. 2000), is neurotoxic in P-glycoprotein deficient CF-1 mice (Lankas et al. 1997), and causes neurologic signs and occasional death in C57BL mouse pups (Skopets et al. 1996). Ivermectin also alters sensitive behaviors, including exploration of a novel open field (129 SvEv mice); the acoustic startle reflex (C57BL/6J but not AKR mice); and the prepulse inhibition of the acoustic startle reflex (C57BL/6J and AKR mice), a measure of sensory gating (Davis et al. 1999). 7.
Prevention and Control
In all cases of ectoparasitism, prevention is key. Mice should only be obtained from vendors shown to have stock free of ectoparasites. Mice obtained from other investigators should come with clean health records. Feral mice should be excluded from the animal facility.
III.
PARASITIC MITES OF LABORATORY MICE A. Demodex musculi
1.
Description and Life Cycle
Demodex musculi is a prostigmatid mite in the family Demodicidae. The order Prostigmata includes a large number of mites that show considerable variation in morphology and habitat selection. The order designation describes the anterior location of the stigmata (Wall and Shearer 2001). D. musculi are minute mites with a characteristic “cigar-shaped” body. The elongated abdomen bears striations on the dorsal and ventral surfaces. Adult males are about 0.13 mm in length, whereas adult females are about 0.15 mm (Hirst 1917). Adult D. musculi are shorter than other Demodex sp. infesting mice (Hill et al. 1999). Nymphal and adult stages have four pairs of short, stout legs located on the thorax. Setae are absent (Wall and Shearer 2001). The entire life of D. musculi is spent in the hair follicles of the host. Life-cycle stages consist of egg, larva, two nymphal stages, and adult male and female. Relatively little is known of the life cycle of this mite, but it is assumed to be quite similar to that of other members of the genus. Adult females lay 20 to 24 eggs in the hair follicle. Eggs hatch, releasing larvae, which develop through the nymphal stages, to the adult stage. The complete life cycle requires 18 to 24 days (Wall and Shearer 2001). D. musculi is likely transmitted from dam to pups during nursing in a manner similar to D. canis (Wall and Shearer 2001). Because mites live within hair follicles, direct transmission may be less common between adult mice, but it does occur (Hill et al. 1999). Relatively little is known of this and other important aspects of the biology of D. musculi.
569
23. ARTHROPODS
2.
Susceptible Hosts
Like other members of the genus, D. musculi is considered to be very host-specific and has only been reported to infest mice. Infestation with D. musculi is likely extremely rare. Until recently (Hill et al. 1999), this parasite had not been reported in mice for nearly a century (Hirst 1917). The extent of host susceptibility is unknown. It should be noted that at least four additional Demodex sp. have been reported from various species of mice (Bukva 1985, 1994; Lukoschus and Jongman 1974; Nutting et al. 1973). However, other than D. flagellurus, these have not been recovered from laboratory mice. 3.
Pathobiology
In other host species infested with Demodex sp., pathologic changes are associated with T cell defects. In contrast, infestations are generally nonpathogenic in immunologically competent hosts. A similar relationship apparently exists for mice infested with D. musculi. Recently, D. musculi was reported in a colony of transgenic mice (Hill et al. 1999). Mouse strains included the B6,CBA-TgN(CD3E)26Cpt(CD3E), a homozygous strain lacking mature T lymphocytes and natural killer cells; the B6,SENCARB-TgN(pk5prad1)7111Sprd (Prad 1) mouse, which is immunologically competent but which overexpresses human cyclin D1 and manifests severe thymic hyperplasia; and the double-Tg F1 offspring of these two lines. In addition, D. musculi was experimentally transmitted to SCID (Severe combined immunodeficient) and nu/nu (CD-1) mice. D. musculi was not recovered from immunologically normal sentinel mice in the same room or from mice of other strains elsewhere in the facility (Hill et al. 1999). In that report, infested mice did not show evidence of dermatitis. Because D. musculi is rarely diagnosed, little is known of its pathologic potential. Likewise, minimal tissue reaction has been observed in the tissues of mice infested with other Demodex sp. (Bukva 1985, 1994; Lukoschus and Jongman 1974; Nutting et al. 1973). 4.
Clinical Symptoms
Infestations with D. musculi are asymptomatic. 5.
Diagnosis
Mite infestation may be diagnosed on plucked hairs, or in deep scrapings from the dorsum of the host. The scraping must be deep enough to include the hair follicle. 6.
Treatment
Historically, numerous acaricides have been used to eliminate Demodex sp. from a variety of host animal species such as the dog. Because of the paucity of reports on demodicosis in mouse colonies, scant information is available on treating the condition. It is likely that treatments described for mice infested
with other genera of mites will also effectively eliminate D. musculi. A diagnosis of D. musculi should prompt consideration of an underlying immunodeficiency. 7.
Prevention and Control
The prevalence of D. musculi is unknown, since adequate diagnostic examinations are infrequently performed. Lack of prevelence data complicates prevention and control recommendations. As for other parasites, purchasing animals only from reputable vendors, using similarly reputable animal carriers and housing mice in a manner that precludes contact with feral rodents will minimize the potential for exposure to D. musculi. Laboratory animal professionals should consider adding checks for D. musculi to their facility health monitoring. Where this is deemed appropriate, immunologically incompetent mice should be examined.
B. 1.
Myobia musculi
Description and Life Cycle
Myobia musculi is a nonburrowing mite in the family Myobiidae. The mite is small and about twice as long as wide, with females 0.40 to 0.50 mm in length and males about 0.28 to 0.30 mm (Fig. 23-2). The sexes are quite similar in appearance, differing only in size, setation, and genitalia. Myobia may be easily differentiated from other mites on the mouse, except Radfordia, by the characteristic shape, especially the lateral margins of the idiosoma, which form bulges between the legs. Differentiation from Radfordia may be accomplished by examining the tarsus of the second pair of legs. In Myobia, a true claw is lacking; however, the terminal tarsal appendage (empodium) ends with a single clawlike structure, the empodial claw. In contrast, the terminal tarsal structure (of leg II only) in Radfordia ends with two terminal tarsal claws. Stages in the life cycle include the egg, larva, two nymph stages, and adults. The eggs are oval, about 0.25 mm long, and attached at the lower pole to hair shafts near the base (Gambles 1952). The eggs hatch in about 7 to 8 days (Friedman and Weisbroth 1977). The larvae are identified by having three pairs of legs. The larval period lasts 10 days, with eight-legged nymphal forms appearing on the eleventh day posthatching. Adults may be observed as early as the fifteenth day and are capable of laying eggs within 24 hours (Friedman and Weisbroth 1977). The complete life cycle thus requires 23 days. Adults do not suck blood but instead feed on interstitial fluid. Access to the interstitial fluids of the host is also an important route by which the host is exposed to parasite antigens. Transmission of Myobia from host to host is primarily by direct transfer of female mites. Neonates may become infested as early as the seventh to eighth day as the hair coats appear. The mites are thermotactic and crawl out to the ends
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A
B
C
D
Fig. 23-2 (A) Myobia musculi, male. (B) Myobia musculi, female. (C) and (D) Ulcerative lesion of the skin in myobic acariasis of a C57BL strain mouse (C) and random-bred Swiss-Webster mouse (D). Figures C and D reprinted from The mouse in biomedical research, Vol. 2, H. L. Foster, J. D. Small, and J. G. Fox (Eds.), Arthropods, Weisbroth S.H., Pages 385–402, Copyright 1982, Academic Press, with permission from Elsevier.
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23. ARTHROPODS
of the hairs of dead hosts. There they await the passage of new hosts or crawl out in all directions to contaminate the environment. New infestations are characterized by an initial increase in mite populations, followed by a decrease as host defense mechanisms are mobilized (Friedman and Weisbroth 1975a,b; Weisbroth et al. 1974). Mite populations decrease until an equilibrium level is reached after 8 to 10 weeks, where the host cannot eradicate the parasite, but parasitic populations cannot significantly expand. The equilibrium population level may be carried for long periods of time, even years. Cyclic fluctuations of 20 to 25 days in the equilibrium population level may represent waves of egg hatches (Friedman and Weisbroth 1977; Weisbroth et al. 1974). 2.
Clinical Symptoms
Clinical signs associated with M. musculi infestation are also variable (Cook 1953) and become more severe as mice age (Galton 1963). When present, clinical signs range from mild alopecia and reddening of the skin to extreme pruritus with self-excoriation and deep ulceration (Fig. 23-2) (Iijima et al. 2000). Alopecia begins in the flank area but may become generalized (Galton 1963). Deep ulcerations may be complicated by secondary infection (Galton 1963; Weisbroth et al. 1974). Systemic alterations may include decreased lifespan (Whitely and Horton 1962), decreased body weight (Galton 1963), development of an IgE antibody response (Iijima et al. 2000), and decreased reproductive indices (Weisbroth et al. 1976).
Susceptible Hosts
M. musculi infests wild and laboratory mice and, to a lesser extent, rats and possibly other closely related rodents. Though once considered nearly ubiquitous in mouse colonies, infestations are now uncommon. In a large survey recently conducted at a commercial diagnostic laboratory, infestation was detected in only 0.12% of mice submitted (Livingston and Riley 2003). Mouse strains differ in susceptibility and clinical response to mite infestation. Strains derived from C57BL/6 mice are genetically predisposed to more severe forms of dermatitis (Dawson et al. 1986). More recently, NC/Jic mice were reported to experience atopic dermatitis associated with M. musculi infestation (Iijima et al. 2000). 3.
4.
5.
Diagnosis
A very effective method of diagnosing M. musculi infestations involves applying a 5.5-cm by 10-cm strip of transparent shipping tape to the back of an euthanatized mouse. The tape is left in place for at least 6 hours (West et al. 1992). Other, less reliable methods include direct examination of the pelage of dead or anesthetized mice under a bright light and dissecting microscope as described for Polyplax serrata. The hair over the dorsum, nuchal crest area of the skull, the cervical dorsum, and the area between the scapulae generally yield the greatest number of parasites. The hair in these areas should be parted and examined for moving or stationary mites (Gambles 1952; Weisbroth et al. 1974).
Pathobiology
Gross skin changes vary from unapparent, to mild dermatitis, to more severe lesions that include ulceration and pyoderma. Lesions are more common on the head, neck, shoulders, and flank (Cook 1953; Galton 1963). There appears to be no direct relationship between mite numbers and severity of lesions (Weisbroth et al. 1976). In uncomplicated chronic infestations, the epithelium is mildly hyperkeratotic. There is an increase in the presence of chronic inflammatory cells underlying the epithelium and an increased rate of epithelial cell mitosis (Galton 1963; Whitely and Horton 1962, 1965). More severe lesions include ulceration and pyoderma (Weisbroth et al. 1976). Lesions are commonly observed in mice of the C57BL/6 strain, and some of its sublines (Dawson et al. 1986). Histologic as well as clinical characteristics support an immediate hypersensitivity component (Iijima et al. 2000; Weisbroth et al. 1976). The “normal” haired skin adjacent to ulcerative zones resembles that described above for chronic, uncomplicated cases. Systemic pathology has also been reported and may include localized lymphadenopathy, variable splenic hypertrophy, epicarditis, pleural thickening, secondary amyloidosis, hypergammaglobulinemia, hypoalbuminemia, and decreased mean hemoglobin concentration (Csiza and McMartin 1976; Galton 1963; Weisbroth et al. 1976).
6.
Treatment
Insofar as treatment programs are concerned, it is important to note that if an acaricide is ineffective on in ova forms, and unless sufficient residual activity remains, a second application should take place sometime after day 8, when all eggs will have hatched, but before day 16, when new adults may have laid new eggs. If needed, a second application should be applied on day 10–12 (Friedman and Weisbroth 1977). Few acaricidal treatments are 100% effective. Therefore, the survival of even a few mites constitutes a potential source of colony reinfestation. For this reason mite infestations may be difficult to eradicate. In many cases, mite infestation is a recurring problem, with complete resolution seemingly unattainable. Final eradication may require rederivation of the colony. Historically, many compounds have been used in attempts to eradicate mite infestations. Currently used acaricides are generally as, if not more, effective than older treatments and are less toxic to the mouse host. One of the more commonly used treatments involves ivermectin (1%), diluted 1:100 with a mixture of propylene glycol and water (1:1) (Baumans et al. 1988). Others (Iijima et al. 2000), including this author, simply dilute the ivermectin in distilled water. The mixture is sprayed, or misted, over the mice and falls on the mice, bedding, and feed.
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Three weekly treatments using this formulation did not eradicate mites from the mouse colony but did result in negative ectoparasite examinations for up to 18 weeks after the last treatment (Baumans et al. 1988). Ivermectin (200 µg/kg body weight) has also been administered subcutaneously for the treatment of mite infestation (Wing et al. 1985). In that study, two subcutaneous injections given one week apart eliminated mites for up to 35 days, which was the duration of the study. One injection reduced, but did not eliminate, the infestation. Others have reported that a single injection of a higher dose (300 µg/kg) will eradicate the infestation (Vachon and Aubry 1996). However, in that study, follow-up examinations were performed for only a few weeks. Lastly, ivermectin administered in drinking water at 1 ml/l (10 µg ivermectin/ml), 2.5 ml/l (25 µg ivermectin/ml), or 5 ml/l (50 µg ivermectin/ml) for 4 consecutive days successfully eliminated infestation with M. musculi and Myocoptes musculinus (Papini and Marconcini 1991). Systemic effects of the avermectins were noted in the section on Polyplax serrata. Permethrins have also been used to control mite infestations when used regularly as a dip or as a dust applied to the bedding or the mice (Bean-Knudsen et al. 1986). Lastly, chlorpyrifos (Dursban) has been applied to bedding and found effective when 6 g Dursban were added at cage changing twice per week for 3 weeks (Pence et al. 1991). At that dosage,
A
mite infestations were eradicated, and no clinical signs of toxicity were observed. However, in some mice, brain acetylcholinesterase levels declined but then returned to normal following completion of the treatment regimen (Pence et al. 1991).
7.
Prevention and Control
Because mite infestations may be difficult to eradicate and because infestation may alter host physiology, prevention is preferred over treatment. Mite infestation may be prevented by purchasing mice only from reputable sources, carefully examining preshipment health records, and excluding feral rodents from the animal facility.
C. 1.
Myocoptes musculinus
Description and Life Cycle
Myocoptes musculinus is a nonburrowing mite in the family Myocoptidae. Adult females measure about 0.30 to 0.38 mm in length, while adult males are shorter, measuring about 0.16 to 0.21 mm (Fig. 23-3) (Gambles 1952; Watson 1960).
B
Fig. 23-3 (A) Myocoptes musculinus, male. (B) Myocoptes musculinus, female. From: Flynn, R. J. 1955. Ectoparasites of mice. Proc. Anim. Care Panel 6, 75–91. Reprinted with permission from AALAS.
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23. ARTHROPODS
M. musculinus is easily differentiated from Myobia musculi by the characteristic morphology of the third and fourth pairs of legs. In the adult female, the third and fourth pairs of legs are dark brown and heavily chitinized, a modification for clasping the hairs of the host. In the males, the third pair of legs is similarly modified, while the fourth pair of legs is greatly enlarged, oriented posteriorly, and used for grasping the female during copulation. Life-cycle stages include the egg, larva, two nymphal stages, and adults. The eggs average 0.20 x 0.045 mm in size and hatch within 5 days (Gambles 1952; Watson 1960). Myocoptes eggs are usually attached to the proximal part of the hair shaft, in contrast to Myobia sp. eggs, which are attached closer to the base of the shaft (Watson 1960). Larvae have three pairs of legs, while nymphs and adults have four pairs of legs. The life cycle is completed in approximately 14 days (Watson 1960). It has been generally recognized that M. musculinus is a more ambulatory species than M. musculi and tends to spread out over greater areas of the body. Particularly in mixed infestations with M. musculi, M. musculinus has some predilection for the skin of the inguinal areas, abdominal ventrum, and back. In monospecific infestation, it will occupy the face, head, and neck as well. In heavy mixed infestations, M. musculinus has some tendency to crowd out populations of M. musculi (Gambles 1952; Watson 1961). Transmission has been shown to require close, direct contact, and can occur in 24 hours or less when they are caged with infested mice. Therefore, contaminated bedding is not considered an effective fomite for transmission (Watson 1961). Infestation of neonates may occur within 4 to 5 days, with mites attaching to the vibrissae of the young (Watson 1961). All life stages of M. musculinus appear to be active in migrations to new hosts.
5 weeks of age and detectable serum IgE antibodies by 6 weeks of age (Laltoo et al. 1979). Evidence of cutaneous allergy and immunological disorder has also been demonstrated in BALB/c and NC/Kuj mice, with extensive mast cell infiltration in cutaneous lesions and lymphoid tissues, elevated serum IgE, IgA, and IgG1 but reduced IgM and IgG3 levels; hypergammaglobulinemia; lymphocytopenia; granulocytosis; and increased in vitro IL-4 and decreased IL-2 production (Jungmann et al. 1996a,b; Morita et al. 1999). 4.
M. musculinus has been described as a surface dweller that feeds on superficial epidermal layers (Watson 1961). Young or lactating mice may be more severely affected (Cook 1953). The signs of infestation may be unapparent or may involve patchy to generalized alopecia, pruritus, erythema, cutaneous ulcers, lymphadenopathy, slowed growth, and wasting (Cook 1953; Gambles 1952; Jungmann et al. 1996a). 5.
Susceptible Hosts
M. musculinus infests mice. A single report of M. musculinus infestation of guinea pigs has been described (Sengbusch 1960). Historically, the prevalence of this parasite was high in laboratory animal facilities. However, improvements in animal procurement, husbandry, and pathogen control have greatly reduced prevalence. In a large survey recently conducted at a commercial diagnostic laboratory, infestation was detected in only 0.10% of mice submitted (Livingston and Riley 2003). Mouse strains differ in their susceptibility to myocoptic dermatitis, with mice from C57BL/6 and NC lines generally considered more susceptible and mice from BALB/c lines considered less so. The literature does not show complete agreement on this point, however (Jungmann et al. 1996a; Morita et al. 1999). 3.
Pathobiology
Chronic infestation with M. musculinus induces epidermal hyperplasia and an increase in the mitotic rate of adult skin, but not that of adolescent mice (Watson 1961). An inflammatory infiltrate of macrophages and lymphocytes was seen in the subcuticular tissues underlying infested epidermis. Infested neonatal SWR mice developed positive skin tests to mite antigens by
Diagnosis
Infestations may be reliably diagnosed by the dorsal tape test as described for M. musculi or by direct examination of the pelage of dead or anesthetized mice under a bright light and dissecting microscope as described for Polyplax serrata. The hair over the inguinal areas, abdominal ventrum, and dorsum generally yields the greatest number of parasites. The hair in these areas should be parted and examined for moving or stationary mites. 6.
2.
Clinical Symptoms
Treatment
Treatment regimens for M. musculinus infestation are essentially as described for M. musculi. Two subcutaneous injections of ivermectin (200 µ/kg body weight), given one week apart, effectively eliminated mites (Wing et al. 1985). Ivermectin administered in drinking water at 1 ml/l (10 µg ivermectin/ml), 2.5 ml/l (25 µg ivermectin/ml), or 5 ml/l (50 µg ivermectin/ml) for 4 consecutive days also successfully eliminated infestation with M. musculinus in one report (Papini and Marconcini 1991). However, in another report, administration of ivermectin in the drinking water (32 mg/L) for three 10-day periods separated by a 7-day rest period did not result in complete elimination of mites until 9 weeks after the last treatment (Conole et al. 2003). Likewise, spraying mice with 1% ivermectin diluted 1:100 did not eradicate mites from the mouse colony after three weekly treatments but did result in negative ectoparasite examinations for up to 18 weeks after the last treatment (Baumans et al. 1988). As for many other acariases, final eradication may occasionally require rederivation of the colony. 7.
Prevention and Control
Mite infestation may be prevented by purchasing mice only from reputable sources, carefully examining preshipment health
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records, and excluding feral rodents from the animal facility. Quarterly monitoring of sentinel animals, along with careful examination of all mice with dermal lesions, are important components of the veterinary care program.
of the life cycle may be found in an infested hair follicle (Flynn 1959). Common sites of infestation include the head, interscapular area, and lumbar region. 2.
D. 1.
P. simplex has been reported from Mus musculus and Mastomys natalensis, the multimammate rat. Several years ago, the mite was commonly seen in laboratory mice in various parts of the world (Cook 1956; Flynn 1955). However, in a large survey recently conducted at a commercial diagnostic laboratory, infestation was not detected in over 14,000 mice submitted (Livingston and Riley 2003). Therefore, P. simplex may currently be regarded as rare. It has not been reported as a parasite of laboratory mice in many years. Little information is available concerning the effect of mouse strain or stock on susceptibility to infestation. In one study, Swiss stock were infested, while those of C3H, BALB/c, DBA, A, and C57 lines were not (Flynn 1955). In another study, mice of the “Porton” strain were infested (Cook 1956). This strain is believed to have originated from albino Swiss mice (Cook 1953).
Psorergates simplex
Description and Life Cycle
Psorergates simplex is a nonburrowing, follicle-dwelling mite in the family Psorergatidae. Adult mites are nearly round and measure roughly 100 to 120 µm (Tyrrell 1883). Like other members of the order Acarina, nymphs and adults have eight legs. The mite can be distinguished from other, similarly sized mites by the presence of a large, inwardly directed, curved spine located on each femur. The sexes may also be differentiated. The adult female bears two pairs of very long, posterior setae, while the adult male bears only one pair (Wall and Shearer 2001). Surprisingly little is known of the life cycle of P. simplex. Stages in the life cycle include the egg, larva, nymph, and adult (Fig. 23-4). Transmission is by direct contact. All stages
A
Susceptible Hosts
B
Fig. 23-4 (A) Psorergates simplex-infested skin inverted to show pouches (arrow). (B) Psorergates simplex, pouch contents: egg (E), adult female (F), adult male (M), larva (L), nymph (N). Reprinted from The mouse in biomedical research, Vol. 2, H. L. Foster, J. D. Small, and J. G. Fox (Eds.), Arthropods, Weisbroth S. H., Pages 385–402, Copyright 1982, Academic Press, with permission from Elsevier.
575
23. ARTHROPODS
3.
Pathobiology
Infestation of the hair follicles with P. simplex may result in formation of small (up to 2 mm), whitish, cyst-like dermal nodules on the legs, back, trunk, abdomen, shoulders, and head (Fig. 23-4) (Flynn 1959; Flynn and Jaroslow 1956). These are thought to occur as a result of epidermal growth to accommodate the internal pressure of space-occupying mites. Histologically, the structures resemble comedones and consist of invaginated sacs of squamous epithelium filled with mites, their products, and keratinaceous debris. Typically, the epidermal lining is intact. Inflammation is variable (Flynn 1955; Flynn and Jaroslow 1956). 4.
Clinical Symptoms
Skin lesions produced by this mite are often unapparent in live hosts. Nodule formation tends to be more common in the skin of the head, shoulders, and lumbar areas but may occur anywhere. Nodules are larger in loose skin of the trunk (2 mm) and smaller where the skin is tighter (1 mm), as in the face and legs. Because lesions form at the base of the hair follicles, they may best be observed on the inverted or subcuticular surface of the skin (Flynn 1955). In contrast, the auricular form, although it develops slowly, may have a severe, mange-like appearance, with crusting of one or both surfaces of the pinna (Cook 1956). In one study, auricular lesions were not observed in mice of the Porton strain less than 12 weeks old, and those were only identifiable with the aid of a hand lens. Grossly, macroscopic lesions were not typically observed until mice reached about 5 to 6 months old (Cook 1956). 5.
Diagnosis
P. simplex can be diagnosed by examination of the inverted subcuticular surface of the pelt for characteristic nodules. Pouch contents may be expressed by the pressure of a scalpel blade or by scraping, and the contents can be mounted under a cover slip in water, glycerine, or 10% KOH for microscopic examination.
Quarterly monitoring of sentinel animals, including careful examination of the inverted subcuticular surface of the pelt during routine health surveillance necropsies, are important components of a preventive health program. E. 1.
2.
Susceptible Hosts
Radfordia sp. has been identified from laboratory mice in many parts of the world (Flynn 1955, 1963; Flynn et al. 1965; Seamer and Chesterman 1967). Historically, infestation rates were high. Improvements in animal husbandry have so reduced the incidence of ectoparasitism that Radfordia sp. is rarely diagnosed. In a large survey recently conducted at a commercial diagnostic laboratory, infestation with R. affinis and R. ensifera was detected in only 0.01% and 0.02%, respectively, of mice submitted (Livingston and Riley 2003). Pathobiology and Clinical Symptoms
Treatment
Little information is available on the treatment of psorergatic mange in any animal species. It is likely that treatment regimens utilizing ivermectin, as described for other mites discussed in this chapter, will also effectively eliminate P. simplex. It is unknown whether final eradication may occasionally require rederivation of the colony. 7.
Description and Life Cycle
Radfordia affinis and R. ensifera are nonburrowing mites in the family Myobiidae (Fig. 23-5). R. affinis is thought to infest only mice, while R. ensifera is thought to infest only rats. However, these host associations are not strict specificities, but should more accurately be considered preferences. Mice may be infested with both mite species (Livingston and Riley 2003). Radfordia sp. is biologically and morphologically similar to M. musculi. Scrutiny of the tarsal terminus of the second pair of legs enables differentiation of Radfordia sp. from M. musculi. Radfordia sp. has two tarsal claws of unequal length (Fig. 23-5), where M. musculi has a single empodial claw. In turn, R. affinis may be differentiated from R. ensifera again by reference to the tarsal claws of the second pair of legs. The claws are paired and unequal in length in R. affinis and paired and equal in length in R. ensifera (Ewing 1938). Little is known of the life cycle of Radfordia sp. It is assumed that the life cycle is similar to that of Myobia musculi.
3. 6.
Radfordia sp.
Virtually nothing is known of the pathobiology or clinical symptoms of Radfordia sp. It is likely that lesion development and clinical signs are similar to those described for M. musculi. 4.
Diagnosis
Radfordia sp. infestations may be reliably diagnosed using the dorsal tape test as described for M. musculi (West et al. 1992).
Prevention and Control
P. simplex is rare in modern rodent facilities. Infestation may be prevented by purchasing mice only from reputable sources and by excluding feral rodents from the animal facility.
5.
Treatment, Prevention, and Control
Little is known of these aspects of the biology of Radfordia sp., but it is likely that approaches developed for M. musculi will also
576
D AV I D G . B A K E R
A
B
Fig. 23-5 (A) Radfordia affinis, male. (B) Radfordia affinis, female. Arrow indicates two terminal tarsal claws on leg II, versus one claw on Myobia musculi. Reprinted from The mouse in biomedical research, Vol. 2, H. L. Foster, J. D. Small, and J. G. Fox (Eds.), Arthropods, Weisbroth S. H., Pages 385–402, Copyright 1982, Academic Press, with permission from Elsevier.
be successful for Radfordia sp. In one report, microdot delivery of 1% ivermectin (2 mg/kg body weight) to the dorsal skin, given three times at approximately 2-week intervals, eliminated R. ensifera from a colony of Long Evans rats (Kondo et al. 1998).
F. 1.
Trichoecius romboutsi
Description and Life Cycle
Trichoecius romboutsi is a nonburrowing mite in the family Myocoptidae. It was originally described from Holland in 1946 and named Myocoptes romboutsi (Van Eyndhoven 1946). The mite was redescribed in 1970 and moved to the genus Trichoecius (Fain et al. 1970). The life cycle and many other aspects of its biology are poorly known. Life-cycle stages include the egg, larva, two nymphal stages, and adult (Fain et al. 1970). Males are approximately 0.16 to 0.19 mm in length, and females 0.20 to 0.28 mm (Fig. 23-6) (Fain et al. 1970). T. romboutsi resembles M. musculinus in appearance but is smaller. Indeed, it has been pointed out that this resemblance may account for T. romboutsi being overlooked in diagnosis (Flynn 1973).
2.
Susceptible Hosts
The mouse is the only known host for T. romboutsi. The mite was found in a survey of commercial mouse breeders in the United States in 1955 (Flynn 1955). However, in a similar survey 10 years later (Flynn et al. 1965), the mite was not observed. Still more recently, T. romboutsi was not detected in a large survey conducted at a commercial diagnostic laboratory (Livingston and Riley 2003). 3.
Pathobiology
Little is known of the pathogenicity of T. romboutsi. However, because of its biological similarity to M. musculinus, it is likely that T. romboutsi may cause dermatitis, epidermal hyperplasia, and cellular and serologic indicators of cutaneous allergy in a similar manner. 4.
Clinical Symptoms
There is also a scarcity of information on the clinical symptoms caused by T. romboutsi. It is likely that clinical symptoms are similar to those induced by M. musculinus and include
577
23. ARTHROPODS
A
B
Fig. 23-6 (A) Trichoecius romboutsi, male. (B) Trichoecius romboutsi, female. From: Flynn, R. J. 1955, Ectoparasites of mice. Proc. Anim. Care Panel 6, 75–91. Reprinted with permission from AALAS.
patchy to generalized alopecia, pruritus, erythema, cutaneous ulcers, lymphadenopathy, slowed growth, and wasting. 5.
Diagnosis, Treatment, Prevention, and Control
These aspects of infestation with T. romboutsi are likely to be similar to those described for M. musculinus.
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Shaughnessy, H.J. (1963). Tularemia. In Diseases transmitted from animals to man (T. G. Hull, Ed.), 5th ed., pp. 588–604. Thomas, Springfield, Illinois. Skopets, B., Wilson, R. P., Griffith, J. W., and Lang, C. M. (1996). Ivermectin toxicity in young mice. Lab. Anim. Sci. 46, 111–112. Sosnina, E. F., Nazarova, I. V., and Sadekova, L. K. (1981). Lice (Anoplura) of small mammals in the Volga-Kama Preserve. Parazitologiia 15, 157–162. Sparrow, S. (1976). The microbiological and parasitological status of laboratory animals from accredited breeders in the United Kingdom. Lab. Anim. 10, 365–376. Toth, L. A., Oberbeck, C., Straign, C. M., Frazier, S., and Rehg, J. E. (2000). Toxicity evaluation of prophylactic treatments for mites and pinworms in mice. Contemp. Topics Lab. Anim. Sci. 39, 18–21. Tyrrell, J. B. (1883). On the occurrence in Canada of two species of parasitic mites. Proc. Can. Inst. 1, 332–342. Uchikawa, K., and Rack, G. (1979). Eulaelaps stabularis (Kock, 1839) and Eulaelaps oudemansi Turk, 1945 (Mesostigmata: Haemogamasidae). Acarologia 20, 163–172. Vachon, P., and Aubry, L. (1996). The use of ivermectin for the treatment of mites, Myobia musculi and Myocoptes musculinus in a colony of transgenic mice. Can. Vet. J. 37, 231–232. Van Eyndhoven, G. L. (1946). Diagnoses of two epizootic mites. Entomol. Ber. 12, 30–31. Wall, R., and Shearer, D. (2001). Veterinary ectoparasites: Biology, pathology, and control. Blackwell Science Ltd., London. Watson, D. P. (1960). On the adult and immature stages of Myocoptes musculinus (Koch) with notes on its biology and classification. Acarologia 2, 335–344.
579 Watson, D. P. (1961). The effect of the mite Myocoptes musculinus (C. L. Koch, 1840) on the skin of the white laboratory mouse and its control. Parasitology 51, 373–378. Weisbroth, S. H., Friedman, S., and Sher, S. (1976). The parasitic ecology of the rodent mite, Myobia musculi. III. Lesions in certain host strains. Lab. Anim. Sci. 26, 725–735. Weisbroth, S. H., Friedman, S., Powell, M., and Scher, S. (1974). The parasitic ecology of the rodent mite Myobia musculi. I. Grooming factors. Lab. Anim. Sci. 24, 510–516. West, W. L., Schofield, J. C., and Bennett, B. T. (1992). Efficacy of the “micro-dot” technique for administering topical 1% ivermectin for the control of pinworms and fur mites in mice. Contemp. Top. Lab. Anim. Sci. 31, 7–10. Whitely, H. J., and Horton, D. L. (1962). The effect of Myobia musculi on the epidermis and hair regrowth cycle in the aging CBA mouse. J. Pathol. 83, 509–514. Whitely, H. J., and Horton, D. L. (1965). Further observations on the effect of Myobia musculi on the skin of the mouse. J. Pathol. 89, 331–335. Wing, S. R., Courtney, C. H., and Young, M. D. (1985). Effect of ivermectin on murine mites. J. Am. Vet. Med. Assoc. 187, 1191–1192. Yunker, C. E. (1964). Infections of laboratory animals potentially dangerous to man: ectoparasites and other arthropods, with emphasis on mites. Lab. Anim. Care 14, 455–465. Zumpt, F., and Till, W. M. (1956). Notes on Haemolaelaps glasgowi (Ewing) and related forms in the Ethiopian region, with descriptions of four new species (Acarina: Laelaptidae). Z. Parasitenkd. 17, 282–291.
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Chapter 24 The Tumor Pathology of Genetically Engineered Mice: A New Approach to Molecular Pathology Robert D. Cardiff, Robert J. Munn, and Jose J. Galvez
I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Disclaimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Principle 1 Oncogenic events initiating tumorigenesis profoundly influence the morphological phenotype of the tumor. . . . . . . . . . . . . . . . . . . . . . . Axiom I: Spontaneous, virus-induced, and chemical-induced mouse tumors do not mimic the microscopic structure of human cancers. . . . Retrovirus-Related Background Pathology . . . . . . . . . . . . . . . . Nonviral Spindle Cell Tumors I . . . . . . . . . . . . . . . . . . . . . . . . . Spontaneous Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemically Induced Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . Axiom II: Genetically engineered mouse (GEM) tumors are unique . . . . Corollary A: Oncogenic alterations in molecular function lead to gene-specific microscopic structural changes. Corollary B: Oncogenic alterations in molecular function lead to molecular pathway-specific microscopic changes. Corollary C: Some genes have a stronger influence than others on the microscopic structure of the tumor. . . . . Corollary D: Expression levels of the oncogenic transgenes influence the microscopic structure of the tumor. Corollary E: Tumor suppressor genes only indirectly affect the microscopic structure of the resulting tumors. . . Axiom III: Some molecular alterations have minimal influence on the microscopic structure of tumors. . . . . . . . . . . . . . . . . . . . . . . . Corollary A: The promoter has minimal effect on the microscopic structure of the tumor. . . . . . . . . . . . . . . . . . . . . Corollary B: Mutational changes in the initiating oncogenic transgene will not change the fundamental microscopic structure of the resulting tumor. . . THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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Corollary C: The site of oncogene insertion has little or no effect on the microscopic structure of the resulting tumor Principle 2: Tumorigenesis is influenced by tissue context . . . . . . . . . . . . . . . . . Axiom I: The microscopic structure of tumors initiated by weak oncogenes can be influenced by tissue context. . . . . . . . . . . . . . . . . . . . . . Axiom II: The tissue and other factors will influence the tumor biology but not the tumor morphology. . . . . . . . . . . . . . . . . . . . . . . . . . Corollary A: GEM tumors exhibit molecular relationships and interdependencies. . . . . . . . . . . . . . . . . . . . . . . . Axiom III: The microscopic structure of tumors is not influenced by external factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corollary A: Microorganisms can be co-factors in GEMassociated tumors, but they do not influence the microscopic structure of the tumors. . . . . . . . . . Corollary B: The microscopic structure of the neoplasm is not affected by the background strain. . . . . . . . . . . . Corollary C: Host modifier genes do not affect the microscopic structure of transgenic tumors. . . . . . . . . . . . . . Principle 3: Neoplastic progression is a multistep process associated with sequential morphological changes. . . . . . . . . . . . . . . . . . . . . . . . . . . Axiom I: Neoplastic progression is associated with a sequential continuum of microscopic changes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corollary A: GEM neoplasia progresses through the same series of structural changes as human neoplasia. . . . . Corollary B: Premalignant lesions are atypical focal hyperplasias associated with high risk of malignant transformation. . . . . . . . . . . . . . . . . . Corollary C: Biological potential is best defined by transplantation. . . . . . . . . . . . . . . . . . . . . . . . . . Axiom II: Regional or systemic metastasis is a more reliable criterion of malignant potential than microinvasion. . . . . . . . . . . . . . . . . . . Corollary A: The primary route of metastasis in the mouse is via tumor emboli. . . . . . . . . . . . . . . . . . . . . . . Principle 4: Mouse tumors mimic many aspects of human cancers. . . . . . . . . . Axiom I: GEM tumors accurately model most aspects of human carcinogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Comparative Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Use of “Controlled” Vocabulary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Digital Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Morphometrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Conditions That Affect Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . III. Summary and Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION A.
Background
Genetically engineered mice (GEM) develop novel disease patterns that create new challenges for comparative pathology which our predecessors could not have envisioned (Cardiff, et al. 2004; Foster et al. 1982; Mohr et al. 1996). The fundamental genetic alterations introduced into GEM have unique effects on the microscopic structure of tumors. For the first time in history, we have an accurate picture of how specific abnormalities of
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molecular function influence tumor morphology. Gene-specific tumor phenotypes can now be recognized (Figs. 24-1 and 24-2). The original observations were made almost two decades ago, but the numbers and types of GEM models of tumor pathology have expanded to include virtually all murine organ systems (Cardiff et al. 1988a). Most important, the microscopic patterns of human and GEM cancers are nearly identical when carrying the same genetic aberrations (Fig. 24-1). The currently available models provide the basis for organizing these newer observations and for assessing their significance, their ramifications, their internal contradictions, and their applicability to existing scientific paradigms. This chapter is the first step in an
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Fig. 24-1 Mouse-Human Comparisons Axiom: Genetically engineered mouse (GEM) tumors are unique. The microscopic patterns of classical MMTV-induced mouse mammary tumors (Figs. 24-1A and 24-1B) are structurally different from the histological patterns of GEM tumors (Figs. 24-1C and 24-1D). Remarkably, the GEM tumors more closely resemble human breast cancer (Figures 24-1E and 24-1F) than the virus-induced tumors (Figs. 24-1A and 24-1B). Digital image Fig. 24-1A illustrates the microacinar pattern described as Type A by Thelma Dunn (Dunn 1953; Sass and Dunn 1979). Note the small clusters of cells surrounded by myoepithelium. Fig. 24-1B illustrates the more solid, cord-like Type B pattern of Dunn. Note the peripheral palisade of cells adjacent to the vascular channels. Fig. 24-1C illustrates a tumor from a Tm(Cdh1-/-) mouse with loss of e-Cadherin function. The loss of e-Cadherin results in dyscohesive tumor cells that tend to infiltrate in single file through a dense connective tissue stroma. Fig. 24-1D illustrates a field from a Tg(Erbb2) with high levels of doxycycline-induced Erbb2 expression with irregular cords and nests in a dense connective tissue stroma. Fig. 24-1E shows the classical single-file pattern of a human lobular carcinoma of the breast associated with silencing of e-Cadherin. Compare this pattern with the pattern of the analogous mouse tumor (Fig. 24-1C). Fig. 24-1F illustrates a comparable pattern in a human breast cancer with amplification of the Erbb2 amplicon and overexpression of Erbb2. Compare with the mouse tumor (Fig. 24-1D). The Erbb2 overexpression results in clumps of cells with abundant cytoplasm and oval nuclei with a delicate chromatin pattern in both GEMs and humans. All images were captured using the Zeiss AxioCam with a 40X objective. The scale bar for all images is in Fig. 24-1F. Details for each image are in Table 24-2. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
iterative process that seeks to summarize, organize, and assess the new functional-structural knowledge base and how it applies to naturally occurring cases in other animals and humans. The current interpretations of pathology harken back to Virchow’s cellular basis of disease, Omnis cellula et cellula (Foster et al. 1982). Our diagnostic lexicons are still organized primarily by organs and tissues. The WHO, SNOMED-RT, CPT, and similar controlled vocabularies, or coding systems, ask that neoplastic diseases be named or coded using process followed by the organ or tissue (e.g., Adenocarcinoma, Colon). Subclassifications of organ-specific neoplasms generally modify tumor names according to cell type or presumed cell of origin (e.g., Mucinous Adenocarcinoma, Colon). These terms reflect how we think and are the generally accepted, clinically relevant views of neoplastic diseases. The limitations of this approach have been discussed by others (Berman 2004). The molecular revolution and the sequencing of genomes have brought tremendous new insights into disease mechanisms but have had relatively little impact on the discipline of anatomic pathology. Pathologists give credence to molecular pathology, but this new knowledge has thus far not resulted in fundamental changes in the organ- and cell-centric approach to pathology. We continue to think and teach primarily in terms of the organs and cells of origin. Although the organ systems approach is not necessarily inaccurate or incorrect, the time has come to assess the structure of neoplastic disease from the perspective of the genes and their products. As director of the National Cancer Institute, Dr. Richard Klausner encouraged the reclassification of cancers with a program called the Director’s Challenge (Berman 2004). The program supported efforts to classify a number of human malignancies based on their molecular profiles. Although based on the new genomics and greeted with enthusiasm (Gabrielson et al. 2001), the Director’s Challenge has not resulted in a new classification of neoplastic diseases in humans (Berman 2004). The current molecular classifications are somewhat limited to
the recognition that certain molecular changes correlate with specific types of tumors in a given organ (Anbazhagan et al. 1999; Golub et al. 1999; Jain 2004; Parmigiani et al. 2004; Ross et al. 2004; Ross et al. 2003; Sorlie 2004). Perhaps our thinking in this realm has been hindered by the seemingly hopeless molecular, morphological, and biological complexity of spontaneous human and animal neoplasms (Berman 2004). Such a multistep, multimolecular process creates morphological alterations that are difficult both to identify and to relate to the morphological changes associated with individual genes. GEM technology has opened new possibilities for the comparative pathologist because these animals allow us to evaluate the effect of individual molecules and combinations of molecules on the structure of tumor cells. In contrast to the seemingly hopelessly complex case of human tumors, the GEM tumors offer a controlled experimental environment leading toward meeting the Director’s Challenge. Perhaps the structural and functional insights gained from such a vantage point will improve the prevention and treatment of neoplastic diseases in all species by facilitating earlier and more accurate diagnoses (Berman 2004). This chapter will marshal the evidence justifying the creation and organization of the new tumor pathology, based, however, on four established principles of comparative tumor biology. Principle 1: Oncogenic events initiating tumorigenesis profoundly influence the morphological phenotype of the tumor. Principle 2: Tumorigenesis is influenced by tissue context. Principle 3: Neoplastic progression is a multistep process associated with sequential morphological changes. Principle 4: Mouse tumors mimic many aspects of human cancers. The new concepts concerning structure and function will be discussed within the framework of these four principles so that the reader can maintain orientation while evaluating the concepts. This chapter will consider how specific molecular changes in
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Fig. 24-2 Mouse Mammary Tumors with Signature Phenotypes Corollary: Oncogenic alterations in molecular function lead to gene-specific microscopic structural changes. This panel of six digital images illustrates the unique microscopic appearances associated with GEM-associated mammary tumors, each image with unique cytology and morphological patterns. Fig 24-2A. Mammary tumor in a Tg(Myc) mouse showing a glandular pattern with dark staining cells with large nuclei and very prominent nucleolus. Fig 242B: Mammary tumor in a Tg(Erbb2) mouse with a solid nodular pattern and distinct peripheral and central zones. The nuclear chromatin is relatively diffuse and pale. Fig 24-2C: Mammary tumor in a Tg(Ras) mouse. Note the solid nodular pattern with the distinctive peripheral nuclear palisade and the small size of the nuclei relative to those in 24-2A and 24-2B. Fig 24-2D: Mammary tumor in a Tg(Wnt1) mouse featuring a complex ductal architecture with two distinct cell layers. The outer layers contain paler myoepithelial cells. Fig. 24-2E: Mammary tumor in a Tg(C(3)SV40-Tag) mouse showing the relatively small cells with dense, overlapping nuclei and very scanty cytoplasm. Fig 24-2F: Mammary tumor in a Tm(e-Cadherin-/-) mouse with tumor cells infiltrating the dense connective tissue in single file and the solid clumps of cells. All images were captured using the Zeiss AxioCam with a 40X objective. The scale bar for all images is in Fig 24-2F. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
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Fig. 24-3 Myc-Related Signature Tumors Corollary: Some genes have a stronger influence than others on the microscopic structure of the tumor. Myc is an example of a dominant transgene associated with distinctive cytology but different patterns in most organs. Digital photomicrographs of Myc-associated mouse adenocarcinomas in prostate (Fig. 24-3A), mammary gland (Fig. 24-3B), liver (Fig. 24-3C), and lymphoma in a lymph node (Fig. 24-3D). Each neoplasm is composed of cells with prominent nucleoli and amphophilic or blue cytoplasm. The lymphoma cells (Fig. 24-3D) are dyscohesive with prominent nucleoli (Fig. 24-3D, inset). Myc is a dominant oncogene. All images were captured using the Zeiss AxioCam with a 40X objective. The scale bar for all 40x images is in Fig. 24-3D. The inset in Fig. 24-3D is expanded by a factor of two. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
the genome create unique microscopic tumor phenotypes in almost all mouse organ systems (Cardiff 1996). Many, but not all, of these unique tumors have characteristic, “signature” phenotypes that can be recognized in different organ systems (Figs. 24-1–24-7). When considered collectively, the tumors arising from genetic alteration of a given molecular pathway will share morphological characteristics belonging to that signal transduction pathway (Fig. 24-6). Tumors initiated by a given genetic change will undergo neoplastic progression that is similar to that observed in the so-called spontaneous tumors of mice, humans and other animals that have mutations in the same molecular pathway (Figs. 24-8–24-10). In the case
of GEM bearing different oncogenic transgenes, one transgene will dominate the morphological pattern of the tumor. The transgene dominance forms a recognizable molecular-structural hierarchy (Figs. 24-3 and 24-4). In contrast, tumor suppressor genes will generally produce a mixture of tumor phenotypes when they are silenced or knockedout (Fig. 24-7). The initiating oncogene will generally dominate the phenotype when paired with tumor suppressor gene knockouts or with other oncogenes (Fig. 24-7). Host modifiers, such as genetic background, age, gender, and immunological status, rarely have a significant effect on tumor morphology but do influence the biology of the tumor (Cardiff 1996).
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Fig. 24-4 SV40-Tag Related Signature Tumors Corollary: Some genes have a stronger influence than others on the microscopic structure of the tumor. SV40-Tag is a dominant gene that results in similar tumor phenotypes in most organs. Digital photomicrographs of SV40-Tag-associated malignancies in the brain (Fig. 24-4A), lung tumor to subcutaneous tissue (Fig. 24-4B), intestine (Fig. 24-4C), and prostate (Fig. 24-4D). A similar pattern of cells with scanty cytoplasm and light pink “stroma” is observed in all frames. When stained for neuroendocrine markers, these tumors are frequently positive. All images were captured using the Zeiss AxioCam with a 40X objective. The scale bar for all images is in Fig. 24-4D. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
These new concepts of molecular-centered pathology are working hypotheses that will be modified as additional models are studied. Hopefully, the new concepts, articulated here, will lay the foundation for future studies. Examples of the principles and concepts will be provided, illustrated, and discussed, and the limits of our knowledge will be evaluated. The application of the principles and concepts from one to all organ systems will be discussed along with the exceptions. The experienced comparative pathologist may find this molecule-centered approach to comparative pathology somewhat
frustrating because, as a new field, the evidence is incomplete. As a result of these limitations, the focus on the molecular origin, reviewed herein, also reveals the limits of our present knowledge. Unfortunately, the current GEM modelers do not generally design experiments that will fill these gaps in our knowledge of structure. Consequently, the picture is incomplete and may raise more questions than give answers. It will be incumbent upon future comparative pathologists to fill in the gaps, either by designing specific experiments or by accumulating and integrating disparate experiences. Our challenge is to create the “New Pathology.”
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Fig. 24-5 Ras-Related Signature Tumors Axiom: The microscopic structure of tumors initiated by weak oncogenes can be influenced by tissue context. Ras is an excellent example of a relatively weak transgene that results in different tumor phenotypes in each affected organ. Digital photomicrographs of Ras-associated lesions in brain (Fig. 24-5A), lung (Fig. 24-5B), bowel (Fig. 24-5C), and prostate (Fig. 24-5D). The brain lesion illustrates an oligodendroglioma (Fig. 24-5A), the lung a papillary adenoma (Fig. 24-5B), the gut an adenocarcinoma with a glandular pattern (Fig. 24-5C) and, in the prostate, an adenocarcinoma with intestinal metaplasia (Fig. 24-5D). All images were captured using the Zeiss AxioCam with a 40X objective. The scale bar for all images is in Fig. 24-5D. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
B.
Disclaimers
This chapter is based primarily on our own experience because the images found in published accounts are usually highly selective and individualized, and sometimes illustrate neither the key features of the lesion nor the biology of the lesion. Furthermore, the descriptions are not always accurate representations of the processes. Our experience is based on the University of California Mutant Mouse Pathology Laboratory Archive, which includes slides from over 17,000 GEM, over 300 GEM model systems from investigators in 14 countries, and the 10 organ-site workshop slide sets produced by the
National Cancer Institute’s Mouse Models of Human Cancers Consortium (Boivin et al. 2003; Borowsky et al. 2003, 2004; Cardiff 2003, 2004; Cardiff et al. 2000a; Kogan et al. 2002; Nikitin et al. 2004; Shappell et al. 2004; Weiss, et al. 2002). This experience has allowed us to compare tumors from one model with another and one organ system with another. This type of comparative study is relatively unique in GEM tumor pathology. Since most of the literature is based on the study of individual models, the pathology reported is also limited to that GEM model and that organ system. Therefore, the literature cited in this chapter is restricted either to specific examples that are generally the first papers on the subject
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F
Fig. 24-6 Pathway Pathology Corollary: Oncogenic alterations in molecular function lead to pathway-specific microscopic structural changes. Pathway pathology was first demonstrated in the mouse mammary gland by comparing tumors from transgenic mice from the Erbb and the Wnt pathways. Key features that distinguish mouse mammary tumors of the Erbb pathway (Figs. 24-6A and 24-6B) from the Wnt pathway (Fig. 24-6C–Fig. 24-6F) are: Erbb2 tumors are solid without acinar, ductular, or squamous differentiation (Fig. 24-6A) and do not stain with anti-K14 or other myoepithelial markers (Fig. 24-6B). In contrast, the Wnt1 pathway tumors may have microacinar patterns (Fig. 24-6C), keratin 14 positive myoepithelial cells (Fig. 24-6D), and branching duct dysmorphogenesis and squamous metaplasia (Figs. 24-6E and 24-6F). The branching ducts can be visualized using antikeratin 14 (Fig. 24-6F) (Arrow). The squamous elements are also highlighted with anti-K14 (asterisk). The WntI pathway tumors are identical to the spontaneous MMTV-induced tumors in feral and wild-type laboratory mice (Figs. 24-1A and 24-1B). All images were captured using the Zeiss AxioCam with a 40× or 10× objective. The scale bar for each pair of images is in Figs. 24-6B, D and F, respectively. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
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A
B
C
D
Fig. 24-7 Tumor Suppressor Genes Corollary: Tumor suppressor genes only indirectly affect the microscopic structure of the resulting tumors. GEM with tumor suppressor genes have a variety of tumor phenotypes that are related to the complementary or initiating oncogene. Digital images illustrating the relation of tumor suppressor genes to the structure of neoplasms. Compare the characteristic morphology of a Tg(Erbb2)-related mouse mammary tumor (Fig. 24-7A) with the neoplasms in Trp53 knockout mice (Figs. 24-7B, C). The uniform solid, nodular pattern with the typical zonal variation of the Tg(Erbb2)-related tumor cells (Fig. 24-7A) is markedly different from bigenic Tg(Erbb2)xTg(Trp53172+/–) mice (Fig. 24-7B) that have larger size, greater nuclear variation, and higher mitotic activity. The mammary tumor from the monogenic Tg(Erbb2) has a near diploid DNA. The tumor from the bigenic mouse is aneuploid. A tumor from a Tm(Trp53-/-) mouse has a completely different glandular pattern (Fig. 24-7C). The complementary oncogenes in this tumor are unknown. Fig. 24-7D demonstrates the back-to-back glandular pattern in a prostatic tumor from a Tm(Pten+/-) mouse. These tumors characteristically activate AKT1. As a result, this pattern is similar to other AKT1-related prostate tumors. All images were captured using the Zeiss AxioCam with a 40X objective. The scale bar for all images is in Fig. 24-7D. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
or to general review articles. Comprehensive surveys of specific GEM models should be sought elsewhere. Table 24-1 provides the reader with a list of the major genes used for engineering in the studies quoted here, with proper names, genetic symbols, and OMIM (Online Mendelian Inheritance in Man) identification code numbers; Table 24-2 provides a guide to some of the promoters commonly used for transgenesis; and Table 24-3 matches the genes to specific illustrations. The reader is encouraged to use the Internet sites maintained by the National Library
of Congress such as OMIM (http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=OMIM) or LocusLink (http://www.ncbi.nlm. nih.gov/LocusLink/), the Davis Human/Mouse Homology Relationships (http://www.ncbi.nlm.nih.gov/Homology/Davis/), and the Jackson Laboratory MGI site (http://www.informatics. jax.org/). These sites offer curated current information that is more invaluable and up-to-date than any hardbound textbook. Since this monograph is not an atlas, the black and white illustrations provided here are insufficient to present the
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TABLE 24-1
LIST OF GENES USED IN TEXT Gene function
Full OMIM name
Cell Cycle Growth Factor/cytokine Growth Factor/cytokine Growth Factor/cytokine Growth Factor/cytokine Growth Factor/cytokine Homeobox Gene Nuclear Factor: Transcription Nuclear Factor: Transcription Nuclear Factor: Transcription Nuclear Factor: Transcription Protease Signal Transduction: Development Signal Transduction: erbB
Cyclin D1 Colony-stimulating factor 1 Fibroblast growth factor Interleukin 10 Transforming growth factor, beta-1 Vascular endothelial growth factor Homeobox 3A V-ETS avian erythroblastosis virus E26 oncogene homolog 2 V-FOS FBJ murine osteosarcoma viral oncogene homolog V-MOS moloney murine sarcoma viral oncogene homolog Myelocytomatosis viral oncogene homolog Plasminogen; PLG Murine mammary tumor virus integration site 3 V-AKT murine thymoma viral oncogene homolog 1 V-ERB-B2 avian erythroblastic leukemia viral oncogene homolog 2 Human ERBB2 homolog Neuroblastoma gene V-HA-RAS harvey rat sarcoma viral oncogene homolog Insulin-like growth factor 1 receptor V-KI-RAS2 kirsten rat sarcoma 2 viral oncogene homolog Phosphatase and tensin homolog Polyomavirus middle T Catenin, beta-1 Casein kinase II, alpha-2 Glycogen synthase kinase 3-beta Wingless-type MMTV integration site family, member 1 Cadherin 1 Integrin, beta-1 Breast cancer 1 gene Breast cancer 2 gene Tumor protein p53 Phosphatase and tensin homolog Retinoblastoma SV40-T121 SV40-TAG
Signal Transduction: erbB Signal Transduction: erbB Signal Transduction: erbB Signal Transduction: erbB Signal Transduction: erbB Signal Transduction: erbB Signal Transduction: erbB Signal Transduction: erbB Signal Transduction: wnt Signal Transduction: wnt Signal Transduction: wnt Signal Transduction: wnt Structural Gene Structural Gene Tumor Suppressor Tumor Suppressor Tumor Suppressor Tumor Suppressor Tumor Suppressor Tumor Suppressor Tumor Suppressor
Text citation
Symbol*
OMIM**
Chromosome no. Human Mouse
cM***
p.598 p.605 p.595 p.604 p.605 p.604 p.600 p.605
Ccnd1 Csf1 Fgf3 Il10 Tgfb1 Vegfa Nkx3-1 Ets2
168461 120420 164950 124092 190180 192240 602041 164740
11q13 1p21-p13 11q13 1q31-q32 19q13.1 6p12 8p21 21q22.3
7 3 7 1 7 17 14 16
72.3 0.51 72.4 69.9 0.65 24.2 0.3 19.6
p.595
Fos
164810
14q24.3
12
0.4
p.595
Mos
190060
8q11
4
0
p.597
Myc
190080
8q24.12-q24.13
15
32
p.605 p.595
Plg Notch4
173350 164951
6q26 6p21.3
17 17
73 18.72
p.599
Akt1
164730
14q32.3
12
57
p.599
Erbb2
64870
17q21.1
11
57
p.597 p.597 p.596
Her2 Neu Hras1
64870 64870 190020
17q21.1 17q21.1 11p15.5
11 11 7
57 57 72
p.598 p.597
Igf1r Kras2
47370 190070
15q25-q26 12p12.1
17 6
33 71.2
p.600 p.597 p.599 p.598 p.598 p.595
Pten PyV-mT Catnb Csnk2a2 Gsk3b Wnt1
601728 not listed 116806 115442 605004 164820
10q23.31 Viral transgene 3p22-p21.3 16p13.3-p13.2 3q13.3 12q12-q13
19
24.5
9 8 16 15
72 50
p.601 p.605 p.601 p.601 p.604 p.600 p.597 p.597 p.697
Cdh1 Itgb1 Brca1 Brca2 Trp53 Pten Rb1 SV40-T121 SV40-Tag
192090 135630 113705 600185 191170 601728 180200 not listed not listed
16q22.1 10p11.2 17q21 13q12.3 17p13.1 10q23.31 13q14.1-q14.2 Viral transgene Viral transgene
8 8 11 5 11 19 14
53.3
56.8
60.5 84 39 24.5 41
* Mouse symbol used. ** Numbers in column refer to number codes assigned in Online Mendelian Inheritance In Man (OMIM). In ambiguous examples, the first or most frequently used gene is cited. *** CentiMorgans from centromere on mouse chromosome.
complete picture of the evidence discussed. Therefore, in cooperation with the editors and the publisher, we are furnishing a web site with digitized whole slide images (WSI) that illustrate the basic structural evidence used for this chapter. The digitized color WSI corresponding to the gray-scale panels found in this
chapter can be found at http://imagearchive.compmed.ucdavis. edu/publications/cardiff/. The reader can “click” on any of the panels to view a WSI that can be manipulated to view any field at any magnification. A WSI archive from the Mouse Models of Human Cancers Consortium slide collection can also be found at
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TABLE 24-2
SELECTED TISSUE-SPECIFIC PROMOTERS USED IN GENETICALLY ENGINEERED MOUSE (GEM) CANCER MODELS* Target tissue
Promoter/gene name
Promoter acronym
Description
Mammary
Whey acidic protein Mouse mammary tumor virus long terminal repeat Rat prostatic steroid-binding protein “C3(1)” β-Lactoglobulin Rat probasin (short, long, and composite) Rat prostatic steroid-binding protein Prostate-specific antigen (human) Cryptidin-2 Keratin 14 Keratin 5 Keratin 14 Tyrosinase Metallothionein
WAP† MMTV-LTR C(3)1/PSBP BLG PB, LPB, ARR2PB “C3(1)” C3(1)/PSBP PSA CR-2 K14 K5 K14
Epithelium-hormone responsive Epithelium-estrogen/corticosteroid responsive Mammary, prostate, and lung Highest with lactation High in dorsolateral gland epithelium-androgen responsive Mammary, prostate, and lung Prostate, salivary gland, and GI tract Neuroendocrine cells Squamous epithelium Basal squamous cells Squamous epithelium Melanocytes Zinc inducible-ubiquitous
α-Fetoprotein Liver activator protein Intestinal trefoil factor Keratin 19 Human intestinal trefoil protein pS2 Keratin 19 Endocrine promoters Metallothionein Keratin 19 Inhibin, α-subunit Ovary–specific promoter Mullerian inhibiting substance II receptor Clara cell secretory protein Clara cell secretory protein 10 kd Glial fibrillary acidic protein P0 glycoprotein Nestin
AFP LAP ITF K19 PS2 K19 Various MT K19
Lymphocyte-specific protein-tyrosine kinase Ig heavy chain Eµ CD2 Metallothionein Multidrug resistance-associated protein S Tec protein tyrosine kinase Cathepsin G
LCK (E-mu) CD2 MT MRPS Tec
Prostate
Cervix Skin
Gastrointestinal tract Liver Colon Stomach Pancreas
Ovary
Lung Brain/nerve
Hematopoietic system Lymphoid
Non-lymphoid
MT
OSP-1 MISIIR CCSP CC10 GFAP P0
Hepatocytes Hepatocytes Intestine Apical colon, stomach, and pancreas Stomach, pancreas, and duodenum Apical colon, stomach, and pancreas Islets of Langerhans Zinc inducible ubiquitous Apical colon, stomach, and pancreas Granulosa cells Granulosa cells Ovarian surface epithelium Clara cells Clara cells Glial cells (principally) Schwann cells Neurogenic cells T cells B cells T cells Zinc inducible ubiquitous Myeloblastic stage Higher in hematopoietic cells Myeloblasts to myelocytes
*This table includes only selected promoters. Many of the most common promoters used to generate GEM cancer models are listed. Inclusion or exclusion from this list is not intended to imply any greater or lesser usefulness. †All acronyms for genes used here use the conventions for genetic symbols found on Online Mendelian Inhertance in Man (OMIM) (http://ncbi.nlm.nci. gov/omim) unless otherwise specified. Modified from Borowsky et al. 2003.
http://imagearchive.compmed.ucdavis.edu. This WSI technology is described later.
C.
Modeling
This chapter will not discuss in any detail the molecular modeling that goes into the construction of GEM. Some terms, however, will recur and need to be fresh on the reader’s mind.
It is important to remember that genetic engineering can introduce gain of function or loss of function. Transgenesis is usually a gain-of-function strategy that involves the insertion of a foreign gene as a purified, linearized DNA construct into the pronucleus of a fertilized egg (Borowsky et al. 2003, 2004). The DNA construct contains a promoter sequence to drive the expression of the gene. The promoter is generally selected on the basis of its tissue specificity. However, almost all promoters are “leaky” and have resulted in tumors occurring in
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TABLE 24-3
IMAGE SOURCES, DIAGNOSIS Gene
Genotype
Pub MedID Investigator
Institute
Diagnosis
Figure No.**
cdk4 (R24C knock-in)
Tm(cdk4R24C xP8−/−)
15315761
Dubus
Tm(cdk4R24C xP8−/−)
15315761
Dubus
Tumor with adjacent tumor embolus, mammary gland Mammary tumor embolus in lung
9C*
cdk4 (R24C knock-in)
9D
Chk2 c-Myc c-Myc c-Myc c-Myc(TRE-Myc) c-Myc(TRE-Myc) e-cadherin−/− e-cadherin (Cdh1/Trp53−/−) Erbb2
Tg(MMTV-LTR-Chk2) Tg(MMTV-LTR-c-Myc) Tg(Wap-c-Myc) Tg(ARR2PB—c-Myc) Tg(LAP-c-Mycdeinducible) Tg(LAP-c-Mycdeinducible) Human Tm(Cdh1−/− × Trp53−/−)
8994298 7624126 7641211 14522256 15475948 15475948
Haber Leder Sandgren Sawyers Felsher Felsher Borowsky Jonkers
Univ. Bordeaux Univ. Bordeaux MGH Harvard U.Wisc. UCLA Stanford Stanford UCDavis NKI
Papillary bronchial adenoma, lung Malignant lymphoma, large cell type Myc-type adenocarcinoma, mammary gland Adenocarcinoma, prostate Hepatocellular carcinoma, liver Dormant hepatocellular carcinoma, transplant Lobular carcinoma, human breast Multiple carcinomas, mammary gland
9F 3D, 3Dinsert 2A, 3B 3A 3C 10D 1E 1C, 2F
Tg(MMTV-LTRErbb2rtTa inducible) Human Tg(MMTV-LTR-Erbb2NDL)
12498714
Chodosh
U.Penn
Erbb2-type carcinoma, mammary gland
1D
10632352 10205169
Waldman Muller
UCSF McGill
1F 9B, 9E
9154814
Rosen
Baylor
Erbb2(Neu) Erbb2(YD) MMTV-Infection
Tg(MMTV-LTRErbb2xTrp53−/−) Tg(MMTV-LTR-Erbb2Neu) Tg(MMTV-LTR-Erbb2YD) Wild Type FVB/N
1359541 12808151 1887859
Shyamala Massague Leder
UCB/LNLB MKSCC Harvard
MMTV-Infection
Wild Type FVB/N
1887859
Leder
Harvard
Trp53−/−
Tm(Trp53−/−)
1552940
Schmidt
MGH
Ptenloxp Ptenloxp Ptenloxp PyV-mT PyV-mT PyV-mT Hras1 Hras1 Ras(Kras2) Ras(Kras2) Ras(v-Ras) SV40-T121
Tm(Pten−/−xTrp53−/−) Tm(Nkx3-1−/−xTrp53−/−) Tm(PbxTrp53−/−) Tg(MMTV-LTR-PyV-mT) Tg(AAR2Pb-PyV-mT) Tg(MMTV-LTR-PyV-mT) Tg(SPB-Hras1) Tg(Villin-Hras1xApc−/−) Tg(Kras2V12floxpxadeno-cre) Tg(Kras2V12floxpxadeno-cre) Tg(Zglob-v-Ras) Tg(LPV-SV40-T121)
12036903 12873978 12163417 15299077 15328372 15065094 12145803 11407943 11641780 1887859 1317542
Cunha Abate-Shen Hong Wu MacLeod Borowsky Gregg Scherl Robine Jacks Meuwissen Leder Van Dyke
UCSF CABM UCLA UCSD UCD UCD AECOM Vanderbilt MIT NKI Harvard UNC
SV40-Tag SV40-Tag SV40-Tag SV40-Tag Wnt1
Tg(SPC-1-SV40-Tag) Tg(sPB-SV40-Tag) Tg(ITF-SV40-Tag) Tg(C(3)-SV40Tag) Tg(MMTV-LTRWnt1rtTa inducible) Tg(MMTV-LTR-Wnt1x beta-catenin) Tg(MMTV-LTRWnt1rtTa inducible)
11751631 7724580 15383629 7972041 12600942
Varmus Balmain Gum Green Chodosh
NCI UCSF UCSF NCI U.Penn
Carcinoma, NOS, human breast Erbb2-type nodular carcinoma, mammary gland Erbb2-type carcinoma with aneuploidy, mammary gland Erbb2-type carcinoma, mammary gland Erbb2-type carcinoma, mammary gland Dunn type B adenocarcinoma, mammary gland Dunn type A adenocarcinoma, mammary gland Adenocarcinoma, glandular pattern, mammary gland PIN, subcapsular renal transplant adenocarcinoma PIN IV with microinvasion, prostate Adenocarcinoma, mammary gland Adenocarcinoma, prostate Adenocarcinoma, mammary gland Intestinal metaplasia, prostate Adenocarcinoma, small bowel Oligodendroglioma, brain Papillary adenocarcinoma, lung Small cell carcinomas, mammary gland Neuroendocrine tumor with papilloma, choroid plexus Neuroendocrine tumor, lung Neuroendocrine tumor, prostate Neuroendocrine tumor, small intestine Carcinoma, mammary gland Wnt1-type carcinoma, mammary gland
11526497
Leder
Harvard
Wnt1-type carcinoma, mammary gland
12600942
Chodosh
U.Penn
Recurrent, EMT tumor, mammary gland
Erbb2 Erbb2 (NDL) Erbb2(c-erbb2)
Wnt1 Wnt1/Trp53
*To directly view the whole slide images go to http://imagearchive.compmed.ucdavis.edu/publications/cardiff/. **To view color panels and the whole slide images go to http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
7B 6A, 6B 2B, 7A 1B 1A 7C 10B 7D 8A, 8C, 8D 10A 8B 9A 5D 5C 5A 5B 2C 4A 4B 4D 4C 2E 2D 6C, 6D, 6E, 6F 10C
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unexpected sites. The literature abounds with examples of “leaky promoters.” One such example is the zeta-globin promoter chosen to study hematopoiesis (Leder et al. 1990). In addition to blood cells, the promoter was expressed in the epidermis and, in concert with the Hras1 oncogene, resulted in squamous papillomas (Cardiff et al. 1993a). The C3(1) promoter was isolated as a prostate specific promoter, but various versions have also resulted in mammary and pulmonary cancers (Green et al. 2000; Tehranian et al. 1996; Yoshidome et al. 1998). The transgene whose function is to be tested is generally a DNA sequence created by reverse transcription from messenger RNA and does not contain introns. A poly A sequence is usually added to enhance expression. A second common method for genetic engineering generally designed for loss-of-function studies is the “knockout” with disruption of a single gene, usually a tumor suppressor gene. Early knockout mice were traditionally created using homologous recombination in embryonic stem (ES) cells (Borowsky et al. 2003). Modified DNA is transduced into the ES cells, and generally some type of selectable marker is used to screen for positive or negative clones. More recently, the loxP recognition site for Cre recombinase has been incorporated into the constructs. When these animals are bred with animals containing Cre recombinase, the DNA between the two loxP sites is excised creating a deletion (knockout) of the gene. Since the most readily available ES cells are derived from the mouse strains designated 129, many of the knockout mice are hybrids (Borowsky et al. 2003). Advances in genetic engineering have led to more sophisticated strategies that allow some site specific or temporal control of the engineered DNA (Borowsky et al. 2003). For example, an oncogenic transgene can be placed behind its native promoter providing a gain of function by “knocking in” the gene (Andrechek et al. 2000a). Furthermore, genetic engineering has been used to create systems that are inducible using exogenous compounds such as doxycycline or tamoxifen (Gunther et al. 2002; Wen et al. 2003). The exogenous systems provide temporal and positional control over the expression of an oncogene or a tumor suppressor gene, allowing the investigator to control the gene at will. These genetic manipulations have created inducible oncogenes and, when combined with the lox/cre systems, conditional knockouts. The details of these systems are covered elsewhere (Borowsky et al. 2004; Cardiff 2004; Galvez et al. 2004) and presented in this and other monographs (Holland 2004).
D.
Nomenclature
The combination of abbreviations for mouse strains, proteins, and genes constitute a daunting “alphabet soup” of confusing letters (Barthold 2002). We will introduce a few conventions that may help the reader who may be inexperienced in the
world of GEM. Tables 24-1 and 24-2 have been provided to show some of the more commonly used genes and promoters, indicating their abbreviations and spelling out their full names and some specificities or functions. The genes in these tables can be matched with the images illustrating the specific microscopic pattern associated with selected genes (Table 24-3). We will adhere to the convention of using the prefix Tg for all transgenics. We will use the prefix Tm for targeted mutations involving either knockins or knockouts. Where possible, we will adhere to the convention that the knockout gene is designated as a superscript Tm and knockins are designated as a superscript allele. All genes will be designated by their official italicized symbol. For example, Tg(Myc) indicates a transgenic mouse bearing a Myc gene as a construct. Tm(PtenTm) indicates a mouse with a knockout of at least one Pten allele. The reader is reminded that some homozygous knockouts are embryonic lethals or have developmental defects. Thus, the investigators maintain the animals as heterozgotes and count on loss of heterozygosity in the target organ to produce the desired result. The morphological end points are the same. Thus, we will distinguish between heterozygosity, hemizygosity, or homozygosity only when it is pertinent to the specific study or specific morphological outcome. Tm(Erbb2Neu) indicates a knockin substitution of the Neu gene sequence for the Erbb2 gene. Since the morphological end points in most of these systems are the same, we will not specify homozygosity or heterozygosity, or use the term null when the specific genotype is uncertain. Gene products will be indicated by nonitalicized symbols. For example, Myc is the protein product of the Myc gene that might be expressed in the Tg(Myc) transgenic. In most cases, we will be comparing examples from numerous founders and laboratories. Therefore, we will only rarely refer to the promoter, mouse strain, or laboratory of origin. In some cases, the specific model is commonly referred to using a “nickname,” such as TRAMP or LADY (Tg(SV40-Tag); Gingrich et al. 1996; Greenberg et al. 1995; Kasper et al. 1998; Masumori, et al. 2001). Such terms can be misleading for the casual reader because they could also indicate some arcane protein product. We will use the nickname and the standard symbols for these types of systems. Many GEM have been produced using F1 hybrids or outbred Swiss mice (CD1 or CF1) (Barthold 2002). This has led to considerable heterogeneity of genetic constitution. However, in more recent years, most investigators have tried to develop their GEM on an inbred background. The most common inbred mouse strains used in genetic engineering are FVB, C57BL/6, and 129. The origins of the particular colonies are rarely recorded in the literature. The 129 strain is particularly problematic because it has been genetically corrupted, requiring revision of nomenclature (Barthold 2002). This is even more complicated because the origins of ES cells are often not defined in the literature. In this chapter, the strain
2 4 . T H E T U M O R PAT H O L O G Y O F G E N E T I C A L LY E N G I N E E R E D M I C E
designation will be coupled with the words “mouse” or “strain” to limit the confusion with potential gene or protein symbols. The reader will also note that the standard symbols are not generally used by investigators, nor are they enforced by journal editors. Therefore, the literature itself is filled with arbitrary, investigator-derived jargon that is frequently inaccurate and misleading. For example, it is not clear what the mouse strain TG.AC stands for unless you know that it is a Tg(Hras1) or, more accurately, a FVB/N-Tg(Hzb2-Hras1) (Cardiff et al. 1993a). Therefore, it may be difficult to refer our genetic symbols to the specific papers and specific models that do not use standard genetic nomenclatures and symbols.
PRINCIPLE 1:
ONCOGENIC EVENTS INITIATING
TUMORIGENESIS PROFOUNDLY INFLUENCE THE MORPHOLOGICAL PHENOTYPE OF THE TUMOR (FIGURES 24-1 TO 24-7). Axiom I:
Spontaneous, virus-induced, and chemical-
induced mouse tumors do not mimic the microscopic structure of human cancers (Fig. 24-1). To fully appreciate the impact of genetic engineering on murine tumorigenesis, one must have an appreciation of the types of “spontaneous” and carcinogen-induced tumors previously described in mice. The molecular pathogenesis of most spontaneous neoplasms is not known. The biology of murine tumors, whether spontaneous or experimentally induced, has been profoundly influenced by endogenous oncogenic retroviruses, by age, by hormonal milieu, by genetic background, and by exogenous factors such as retroviruses and chemical carcinogens. Other factors that give mouse tumor biology a unique “flavor” will also be considered in this section. While carcinogen-induced and viral-induced tumors of the lymphocytes, myelocytes, liver, bowel, and connective tissue resemble some types of human cancers, the microscopic appearances of spontaneous solid epithelial tumors of mice rarely resemble human cancers. For example, the microscopic structure of traditional mouse mammary tumors rarely resembles that of human breast cancers (Fig. 24-1) (Cardiff et al. 1999). GEM tumors, however, are quite different from the tumors produced by other forms of carcinogenesis and often closely resemble cancers that occur commonly in humans. Many examples can be offered in defense of this proposition. The mammary gland, the prostate, and the lung are emphasized in the following sections.
Retrovirus-Related Background Pathology (Fig. 24-1).
In the early 1900s, inbred laboratory mice were originally developed to study coat color but were rapidly adapted to
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study the genetics of cancer. Led by Dr. C. C. Little, spontaneous mammary tumor-bearing mice were first selected for inbreeding to yield tumor-prone, genetically homogeneous animals (Barthold 2002; Laboratory 1953). Mouse mammary tumors were later classified by Thelma Dunn as type A, B, C, and P (Figs. 24-1A and 1B) (Sass and Dunn 1979). However, these mouse mammary tumors do not at all resemble human breast cancer (Cardiff et al. 1999). In retrospect, these virusinduced tumor types are identical to the tumors found much later in transgenic mice bearing wnt pathway transgenes (Rosner et al. 2002) (Figs. 24-1, 24-2D, and 24-6). The tumors typically have an organized myoepithelium not found in human cancer (Cardiff et al. 2000a) (Figs. 24-6D, and 24-6F). A surgical pathologist trained in human pathology would regard the tumors as benign because of the organized myoepithelium and expansile margins. They would be surprised to learn that 60% of these tumors are metastatic. In retrospect, all these mammary tumors are induced by the mouse mammary tumor virus (MMTV). It has relatively recently been recognized that the “spontaneous” mammary tumors arise from activation of either Wnt1, Notch4, or Fgf3 proto-oncogenes by insertion of the MMTV proviral DNA (van Leeuwen et al. 1995). MMTV DNA is inserted either upstream or downstream from the proto-oncogene and causes inappropriate expression of these genes, a process called insertional activation. The process has been experimentally recapitulated using Fgf or Wnt as a MMTV-LTR promoted transgene (Cardiff 1996). The resulting transgenic tumors have structural phenotypes that fit into the Dunn classification (Figs. 24-1A, 24-1B, and 24-2D). Furthermore, when a transgenic Tg(Wnt1) mouse is infected with MMTV, the resulting tumors show activation of a Fgf3 (MacArthur et al. 1995). In addition, when a transgenic Tg(Fgf3) mouse is infected with MMTV, the resulting tumor has activation of a Wnt gene (Lee et al. 1995; MacArthur et al. 1995). The other common “spontaneous” tumors of the laboratory mouse are the leukemias induced by the exogenous or endogenous murine leukemia viruses (MuLV). These appear quite frequently in all strains of laboratory mice, but some strains, such as AKR, were specifically selected and bred for their association with MuLV-induced leukemia (Kogan et al. 2002). These leukemias are generally lymphocytic or myeloid. However, a number of neoplastic hematopoietic cell types have been recognized and classified (Morse et al. 2002). Sarcomas are a third category of retrovirus-induced lesions. However, it is uncertain whether all spindle cell tumors are sarcomas or whether sarcomas are all virus-induced. Nevertheless, several types of sarcoma viruses have been isolated from different mouse strains and, like the chicken sarcoma viruses, involve the transduction of cellular genes such as Fos (Verma et al. 1987) and Mos by exogenous leukemia retroviruses (Yew et al. 1993). Several other sarcomas are the result of leukemia virus transduction of genes from other species such as rats, cats, and gibbon apes (Gardner et al. 1970, 1971;
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Kawakami et al. 1973; Kirsten and Mayer 1969; Kumar et al. 1990; Naharro et al. 1983; Snyder et al. 1969). Nonviral Spindle Cell Tumors I (Figure. 24-10C).
More recently, the epithelial-mesenchymal-transition (EMT) tumors have drawn a great deal of attention from the GEM modeling community because many of the “spindle cell” tumors are not of mesenchymal origin but, rather, represent a transition of mouse epithelium into a spindle cell phenotype (Fig. 24-10C) (Landesman-Bollag et al. 2001; White et al. 2001). It is particularly problematic in some transgene-induced tumors in which the transgene is no longer expressed but the GEM strain has increased tumor incidence (Landesman-Bollag et al. 2001; White et al. 2001). Thus far, these tumors can be identified by the increased expression of the transcription factors, Snail or Slug. They can also be recognized by the pathologist by their dual staining for low-molecular-weight keratin and vimentin or smooth muscle actin. The mechanism for the EMT tumors in these mice is not currently understood. However, the EMT tumor is also a notorious phenomenon in tissue culture of mouse cells and their tumor cell explants. This does not mean that all spindle cell tumors are EMT tumors or retrovirus-induced. Transformed myeloid cells can be another source of spindle cell tumors (Cardiff et al. 1993a). For example, mice transgenic for ras (Tg(Ras)) with enlarged spleens and myeloid hyperplasia, a common event in these mice, frequently develop spindle cell lesions beneath epidermal papillomas. The spindle cell populations seem to relate to the inflammatory infiltrates that initially appear innocently beneath the papillomas. These spindle cell tumors do not express keratin and are rich in CSF-GM (Leder Personal Communication). These data imply that the pathologist must evaluate the spindle cell tumors on an individual basis and not assume that all are simply “fibrosarcomas.” Spontaneous Tumors
Many mouse strains have “background” tumors that do not appear to be the result of oncogenic viruses. Many of these tumors are described in other chapters found within this series (Foster et al. 1982) or elsewhere (Maronpot et al. 1999; Mohr et al. 1996). These “spontaneous” or “background” tumors deserve special consideration in the context of GEM tumors because the uninitiated pathologist might interpret them as being the result of the genetic manipulation rather than the background of the animal. In most cases, the interpretative problem arises because these tumors develop in secondary organs and not the target organ. The background pathology of aging mice is discussed in monographs (Frith and Ward 1988; Mohr et al. 1996) and reviews (Gardner et al. 1973). However, relatively few studies exist on the background pathology of some mouse strains commonly used for transgenesis (Mahler et al. 1996).
Chemically Induced Tumors
Mice have long been used to test chemical carcinogens and other toxic substances. The tumors associated with these experiments are described in detail elsewhere in this series. Their general characteristics need to be considered here in the context of GEM. In general, mammary tumors associated with chemical carcinogens are either poorly differentiated or adenosquamous carcinomas (Ashley et al. 1980; Cardiff et al. 1988c; Gardner et al. 1985). Many have been induced by mutations of Ras (Cardiff et al. 1988c, 1993a).These tumors do not resemble the tumors found in the GEM signature phenotypes or in MMTV-infected mice. Carcinogen-induced hepatomas have various levels of differentiation (Bannasch et al. 1989) that do not resemble the hepatoblastomas created with Tg(Myc) (Fig. 24-3C) (Shachaf et al. 2004). The comparative list is extensive. We will comment on comparisons when appropriate elsewhere in the text. Axiom II: Genetically engineered mouse (GEM) tumors are unique. (Figures 24-1–24-7).
The tumors produced in GEM using genes related to human cancers can produce cancers that can be remarkably similar to their human counterparts (Cardiff 1996, 2001, 2003; Cardiff et al. 1993b, 1999, 2000a) (Fig. 24-1). The single most important discovery emerging from the GEM tumor pathology, however, is that dysregulation of most oncogene results in tumors with a unique or signature phenotype (Cardiff 1988; Cardiff et al. 1988b, 1991, 1992. Signature phenotypes were initially recognized in the mammary gland where unique phenotypes for Tg(Myc) (Figs. 24-2A and 24-3B). Tg(Neu) (Fig. 24-2B) and Tg(Ras) (Fig. 24-2C) transgenic mice and their transgenes were described (Cardiff et al. 1991). Since the GEM modeling has been most frequently applied to the mammary gland, a broader experience is available in mammary biology than most other organ systems (Figs. 24-1C, 24-1D, 24-3, 24-4 and 24-5). Subsequently, however, signature phenotypes have been observed in association with the transgenes in other organ systems. The list is not comprehensive, but it is sufficiently complete to identify the effects of several specific transgenes in a variety of organs. Corollary A: Oncogenic alterations in molecular function lead to gene-specific microscopic structural changes (Figures 24-2–24-7).
Gene-specific phenotypes were first described in GEM transgenic mammary tumors (Fig. 24-2) (Cardiff et al. 1991). The first three mammary oncogenes studied were Ras, Neu, and Myc. In the mammary gland, these three transgenes induce distinct histological phenotypes and remain the most dramatic examples of signature phenotypes, particularly since they closely resemble human breast cancer and are quite distinct from those seen heretofore in mice. Experimental crosses with mice bearing one or combinations of two or three of the transgenes
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resulted in various degrees of acceleration of mammary tumorigenesis (Cardiff et al. 1991). It was also clear that the tumors had unique morphological phenotypes. Subsequently, the molecular expression profiles have confirmed that the Tg(Myc), Tg(SV40-Tag), and Tg(Erbb2) tumors form distinctly separate morphologic clusters (Desai et al. 2002; Fargiano et al. 2003). The Myc oncogene, a dominant oncogenic transgene, is overexpressed in a variety of human cancers (Schmidt 1999, 2004). Since Tg(Myc) was successfully used early in the development of transgenic mouse models of human cancers, Myc has induced tumors in almost all organ systems, serving as proof of principle (Fig. 24-3). Myc is a transcription factor that controls a number of events within the cell that are concerned primarily with growth and apoptosis (Schmidt 1999, 2004). Myc was first discovered as a transduced gene in the avian myelocytomatosis virus, hence, the “name” Myc (Sheiness and Bishop 1979; Sheiness et al. 1980; Vennstrom et al. 1982). It was then found that Myc was activated by insertion of the exogenous avian leukosis proviral DNA to cause the commonly occurring lymphomatosis of domestic chickens (Hayward et al. 1981). Myc was one of the first oncogenes found in human cancer in association with Burkitt’s lymphoma (Taub et al. 1982). The Tg(Myc) tumor phenotype in GEM is characterized by large cells with large pleomorphic nuclei with a coarse clumped chromatin and prominent nucleoli (Fig. 24-3) (Cardiff et al. 1991). The cytoplasm is amphophilic, implying abundant RNA. These mammary tumors most frequently have a glandular pattern quite distinct from that of MMTV-induced mammary tumors and other kinds of GEM mice. The same cytological pattern is found in Tg(Myc)-induced tumors of the mammary gland (Figs. 24-2A and 24-3B), liver (Fig. 24-3C), lung, and lymphoid tissues (Fig. 24-3D, 24-3 and 24-10). Many Tg(Myc)-induced tumors have characteristic small clusters of apoptotic cells. The large blue cell phenotype of Tg(Myc)-based tumors contrasts dramatically to the Tg(Ras) signature phenotype (Cardiff et al. 1991). The signature Tg(Ras) tumor has relatively small cells with oval to round nuclei and a delicate chromatin pattern with bright red cytoplasm (Figs. 24-2C and 24-5). The characteristic Tg(Ras) mammary tumors tend to be oriented around blood vessels, providing a papillary pattern that morphologically resembles transitional cell carcinomas of the human bladder. The third signature phenotype is the Tg(Neu) phenotype composed of cells intermediate in size that have large round nuclei with a delicate chromatin pattern and a small nucleolus (Fig. 24-2B) (Cardiff et al. 1991). The cytoplasm is a lighter pink than the Tg(Ras) tumor cell cytoplasm. The Tg(Neu) mammary tumors tend to form nodular, expansile masses. The nodules are characteristically “zonal” with increasing differentiation toward the center (Deckard-Janatpour et al. 1997; DiGiovanna et al. 1998).
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The Tg(Neu) mice originated in several laboratories that received similar but different clones of Neu DNA (Bargmann et al. 1986; Bouchard et al. 1989; Muller et al. 1988). Since the two original lines were developed, numerous additional molecular constructs have been developed and tested in transgenic mice. There are some subtleties in the specifics of the tumors arising in the different Tg mice. However, all of the different constructs result in a very similar tumor phenotype that will be referred to in this chapter as the Erbb phenotype. Although the experience with Tg(Neu) and the mammary gland phenotype is extensive, Neu produces tumors in many other systems. On the other hand, Polyoma Virus middle T (PyV-mT) behaves as a surrogate molecule by constitutively activating the Erbb pathway, and it produces tumors in a variety of organ systems (Gottlieb and Villarreal 2001). The Erbb tumor phenotype in all of the epithelial organ systems is a somewhat undifferentiated glandular pattern (Rosner et al. 2002; Tehranian et al. 1996). The PyV-mT-induced tumor cells have relatively more pleomorphic nuclei than the Tg(Erbb2) tumors and less cytoplasm that tends to be more amphophilic. When used correctly, Neu refers to the gene isolated from rat neuroblastomas, but many authors use it interchangeably with the mouse (Erbb2) and the human (HER-2) genes. While the Tg(PyV-mT) tumors tend to be only slightly different as compared to the Tg(Erbb2) tumors, they are quite distinct from the deep blue Tg(Myc)-induced tumors. The signature phenotypes of the different transgenes are diagnostic in the mammary glands and tumors of GEM. However, the pathologist also needs to recognize that each transgene may produce a range of tumor phenotypes (Cardiff and Muller 1993b). Varying proportions of the tumors in each transgenic line fulfill the criteria of the “signature” phenotype for that specific gene (Cardiff and Munn 1995). The tumors that deviate from the signature should prove extremely informative, revealing important secondary changes and interesting new interdependencies between the different pathways (Saez et al. 2004). As might be expected because they are members of the same pathway, the Tg(Ras) and Tg(Neu) phenotypes overlap so that a subset of Tg(Ras) tumors are indistinguishable from the Tg(Neu) phenotype (Cardiff and Muller 1993b; Cardiff et al. 1991). In a like manner, the Tg(Myc) phenotype has a considerable range of organizational patterns that tends to overlap with other highly proliferative tumor types. It may turn out that all very proliferative tumor types overexpress Myc. Tg(SV40-Tag) is another dominant oncogenic transgene that has been expressed in many different organ systems behind various promoter systems (Figs. 24-2E and 24-4). Therefore, an extensive multi-organ experience is available with the SV40Tag oncogene. SV40-Tag is a viral gene whose product binds and inactivates both Trp53 and Rb1, classical tumor suppressor genes (Ali and De Caprio 2001; Furth 1998). Tag is a powerful antitumor suppressor protein (Ali and De Caprio 2001). One mutational variant, T121 is designed to bind Rb1 but not Trp53 (Saenz Robles et al. 1994).
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The Tg(SV40-Tag) oncogene characteristically produces hyperplasias and tumors with small oval to round nuclei with very dense chromatin and relatively scanty cytoplasm (Fig. 24-4). These dysplasias become diffuse in the prostate to occupy almost the entire luminal epithelium (Shappell et al. 2004). One of the remarkable aspects of the Tg(SV40-Tag) phenotype is that the invasive, metastatic tumors are frequently of neuroendocrine differentiation (Evangelou et al. 2004; Powell et al. 2003, Shappell et al. 2004). The neuroendocrine tumors have rather pleomorphic nuclei, with a typical “salt and pepper” chromatin pattern and variable but scanty cytoplasm that, in well fixed samples, appears fibrillar (Fig. 24-4). The tumors appear undifferentiated but occasionally form pseudorosettes that may be confused with glands. The diagnosis can be confirmed with either antisynaptophysin or anti–low molecular-weight keratin stains such as anti-K8 or anti-K18. The antikeratin antibodies typically reveal a discrete paranuclear granule that is characteristic of neuroendocrine tumors in both humans and mice. Tg(SV40-Tag)-related neuroendocrine tumors have been observed in the prostate (Fig. 24-4D), lung (Fig. 24-4B), pancreas, and gut (Fig. 24-4C) (Borowsky et al. 2004; Evangelou et al. 2004; Hager et al. 1999; Kasper et al. 1998; Masumori et al. 2001; Nikitin et al. 2004; Powell et al. 2003; Shappell et al. 2004). The morphology of Tg(SV40-Tag) tumor cells in these organ systems is similar to the tumor cells induced by the transgene in the mammary gland and some central nervous system tumors (Borowsky et al. 2004; Cardiff 2004; Galvez et al. 2004). The pathology of the Tg(SV40-Tag) tumors has additional characteristics that have not been thoroughly studied and should be mentioned in the current context. The stromal proliferation associated with Tg(SV40-Tag) is another characteristic of these tumors (Shappell et al. 2004). The stromal proliferation in the prostate expresses the antigen. Other promoters in other organ systems have led to unexpected Tg(SV40-Tag)-related smoothmuscle proliferations. However, it is not clear whether these proliferations always express Tag antigen. Other transgenes have characteristic mammary tumor phenotypes, but the transgenes have not been utilized in a sufficient number of different organ systems to determine whether their mammary phenotype will be expressed in other locations. The two classical groups are the Tg(Wnt1) and Tg(Erbb2)-related tumors (Figs. 24-2 and 24-6) (Rosner et al. 2002). The Tg(Erbb2) tumors, as noted above, have a characteristic pattern and cytology. The Tg(Wnt1) tumors, as might be predicted, have the classical phenotypes of the MMTV-induced tumors described by Thelma Dunn (Figs. 24-1 and 24-2D) (Sass et al. 1979). Interestingly, a number of genes in the Wnt pathway share one or more characteristics of these tumors and will be discussed below. Although tumors in other GEM have been found to have unique phenotypes, limited numbers of examples are available, or the oncogenic transgenes have not been applied to enough different organ systems to validate the phenotype. Many of the transgenes seem to adopt the phenotype of the signal transduction pathway that they activate.
Corollary B: Oncogenic alterations in molecular function lead to molecular pathway-specific microscopic changes (Figure 24-6).
As an increasing number of GEM have become available, it has become obvious that the oncogenic transgenes belonging to the same signal transduction pathways share a number of morphological characteristics (Fig. 24-6) (Rosner et al. 2002). Recognition that the tumor phenotype can resemble other tumors in the same signal transduction pathway is important because common features in any tumor group may provide a clue to the underlying genetic aberration. Molecular expression profiling has demonstrated that Tg(Ras), Tg(PyV-mT), and Tg(Erbb2) form a gene expression cluster that is distinct from the Tg(Myc) and Tg(SV40Tag) tumors (Desai et al. 2002; Fargiano et al. 2003). Genetic crosses of Tg(Myc), Tg(Wnt1), or Tg(Erbb2) GEM with cyclin D1 (Ccnd1) knockout mice (Tm(Ccnd1-/-)) demonstrate that tumorigenesis is abrogated in the Tg(Erbb2), Tm(Ccnd1-/-) null mice but not in the Tm(Ccnd1-/-) null mice crossed with Tg(Myc) or Tg(Wnt1) GEM (Yu et al. 2001). These data provide molecular and genetic substantiation for the different phenotypes observed in these GEM. Thus, pathologists need to recognize these distinct patterns. The most extensively studied molecular pathways are the Erbb2/Ras and the Wnt/Fgf pathways (Rosner et al. 2002). The Tg(Erbb2/Ras) pathway mammary tumors are characterized by solid, nodular growth patterns that clearly originate in the mammary side buds. The tumors tend to be solid expansile nodules and noninvasive. The nodules tend to form concentric zones of cells suggesting a layerlike differentiation from the periphery to the center. These tumors do not have myoepithelium and rarely undergo squamous metaplasia (Figs. 24-6A and 24-6B). Tumor-bearing animals belonging to this group include Tg(Erbb2), Tg(Neu), Tg(PyV-mT), Tg(Ras), and Tg(Igf1r) (Rosner et al. 2002). In contrast, the Tg(Wnt/Fgf) mammary tumors have a more complex growth pattern that appears to begin with a ductal dysmorphogenesis seen as multibranched ductal foci (Figs. 24-6E and 24-6F) (Rosner et al. 2002). The terminal ends of these ducts differentiate into microacinar (type A of Dunn) (Figs. 24-6C and 24-6D), adenosquamous, solid embryoid (type B of Dunn), or pilar patterns (Figs. 24-6E and 24-6F) (Rosner et al. 2002). These types of tumors have been associated with Tg(Wnt1) (Cardiff et al. 2000a, 2000c, 2001; Li et al. 2003; Rosner et al. 2002), Tg(Fgf3), Min mice (C57BL/6J-ApcMin/J) (Moser et al. 2001), Tg(Catnb) (beta-catenin) (Miyoshi et al.2002b), Tg(Gsk3b) (glycogen synthase kinase1), and Tg(Csnk2a2) (casein kinase 2a) (Landesman-Bollag et al. 2001; Miyoshi et al. 2002a; Rosner et al. 2002). The different transgenes produce tumors resembling different subsets of the Tg(Wnt) tumors and those tumors occurring in MMTV-infected mice. For example, the MIN mice treated with chemical carcinogens develop almost solely pilar tumors with very distinctive production of extracellular hard keratin characteristic of hair and are thus named “pilar” tumors (Rosner et al. 2002). The related
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Tg(Catnb) (beta-catenin) mice also produce a distinctive pilar configuration (Miyoshi et al. 2002b). In contrast, Tg(Wnt1) tumors are primarily Dunn type A or B (Figs. 24-2D, 24-6C and 24-6E) (Rosner et al. 2002) with prominent, well-organized myoepithelium (Figs. 24-6D and 24-6F). Perhaps the most telling observations come from the doxycycline-inducible, MMTV-LTR-promoted Tg(Wnt1) models (Gunther et al. 2003) (Fig. 24-2D). These mice develop mammary tumors at a rapid rate, and each mammary fat pad has multiple tumors. The tumors in any one fat pad will have the full range of Tg(Wnt1) phenotypes. Some fat pads have as many as five different phenotypes. Each tumor has its own pattern. However, all tumors share the general characteristics of Tg(Wnt1) tumors. Thus, it is clear that Tg(Wnt1) alone is capable of producing the entire set of tumor phenotypes that can be found in the MMTV-induced tumors classified many years ago by Thelma Dunn (Dunn 1953; Sass and Dunn 1979). It will be interesting to determine what secondary events control the morphogenesis of the individual tumor types. Do other molecular pathway tumors exist? Unfortunately, insufficient examples of other pathways in a single organ system are currently available to answer the question. Hence, the rules governing those pathways are not known. However, the Pten/Akt1 pathway in the mouse prostate is an exception (Park et al. 2002). As it turns out, the initial prostate tumor models were all stimulated by Tg(SV40-Tag), which produced a typical tumor phenotype documented in the previous section. The next generation of experimental systems concentrated on phosphorylation of the pAKT-1 protein. Therefore, Pten, Nkx3-1, Akt1, Fgf3, and similar oncogenes or tumor suppressor genes stimulate the premalignant transformation of prostate epithelium through pAKT-1, resulting in premalignant lesions and adenocarcinomas that are indistinguishable from each other (Figs. 24-7D and 24-8C) (Abate-Shen et al. 2003; Freeman et al. 2003; Kim et al. 2002b; Song et al. 2002). Even when crossed with functional knockout Tg(SV40-T121) mice, Tm(Pten-/-) animals develop a subset of lesions with an unusual Akt-signature papillary lesion (Hill et al. Personal Communication). The Pten/Akt prostate cancer phenotype is characterized by cells with large oval nuclei with a relatively open chromatin and distinct nucleolus. The cytoplasm is abundant and pale pink. This pattern can be easily distinguished from the SV40-Tag type of prostate cancer cell that has smaller nuclei with more diffuse hyperchromatic chromatin and relatively scanty cytoplasm. Now that pathway pathology has been identified, additional pathways will, no doubt, be found (Rosner et al. 2002). However, this effort will require the coordinated creation and examination of multiple model systems in GEM and other species. Corollary C: Some genes have a stronger influence than others on the microscopic structure of the tumor (Figures 24-3, 24-4, and 24-5).
Some transgenes appear more dominant than other oncogenes, resulting in more characteristic changes in the
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microscopic structure. Patterns of dominance have been illustrated primarily in bigenic and trigenic crosses. Such genetic crosses have not as yet led to remarkably new tumor phenotypes. Indeed, initial experiments with bi- and tri-transgenic animals suggest a specific hierarchical order in phenotype dictated by the dominant transgene (Cardiff 1996). The first example was from the Leder Lab which compared Tg(Myc), Tg(Erbb2neu), and Tg(Ras) GEM in various bi- and tri-transgenic crosses (Cardiff 1996; Cardiff et al. 1991). All crosses with the myc transgene resulted in tumors with the Tg(Myc) phenotype. The Tg(Erbb2) phenotype was dominant over the Tg(Ras). Thus, a hierarchy of transgenes was suggested, with Myc the most dominant. The finding of k-Ras mutations in Tg(Myc) phenotype tumors reinforces the dominance of Myc (D’Cruz et al. 2001). However, the temporal expression of Myc could also be a factor in determining the morphological pattern of the tumor. As the Human Genome Project continues to uncover more potential oncogenes, an increasing number of combinations of oncogenes can be anticipated in bi- and tri-transgenic mice. Crosses of oncogenic transgenic Tg mice with knockout mice tend to reproduce the phenotype of the activating transgene but with an increased nuclear pleomorphism and level of dedifferentiation. Further combinations will be discussed below in the context of other onco- and tumor suppressor genes. As suggested above, the Myc oncogene appears to be the single most dominant oncogene yet tested (Fig. 24-3). In humans, Myc activation by chromosomal translocation was shown to play a critical role in the pathogenesis of Burkitt’s lymphoma. Myc was among the first to be discovered and defined. Myc is one of the most ubiquitously activated oncogenes in tumors of all organs. It has been inserted as a transgenic oncogene behind numerous promoters, giving ample opportunity for comparative morphology. The characteristic patterns are recognizable in most organ systems. However, Tg(Myc) expressed in the mouse prostate evokes a different pattern as discussed below (Fig. 24-3A) (Ellwood-Yen et al. 2003). The Tg(SV40-Tag)-related tumors are a second intriguing dominant phenotypic group (Fig. 24-4). The gene product (T-antigen) is a powerful viral oncogene that interferes with the function of both Rb1 and Trp53. SV40-Tag has been used as an oncogenic transgene in many organ systems. In general, Tg(SV40-Tag), behind the appropriate promoter, produces welldifferentiated epithelial tumors with small, hyperchromatic nuclei and very scanty cytoplasm. The prototypic lesion is the hyperplasia found in the prostate of the “TRAMP” and “LADY” variations of the Tg(SV40-Tag) models. Similar lesions are induced with Tg(SV40-T121) models that specifically inactivate Rb1 but not Trp53. Significantly, the undifferentiated tumors arising in the SV40-Tag models are usually neuroendocrine neoplasms (Gum et al. 2004). Neuroendocrine differentiation has been observed with SV40-Tag behind probasin, big probasin, C(3)1, and cryptidin promoters (Garabedian et al. 1998; Masumori et al. 2001; Shappell et al. 2004; Yoshidome et al. 1998), suggesting a specific phenotype. Indeed, SV40-Tag
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behind the Clara cell promoter results in neuroendocrinetype tumors of the mouse lung (Linnoila et al. 2000). The gut promoter, Trefoil, in front of SV40-Tag, induces neuroendocrine tumors of the small intestine (Gum et al. 2004). The C(3)1 promoter, designed for prostate targeting, also produces poorly differentiated ductal carcinoma in situ (DCIS) and tumors of the mammary gland that have oat cell characteristics. These lesions, however, have not been adequately tested for neuroendocrine differentiation (Yoshidome et al. 1998). The exception appears to be in the brain where Tg(SV40-Tag) classically induces papillary tumors of the choroid plexus (Brinster et al. 1984). However, careful examination of these tumors suggests that the more poorly differentiated tumors have the small-cell, neuroendocrine appearance. Pten is another type of tumor suppressor gene (Kishimoto et al. 2003). The protein controls the phosphorylation of Akt1 (pAkt1), a pivotal signal transduction protein. One characteristic of Tm(Pten) knockout tumors is overexpression of Akt1 (Franke et al. 2003). The Tm(Pten) knockout prostate tumors produce back-to-back glands that can invade the surrounding stroma (Fig. 24-7D) (Abate-Shen et al. 2003). The cells have large pleomorphic nuclei with vesicular chromatin and prominent nucleoli. The cytoplasm is relatively abundant and pale staining. The prostatic tumors in GEM with knockout of the homeobox gene Nkx3-1 (Tm(Nkx3.1)) have a very similar pattern (Abate-Shen et al. 2003; Kim et al. 2002a; Kim et al. 2002b). These characteristics are shared by all of the Akt1-related prostate oncogenes and imply that the Tm(Pten) and Tm(Nkx3.1) knockouts also act through Akt. In fact, other oncogenic transgenes that target the prostate also activate Akt and produce a similar phenotype (Majumder et al. 2003; Powell et al. 2003). This concept has been dramatically illustrated by the crosses of Tg(SV40-Tag) with Tm(Trp53-/-) and Tm(Pten+/–) heterozygous mice. The crosses with Tm(Trp53-/-) mice result in classical Tag hyperplasias, while the crosses with Tm(Pten) knockouts result in a mixture of Tag-induced hyperplasias and papillary lesions that appear suspiciously like Pten hyperplasias (Hill et al. Personal Communication). Other examples of context-related Pten phenotypes will be discussed below. Corollary D: Expression levels of the oncogenic transgenes influence the microscopic structure of the tumor.
It seems intuitively obvious that the transgene expression level should govern the tumor structure. Some investigators produce a number of founder strains for their specific transgene but report only on the “highest” expressing line. Some investigations suggest that expression levels can influence tumorigenesis (Siegel 1999). However, little experimental evidence is available to support the hypothesis that expression levels have a specific effect on the structure of the tumor. Some insight is afforded by recent models that depend, in part, on the dose of the inducer (D’Cruz et al. 2001; Gunther et al. 2003; Morris et al. 1989).
Generally, the levels of expression of most oncogenic transgenes do relate to the kinetics of tumor development. In most cases, the tumor phenotype undergoes subtle changes in pattern, but the cytological features are retained. Occasionally, the level of expression drastically alters the tumor phenotype. The exact “rules” that control the phenomenon still need to be developed. The viewpoint that the level of expression has some control over tumor phenotype is supported by the nature of the early neoplastic lesions in most organs. Although the expression of transgenes can frequently be detected before tumors are identified, the level of the expression is usually quite low as compared to the subsequent tumor by in situ hybridization (ISH) or immunohistochemistry (IHC). With these techniques, the transgene antigen and RNA colocalize and are limited to the morphologically dysplastic tissues (Deckard-Janatpour et al. 1997; Tulchin et al. 1995), suggesting either that additional events or heightened transgene expression are required for progression to full-blown cancer. Support for the “additional event” hypothesis comes from the recent observations comparing premalignant outgrowths with the tumor arising from the same outgrowths (Maglione et al. 2001, 2004). In the Tg(PyV-mT) mammary gland transplantation system described below, the premalignant stage frequently had higher levels of transgene expression than the tumors, suggesting that the malignant phenotype is not dependent on level of transgene expression (Maglione et al. 2004). This study, however, did not include any histopathologic analysis of the tumors. Another line of evidence comes from the recurrent tumors in inducible Tg(Myc) animals (D’Cruz et al. 2001; Shachaf et al. 2004) (Fig. 24-10) in which many of the tumors are associated with a mutant k-Ras (D’Cruz et al. 2001). Recently, several laboratories have developed inducible, dose-dependent systems that provide insight into the effects of dose on mammary tumor structure (D’Cruz et al. 2001; Gunther et al. 2003; Moody et al. 2002). For example, under low doses of doxycycline, the Tg(Erbb) tumors have recognizable cytology with round to oval nuclei and relatively abundant pink-orange cytoplasm (Moody et al. 2002). By contrast, under higher doses of doxycycline, the tumor patterns are quite different and variable. The early high-dose tumors tend to have smaller nodules without zonation. The larger high-dose tumors remain subdivided with sheets of cells rather than the characteristic nodular, zonal pattern of the typical Tg(Erbb) tumors. Neovascularity is a striking feature. The high-dose tumors also have more microinvasion and fibrosis. The Tg(Ras) mammary tumors induced with low-level doxycycline, and thus, with low-level Ras expression, have the typical Tg(Erbb) phenotype complete with the characteristic zonation (unpublished observations). Recurrent tumors have the same histology. However, Ras tumors induced by high levels of doxycyline are an unusual mixture of epithelium and spindle cells. IHC suggests that the bulk of the spindle cells are neither myoepithelial nor luminal. These masses don’t resemble any other tumor that we have seen thus far.
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In general, the levels of transgene expression are very important in determining the tumor pattern, but the cytology will remain recognizable regardless of the expression level. This conclusion is tentative because there is relatively little extant data concerning the effect of different levels of transgene expression on tumor cell structure. Several hypotheses can be considered. Perhaps tumorigenesis depends on a threshold level of gene expression. Once the threshold is reached, a tumor is induced. Another possibility is that the absolute level of transgene expression will have specific effects on tumor phenotype. Experiments need to be designed to specifically address the issue of expression levels. Corollary E: Tumor suppressor genes only indirectly affect the microscopic structure of the resulting tumors (Figure 24-7).
Compared to the transgenic oncogene models of GEM, the knockout, or silencing, of tumor suppressor genes leads to a greater range of tumor phenotypes. Many of the early studies were difficult to interpret as tumor suppressor knockout tumors were initially induced using germ cells. For example, many of the “tumors” in the mammary fat pad of Tm(Trp53-/-) mice are hemangiomas, hemangiosarcomas, or spindle cell tumors (Donehower et al. 1992) and are not true mammary tumors. More recently, however, conditional knockouts have been used to target specific tissues and organs. Even then, the typical mammary tumors in these knockouts are poorly differentiated without an identifiable signature phenotype (Lin et al. 2004). These targeted knockouts give a much clearer idea of the effect of the loss of tumor suppressor gene expression in specific organs. Based on these experiences, it might be argued that the morphological hallmark of tumor suppressor genes, such as Trp53, Rb1, Brca1, and Brca2, is, instead, the phenotype of whatever “secondary hit” is associated with tumor progression (Cardiff 1996; Cardiff et al. 2000a). This principle can be illustrated by having the tumor suppressor gene knockout specifically paired with an oncogenic transgene. In these cases, the tumors have the phenotype of the transgene but with more nuclear pleomorphism (Cardiff et al. 2000a) and increased aneuploidy (Li et al. 1997) (Figs. 24-7A, 24-7B, and 24-7C). Since most, if not all, tumors induced by powerful oncogenic transgenes result in limited genetic instability and near-diploid DNA content, the pleomorphic tumor phenotype and genetic instability of the tumor are probably the result of loss of the suppressor gene control of the cell cycle (Borowsky et al., Personal Communication). These principles are best illustrated when p53 Tg(Trp53tm) mice are crossed with Tg(Erbb2Neu) GEM. Pure Neu tumors were diploid (Fig. 24-7A), whereas the tumors in bigenic mice were aneuploid (Fig. 24-7B) (Li et al. 1997). Morphologically, the bigenic tumors have the typical Erbb2 signature cytology with solid nests of tumor cells with abundant pink-orange cytoplasm. However, when compared to Erbb2-type tumors, the nuclei are larger and more pleomorphic (Fig. 24-7B), and mitotic figures
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appear randomly throughout the tumors rather than limited to a peripheral area. Furthermore, the nodules of cells rarely have the zonal layering seen in the well-differentiated Tg(Erbb) tumors (Figs. 24-2B and 24-7A). The pattern observed in these crosses can be compared with a mammary tumor from a pure Tm(Trp53-/-) (Fig. 24-7C). Another example can be found in Figs. 24-1D and 24-2F illustrating e-cadherin on a Trp53 null background (Tm(Cdh1-/xTrp53-/-)) (Jonkers and Derksen, Personal Communication). The Tm(Cdh1-/-xTrp53-/-) pattern is strikingly different from those in Fig. 24-7. Similarly, Trp53 null mice were crossed with exogenous MMTV-infected C3H/OuJ mice (Chatterjee et al. 2002). Consistent with MMTV infection, the resulting tumors clearly had the Wnt pathway phenotype. However, the loss of Trp53 increased the nuclear pleomorphism and led to progressively less differentiated phenotypes. Another line of evidence deserves recognition here: the silencing of two tumor suppressor genes. The combination of Trp53 and Rb1 knockout, whether produced by Tg(SV40-Tag) or double conditional knockout, apparently leads to a tumor with neuroendocrine characteristics like those induced in different organ systems with SV40-Tag (Galvez et al. 2004; Linnoila et al. 2000; Meuwissen et al. 2003; Minna et al. 2003; Nikitin et al. 2004). Not all tumor suppressor genes lead to aneuploid tumors with genetic instability. For example, Pten is considered a tumor suppressor gene (Kishimoto et al. 2003). However, in the prostate, Pten behaves like an oncogene and activates pAKT-1, leading to a somewhat specific phenotype with large pleomorphic cells with a pale cytoplasm (Abate-Shen et al. 2003) (Figs. 24-7D and 27-8C). These cells are positive for pAKT-1 antigen by IHC (Abate-Shen et al. 2003). All prostate cancers associated with AKT1 activation have this appearance.
Axiom III:
Some molecular alterations have minimal
influence on the microscopic structure of tumors. Corollary A: The promoter has minimal effect on the microscopic structure of the tumor.
Tissue-specific and conditional promoters have been developed to target the same genes to different organs. These constructs give the investigator tremendous control over dose and temporal and spatial expression patterns during neoplastic progression. No doubt, these variables will provide additional insights into the neoplastic process and the structure of tumors. Our major concerns in this section, however, are how tissuespecific promoters influence the tumor structure when the same oncogene is expressed behind different promoters targeting the same organ. Three mammary oncogenes—Tg(Ras), Tg(Myc), and Tg(Erbb2Neu)—have been expressed by several different promoters to target the mammary gland. Originally, they were placed
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behind the MMTV-LTR promoter (Pattengale et al. 1989). All three oncogenes have also been placed behind WAP and other promoters (Andrechek et al. 2003; Andres et al. 1987; Sandgren et al. 1995; Schoenenberger et al. 1988). In those examples that we have examined, these oncogenes retain their original phenotype regardless of the promoter. For example, Tg(Myc) tumors have the same appearance behind WAP and MMTV-LTR (Cardiff et al. 2000a, 2000c). Erbb2Neu has been expressed by the native Erbb2 promoter (Andrechek et al. 2000a) and the tumors retain the original Tg(Erbb) tumor phenotype. When Myc, Wnt1, Ras, or Neu were expressed by the MMTV-LTR and inducible promoters, each oncogene produced its recognizable, unique mammary tumor phenotype (D’Cruz et al. 2001; Gunther et al. 2003; Moody et al. 2002). Recently, Trp53 conditional knockouts have demonstrated similar phenotypes, but with increased numbers of estrogen receptor positive (ER+) cells when placed behind the WAP as compared with the MMTV-LTR promoter (Lin et al. 2004). This finding raises the possibility of subtle functional differences in tumor phenotypes when different promoters are used. Other organ systems have their own tissue-specific promoters. For example, new models of neural tumors have been created using common oncogenes with brain-specific promoters such as Nestin (Weiss et al. 2002). These GEM have most frequently resulted in poorly differentiated malignancies. However, replicas of oligoneuromas, medulloblastomas, and glioblastomas are also being produced using tissue-specific promoters (Weiss et al. 2002). Beta-catenin (Catnb) is another example. In the mammary gland, Tm(Catnb) conditional knockout results in alveolar squamous metaplasia and formation of hair keratins referred to as pilar growth (Miyoshi et al. 2002a, 2002b; Rosner et al. 2002). Expressed by a different promoter targeted to the prostate gland, the conditional knockout also results in squamous metaplasia of the prostatic ducts (Bierie et al. 2003). Thus, the same knockout gene under the control of different targeting systems targeting different organs results in the same kind of epithelial metaplasia (Bierie et al. 2003). Corollary B: Mutational changes in the initiating oncogenic transgene will not change the fundamental microscopic structure of the resulting tumor.
Major efforts have been made to modify gene function by the introduction of site-specific mutations into the oncogenes (Andrechek et al. 2000b; Dankort et al. 2001; Guy et al. 1992a, 1992b, 1994, 1996; Webster et al. 1998). Thus far, these alterations have changed the tumor kinetics or mutation rates but have rarely had a major influence on the morphology of the end-point tumor. Many mutational variants of PyV-mT and Erbb2 have been placed into transgenic mice (Andrechek et al. 2000b; Dankort et al. 2001; Guy et al. 1992a, 1992b, 1994, 1996; Webster et al. 1998). Such mutations include PyV-mT PI-3′Kinase (Hutchinson et al. 2001), the Grb/Shc (Guy et al. 1994), and
the Src binding sites (Webster et al. 1998). The biology of mammary tumorigenesis was altered by these mutations. For example, the inactivation of PI-3′Kinase binding sites, a double base (Db) mutant at amino acids 315 and 322, resulted in tumors with a low metastatic potential. Morphologically, the metastatic wild-type tumor and nonmetastatic Db mutant tumors had similar cytology (Cheung et al. 1997). However, the early proliferations associated with the nonmetastatic Db mutant tumors include a particularly fibrotic stroma, whereas fibrosis is not a prominent feature of the wild-type tumors (Hutchinson et al. 2001). In a like manner, the Muller Laboratory mutated five of the tyrosine docking sites in the Erbb2 gene and then systematically added them back (Dankort et al. 2001). The resulting tumors in all of the different “add-backs” were remarkably morphologically similar. With rare exception, the Erbb2 tumors maintain their phenotype whether or not they contain activating mutations. One reason for this event is that the genes undergo similar amplifications and mutations. The truncation of the Erbb transcript is one example. The bottom line is that all of the different mutations in the Erbb pathway tumors have provided remarkably similar tumor morphology. Corollary C: The site of oncogene insertion has little or no effect on the microscopic structure of the resulting tumor.
Promoters and insertion sites apparently have little effect on tumor morphology produced by the major oncogenic transgenes. Multiple lines of Tg(Erbb) have been created as described above. Certainly the inserts are not all in the same sites. Yet, the morphologies of the tumors are indistinguishable. Myc has also been used frequently as a transgene in different experimental environments. Again, the multiple mouse strains and the different promoters result in very similar tumors of the mammary gland. The large blue cell Tg(Myc) tumor is recognizable in almost every experimental setting. However, very few transgene insertion sites have been molecularly characterized. While the promoter and the insertion sites do not appear to have a specific effect on the tumor morphology, the adjacent DNA clearly does have an effect. One dramatic example of this can be found in GEM with Burkitt’s-like lymphoma (Kovalchuk et al. 2000). The Myc transgene is generally associated with a highly proliferative lymphoid cell population. However, the typical “starry sky” appearance found in the human disease had not been consistently observed in mice until Morse’s group inserted a portion of the human Burkitt’s tumor cell intron located near the Myc oncogene translocation site. Then, all the morphological characteristics of Burkitt’s lymphomas were produced (Kovalchuk et al. 2000). Clearly, the adjacent DNA played an important role in this tumor phenotype. The other argument concerning insertion sites involves the numerous transgene insertions that have not resulted in tumorigenesis. These negative experiments are rarely recorded in the literature and are seldom pursued. Furthermore, some investigators have observed the silencing of transgenes with successive
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Tg mouse generations (Tehranian et al. 1996). Therefore, it would be premature to contend that the insertion site has no effect on transgene tumor phenotype.
PRINCIPLE 2:
TUMORIGENESIS IS INFLUENCED
BY TISSUE CONTEXT (FIGURE 24-5). Axiom I:
The microscopic structure of tumors initiated
by weak oncogenes can be influenced by tissue context. As previously discussed, the Myc oncogene may represent the single most dominant oncogene (Fig. 24-3). Although the characteristic Tg(Myc) tumor patterns are recognizable in most organ systems, Tg(Myc) expressed in the mouse prostate evokes a different pattern (Fig. 24-3A) (Ellwood-Yen et al. 2003). Myc-driven prostatic tumors have oval, relatively uniform nuclei with a delicate vesicular chromatin and relatively abundant pale blue chromatin. Although these tumors form back-to-back glands and resemble the cytology of human prostatic tumors, they do not have the nuclear pleomorphism and clumped chromatin seen in Tg(Myc) models in other organ systems (Ellwood-Yen et al. 2003). It is not clear whether this more delicate cytological pattern is related to the level of gene expression, the organ system, or the temporal expression of Myc in the prostate cell cycle. Like other tumor suppressor genes, Tm(Pten) knockout is also associated with a variety of neoplasms, including hemangiomas, sarcomas, and mammary tumors (Kishimoto et al. 2003). The mammary tumors merit special mention because, in contrast to prostate tumors, Tm(Pten) knockout tumors of the mouse mammary gland are typically papillary myoepithelomas (Freeman et al. 2003). This observation illustrates that the organ site can influence the transgene tumor phenotype. However, the Pten knockouts are not targeted to the mammary gland and are not behind a mammary specific cre-lox system. Ras was one of the first oncogenes discovered and used in transgenesis. Ras is complementary to, and dominated by, the Myc gene (Cardiff et al. 1991). Tg(Ras) tumor patterns vary from organ to organ and illustrate that relatively “weak” transgenes are influenced by the organ context (Fig. 24-5). Ras oncogenes have been used for transgenic oncogenesis in almost every organ system. In the mammary gland, the Ras transgenes tend to form papillary or solid tumors that are closely related to the Erbb pathway tumors (Cardiff et al. 1991). However, the context of the Ras transgenesis or the specific Ras mutation is critical. For example, high levels of expression result in complex, poorly differentiated mammary neoplasms (Strange et al. 1989). Lower level induction with doxycycline produces typical papillary and nodular zonal tumors characteristic of the Erbb pathway. Historically, mutational activation of Ras by chemical carcinogens 7,12dimethylbenz[a]anthracene (DMBA) or 3-methylcholanthrene
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(MCA) led to tumors with adenosquamous differentiation (Cardiff et al. 1988c; Gardner et al. 1985). Transduction with replication defective viruses containing H-vRas led to abnormal mammary outgrowths, while infection with replicationcompetent sarcoma virus carrying Ras led to undifferentiated spindle cell tumors (Strange et al. 1989). Thus, the level of Ras expression has a dramatic effect on the tumor phenotype. In mouse lungs, the Tg(Ras)-induced tumors form typical papillary adenomas and carcinomas (Fig. 24-5B) (Galvez et al. 2004; Nikitin et al. 2004) that closely resemble the spontaneous papillary tumors observed in wild-type A/J and FVB/N mice. This observation is consistent with the presence of the A/J Ras allele in these strains (Lin et al. 1998; Matzinger et al. 1997). In the epidermis, Ras induces squamous papillomas and squamous cell carcinomas (Borowsky et al. 2004). In the brain, the tumor type depends on the promoter (Fig. 24-5A) (Borowsky et al. 2004). The Ras prostate phenotype is particularly interesting because, placed behind probasin, Tg(Ras) produces Pten(Akt) type of papillomas and a peculiar intestinal metaplasia featuring mucus-producing goblet cells in luminal epithelium (Fig. 24-5D) (Scherl et al. 2004). This finding wellexemplifies the unexpected consequences of transgenesis. Strangely, when overexpressed in the intestine, ras results in an invasive adenocarcinoma without extensive mucus production (Fig. 24-5C) (Janssen et al. 2002). Beta-catenin overexpression results in mammary gland tumors that form skin-like squamous lesions with a peculiar squamous metaplasia that resembles hair follicles (pilar differentiation) (Miyoshi et al. 2002a, 2002b). Since the mammary gland develops as a modified sweat gland, pilar differentiation is not unexpected. However, beta-catenin expression in the prostate is also associated with squamous metaplasia (Bierie et al. 2003). Thus, to summarize, the tissue context has a powerful effect on the tumors induced by relatively weak oncogenes and less effect on more dominant oncogenes.
Axiom II:
The tissue and other factors will influence the
tumor biology but not the tumor morphology. Corollary A: GEM tumors exhibit molecular relationships and interdependencies.
Although tumorigenesis requires numerous interacting, complementary molecular events, the morphology of resulting tumors does not appear to be affected by these events. Examples of what is meant by molecular relationships and dependencies include the requirement for cyclinD1 for mammary tumorigenesis in the Erbb but not the Wnt/Myc pathways (Yu et al. 2001). Another example is the Wnt1-Fgf3 interdependency. When an Fgf-induced transgenic is infected with MMTV as a gene-targeting vector, the resulting tumors have the provirus inserted into a Wnt site and vice versa, and Fgf insertion activation is found in MMTV-induced tumors in Tg(Wnt) mice (MacArthur et al. 1995). The Tg(Fgf3), Tg(Wnt1), and bigenic
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Tg(Fgf3)xTg(Wnt1) GEM all have tumors with similar morphological patterns. Apparently, Myc and Ras form a similar interdependency. Although the bigenic Tg(Myc), Tg(Ras) cross is dominated by the Myc tumor phenotype, Ras has a related interdependency in that the persistent and recurring doxycycline-induced Tg(Myc) tumors have a mutated k-Ras. That is, the k-Ras mutation is usually present in the primary tumor. The morphology of the tumor is more related to the initiating oncogenes than to the secondary event. Other interdependencies have been demonstrated that are not based on oncogenes or tumor suppressor genes. For example, knockout of integrin beta 1 (Tm(Itgb1)) resulted in the abrogation of Tg(MMTV-PyV-mT) mammary tumors when the two mice were mated (White et al. 2004). Since these bigenic animals are apparently chimeras, a few cells with the native wild-type integrins can give rise to the occasional tumor (White et al. 2004). Tg(MMTV-Erbb2-ndl), Tg(Vegfa) mice have accelerated tumorigenesis (Oshima et al. 2004). The tumors have the features of the signature Erbb2 tumors but with accentuated vasculature. Axiom III:
The microscopic structure of tumors
is not influenced by external factors. The principles developed above have been derived primarily from mammary gland transgenesis using dominant oncogenes. The central question is whether these, or any, genes have sufficient influence that their tumor phenotype is recognizable in the context of different organs, different genetic backgrounds, and different microenvironments. For the dominant oncogenes such as Tg(Myc) and Tg(SV40-Tag), the answer is largely affirmative. The tumor morphology is recognizable in various organs (Figs. 24-3 and 24-4). Thus, from a gene-centric viewpoint, a gene expression hierarchy exists that prevails across tissue and organ systems. As suggested previously, some genes, such as Myc, are dominant and leave their signature phenotypes in all systems. Other genes are less so. However, certain patterns prevail in other organs. Although the rules are not completely understood, an empirical classification based on current examples can be started. Oncogene dominance is judged on the basis of bigenic and trigenic crosses, where available, and the effect on tumor phenotypes in different organ systems. The most dominant genes in terms of tumor phenotypes are Tg(Myc), Tg(SV40-Tag), Tg(Akt1), and Tg(ErbbNeu). The less dominant genes providing contrast are Tg(Ras) and Tm(Trp53). Corollary A: Microorganisms can be co-factors in GEM-associated tumors, but they do not influence the microscopic structure of the tumors.
Opportunistic microorganisms, other than the retroviruses, can influence tumorigenesis. These discoveries are generally regarded with horror as “train wrecks” rather than as
serendipitous opportunities. Therefore, most of the inadvertent infections were cured by the administration of antibiotics or rederivation of the mice. However, the possible role of the microorganisms as co-factors in the neoplastic process remains unknown. The classical example of microbial co-factors can be found in the Tm(Il10-/-) knockout mice that developed intestinal neoplasms (Boivin et al. 2003). The initial publications suggested that the immunodeficiency created in the knockout was a direct cause of the neoplasm (Berg et al. 1996). However, others found that the tumor incidence was reduced to zero when the animals were treated for Helicobacter sp. Subsequently, it was demonstrated that introduction of Helicobacter sp. into a defined pathogen-free colony of Tm(Il10-/-) mice stimulated the development of inflammatory bowel disease (hyperplastic typhlocolitis) and tumors (Kullberg et al. 1998). It is not clear, however, whether the microorganisms are co-factors similar to human gastric cancer or whether the intestinal lesions simply represent a dysplasia occurring during repair of bowel inflammation. Microscopic examination of the prolapsed rectum of animals with Helicobacter infections reveals severely dysplastic epithelial proliferations that herniate through the muscularis (Boivin et al. 2003). Although histological criteria for microinvasion in the gastrointestinal tract have been suggested, they have not been validated by experimental evidence (Boivin et al. 2003). Corollary B: The microscopic structure of the neoplasm is not affected by the background strain.
Although genetic backgrounds are critical in tumorigenesis, we have not yet observed cases in which different morphological patterns are directly attributable to the mouse strain. Three strains have been most commonly used to produce GEM: FVB/N, C57BL/6, and 129. C57BL/6 mice are the most commonly used, comprising over 90% of all mice sold in the United States. They are relatively resistant to spontaneous tumorigenesis and are widely used for toxicological studies. The FVB/N is a derivative of the Swiss mouse that superovulates and has proven to be relatively susceptible to spontaneous tumorigenesis. It is most commonly used for mammary tumorigenesis. The 129 strain was developed because they are a ready source of embryonic stem cells used in targeted mutagenesis. In comparing transgenic tumors in several strains, we have not found significant differences in the tumor phenotype. For example, PyV-mT mammary tumors are morphologically similar in B6, FVB/N, BALB/c, and in some hybrids (Le Voyer et al. 2000). Prostatic tumors and hyperplasias have different tumor kinetics in B6 as compared to FVB mice, but the tumors are indistinguishable (unpublished data). The original transgenic mammary tumors, Tg(Myc), Tg(Ras), and Tg(Neu), were created in CD1XB6 F1 hybrids (Muller et al. 1988; Sinn et al. 1987). They are identical to tumors induced by the same oncogenes in FVB/N GEM.
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On the other hand, investigators are also using GEM to search for genetic modifiers of tumorigenesis (Dragani 2003). By their very nature, these experiments involve lengthy and complex breeding programs. One successful example was the description of the Aurora gene in skin carcinogenesis (EwartToland et al. 2003). The breeding experiments, however, did not substantially modify the microscopic morphology of the tumors. Extensive outcrossing of Tg(PyV-mT) mice onto multiple-strain backgrounds provides another example (Le Voyer et al. 2000). Although the latency and metastatic rates were modified in these animals, there was little morphological change. These experiments are being continued in hopes of defining the modifier genes. Another type of experiment that potentially deals with host modification has been the crossing of a specific GEM with MMTV-infected strains. In most of these cases, the experiments are designed for oncogene discovery rather than host modifier genes. Thus far, these studies primarily have identified previously known collaborating genes and little change in the MMTV tumor phenotype. However, when crossed with Tm(Trp53) null mice, the mammary tumor phenotype retained the basic Wnt characteristics, which, in multiple back-crosses, became progressively less differentiated (Chatterjee et al. 2002). These examples deal with poorly defined genetic background and not with specific molecular modifications of the host strain. Although the biology of the tumors is frequently affected, the microscopic structure is usually unaffected. The types of molecular dependencies and relationships are detailed in the next section. Corollary C: Host modifier genes do not affect the microscopic structure of transgenic tumors.
The flexibility of transplant biology and genetic engineering afforded by the mouse models has illuminated the role of host genes that is difficult to document in humans. For example, the ability to cross transgenic mice bearing oncogenic genes with knockout mice has uncovered the influence of host factor genes such as Ets2, Csf1, plasminogen, Tgfb1, and proteases (Lin et al. 2004; Oshima et al. 2004; Siegel et al. 2003; Sternlicht et al. 1999). The influence of these host genes clearly can be defined by transplanting the wild-type tumor into knockout hosts that do not contain the gene in question (Oshima et al. 2004). A dramatic example of host modifiers has been provided by the knockout of the integrin 1b gene in Tg(PyV-mT) tumorbearing mice in which there was an almost complete ablation of tumorigenesis (White et al. 2004). However, the tumors that did progress had the phenotype expected for the Tg(PyV-mT). Less dramatic influence on tumor kinetics was observed with the dysregulation of Ets2, which slowed the development of tumors but had only marginal effects on the tumor phenotype (Man et al. 2003; Neznanov et al. 1999). When suspected host modifiers are included in the epithelium or other oncogenic target tissue, one may have dramatic
effects on the tissue characteristics but retain the cytological features. For example, Tg(Erbb) mutants crossed with mice expressing mammary vegfa developed tumors at an accelerated rate (Oshima et al. 2004). The tumors were remarkable in that they had huge vascular sinuses that subdivided the tumors into small nests rather than large nodules. These bigenic mice resemble some of the tumors in the high expression, doxycycline induced Tg(Erbb) models (Moody et al. 2002). A more subtle example is the bigenic Tg(Erbb,Tgfb1) mice whose mammary tumors are identical to the standard Tg(Erbb) tumors but that have a different pattern of pulmonary metastases (Siegel et al. 2003).
PRINCIPLE 3:
NEOPLASTIC PROGRESSION IS A
MULTISTEP PROCESS ASSOCIATED WITH SEQUENTIAL MORPHOLOGICAL CHANGES (FIGURES 24-8, 24-9 AND 24-10). Axiom I:
Neoplastic progression is associated with a
sequential continuum of microscopic changes. Pioneering studies by Trout and Papinicolau clearly defined clinical cancer of the uterine cervix as a multistep process, giving hope that early detection and intervention could cure the disease. History has proven this true. The early essays by Leslie Foulds crystallized the concepts of neoplastic progression which were based primarily on the mammary gland of the RIII mouse (Foulds 1951, 1958). However, by that time Shubik had defined a two-step process of initiation and promotion on skin carcinogenesis (Shubik 1984). Potential precursors to overt cancer in the mouse mammary gland were identified as early as 1906 (Apolant 1906; cited in Blair 1968; Medina 1996) and associated with overt mammary cancer in 1911 by Haaland (1911) (cited in Blair 1968; Medina 1996). DeOme and colleagues defined mouse mammary gland preneoplasia as a focal atypical lesion that stood out from the background and provided an operational definition of the early stages of neoplastic progression (Cardiff 1984; Cardiff et al. 2002). The stages of progression in the colon have been elegantly related to sequential genetic mutation by Vogelstein and colleagues (Vogelstein and Kinzler 2004). This type of molecular and morphological modeling has become the standard in mouse as well as human cancer. The models have become increasingly more complex as additional genes and host factors have been added to the schemata (Hanahan and Weinberg et al. 2000). The challenge for the comparative pathologist is to recognize the early lesions in the different models and to correlate them with the genetic and biological changes. The key question is whether morphological markers can be identified that predict biological potential. Thus far, such markers have eluded us (Cardiff et al. 2000b).
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SM SM
SM
SM
A
B
SM
VE
C
D
Fig. 24-8 Neoplastic Processes Axiom: Neoplastic progression is associated with a sequential continuum of microscopic changes. The interpretation of each phase of the process requires close attention to histologic detail. This panel compares different stages of the neoplastic process with nonneoplastic conditions. Digital photomicrographs illustrating herniation (Fig. 24-8A), microinvasion (Fig. 24-8B), in situ premalignancy (Fig. 24-8C), and tumor emboli (Fig. 24-8D). Fig. 24-8A is a view of the ureter that shows herniation through the smooth muscle layer (SM) and into the surrounding serosa. The cells inside the mucosa and outside the muscle have the same nuclear features, and little or no host inflammatory response to the herniated mucosa is observed. Contrast this pattern to that of the microinvasion in the prostate (Fig. 24-8B) where tumor cells bulge through the fibromuscular tunica (SM). The cells at the advancing margin have larger, more pleomorphic nuclei and the surrounding stroma has dilated vessels and numerous inflammatory cells. Fig. 24-8C illustrates an in situ carcinoma of the mouse dorsolateral prostate with a small zone to the left of relatively normal epithelium inside a normal smooth muscle layer (SM), the single layer of cells at the center with larger nuclei and more abundant cytoplasm (mPIN grade I), and cells at the right-hand side heaped up in multiple layers with a cribriform pattern (mPIN III). Panel Fig. 24-8D shows two intravascular tumor emboli. Note the larger embolus in an endothelium-lined vessel (VE) containing red blood cells. All images have been captured using a Zeiss AxioCam with a 40x objective. The scale bar for all images is in Fig. 24-8D. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
Corollary A: GEM neoplasia progresses through the same series of structural changes as human neoplasia.
The explosive growth rate of multiple tumors in any individual GEM originally suggested a one-step “simultaneous” transformation (Muller et al. 1988). However, even the most accelerated tumorigenesis in GEM is associated with multiple
secondary events (Maglione et al. 2001). Each step is recognized as a discrete, increasingly abnormal focus (Fig. 24-10). Even the inducible tumor models are associated with secondary mutational events (D’Cruz et al. 2001). Clearly, neoplastic progression in GEM occurs as multiple complementary and sequential events similar to those that occur in other models of neoplasia.
Tu
A
B VE VE
TE
TE
Br
C
D
Br
E
F
Fig. 24-9 Metastatic and Nonmetastatic Conditions Axiom: Regional or systemic metastasis is a more reliable criterion of malignant potential than microinvasion. Tumors of the mouse may spread regionally or systemically. Corollary A: The primary route of metastasis in the mouse is via tumor emboli. The majority of systemic metastases are pulmonary and intravascular. Pathologists need to distinguish between intravascular lesions that do not colonize the lung, true invasive metastases, and intrinsic tumors of the lung. The tumor cells from a local mammary tumor (Tu) are infiltrating the lymph node as sheets of large pale cells (Fig. 24-9A). A mass of metastatic mammary tumor cells is found invading the pulmonary parenchyma (Fig. 24-9B). The tumor embolus adjacent to the mammary tumor in Fig. 24-9C has an endothelial layer covering the tumor (TE) and another lining the vessel (VE). The lung from the same animal also has an intravascular tumor embolus lined by two endothelial layers (TE and VE) (Fig. 24-9D). A small tumor embolus (E) that is entrapped in a sub-bronchial (Br) blood vessel is illustrated in Fig. 24-9E. The vessel wall is not infiltrated. A large primary lung papillary adenoma is observed within the lumen of a bronchus (Fig. 24-9F). Note the distinct papillary pattern in Fig 24-9F that is quite different from the metastatic tumor cells in Fig. 24-9B, Fig. 24-9D, and Fig. 24-9E. All images have been captured using a Zeiss AxioCam with a 20x objective. The scale bar for all images is in Fig. 24-9F. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
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Recently, the concept of tumor “stem cells” has reintroduced the possibility of one-step transformation (Al-Hajj and Clarke 2004; Harrington 2004; Warner et al. 2004). This idea arose long ago from observations with totipotential ovarian teratocarcinomas. A teratocarcinoma cell could be cloned and injected into normal mouse embryos to create allophenic chimeras. In this remarkable situation, tumor cells could be shown to be differentiating into mature, adult structures such as hair (Martin 1980). Recently, these concepts have been popularized and expanded in the new field of stem cell biology. In the context of tumor transgenesis, de-inducible oncogenes demonstrated that tumor phenotype is dependent upon expression of a single oncogene (D’Cruz et al. 2001; Gunther et al. 2003; Moody et al. 2002). When “switched off,” the oncogene expression and the palpable tumor disappeared. It is tempting to conclude that tumorigenesis in this instance is the result of the expression of a single oncogene. However, subsequent analysis of primary and recurrent tumors suggests that additional mutations preexist in the primary tumor and are predictive of recurrence (D’Cruz et al. 2001). This notion is consistent with the observation that in normal transgenic mice, the highest levels of the transgene appear in areas of dysplasia and not in normally developed tissues (Deckard-Janatpour et al. 1997). Another interpretation is afforded by the observation of hepatoblastomas transplanted into subcutaneous tissues (Shachaf et al. 2004). In this case, “switch off” of the Tg(Myc) transgene leads into differentiation of quasi-mature hepatocytes with bile ducts and bile canaliculi (Fig. 24-10D). However, these differentiated cells retain some stem cell antigens such as K19 and might be regarded as dormant tumor stem cells (Shachaf et al. 2004). Whether these inducible/de-inducible models are relevant to spontaneous human tumors remains to be seen. However, these models open up a novel experimental approach to further our understanding of cancer. Bereft of such intriguing model systems, the comparative pathologist is left with the microscopic evidence. Morphological events associated with increasing tumor size have been described in many organ systems, including the exocrine pancreas (Hingorani et al. 2003a, 2003b), pancreatic islet cells (Teitelman et al. 1988), mammary gland (Cardiff et al. 2000b), prostate (Gingrich et al. 1999; Park et al. 2002; Shappell et al. 2004), intestinal tract (Boivin et al. 2003), cervix (Arbeit 2003), lung (Nikitin et al. 2004), and other organs. Most of these studies draw attention to atypical foci that stand out from the background. In most cases, the investigators can also associate the insitu lesions with invasive carcinomas. This morphological approach utilizes traditional “guilt by association” embellished by recognizable sequential morphological changes that suggest progression. It was recommended, however, that the morphological evidence be confirmed by ancillary changes in DNA and cytoplasmic differentiation (Park et al. 2002). Thus far, the progressive nature of neoplasia in GEM has remained largely circumstantial and not experimental.
While it is very tempting to regard morphological change as the sine qua non evidence of biologic behavior, this is an erroneous strategy because it does not use the full potential of mouse biology. For example, surgical pathologists with a background in human pathology would regard most of the Wnt-related mouse mammary tumors as benign adenomas because they have an organized myoepithelium. However, the experienced comparative pathologist regards the tumors as malignant because up to 60% of the MMTV-induced Wnt-type mammary tumors are metastatic and the metastases also have a well-organized myoepithelium (Vaage 1989). In contrast, the surgical pathologist tends to overcall the “dysplasias of repair” that are so prominent in many of the mouse models. The Helicobacter sp. proliferative lesions appearing in the large intestine of mice with immunological deficiencies are readily “cured” by adequate treatment with antibiotics (Kullberg et al. 1998). The point here is that the biologic behavior of the lesion is the single most significant factor. The morphology can be easily misinterpreted. There is no excuse for not testing any given hypothesis concerning neoplastic potential in mice by transplantation of the suspect tissue. Corollary B: Premalignant lesions are atypical focal hyperplasias associated with high risk of malignant transformation.
The basic concepts of initiation, promotion, and premalignancy have been developed in the mouse by direct experimental observation and transplantation (Cardiff et al. 2000a, 2000b). Neoplastic progression in human pathology is based on a hypothetical progression of naturally occurring lesions associated with complex demographic factors, a process called guilt by association (Cardiff et al. 2004). In human cancer, the initial observations generally associate the focal lesions with atypia and an observable invasive cancer (Figs. 24-8 and 24-10). Given this association, can we associate the presence of the focal atypias with the eventual development of cancer? If this can be proven in humans through epidemiological evidence, it becomes the basis for clinical treatment. In experimental pathology of the mouse, the pathologist must also learn to recognize focal atypias and render a reasonable judgment about their biological potential. In most cases, the comparative pathologist does so using guilt by association; that is, coupling evidence of progressive morphological alteration with a clear-cut association with malignancy. The best examples come from tissues adjacent to a grossly malignant neoplasm. Very frequently, the nonmalignant tissue adjacent to a tumor will reveal precursor lesions illustrating the evolution of the tumor (Abate-Shen et al. 2003; Park et al. 2002). For this reason, sampling of tumors should include at least an equal area of “normal” tissue adjacent to the tumor. Of course, in some early GEM models, potential precursor lesions were identified. Unfortunately, the animals were sacrificed before the development of overt invasive tumors. In these situations, one has to rely on supposition of progression without
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morphological proof. The early studies of non-Tag mouse Prostatic Intraepithelial Neoplasia (mPIN) illustrate the principles (Park et al. 2002). Atypical focal lesions stood out from the background as larger cells with large atypical nuclei and an abnormal growth pattern. They could also be identified as focal lumps when examined in subgross whole mounts (Kim et al. 2002a; Maglione et al. 2001). The early lesions in mPIN were characterized by increasing size and nuclear atypia as the mouse aged (Park et al. 2002). The lesions eventually filled and distorted the duct lumina with a loss of the fibromuscular sheath surrounding the ducts. These focal lesions, typical of all GEM associated with the Pten/Akt pathway, were categorized and subdivided into a continuum of morphological changes that were predictive of progressive biological severity (Park et al. 2002). Vascular tumor emboli plus regional and distant metastases were found in association with the more severe Grade IV lesions (Abate-Shen et al. 2003). Overt invasive tumors were subsequently identified in older animals, validating the grading scheme (Abate-Shen et al. 2003). The weakness of the guilt-by-association approach is that it is ultimately a statistical association and not a biological occurrence. Pathologists have not been able to identify phenotypic markers that are predictive of biological behaviors such as tumor risk or metastatic rate. This is true even in the wellcharacterized spontaneous mouse and GEM mammary tumor systems. Years of searching have failed to reveal any morphological features of premalignancy that predict the relative risk of malignant transformation (Medina 2000). “Guilt by association” has also led to disappointing misinterpretations. For example, “low-grade” PIN is no longer considered a risk factor in human prostate cancer (Shappell et al. 2004). Some forms of DCIS are not the basis for intervention in human breast cancer (Cardiff et al. 2004). In these cases, it is not clear whether these lesions are ever progressive. Therefore, the adoption of simple guilt-by-association morphological criteria for focal dysplastic lesions of the mouse is likely to be misleading and ignores the biology: the real value of the mouse. For example, the Grade IV mPIN lesions associated with metastatic mouse prostate adenocarcinoma are still in situ and are probably not the ultimate source of the metastatic disease. One has to consider sampling error, but few investigators have done serial sections to confirm the presence of invasive cancer elsewhere in the prostate. Unfortunately, many of the invasive prostatic tumors in GEM are not associated with metastatic disease. Therefore, some uncertainty persists owing to the indirect association in the interpretation of these prostatic lesions. Pathologists also should be wary of lesions, diffuse and focal, that show little or no progression. For example, the mammary glands of multiparous mice frequently have focal areas of inflammation associated with squamous metaplasia. The residual columnar epithelium frequently has large hyperchromatic nuclei and irregular outlines that are regarded as the
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“dysplasia of repair” lesions that are associated with blockage of the ducts and milk stasis. They are called inflammatory nodules or squamous nodules and rarely are associated with tumors. Years ago, these nodules were tested by transplantation, and they did not have a high risk of malignant transformation. Diffuse hyperplasia is rarely associated with progression. For example, probasin-driven SV40-Tag induces complex and diffuse hyperplasias of the prostatic epithelium (Masumori et al. 2001; Shappell et al. 2004). Although some examples of microinvasive adenocarcinoma have been identified in these animals, the majority of invasive lesions are neuroendocrine tumors that either represent a neoplastic transdifferentiation (an alternate pathway of the epithelium) (Evangelou et al. 2004; Shappell et al. 2004) or a tumor originating in the neuroendocrine cell population. In this specific case, evidence of progressive change of the surface epithelium has been offered from the cryptidin-driven Tg(SV40-Tag) model (Garabedian et al. 1998). The well-differentiated microinvasive tumors in these Tag-associated mice have morphology that resembles the Pten/Akt pathway and have an upregulated phospho-Akt1 at the protein level. Experimental transplantation of the bulk Tag epithelium suggests that it does not progress to cancer (Couto, Personal Communication). Another type of lesion begins as small foci but without progressive changes in the cytology of the cells. The Tg(Erbb2)based mammary gland models develop small dysplastic solid nodules early in the evolution of the neoplastic disease. These appear to expand into larger foci without much cytological progress. They could be examples of early transformation. Moreover, the grossly palpable tumors in these models are seldom aneuploid. Transplantation of these lesions has proven that they are premalignant in Tg(PyV-mT) mice (Garabedian et al. 1998; Maglione et al. 2001, 2004). Corollary C:
Biological potential is best defined by transplantation.
Operational criteria for neoplastic progression are well defined in the mouse and have been in existence for over 50 years (Cardiff 1984; Cardiff et al. 2000b, 2002, 2004). These criteria now need to be applied to the novel GEM models (Cardiff et al. 2000b). Preneoplastic lesions are focal areas of atypia that can be identified in situ, isolated, dissected, and transplanted into the same (orthotopic) and other (ectopic) organ sites in syngeneic mice (Fig. 24-10A) (Cardiff et al. 2000b). Normal mammary tissues can be transplanted for several generations, but growth is self-limited and the transplants ultimately senesce in orthotopic sites. Normal tissue will not survive when transplanted into ectopic sites. Mammary neoplasms that are benign or premalignant can be serially transplanted and will grow in orthotopic sites but will not grow in ectopic sites. The neoplastic growth of premalignant mammary tissue is immortalized and can be grown indefinitely in the orthotopic site by serial transplantation. In contrast, a malignant neoplasm, by definition, can grow indefinitely in both orthotopic and ectopic sites.
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SV
Tu
MIN-O
A
B
K
Ep
C
D
Fig. 24-10 Transplantation and Persistent Induced Tumors Corollary: Biological potential is best defined by transplantation. Three of many transplantation techniques are illustrated here. Digital micrographic images illustrating transplantation of premalignant mouse mammary tissues into the gland-cleared fat pad (Fig. 24-10A). The image contains the transplanted abnormal premalignant Mammary Intraepithelial Neoplasia-Outgrowth (MIN-O) separated by host fat cells in the lower right field and the emerging solid tumor (Tu) in the upper left field. Note the increased fibrosis around the tumor. Fig. 24-10B shows a transplant of mouse prostate recombined with rat seminal vesicle mesenchyme implanted beneath the renal capsule of a nude mouse. Nontransformed, normal mouse epithelium recapitulating the structure of a normal seminal vesicle is present in the upper right field (SV). The seminal vesicle is surrounded by a normal appearing fibromuscular tunica maintaining an interface between the seminal vesicle and the host kidney (K) in the lower central field. The abnormal nests of cells exemplifying the dysplasias found in severe mPIN are seen at the upper left. Most of the nests are surrounded by dense connective tissue and a partially formed fibromuscular tunica. However, one nest is not surrounded by stroma but has a different and direct interaction with the host kidney (arrows). Fig. 24-10C and 24-10D illustrate recurrent or persistent tumors in doxycycline-inducible GEM models. Image 10C illustrates a persistent epithelialmesenchymal-transition (EMT) tumor from a doxycycline induced Tg(Wnt1) mouse. The tumor grew after the wnt expression was de-induced and illustrates the combination of epithelial nests (Ep) and stroma that is diagnostic of EMT tumors. Image 10D shows the “dormant” or persistent tumor following subdermal transplantation of a poorly differentiated hepatoblastoma induced by myc. Following repression of the myc, the tumor regressed, but the persistent cells began forming ducts and cords resembling normal liver. All images were captured with a Zeiss AxioCam using 10× and objective 3. The scale bar indicates the scale for each image. The color version and annotated whole slide images of this panel can be viewed at http://imagearchive.compmed.ucdavis.edu/publications/cardiff/.
The dividing line between a benign neoplasm and a premalignant neoplasm is the rate at which malignant neoplasms emerge from the transplant. The premalignant lesion is defined by the high risk of malignant transformation. Two organ systems that have been traditionally used for these definitions have been the mammary gland and the skin. However, transplant systems have also been devised to operationally define neoplastic
progression in other organ systems such as the prostate (Fig. 24-10B) (Wang et al. 2000). Mammary gland premalignancy was originally defined as preneoplasia in the 1960s by DeOme and his students (Cardiff et al. 2002). Although many papers have appeared describing premalignancy in the GEM mammary glands (Cardiff et al. 2000b), only two transplant systems have been developed to
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test the biology of premalignancy in GEM (Maglione et al. 2001, 2004). One involves transplanted Trp53 null mammary premalignant epithelium and the other Tg(PyV-mT ) premalignant mammary epithelium (Maglione et al. 2001, 2004; Medina et al. 2002). Together, they provide proof of the principle that tumorigenesis requires multiple steps for evolution to invasive malignancy. A surprising result of these experiments is that, although the premalignant lesions first identified in the Tg(PyV-mT) founders are somewhat homogeneous dysplasias, the serial transplants are quite heterogeneous in morphological appearance, latency periods, and metastatic rates (Maglione et al. 2004). Preliminary analysis suggests that the major molecular changes occur in the transition from benign to premalignant (Maglione et al. 2004). The transition from premalignant to invasive is associated with relatively few molecular changes (Namba et al. 2004). This observation implies that, even in these transgenic lesions, the biological characteristics (potential) is already predetermined or encoded in the premalignant cells (Maglione et al. 2004; Namba et al. 2004).
Axiom II:
Regional or systemic metastasis is a more
reliable criterion of malignant potential than microinvasion (Figures 24-8 and 24-9). Invasive growth pattern is one of the traditional morphological criteria of malignancy. It certainly holds in mouse tumor biology. However, several caveats must be recognized. First, many, or most, mouse tumors appear to grow by expansion rather than by invasion (Cardiff et al. 2000a). This pattern is misleading since many of the “expansile” tumors are indeed metastatic. Another source of misinterpretation lies in tangential sections through the irregular boundaries of epithelial tissues (Shappell et al. 2004). Usually, tangential sections can be recognized by the asymmetrical outlines of the organ. In organs such as the mouse prostate, proof of invasion requires increased nuclear pleomorphism and a visible host inflammatory response at the site of the invasion (Fig. 24-8) (Shappell et al. 2004). Of course, potentially tangential sections should be serial sectioned to verify that the glandular tissue is disconnected from the adjacent mucosa. Furthermore, an old, recently rediscovered observation may explain the origins of “metastasis” in many models. Some metastases may not necessarily be associated with invasive growth (Vaage 1989), a phenomenon recently termed “invasionindependent intravascular metastasis” (Oshima et al. 2004; Sugino et al. 2002). This idea suggests that pulmonary metastases often arise from fragments of tumor pinching off as emboli in intra-tumoral vessels and not spreading to the lungs by invasion through the stroma (Figs. 24-8D and 24-9C, D and E). Another frequent source of overinterpretation is the herniation of dysplastic epithelium through the muscularis of various organs (Fig. 24-8A) (Boivin et al. 2003; Shappell et al. 2004).
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Herniation is a very common phenomenon in mice with inflammatory lesions and must be distinguished from actual invasion. The criteria recommended for evaluation of such lesions in the gut have general applicability (Boivin et al. 2003). The herniation is recognized as microinvasive only if the lesion has significant cytological change as compared to adjacent epithelium and if it has evolved a host response in terms of increased fibrosis and/or inflammation (Fig. 24-8B). As emphasized above, the final test should be biological behavior rather than morphological appearance. Corollary A: The primary route of metastasis in the mouse is via tumor emboli (Figure 24-9).
The majority of the metastases arising at any site from mouse primary tumors appear in the lungs (Fig. 24-9B). This does not necessarily mean that metastases do not occur in other organs or tissues (Fig. 24-9A). Involvement of the lungs, however, is so frequent and dramatic that most investigators do not bother examining the other organs. It must be pointed out that spontaneous papillary adenomas of the lung are often erroneously counted as metastases (Fig. 24-9F). As mentioned previously, because so many metastases arrive at the lung as tumor emboli, it is not clear whether these are biologically relevant. Many such emboli probably do not invade and colonize the lung (Vaage 1989). We recommend that these tumor emboli be recorded but counted separately from emboli that colonize the lung. The morphological criteria to note are either growth within the vessel to expand the vessel diameter (Figs. 24-9D, 24-9E) or actual invasive growth through the vessel wall into the surrounding parenchyma (Fig. 24-9B). By separating the two phenomena, we have been able to demonstrate the influence of genes that modify metastatic behavior (Siegel et al. 2003). It is very important to note the exceptions to the principle of pulmonary metastases. Sporadic metastases have been found in other organs such as liver or regional lymph nodes (Fig. 24-9A). In fact, metastases to regional lymph nodes seem to be more frequent than distant spread to lungs in some mice such as the Tm(Pten) conditional prostate knockouts (AbateShen et al. 2003). Metastatic growths in the liver are common with some tumor systems. However, bone marrow metastases, a favorite location in human prostate cancer, have been discouragingly rare in transgenic mice. For example, the Tg(SV40-Tag) (TRAMP) prostate cancer model only has about 10% bone marrow metastases (Gingrich et al. 1996). The dearth of bone marrow metastases raises the question of “seed versus the soil.” The mouse spleen, in contrast to humans, is a major hematopoietic organ and yet is not generally a site for metastasis. One could suggest that the mouse cells simply do not have the ability to home-in on the bone marrow or the spleen. In classical tumor recycling experiments, tumor cells can colonize only specific organs (Nicolson 1993), suggesting that the “seed” needs to be competent. Recently, left ventricular injections of tumor cells into mice with transplants
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of exogenous bone and bone marrow suggested that the “soil” is also critical (Urashima et al. 1997). However, the latter experiments were not done in immunologically intact mice. Thus, they may not provide an accurate assessment of the “seed versus soil” controversy. Another issue in evaluation of metastatic disease in GEM relates to the biology of the tumors in these animals. Most of the tumors in GEM organs are multifocal. Therefore, it is difficult to establish the origin of a given metastasis. Even in the most highly metastatic models, only a small proportion of the tumors have metastatic potential. It is uncertain, therefore, whether any given tumor is metastatic. At the present time, most investigators are concerned only with whether the model has metastases. On the other hand, any study of the molecular characteristics of metastatic tumors must include the original and the metastatic tumor for comparison. Perhaps this issue is best solved using solitary tumor transplants as the source of the metastatic disease. The flip side to the question of which tumor has caused the metastasis is the contrary situation in which metastases have been identified but the source cannot be identified. The prostate models are one such example in which local tumor emboli metastasize to the lymph nodes and lungs but in which all the lesions identified in the prostate are in situ (mPIN), without evidence of invasive growth. This discrepancy is almost certainly a sampling error. However, it could also result from overly rigorous criteria for “invasion.” Another subtle source of metastatic disease is the “noninvasive intravascular” metastasis discussed above (Figs. 24-8 and 24-9) (Sugino et al. 2002). This process involves the pinching off of neoplastic papillae that project into the vascular sinuses and are surrounded by endothelium. Since mouse tumors frequently have large vascular sinuses deep within the neoplasm, the source of the emboli may be central rather than peripheral. These tumor emboli then float through the vascular system until they wedge into a smaller caliber blood vessel. These lesions can be identified in the pulmonary vessels by the double layer of endothelium surrounding the emboli. One layer is from the pulmonary vessel and the other from the endothelium surrounding the periphery of the tumor (Figs. 24-9C and 24-9D).
PRINCIPLE 4:
MOUSE TUMORS MIMIC MANY
ASPECTS OF HUMAN CANCERS (FIGURE 24-1). Axiom I:
GEM tumors accurately model most
aspects of human carcinogenesis. The GEM has given us a new basis for understanding the correlation between structure and function. This correlation also provides a basis for comparing human and murine tumors
leading to a new outlook for comparative tumor pathology. The new synthesis does not change the fundamental premises of tumor biology but does challenge the pathologist to examine the morphological organization of the tumor cell in a different way. The new pathology is based on molecular events that provide unique insights into their structural consequences. The specific molecular event clearly results in specific microscopic tumor phenotypes. Human and mouse tumors with overexpression of Erbb2 and silencing of e-Cadherin result in distinctive patterns in both species (Fig. 24-1) (Table 24-3). The tumor patterns are more closely related to their molecular origin than to the species of origin. Other examples of direct comparisons of human and mouse tumors can be found in other reviews of the subject (Boivin et al. 2003; Borowsky et al. 2003, 2004; Cardiff and Wellings 1999; Cardiff 1996, 2001, 2003; Cardiff et al. 1988b, 1992, 1995, 2000, 2001, 2002; Galvez et al. 2004; Holland 2004; Kogan et al. 2002; Shappell et al. 2004). This exciting new era has created a much greater demand for the skills of comparative pathologists who can bridge the gap between human and animal diseases and relate biology with structure and function (Cardiff et al. 2004). The existing scientific concerns about the validity of the GEM models need to be addressed. The era of GEM pathology also requires a new approach to traditional pathology. In the past, it has been sufficient for the pathologist to identify or verify the process. Previous resource monographs have been useful to pathologists but are rather arcane because they have little to do with the experimental details. The diagnostic “naming” of tumors is no longer sufficient. With the emergence of GEM tumors that are intended to accurately “model” human disease, pathologists are now called upon to “validate” models. This demand requires much more careful study of the lesions, and an understanding of exactly what is meant by “validation.”
II.
COMPARATIVE PATHOLOGY
This chapter began with the concept of using pathology for validation of GEM models of human disease. The validation can occur at many different levels. In GEM tumor biology, the engineering itself represents a validation step for each engineered gene. Does the gene in question create a specific malignant neoplasm when it is either overexpressed or ablated in the target tissue? The answer in the case of many genes, such as Myc and Neu, is unequivocally, yes (Table 24-1). But how far can we carry the similarity between human and mouse? If modifications of the anatomic foundational ontology of Rosse are used as a guideline, it is clear that GEM models can be validated in most instances (Cardiff 2001; Cardiff et al. 2004; Green et al. 2002; Rosse and Mejino 2003). That is, the overexpression of a gene such as Myc causes similar appearing neoplasia in both human and mouse, and the neoplasia arises in the same organs or the same cells. For example, the
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Myc- and neu-induced tumors of the mouse mammary gland resemble in great morphological detail some human breast cancers. The specific examples provided in this chapter show the difference between the standard MMTV-induced mammary tumors (Figs. 24-1A, 24-1B) and GEM-related mammary tumors (Figs. 24-1C and 24-1D). The two examples of GEM tumors, associated with silencing of the e-cadherin gene (Fig. 24-1C) and the overexpression of Erbb2 (Fig. 24-1D), more closely resemble the human lobular breast cancer with silenced e-cadherin (Fig. 24-1E) and with amplification of Erbb2 (Fig. 24-1F). Numerous other examples of morphological similarities between mouse and human tumors have been provided in almost all organ systems (Borowsky et al. 2003; Cardiff 1996, 2001, 2003; Cardiff and Wellings 1999; Cardiff et al. 1977, 1992, 1995, 2000a, 2000c, 2001, 2004; Holland 2004). In this author’s experience, even sophisticated professional audiences cannot reproducibly distinguish between microscopic images of selected GEM and human tumors. The equally sophisticated readers will recognize that these comparative images are simulations that illustrate a point, but when the limited field of the image is placed in the context of the whole animal, one can easily distinguish between human and mouse. The mouse, of course, is the model, the human is not. Therefore, with these limitations, the GEMs are valid models of the human disease. The gene, the tissue, and the microscopic anatomy are similar. However, the modern investigator needs an even more detailed validation. What about host response, and the tumor stroma or the vasculature? What about the tumor biology? Our opinion remains that the biology of the tumor is the single most important factor in assessment of the identified microscopic lesions. Throughout this chapter, the misrepresentations of the biology based on misinterpretations of the microscopic structure have been emphasized. The ethical and legal boundaries compel the medical oncologist/scientist to rely on the inferences of “guilt by association,” rather than experimental evidence, in trying to relate function and structure in human cancer. In contrast, the comparative oncologist has the opportunity to verify the empirical inferences with experimental science. Thus, each inference can be tested using rigorous experimental biology. The test-by-transplantation is but one such approach available to the GEM pathologist and should be used much more often.
A.
Validation
Validation is the new challenge for the comparative pathologist (Cardiff et al. 2004). The standard classification of tumors and disease no longer provides sufficient guidance for the scientific community. As GEM modeling has become increasingly sophisticated, the focus has changed to determine the extent that the genetic alteration has modeled human disease. This focus requires increasing attention to the
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morphological details of the neoplastic process. Therefore, it is incumbent on the comparative pathologist to have an understanding of what questions their fellow scientists seek to answer. The first step of “validation” can be defined as the matching of the attributes (characteristics) of the GEM model to the attributes of the human disease (Cardiff et al. 2004). As implied, validation requires comparison at multiple levels and multiple characteristics. In the broader sense, validation requires what pathologists are best at — the integration of structure and function. The second implication of “validation” is that the types of data recorded extend beyond diagnostic notation to more detailed characteristics of the neoplastic progression. Such detailed accounts were generally recorded in the classical descriptions of diseases before the era of photographic illustrations. A review of records from our institution and elsewhere suggests that descriptive skills of most pathologists have diminished. Many of the descriptions lack even diagnostic or prognostic information to the point that clinicians rely more on “markers” other than pathology to supply the needed information.
B.
Use of “Controlled” Vocabulary
The information age clearly launched an era that offers almost unlimited potential for data storage, retrieval, and analysis. This potential is only now being used for comparative pathology. In order to make maximum use of these resources, experienced pathologists will need to accurately record their observations in a form that the computer can “understand” and use. Two important formats are synoptic reports and controlled vocabularies (Cardiff et al. 2004). While controlled vocabularies already exist in the form of various formal diagnostic terminologies, such as SNOMED-RT, these terminologies are neither sufficient for GEM nor do they have the detail required for validation. Specifically, notation of molecules and genes that are affecting the structure need to be recorded in a formal manner. Modifications of the current vocabularies will need to be developed. Synoptic reports are “cued” report forms that guide the user in the selection of diagnostic terms and descriptive characteristics. They have been used for over a decade in clinical trials. They remind the pathologist which critical information is needed and assure the investigator that the information is properly recorded. However, the current report forms are designed for clinical trials and need to be altered for the needs of comparative pathologists who are validating models. An effort is under way by several groups to develop customized electronic synoptic reports for comparative pathology. The added value to synoptic reports is that they can enforce the use of controlled vocabulary by presenting the user with a limited list of selected terms. When properly constructed, synoptic reports will encourage a standardized collection of
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characteristics needed for validation. One may be guided by the recent reviews outlining the six essential genetic aberrations and many other factors that influence the neoplastic process (Hager et al. 1999; Hanahan and Weinberg 2000). A checklist can be devised as a reminder to examine all of the pertinent features, since each feature is important. Each feature requires validation. However, a checklist alone is not adequate in the absence of comparisons with other models and other species. There is no substitute for a broad experience-base. The issue is how to share and compare. The limitation to controlled vocabularies and synoptic reports is that they encourage the pathologist and investigator to record prescribed descriptions rather than to recognize novel phenomena. Therefore, each field in the synoptic report needs to have an “other (Please specify)” tag in the pull down, cued menu. However, if these novel phenomena are repeated, a mechanism for transferring them to the permanent vocabulary must also be in place. Accordingly, each GEM should have the details of the genetic engineering attached to the results, and the diagnostic terminology should include the molecular phenotype. By molecular phenotype, we mean one of the known “signature” phenotypes. For example, when the tumor is induced by the Myc transgene and has the microscopic characteristics of Tg(Myc) tumors, it should be noted as a Myc-type tumor. This type of diagnostic nomenclature serves several purposes. First, it encourages the pathologist to be familiar with molecular phenotypes and to set criteria for accepted variations. Second, if the tumor is induced by a Tg(Myc) transgene but does not have the microscopic signature of Tg(Myc) tumors, the specific tumor warrants further investigation. Such aberrant tumors should not be used for expression analysis with the assumption that it is a standard Tg(Myc) tumor. Finally, one of the primary uses of the GEM will be to study therapeutic intervention. It will become increasingly important to identify which tumors escape targeted therapies. For example, rapamycin is an inhibitor of mTOR. GEM tumors that signal through mTOR such as the Tg(Erbb) tumors are readily inhibited or ablated by rapamycin therapy. However, some tumors escape therapy. What is the microscopic phenotype of the tumors that escape therapy? What does this tell us about the nature of the tumors? Again, with an appropriately structured synoptic report, the pathologist will be prompted to observe features that are helpful to the investigator. It should now be clear that the report needs to include the molecular phenotype as well as novel observations.
C.
Digital Imaging
Pathology is an image-intense discipline. Our libraries are filled with atlases and 35-mm slide collections. The pathology journals are filled with photographs illustrating phenomena. In earlier eras, we provided drawings of the microscopic phenomena.
We have always regarded these as simulations of reality and have rapidly adopted each technical advancement for our use. When the Internet became a reality, TelePathology was born, first using analog images and now digital imaging. The early experience made the pathology community even more eager for either the display of selected still images or the projection of analog images using remote control (Cramer 2004; Weinstein et al. 1989). However, in recent years, others have worked toward the digital capture, compression, storage, and display of images of the whole slide. The WSI (whole slide imaging) technology provides images that can be used to view the critical information in the context of the entire slide. The development of digitized WSI promises to revolutionize the capture, storage, retrieval, and display of microscopic images (Cardiff et al. 2004). Current technology captures digital images of the entire slide at resolutions up to 50,000 dpi. The images are compressed using a variety of formats. Using one of several browsers, the images can be presented over the Internet allowing the viewer to scan any part of the slide image at any magnification. These slides have become available as massive archives for comparative pathology (http://imagearchive. compmed.ucdavis.edu/cardiff/). In the future, large slide collections will be stored and even accessed remotely. The WSI system being used at the University of California, Davis, is manufactured by Aperio Technologies, Inc. The scanning device (ScanScope®) typically scans and captures the slide in 2 to 5 minutes. After acquisition the image is compressed (typically at a ratio of 1:20) and stored in a proprietary, multilayered JPEG format. The slides are presented via the web using a WSI image server and Flash® plug-in developed by Zoomify.com®. In addition to simply viewing the image, at any field or magnification, the viewer also allows the user to utilize nondestructive annotation of the images. Nondestructive annotation is accomplished by the “layering” of points of interest (POI) and labels at specific locations and magnifications within each image. In addition to labels and POI, each annotation can have multiple textual notes associated with it to further explain its importance. Because the annotations are nondestructive, they can be turned on or off at the discretion of the user. Importantly, the underlying image is not altered by the annotation process. In fact, the annotations are stored in a separate XML format that is independent of the image. The reader is encouraged to visit the Publication Supplement page at http://imagearchive.compmed.ucdavis.edu/publications/ cardiff/. Each of the panels presented in this chapter is black and white. The color version of each panel can be viewed at the Publication Supplement URL given above. By pointing and clicking on the specific image frame within the panel, the reader will be able to view the WSI. The field used to provide the image for the frame can then be located as a “point of interest.” Table 24-3, included here to guide the interested reader, will also appear at the Publication Supplement with links to the specific WSI and to the appropriate literature citation.
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D.
Morphometrics
The demand for validation of the GEM model’s various attributes of human disease raises another interesting issue. Perhaps validation is best performed by objective measurements. One would argue that mathematics is the language of science. While the skill of the expert pathologist with exquisite descriptive language cannot be replaced by the computer, it is quite possible that many of the current quandaries and controversies would be resolved if phrases such as “increasing nuclear pleomorphism” could be defined mathematically. Is the criterion for “degree” of increase the same in California as it is in New York? What are the standards? What is a “Grade 1” in Europe versus a “Grade 1” in Canada or elsewhere? How reproducible is the pathology among different experts? Opinion is replaced by measurement. One advantage of capturing and archiving digital images is that they can be retrieved and analyzed morphometrically. As morphometric analysis of digital images improves, we will have vast archives of digital whole slide images that can be subjected to analysis. This will permit quantification of subtle characteristics such as neoangiogenesis. More important, the investigator will be able to normalize the characteristic on the basis of other attributes such as nuclear size. These data will permit multivariant analysis that reveals novel relationships.
E. Conditions That Affect Tumorigenesis Comparative pathologists are the gatekeepers guarding the pathobiology of GEM. As such, they need to be aware of various host conditions that may influence the occurrence and interpretation of tumors. For example, several research programs use GEM to search for genetic modifiers. They require input from pathologists to prevent misinterpretations (Balmain and Nagase 1998; Dragani 2003; Nagase et al. 1999). The pathologist is expected to detect subtle phenomena, distant from the target tumor, that might reveal important biological insights. The microbiological flora could be important in interpreting the biology of the neoplastic process but is rarely considered in the natural history of neoplastic disease. The following section discusses some other examples of conditions that may have unexpected consequences for the biology of tumors. Spontaneous pulmonary adenomas are found in as many as half of elderly FVB females (Mahler et al. 1996). Since most metastases are also found in the mouse lung, the adenomas are frequently misinterpreted as metastases by the unwary (Fig. 24-9F). The spontaneous adenomas are papillary and often involve the bronchiolar mucosa, which differentiates them from the intravascular tumor emboli. Spontaneous pituitary adenomas are found in as many as 50% of 18-month-old females in some FVB/N mouse colonies and represent another example of perplexing tumors that have far-reaching and not fully appreciated implications
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(Mahler et al. 1996; Wakefield et al. 2003). These tumors are almost all prolactinomas that stimulate a virginal mammary lobuloalveolar hyperplasia and an increase in mammary adenocarcinomas with or without metastasis (Nieto et al. 2003; Wakefield et al. 2003). A careful evaluation revealed that all of the virgin FVB/N females in one colony had pituitary abnormalities by 12 months of age. These ranged from pituitary cysts to huge adenomas (Wakefield et al. 2003). Two spontaneous pituitary lesions that are difficult to interpret are chromophobe hyperplasias and small, developing adenomas with no gross pituitary enlargement (Capen et al. 2001). However, careful examination of the gland reveals increased monomorphic populations of chromophobe cells. The adenomas appear as small expansile tumors. The hyperplasias are even more difficult to recognize, given the zonal distribution of cells in the mouse pituitary. However, both of these subtle lesions are associated with mammary hyperplasia. A little correlative experience with these lesions teaches the boundaries. Arguably, the cells in the pituitary hyperplasias have slightly larger nuclei than the normal chromophobe cells. The exact incidence of mammary tumors in FVB/N mice with these spontaneous pituitary neoplasms is not known and may well vary from colony to colony. However, the most common pituitary-associated tumors are adenosquamous mammary carcinomas (Huseby et al. 1985; McGrath et al. 1981; Wakefield et al. 2003). However, a broad range of mammary tumors may occur. Papillary and micropapillary carcinomas are the second most common, followed by glandular adenocarcinomas. Previous studies of the BALB/c strain treated with pituitary explants suggest that pituitary overstimulation can lead to adenosquamous carcinomas (Huseby et al. 1985; McGrath et al. 1981). However, we have been surprised by the range of phenotypes found in the pituitary-related mammary tumors found in FVB/N mice. Most importantly, these tumors do not resemble GEM mammary tumors of the more common oncogenic transgenes. The nuclei are generally oval with a delicate chromatin. Most of the tumors are well-differentiated. Most transgenic studies of mammary development and tumorigenesis are based on FVB mice, yet very few of these studies have the appropriate controls. Most use “historical” controls that do not account for the current conditions in the investigator’s colonies. As a result, some conclusions about gene contribution to mammary development might be suspect. As a result of the spontaneous pituitary neoplasms described above, we now recommend that all long-term mammary studies be accompanied by age and parity controls that include littermate controls (Nieto et al. 2003; Wakefield et al. 2003). Most common spontaneous mouse ovarian tumors are teratomas or granulosa cell tumors (Galvez et al. 2004). These tumors are particularly common in the strains of 129 mice (Stevens 1980). Because many GEM used for targeted mutations were initially created using ES cells and because of the genetic contamination of the parental 129 strain, it is difficult to be certain which variant 129 strain is being used in any given
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ES cell-derived GEM. Moreover, journal editors and reviewers have created further confusion by failing to insist that exact strain nomenclature be used in publication. Since prostate tumors and hyperplasias in wild-type laboratory mice are extremely rare, prostate abnormalities occurring in GEM are probably due to genetic manipulation (Shappell et al. 2004). On the other hand, the advent of GEM mice has led to interesting but frequently overlooked pathology. For example, we have found some examples of prostatic hyperplasia associated with interstitial cell (Leydig cell) hyperplasias, particularly in GEM with Fgf transgenes. Dilation and engorgement of seminal vesicles can be extensive and even unilateral in some wild-type males and are a common agerelated pathology of C57BL/6 mice. Enlarged, engorged seminal vesicles have also been associated with hyperplasia of the ampullary glands and not the prostatic glands (Donjacour et al. 1998).
III.
SUMMARY AND IMPLICATIONS
A systematic analysis of the microscopic anatomy from a molecule-centric rather than a tissue- or cell-specific view has been provided herein. By necessity, this account has required the comparison of cellular and tissue phenotypes to the genes that initiated the neoplasm. The dysregulation of a dominant oncogene (e.g. Myc) often results in an identifiable microscopic tumor phenotype independent of the tissue or cell of origin (Fig. 24-3). Usually, the tumor phenotype of dominant transgenes is not affected by the promoter or the site of transgene integration (Figs. 24-3, 24-4, Table 24-3). The background mouse strain appears to alter the biological characteristics of the tumors but does not have much effect on the microscopic structure of the tumor. By contrast, less dominant oncogenes (e.g. Ras) will result in identifiable structural changes that depend on the tissue targeted by the promoter system (Fig. 24-5). The knockout of the classical tumor suppressor genes, because of their role in control of cell cycle (Lowe et al. 2004), results, most often, in genetic instability and thus, adoption of the structural characteristics of the initiating oncogenes (Fig. 24-7). Combinations of oncogenic transgenes and knockouts have also suggested that some oncogenes such as Myc are dominant and overshadow the contributions of other oncogenes. Genes such as SV40-Tag are dominant in the sense that they result in very similar morphological changes in various organs (Fig. 24-4). However, a full panel of bigenic and trigenic oncogenic crosses has not been studied in all organ systems. Thus, the hierarchy of dominant oncogenes remains unclear as do the rules that control the morphology of the resultant tumors. Although the connection between oncogene function and the end-point tumor is very clear, the nature of the molecular, microscopic, or ultrastructural events within the tumor cells has
not been extensively studied. Only scattered electron microscopic observations of tumors from transgenic mice have been recorded, and they do not represent, in any sense, a systematic comparison of tumor phenotypes. For example, one can assume that the amphophilic cytoplasm of the Tg(Myc) tumors is due to high levels of cytoplasmic nucleic acids. Furthermore, knowing that Myc can regulate ribosomal function, one can assume that the cytoplasmic nucleic acid is in the form of ribosomes. However, there are no published electron micrographic studies documenting increased numbers of ribosomes in Myc tumors. In a like manner, several ultrastructural studies have documented the relatively organelle free cytoplasm of Neu-induced mammary tumors (Di Carlo et al. 1999). However, these studies do not indicate an awareness of the zonal organization of the Erbb-type tumors and leaves unclear which cell sample is being imaged (Deckard-Janatpour et al. 1997; DiGiovanna et al. 1998). Our growing knowledge of GEM molecular phenotypes, however, should be relevant to human cancer. The microscopic similarities of human and mouse tumors and their correlation with the molecular aberrations are the most remarkable observations of GEM comparative pathology (Fig. 24-1). These correlations should form the basis for understanding the effect of function on structure. The correlations provided here support the concept that structure and function are very similar in all species. Therefore, neoplasms initiated by the same genetic abnormalities will lead to similar tumor phenotypes. These similarities will become the bases for a revised classification based on the combination of structure and function. Some progress has been recorded in molecular classifications of cancers of the breast, lung, and hematopoietic system (Anbazhagan et al. 1999; Golub et al. 1999; Jain 2004; Parmigiani et al. 2004; Ross et al. 2003, 2004). These types of studies should eventually lead to a molecular classification of human neoplastic disease. We have emphasized the commonality of tumor phenotype in GEM representative of the organs most commonly involved in human cancers. The majority of the oncogenes and tumor suppressor gene-induced tumors in this collection are from either the ectodermal or endodermal epithelium. Relatively few studies of the mesodermal tumors in GEM are available using the same genes. Although GEM studies are not yet sufficiently comprehensive to lead to a universal molecular classification of neoplasms, with further GEM studies other commonalities will emerge to provide the basis for a more inclusive molecular classification (Berman 2004). We hope that this chapter will establish the principles underlying the morphological changes associated with the activation of specific oncogenes and knockout of specific tumor suppressor genes. The merger of function and structure afforded by molecular tumor pathology implies that we will begin to appreciate that the biology of the specific genetic alterations will also share common features. For example, the tumor latencies of breast cancer and lymphomas are shorter in Myc-induced tumors than
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for Ras-induced tumors (Cardiff et al. 1991). The combination of Ras and Myc accelerates mammary tumorigenesis but the combination of Ras and Erbb2, members of the same pathway, does not (Cardiff et al. 1991). Unfortunately, the latencies and metastatic rates for different tumors and for the same tumors in different GEM strains have not been recorded in a sufficiently uniform format for detailed analysis. The success of molecular phenotyping has permitted the molecular characterization of tumors whose microscopic structure is already identified (Anbazhagan et al. 1999; Golub et al. 1999; Jain 2004; Parmigiani et al. 2004; Ross et al. 2003, 2004). The real challenge is going to be in the solid tumors of humans with levels of differentiation that are not well understood. Will we be able to microscopically examine a human tumor and be able to consistently identify the oncogenes involved? Will they be identical to those that we see in the GEM? Or, more likely, will the human tumors have unique molecular phenotypes and, hence, their own signatures? How will we reach this level of knowledge in human and other species? Clearly, the responsibility will belong to comparative pathologists. We must be alert to the opportunities and be certain that our work is promulgated. However, our cause will be enhanced by making certain that members of our own discipline are well trained and achieve full membership in the research team. In the future, as pathologists from both the medical and veterinary professions work together with basic scientists, the new molecular-based pathology will be combined with traditional views of pathology and will yield new insights into tumorigenesis and the correlation between structure and function.
ACKNOWLEDGMENTS Dr. Cardiff wrote and takes sole responsibility for the text. The work described in this chapter is the sole responsibility of the first author. However, he is indebted to his numerous collaborators who have provided the samples, the scientific stimulus and the many discussions that are the basis for this chapter. Robert J. Munn is responsible for all of the digital images. Dr. Galvez created and developed the web site. The authors are indebted to Drs. C. M. Miller, M. B. Gardner, S. W. Barthold, A. D. Borowsky, and B. Tarnowski who cheerfully took the time to carefully edit the manuscript and provide many thoughtful suggestions that improved the organization, rigor, and clarity of the text. Dr. Cardiff is deeply indebted to R. J. Munn who worked closely with him to design the panels and to capture and edit the images. Dr. J. J. Galvez and R. J. Munn are also responsible for the images, organization, and web pages that supplement this chapter. We appreciate the willingness of investigators to share unpublished data and images with us. In particular, Dr. Borowsky (CCM, University of California–Davis) contributed the framework for Table 24-1
and the slide for the image in Fig. 24-8B. Drs. Jos Jonkers and Patrick Derksen (Netherlands Cancer Institute, Amsterdam) contributed the slides used for images in Figs. 24-1C and 24-2F and shared the experimental detail of the genotype prior to publication. Drs. P. Dubus (University of Bordeaux), J. Iovanna (Centre de Recherche INSERM, Marseilles), and M. Barbacid (Centro Nacional de Investigaciones Oncologicas, Madrid) contributed slides used for images in Figs. 24-8C and 24-8D and shared their personal observations concerning the slides prior to publication. Dr. Borowsky also contributed data on PyV-mT cell lines while the paper was in press. Drs. Terry Van Dyke and Reggie Hill (University of North Carolina, Chapel Hill) contributed slides and experimental insights with the SV40-T121 while still in press. Finally, this work has been supported, in part, by grants and contracts R01 CA089140, 22XS037A, U01 CA10102, and U01 CA084294 from the National Cancer Institute and National Centers for Research Resources (UR42-RR1495) from the NIH. REFERENCES Abate-Shen, C., Banach-Petrosky, W. A., Sun, X., Economides, K. D., Desai, N., Gregg, J. P., et al. (2003). Nkx3.1; Pten mutant mice develop invasive prostate adenocarcinoma and lymph node metastases. Cancer Res. 63(14), 3886–3890. Al-Hajj, M. and Clarke, M. F. (2004). Self-renewal and solid tumor stem cells. Oncogene 23(43), 7274–7282. Ali, S. H., and DeCaprio, J. A. (2001). Cellular transformation by SV40 large T antigen: interaction with host proteins. Semin. Cancer Biol 11(1), 15–23. Anbazhagan, R., Tihan, T., Bornman, D. M., Johnston, J. C., Saltz, J. H., Weigering, A., et al. (1999). Classification of small cell lung cancer and pulmonary carcinoid by gene expression profiles. Cancer Res. 59(20), 5119-5122. Andrechek, E. R., Hardy, W. R., Siegel, P. M., Rudnicki, M. A., Cardiff, R. D., and Muller, W. J. (2000a). Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis. Proc. Natl. Acad. Sci. USA 97(7), 3444–3449. Andrechek, E. R., Laing, M. A., Girgis-Gabardo, A. A., Siegel, P. M., Cardiff, R. D,. and Muller, W. J. (2003). Gene expression profiling of neu-induced mammary tumors from transgenic mice reveals genetic and morphological similarities to ErbB2-expressing human breast cancers. Cancer Res. 63(16), 4920–4926. Andrechek, E. R., and Muller, W. J. (2000b). Tyrosine kinase signalling in breast cancer: tyrosine kinase-mediated signal transduction in transgenic mouse models of human breast cancer. Breast Cancer Res. 2(3), 211–216. Andres, A. C., Schonenberger, C. A., Groner, B., Hennighausen, L., LeMeur, M., and Gerlinger, P. (1987). Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice. Proc Natl Acad Sci USA 84(5), 1299–303. Apolant, H. (1906). Die epithelialen Geschwulste der Maus. Arbeiten aus dem Koniglichen Institut fur Experimentelle Therapie zu Frankfurt., 7–62. Arbeit, J. M. (2003). Mouse models of cervical cancer. Comp. Med. 53(3), 256–258. Ashley, R. L., Cardiff, R. D., Mitchell, D. J., Faulkin, L. J., and Lund, J. K. (1980). Development and characterization of mouse hyperplastic mammary outgrowth lines from BALB/cfC3H hyperplastic alveolar nodules. Cancer Res. 40(11), 4232–4242. Balmain, A., and Nagase, H. (1998). Cancer resistance genes in mice: models for the study of tumour modifiers. Trends Genet. 14(4), 139–144. Bannasch, P., Enzmann, H., Klimek, F., Weber, E. and Zerban, H. (1989). Significance of sequential cellular changes inside and outside foci of altered hepatocytes during hepatocarcinogenesis. Toxicol. Pathol. 17(4 Pt. 1), 617–628; discussion 629.
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Chapter 25 Spontaneous Diseases in Commonly Used Mouse Strains Cory Brayton
I.
Introduction A. Variables That Affect Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Resources: Internet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Coat Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Sexual Dimorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Age-Related Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Inbred Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Strain 129 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. AKR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. BALB/c, BALB/cBy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. C3H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. C57BL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. DBA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. FVB/N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. SJL/J . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Non-Inbred Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. B6;129 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. B6C3F1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Swiss Mice—Derivative Inbred Strains and Outbred Stocks . . . . . . . . IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Key to Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 25-1 Attributes of some common mouse strains and stocks . . . . . . . . . . . Table 25-2 Sources and origins of some Swiss-derived mice and non-Swiss stocks available in North America . . . . . . . . . . . . . . . . . Table 25-3 Spontaneous nonneoplastic conditions in some commonly used mouse strains or stocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 25-4 Spontaneous neoplasms in some commonly used mouse strains or stocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Table 25-5 Glossary by system: Nonneoplastic changes . . . . . . . . . . . . . . . . . . Table 25-6 Glossary by system: Neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
This chapter discusses the range of pathologic changes that can occur in commonly used mouse strains or stocks in the absence of intended experimental interventions. While there are ample and systematically collected data on B6C3F1 and CD-1® mice from toxicology testing, data on inbred strains that are used more commonly in other research areas are less available and more dispersed. When evaluating experimental results from mice of specific strains or stocks, or when attempting to characterize phenotypes of mutant mice derived from them, it is important to be aware of the background pathology or phenotypes in order to determine if lesion incidences are increased or decreased compared to what occurs in nonmanipulated, or wild-type animals of the same strain or stock. Similarly, when designing studies or developing genetically based mouse models of specific conditions, background genotypes and phenotypes may provide substantial advantage or disadvantage to achieving specific aims. (Sher et al. 1982; Brayton et al. 2001; Linder 2001; Barthold 2002, 2004) Although the concepts were expressed by Mendel decades earlier, the terms gene, genotype, and phenotype were introduced by the Danish botanist Wilhelm Johannsen in 1909. (Wanscher 1975; Mayr 1982; Henig 2001) Gene is from the Greek word genos (meaning race or offspring) and refers to a unit of heredity. Genotype is from the Greek genos plus tupos (meaning type) and refers to the genetic constitution of an individual organism. Phenotype is from Greek phainen (meaning to show) plus tupos and refers to the set of observable characteristics of an individual resulting from the interaction of its genotype with the environment (Jewell 2001). In other words, phenotype reflects the nature and the nurture of the organism. Genetics or genetic constitution, including any spontaneous genetic alteration or intended manipulation, comprises the nature of the individual (or genetically identical individuals), and environmental and experiential factors, including infectious agents, comprise their nurture.
A. 1.
Variables That Affect Phenotype
Nature: Genetics
Mice of fully inbred strains should be genetically identical and homozygous at all loci (genes). Some inbred strains were
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initiated in the 1920s and have undergone hundreds of generations of inbreeding (sibling mating). Inbred strains continue to demonstrate important susceptibilities for which they were developed initially, such as lung tumors in A strain, lymphoma (leukemia) in AKR and SJL/J, and mammary and liver tumors in C3H. (Murphy 1966) Despite the perpetuation of historically important susceptibilities, it is not surprising that due to genetic drift or to genetic contamination, contemporary strains and substrains differ genetically from mice evaluated 20 or 50 years ago. (Beck et al. 2000; Wotjak 2003) While historical data can provide important insight for study design, they do not obviate adequate numbers of relevant concurrent control animals. (Haseman et al. 1994, 1997; Greim et al. 2003) Genetic drift refers to spontaneous neutral mutations (i.e., not subject to selective forces) that disappear or become fixed in a population (Silver 1995), but spontaneous mutations also can impact phenotype and confound research, or lead to new insights or to development of important models. A well-known example of a spontaneous mutation in inbred C3H/HeJ mice is in the Tlr4 (Toll-like receptor 4, formerly Lps, lipopolysaccharide) gene. The mutation confers endotoxin resistance and altered susceptibility to infections but is absent in various other C3H/He substrains. (Apte et al. 1977; Poltorak, et al. 1998; Branger Knapp, et al. 2004; Branger, Leemans, et al. 2004; Schurr et al. 2005) Nude (Foxn1nu) and scid (Prkdcscid) are examples of spontaneous mutations that have led to the development of important research tools, especially in research on immunology and transplantable cancers. The 129 mice, with at least 16 recognized substrains, provide a well-documented example of genetic contamination and substrain divergence. (Simpson et al. 1997) Inbred mice raised in a different environment, and perhaps of only slightly different lineage than those bred and raised at another study site or academic institution, can exhibit substantial differences in the incidence or expression of disease or other phenotypes, and such differences may impact some studies. (Wahlsten 1982; Everett 1984; Engelhardt et al. 1993) Such findings strongly suggest that “wild-type” mice, obtained from a commercial breeder or other source, should not be used as “controls” in studies of genetically engineered mice developed from laboratory colonies. Genetic sex impacts various anatomic and physiologic phenotypes, responses to infection or to other environmental factors, and to experimental manipulations. Sexual dimorphisms may manifest as differences in size, morphology, metabolic
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activities, responses to hormones, drug sensitivity, rates of maturation, and other features (Pinkstaff 1998), and may vary with mouse strain (background genetics) and other factors. Some important sexual dimorphisms are described in greater detail in a later section (E.Sexual Dimorphisms) and in specific strain sections. 2.
Nurture: Microbial Impacts on Pathology and Phenotype
Infectious or infesting agents are environmental factors that can cause pathology and impact phenotypes, whether they occur naturally or are introduced intentionally. The induced pathology or phenotype frequently is strain dependent and varies with other factors, including sex and age of the host, virus strain and dose, route of infection, and copathogens. Among the many examples are mouse hepatitis virus (MHV), Mycoplasma pulmonis, Helicobacter spp., pinworms, and fur mites. Depending on mouse age and strain, virus dose and strain, and route of infection, the various strains of MHV can induce enteric and hepatic phenotypes (enteritis and hepatitis), as well as encephalitis, hydrocephalus, or demyelinating diseases that could alter the results of many types of studies. (Virelizier et al. 1975; Knobler et al. 1982; Tardieu et al. 1982; Lavi et al. 1986; Barthold 1987; Barthold et al. 1986, 1987) Mycoplasma pulmonis (Lindsey and Cassell 1973; Cassell et al. 1974; Romero-Rojas et al. 2001) and Sendai virus (Ward 1974; Brownstein et al. 1986; Brownstein and Winkler 1987) acting singly or in concert with other agents induce respiratory phenotypes that can interfere with studies of the respiratory or immune systems. Helicobacter hepaticus also induces enteric and hepatic phenotypes, including typhlocolitis and hepatitis in various mouse strains, and accelerates development of liver tumors in A/J mice (Ward et al. 1994; Diwan et al. 1997; Livingston et al. 2004). Pinworms inhibit development of the diabetic phenotype in NOD mice (Gale 2002), stimulate mucosal immunity, and may induce or accelerate lymphoma development in athymic nude mice (Beattie et al. 1980). Fur mites induce skin and immune phenotypes, including dermatitis with atopy, with ulceration and secondary infections being likely in certain strains including C57BL and BALB/c, and with altered immunoglobulin levels, white blood cell counts, and differentials. (Csiza and McMartin 1976; Dawson et al. 1986; Jungmann, Freitas, et al. 1996; Jungmann, Guenet, et al. 1996; Iijima et al. 2000) The mouse retroviruses are microbial agents of special interest because their genetic interactions blur the distinction between nature and nurture influences, reviewed in this series (Morse 2007). Mouse retroviruses influence a variety of phenotypes as inserted or endogenous viruses (proviruses) and as more conventionally transmitted exogenous viruses. More than 100 B-type mouse mammary tumor virus (MMTV) and C-type murine leukemia virus proviruses have been detected in the mouse genome, more than one per chromosome, with strain-dependent type, number, and distribution, and estimated 40 to 60 proviruses
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per strain, and they have been useful as genetic markers. (Coffin et al. 1989; Frankel et al. 1990; Morse 2007) Retroviruses play important roles in mouse mammary tumors (notably in C3H mice) (Hageman et al. 1968; Bentvelzen et al. 1970; Hook et al. 2000; Jude et al. 2003), in lymphoma (notably in AKR, C58, and SJL/J mice) (Gross 1956; Rowe and Pineus 1972; Mucenski et al. 1988; Coffin et al. 1989; Hiai 1996; Rajan et al. 1998; Erianne et al. 2000), and other tumors (Kang et al. 1993). Mtv genes or loci represent insertions of portions of mammary tumor viruses (Mtv1-56). Endogenous ecotropic murine virus loci have been designated Emv (Emv1-88). Endogenous xenotropic mink cell focus (MCF) inducing leukemia virus loci have been designated Xmmv (Xmmv1-79). Endogenous xenotropic murine virus loci have been designated Xmv (Xmv1-66) (Blatt et al. 1983; Wejman et al. 1984; Frankel et al. 1989). Intracisternal A particles (IAP) are defective murine retroviruses that are encoded by endogenous genetic elements, lack an extracellular phase, and exist at a copy number of about 1000 to 2000 per cell. Their loci may be designated Iap, Iapl, or Iapls (IAP lymphocyte specific), Iapts (IAP tumor specific), or they may retain earlier designations, for example, Avy (Amariglio and Rechavi 1993; Chang-Yeh et al. 1993; Morgan et al. 1999; Fehrmann et al. 2003; Morse 2007). Retroviral insertions in functional genetic loci have created mutant alleles, including rd1 (retinal degeneration 1) allele in the Pde6b gene (Bowes et al. 1993) (this allele also has a nonsense mutation (Pitter et al. 1993)) d (dilute) allele in the Myo5a gene (Jenkins et al. 1981; Seperack et al. 1995), and hr (hairless) allele in the Hr (hairless) gene (Coffin et al. 1989; Cachon-Gonzalez et al. 1994). In addition, insertions near a gene may influence expression levels or sites of expression of a gene, as with the effects of vy (viable yellow) on a+ (wild-type nonagouti) gene expression (Perry et al. 1994; Argeson et al. 1996; Morgan et al. 1999), or with transcriptional activation of oncogenes in the vicinity of MMTV and other retroviral integration sites (Rajan et al. 1998; Hook et al. 2000; Kim et al. 2003). Fostering and rederivation are widely used strategies to eliminate pathogens from mice (Lipman et al. 1987; Marcotte et al. 1996; Suzuki et al. 1996; Maggio-Price et al. 1998; Watson et al. 2005). Fostering pups from exogenous (milk-borne) MMTV (Bittner agent)-carrying dams onto dams that do not carry MMTV eliminates exogenous MMTV from offspring and impacts mammary phenotype by reducing (but not eliminating) mammary tumors in susceptible strains, notably C3H. (Hageman et al. 1968) Fostering can eliminate Helicobacter spp. from offspring, thus eliminating Helicobacter-related hepatic and enteric phenotypes. (Truett et al. 2000; Singletary et al. 2003) Cesarean rederivation followed by barrier maintenance to eliminate pathogens reduced pristane-induced plasmacytomas in BALB/c mice. (Byrd et al. 1991) While undesirable flora (along with related phenotypes) can be eliminated, fostering, or cesarean or embryonic rederivation may introduce other factors, and impact behavioral and other phenotypes. (Maxson and Trattner 1981; Cowley et al. 1989)
626 3.
Nurture: Nonmicrobial Environmental Factors
Noninfectious environmental factors, including experiential or social factors, also impact phenotype. Examples include parity and age of dam, intrauterine position of fetuses, in utero stressors, population density during and after pregnancy, lighting, temperature, caging, bedding, exercise, and diet. Multiparous or breeder mice have substantially different incidences of certain lesions or phenotypes (e.g., ovarian and uterine changes and lymphoma) compared to virgin mice (Tables 25-3, 25-4) (Frith et al. 1983). Examples of prenatal influences on phenotypes include: in utero proximity of female mice to male siblings affecting the time of vaginal opening (Zielinski et al. 1991) and the sex ratio of the female siblings’ offspring (Vandenbergh and Huggett, 1994, 1995); in utero proximity to male siblings and prenatal environmental stressors such as crowding, heat or light stress being associated with increased anogenital distance (indicating masculinization) in females (Ryan and Vandenbergh 2002; Zielinski et al. 1991); and intrauterine location (near cervix or ovaries), age of dam, and litter size influencing frequencies of cleft lip and palate, open eyelids, and fetal resorption in susceptible A/J mice (Kalter 1975). A wide variety of social and in-cage factors have been shown to impact phenotypes. High population density (crowding) affects reproductive parameters, behaviors, steroid hormone levels, and other traits (Peng et al. 1989; Chapman et al. 1998, 2000; Nagy et al. 2002; Ishida et al. 2003). Compared to grouphoused mice, individually housed mice have reduced amyloid incidence (Lipman et al. 1993), reduced animal loss due to fighting, and increased body weight and liver tumor incidence (Haseman et al. 1994). Compared to enriched environments, mice housed in standard cages have impaired brain development and abnormal repetitive behaviors (stereotypies). (Wurbel 2001, 2002; Wolfer et al. 2004) Compared to suspended wire caging, mice on solid bottom polycarbonate caging have had improved survival and reduced tumors on long-term studies in (Rao and Crockett 2003) and reduced urinary obstruction in AKR mice (Everitt et al. 1988). Cedar-shaving contact bedding, popular for its aroma and inhibitory effects on ectoparasites, induces liver enzymes, alters drug responses (Sabine 1975), increases the liver:body weight ratio (CunliffeBeamer et al. 1981), and may enhance mammary and liver tumor growth (Vlahakis 1977; Everett 1984). Cage shelf level may influence onset or incidence of tumors (Greenman et al. 1984). Increased ambient light exposure due to position of a cage on a rack may increase the incidence and severity of retinal atrophy (Greenman et al. 1982) or cataracts (Haseman et al. 1989). This issue may be less of a concern today when many facilities use microisolator caging or ventilated racks with lower in-cage light levels than in suspended wire or uncovered cages. Photoperiod or light cycle can influence tumor growth and immune responses (Waldrop et al. 1989; Yellon and Tran 2002; Lang et al. 2003) and responses to additional stimuli or drugs, and varying photoperiod is a stressor to which mice do
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not adapt (Sakellaris et al. 1975; Kavaliers and Hirst 1983). Cool ambient temperatures or cold stress may reduce tumor growth (Everett 1984; Yamamoto et al. 1995). Cold stress immunomodulates and influences response to infections and toxins. (Kizaki et al. 1996; Sima et al. 1998; Aviles et al. 2001; Cichon et al. 2002; Yamamoto et al. 1995) Exercise may delay growth and improve or accelerate regression of experimental tumors (Zielinski et al. 2004) or reduce experimental metastases (Davis et al. 1998). Increased body weight has been associated with increased tumor incidence, especially of hepatic tumors. (Haseman et al. 1994) Diet or caloric restriction increases longevity and reduces body weight, tumor incidence, and ulcerative dermatitis (Bronson and Lipman 1991; Blackwell et al. 1995). A change of diet initiated by the National Toxicology Program (NTP) in 1994 from NIH 07 diet, formulated to promote reproduction and growth, to NTP 2000 diet, increased survival and reduced tumor incidence in B6C3F1 mice in long-term studies. (Rao et al. 1989, 1990, 2003) Dietary phytoestrogens influence uterine weight and time of vaginal opening (Thigpen et al. 2002, 2003) and are implicated in the development of vulvar carcinomas. (Thigpen et al. 2001) Purified diets increased cardiac calcinosis in C3H mice compared to natural ingredient diet, and high-fat purified diet increased mortality. (Everitt, Olson, et al. 1988; Everitt, Ross, et al. 1988) Powdered diets reduce wear on teeth and may contribute to malocclusion and dental dysplasia. (Losco 1995)
B.
Resources: Internet
More and more information is becoming available via the Internet. Major vendors provide useful information, including hematology, serum chemistry, pathology, and husbandry data for their major stocks and strains. The Jackson Laboratory provides data regarding many of the hundreds of strains that they distribute or have cryopreserved. Some useful web sites include the following: ●
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“The Coat Colors of Mice” by Wilys Silvers (Silvers 1979) is available and searchable online. http://www.informatics. jax.org/wksilvers/ Eumorphia is a European initiative that is developing and standardizing approaches in phenotyping, mutagenesis, and informatics to improve characterization of mouse models for the understanding of human physiology and disease. http://www.eumorphia.org/ Michael Festing’s information on inbred strain characteristics describes the history, development, and various characteristics, with many references, of more than 400 major mouse strains. http://www.informatics.jax.org/external/festing/search_ form.cgi Mouse Genetics by Lee Silver (Silver 1995) is available and searchable online. http://www.informatics.jax.org/silver/
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Mouse genome informatics, homepage http://www.informatics. jax.org/, with links to search genes, markers, phenotypes, alleles, strains, and polymorphisms. The mouse nomenclature homepage is the authoritative source of official names for mouse genes, alleles, and strains. Nomenclature follows the rules and guidelines established by the International Committee on Standardized Genetic Nomenclature for Mice, which are implemented through the Mouse Genomic Nomenclature Committee (MGNC). http://www.informatics.jax.org/mgihome/nomen/ The mouse phenome database web site aims to establish a collection of baseline phenotype data on commonly used and genetically diverse inbred mouse strains through a coordinated international effort (Bogue 2003). As of 2004, 40 priority strains have been identified, and standardized protocols for phenotype evaluation are published on the web site http://aretha.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/ aboutmpd The Mouse Tumor Biology (MTB) Database provides descriptions of tumors (organized by strain and organ of tumor origin), tumor frequency, and latency data, genetic factors associated with tumors and tumor development, tumor pathology reports and images, and references, both published and unpublished. http://tumor.informatics.jax.org/ FMPro?-db=TumorInstance&-format=mtdp.html&-view NIAID Veterinary pathology homepage http://www.niaid.nih. gov/dir/services/animalcare/VetPathology/VetPathologyindex.html with links to information on the pathology of genetically engineered mice, mouse immunohistochemistry, Helicobacter information, and other resources. The NCI Mouse Models of Human Cancers Consortium (MMHCC) web site provides information including recent publications and protocols on mouse models of human cancer. http://emice.nci.nih.gov/ Pathbase, the European mutant mouse pathology database, provides an ontology and is developing an image database for mouse pathology. http://www.pathbase.net/ The RENI (Registry Nomenclature Information system) web site, WebRENI, provides access to standardized and international nomenclature and diagnostic criteria of proliferative lesions in rats and mice. Although this is aimed primarily for toxicology applications, the information is relevant to other areas as well. http://www.item.fraunhofer.de/reni/ index.htm
The Mouse Phenome Project is an international collaboration headquartered at the Jackson Laboratory to promote quantitative phenotypic characterization of a defined set of mouse strains under standardized conditions and to make data publicly available through a web-accessible database (www.jax.org/phenome). Recent efforts define strain-specific characteristics or phenotypes according to specific protocols for collection, processing, evaluation, and reporting (Kile et al.
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2003; Deschepper et al. 2004; Zheng et al. 1999; Paigen et al. 2000; Peters et al. 2002; Reinhard et al. 2002; Wahlsten et al. 2003). Forty inbred strains were selected and recommended for testing for the project, and they were grouped into four priority categories (Bogue 2003). Group A strains are prioritized because these are ●
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widely used with available genetic and phenotypic information, providing useful data for comparison and validation. common progenitors in transgenesis or mutagenesis studies. progenitors of recombinant inbred, consomic, or congenic strains. generally easy to maintain with good reproductive performance. genetically diverse.
There are more than 450 inbred mouse strains (Beck et al. 2000; Paigen 2003). Excluding the strains derived from wild mice (Mus castaneus, Mus spretus), prioritized inbred strains in group A of the mouse phenome project are emphasized here. These are 129S1/SvImJ, A/J, BALB/cByJ, BALB/cJ, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ, and SJL/J. Because of their importance to different areas of research and their relationships to group A strains, AKR, B6;129, B6C3F1, and outbred Swiss stocks also are discussed.
C.
Nomenclature
Correct nomenclature of mice provides information on the origin and current source of the mice, breeding strategies, and mutation type, strategy, and developer of the mutant. (Linder 2003; Wotjak 2003) Unfortunately, correct nomenclature and substrain information are not always included in publications. Failure to use internationally accepted nomenclature can make it difficult or impossible to understand the study, to replicate it, or to continue related studies. In our discussion, when only the root strain name is used in the cited reference or when information on several similar substrains is summarized, only that root name will be used. Nomenclature details are discussed elsewhere in this series (Eppig 2007), but the terms used here are defined briefly. Except where indicated, information is from the Mouse Genomic Nomenclature Committee (MGNC) web site, http://www.informatics.jax.org/mgihome/nomen/, which includes revisions approved at the November 10, 2003, meeting of the International Committee on Standardized Genetic Nomenclature for Mice. Approved strain abbreviations used below are listed in Table 25-1, and additional abbreviations are listed in Eppig (2007) in this series and on the MGNC web site. Although the terms strain and stock frequently are used interchangeably, strain will refer to genetically defined, non-hybrid mice (e.g., fully inbred or fully backcrossed mice), and stock or line will refer to genetically mixed (e.g., hybrid, incipient
628 inbred, incipient congenic) mice and genetically undefined (e.g., outbred or random bred) mice. Inbred strains should be genetically homogeneous, homozygous at all alleles, and can be traced to a single ancestral pair. They can be termed inbred if they have been brother × sister mated (sibling mated) for 20 or more consecutive generations. Related inbred strains have a common origin but were separated before 20 generations of inbreeding, when the strain of origin would be expected to be heterozygous at some many loci (residual heterozygosity). Substrains are derived from an inbred strain after it is fully inbred (i.e., after 20 generations of inbreeding). Parental strains and substrains genetically diverge with time, owing to residual heterozygosity at the time of separation, to mutation and genetic drift, and sometimes to genetic contamination (e.g., 129× substrains). Substrains usually have the root symbol of the original strain, followed by a forward slash and a substrain designation. Substrains of substrains develop through continued maintenance by a different investigator or by establishment of a new colony. Further substrain designations are added, without the addition of another slash. For example, C3H/HeJ is Heston’s (He) substrain of C3H, subsequently maintained at the Jackson Laboratory (J). Laboratory codes (e.g., He, J) should be accumulated because genetic differences will accumulate with time. Registered Laboratory codes are searchable on the nomenclature web site. F1 hybrid mice are the first-generation progeny of two inbred strains. They are genetically identical, heterozygous for any alleles that differed between the parental strains, and are designated by the upper-case abbreviations of the two parents (maternal strain listed first), followed by F1. For example, B6C3F1 are offspring of a C57BL6 mother and C3H or C3H/He father, with all males carrying a C57BL/6 X chromosome and a C3H or C3H/He Y chromosome. (Maronpot et al. 1999) Recombinant inbred (RI) strains are created by crossing two inbred strains, followed by 20 or more consecutive generations of brother × sister mating (inbreeding). These are designated by upper case abbreviations of both parental strain names, with the dam’s strain abbreviation first and separated from the sire’s strain abbreviation by an upper case letter X with no intervening spaces. RI strains are especially useful to study or map traits that are divergent in parental strains. For example, AKXD RI strains, derived from AKR × DBA/2, are used to study the genetics of lymphoma susceptibility, with AKR being the highly susceptible strain and DBA/2 having a relatively low incidence of lymphoma (Gilbert et al. 1993). AXB and BXA RI strains, derived from A × C57BL/6 and C57BL/6 × A, respectively, are used to study the genetics of cerebral ventricular size, with C57BL/6 having relatively large ventricles compared to A. (Zygourakis and Rosen 2003) AXB RI strains also are used to study the genetics of susceptibility to cleft palate, with A being the more susceptible strain. (Diehl et al. 1997) Congenic strains are produced by repeated backcrosses to the inbred recipient strain, with selection for a particular
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marker from the donor strain. Fully congenic strains, achieved by 10 generations of backcrossing (N10) or the equivalent of marker-assisted backcrossing (Markel et al. 1997; Berry and Linder 2007), are designated by the full or abbreviated symbol of the recipient strain separated by a period from an abbreviated symbol of the donor strain, in which the allele or mutation originated. The mutation or allele is italicized and separated from the donor strain name by a hyphen. For example, the name B6.129P2-Apoa1tm1Unc indicates that this congenic strain on a C57BL/6 background carries a targeted mutation in ApoA1, created on an ES cell line derived from 129P2 mice, developed at the University of North Carolina (Laboratory code Unc) (MGI database query 04/04/04). By N5, mice are accepted as incipient congenic and may be given congenic nomenclature, as long as the number of generations of backcrossing is documented in information accompanying the strain. When a congenic strain is maintained by brother × sister matings (F generations) after backcrossing (N generations), the number of brother × sister generations follows the number of backcross generations, for example, B6.129 (N10F6). Incipient inbred refers to mice that are being sibling mated (inbred) but have not yet achieved 20 generations of inbreeding (F20). When derived from only two parental strains (one of which may be a gene-targeted ES cell line), their name uses upper-case abbreviations (similar to congenic names) but separated by a semicolon instead of a period. Thus B6;129 indicates that the mice derive from a C57BL/6 mouse and a 129 mouse (or a 129 ES cell line), and are currently being inbred. However, this designation frequently is used for animals that have been backcrossed and inbred but are not yet fully congenic or fully inbred. The genetically mixed status of such stocks should be clarified by indication of backcrossing and inbreeding generations, for example, B6;129 (N3F6). Outbred stocks are genetically undefined; that is, no two individuals from an outbred stock should be genetically identical. Outbred names use the Laboratory code of the institution holding the stock, followed by a semicolon and the common root stock name. (ILAR 1979; NIH 1982; Berry et al. 2005; Eppig 2005) For example, Crl:CD-1 is the Charles River Laboratory’s (CRL) outbred CD-1 stock.
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Coat Color
Coat color usually is the most immediately obvious mouse phenotype. Many of the common inbred strains have coat colors that result from selection for mutations in wild-type coat-color genes or loci. Some coat-color mutations are associated with pathologic phenotypes in addition to the color phenotype. The genetics of coat color are complex, with multiple alleles of multiple genes or loci interacting to produce a color phenotype. For clarity in the subsequent descriptions of common inbred strains, some features of albino, agouti (nonagouti), and black coloration are discussed here. Color genotypes and some other
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attributes of inbred strains discussed in this chapter are summarized in Table 25-1. Except where indicated, information on coat color is from Wilys Silvers’ “The Coat Colors of Mice” (Silvers 1979). Gene names conform to those in MGI database as of October 1, 2005. 1.
Tyrosinase Tyr: Albino (c)
The tyrosinase gene or locus (Tyr), formerly known as c, is on mouse chromosome 7. More than 100 spontaneous and induced mutations (alleles) are recognized, many of which result in albinism or hypopigmentation due to the absence or reduction of melanin in homozygotes. The albino (c) allele of Tyr (Tyrc) is the result of a recessive point mutation in Tyr that causes an amino acid substitution in tyrosinase protein. Albinism is epistatic to all other coat-color determinants. Thus, in the presence of Tyrc/Tyrc (c/c), mice have melanocytes and melanosomes, but with no tyrosinase activity, they lack pigment, regardless of other coat-color determinants. (Searle 1990; Beermann et al. 2004) Albino mice have altered vision compared to pigmented mice, with defects in the visual projections at the optic chiasm, decreased numbers of rod photoreceptors, and spatiotemporal defects in neuronal production. (Balkema and Drager 1991; Jeffery 1997; Beermann et al. 2004) 2.
Tyrosinase Tyr: Chinchilla (c-ch)
The chinchilla (c-ch) allele of Tyr (Tyrc-ch), formerly known as cch, is the result of another spontaneous point mutation in Tyr that causes an amino acid substitution in tyrosinase. This mutation reduces or lightens yellow or black pigmentation by drastically reducing pheomelanin production and also reducing black eumelanin expression, but it does not influence brown eumelanin. (Beermann et al. 2004) 3.
Tyrosinase-Related Protein 1 Tyrp1: Brown (b)
The tyrosinase-related protein 1 gene or locus (Tyrp1) is on chromosome 4. The dominant wild-type allele, B or Tyrp1B, produces black eumelanin. The recessive brown (b) mutation or allele (Tyrp1b) is a point mutation in Tyrp1, and homozygosity results in brown eumelanin. It arose spontaneously in C57BL and was selected to produce the C57BR strain with a/a, b/b color genotype. Homozygosity for b changes the gray color of a wild-type (a+), agouti-colored mouse to a brownish hue, known as cinnamon agouti or cinnamon, due to yellow banded brown (instead of black) hairs. 4.
Nonagouti (a)
The dominant wild-type a+ allele of the nonagouti (a) gene or locus on chromosome 2 encodes a protein that acts as a molecular switch to instruct melanocytes to make either yellow pigment (pheomelanin) or black pigment (eumelanin). The nonagouti
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alleles determine the relative amount and distribution (banding) of pheomelanin and eumelanin in the hairs (Hustad et al. 1995). The wild-type (a+) coloration is characterized by banded hairs, with dorsal agouti hairs having a subapical yellow band on otherwise black (or brown) hair resulting in a black-yellow-black pigment pattern on each hair. Ventral hairs are also banded, typically with yellow tips and black bases, resulting in a lighter colored appearance to the ventrum. There are many alleles of the nonagouti locus with at least 25 recessive, homozygous-viable alleles. Many of these amorphic (loss-of-function) or hypomorphic (reduced function) alleles were induced by irradiation or other mutagens. (Hustad et al. 1995; Miltenberger et al. 2002) The recessive nonagouti (a) allele, for which the locus is named, results in nonbanded (i.e., nonagouti) hairs in a/a mice. C57BL6, a/a mice are black. This mutant allele is the result of a VL30 (virus like element) insertion into the first intron of a+. (Bultman et al. 1994) Although a/a hairs are almost exclusively pigmented with eumelanin, hairs originating on and behind the ears as well as the hairs around the genital papilla and mammae are yellow. Additional coat-color genes can modify the a/a effect on coloration (e.g., a/a, b/b animals are brown). 5.
Nonagouti: White-Bellied Agouti (Aw)
The white-bellied agouti, Aw (also referred to as AL, lightbellied agouti) allele, like a+ is a dominant coloration in wild Mus musculus (M. musculus musculus, M. musculus domesticus, M. musculus molossinus) and may be the predominant coloration in sandy or arid regions. This allele is the result of a retrotransposon (retrovirus like transposable element) insertion into the first intron of a+. Reversion from a (nonagouti) to Aw occurs with a high frequency and is attributed to intrachromosomal homologous recombination involving the inserted elements. (Bultman et al. 1994; Chen et al. 1996) Paler ventral coloration distinguishes the light-bellied agouti (Aw) from the wild-type (a+) phenotype. Aw confers typical agouti dorsal hairs, and white, cream, or tan (yellow) ventrum, depending on the genetic background, with ventral hairs being nonpigmented, yellow pigmented, or predominantly yellow with black bases. Aw is dominant to a+ and lower alleles. 6.
Nonagouti: Lethal Yellow (Ay) and Viable Yellow (Avy)
The lethal yellow Ay and viable yellow Avy alleles result in variably yellow hair pigmentation. The Ay mutation is a 170-kb deletion that removes most of the Raly (hnRNP-associated with lethal yellow) gene (adjacent to a) except for its promoter and noncoding first exon, such that a+ is controlled by the Raly promoter. (Michaud et al. 1994) Heterozygotes for Ay manifest various dominant pleiotropic effects, including a completely yellow coat color, obesity, an insulin-resistant type II diabetic condition, and increased development of multiple spontaneous and induced tumors. Homozygous Ay/Ay embryos die early in gestation. (Bultman et al. 1992; Michaud et al. 1994)
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The Avy (viable yellow) allele results from an intracisternal A particle (IAP) retrotransposon insertion upstream of A that causes ectopic expression of wild-type (a+) protein, resulting in variably yellow fur, obesity, diabetes, and increased susceptibility to tumors. Aiapy, Aiy, Ahvy also result from IAP insertions. (Wolff et al. 1986, 1993; Duhl et al. 1994; Perry et al. 1994; Yen et al. 1994; Morgan et al. 1999) 7.
Myosin Va Myo5a: Dilute (d)
The myosin Va gene or locus (Myo5a) on chromosome 9 encodes myosin Va, which is a major actin-based vesicle transport “motor” that binds to melanosomes via a receptor that includes the melanophilin (Mlph) gene product. The recessive dilute (d) mutation or allele (Myo5ad) is a result of a retroviral insertion resulting from integration of an ecotropic (MuLV) provirus into Myo5a locus. When a component of this receptormotor complex is defective, melanosomes clump in the melanocyte and are transferred unevenly to the developing hair. (O’Sullivan et al. 2004) Homozygous (d/d) mutants probably have more pigment than corresponding non-dilute animals, but the clumped pigment has little effect on light absorption, resulting in the dilution effect. Other mutant alleles in Myo5a result in CNS abnormalities or lethality. (Jenkins et al. 1981; Seperack et al. 1988) 8.
Melanophilin Mlph: Leaden (ln)
The melanophilin gene or locus (Mlph) on chromosome 1 encodes melanophilin, which is involved with melanosome function via its interactions with myosin Va. (Li et al. 2005) The recessive leaden (ln) allele (Mlphln) is the result of a point mutation that arose spontaneously in C57BR and was selected to produce the C57L strain with a/a, b/b, ln/ln color genotype. Homozygosity (ln/ln) results in dilution effects similar to d/d. 9.
Pink-Eyed Dilution ( p)
The pink-eyed dilution, ( p) locus on chromosome 7 (near Tyr), has more than 50 spontaneous and induced alleles (MGI database search 04/04/04). The wild-type dominant allele, P, produces intense pigmentation of both the hair and eyes, whereas the oldest and most common recessive mutant allele, p (pink-eyed dilution), reduces pigmentation of the eyes and coat. Eyes of p/p mice resemble those of albino mice but have small amounts of melanin in the iris and retina and a few melanocytes in the choroid as well. (Staleva et al. 2002) 10.
Kit Ligand Kitl: Steel (Sl)
The Kit ligand gene or locus (Kitl) on chromosome 10 normally encodes the hematopoietic growth factor SCF (stem cell factor), which is a ligand for c-kit. Mutations in this gene, or in
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its receptor’s gene Kit, affect the migration of embryonic stem cells and result in mild to severe defects in pigmentation, hematopoiesis, and reproduction. The steel (Sl) mutation was a spontaneous deletion of most of the coding region of the gene and is homozygous lethal, with KitlSl/KitlSl homozygotes having severe anemia and rarely surviving until birth. Heterozygotes have an overall dilution of hair pigment that is more pronounced on the ventrum; a white spot or blaze in the middle of the forehead and often on the belly; light vibrissae, ears, feet, and tail; white snout tip; macrocytic anemia with red blood cell counts 20% to 30% of +/+ littermates; increased tumor incidence compared to +/+ mice; and increased incidence of testicular teratomas in mice on a 129/Sv background. (Wang and Enders 1996) 11.
Kit Kit: White Spotting (W, Wv)
The Kit oncogene or locus on chromosome 5 encodes a tyrosine kinase receptor (c-kit). Mutations at this locus in the mouse have pleiotropic effects on embryonic development and hematopoiesis. Associated phenotypes, including white coat color, sterility, and anemia, are attributed to the failure of stem cell populations to migrate and/or proliferate effectively during development. Historically and scientifically important mutations of Kit include W and W-v (Chabot et al. 1988; Geissler et al. 1988; Bernstein, Chabot, et al. 1990). The semidominant allele W (dominant white spotting) is a deletion mutation once favored by mouse fanciers because heterozygotes have white spotting, frequently a white belly spot and white tail tip, or a roan pattern, depending on the genetic background. KitW/Kit+ heterozygotes have normal blood and are fully viable and fertile, while homozygotes are white with black eyes, develop macrocytic anemia beginning at 12 days of gestation, and die within the first week after birth. Viable white spotting (W-v) is a point mutation that arose spontaneously on the C57BL/6 background. KitW-v/Kit+ heterozygotes have a white belly spot and white tail tip. Homozygotes are black-eyed white with less severe anemia than KitW/KitW and may be able to reproduce (Geissler et al. 1981). KitW/KitW-v mice are black-eyed white, anemic, mast cell deficient, and sterile. (Geissler and Russell 1983a, 1983b)
E.
Sexual Dimorphisms
The term sexual dimorphism is used to refer to any sex related differences detected between individuals or populations. Examples of sexual dimorphisms in mice include greater brain weight, spinal cord weight, and brain:body weight ratios in female mice (Roderick et al. 1973); size and morphology of mouse kidneys, adrenal glands, and salivary glands; predilections for different lesions or types of pathology; and susceptibilities to various agents.
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The larger size of intact male mouse kidneys, compared to female or castrated male kidneys, has been attributed to a larger renal cortex. (Messow et al. 1980) Larger cells in male proximal convoluted tubular epithelium, as measured by fewer nuclei in similar lengths of proximal convoluted tubules than in females’, have been noted in ICR, C57BL/6, C3H/He, DBA/2 mice but not in BALB/c mice. (Hanker et al. 1980) Kidneys of adult male mice also have a morphologic characteristic that affects variable numbers of renal corpuscles (i.e., glomeruli plus Bowman’s capsules). (Messow et al. 1980) In most mammals, including female mice, the parietal layer of Bowman’s capsule is a single layer of flattened, squamous epithelium. Male mice, however, have slightly larger renal corpuscles than female mice, and have varying numbers of renal corpuscles with cuboidal cells in their parietal epithelium, with the tallest cells usually in the central portions of the capsular crescent and having a distinct microvillous border. (Yabuki et al. 1999) This morphologic feature of male mice corpuscles may resemble, but should not be confused with, a metaplastic change to cuboidal parietal epithelium in human kidneys, which is associated with conditions such as hepatic fatty change and alcoholic liver disease. (Haensly 1988; Haensly et al. 1986) After castration of male mice, their renal corpuscles resemble those of females, and testosterone treatment of females or castrated males increases the percentage of cuboidal cells as well as the diameter of renal corpuscles. The percentages of cuboidal epithelium in male and female mice may be strain dependent, with C3H/He and C57BL/6J male and female mice having more cuboidal cells than BALB/c and DBA/2 mice in one study, and ICR mice having larger corpuscles than these four inbred strains (Yabuki et al. 1999). Another study reports a higher percentage of cuboidal parietal epithelial cells in male DBA/2 compared to C57BL/6 mice, and notes an association with lower susceptibility to chloroform in female and C57BL/6 male mice compared to DBA/2 male mice. (Ahmadizadeh et al. 1984) The oxidoreductase activity of mitochondria in cuboidal capsular cells is similar to that of columnar cells in proximal convoluted tubules, in contrast to the unreactive mitochondria in squamous capsular cells. The brush border of the cuboidal capsular cells has prominent alkaline phosphatase and aminopeptidase activities (Hanker et al. 1980). This proximal convoluted tubule-like differentiation suggests involvement of cuboidal parietal cells in resorption and perhaps in active transport, has been implicated in the proteinuria that is normal in adult male mice (Hanker et al. 1980), and has been associated with susceptibilities to certain toxins, for example, chloroform (Ahmadizadeh et al. 1984). Periodic acid Schiff (PAS) staining of the brush border of the proximal straight tubule epithelium is more intense in the kidneys of adult intact female mice than in males, and PAS-positive cytoplasmic granules are a feature of these cells
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only in females. After castration, male kidneys resemble female kidneys with respect to these features. Treatment with testosterone induces the male-specific features (less intense PAS staining of brush borders and loss of PAS positive cytoplasmic granules), while ovariectomy and estradiol treatment do not. Ultrastructurally, the PAS positive granules correspond to electron-dense bodies and myelinoid bodies, with acid phosphatase activity indicating that they are lysosomes. This sexual dimorphism is not discerned with routine hematoxylin and eosin (H&E) staining. (Yabuki et al. 1999) The adrenal glands of male mice contain less lipid and are smaller than those of female mice. Mouse adrenal glands typically are 1 to 3 mg each, with the left gland slightly heavier than the right, and with considerable variation between strains. Adrenal glands of young mice have a transient X-zone that surrounds the medulla. In males, the X-zone appears at about 10 days postpartum and disappears rapidly at sexual maturity at approximately 5 weeks of age. In females, the zone disappears at first pregnancy, but in virgin female mice, it may be visible for up to 30 weeks and may undergo prominent vacuolization. (Beamer et al. 1983; Frith et al. 1988; Faccini et al. 1990) Thickness and persistence of the X zone may be strain-related and influenced by multiple genes. Thickness of the zona reticularis also may be strain-related and under genetic control. (Tanaka et al. 1994, 1995; Deschepper et al. 2004) Aging female mice of A, C57BL/6 and DBA/2 strains have higher incidences of adrenal subcapsular cell hyperplasia than do males, but these changes can be found with high incidence in both sexes in various strains. Elongate, spindled subcapsular cells are referred to as type A cells. Polygonal, lipid-laden, subcapsular cells that more closely resemble normal cortical cells are referred to as type B cells. (Kim et al. 1997) The submandibular salivary gland1 of intact adult male mice is larger, up to twice as large as that of female mice. Submandibular gland histomorphology in immature mice of both sexes is similar to that in adult female mice, suggesting that the difference in adult males is testosterone-related. (Frith et al. 1988; Pardini and Taga 1996; Pinkstaff 1998) In the adult male, the granular convoluted tubules are larger, with more prominent eosinophilic apical zymogen granules than in the female. (Jayasinghe et al. 1990; Sawada and Noumura 1991) At about 4 weeks of age the duct system differentiates to form granular convoluted tubules. By 6 weeks of age, the volume proportion of granular convoluted tubule in the male is 45% and that in the female is only 12%, with acini occupying only 30% of the male gland compared to 57% of the female gland. At 6 weeks the volume of granular convoluted tubule cells is 40% lower in the female than in the male gland. Granular convoluted tubule cells proliferate to reach a maximum at 6 months of age; then decrease gradually from 6 to 21 months.
1Also
called submaxillary gland in some references.
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Aging submandibular glands develop regressive changes in the granular ducts, with diminishing height of granular duct cells and loss of mature secretory granules. (Chen et al. 1995) Higher amylase activity in male glands corresponds to the higher granularity of their granular convoluted tubules. (Gresik 1975, 1980) Female submandibular glands undergo “masculinization” during pregnancy and lactation, with increases in the volume fraction and area of the granular ducts and in the apical granulation of their cells. The response peaks on the fifteenth day of pregnancy; then volume fraction of the granular ducts diminishes, even though the gland retains the “masculine pattern” throughout lactation. (Rossi et al. 1989) Parotid glands have a more subtle sexual dimorphism than that in the submandibular glands. The male parotid gland is larger than the female’s, with the difference attributed primarily to larger male acinar volume due to more and larger acinar cells, and to larger intercalated duct volumes. (Ribeiro et al. 2001) Branching patterns of the ducts may be strain dependent. (Domon et al. 1987) Similar to human sexual dimorphism in digit-length ratios, in which men have a smaller 2D:4D (index finger:ring finger) ratio compared to women, adult and weanling outbred male mice also have a smaller 2D:4D ratio in the right rear paw, compared to females. (Brown et al. 2002) Mice have a variety of sex predilections or predispositions for different lesions or types of pathology. Compared to male mice, female mice are more likely to develop fibroosseous lesions or changes (also called myelofibrosis) (Sass et al. 1976; Wijnands et al. 1996; Rittinghausen et al. 1997), and pituitary tumors and mammary tumors (Frith and Ward 1988; Faccini et al. 1990), and they are more susceptible to H. hepaticus typhlocolitis (Livingston et al. 2004). Compared to female mice, male mice of some strains or stocks, including C3H and CBA, are more susceptible to liver tumors (Jacobs and Dieter 1978; Poole and Drinkwater 1995), and males but not females are susceptible to chloroform-induced nephropathy, with DBA/2 mice more sensitive than C57BL/6 (Ahmadizadeh et al. 1984). Males are more susceptible to respiratory mycoplasmosis (Yancey et al. 2001), to Mycobacterium marinum and M. intracellulare (Yamamoto et al. 1990, 1991), and to H. hepaticus hepatitis (Livingston et al. 2004).
F.
Age-Related Pathology
Anatomic or pathologic changes (phenotypes) that are identified at higher incidences in older populations can be statistically significant findings in chronic studies, and some (e.g., amyloidosis) impact lifespan. Frequently, these exhibit sex or strain predilections and may also be influenced by environmental factors. Incidental age-related findings in the liver include hepatocyte karyomegaly and cytomegaly (Frith and Ward 1988; Lu et al. 1993; Styles 1993), or polyploidy (Chipchase et al. 2003) and hepatocellular inclusions (Frith and Ward 1988).
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Other findings include fibroosseous changes (also called myelofibrosis in some studies) (Sass et al. 1980; Wijnands et al. 1996), cerebral thalamic mineralization (Morgan et al. 1982), cerebral neuronal lipofuscin (Lamar et al. 1980; Moore and Davey 1995; Moore and Ivy 1995), deposits or accumulations of ceroid or lipofuscin pigments in ovaries or adrenal glands (Maekawa et al. 1996; Yarrington 1996; Davis et al. 1999; Nyska and Maronpot 1999), and adrenal subcapsular cell proliferation (Kim et al. 1997). Amyloidosis is an important age-related, pathologic phenotype that has shortened numerous chronic studies of susceptible strains or stocks of mice. Its incidence and severity can be related to age, genetics, and environmental factors. Although it may be less common and less severe in contemporary cleaner colonies than it was in the 1940s to 1970s, when it occurs amyloidosis can impact various phenotypes such as lifespan and body condition, as well as morphology and function of kidney, intestine, liver, heart, and other tissues. Amyloid deposits in mice are mainly in the small intestines, stomach (glandular), kidney, liver, spleen, heart, thyroid, parathyroid, adrenals, salivary glands, and ovaries, but not in the brain, spinal cord, bone, or bone marrow. (Powers et al. 1976; Frith and Chandra 1991; Higuchi et al. 1991; Hogen-Esch et al. 1993; Lipman et al. 1993; Majeed 1993; Gonnerman et al. 1995; Gruys et al. 1996; Yabuki et al. 2002; Guo et al. 2003) Amyloid deposits are extracellular and stain positively with Congo Red, oil red O, Alcian Blue, and Thioflavine T, but stain affinity can vary with the type of amyloid. (Hogen-Esch et al. 1993; Majeed 1993; Korenaga et al. 2004) Trichrome staining suggests that collagen may be a component of the deposited material at some sites, such as nasal septum. (Haines et al. 2001) Systemic amyloidosis in mice can include senile ApoAII amyloid (AApoAII) as well as secondary or reactive AA amyloid. Senile amyloid AApoAII (also known as senescence accelerated or ASsam amyloid) was initially identified in senescenceaccelerated SAMP mouse lines. Earlier and more extensive deposition of AApoAII(C) amyloid fibrils, especially in liver and spleen, is associated with possession of the c (highly amyloidogenic) allele of the apolipoprotein AII (Apoa2) gene, in A/J, SJL, and some SAMP lines. (Guo et al. 2003; Korenaga et al. 2004) Deposition of AApoAII(A) amyloid fibrils is milder, later, and primarily in intestine, lungs, tongue, and stomach but not in the liver or spleen in strains that carry the less amyloidogenic ApoAIIa allele, including C57BL/6, AKR/J, DBA/2 and some related strains. (Higuchi, Kitagawa, et al. 1991; Higuchi, Naiki, et al. 1991; Kitagawa et al. 2003; Korenaga et al. 2004) Specific pathogen-free outbred Swiss stocks (e.g., CD-1) are susceptible to senile and reactive amyloidosis, with gut, heart, and lung being predilection sites for AApoAII amyloid deposition. (Gruys et al. 1996) In secondary or reactive amyloidosis, AA amyloid protein is derived from serum amyloid A (SAA) protein precursor, which is elevated in the blood during inflammation. (Higuchi et al. 1991) Spleen, liver, gut, and kidney are predilection sites
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for AA-amyloid deposition, and it is often associated with inflammatory lesions of the skin. (Gruys et al. 1996; Solomon et al. 1999) CBA/J, C57BL/6 strains, and some Swiss stocks are among the most susceptible, and A/J mice have been considered to be resistant to secondary or reactive amyloidosis. (Butler and Whitehead 1994) A dominant gene or allele in the Saa gene or gene cluster has been linked to resistance (Gonnerman et al. 1995), but other genes seem to be involved (Butler and Whitehead 1994; Wang et al. 2000). Hematopoietic neoplasms, especially lymphomas and histiocytic sarcomas, are common findings in many chronic studies and consistently represent the most common neoplasms in AKR (Festing and Blackmore 1971; Rowe and Pincus 1972; Karpova and Fomina 2002), C58 (Strand et al. 1974; Nexo and Krog 1977), C57BL/6 (Frith et al. 1975, 1983; Zurcher et al. 1982; Ward et al. 2000), SJL/J (Kaminsky et al. 1985; Lin and Ponzio 1991; Erianne et al. 2000; Rubin 1968; Kumar 1983; Wrone-Smith et al. 1993), and CD-1® Swiss mice (Homburger et al. 1975; Maita et al. 1988; Engelhardt et al. 1993; Giknis et al. 2000; Son 2003), and have been found to be the most common neoplasm in a study of B6;129 mice (Haines et al. 2001). Thus, they should be expected in old mice and in chronic studies, including studies of genetically engineered mice (GEM). Genetically and environmentally relevant control animals may be important to detect deviations from normal incidences of these neoplasms. Definitive identification and typing of lymphomas require immunohistochemistry and/or molecular techniques. (Morse et al. 2002) Historically, the term leukemia was used to refer to various lymphomas in mice, but the term should be interpreted and used with caution in reference to hematopoietic neoplasms in mice. In humans, lymphoid leukemia is a neoplastic disease that involves the blood and/or hematopoietic marrow and spleen from its early stages, while lymphoma presents as a solid tumor. The majority of lymphoid leukemias in mice, as defined by involvement of the blood, represent “spillovers” of lymphoma cells into the blood. These are not primary leukemias but rather lymphomas with leukemic phases. Lymphoid leukemias comparable to those in humans have been induced by genetic manipulations but are an uncommon spontaneous event. In mice, a leukemic phase may occur in about one-third of small lymphocytic or lymphoblastic lymphomas. (Morse et al. 2002) The term thymoma also has been used historically to refer to thymic masses, including lymphomas (e.g., in AKR and C58 mice). However, thymoma is an epithelial neoplasm, and the term should not be used to refer to lymphoid neoplasms of the thymus. Other age-related neoplasms, cardiac, pulmonary, and renal disease, and other conditions are discussed below in sections pertaining to susceptible strains, and their incidences and descriptions are summarized in Tables 25-3–25-6. Various inbred strains (C3H, CBA, A, AKR, C57BL/6, etc.) were developed for and generally retain their susceptibility or relative resistance to specific tumors. Mammary tumors, lymphoma, lung tumors, and liver tumors have long been recognized as the
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most common neoplasms of aging laboratory mouse populations (reviewed in Murphy 1966).
II.
INBRED STRAINS A.
Strain 129
The original 129 strain was initiated by Dunn in 1928 from crosses of coat-color stocks from English fanciers and a chinchilla stock from Castle. Currently, 129 mice comprise at least 16 recognized substrains (Festing et al. 1999). Most 129 substrains carry the dominant white-bellied agouti allele AW. Not all have the agouti pattern because they also may carry albino or chinchilla or the pink-eyed dilution alleles. L. C. Stevens’ (Laboratory code Sv) studies on embryonal carcinoma cells derived from testicular germ-cell tumors (TGCT) led to methodologies for establishing and manipulating embryonic stem (ES) cell cultures derived from his 129/Sv mice. Thus, 129/Sv mice were the original source of ES cells that were manipulated in vitro and injected into blastocysts of C57BL/6J mice to generate chimeric offspring with targeted mutations. (Mintz and Illmensee 1975; Illmensee and Mintz 1976; Doetschman et al. 1988; Baribault and Kemler 1989; Jiang and Nadeau 2001) Chimeras with the targeted mutations in germline cells are bred to produce “knockout” mice that have those targeted mutations (frequently deletions) in all cells. Over time, 129 lines or substrains maintained in different sites have been “contaminated” by intentional or inadvertent breeding with mice of other strains and/or have undergone genetic drift. ES cells generated from mice of different substrains may have significant genetic differences, so it can be important to specify the ES cell line and to use correct and specific nomenclature of 129 mice. (Simpson et al. 1997; Threadgill et al. 1997) In the 1999 revision of the 129 substrain nomenclature, “129” is followed by one of the letters P, S, T, or X, which is followed by additional information (Festing et al. 1999). “P” indicates origin from the parental stock; “S” indicates origin from a line that had carried Sl (steel allele on the Kitl gene, which is lethal when homozygous); “T” indicates that the line carried the Ter (teratoma) gene; and “X” indicates that the line was genetically contaminated (Festing et al. 1999; Simpson et al. 1997). Thus, 129P2/OlaHsd is from the original parental strain, maintained at Harlan UK (Ola), then transferred to and inbred at Harlan Sprague Dawley (Hsd). 129X1/SvJ, originally from Stevens’ laboratory (Sv) and maintained at the Jackson Laboratory (J), was found to be genetically contaminated in 1997. 129S1/SvImJ was developed by the induced mutant resource (Im) at the Jackson Laboratory (J) to serve as a control inbred strain for steel-derived ES cell lines, and is the prioritized 129 strain in the phenome project. 129S4/SvJae is from a line that carried the Sl allele in Stevens’ laboratory (Sv).
634 Subsequently Sl was eliminated when the line was maintained and inbred by Rudolph Janesch (Jae) (Simpson et al. 1997) (JAX® Mice Database query 4/14/04). Below, nomenclature from the original citation is used when the current correct designation is not clear. Heterogeneity within the 129 “strain” impacts research in various ways. Genetic differences between targeting constructs and ES cells can reduce the efficiency of homologous recombination (Simpson et al. 1997). “Control” mice of some 129 substrains may not be histocompatible with targeted mutants developed from other 129 substrains, and thus may not accept grafts from them. (Sechler et al. 1997) Behavioral and other phenotypes differ among the 129 substrains. (Montkowski et al. 1997; Corcoran and Metcalf 1999; Zheng et al. 1999; Cook et al. 2002; Rodgers et al. 2002) 1.
Nonneoplastic Conditions
Hermaphroditism in chimeric mice is discussed here because so many chimeras are generated using ES cells derived from mice of 129 substrains. About half of the chimeras produced by aggregation techniques or by injection of (usually) XY ES cells into XX or XY blastocysts are XY/XY male chimeras, and about half are sex chimeras composed of XY (ES-derived) and XX (blastocyst-derived) cells (Nakayama et al. 1988; Nagy et al. 2003). Most phenotypically male chimeras are XY/XY with normal reproductive tissues, but XY/XX chimeras develop as males and are fertile (McLaren 1975; Jankowska-Steifer et al. 1992), although they may have small testes and poorer reproductive performance. At days 12–14 postcoitus (dpc), XY/XX chimeras have ovotestes with varying proportions of ovarian tissue at the gonadal poles, but apparently ovarian tissue usually regresses during development, usually resulting in testes in the adult. (Bradbury 1987; Nakayama et al. 1988) Infertile phenotypically male adult chimeras may be hermaphroditic XY/XX chimeras with cystic m¨ullerian duct remnants, an ovary as well as a testicle, and/or ovotestes. (Shomer et al. 1997) Aggregation chimeras with more than 30% of XY cells usually develop into males, and females usually develop from chimeras that contain less than 20% of XY cells. Those with 20-25% XY cells develop into true hermaphrodites with ovotestes. (Jankowska-Steifer et al. 1992) In mouse hermaphrodites, ovaries may be more likely to occur on the right side, and testes and ovotestes are more likely to occur on the left side in contrast to human hermaphrodites where the reverse is true. (van Niekerk and Retief 1981; Mittwoch 2000) Strain 129S1/SvImJ mice have relatively high systolic blood pressure and left cardiac ventricle to body weight ratio compared to 10 (non-129) inbred strains (Deschepper et al. 2004). Some 129 strains, including 129/Ola but not 129/J, have a high incidence of hepatic portal vascular peculiarities resulting in portal-systemic shunts that confer resistance to experimental schistosomiasis. C57BL/6J mice have a lower incidence of
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similar shunts. (Coulson and Wilson 1989; Mitchell 1989; Mitchell et al. 1990) Strain 129/J (now 129P3/J) mice have a relatively large brain/ body weight ratio (third highest of 25 strains evaluated) (Roderick et al. 1973), and relatively small forebrain volume and neocortex (Wimer et al. 1969). Reduced or absent corpus callosum has been identified in up to 80% of some groups of 129 mice. (Balogh et al. 1999; Magara et al. 1999; Schalomon and Wahlsten 2002; Wahlsten et al. 1982, 2001, 2003) There is substantial neurobehavioral variation among 129 substrains. Subnormal performance on some neurobehavioral tests is attributed to hypocallosity, but the effect seems to be mild. (Balogh et al. 1999; Rodgers et al. 2002; Schalomon et al. 2002; Wahlsten et al. 2001) Strain 129S6/SvEvTac mice are very resistant to noiseinduced hearing loss (Yoshida et al. 2000). The 129 substrains vary in the progression and severity of hearing impairment (Zheng et al. 1999), with a major age-related hearing loss mutation Cdh23ahl (Ahl, age-related hearing loss 1) identified in 129P1ReJ mice (Johnson et al. 2000). Ahl is a recessive hypomorphic allele of the cadherin 23 (Cdh23) gene that results from a spontaneous point mutation. Age-related hearing loss in mice is a genetically complex quantitative trait. Homozygosity at Cdh23ahl alone or in combination with secondary factors is a primary determinant of age-related hearing loss (AHL) in mice (Johnson et al. 2000; Noben-Trauth et al. 2003; Zheng et al. 1999). Blepharoconjunctivitis (inflammation of the eyelid and conjunctiva) can be common in young 129/J (now 129P3/J) mice (12–15% incidence) and increases to 50% incidence by 20 weeks of age. Initially, there is suppurative conjunctivitis and/or ulceration at the mucocutaneous junction, progressing to suppurative inflammation involving meibomian ducts, with conjunctival ulceration. Various bacterial species, including corynebacteria, lactobacilli, and Pasteurella pneumotropica, may be isolated, but their role as pathogens or opportunists is unclear. (Smith and Sundberg 1996; Sundberg et al. 1991) In a recent study of 98 aging 129S4/SvJae mice, more than 50% of male and female mice survived to 2 years old. (Ward et al. 2000, 2001) Acidophilic macrophage pneumonia and megaesophagus with impaction were the most common nonneoplastic proximate causes of death. Arteritis, abscesses, and mouse urologic (or urinary) syndrome (urinary obstruction) in males; uterine hematomas and thrombi in females; and neoplasms were identified as contributors to death as well. (Ward et al. 2000, 2001) Additional details are summarized in Table 25-3. Acidophilic macrophage pneumonia was considered to be an important contributor to death in about 10% of females and 10% of males, with more than 80% of females and 60% of males having the condition to some degree. Many affected mice also had eosinophilic degenerative changes or “hyalinosis,” characterized by cytoplasmic accumulation of hyalin, intensely eosinophilic material, sometimes with needle-like crystals in
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nonpulmonary epithelial tissues, including nasal cavity, glandular stomach, trachea, gall bladder, and bile duct (see below). The eosinophilic material has been identified as Ym1 and/or Ym2 chitinases. (Ward et al. 2001) Acidophilic macrophage pneumonia, also referred to as acidophilic crystalline pneumonia, is characterized by the presence to varying degrees of plump macrophages, acidophilic macrophages in alveoli and bronchioles. The cells are distended by intracytoplasmic eosinophilic (acidophilic) granular and crystalline material, and may have two or three, or occasionally more, nuclei. Extracellular small (<10 µ long) and large (up to 100 µ long) spicule-like crystals in airways may be inconspicuous, or may be a prominent feature of the condition. The condition was described in Swiss mice in 1942, in a report that cited observations of earlier (1905, 1909) studies (Green 1942). Recently, the acidophilic material in lung and other tissues has been identified as YM1 and/or YM2 chitinases that precipitate (crystallize) at neutral pH. (Guo et al. 2000; Harbord et al. 2002) They may be produced by macrophages, neutrophils and other cells in association with parasitism (Nair et al. 2005; Tsai et al. 2004), fungal infections (Feldmesser et al. 2001; Huffnagle et al. 1998), chronic infections (Harbord et al. 2002), allergy (Nio et al. 2004), and neoplasia (Harbord et al. 2002). Intracellular or extracellular eosinophilic needle-like crystals in airways may or may not be conspicuous and may stain blue with Perl’s reaction (for iron). Acidophilic macrophage pneumonia and hyalinosis in nonpulmonary epithelial tissues (especially in the nose) can be common in B6;129 mice as well as in 129 mice. (Haines et al. 2001; Ward et al. 2000, 2001) Acidophilic macrophage pneumonia with crystals is reported in various studies of C57BL/6 mice and some Swiss stocks (Green 1942; Ward et al. 2000; Harbord et al. 2002) and seems to be less common in BALB/c and CBA mice (Murray and Luz 1990). In these studies (Haines et al. 2001; Ward et al. 2000, 2001), hyalinosis was most common in nasal mucosa, trachea, lung, and stomach of 129S4/SvJae mice, usually not associated with inflammation, and is attributed to production and deposition of YM1 and/or YM2 chitinases (Ward et al. 2001; Nio et al. 2004). In nasal hyalinosis, respiratory epithelium may be more affected than olfactory epithelium. Affected respiratory epithelium usually is in the regions of the nasal glands, and epithelial cells frequently are distended with peripheral displacement of nuclei by eosinophilic hyaline material. Foci of affected olfactory epithelium are primarily near the olfactory/respiratory transition areas, and the cytoplasmic material tends to originate at the basal aspects of lining cells. Extracellular crystals are variably needle-like, rectangular, or square. Gastric hyalinosis is most common in the cardiac glandular stomach at or near the limiting ridge. Grossly discernible areas of plaque-like thickening, sometimes with hemorrhage, are periesophageal, with the largest lesions found in the oldest mice. Histologically, glands are elongated and hyperplastic, and may be focally disorganized with loss of normal differentiation patterns. Some epithelial
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cells contain only a few intracytoplasmic droplets, or cells may be distended and nuclei displaced peripherally by brightly eosinophilic material. Glandular lumina may contain extracellular rectangular eosinophilic crystals. Gall bladders with hyalinosis may be grossly enlarged with thickened opaque walls. Extracellular crystals, when present, tend to be large and rectangular or square. Affected bile ducts have associated mucoid metaplasia and fibrosis. (Ward et al. 2001) Megaesophagus with impaction was considered to be an important contributor to death in 15% of female and 7% of male of 129S4/SvJae mice, with megaesophagus noted in 33% of females and 27% of males. The cause was not determined, and the condition was noted in B6;129 mice in this study as well. (Ward et al. 2000; Haines et al. 2001) The muscularis externa of the adult mouse esophagus normally is composed of two skeletal muscle layers (Zhao and Dhoot 2000; Rishniw et al. 2003). In ICRC strain mice, megaesophagus has been associated with smooth muscle in the abdominal segment of the esophagus instead of only immediately adjacent to its junction with the stomach. (Randelia et al. 1988, 1990) Arteritis was judged to be a contributing cause of death in about 7% of male 129S4/SvJae mice and was a relatively common finding, occurring in multiple tissues including spleen, heart, and urinary bladder, with incidence of 30-50% in males and <10% in females (Ward et al. 2000). The condition in mice also is known as polyarteritis, periarteritis, or systemic arteritis, and it shares histologic features with periarteritis nodosa of rats, as well as with human polyarteritis nodosa. “Autoimmune” MRL and NZB mice have a high incidence and early onset of this condition. Organs commonly involved include the heart, tongue, uterus, testes, mesentery, kidney, and urinary bladder. There are perivascular and intramural accumulations of mononuclear cells, sometimes neutrophils, and there may be perivascular fibrosis and deposition of homogeneous eosinophilic “fibrinoid” material in the media. (Frith and Ward 1988; Plendl et al. 1996; Elwell and Mahler 1999) The condition may be less common in B6;129 mice than in parental 129S4/SvJae (Ward et al. 2000; Haines et al. 2001). Other nonneoplastic conditions in 129S4/SvJae mice that were noted but not considered to be major contributors to death are detailed in Table 25-3. These conditions include cardiomyopathy (with or without mineralization or arteritis); chronic nephropathy (frequently with mineralization); myelofibrosis (fibrotic change in the bone marrow), especially in female mice; melanosis in the meninges; ovarian atrophy (with or without hyaline material), pigment (ceroid-lipofuscin), tubular, or stromal hyperplasia; cystic endometrial hyperplasia; testicular tubular degeneration or mineralization; prostate atypical epithelial hyperplasia; gastric glandular epithelial hyperplasia; pancreatic islet cell hyperplasia; dental dysplasia (incisor teeth); pituitary hyperplasia of pars intermedia and pars distalis; cataracts; increased extramedullary hematopoiesis in spleen; and lymphocytic infiltrates or other inflammatory
636 changes in various tissues, including Harderian gland, salivary gland, kidney, liver, gall bladder, nose, trachea, thyroid, periovarian fat, epididymis, and urinary bladder. (Ward et al. 2000, 2001) 2.
Neoplasia
Testicular teratomas, or TGCT, are congenital anomalies as well as spontaneous neoplasms that develop from totipotent primordial germ cells during early stages of gonadal differentiation, starting at about day 12 of development. (Matin et al. 1998; Lam and Nadeau 2003) By definition, teratomas contain tissues from all three germ layers (endoderm, mesoderm, ectoderm). They usually contain a variety of epithelial types, including cornifying stratified squamous epithelium, ciliated tall columnar epithelium, and intestinal epithelium, sometimes thyroid or pancreatic tissue, along with well-differentiated cartilage, bone, skeletal, or smooth muscle, and variable amounts of well-differentiated nervous tissue resembling cerebral cortex. Benign tumors tend to appear more differentiated with easily recognizable mature tissues (Rehm et al. 2001). 129/Sv mice have been known for their high incidence of these early onset tumors since the 1950s, with 1-10% incidence noted by 3 weeks of age. (Stevens and Hummel 1957; Stevens 1973) The incidence of TGCT is influenced by as many as 15 susceptibility genes including Ter, Sl, Sl-J, Ay, and Trp53 (P53), with Ter having the strongest effect and detectable tumors in most Ter/Ter homozygotes by 3 weeks of age. (Noguchi and Noguchi 1985; Asada et al. 1994; Jiang and Nadeau 2001; Lam and Nadeau 2003) Ter/Ter homozygotes may have incidence of up to 94% TGCT, with up to 75% bilateral tumors, and Ter/+ heterozygotes may have an incidence of up to 17% TGCT, with 10% bilateral tumors. (Matin et al. 1998) Extragonadal teratomas in chimeras derived from 129 ES cells usually are perigenital and on the midline in young chimeras, and likely arise from 129 origin ES cells. (Hardy et al. 1990; Blackshear et al. 1999) Ovarian teratomas have a different pathogenesis and origin, developing from parthenogenetic ovarian oocytes. They are rare except in LT/Sv mice (Stevens 1980, 1984), comprising <10% of 587 ovarian neoplasms in studies involving 41,102 B6C3F1 female mice (Alison et al. 1987). Historically, 129 mice have been considered to have a relatively low lifetime tumor incidence (F 21%, M 7%), primarily lymphoma (F 7%, M 2%), soft tissue sarcomas (F 1%, M 2%), and “benign tumors” (F 2%, M 2%) (Smith et al. 1973). However, lung tumors also have been reported in 4-46% of 129 mice in a survey of multiple studies (Festing and Blackmore 1971). The more recent study of 129S4/SvJae mice identified 78 tumors in 30 of 40 male mice, and 84 tumors in 39 of 48 female mice (see Table 25-4). In this study, lung tumors were the most common neoplasm (F 31%, M 63%) and the most common neoplastic contributor to death (F 4%, M 15%), with a distinct male bias and more adenomas than
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carcinomas. Harderian gland tumors also were common, with a male bias and more adenomas than carcinomas. Liver adenomas were less common but also had a male bias. Ovarian tumors occurred in almost one-third of females and included adenoma, granulosa cell tumor, luteoma, hemangiosarcoma, and theca/Sertoli cell tumor. Hemangiosarcomas were relatively common in males and females and occurred in the epididymis or vas deferens, liver, ovary, skin, spleen, and uterus. Hematopoietic tumors, lymphoma, and histiocytic sarcoma were less common than in C57BL/6 or B6;129 mice, while lung tumors and Harderian gland tumors were more common than in C57BL/6 or in B6;129 mice. (Ward et al. 2000) Less common tumors in this study of 129S4/SvJae mice included adrenal cortical adenoma, pheochromocytoma, renal tubular adenoma, nasal cavity hemangioma, nasal cavity malignant schwannoma, pancreatic islet cell adenoma, parathyroid adenoma, skin papilloma, small intestinal adenoma and adenocarcinoma, gastric squamous papilloma, squamous cell carcinoma testicular teratoma, thyroid follicular cell adenoma, thyroid C cell carcinoma, uterine stromal polyp, and uterine stromal carcinoma. (Ward et al. 2000) (See Table 25-4.) 3.
Related Strains or Substrains
129P1/ReJ-Lama2dy mice carry a muscular dystrophy mutation that arose spontaneously in the 129/ReJ inbred strain at the Jackson Laboratory in 1951 (Nonaka 1998). The single basepair mutation in the laminin alpha2 gene, Lama2 results in merosin deficiency. Lama2dy homozygotes have progressive weakness and paralysis beginning at about three-and-half weeks of age. The hind limbs are affected first, axial and forelimb musculature later. Death usually occurs before 6 months of age, and the mice usually are sterile. Skeletal muscle degenerative changes include proliferation of sarcolemmal nuclei, increased interstitial tissue, and muscle fiber-size variation. There also are Schwann cell defects with abnormal myelination of peripheral nerves. (West and Murphy 1960; John 1990)
B.
A
The A strain was developed in 1921 by L. C. Strong from a cross between Cold Spring Harbor and Bagg albino randombred stocks, and thus they are related to BALB/c. A/J mice derive from A mice transferred to Cloudman in 1928, then to Jackson in 1947. A/HeJ mice derive from A mice transferred to Heston in 1938 and developed for cancer research. (Kalter 1979; Festing 1999; JAX® 2004) A strain mice have been popular for research on cancer, especially lung tumors, and for research on teratology. (Shimkin and Polussar 1958; Shimkin, Wieder, et al. 1968; Kalter 1979; Theiss et al. 1979) A and A/He mice are albino with coat-color genotype a/a Tyrp1b/Tyrp1b Tyrc/Tyrc (a/a, b/b, c/c). A strain mice are
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considered to have an intermediate lifespan in conventional or specific pathogen-free conditions with reported mean lifespans of 558 or 590 days in females and 490 or 512 days in males (Storer 1966; Festing and Blackmore 1971), and longer lifespans in a later study: 688 days in females, 662 days in males (Goodrick 1975). A/J mice have intermediate breeding performance, 22% of dams having at least four litters, with about six pups per litter, 90% weaned:born ratio (Fox et al. 1997). They are of intermediate size with mean female weight of 21g and mean male weight of 26g at 8 weeks old (Fox et al. 1997). A/J mice have intermediate systolic blood pressure and low left ventricle:body weight ratio. (Deschepper et al. 2004) A/J mice have intermediate brain weight and brain:body weight ratio, with relatively small cerebral ventricles (Roderick et al. 1973), and hydrocephalus is relatively uncommon (Sundberg et al. 1991). Recombinant inbred strains developed from A/J and C57BL6/J (with large ventricles) are used to study the genetics of ventricle size. (Zygourakis and Rosen 2003) They are relatively placid and easy to handle. (Wahlsten et al. 2003) Nonneoplastic Conditions
A strain mice have been known for their relatively high incidence of spontaneous congenital malformations including cleft palate, cleft lip, polydactyly, and prenatal open eyelids (Kalter 1968, 1975, 1979; Strong 1978). A/WySn have even higher frequency of cleft lip (20–30%) than A/J or A/HeJ (10%). Cleft lip usually accompanies cleft palate (cleft lip/palate), but nonsyndromic cleft lip is genetically complex and distinct from cleft palate (Juriloff 1982; Wang et al. 1995). Mouse eyelid development begins at 13.5 days postcoitus (E 13.5), with eyelid fusion at E 15–16, protecting the eyes while they develop. Mouse eyelids normally open between postnatal days 12 and 14 (P12–14) (Smith et al. 2002). Experiential or environmental factors can influence these developmental anomalies. In A/J mice, frequencies of cleft lip/palate and fetal resorption are inversely related to maternal age, directly related to litter size, and are sensitive to intrauterine location. Frequency of open eyelids is directly related to litter size, and is sensitive to intrauterine location. Cleft lip/palate is more common in females, and open eyelid is more common in males. Because the percentage of males increases with parity, the frequency of cleft lip/palate in males relative to that in females decreases with parity, and that of open eyelid increases. (Kalter 1975, 1979) A/J mice develop progressive muscular dystrophy with biochemical, histopathological, and ultrastructural features similar to dysferlin-deficient human muscular dystrophies. The condition is due to a retrotransposon insertion in the dysferlin gene. The Dysfprmd mutation arose between the late 1970s and early 1980s (different from the Dysf im mutation in SJL/J mice), and became fixed in A/J production breeding stocks. Muscle pathology progresses with age, with increasing numbers of
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necrotic and regenerating fibers, phagocytosis, and mononuclear cell infiltration of fibers, marked variation of fiber size, hypertrophic fibers, fiber splitting, and fat replacement, by 5 months of age. Perivascular inflammation, endomysial fibrosis, and inflammation are seen at 8 months of age, and progress as well. Proximal limb muscles, quadriceps femoris, and triceps brachii, are earliest and most severely affected, with gastrocnemius, soleus, anterior tibial, biceps brachii, diaphragm, masseter, and pectoral muscles having milder changes, even at late stages of the disease. (Ho et al. 2004) A/J mice have progressive hearing loss with onset between 3 and 5 months of age, and are homozygous for Cdh23ahl (Ahl, age-related hearing loss 1), a primary determinant of agerelated hearing loss (AHL) in mice. (Zheng et al. 1999; Johnson et al. 2000; Noben-Trauth et al. 2003) Despite reports of low femoral bone density in C57BL6/J mice compared to various other strains, not including A/J (Beamer et al. 1996; Turner et al. 2000), A/J bones have been reported to be more brittle than C57BL6/J bones. (Jepsen et al. 2001) Compared to C57BL6/J mice, A/J mice also have lower tibial and femoral bone mass, lower activity levels, and smaller quadriceps muscles in both sexes, smaller testicles and lower testosterone levels in males, and larger ovaries and higher estradiol levels in females (Kaye and Kusky 1995). Aged female A/J mice have high incidence (almost 100% at 13–15 months of age) of adrenal subcapsular spindle cell hyperplasia compared to males with incidence of approximately 20%. The condition may be associated with mast cell infiltration of the cortex. (Kim et al. 1997) Susceptibility of A strain mice to systemic amyloidosis is attributed to their possession of the c (highly amyloidogenic) allele of Apoa2, along with some SAMP lines and SJL mice. Heavy deposition of AApoAII(C) fibrils in liver and spleen is characteristic of affected mice (Korenaga et al. 2004). Lower levels of senile amyloid in A/J compared to some SAMP mice seem to be under polygenic control (Guo et al. 2003). Under conventional conditions, 55% of retired A/J breeders examined between 3 and 28 months of age had amyloid deposits, with similar amounts of senile amyloid AApoAII, and of secondary or reactive amyloid AA protein (Higuchi et al. 1991), although A/J mice have been found to be relatively resistant to development of reactive SAA amyloidosis in some studies (Powers et al. 1976; Wohlgethan and Cathcart 1979; Butler and Whitehead 1994). Neoplasia
A strain mice are known for their high incidence of spontaneous lung tumors and susceptibility to induction of lung tumors, especially compared to more “resistant” strains such as BALB/c, C3H/He, and C57BL/6. Pulmonary adenoma susceptibility (Pas) genes have been identified in A/J and other strains of mice, and the A/J strain has been useful in
638 identifying and mapping Pas and pulmonary adenoma resistance (Par) genes. The genetics of mouse pulmonary adenoma susceptibility is complex, with at least 14 susceptibility and resistance genes recognized. (Festing et al. 1994; Dragani et al. 1996; Manenti et al. 1997, 2002) Reported incidences of spontaneous lung tumors in A and closely related A/He substrains vary from 10% to 90% (Festing and Blackmore 1971; Dixon et al. 1991). Variable sex predisposition with regard to incidence and multiplicity is reported, and the right lobes are involved more frequently than the left (Dixon et al. 1991; Festing et al. 1994). The most common tumor type, previously called bronchoalveolar or bronchioloalveolar adenoma, currently is classified simply as adenoma of the lung, and these may have acinar (solid), papillary or mixed patterns (Nikitin et al. 2004). Grossly, these tumors are yellow-white, discrete nodules ranging in size from 1.0 to 10 mm. Adenomas are usually less than 4 mm in diameter with solid > papillary > mixed patterns. Carcinomas usually are larger than 4 mm in diameter with papillary > mixed patterns. Carcinomas are less common and may metastasize to liver. (Frith 1983; Stoner 1991, 1998; Stoner et al. 1993; Nesnow et al. 1998) Reported incidences of leukemia or lymphoma in A strains vary from 0 to 43% (Hoag 1963; Festing and Blackmore 1971). Rare spontaneous myoepitheliomas arising from myoepithelial cells of various exocrine glands are reported in A/J and A/HeJ mice (Sundberg et al. 1991). In a study of the frequency of neoplasms (percentage of cases with specific diagnoses of all mice submitted for necropsy) in breeding populations of inbred strains over a 13-year period, the most common neoplasms in 281 female A/J mice were lung tumors, lymphoma, myoepithelioma, and lipoma, with frequencies of 0.5-1%. The most common neoplasms in 236 male A/J mice were myoepithelioma and rhabdomyosarcoma, with frequencies of 0.5-1% (Mikaelian et al. 2004). It should be noted that most of the mice evaluated in this type of study would be younger than those evaluated in typical chronic toxicology studies. Susceptibility to Selected Experimental Conditions and Infectious Agents
Notable responses of A strain mice under experimental conditions include their high susceptibility to induction of cleft palate by cortisone and other teratogens (Gasser et al. 1981; Diehl and Erickson 1997), and their high susceptibility to chemical induction of lung tumors (Festing et al. 1998; Malkinson 1999). A/J mice develop diet-induced obesity but are resistant to diet-induced diabetes mellitus in contrast to diabetes prone C57BL/6J mice (Surwit et al. 1988, 1991; Mills et al. 1993). They have low-intermediate serum cholesterol levels on a chow diet (<100 mg/dl), and the greatest increase (to >500 mg/dl) in response to a high-fat diet compared to other inbred strains (Svenson et al. 2003). They are relatively resistant to diet-induced
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atherosclerosis or fatty streaks (Paigen et al. 1985; StewartPhillips et al. 1988, 1989), but are very susceptible to dietinduced cholelithiasis, with various Lith genes implicated in susceptibility to gallstones (Khanuja et al. 1995; Lammert et al. 1991; Paigen et al. 2000). Notable responses of A/J mice to infectious agents include high susceptibility to lethal infection and disease (mousepox) after infection with Ectromelia virus (Bhatt and Jacoby 1987; Jacoby and Bhatt 1987; Brownstein et al. 1989); high susceptibility (especially of males) to hepatitis due to Helicobacter hepaticus (which increases their susceptibility to carcinogeninduced liver tumors) (Ward, Anver, et al. 1994; Ward, Fox, et al. 1994; Diwan et al. 1997; Whary et al. 2001; Whary and Fox 2004); high susceptibility (especially of females) to typhlocolitis due to Helicobacter hepaticus (Livingston et al. 2004); high susceptibility to death due to Listeria monocytogenes infection (Czuprynski, Faith, et al. 2003) and to Mycobacterium tuberculosis infection (Chackerian and Behar 2003).
C.
AKR
The AKR strain was developed by Jacob Furth in the 1920s and 1930s for its high incidence of thymic lymphoma (“leukemia”) (Lynch 1954; Furth 1978; Atchley and Fitch 1993). AKR mice are albino, with color genotype a/a Tyrc/Tyrc (a/a, c/c). They have a short lifespan in conventional or specific pathogen-free conditions, with mean lifespans of 276 or 312 days in females and 326 or 350 days in males due to early onset of thymic lymphoma (Storer 1966; Festing and Blackmore 1971; Karpova et al. 2002). AKR/J mice have intermediate breeding performance despite their short breeding life, with 15% of dams having at least four litters, about six pups per litter, 93% weaned:born ratio (Fox et al. 1997). They are relatively large mice with mean female weight of 31g and mean male weight of 34g at 8 weeks old (Fox et al. 1997). AKR/J mice have intermediate systolic blood pressure and left cardiac ventricle:body weight ratio compared to 10 inbred strains (Deschepper et al. 2004). They have relatively high brain weight (third of 25 strains), but intermediate brain:body weight ratio (fifteenth of 25 strains) (Roderick and Storer 1961). They tend to resist being held and vocalize (squeak) when held (Wahlsten et al. 2003). Nonneoplastic Conditions
AKR/J mice have an anomalous (preduodenal) portal vein, attributed to a single autosomal recessive mutation, but not associated with clinical signs of disease or with other congenital malformations. The portal vein is ventral to the duodenum in most (98%) AKR/J mice, and dorsal to the duodenum in 52 other inbred mouse strains in one mouse colony. (Nakajima et al. 2001)
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AKR/J and C57BL/6J mice have a relatively larger thymus (approximately twofold) with later onset involution compared to most other strains (A/J, DBA/2J, BALB/cJ, CBA/J, C3H/HeJ, 129/J, C57BL/10J). These features may be influenced by more than one genetic locus. (Peleg and Nesbitt 1984; Hsu et al. 2003) Male AKR mice (AKR/NCrlBR) are susceptible to development of urinary obstruction and sequelae known as mouse urologic syndrome or mouse urinary syndrome (MUS), especially when housed in suspended wire caging. MUS has one or more of the following features: bladder distension; peripreputial urine staining, alopecia, and edema; paraphimosis; urethral blockage; ulcerative balanoposthitis; hydronephrosis; pyelonephritis; rectal prolapse; and perineal ulcerative dermatitis. (Everitt et al. 1988) Neoplasia
AKR mice have a high incidence of thymic T cell lymphomas before 1 year of age, and most are dead of the condition by 18 months, with females having higher incidence and/or earlier onset compared to males. (Lynch 1954; Festing and Blackmore 1971; Rowe and Pineus 1972; Furth 1978; Karpova et al. 2002) These neoplasms currently are classified as precursor T cell lymphomas and previously have been called lymphoblastic lymphoma, thymoma, and lymphoid leukemia. These lymphomas result from interactions of multiple endogenous retroviruses. AKR mice are viremic from birth and express several endogenous ecotropic retroviruses (Emv’s formerly Akv’s) in all tissues (Hiai 1996; Morse et al. 2001, 2002). These lymphomas also occur early with high incidence in C58 mice (Strand et al. 1974; Nexo and Krog 1977), Prkdcscid/Prkdcscid (scid) mice (Custer et al. 1985; Bosma et al. 1988), NOD-scid mice (Prochazka et al. 1992), and in HRS/J mice that are heterozygous or homozygous for the hairless mutation (hr) (Mucenski et al. 1988). Also, this is the most common lymphoma type to be induced by viruses, chemical carcinogens, or irradiation (Karpova et al. 2002; Morse et al. 2002). Mice with thymic lymphoma may be dyspneic due to massive enlargement of the thymus that impinges on the lungs and may fill much of the thoracic cavity. Necropsy findings commonly include enlarged spleen and lymph nodes due to tumor involvement. Involvement of liver, kidneys, and bone marrow may occur in advanced stages, or may represent an additional neoplastic process. Histologically, neoplastic cells are monomorphic and medium sized with scant cytoplasm. The “starry sky” pattern is attributed to scattered larger, paler, tingible body macrophages among homogeneous populations of neoplastic lymphoid cells. (Karpova et al. 2002; Morse et al. 2002) Susceptibility to Selected Experimental Conditions and Infectious Agents
Notable responses of AKR strain mice under experimental conditions include strong preference for high-fat diet and
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susceptibility to dietary obesity (Smith et al. 1999, 2000), but with only low to intermediate susceptibility to diet-induced atherosclerosis or fatty streaks (Paigen et al. 1985; Nishina et al. 1993). They are resistant to diet-induced cholelithiasis (Khanuja et al. 1995). Notable responses of AKR strain mice to infectious agents include susceptibility to age-dependent poliomyelitis, with paralysis following infection with the lactate dehydrogenaseelevating virus, which is attributed to their endogenous ecotropic murine leukemia viruses (Anderson, Even, et al. 1995; Anderson, Palmen, et al. 1995; Anderson and Plagemann 1995). AKR mice demonstrate intermediate susceptibility to mouse pox due to Ectromelia virus (Bhatt and Jacoby 1987; Jacoby and Bhatt 1987; Brownstein et al. 1989) and to M. pulmonis (Cartner et al. 1996). Related Strains
Senescence-accelerated mice (SAM) originated from an inadvertent cross between AKR/J and an unknown strain, and multiple strains were developed at Kyoto University in the 1970s. They are considered to be prone (SAMP) or resistant (SAMR) to accelerated senescence. SAMP lines share signs of early senescence, including ruffled coat, lordokyphosis, skin lesions, reduced activity, shortened lifespan, and systemic senile amyloidosis (Kitado et al. 1994; Takeda et al. 1989, 1997, 1999). Abscesses, chronic renal disease (contracted kidney), and lymphoma were identified as the most common causes of death in 9 SAMP and 3 SAMR strains under conventional conditions. Arteritis (angionecrosis, angiitis) also was fairly common, involving alimentary tract, tongue, pancreas, kidney, ovaries, uterine adnexa, and testes. Thymic lymphomas were most common in SAMP7 (56% incidence), SAMP9 (43% incidence), and SAMP8 (24% incidence) mice. Nonthymic lymphomas and histiocytic sarcomas were most common in SAMR5 (15% and 20% incidences, respectively) and SAMR4 (14% and 15% incidences, respectively) mice. Other conditions considered to be significant senescent phenotypes in different SAMP strains included senile amyloidosis in SAMP1; senile and secondary amyloidosis in SAMP2; degenerative temporomandibular joint disease in SAMP3; senile osteoporosis in SAMP 6; cataracts in SAMP9; brain atrophy (especially frontal cortex) and learning deficits in SAMP10; and renal diseases and senile amyloidosis in SAMP11 (Takeda et al. 1997; Takeda 1999). Senile amyloid AApoAII (also known as senescence-accelerated or ASsam amyloid) was initially identified in senescence-accelerated SAMP mouse lines (Matsumura et al. 1982), but amyloidosis may not be seen under specific pathogen-free conditions (Shino et al. 1987).
D.
BALB/c, BALB/cBy
BALB/c and BALB/cBy mice are albino with color genotype a+/a +, Tyrp1b/Tyrp1b Tyrc/Tyrc (a+/a+, b/b, c/c). Both are prioritized
640 strains in the mouse phenome database. They have a common origin, developed by Halsey Bagg in 1913, designated BALB for Bagg ALBino. In 1935, Snell added ‘/c’ to indicate the albino allele. BALB/c mice transferred to various holders and ultimately to Bailey (By) in 1961 at F99 gave rise to the BALB/cBy substrain (Booth and Sundberg 1996; Festing 1999). Their tendency to develop plasmacytomas after injection of mineral oil or pristane makes them useful for generation of monoclonal antibodies (Potter and Maccardle 1964; Anderson and Potter 1969). This tendency varies with the substrain (Anderson et al. 1985) and may diminish after rederivation and maintenance under specific pathogen-free conditions (Byrd et al. 1991). BALB/cJ lifespan is considered to be intermediate in conventional or specific pathogen-free conditions: 561 or 648 days in females, 509 or 816 days in males (Storer 1966; Festing and Blackmore 1971; Goodrick 1975). BALB/c mice have intermediate breeding performance, 28% of dams having at least four litters, about five pups per litter, 92% weaned:born ratio (Fox et al. 1997). They are of intermediate size with mean female weight of 22g and mean male weight of 28g at 8 weeks old. They tend to have an unkempt dirty or oily appearance and exhibit high intrastrain aggression (Fox et al. 1997). Compared to BALB/cJ, BALB/cByJ mice may be slightly bigger and less aggressive and have better breeding performance, with 58% of dams having at least four litters, about seven pups per litter, 91% weaned:born ratio (Fox et al. 1997). They have been characterized as stress reactive, with large adrenal glands compared to ten other inbred strains. (Deschepper et al. 2004) They are relatively placid and easy to handle. (Wahlsten et al. 2003) Nonneoplastic Conditions
In BALB/c mice, dystrophic cardiac calcinosis manifests as epicardial mineralization that increases slightly with age, and primarily or exclusively involves the right ventricular free wall, with higher incidence in males (11%) than in females (4%) (Festing and Blackmore 1971; Frith et al. 1975). Age-related cardiopathy or cardiomyopathy that is more severe in females than in males is reported in some studies. It is characterized by myocardial degeneration or necrosis, fibrosis (scarring), inflammation or mononuclear infiltration, with or without mineralization or arteritis. (Bellini et al. 1976; Price and Papadimtirou 1996) Left auricular thrombosis was reported to be common in earlier longevity studies, with incidence up to 66% in older breeding females. (Meier and Hoag 1961) Polyarteritis is reported in BALB/c mice with an incidence of up to 4% when mice were examined after natural death rather than at timed sacrifice. (Cosgrove et al. 1978; Plendl et al. 1996) Spontaneous corneal opacities may occur in BALB/c mice with an incidence of about 10%. The condition also is referred to as corneal dystrophy or corneal mineralization.
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Higher incidences occur in other strains that develop dystrophic cardiac calcinosis (C3H and DBA/2), and C57BL/6 mice also may develop this condition but with a lower incidence. Shorter cage-change intervals may reduce the incidence of corneal opacities. Ammonia has been proposed as a factor in the pathogenesis of spontaneous corneal opacities in laboratory mice. (Van Winkle and Balk 1986) Histologically, there is mineralization of the basement membrane with varying degrees of edema, inflammation, vascularization of the stroma, and erosion or ulceration. (Frame and Slone 1996) MRL mice may have a high incidence of corneal mineralization, which in this strain has been compared to human band keratopathy in Sjogren’s syndrome (Hoffman et al. 1983; Verhagen et al. 1995). BALB/c mice have a large brain and brain to body weight ratio (second of 25 strains) and large spinal cord (Roderick et al. 1973). Corpus callosum may be absent in 30%-40% of BALB/c mice, and several genes seem to be involved in this trait. (Wahlsten 1982; Livy and Wahlsten 1991; Livy et al. 1997) Hydrocephalus is unusual in BALB/cJ and BALB/cByJ mice, with <0.001% of these mice culled at weaning for the condition (JaxNotes 2003). Both BALB/cJ and BALB/cByJ mice have age-related hearing loss. Only BALB/cByJ mice are homozygous for Cdh23ahl, the age-related hearing loss 1 mutation, which on this background results in progressive hearing loss with onset after 10 months of age. (Willott et al. 1998; Johnson et al. 2000) BALB/cJ and BALB/cByJ develop ulcerative blepharitis and periorbital abscesses from which Pasteurella pneumotropica or other bacteria may be isolated. The condition presents as swollen eyelids and crusting at the medial canthus, which may progress to periorbital swelling. Histologically, there is ulceration of the lid margins near the mucocutaneous junction over the meibomian glands, with associated crust, inflammation, edema, and development of periorbital abscesses (Sundberg et al. 1990; Smith and Sundberg 1996). Bagg albino mice have been reported to have high variability in vertebral formulae compared to other strains (13 or 14 thoracic vertebrae, and 5 to 7 lumbar vertebrae), with 14 thoracic and 5 lumbar vertebrae, being most common (Green 1941, 1978). BALB/cByJ have intermediate femoral bone density (Beamer et al. 1996), and intermediate tibial and femoral bone mass compared to other inbred strains. (Kaye and Ivy 1995). Malocclusion is less common in BALB/cJ and BALB/cByJ mice than in C57BLKS/J, C57BL6/J, DBA/2J, C3H/HeJ, with <0.002% of BALB/cJ or BALB/cByJ culled at weaning for malocclusion (JaxNotes 2003). Imperforate vagina with associated hydrometra and mucometra was diagnosed in more BALB/cJ mice (12 of 35 total) than in any other mouse strain at the Jackson Laboratory over a two-year period. Mice with imperforate vagina are infertile and develop perineal swelling and progressive abdominal distention (due to the enlarging uterus) that may be mistaken for pregnancy (Sundberg and Brown 1994). A 7% incidence of hydrometra
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was reported in a study of virgin female BALB/c mice maintained up to 1001 days (Sheldon and Greenman 1980). Dorsoventral longitudinal vaginal septa also have been associated with infertility and have been found in up to 38% of recently weaned BALB/cJ female mice in the production colony at the Jackson Laboratory. The frequency of septa in two other BALB/c substrains and eight different inbred strains ranged from 0 to 8%. Fewer septate females than nonseptate females were fertile. Breeding studies indicated that more than one recessive gene is involved, and genetic background influences expression of these genes. Histologically, the septum consists of a fibrous partition covered by normal epithelium. (Cunliffe-Beamer and Feldman 1976; Shire 1984) Adrenal subcapsular spindle cell hyperplasia, which may be associated with mast cell infiltration, is common in aged female and male BALB/cJ mice, with almost 100% incidence at 13 to 15 months of age (Kim et al. 1997). Accessory adrenal cortical nodules, consisting of encapsulated cortical tissue, within or outside of the adrenal capsule or in adjacent fat, have been reported in more than 50% of BALB/c or C57BL/6 mice, and are less common in A and C3H mice in some studies. (Sass 1983; Yarrington 1996) Amyloidosis in BALB/c mice is relatively uncommon (Andervont and Dunn 1970), although some studies report a relatively high incidence in group-housed males (Ebbesen 1971). Secondary reactive amyloidosis also is relatively difficult to induce in this strain, but has been induced with multiple oral doses of pristane (Suzuki et al. 1980; Ho and Fu 1987). While amyloidosis has been reported in mice bearing myelomas, it has been identified as secondary AA amyloid rather than the primary light chain (AL) amyloid fibrils of primary amyloidosis associated with human plasma cell dyscrasias (Baumal et al. 1975). In specific pathogen-free BALB/c mice from a study of virgin and breeder mice maintained up to 689 days, (BALB/ cStCrlfC3Hf/Nctr mice (Charles River Laboratories BALB/cSt mice fostered onto gnotobiotic MMTV-negative C3H/He from Sprague Dawley, barrier-maintained in isolators at the National Center for Toxicological Research in Jefferson, AR), the most common nonneoplastic changes were: hepatic fatty metamorphosis (especially in male mice); uterine hemosiderosis and mineralization in multiparous females; uterine polyps and ovarian cysts primarily in virgin mice; testicular atrophy; epicardial mineralization more commonly in males; and adrenal subcapsular spindle cell hyperplasia. Nonneoplastic findings from mice of the oldest group (>500 days) are summarized in Tables 25-3 and 25-4. (Frith et al. 1983). In a report of 2376 nontreated control female, specific pathogen-free BALB/c (also BALB/cStCrlfC3H/Nctr) mice examined up to 1001 days (33 months) of age, uterine stromal polyps occurred in 24% of mice (32% in the oldest age group, 24–33 months), and no other nonneoplastic lesions had an overall incidence (all age groups combined) >10%. In the oldest age group (24–33 months), common findings were foci
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of vaginal dysplasia (31%), ovarian vascular lesions or thrombi (23%), brain (thalamic) mineralization (28%), corneal mineralization (25%), and adrenal subcapsular cell hyperplasia (24%). (Sheldon and Greenman 1980) Neoplasia
In specific pathogen-free BALB/c mice from a study of virgin and breeder BALB/cStCrlfC3Hf/NCtr mice, the most common neoplasms overall (all age groups combined) were lung tumors, lymphoma, Harderian gland tumors, and adrenal adenomas (Frith et al. 1983). Findings from mice of the oldest group (>500 days) are summarized in Table 25-4. Female predisposition for lymphoma has been recognized (Ebbesen 1971), and in this study virgin females had a much higher incidence (34%) than did breeder females, or breeder or virgin males (11–15%) (Frith et al. 1983). In the report of 2376 control female BALB/cSTCrlfC3H/ Nctr mice, hematopoietic tumors (“reticulum cell sarcomas” now classified as lymphoma or histiocytic sarcoma) and lymphomas were most common, with about 30% incidence in all mice maintained up to 33 months (up to 75% incidence in the oldest age group), and were the primary cause of morbidity and mortality. Lung tumors (22%) and Harderian gland tumors (11%) also were common, with 52% and 24% incidence in the oldest age group. Adrenocortical adenomas (33%), liver tumors (17%), and mammary adenocarcinomas (12%) were common in the oldest age group, but each had less than 6% incidence overall (Sheldon and Greenman 1980). The most common hematopoietic neoplasm in these BALB/c females has been called type B reticulum cell sarcoma (or tumor) and follicular center cell lymphoma, and probably is most compatible with follicular B cell lymphoma under current classification (Morse 2007). Some lymphomas in mice over 20 months of age also were of B cell origin but lymphoblastic or immunoblastic. In addition, there were some thymic lymphomas with a bimodal incidence, peaking in young (<6 months) and old (>21 months) female mice (Pattengale and Frith 1983). In a study of the frequency of neoplasms in primarily breeding populations of inbred strains over a 13-year period, the most common neoplasm in BALB/cJ mice of both sexes was myoepithelioma with a frequency of 1-10%. Of 899 female BALB/cJ mice, lymphoma, papilloma, rhabdomyosarcoma, and mammary tumor, and of 349 male BALB/cJ mice, lymphoma, papilloma, rhabdomyosarcoma, and testicular interstitial cell tumor occurred with frequencies of 0.5-1%. Of 516 female BALB/cByJ mice, myoepitheliomas, lymphoma, papilloma, and hemangioma occurred with frequencies of 0.5-1%. Of 256 male BALB/cByJ mice, myoepithelioma, lymphoma, papilloma, and rhabdomyosarcoma occurred with frequencies of 0.5-1%. (Mikaelian et al. 2004) These findings corroborate an earlier study of BALB/cJ and BALB/cByJ breeding colonies, with regard to frequency of tumor types and relatively
642 low tumor incidences in breeding populations (i.e., younger, non-virgin mice compared to chronic toxicity or carcinogenicity studies). (Booth and Sundberg 1996) Myoepitheliomas arise from myoepithelial cells of various exocrine glands, especially the salivary glands. They are relatively rare in mice but occur more commonly in BALB/cJ and BALB/cByJ than in other strains. They are most common in the submandibular (sometimes referred to as submaxillary) or parotid salivary glands and are rare in the sublingual gland. The tumors can become large and appear to be cystic. Histologically, they are biphasic tumors, composed of large pleomorphic cells including elongated or spindle-shaped mesenchymal-type cells, mixed with areas of polyhedral epithelial-type cells. Areas of degeneration and necrosis can result in pseudocysts filled with mucus and necrotic cell debris. The neoplastic cells may palisade around blood vessels. Larger tumors may metastasize to the lung (Frith et al. 1981; Frith and Ward 1988; Sundberg et al. 1991; Booth and Sundberg 1996; Botts et al. 1999; Mikaelian et al. 2004). Tumor growth may be associated with an intense myeloproliferative response (Delaney 1977). Mammary glands have myoepithelial cells, and mammary myoepitheliomas can be induced with 7,12-dimethylbenz[a]anthracene (DMBA), but seem to be very rare spontaneous lesions (Rehm 1990). Mammary tumors in breeding populations usually are papillary adenocarcinomas that not infrequently metastasize to lung. These tumors are characterized as multilobulated, papillary, cystic, and locally invasive (Booth and Sundberg 1996). The term adenoacanthoma historically has been used to refer to carcinomas with squamous, cornifying components, sometimes called adenosquamous carcinomas, with adenosquamous being the currently preferred descriptor of neoplasms with glandular plus squamous differentiation. These typically occur in old retired breeders, and are reported to be more common in BALB/c than in other common strains (Medina 1982; Rehm and Leibert 1996; Cardiff and Wellings 1999). Hemangiomas and hemangiosarcomas are relatively rare in mice but occur in BALB/cJ and BALB/cByJ. These endothelial origin neoplasms are found in skin, seminal vesicles, liver, muscle tissue, cerebellum, heart, and other tissues. Cutaneous hemangiomas are dark red to purple raised lesions that bleed profusely when cut. Cavernous hemangiomas are dilated cavernous spaces that are lined by endothelial cells and filled with red blood cells. Capillary hemangiomas are circumscribed accumulations of small cleft-like spaces that are lined by typically plump endothelial cells and may contain red blood cells or be vacant or flattened. Mitotic figures are rare in hemangiomas. Hemangiosarcomas are noncircumscribed and locally invasive. They typically consist of numerous small irregular, vessel-like structures that may contain few or many red blood cells, and are lined by plump cells that may have bizarre mitotic figures. In some cases vascular neoplasms may be difficult to distinguish from nonneoplastic distended vascular spaces
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referred to as telangiectasis or peliosis (usually in the liver) (Frith and Highman 1983; Booth and Sunbderg 1995). Rhabdomyosarcomas are rare in mice but seem to be more common in BALB/c and BALB/cBy than in other strains. These skeletal muscle neoplasms arise as nodules in skeletal muscle. Neoplastic cells are pleomorphic, with elongate, straplike cells with multiple nuclei in tandem array, and smaller plump to spindled, haphazardly oriented cells. Cross striations in neoplastic cells distinguish this from other sarcomas, but can be very difficult to discern (Booth and Sunbderg 1996; Sundberg et al. 1991, 1996). Adrenal cortical tumors are not common in mice but are more common in BALB/c than in outbred Swiss mice or in B6C3F1 mice. Adenomas occurred in almost 6% of female BALB/c mice examined between 24 and 33 months of age. Adrenal cortical carcinomas are less common, and pheochromocytomas are rare but may occur in BALB/c mice also (Sheldon and Greenman 1980; Russfield 1982; Tischler and Sheldon 1996). Neonatal gonadectomy-induced adrenal cortical tumors in various strains of mice (Russfield 1982). In descending order, the most common ovarian tumors in BALB/c mice are luteoma, tubulostromal adenoma (tubular mesothelioma), granulosa cell tumor, thecoma, and cystadenoma (see Table 25-6 for descriptions). Ovarian tumors were rare before 1 year of age but increased with age after 1 year (Frith et al. 1981). Leydig cell tumors (interstitial cell tumors) of the testicle are rare in mice, with only 6 identified in 6500 male mice of various strains necropsied over a period of 8.5 years in one study. All 6 tumors developed spontaneously in BALB/cJ or BALB/cByJ mice. Tumors were unilateral, with no right or left predilection (Mahler and Sundberg 1997). In a chronic study, the incidence in virgin and breeder males was about 2% (Frith et al. 1983). Histologically, spontaneous Leydig cell tumors are well differentiated and of the solid, diffuse type, composed of round homogeneous cells with eosinophilic granular cytoplasm. “Tumors” may be difficult to distinguish from interstitial hyperplasia. Hyperplastic foci have been distinguished by size (<3 seminiferous tubules diameter) and non compression, compared to adenomas that are larger (>3 seminiferous tubules diameter), compress adjacent tissue, and exhibit some cellular pleomorphism. Small, well-circumscribed tumors are adenomas, and large tumors that are invasive or metastasize are referred to as carcinomas (Frith and Ward 1988; Prahalada et al. 1994; Gordon et al. 1996). Susceptibility to Selected Experimental Conditions and Infectious Agents
Notable responses of BALB/c mice under experimental conditions include their high susceptibility to a two-stage skin carcinogenesis protocol involving DMBA initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion
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(Sundberg et al. 1997); high susceptibility to carcinogeninduced and MMTV-induced mammary tumors (Pazos et al. 1992; Swanson et al. 1996) (reviewed in Medina 1982); and high susceptibility to acetylaminofluorene-treated AAFinduced urinary tract tumors, including transitional cell carcinomas, squamous cell carcinomas, and undifferentiated carcinomas in the urinary bladders, and renal cell adenomas and renal cell carcinomas (Frith et al. 1980; Shinohara and Frith 1980). Spontaneous renal tumors, usually cortical adenomas, are rare; adenocarcinomas are even rarer and may metastasize to the lung (Sheldon and Greenman 1980). Compared to other strains, BALB/c mice are relatively susceptible to carcinogen-induced osteosarcomas (Frith and Wiley 1982); brain tumors (Morgan et al. 1984) have intermediate susceptibility to lung tumor induction by carcinogens (Manenti et al. 1997, 2002; Malkinson 1999). BALB/cJ mice are resistant to diet-induced atherosclerosis (Paigen et al. 1985; Phelan et al. 2002). BALB/c mice have been widely used for studies of infectious diseases. Notable responses of BALB/c mice to infectious agents include susceptibility to acute lethal mousepox (Ectromelia virus) (Bhatt and Jacoby 1987; Jacoby and Bhatt 1987; Brownstein et al. 1989); some mouse hepatitis viruses (Barthold et al. 1986; Barthold 1987; Barthold and Smith 1987); encephalomyocarditis virus (EMCV), diabetes, and myocarditis (Tomioka et al. 1985, 1986; Hirasawa, Han, et al. 1992; Hirasawa and Ogiso 1995); mouse cytomegalovirus myocarditis (MCMV) and congestive heart failure (Lawson et al. 1990; Lenzo et al. 2002); murine gammaherpes virus, arteritis, and lymphoproliferative disease (Mistrikova et al. 1994, 2000; Raslova et al. 2000); H. hepaticus hepatitis (Ward et al. 1994); Borrelia burgdorferi myocarditis (Barthold et al. 1990; Matyniak and Reiner 1995); L. monocytogenes gastritis (Park et al. 2004); cariogenic streptococci (Kurihara et al. 1991; Suzuki and Kurihara 1998); and mite-associated ulcerative dermatitis (Jungmann, Freitas, et al. 1996; Jungmann, Guenet, et al. 1996). BALB/c mice have been noted to be relatively resistant to polyomavirus-induced tumors (Freund et al. 1992); experimental lethal mouse adenovirus 1 (MAV1) hemorrhagic encephalomyelitis (Guida et al. 1995; Kring et al. 1995; Charles et al. 1998; Kajon et al. 1998; Spindler et al. 2001); Helicobacter felis gastritis (Mohammadi et al. 1996; Sakagami et al. 1996); and Borrelia burgdorferi arthritis (Barthold et al. 1990; Matyniak and Reiner 1995). Related Strains or Substrains
BALB/cWt mice have a relatively high incidence of true hermaphroditism (3%), related to a spontaneous, chromosomal mosaicism. Their ovotestes contained more testicular than ovarian tissue, and the ovarian tissue was more frequently located at the gonad poles, particularly the cranial pole. There was no difference between left and right sides with regard to gonad type,
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but more single-sex gonads were found on the left than on the right side (Eicher et al. 1980; Whitten et al. 1979, 1991).
E.
C3H
The C3H and CBA strains were developed by Strong in 1920 from a cross of an early A albino female with a DBA male, then selection for a high mammary tumor incidence, resulting in C3H, and selection for low mammary tumor incidence, resulting in CBA (see below Related Strains and Substrains) (Heston 1978; Strong 1978). C3H/He, initiated by Heston in 1941 for cancer research, is the most common substrain; it is prioritized in the phenome database and is emphasized here. Most C3H and C3H/He substrains currently available have been cesarean derived to eliminate exogenous mouse mammary tumor virus (MMTV), but still carry multiple endogenous Mtv’s, and develop later onset mammary tumors (Heston 1958, 1978; Groner and Hynes 1980; Cardiff and Wellings 1999; Morse 2007). The Jackson Laboratory strains of C3H/HeJ and C3H/HeOuJ mice were rederived in 1999 during planned efforts to increase the overall health status of the mice, and MMTV was not reintroduced (JAX® 2004). C3H mice are agouti colored with color genotype a+/a+. C3H mice have an intermediate lifespan in conventional or specific pathogen-free conditions, with mean lifespans of 676 days in females and 590 days in males (Storer 1966; Festing and Blackmore 1971). C3H/HeJ mice have poor intermediate breeding performance, with only 26% of dams having at least four litters, about five pups per litter, 85% weaned:born ratio. (Fox et al. 1997). They are intermediate in size, with the lowest systolic blood pressure of 11 (non-wild) inbred strains (Deschepper et al. 2004). They have relatively high lung volume and alveolar size compared to other inbred strains, with approximately 50% more lung volume than C57BL6 (Tankersley et al. 1999; Soutiere et al. 2004). Variability in vertebral formulae among C3H substrains has been reported, with 13 thoracic and 5 lumbar vertebrae strongly predominating in C3H substrains and with 13 thoracic and 6 lumbar vertebrae strongly predominating in C3H/He substrains (Green 1953, 1978). Nonneoplastic Conditions
Between 1960 and 1968 the C3H/HeJ substrain developed a mutation in the Toll-like receptor 4 (Tlr4) gene, then called Lps (for lipopolysaccharide), rendering them resistant to endotoxin but paradoxically susceptible to infections by some gram-negative bacteria, including Salmonella typhimurium (O’Brien et al. 1980, 1985; Festing 1999). The Tlr4 mutation also alters the response to exogenous MMTV and reduces the incidence of mammary tumors in C3H/HeJ mice (see below, Neoplasia) (Outzen et al. 1985; Jude et al. 2003).
644 C3H and C3H/He mice develop dystrophic cardiac mineralization or dystrophic cardiac calcinosis that is more common and more severe in females than in males. While frequently not associated with clinical disease, mortality with pleural effusion and severe myocardial mineralization has been reported in pregnant or lactating mice. Myodegeneration, necrosis, and mineralization can occur throughout the myocardium, of both ventricular walls and the interventricular septum (Dunn 1954; Vargas et al. 1996; van den Broek et al. 1997; Brunnert 1997; Brunnert et al. 1999). Several dystrophic cardiac calcinosis (Dyscalc) loci on different chromosomes are implicated in this complex trait (Colinayo et al. 2002). Various dietary deficiencies or imbalances have been associated with earlier onset and increased severity of cardiac calcinosis, and with mineralization in other tissues, including renal arteries and tubules; pulmonary arteries and septa; and rectus capitis and posterior femoral muscles. (Highman and Daft 1951; Staff 1975; Everitt et al. 1988) Increased dietary soy oil may enhance resistance to the condition (McElwee et al. 2003). Spontaneous corneal opacities, also called corneal dystrophy, or corneal mineralization, may occur in C3H mice with an incidence of about 16% (Van Winkle and Balk 1986). Larger spleens, lower platelet count, and higher megakaryocyte ploidy in C3H, compared to C57BL/6 mice, have been attributed to shorter platelet lifespan and increased platelet production (McDonald et al. 1992; McDonald and Jackson 1994). C3H mice tend to have higher megakaryocyte ploidy, and males have higher ploidy than females, with more 32N and 64N megakaryocytes compared to other strains that have predominantly 16N megakaryocytes (McDonald et al. 1992; McDonald and Jackson 1994). C3H, C3H/He, and related strains are homozygous for Pde6brd1, the rd1 mutation in the phosphodiesterase 6B gene. This spontaneous recessive mutation resulted from a retroviral insertion (Bowes et al. 1993), as well as a nonsense mutation that truncates the normal gene product. (Pitter et al. 1993) The condition was orginally described as “rodless” retina by Keeler in 1924. Mice with this condition have outer segment degeneration by postnatal day 14 (P14) when their eyes open. Mouse photoreceptors are 95% rods, and there is preferential loss of rods, with only 2% of rods and 75% of cones remaining at P17. By P20, almost no photoreceptor nuclei are discerned. In older mice there is loss of retinal vasculature and of pigment in the retinal pigmented epithelium (RPE), foci of thinning of the inner nuclear layer, and loss of ganglion cells and nerve fibers (Dunn 1954; Pittler et al. 1993; Chang et al. 2002; Smith 2002). Affected mice should be expected to perform poorly in neurobehavioral tests requiring vision. Although the absence or loss of photoreceptors may affect circadian phenotype (Yoshimura and Ebihara 1998; Lupi et al. 1999), mice that lack rod and cone photoreceptors still use their eyes to detect light to regulate circadian rhythms, suppress pineal melatonin, modify locomotor activity, and modulate pupil size. A subset of retinal ganglion cells is photosensitive, and melanopsin may be
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the photopigment that mediates these responses. (Foster et al. 1991; Foster and Hankins 2002; Panda et al. 2003) Hearing sensitivity is normal, and cochlear function is excellent in C3H/HeJ and C3H/HeSnJ up to 14 months of age. By 18 months of age, thresholds are slightly elevated at most frequencies. At 30 months, C3H/HeSnJ mice have poor hearing as a result of extensive sensorineural degeneration. LPS-sensitive C3H/HeJ mice may have up to 33% incidence of otitis media in contrast to no otitis media in normally LPS-responsive C3H/HeSnJ. Conductive loss due to otitis media increases auditory brain stem response thresholds. (Trune et al. 1996; Mitchell et al. 1997) Hamartomas and choristomas are rare, noninherited tumor-like conditions that are more common in C3H/HeJ and C57BL/6J than in other inbred strains. They are soft, raised masses on the dorsal midline, primarily above the sutures of the skull. They may be noticed because of abnormally long hair, change in direction of the hairs, or change in hair color. Histologically, normal adipose tissue in the reticular dermis and subcutis sometimes extends through the cranial sutures, entering the brain, or expanding into the ventricles. Large masses may contain normal-appearing thyroid, intestine, respiratory epithelium-lined cysts, squamous epithelial cysts, bone and marrow, cartilage, glands, and angiomatous anomalies. Overlying epidermis is intact. Breeding studies did not yield affected offspring, indicating that this is a congenital, noninherited abnormality. This condition resembles “lipomatous” hamartomas, a congenital defect in human beings. (Adkison and Sundberg 1991) Alopecia areata develops spontaneously in C3H/HeJ mice from about 5 months of age in females and later in males. Alopecia areata in humans is a nonscarring, inflammatory hair loss condition with a suspected autoimmune pathogenesis. Frequency of disease can approach 20% by 18 months of age (McElwee et al. 1998, 1999, 2003). Several genetic loci have been linked to this condition in mice. (Sundberg et al. 1995, 2003) C3H/HeJ mice have high femoral bone density (Beamer et al. 1996). Recombinant inbred (RI) strains of mice derived from C57BL/6J (low femoral bone density) and C3H/HeJ progenitor strains indicate polygenic control of bone density and microstructural, biomechanical, and geometrical properties. Femoral biomechanical properties are associated with femoral width in the anteroposterior (AP) direction and cortical thickness, which are geometric properties with complex genetic regulation. Although the C57BL/6 and C3H mice have similar vertebral strength, their vertebral structures differ: C57BL/6 mice have good trabecular bone structure and modest cortical bone mineral content (BMC), compared to C3H, which have high cortical BMC and a deficiency in trabecular structure. Vertebral strength does not correlate consistently with femoral strength among the RI strains, suggesting that genetic regulation of bone strength is specific to anatomic site. (Turner et al. 2000)
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Adrenal subcapsular spindle cell hyperplasia can be common in both female and male C3H/HeJ mice (almost 100% incidence at 13–15 months of age) and was not associated with mast cell infiltration of the cortex in this strain. (Kim et al. 1997) Neoplasia
C3H and C3H/He mice that were not fostered onto a nonMMTV-bearing strain and thus carried the exogenous retrovirus, MMTV (Bittner agent), historically have had very high incidences of early-onset mammary adenocarcinomas: 95100% in breeding females, slightly lower in virgin females, and less than 1% in males. The latent period to develop mammary tumors in unfostered C3H substrains ranged from 276 to 566 days, depending on breeding status and environmental stress (Riley 1966; Riley 1975). Despite lacking exogenous MMTV, fostered or rederived C3H virgin and breeding females develop mammary tumors later in life. (Festing and Blackmore 1971) In C3HfC57BL mice (C3H mice fostered onto MMTV-free C57BL/6 females), the incidence of mammary tumors is only 40% at 18.8 months, compared to 99% at 7.2 months in unfostered C3H in the same study. (Heston and Vlahakis 1971) Other strains fostered onto MMTV-bearing mice develop high incidences of mammary tumors, athough not as high as in MMTVbearing C3H mice (Medina 1982). In about 1968, a drastic decrease in mammary tumor incidence was reported in exogenous MMTV-infected C3H/HeJ mice but not in the exogenous MMTV-infected C3H/HeN mice. The exogenous MMTV in C3H/HeJ mice was a recombinant between the wildtype MMTV(C3H) carried by C3H/HeN mice and endogenous nontumorigenic Mtv1 present in all C3H stocks. In the presence of mutant Tlr4 in C3H/HeJ mice, wild-type MMTV(C3H) virus is eliminated by the cytotoxic immune response, promoting selection of the recombinant MMTV variants. (Outzen et al. 1985; Hook et al. 2000; Jude et al. 2003). Most spontaneous mammary tumors in mice can be classified as type A, B, or C adenocarcinomas, according to Thelma Dunn’s original 1959 classification, and they do not resemble most human breast cancers. Type A (acinar) or microacinar adenocarcinomas are composed of small acini lined by a single layer of cuboidal cells. These also have been referred to as adenoma and tubular carcinoma. Type B ductal tumors are most common and have more variable histologic features, with well- and poorly differentiated regions of neoplastic cells in cords or sheets or papilloma-like configurations. They can arise from carcinogen-induced ductal hyperplasias. Type C (cystic) tumors are less common than A or B tumors and feature cystic epithelial structures in more abundant stroma. Mammary tumors with some squamous differentiation (adenoacanthomas or adenosquamous carcinomas) may occur in old retired breeders of low mammary tumor incidence strains (e.g., BALB/c) and can be induced with carcinogens. Spontaneous mammary adenocarcinomas most commonly metastasize to the lung
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without extravasation, and the incidence of metastasis may exceed 50%. Dunn’s original 1959 classification remains relevant and applicable to many of the spontaneous tumors of mice, but more recent classifications are more relevant to neoplasms that have been induced genetically to recapitulate human breast cancers. (Medina 1982; Vaage and Harlos 1987; Rehm and Leibelt 1996; Cardiff and Wellings 1999; Cardiff et al. 2000) Activation of the Wnt signaling pathway by MMTV proviruses plays an important role in many mouse mammary tumors, and mammary tumors of transgenic mice that overexpress some of the Wnt genes histologically resemble the Dunn’s type A tumor. (Lane and Leder 1997; Cardiff and Wellings 1999; Li et al. 2000; Landesman-Bollag et al. 2001) Hyperplastic alveolar nodules (HAN) are common preneoplastic findings in spontaneous and carcinogen-induced mammary tumors in mice. This hyperplastic mammary tissue is immortal and can be serially transplanted, with development into focal proliferations and neoplastic lesions. Grossly HAN are 1–5 mm nodules, frequently outlined by yellow pigment. Histologically, they are foci of lobuloalveolar hyperplasia, characterized by closely crowded acini that are lined by a single layer of epithelium and lack significant dysplasia. (Medina 1982; Rehm and Leibelt 1996; Cardiff and Wellings 1999) Plaques are epithelial proliferations that occur in mouse mammary glands during pregnancy or after hormone induction, but regress after withdrawal of the stimulus. These were formerly known as type P or pregnancy-dependent tumors. Histologically, they consist of radiating ducts surrounded by dense connective tissue. After repeated hormone stimulation, some may become hormone-independent tumors. Inflammatory nodules and squamous or keratinized nodules also occur in mouse mammary glands, but do not seem to progress to cancer. (Medina 1982; Cardiff et al. 1999) Fostered and unfostered substrains have a high incidence of liver tumors (Festing and Balckmore 1971) and are used as a model of genetic predisposition to hepatocellular tumors (Dragani et al. 1995). Like B6C3F1 mice, males have a higher incidence and multiplicity than females. (Andervont 1950; Frith et al. 1982; Poole and Drinkwater 1995, 1996) C3H/HeJ mice carry multiple susceptibility loci, designated Hcs. (hepatocarcinogen sensitivity). (Drinkwater and Ginsler 1986; Dragani et al. 1995) Hepatocellular adenomas in mice also have been referred to as type A nodules or type A tumors and hepatomas. Benign hepatocellular neoplasms typically are distinctly demarcated or circumscribed nodules, 1 to 10 mm diameter, that lack lobular organization, compress adjacent parenchyma, and may bulge from the liver surface. They do not invade adjacent parenchyma or vessels, and they do not metastasize. They consist of a uniform population of well-differentiated cells that resemble normal hepatocytes but may be larger or smaller than adjacent normal hepatocytes, and can have more basophilic, eosinophilic, or vacuolated cytoplasm. Hepatocellular carcinomas may arise
646 within adenomas. (Frith and Ward 1979; Frith 1982, 1994; Harada et al. 1999) Hepatocellular carcinomas in mice also have been referred to as type B nodules or type B tumors. They usually have distinct trabecular or adenoid patterns, with solid patterns less common, and the cells can be poorly differentiated to well differentiated. Borders with adjacent parenchyma can be sharp but with areas of invasion into parenchyma. Moderately to well-differentiated hepatocellular carcinomas are composed of larger hepatocytes that vary in size and shape in trabecular or solid patterns. Poorly differentiated tumors are composed of cells with less cytoplasm and more immature nuclei. Mitotic figures can be common, and some tumors have extremely large anaplastic cells. Metastases are typically to the lung; careful examination may reveal pulmonary metastases in up to 40% of male B6C3F1 or C3H mice with hepatocellular carcinoma that are allowed to live out their lifespan. Metastases usually occur only when tumors are large (>10 mm) (Frith and Ward 1979; Frith et al. 1982, 1994; Harada et al. 1999). Hepatoblastomas are rare spontaneous or induced liver tumors that possibly arise from poorly differentiated areas of hepatocellular carcinomas, as they usually are found within or adjacent to carcinomas. Cholangiomas or cholangiocarcinomas, of bile duct origin, also are rare spontaneous tumors but may be induced with carcinogens. (Frith et al. 1994; Harada et al. 1999) Lung tumors, lymphoma, Harderian gland tumors, and osteosarcoma have been significant findings in some studies of C3H mice, but are less common than mammary and liver tumors (Dunn 1954; Festing and Blackmore 1971) In descending order, the most common ovarian tumors in C3H mice are tubular mesothelioma (adenoma), thecoma, luteoma, granulosa cell tumor, and cystadenoma. Most of the ovarian tumors were rare before 1 year of age, but they increased with age after 1 year. (Frith et al. 1981) Pheochromocytomas, neoplasms of the adrenal medulla, are not common in mice, but may be more common in C3H than in other inbred strains and may be induced by polyomavirus in C3H mice. Although these neoplasms tend to be more pleomorphic in mice than they are in rats (in which they are much more common), they typically consist of relatively uniform polyhedral cells resembling normal medullary secretory cells supported in delicate fibrovascular stroma. Large tumors may have conspicuous blood-distended capillaries. Tumors that invade the adrenal capsule or spread beyond the adrenal gland are diagnosed as malignant. (Frith 1983; Tischler and Sheldon 1996) Susceptibility to Selected Experimental Conditions and Infectious Agents
Notable responses of C3H or C3H/He mice under experimental conditions include their high susceptibility to hepatocarcinogenesis (Drinkwater and Ginsler 1986; Lee
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and Drinkwater 1995); susceptibility to estrogen-induced endometrial hyperplasia, vaginal keratinization (cornification), mammary, endometrial, and cervical tumors, osteofibrosis, hyperostosis, and osteosarcoma (Highman et al. 1980; Greenman et al. 1983, 1984). C3H/HeJ mice are resistant to dietinduced atherosclerosis, and crosses with susceptible C57BL/6J mice have been used to map atherosclerosis susceptibility genes or loci. (Paigen et al. 1985, 1987) Notable responses of C3H mice to infectious agents include susceptibilities to acute, lethal mousepox (Ectromelia virus) (Bhatt and Jacoby 1987; Jacoby and Bhatt 1987; Brownstein et al. 1989); to some neurotropic mouse hepatitis virus (MHV) variants, developing encephalitis, vasculitis, and demyelination (Virelizier et al. 1975; Pereira et al. 1984; Woyciechowska et al. 1984; Barthold et al. 1986); to polyomavirus-induced tumors (Dawe et al. 1987; Freund et al. 1992; Lukacher et al. 1995) and necrotizing arteritis in some substrains (Dawe et al. 1987); to severe murine respiratory mycoplasmosis (M. pulmonis) (Cartner et al. 1995; Faulkner et al. 1995; Cartner et al. 1996); to H. hepaticus hepatitis (Ward et al. 1994); to H. felis gastritis (Mohammadi et al. 1996; Sakagami et al. 1996); and to Lyme borreliosis arthritis and myocarditis (Armstrong et al. 1992). C3H mice have been found to be relatively resistant to experimental lethal mouse adenovirus 1 (MAV1) hemorrhagic encephalomyelitis (Guida et al. 1995; Kring et al. 1995; Charles et al. 1998; Kajon et al. 1998; Spindler et al. 2001) and to mouse cytomegalovirus myocarditis (Price et al. 1991; Price and Papadimtirou 1996). C3H strains can demonstrate varying susceptibilities to mycobacterioses (Closs 1975; Closs and Hanger 1975; Yamamoto et al. 1991; Tanaka et al. 1994; Veazey et al. 1995; Kamath et al. 2003; Branger et al. 2004), and to other infections. High susceptibility of C3H/HeJ but not other C3H strains to Salmonella typhimurium is attributed to their Tlr4 mutation (O’Brien et al. 1980, 1985; Weiss et al. 2004). In some studies, the strain or substrain of C3H is not clear, and possession of functional Tlr4 may play a role. Related Strains or Substrains
C3H/He-Avy The Avy viable yellow mutation arose spontaneously in C3H/HeJ (see above, Coat Color). C3H/He- Avy mice vary from yellow to sooty in color. They are obese and highly susceptible to tumors, especially mammary and liver tumors. (Heston et al. 1968; Vlahakis 1975; Wolff et al. 1986; Wolff 1993; Yen et al. 1994) CBA The CBA and C3H strains have similar origins, but CBA was selected for low mammary tumor incidence and is known for its longevity (Heston 1978; Strong 1978). CBA mice are agouti colored (a+/a+) and homozygous for Pde6brd1. CBA mice have only modest sensorineural hearing loss late in
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life (Willott et al. 1994) and may be used as “normal hearing” controls in some surveys (Zheng et al. 1999). In an aging study of group-housed mice with up to 15 mice per cage (initially), CBA were the longest lived of five inbred strains (CBA, C57BL/6, NZB, RFM), with maximum female lifespan of 40 months and a maximum male lifespan of 36 months. Mice were necropsied after spontaneous death or after euthanasia when found moribund. The most common nonneoplastic findings were mild glomerulonephritis (F 66%, M 84%), dystrophic calcinosis (F 78%, M 75%), cystic endometrial hyperplasia (F 45%), testicular atrophy (77%), thyroid follicular cysts (F 51%, M 36%), and thymus cysts in males (16%). Amyloidosis, arteritis, and acidophilic macrophage pneumonia were not common in contrast to C57BL/6 mice on the same study. The most common neoplasms were liver tumors (F 19%, M 34%); lung tumors (F 7%, M 11%); ovarian tubular adenoma (71%); ovarian granulosa theca cell tumors (12%); and testicular interstitial cell tumors (11%). Lymphoma and histiocytic sarcoma were unusual in contrast to C57BL/6 mice on the same study (Zurcher et al. 1982). C3H and CBA mice have been known for their susceptibility to spontaneous and induced liver tumors, with a pronounced male predisposition (Andervont 1950). Male CBA/J mice were more susceptible to ethylnitrosourea (ENU)-induced liver tumors (averaging 45 tumors per animal) than were female CBA/J, or male or female C57BR/cdJ, P/J, SM/J, SWR/J, and C57BL/6J mice. Also compared to these strains, male and female CBA/J mice were moderately susceptible to ENU-induced lung tumors. (Kemp and Drinkwater 1989)
F.
C57BL
Mice of the major C57BL substrains are black (nonagouti) a/a. The most common substrains are C57BL/6 and C57BL/10. They are named for their origin from black offspring of female number 57 to male number 52 of Abbie Lathrop’s stock in the original mating by Clarence Cook Little in 1921. The same cross gave rise to strain C57BR. Female 58 mated with the same male gave rise to strain C58 (Russell 1978). C57BL/6J DNA was used in the mouse genome project (Gregory et al. 2002; Waterston et al. 2002), C57BL/6 blastocysts are commonly used for injection of manipulated embryonic stem (ES) cells to give rise to targeted mutant or knockout mice. C57BL/6 mice are often used as the host, recipient, or background strain for generation of congenics carrying spontaneous or induced mutations. C57BL/6J mice have been considered to be refractory to many tumors and are used in a wide variety of research areas including cardiovascular biology, developmental biology, diabetes and obesity, osteoporosis, genetics, immunology, neurobiology, and sensorineural research. C57BL/6 mice have a long lifespan in conventional or specific pathogen-free conditions with mean lifespans of 730 to
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895 days in females and 763 to 888 days in males (Storer 1966; Festing and Blackmore 1971; Goodrick 1975, 1977). Depending on substrain, C57BL mice usually have good breeding performance. C57BL/6J mice have good breeding performance, 55% of dams having at least four litters, about seven pups per litter, 87% weaned:born ratio. (Fox et al. 1997) They are intermediate in size with mean female weight of 19g and mean male weight of 25g at 8 weeks of age (Fox et al. 1997). C57BL/10J mice have poorer breeding performance, with only 7% of dams having at least four litters, about seven pups per litter, 90% weaned:born ratio. (Fox et al. 1997) They are similar in size with mean female weight of 21g and mean male weight of 27g at 8 weeks of age (Fox et al. 1997). C57BL/KSJ mice have intermediate breeding performance, with 23% of dams having at least four litters, about six pups per litter, 76% weaned:born ratio. (Fox et al. 1997) C57BL/6J mice have relatively low lung volume and small alveolar size (Tankersley et al. 1999; Soutiere et al. 2004) and relatively small kidneys and adrenal glands compared to other common inbred strains (Deschepper et al. 2004). Nonneoplastic Conditions
Hydrocephalus is more common in C57BL sub strains than in other common inbred strains with 0.36% and 0.029% of C57BL/KsJ and C57BL/6J, respectively, culled at weaning for domed cranium indicative of hydrocephalus (JaxNotes 2003), and 0.12% and higher incidences noted in C57BL/10SnJ and related strains in an earlier study (Sundberg et al. 1991). C57BL6/J have intermediate brain size among inbred strains (Roderick et al. 1973) but have larger cerebral ventricles, and crosses with A/J (small cerebral ventricles) have been used to identify and map genes relating to ventricular size (Holzgraefe and Spoerri 1980; Zygourakis and Rosen 2003). The C57BL/6J cerebellum (approximately 60 mg) is about 18% larger than DBA/2J cerebellum (50 mg). Cerebellar folial patterns of mice vary among inbred strains, and C57BL/6J possess an intraculminate fissure between vermian lobules IV and V in contrast to DBA/2J that lack it. Recombinant inbred BXD strains have been used to identify several genetic loci that are involved in cerebellum size and architecture. (Cooper et al. 1991; Neumann et al. 1993; Airey et al. 2001) C57BL/6J mice may develop neurodegenerative lesions of the hippocampus that are suggestive of a tauopathy. Abnormal granular deposits are predominantly in the hippocampus in mice from 6 months of age and tend to increase in size and density up to 30 months of age. Histologically, the clusters of granules are not readily apparent on routine H&E-stained sections, but stain positively with periodic acid Schiff (PAS) reagent or Gomori’s methenamine silver stain. Immunohistochemistry has identified various materials, including laminin and heparan sulfate proteoglycan suggestive of neuritic plaques, as well as tau protein and alpha synuclein, which is also present in Alzheimer’s-type
648 neurodegenerative conditions. Similar lesions are reported in AKR-derived SAM strains but were absent in DBA/2J and BALB/cJ. (Krass et al. 2003; Ye et al. 2004) Lipofuscin also can be found in neurons, neuroglia, and endothelial cells, especially in the hippocampus of aging mice. (Lamar et al. 1980) Eye abnormalities were noted early in the history of C57BL/6 and C57BL/10 mice. Microphthalmia and anophthalmia were reported to be 6.2 times more common in females than in males and 5.8 times more common in the right than in the left eye. (Smith et al. 1994; Smith 1995) A nophthalmia is highly unusual, and diagnosis requires careful histopathologic examination. Since some studies are based on gross examination alone, the incidence of anophthalmia may be overreported. C57BL microphthalmic eyes may have profound retinal abnormalities and/or cataracts that may rupture, extruding lens material. Affected mice often develop ocular infections, due to poor drainage of tears and debris from around small or absent eyes. (Smith et al. 1994; Smith 1995) C57BL/6 mice in some colonies develop corneal opacities with corneal mineralization in <5% of mice, a substantially lower incidence than in BALB/c, C3H/He, and DBA/2 mice on the same study. (Van Winkle and Balk 1986) In an epizootic of keratoconjunctivitis in 19- to 20-month-old male C57BL/6J mice, a Corynebacterium species agent was isolated most commonly, and other bacterial opportunists were identified as well. (McWilliams et al. 1993) C57BL/6 mice are homozygous for Cdh23ahl (Ahl, age-related hearing loss 1) and suffer presbyacusis, an age associated progressive hearing loss that manifests as deafness linked to cochlear degeneration. C57BL/6 deafness is later onset (after 2 months of age), with slower progression than in DBA and BALB/c strains, and C57BL/6 mice also are susceptible to noise-induced hearing loss. (Willott et al. 1994; Johnson and Zheng 1997; Johnson et al. 2000, 2002; Keithley et al. 2004) Cochlear pathology includes disruption and loss of both outer and inner hair cells, culminating in degeneration and collapse of the organ of Corti, which is replaced by a single epithelial layer. (Li and Hultcrantz 1994; McFadden et al. 2001; Bartolome et al. 2002) C57BL/Ks and C57/LJ have significant hearing impairment by 3 months of age. (Zheng et al. 1999) C57BL/6J and AKR/J mice have a relatively larger thymus (approximately twofold) with later onset involution than most other strains (A/J, DBA/2J, BALB/cJ, CBA/J, C3H/HeJ, 129/J, C57BL/10J). Studies on recombinant inbred strains derived from C57BL/6J and DBA/2J (BXD strains), and C57BL/6J and A/J (AXB and BXA strains) indicate that large thymus size is a dominant trait in F1 and is influenced by more than one genetic locus. (Peleg and Nesbitt 1984; Hsu et al. 2003) Malocclusion is more common in C57BL/6J and some related strains or substrains than in other common strains, with 0.046% of C57BL/6J mice culled at weaning for malocclusion in 2002 at the Jackson Laboratory (JaxNotes 2003). Malocclusion in mice ultimately manifests as long maxillary incisors that grow out from and curl back into the maxilla, and long mandibular incisors that tend to grow upward from the mandible. Mice with
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this condition cannot eat a hard-pelleted diet. Mice that are small or seem to be “runted” at weaning should be examined for malocclusion because regular trimming of the continuously growing incisors may permit survival of mice, or the animal should be culled from a breeding population. Trauma and genetics have been implicated in the development of the condition. (Losco 1995; Petznek et al. 2002) C57BL6/J mice develop age-related osteoporosis and osteoarthritis (Wilhelmi and Faust 1976). Female C57BL6/J mice have the lowest cortical femoral bone density of females of multiple inbred strains (AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, NZB/B1NJ, SM/J, SJL/BmJ, SWR/BmJ, 129/J). Crosses with C3H/HeJ (high bone density) are used to study the genetics of bone density. (Beamer et al. 1996, 2001; Shultz et al. 2003; Turner et al. 2003) Interestingly, compared to A/J, DBA/2J, and BALB/cByJ mice, C57BL6/J mice have been found to have high tibial and femoral bone mass. C57BL6/J mice are more active than A/J mice with larger quadriceps muscles, and male C57BL6/J have larger testicles and higher testosterone levels, while female have smaller ovaries and lower estradiol levels than male and female A/J mice respectively. (Kaye and Kusy 1995) A/J bones are more brittle than C57BL6/J bones. (Jepsen et al. 2001) Hamartomas and choristomas are rare, noninherited tumorlike conditions that are more common in C3H/HeJ and C57BL/6J than in other inbred strains. These present as tufts of hair on the dorsal cranial midline. The tufts overlie an opening in the sagittal suture that contains fatty tissue. (Adkison and Sundberg. 1991) Overgrooming, or barbering, is common in C57BL6/J and related strains. Areas of alopecia frequently have a distinctive pattern common to several mice within a cage. The barberer is unaffected unless more than one mouse is barbering. Removal or separation of the barbering mice may resolve the condition and allow regrowth of hair (JaxNotes 1987). It has been proposed that dominant mice barber the hair of recipients (the Dalila effect), and the whiskers may be plucked, not nibbled. (Sarna et al. 2000) There is a female bias, and this mouse behavior has been posed as a model of human trichotillomania and obsessivecompulsive spectrum disorders. (Garner et al. 2004) Ulcerative dermatitis was noted early in the history of the strain (Dunn 1954) and may occur in up to 21% of animals in some colonies. The condition is progressive, and the lesions may become very severe and associated with secondary conditions such as reactive lymphadenopathy, leukocytosis, and amyloidosis. (Sundberg 1996) Dermatitis with severe ulceration can complicate studies by requiring early termination of mice for humane reasons. In a long-term survival study of ad libitum-fed and diet-restricted mice (Blackwell et al. 1995), ad libitum-fed mice had a much higher incidence than did diet-restricted mice. Compared to only 2% and 0.3% of diet restricted female and male mice, 26% of female and 13.5% of male ad libitum-fed mice suffered severe dermatitis, and many had to be withdrawn from the study. The condition frustrates clinical veterinarians and scientists, and the etiology remains
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obscure, with genetic, autoimmune or allergic, and environmental allergic factors implicated. Imperforate vagina with associated hydrometra and mucometra was diagnosed in 6 C57BL/6J mice (of 35 cases total) at the Jackson Laboratory over a two-year period. (Sundberg and Brown et al. 1994) Longitudinal vaginal septa in C57BL strains have marked subline differences in incidences, ranging from 1% in C57BL/6By to about 26% in two lines congenic with C57BL/10ScSn. Imperforate vagina occurred at a low frequency in four C57BL stocks in which septa were regularly found. The septum was about 15 times more common than imperforate vagina. (Shire 1984) Adrenal subcapsular spindle cell hyperplasia, which may be associated with mast cell infiltrates, is common in aged female C57BL/6J mice (almost 80% at 13–15 months of age), in contrast to male C57BL/6J mice that do not develop the condition. (Kim et al. 1997) Accessory adrenal cortical nodules, consisting of encapsulated cortical tissue, within or outside of the adrenal capsule or in adjacent fat, may occur in more than 50% of C57BL/6 or BALB/c mice and are less common in A and C3H mice. (Sass 1983; Yarrington 1996) Melanosis of the spleen and other tissues is common in black mice. Black areas may be visible grossly, especially in the cranial pole of the spleen. Melanin can be distinguished from hemosiderin histochemically by the Perl’s or Prussian Blue stain, which stains hemosiderin blue. (JaxNotes 1989; Veninga et al. 1989; Sundberg 1991) Black pigment, melanin, also occurs frequently on heart valves, dura, and interstitially in Harderian glands and parathyroid glands in pigmented strains. (Dunn 1954; Elwell and Mahler 1999; Haines et al. 2001) In the brain there may be abundant melanin in melanocytes in meninges and along cerebral capillaries. (Klein-Szanto et al. 1991) C57BL/6J are susceptible to acidophilic macrophage pneumonia, although usually at less than 10% incidence. (Zurcher et al. 1982; Murray and Luz 1990; Ernst et al. 1996) Infection with agents such as Cryptococcus neoformans can increase incidence substantially. (Huffnagle et al. 1998) Their predisposition to this condition may be related to their susceptibility to certain agents and may complicate studies of respiratory phenotypes. (Feldmesser et al. 2001) C57BL6 mice are considered to be relatively susceptible to senile (AApoAII) amyloidosis as well as to secondary (SAA) amyloidosis. (Higuchi et al. 1991; Elliott-Bryant et al. 1997) Along with AKR/J and DBA/2 mice, C57BL/6 mice carry the a allele of the ApoAII gene, which confers less susceptibility to senile amyloidosis than does the c allele. (Kitagawa et al. 2003; Korenaga et al. 2004) In some studies, amyloidosis has been the most common age-related nonneoplastic finding, with more than 80% of C57BL/6 males affected. (Zurcher et al. 1982) Stressors, such as group housing and infections, are implicated in the development of amyloidosis with greater than 30% incidence. (Henderson and Giddens 1970; Lipman et al. 1993) In specific pathogen-free C57BL/6 mice from a study of virgin and breeder C57BL/6JfC3Hf/NCtr (C57BL/6J fostered onto gnotobiotic C3Hf/He mice) mice and barrier maintained
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up to 689 days, common nonneoplastic changes included hepatic fatty metamorphosis especially in males; mild chronic inflammatory changes; lymphoid accumulations or mononuclear aggregates in liver, lung and lacrimal gland; proteinaceous renal casts; cystic endometrial hyperplasia; ovarian atrophy, cysts and ceroid pigment especially in virgin mice; and pituitary cysts. The incidence of amyloidosis in these specific pathogenfree mice was very low and similar to that in BALB/c mice in the same study (Frith et al. 1983). Additional results from this study are summarized in Tables 25-3 and Table 25-4. In an earlier study of almost 400 group-housed mice with up to 15 mice per cage (initially), maintained until their maximum age of 31 months, the most common nonneoplastic findings were glomerulonephritis (F 100%, M 100%), amyloidosis (F 73%, M 83%), acidophilic macrophage pneumonia (F 16%, M 30%), arteritis (polyarteritis) (F 36%, M 16%), cystic endometrial hyperplasia (F 52%), and hydronephrosis (F 9%, M 6%). (Zurcher et al. 1982) Neoplasia
In specific pathogen-free C57BL/6 mice from the study of virgin and breeder C57BL/6JfC3Hf/NCtr mice, the most common neoplasms were lymphoma, hemangiosarcoma, and pituitary adenoma. Histiocytic sarcoma was more common, and Harderian gland and lung tumors were less common than in BALB/c mice on the same study (Frith et al. 1983). Additional results from this study are summarized in Tables 25-3 and 25-4. In other chronic studies, lymphoma usually is the most common neoplasm in C57BL/6 mice, with incidences of up to 31% in females and lower incidences in males. Histiocytic sarcoma (female bias), lung tumors (male bias), liver tumors (male bias), pituitary tumors (female bias), and testicular interstitial cell tumors are among the most common neoplasms, with Harderian gland tumors, hemangiomas, and thyroid follicular adenomas also noted. (Frith et al. 1975, 1983; Zurcher et al. 1982; Ward et al. 2000) Diet restriction has been shown to increase lifespan, reduce tumor incidence, and delay tumor onset in multiple studies. (Volk et al. 1994; Blackwell et al. 1995; Sheldon et al. 1995) In a long-term survival study of ad libitum-fed and diet-restricted C57BL/6 mice, 40% diet restriction increased mean lifespan 15% and 25% beyond 27.5 months and 26.9 months in males and females, respectively, and extended the maximal lifespan by 18% in both sexes, reduced the overall incidence and number of neoplasms, and delayed onset of most tumors. Histiocytic sarcomas were the most common apparent cause of death and the most common tumor in diet-restricted mice found dead or moribund at any age, occurring in almost one-third of the mice (337/991). The diet-restricted female mice had fewer pituitary neoplasms (37% vs. 1% in diet-restricted mice); lymphomas (29% vs. 9% in diet-restricted mice); and fewer thyroid neoplasms (8% vs. 0.4% in diet-restricted mice), but incidence of histiocytic sarcoma increased. Ad libitum-fed female mice had an overall histiocytic sarcoma incidence of 25%,
650 while other groups (restricted male and female, and ad libitum male) had similar incidences of about 40%. The diet-restricted male mice had fewer liver tumors (10% vs. 1% in diet-restricted mice). (Blackwell et al. 1995) The most common spontaneous lymphoma in C57BL/6 mice has been classified as a type B reticulum cell tumor, follicular center cell lymphoma, or mixed-cell lymphoma, and probably is most compatible with follicular B cell lymphoma under current classification. It is unusual before 12 months of age, and it typically involves spleen, mesenteric lymph nodes, and liver; it may also involve small intestine Peyer’s patches or other gut or mucosa associated lymphoid tissue (GALT or MALT). The primary cell type of this neoplasm is relatively pleomorphic, small to large with more cytoplasm than mature lymphocytes, and is admixed with plasma cells and lymphocytes (Ward et al. 2000). Definitive identification and typing of lymphomas requires immunohistochemistry and/or molecular techniques (Morse et al. 2002). Histiocytic sarcomas are rare until 12 months of age and often are slightly more common in females than in males. Previous classifications of this neoplasm include Dunn’s (1954) reticulum cell neoplasm, type A, or malignant lymphoma histiocytic type, and histiocytic lymphoma. The liver is the most commonly involved organ in male mice; in females the uterus and vagina, as well as the liver, often are involved, suggesting these tissues as sites of origin. Organs that are less frequently involved include the spleen, lymph node, bone marrow, lung, kidney, and ovaries. Pulmonary involvement occurs in a high percentage of cases with liver involvement. (Kogan et al. 2002; Lacroix-Triki et al. 2003) Pituitary tumors, predominantly of mammotrophs or prolactinsecreting cells, arising from the pars distalis have been reported in more than 80% of female C57BL/6J greater than 22 months old and occur primarily in the lateral zones (Schechter et al. 1981; Clayton et al. 1984; Gordon et al. 1987). The most common ovarian tumor in C57BL/6 (as well as in B6C3F1) mice is tubular mesothelioma (adenoma). (Frith et al. 1981) Susceptibility to Selected Experimental Conditions and Infectious Agents
Notable responses of C57BL/6 mice under experimental conditions include their high susceptibility to diet-induced atherosclerosis, carrying susceptibility alleles at Tnfsf4 (Ath1) and other loci (Paigen 1995; Phelan et al. 2002; Wang et al. 2005); to diet-induced gallstones (Khanuja et al. 1995; Paigen et al. 2000); to diet-induced obesity, non-insulin–dependent diabetes and hypertension (Schreyer et al. 1998; Surwit et al. 1988, 1991; Mills et al. 1993; Petro et al. 2004); and their preference for high-fat diets (Smith et al. 2000). They also are relatively susceptible to irradiation-induced lymphoma (Morse et al. 2002; Shimada et al. 2003) and to experimental autoimmune encephalomyelitis (EAE) (Okuda et al. 2002; Palaszynski et al. 2004); and they are relatively sensitive to exogenous estrogens
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(Greenman et al. 1990; Perez et al. 1996; Griffith et al. 1997; Roper et al. 1999). C57BL/6 mice may be sensitive to estrogens or estrogenic compounds, demonstrating increased uterine weight (uterotrophic response) compared to some other strains (Perez et al. 1996; Griffith et al. 1997; Roper et al. 1999) and developing estrogen-induced thyroid follicular cell adenomas (Greenman et al. 1990). Estrogenic effects in mice include endometrial hyperplasia and eosinophil infiltration (Cooke et al. 1997; Griffith et al. 1997); vaginal epithelial proliferation, stratification, and cornification (Buchanan et al. 1998); pituitary prolactinoma; ovarian changes, including reduced corpora lutea and increased ovarian tumors (Walker and Kurth 1993); bone changes, including proliferation of osseous trabeculae (hyperostosis), osteofibrotic areas (myelofibrosis) in the sternum, and osteosarcoma (Highman et al. 1981; Greenman et al. 1983). C57BL/6 mice are relatively resistant to induced seizures and neuropathology compared to other inbred strains (Ferraro et al. 1995, 2002; Golden et al. 2001; McKhann et al. 2003); to DEN-induced liver tumors (Drinkwater and Ginsler 1986; Lee and Drinkwater 1995); and to some skin carcinogenesis protocols (Sundberg et al. 1997). C57BL/6 mice are known for their resistance to many infectious agents and disease, including mousepox (Ectromelia virus) (Bhatt and Jacoby 1987; Jacoby and Bhatt 1987; Brownstein et al. 1989); Sendai viral pneumonia (Brownstein, Smith, et al. 1981, 1987); polyomavirus-induced tumors (Freund et al. 1992); EMCV-induced diabetes or myocarditis (Hirasawa et al. 1992, 1995, 1996, 1999); murine respiratory mycoplasmosis (M. pulmonis) (Davidson et al. 1988; Cartner et al. 1996; Lai et al. 1996); H. hepaticus hepatitis (Ward et al. 1994; Whary and Fox 2004); Lyme borreliosis (Barthold et al. 1990; Armstrong et al. 1992; Ma et al. 1998); Listeria monocytogenes mortality (Czuprynski et al. 2003) but not gastritis (Park et al. 2004); and Mycobacterium tuberculosis (Medina and North 1998; Kramnik et al. 2000; Chackerian and Behar 2003; Mitsos et al. 2003). C57BL/6 mice are relatively susceptible to H. felis gastritis (Fox et al. 1996; Mohammadi et al. 1996; Sakagami et al. 1996; Walker et al. 2002; Court et al. 2003); experimental lethal mouse adenovirus 1 (MAV1); hemorrhagic encephalomyelitis (Guida et al. 1995; Kring et al. 1995; Charles et al. 1998; Kajon et al. 1998; Spindler et al. 2001); Cryptococcus neoformans pneumonia (Huffnagle et al. 1998; Feldmesser et al. 2001); and mite-associated ulcerative dermatitis (Friedman and Weisbroth 1975; Csiza and McMartin 1976; Weisbroth et al. 1976; Dawson et al. 1986). Related Strains or Substrains
C57BLKS/J (formerly C57BL/KSJ) C57BLKS was derived from C57BL/6J strain maintained by Dr. N. Kaliss (Ks) in 1947. Eighty-four percent of the alleles in this strain
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are shared with C57BL/6J, and 16% are shared with DBA/2J, indicating genetic contamination early in the strain’s history. Spontaneous hydronephrosis may occur in more than 60% of male C57BL/KsJ mice by 15 weeks of age. (Wright and Lacy 1988; Weide and Lacy 1991) They are more susceptible to diabetes and atherosclerosis than are C57BL/6J mice. (Mu et al. 1999) The diabetes (db) allele is a point mutation in the leptin receptor (Lepr) gene that arose spontaneously in this strain. The diabetes (Leprdb) and obese (Lepob) mutations in the leptin receptor gene and in the leptin gene, respectively, result in much more severe diabetes phenotypes on the C57BLKS/J inbred background than on the C57BL/6J inbred background. (Sulakhe 1987; Leiter et al. 1989, 1999; Davis et al. 2005) C57BLKS/J mice exhibit age-related hearing loss by 3 months of age (Zheng et al. 1999). C57L C57L are gray mice (a/a, b/b, ln/ln) that carry the Leaden (ln) coat-color mutation, now is recognized as the Mlphln allele in the melanophilin gene. They were developed from a spontaneous mutant in a C57Br strain at the Jackson Laboratory in 1933. (Silvers 1979; Festing 1999; JAX® 2004) Among inbred strains, they have been found to be extremely susceptible to diet-induced gallstones, and carry susceptibility alleles at Lith1 and other sites associated with gallstone susceptibility. (Khanuja et al. 1995; Paigen et al. 2000) However, their susceptibility is substantially reduced by elimination of cholelithogenic helicobacters. (Maurer et al. 2005) They have a high incidence of lymphoma, approximately 25% incidence at 21 months of age, which resembles the B cell lymphoma of SJL mice. (Erianne et al. 2000; Morse et al. 2002) C57L/J mice are susceptible to experimental allergic encephalomyelitis (EAE). (Lindsey 1996) C58 C58 are black mice (a/a), derived from female number 58 and male number 52 of Abbie Lathrop’s stock in 1921. C58/J mice are used largely because of their high incidence of thymic precursor T cell lymphoma (leukemia) by one year of age (Nexo and Kog 1977; Duran-Reynals et al. 1986; Mucenski et al. 1988; JAX® 2004). B6;129 mice and B6C3F1 hybrid mice are discussed below, after inbred strains.
G.
DBA
DBA is the oldest of all inbred strains of mice. It was developed by C. C. Little in 1909 during coat-color experiments from stock segregating for the recessive coat-color genes d, b, a (dilute, brown, nonagouti) and it was named for its color genotype (now Myo5ad /Myo5ad, Tyrp1b /Tyrp1b, a /a, discussed above) (Russell 1978). The mice are uniformly nonagouti, which results in a washed out (dilute), brown coat color (Silvers 1979). In 1929, 30 crosses were made between DBA substrains, and several new substrains were established,
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including the widely used substrains DBA/1 and DBA/2. There are substantial differences between substrains (including the MHC H2 haplotype) probably because of substantial residual heterozygosity at the time of the crosses (Festing 1999; JAX® 2004). DBA/2J is prioritized in the mouse phenome database (Bogue 2003). DBA/1 DBA/1 mice are relatively long-lived (F 750 d, M 728 days) (Storer 1966). They have intermediate breeding performance, 81% of dams having at least four litters, about five pups per litter, 82% weaned:born ratio. They are of intermediate size with mean female weight of 21g and mean male weight of 25g at 8 weeks old. (Fox et al. 1997) Nonneoplastic Conditions
Imperforate vagina with associated hydrometra and mucometra was diagnosed in 5 DBA/1J mice (of 35 cases total) at the Jackson Laboratory over a two-year period. (Sundberg and Brown 1994) Neoplasia
DBA/1 mice have been noted to have low gross tumor incidence especially in males with lung tumors identified in 3% of males, and 1% of breeding females, and lymphoma (lymphatic leukemia) in less than 1% of males. (Storer 1966) But high incidences of lymphoma, 90% in breeding females and 61% in virgin females (Hoag 1963), and of lung tumors in both males and females (2–27%) have been reported as well (Festing and Blackmore 1971). Susceptibility to Selected Induced Conditions and Infectious Agents
DBA/1 mice probably are best known for their susceptibility to type II collagen-induced arthritis, which results in changes similar to those occurring in human rheumatoid arthritis (Stuart and Dixon 1983; Terato et al. 1985; Osman et al. 1999). DBA/2 DBA/2 is a widely used inbred strain that has been especially valuable in cardiovascular biology, neurobiology, and sensorineural research. Its characteristics are often contrasted with those of the C57BL/6J inbred strain. BXD (also referred to as BXD2) recombinant inbred mice are used to map genetic loci relevant to traits such as thymus size (Peleg and Nesbitt 1984), cerebellar folial pattern (Neumann et al. 1993; Airey et al. 2001), atherosclerosis susceptibility (Paigen et al. 1990; Colinayo et al. 2003), hearing loss (Willott et al. 1998), seizure susceptibility (Neumann and Collins 1991), and susceptibility to infectious diseases (Melvold et al. 1990; Lee et al. 2001). DBA/2 mice have been reported to have an intermediate mean lifespan in conventional or specific pathogen-free
652 fostered conditions (F 750 or 686 days, M 433 or 487 days), with shorter male lifespan especially in conventional conditions. (Storer 1966; Festing and Blackmore 1971; Peleg et al. 1984) DBA/2J have poorer breeding performance than do DBA/1J mice, with only 48% of dams having at least four litters, about five pups per litter, 83% weaned:born ratio (Fox et al. 1997). They are of intermediate size with mean female weight of 22g and mean male weight of 29g at 8 weeks old (Fox et al. 1997). DBA/2J have the lowest brain weight of 25 inbred strains, but intermediate brain:body weight ratio (eleventh of 25) (Roderick et al. 1973). DBA/2J are relatively easy to handle and may squeak when held, but are not as vocal as AKR/J (Wahlsten et al. 2003). Nonneoplastic Conditions
DBA/2J mice develop spontaneous calcified heart lesions (cardiac calcinosis) that progress with age, with up to 90% of individuals affected by one year of age. (Dunn 1954; van den Broek et al. 1998; Brunnert et al. 1999) Early lesions may include subepicardial eosinophilic myocarditis of the right ventricular free wall (Hirasawa et al. 1998). Dystrophic calcification or mineralization also can occur in aorta, testes, tongue, skeletal muscle, cornea, kidney, stomach, small intestine, and ovary, with incidence and severity increasing with age, and without apparent sex differences (in non-gonadal tissues) (Rings et al. 1972; Maeda et al. 1986; Yamate et al. 1987, 1990). In the tongue, calcification especially involves the superficial longitudinal muscles, and calcified nodules may lift the mucosa into polypoid lesions (Imaoka et al. 1986). Several dystrophic calcification (Dyscalc) loci are implicated in cardiac mineralization in DBA/2 and C3H mice (Colinayo et al. 2002). Corneal changes including ulcers and erosions, acute keratitis, stromal neovascularization, and mineralization of the basement membrane zone, occur at higher incidence in DBA/2 (29.1%) than in other susceptible strains C3H (16.2%), CF1 (16.2%), and BALB/c (10.0%) (Dunn 1954; Van Winkle et al. 1986). DBA/2J mice develop a progressive form of secondary angle-closure glaucoma that appears to be initiated by iris atrophy and synechiae. It may be a useful model of pressure-related ganglion cell death and optic nerve atrophy, and to evaluate strategies for neuroprotection. They develop pigment dispersion, iris transillumination, iris atrophy, anterior synechiae, and elevated intraocular pressure (IOP). IOP is elevated in most DBA/2 mice by the age of 9 months. These changes are followed by the death of retinal ganglion cells, optic nerve atrophy, and optic nerve cupping. The prevalence and severity of these lesions increase with age. By 22 months of age, most mice have optic nerve atrophy with cupping. (John et al. 1998; Chang et al. 1999; Anderson et al. 2002) DBA/2 mice have progressive hearing loss that is already severe by 3 months of age (Zheng et al. 1999). They carry three recessive alleles similar to those found separately in C57BL/6J,
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BALB/cBy and WB/ReJ, including Cdh23ahl the age-related hearing loss 1 mutation. (Willott Erway 1995; Willott Turner 1998; Johnson et al. 2000) Malocclusion in DBA/2J was less common than in some C57BL strains and more common than in other strains, with 0.0130% of DBA/2J mice culled at weaning for malocclusion, in 2002 at the Jackson Laboratory. (JaxNotes 2003) Imperforate vagina with associated hydrometra and mucometra was diagnosed in three DBA/2J mice (of 35 cases total) at the Jackson Laboratory over a two year period (Sundberg and Brown 1994). Adrenal subcapsular spindle cell hyperplasia with associated mast cell infiltration is more common in aged female DBA/2J mice (almost 100% at 13 to 15 months of age) than in male DBA/2J mice, with incidence of about 70%. (Kim et al. 1997) Neither senile (AApoAII or ASSAM) nor reactive (SAA) amyloidosis is likely to develop in DBA/2 mice, despite their possession of intermediate susceptibility allele (ApoAIIa) to senile amyloidosis. (Higuchi et al. 1991) Spontaneous Neoplasia
In a study of the frequency of neoplasms in primarily breeding populations of inbred strains over a 13-year period, the most common neoplasm in DBA/2J mice of both sexes was lymphoma with frequency of 0.5-1%, and mammary tumors in females were found with a similar frequency. (Mikaelian et al. 2004) There are no other recent systematic studies of spontaneous pathology in DBA/2 mice. Studies conducted prior to 1973 showed notable incidence of leukemia or lymphomas 2-12% in females, 0-10% in males; mammary tumors in unfostered substrains 31%-48% in virgin females, 72% in breeding females, 0-1% in males (Hoag 1963; Smith et al. 1973); liver tumors 6-35%; and lung tumors 1-23% (Festing and Blackmore 1971). Susceptibility to Selected Experimental Conditions and Infectious Agents
Notable responses of DBA/2 mice under experimental conditions include their high susceptibility to audiogenic seizures between 14 and 42 days of age (Seyfried et al. 1980, 1981; Neumann et al. 1991); to chloroform-induced renal injury in males (Ahmadizadeh et al. 1984); to N,N-diethylnitrosamine (DEN)–induced hepatocarcinogenesis in males (Lee et al. 1995); and to some skin carcinogenesis protocols (Sundberg et al. 1997). DBA/2 mice are relatively resistant to diet-induced atherosclerosis or fatty streaks (Paigen et al. 1985; Nishina et al. 1993) and to diet-induced cholelithiasis (Khanuja et al. 1995). Notable responses of DBA/2 mice to infectious agents include their high susceptibility to lethal mousepox (Ectromelia virus) (Bhatt and Jacoby 1987; Jacoby and Bhatt 1987; Brownstein et al. 1989) and to Sendai virus pneumonia and death (Brownstein
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et al. 1981; Brownstein and Winkler 1986). They also are susceptible to some mouse hepatitis viruses (Le Prevost et al. 1975); encephalomyocarditis (EMCV) myocarditis, diabetes, and demyelination (Matsumori et al. 1982; Takeda et al. 1991, 1995; Hirasawa et al. 1999); experimental lethal mouse adenovirus 1 (MAV1) hemorrhagic encephalomyelitis (Guida et al. 1995; Kring et al. 1995; Charles et al. 1998; Kajon et al. 1998; Spindler et al. 2001); murine respiratory mycoplasmosis (M. pulmonis) (Davidson et al. 1988; Lai et al. 1996); H. felis gastritis (Mohammadi et al. 1996; Sakagami et al. 1996; Whary and Fox 2004); and cariogenic streptococci (Kurihara et al. 1991; Suzuki and Kurihara 1998). DBA/2 mice have been noted to be relatively resistant to polyomavirus-induced tumors (Freund et al. 1992).
H.
FVB/N
The FVB/N strain was established at NIH (Laboratory code N) in 1975 by inbreeding a group of HSFS/N (Histamine Sensitivity Factor Sensitive) Swiss mice selected for their possession of the b allele Fv1 gene, which confers susceptibility to Friend leukemia virus B strain. Their fertilized eggs contain prominent pronuclei that facilitate microinjection of DNA in transgenesis, and FVB/N zygotes survive well after microinjection. The phenotype of large pronuclei in the zygote is a dominant trait associated with the FVB/N oocyte but not the FVB/N sperm (Taketo et al. 1991). As a result of their utility and popularity for the production of transgenic mice, FVB/N mice now are available from most major vendors, for example, FVB/NCrl, FVB/NHsd, FVB/NJ, and FVB/NTac. FVB/N mice are albino with color genotype a+/a+, Tyrc/Tyrc (a+/a+, c/c). They are relatively long lived with 60% survival to 24 months of age (Mahler et al. 1996). The strain is characterized by vigorous reproductive performance and consistently large litters (Taketo et al. 1991). Nonneoplastic Conditions
Like some other Swiss strains and stocks as well as C3H mice, FVB/N mice are homozygous for Pde6brd1, which results in early-onset retinal degeneration (Taketo et al. 1991). While blind mice should be expected to perform poorly in learning tasks that require vision, FVB/N mice also appear to have a spatial learning deficit that is independent of their visual deficits. In addition, they are thigmotactic and hyperactive, and display higher levels of anxiety and aggression compared to C57BL/6J mice. (Mineur and Crusio 2002) FVB/N mice suffer a sometimes lethal epileptic syndrome, with apparently spontaneous seizures, or with seizure induction by tail tattooing, fur clipping, and fire alarms. Females are affected almost eight times more commonly than males. Seizure activity may include facial grimace, chewing, ptyalism
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653
with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions progressing to tonic convulsions and death. Mice may be found dead with characteristic histopathology, including neuronal necrosis and astrocytosis in the cerebral cortex, hippocampus, and thalamus. Centrilobular hepatic necrosis attributed to terminal hypoxia in seizuring animals may occur. (Goelz et al. 1998) Multiparous and virgin female FVB/N mice develop persistent mammary hyperplasia that may be associated with hyperplasia or adenoma of prolactin-secreting chromophobe cells of the pituitary pars distalis. Histologically, mammary glands have lobuloalveolar hyperplasia with distended secretory alveoli and ducts that resemble glands during pregnancy or delayed involution. Multiparous animals have higher incidence of the condition than virgin females, with all multiparous females affected in some cohorts. Hyperplastic glands frequently have small nodules of squamous epithelium. (Nieto et al. 2003; Wakefield et al. 2003) Spontaneous Neoplasia
In a 24-month study of FVB/N mice, survival to 24 months was approximately 60% in both sexes, and the incidence of mice with tumors at the end of the study was 55% in males and 66% in females. In females, lung tumors, pituitary gland adenomas, ovarian tumors (combined types), lymphomas, histiocytic sarcomas, Harderian gland adenomas, and pheochromocytomas were most common (found in more than 5% of female mice). In males, lung tumors, liver tumors, skin tumors, and Harderian gland adenomas were most common (found in more than 5% of male mice), and are summarized in Table 25-4. Compared with other inbred strains, FVB/N mice may have relatively high incidence of lung tumors and relatively low incidence of liver tumors and lymphomas. An unusual skin tumor on the pinna or tail of five mice in the study (3% of females and 10% of males) was a spindle cell neoplasm that was diagnosed as a neural crest tumor. (Mahler et al. 1996) This tumor profile, as well as FVB/N susceptibility to mammary gland hyperplasia, should be considered in the interpretation of neoplastic phenotypes in FVB/N-derived transgenic lines. The FVB/N strain may have relatively low incidence of mammary carcinoma despite their predisposition to mammary hyperplasia. Only six carcinomas were diagnosed in mammary glands from almost 500 wild-type control FVB/N mice, two were adenosquamous, three were squamous cell carcinoma, and one was an adenocarcinoma. (Nieto et al. 2003) By 13 months of age more than 40% of virgin females may have mammary hyperplasia and elevated prolactin levels. Between 18 and 23 months of age, 52% of virgin females had pituitary pars distalis hyperplasia, and 19% had adenomas. Multiparous mice 18 to 23 months of age had 83% incidence of pituitary adenomas. (Wakefield et al. 2003) Such changes should be considered when designing breast cancer and endocrine studies.
654
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Susceptibility to Selected Experimental Conditions and Infectious Agents
Notable responses of FVB/N mice under experimental conditions include their high seizure susceptibility (Royle et al. 1999; Schauwecker 2003; Mohajeri et al. 2004); and their susceptibility to some skin carcinogenicity protocols (Hennings et al. 1993). FVB/N mice are relatively resistant to collageninduced arthritis (Osman et al. 1999). FVB/NJ mice had the highest serum cholesterol levels on chow diet and only modest increases in response to high-fat diet compared to other inbred strains (Svenson et al. 2003). They are relatively resistant to atherosclerosis compared to C57BL/6J mice (Idel et al. 2003; Teupser et al. 2003). FVB/N mice are susceptible to Theiler’s murine encephalomyelitis virus (TMEV), induced chronic inflammation, and primary demyelination. (Azoulay et al. 1994)
I.
SJL/J
SJL/J mice are albino with color genotype p/p Tyrc/Tyrc (p/p, c/c). SJL (for Swiss James Lambert) mice derived from three sources of Swiss Webster outbred stock that were brought to the Jackson Laboratory between 1938 and 1943. These were pen-bred until 1955 and were then inbred by James Lambert at the Jackson Laboratory (Festing 1999; JAX® 2004). The strain has been popular for high incidence of lymphomas (formerly called reticulum cell sarcoma type B) that resemble Hodgkin’s disease (Crispens 1973). Although SJL/J mice are relatively easy to handle, (Wahlsten et al. 2003), they are known for their aggressive behavior with conspecifics. Most males will be killed by 4 to 5 months of age unless caged separately. (Crispens 1973; Weinhold and Ingersoll 1988; Lumley et al. 2004) Nonneoplastic Conditions
Like some other Swiss strains and stocks as well as C3H mice, SJL/J is homozygous for the retinal degeneration allele Pde6brd1, resulting in early-onset retinal degeneration. (Festing 1999; JAX® 2004; Serfilippi et al. 2004) A spontaneous recessive mutation in the Dysferlin gene Dysfim arose in SJL/J mice and is maintained on that strain. This allele reduces levels of functional dysferlin protein resulting in an inflammatory myopathy (im) model for limb girdle muscular dystrophy 2B. Progressive loss of muscle mass and strength results in hind limb clasping and inability to spread hind limbs and digits when suspended by their tails by about 8 months of age. Corresponding muscle pathology includes muscle fibers with central nuclei, variation in fiber size, fiber splitting, inflammatory infiltrate, necrosis, and eventual replacement of muscle fiber with fat. While muscle weakness can be detected as early as 3 weeks of age, the greatest pathology occurs after 6 months of age. SJL/J mice also have an increased rate of
B R AY T O N
muscle regeneration after injury when compared to BALB/c mice. (Bittner et al. 1999; Vafiadaki et al. 2001; Confalonieri et al. 2003) A/J mice also develop muscular dystrophy due to their possession of a different allele (prmd mutation) in the same gene (Ho et al. 2004). Imperforate vagina with associated hydrometra and mucometra was diagnosed in 4 SJL/J mice (of 35 cases total) at the Jackson Laboratory over a two-year period. (Sundberg and Brown et al. 1994) SJL/J mice carry the high amyloidogenicity c allele of the Apoa2 gene and are highly susceptible to senile amyloidosis (Higuchi et al. 1991; Kitagawa et al. 2003). Neoplasia
By one year of age, SJL/J mice have high incidence (up to 90% by 18 months) of B cell lymphomas that have been classified as germinal center B cell lymphoma, pre-B cell lymphomas, and reticulum cell sarcoma. These lymphomas have been associated with low natural killer cell activity as well as with high expression of several endogenous retroviruses in this strain (Kaminsky et al. 1985; Lin and Ponyio 1991; Erianne et al. 2000). The tumors are preceded by lymphoid hyperplasia in the spleen, Peyer’s patches, or gut-associated lymphoid tissue (GALT), and mesenteric lymph nodes. After 6 months of age, tumors first appear in GALT and mesenteric lymph nodes and later in the spleen, liver, thymus, and other lymph nodes. Advanced cases have massively enlarged mesenteric lymph nodes and spleen, and very prominent GALT. Histologically, cells are pleomorphic or of apparently mixed-cell types, although early tumors may be more plasmacytoid. (Chow and Ho 1988; Mucenski et al. 1988; Tang et al. 1998a, 1998b; Morse et al. 2002; Wajchman et al. 2002) Because of these tumors, SJL/J mice have been used to model human Hodgkin’s disease and low-grade B cell non-Hodgkin’s lymphoma. (Rubin 1968; Kumar 1983; Wrone-Smith et al. 1993) In a study of the frequency of neoplasms in primarily breeding populations of inbred strains over a 13-year period, the most common neoplasms in SJL/J mice of both sexes were lymphoma, plasmacytoma, and histiocytic sarcoma, with frequencies of 0.5-1%. (Mikaelian et al. 2004) Susceptibility to Selected Experimental Conditions and Infectious Agents
Notable responses of SJL/J mice under experimental conditions include their high susceptibility to autoimmune conditions such as experimental allergic encephalomyelitis (EAE) (Encinas et al. 1996; Ding et al. 1997; Butterfield et al. 1998; Maron et al. 1999; Matarese et al. 2001) and to experimental allergic myositis (which may be complicated by their muscular dystrophy phenotype) (Matsubara et al. 2001). SJL/J mice are relatively resistant to diet-induced atherosclerosis or fatty streaks (Paigen et al. 1990) and diet-induced cholelithiasis (Khanuja et al. 1995).
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Notable responses of SJL/J mice to infectious agents include high susceptibility to experimental lethal mouse adenovirus 1 (MAV1) hemorrhagic encephalomyelitis (Spindler et al. 2001), susceptibility to Theiler’s murine encephalomyelitis virus (TMEV), inflammation and demyelination (Kang et al. 2002; Njenga et al. 2004); and intermediate susceptibility to mousepox (Bhatt and Jacoby 1987; Jacoby and Bhatt 1987; Brownstein et al. 1989). They are susceptible to Lyme borreliosis arthritis but not myocarditis (Barthold et al. 1990). Their resistance to MHV-induced demyelination and mortality has been attributed to lack of a viral receptor on target tissues. (Knobler et al. 1982; Barthold et al. 1987; Ohtsuka and Naguchi 1997; Ohtsuka et al. 1998)
III.
NON-INBRED MICE A.
B6;129
Genetically mixed, partially backcrossed, and/or partially inbred B6;129 mice are a common by-product of the generation of targeted mutant (knockout) mice from injection of 129-derived ES cells into C57BL/6 blastocysts. When inbred for homozygosity of the targeted mutation, genetically relevant heterozygous and homozygous, “wild-type” control mice are eliminated, and determination and generation of appropriate control animals may be difficult. Depending on the extent of backcrossing and inbreeding, considerable genetic variability within and between litters should be expected. (Ward et al. 2000; Haines et al. 2001; Linder 2001; Barthold 2004; Berry and Linder 2007) Nonneoplastic Conditions
In a study of the pathology of 99 B6;129 mice (derived from C57BL/6J and 129S4 /SvJae mice), the most common nonneoplastic lesions included amyloid-like material in the nasal septum; otitis media; epididymal epithelial karyomegaly; melanosis (usually in brain, parathyroid, and spleen); membranoproliferative glomerulonephritis, hyalinosis with extracellular crystals in several tissues, especially nasal respiratory and olfactory epithelium; islet cell hyperplasia; and esophageal dilation. These results are summarized in Table 25-3. Similar to other reports (Monticello et al. 1990; Hogen-Esch et al. 1996), this study noted that the amyloid-like material in the nasal septum stained with a trichrome stain indicating that the material contained collagen. In the cases of otitis, organisms usually could not be detected with special stains. Three male mice were sacrificed before the end of the study because of the severity of their ulcerative dermatitis. Other nonneoplastic contributors to death, each affecting fewer than 5% of the animals, were infection/septicemia secondary to skin ulceration, inhalation pneumonia, and arteritis. (Haines et al. 2001)
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Compared to parental 129S4 /SvJae mice, hyalinosis in B6;129 mice was much more likely to occur in the nose and gall bladder, and less likely in stomach, bile duct, and trachea. Acidophilic macrophage pneumonia was much less common in B6;129 than in 129S4 /SvJae mice in this study. Histologic features of hyalinosis were identical, characterized by cytoplasmic accumulation of hyaline intensely eosinophilic material, identified as a chitinase, sometimes with extracellular square, rectangular, or needle-like crystals. (Ward et al. 2001) Neoplasia
In the same study, by 104–105 weeks of age. 30 of 49 (61%) of the females and only 14 of 50 (28%) of the males were alive. Lymphoma was the most common contributing cause of morbidity or mortality and was identified in 8 of 19 females and 9 of 36 males that died or were sacrificed. Lymphoma also was the most common neoplasm (F 67%, M 42%) with the most common sites being mesenteric lymph nodes (where it most often appeared to originate), GALT (Peyer’s patches), and spleen. It is characterized as a B cell lymphoma, with similarities to T cell rich, B cell lymphomas in humans. Other relatively common tumors included liver tumors, lung tumors, thyroid follicular tumors, ovarian tumors, and uterine tumors (summarized in Table 25-4). Other than lymphoma, neoplastic contributors to death, each affecting fewer than 5% of the animals, were histiocytic sarcoma, duodenal adenoma, and hemangiosarcoma. (Haines et al. 2001) Relative to their parental strains, these B6;129 mice have similar (high) lymphoma incidences compared to C57BL/6J, and much higher incidences than 129S4/SvJae, with a female bias in all three types of mice. C57BL/6J mice have more histiocytic sarcomas than B6;129 or 129S4/SvJae. 129S4/SvJae mice have more lung tumors and Harderian gland tumors (with a male bias) than C57BL/6J or B6;129. All three types of mice have similar incidence of liver tumors with a male bias. (Ward et al. 2000)
B.
B6C3F1
B6C3F1 hybrid mice are genetically identical, dark agouti mice, color genotype (a+/a). This hybrid has been used widely in toxicology, and there is a substantial body of literature on the pathology of naïve control mice. They are robust with long lifespan and generally lower susceptibility to spontaneous pathologies than either parental strain. (Maronpot et al. 1999) This reference is an excellent resource regarding the spectrum of pathology in these mice. Substantial variability in the incidence of lesions between and within laboratories has been attributed to variations in sources, environments, and evaluators. (Sher et al. 1982; Everett 1984; Haseman et al. 1994, 1997; Greim et al. 2003) A recent chronic (two-year) National Toxicology Program (NTP) study,
656 which includes nonneoplastic findings, was selected for discussion below (NTP 2000). Nonneoplastic Conditions
The most common nonneoplastic findings (summarized in Table 25-3) were foci of cellular alteration in the liver, pancreatic islet cell hyperplasia, liver necrosis, pituitary hyperplasia of the pars distalis (in females only), thyroid follicular cell hyperplasia, chronic nephropathy (especially in males), splenic extramedullary hematopoiesis, thymic lymphoid depletion, thymic lymphoid hyperplasia, ovarian atrophy, ovarian cysts, uterine cystic endometrial hyperplasia, hydrometra, and cystic preputial gland ducts. (NTP 2000) Less common findings included hepatocellular vacuolization, hepatic centrilobular hypertrophy, hepatic extramedullary hematopoiesis, pancreatic exocrine atrophy, pancreatitis, gastric squamous epithelial hyperplasia, chronic mesenteric inflammation, arteritis, adrenal subcapsular cell hyperplasia, lymphoid hyperplasia in lymph nodes and spleen, macrophage or plasma cell infiltration of lymph nodes, pigment in the lymph nodes, myeloid hyperplasia in marrow, lymphoid, reticular, erythropoietic and/or granulopoietic hyperplasia in spleen, ulcerative dermatitis, pulmonary alveolar epithelial hyperplasia, retinal degeneration, chronic renal inflammation, hydronephrosis, renal pigment (hemosiderin), ovarian hemorrhage, angiectasis, or hematocyst, epididymis sperm granuloma, testicular atrophy, and preputial gland inflammation. (NTP 2000) Hepatocellular vacuolization due to fatty change can be a common finding in control mice in some studies, and it may be reported using a variety of terms such as steatosis, lipidosis, or fatty metamorphosis. It is especially common in old obese control mice, and it is more prevalent in male than in female mice. It may also occur in response to a toxicant. The clear empty vacuoles may compress nuclei to the cell periphery, and represent lipid that was removed during tissue processing. (Frith and Ward 1988; Harada et al. 1996, 1999) Altered hepatic foci or foci of hepatocellular alteration increase with age in mice. Eosinophilic and clear cell foci are more common than basophilic foci in B6C3F1 mice, and all types of foci are more common in male than in female mice. There is no obvious disruption of the liver architecture or compression of adjacent normal parenchyma. They have been associated with administration of carcinogens and with development of hepatocellular neoplasms, but their biological and toxicological significance remains unclear. (Frith and Ward 1979; Harada et al. 1996, 1999) Amyloidosis is notably absent in this hybrid, especially in contrast to CD-1® mice, which also have been popular in chronic toxicology studies. (Sher et al. 1982; Maita et al. 1988; Engelhardt et al. 1993; Majeed 1993) Obstructive urinary tract disease, or mouse urologic syndrome (MUS), has been recognized as an important cause of death
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of control and treated B6C3F1 male mice on some studies. (Bendele and Carlton 1986, 1998) Fibroosseous changes (myelofibrosis, metaphyseal osteosclerosis) can occur with an incidence >33% in 2-year-old female B6C3F1 mice, compared to <1% in males. Histologically, there are foci of replacement of marrow by eosinophilic matrix with spindled fibroblast-like cells, osteoclasts, and marrow cells. In advanced lesions the marrow cavity may be replaced by bony trabeculae. (Sass et al. 1980; Wijnands et al. 1996) Estrogenic compounds may also induce endosteal bone proliferation in mice. (Highman et al. 1981) Because they are heterozygous for the recessive rd1 allele, B6C3F1 mice do not develop rd1/rd1-like retinal degeneration. Neoplasia
Consistent with earlier studies (Sher et al. 1982; Everett 1984), the most common neoplasms in this study were liver tumors, lung tumors, and lymphoma, with a distinct male bias for liver tumors. Compared to the parental strains, the relatively high incidence and male predisposition for liver tumors are similar to C3H or C3H/He strains, and contrast with the low incidence of liver tumors in C57BL/6 (Harada et al. 1999; NTP 2000). Additional incidence details are provided in Table 25-4. The most recent summary of NTP historical control data, involving 1,158 female mice and 1,159 male mice, treated with various control vehicles via various routes, in 20 chronic (twoyear) studies initiated between 1992 and 1998, corroborates these data. Mice in this report received NTP 2000 diet ad libitum, female mice were housed five per cage, and male mice were housed individually. In female mice, the most common neoplasms and their mean incidences were liver tumors at 22% (adenomas > carcinomas); lymphoma at 16%; pituitary gland adenomas at 11%; lung tumors at 8% (adenomas > carcinomas); Harderian gland tumors at 8% (adenomas >> carcinomas); and hemangioma/hemangiosarcoma at 4%. In male mice, the most common neoplasms and their mean incidences were liver tumors at 47% (adenomas > carcinomas); lung tumors at 26% (adenomas > carcinomas); Harderian gland tumors at 11% (adenomas >> carcinomas); hemangioma/hemangiosarcoma at 6%; and lymphoma at 5%. (NTP 2003) Individual housing of males to reduce loss due to fighting and sequelae has resulted in longer male survival, but also has been associated with increased body weight, reduced incidence of dermal-subcutaneous tumors, marked increase in incidence of liver tumors, and smaller increase in incidence of lung tumors, compared to earlier studies of group-housed male mice. (Haseman et al. 1994; Rao and Crockett 2003) Hemangiosarcomas were more common than hemangiomas and occurred in skin, spleen, ovary, bone marrow, lymph nodes, uterus, testes, preputial gland, epididymis, urinary bladder, heart, intestine, kidney, liver, lung, and muscle. (NTP 2003)
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Mammary tumors and histiocytic sarcomas are not common in these mice compared to some other strains. (Maronpot et al. 1999; NTP 2000, 2003) Ovarian tumors can be common on aging studies, with most ovarian tumors rare before 1 year of age. The most common ovarian neoplasms in B6C3F1 mice were cystadenomas (24%), tubulostromal adenomas (24%), benign granulosa cell tumors (21%), and benign teratomas (8%). (Alison and Morgan 1987; Alison et al. 1987; Maronpot et al. 1999) Less common neoplasms include skin neurofibrosarcoma or schwannoma, papilloma, squamous cell carcinoma, fibrous histiocytoma, melanoma, sebaceous adenoma, basal cell tumor; oral papilloma, squamous cell carcinoma; forestomach papilloma, squamous cell carcinoma; intestine adenoma/carcinoma, leiomyoma/leiomyosarcoma, fibrous histiocytoma; liver hepatoblastoma, cholangioma, cholangiocarcinoma; kidney tubule adenoma/carcinoma; adrenal cortex adenoma/carcinoma; adrenal pheochromocytoma; pancreas islet adenoma; thyroid follicular adenoma/carcinoma, C cell adenoma/carcinoma; mast cell tumor; brain meningioma, oligodendroglioma, glioma, or astrocytoma; pituitary pars intermedia tumors; ovary cystadenoma, luteoma, thecoma, teratoma, granulosa cell tumor; uterus adenoma/carcinoma, uterus stromal polyp/sarcoma, leiomyoma/leiomyosarcoma; testicular Sertoli cell tumor; prostate adenoma/carcinoma, seminal vesicle adenoma/carcinoma, coagulating gland adenoma; mammary fibroma, fibroadenoma; clitoral/preputial gland adenoma/carcinoma; salivary adenoma/ carcinoma; Zymbal gland adenoma/carcinoma; neural crest tumor; mesothelioma; osteosarcoma/osteoma; chondrosarcoma/ chondroma; rhabdomyosarcoma; fibrosarcoma. (NTP 2000, 2003) Susceptibility to Selected Experimental Conditions and Infectious Agents
B6C3F1 mice may be more susceptible to urethane-induced tumors and mortality compared to B6CF1 (C57BL/6J X BALB/c F1) mice (Dragani et al. 1984), more susceptible to ENU-induced renal tumors compared to outbred Swiss mice (Lombard et al. 1974; Vesselinovitch et al. 1974), and less sensitive to some skin tumor induction protocols compared to Swiss and SENCAR mice (NTP 1996).
C.
Swiss Mice
So-called Swiss mice derive from two males and seven females obtained by Clara Lynch from Andre de Coulon in Lausanne, Switzerland, in 1926. The mice traveled from Paris to the Rockefeller Institute in New York City in her purse and/or in a shoebox, which she kept in her state room on the transatlantic voyage (Krause 1978). The habit of referring to these mice and their descendants as Swiss mice in Dr. Lynch’s laboratory at the Rockefeller Institute (now the Rockefeller
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University) persists today, with multiple derived strains or stocks still referred to as Swiss mice. (Lynch 1969) Outbred or non-inbred albino mice are available from major commercial breeder/suppliers of mice, and Swiss-derived stocks are the most common stocks currently available in North America. They have been popular largely because of characteristics attributed to heterosis or hybrid vigor, such as relatively long lifespans, high disease resistance, early fertility, large and frequent litters, low neonatal mortality, rapid growth, and large size. Importantly, they cost less than inbred mice. While some argue that the genetic heterogeneity of outbred mice is advantageous to some areas of research and for toxicity and carcinogenicity studies because it is similar to the heterogeneity in human populations (Miller and Austad et al. 1999; Miller, Burke, et al. 1999), it is well established that inbred strains or F1 hybrid mice are extremely valuable to many fields of research precisely because of their homogeneous and defined genetic constitutions (Festing 1999, 1999; Beck et al. 2000; Silver 1995). In addition, the expected heterogeneity of many outbred mouse stocks or strains is limited by the narrow genetic base of the founders, early selective breeding and inbreeding programs (Parker and Tyl 2003), and subsequent rederivation efforts that “reconstituted” an outbred stock from only a few or even from only one breeding pair (Charles River Laboratories 2004; Berry and Linder 2007). Swiss mice with similar names may have been separated from the stock of origin at very different dates, derived from only a few mice, and maintained by different breeding methods. In addition, all outbred albino mice are not Swiss or Swiss origin. Despite the similarity in their names to Swiss Crl:CD-1® and to Swiss Crl:CFW® mice, Crl:CF-1® mice derive from non-Swiss mice that were believed to be unrelated wild mice. Earlier nomenclature conventions used parentheses, for example, Ha(ICR) instead of colons, and this convention is maintained below if it was used in the source reference. After their arrival at the Rockefeller Institute, Dr. Lynch’s Swiss mice were inbred (sibling mated) until 1951 when that line was discontinued. At first they were used chiefly for cancer research, and then they became especially important in serologic testing for Yellow Fever in the 1940s through the 1960s, and were used for research on other infectious diseases (Lynch 1969). Mice were given to various laboratories during the period of inbreeding. Dr. Leslie Webster, at the Rockefeller Institute, obtained some of these mice in 1932, shortly before many were lost in an epidemic of mouse typhoid (probably salmonellosis). Dr. Webster inbred these mice for 10 to 12 generations, and then random bred them. Dr. Webster’s ‘inbred’ and random bred Swiss mice were transferred to commercial dealers and other laboratories in 1936–1937, and their descendants usually bear reference to his name, for example, Swiss Webster or SW (Lynch 1969; Hauschka and Mirand 1973).
658 In 1947, Dr. Theodore Hauschka (Laboratory code Ha) at the Institute of Cancer Research (ICR) at Fox Chase Cancer Center, Philadelphia, purchased 100 females and 30 males from John Landis who had obtained his breeding stock from 6 different sources of ‘Swiss Webster mice’ (mice from Webster’s lines of Swiss mice). Dr. Hauschka selected for high production and high growth rate characteristics and achieved exceptional production parameters with average 15 pups per litter, >99% pups weaned, <23 day interval between litters. Hauschka also was concerned about maintaining heterozygosity in his Ha(ICR) outbred stocks and avoided sib mating at ICR and later at Roswell Park Memorial Institute (RPMI). Based on segregation at 8 of 28 loci, he estimated 28.5% residual heterozygosity in Ha(ICR) mice after 20 years of mating (approximately 60 generations). (Hauschka and Mirand 1973) Although today’s commonly available Swiss stocks descend from Lynch’s original mice, and probably through Webster’s colonies, they are distinguished nominally by whether they descended through ICR (ICR mice), or through NIH (NIH Swiss mice), or more or less directly from Webster’s colonies (Swiss Webster mice). The histories of some of the stocks that are commonly available in the United States are summarized in Table 25-2. Non-Swiss albino stocks deserve mention here because they have similar names to some Swiss stocks but they have different origins and for the purposes of most studies should not be used interchangeably. Crl:CF-1® mice are probably of wild albino origin, obtained by Carworth Farms (CF) and intensively inbred for over 20 generations, then reduced to a single pair and outbred from that point forward as Carworth Farm’s CF-1 colony. They were cesarean derived by Charles River Laboratories’ in 1974 from a “representative cross section of the Carworth Farm’s CF-1 colony” (Charles River Laboratories 2004). Harlan’s nonSwiss albino, Hsd:NSA(tm) (CF-1®) (NSA™) derive from an unknown number of CF-1® mice that were obtained from Charles River Laboratories before 1980 (Harlon 2004). Disregarding the possibility of cross contamination along any branches of their pedigrees, contemporary sources of outbred Swiss and non-Swiss albino mice have experienced genetic bottlenecks related to intended periods of inbreeding or selective breeding, and to rederivations from one or only a few breeding pairs. Variability in experimental results and in spontaneous lesions (including greater than twentyfold difference in amyloidosis incidence, and two–threefold differences in tumor incidences) has been recognized between different sources of outbred Swiss mice with similar or identical names. Although some variability is attributable to different evaluators and different methods of evaluation or scoring, several surveys of multiple chronic studies contend and document that substantial variability may be attributable to the mice themselves. These surveys emphasize that caution should be exercised in selecting sources and types of outbred mice for research models. (Sher 1974; Engelhardt et al. 1993) CD-1® mice are used as the primary example of Swiss mice below because of the availability of literature on these mice.
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Nonneoplastic Conditions
“Retinal dystrophy” (retinal degeneration) was noted in an inbred SW line and in about 13% of a random bred Swiss stock by the mid 1960s (Lynch 1969). Several inbred Swiss-derived strains (FVB/N, SJL/J, SW) (Festing 1999) are homozygous for Pde6brd1. Retinal degeneration compatible with homozygosity for rd1 was not discerned in more than 3000 of Crl:CD-1® mice examined in one study (Hubert et al. 1999). In a recent survey of several outbred stocks (see Table 25-2), the trait was not discerned in Crl’s CD-1® mice, but it was identified in about half of Hsd:ICR mice that derive from Crl:CD-1®, and in almost all of Crl:CFW stock examined. In two stocks of nonSwiss albino mice (Crl:CF-1® and Hsd:NSA™ mice derived from them), this condition was found in only 1 of 120 mice. (Serfilippi et al. 2004) Corneal dystrophy or mineralization may occur in about 4% of aged CD-1® mice. (Van Winkle et al. 1986) Cataracts may occur in up to 25% of CD-1® mice by 18 months of age. (Taradach and Greaves 1984) In a study specifically of ocular lesions in about 3,000 4- to 5-week-old, CD-1® mice, using indirect ophthalmoscopy (IO) and slit lamp, findings included lenticular opacities or other changes in 19% of mice, and hyaloid artery remnants, vitreous bodies, or hemorrhage in up to 17% of mice. Abnormalities with an incidence <4% included mineralized and nonmineralized corneal opacities (visible with IO with or without additional lens), iridal changes (ectopic pupil due to coloboma, persistent papillary membrane, synechiae, miosis), colobomatous fundus, retinal fold or retinal atrophy, and a few cases of chorioretinal atrophy, hemorrhage, abnormal retinal vasculature pattern, incomplete palpebral fissure, microphthalmia, exophthalmia, and ophthalmic hemorrhage. (Hubert et al. 1999) A condition referred to as “progressive necrosing dermatitis of the pinna,” sometimes associated with ulcerative dermatitis extending over the neck and shoulders, with a predilection for male mice, has been noted in CD-1® mice from various colonies, with prevalences of 2 to 42% at different study sites. (Slattum et al. 1998) Amyloidosis has been an important contributor to morbidity and mortality in many studies of Swiss mice, with incidences varying from less than 1% to 54% in females and from 2%56% in males. (Homburger et al. 1975; Frith and Chandra 1991; Engelhardt 1996; Engelhardt et al. 1993; Majeed 1993) CD-1® mice are susceptible to senile (ApoA2) as well as to reactive (AA) amyloidosis. Immunohistochemical identification of the amyloid types reveal senile ApoAII amyloid primarily in intestine, heart and lung, and reactive AA amyloid deposits primarily in spleen, liver, kidney, and gut in mice with chronic dermatitis. (Gruys et al. 1996) Disease status, intrastrain aggression, selftrauma and skin lesions have been implicated in amyloidosis in Swiss mice. (Homburger et al. 1975; Maita et al. 1988; Engelhardt et al. 1993) Incidence of amyloidosis in mice less than 1 year old may be <3%. (Majeed 1993) A study of the progression of amyloidosis
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in CD-1® mice demonstrated amyloid only in duodenum, jejunum, ileum, mesenteric lymph node, and ovaries at 8 months of age. By 12 months, amyloid also was found in the adrenal gland, gall bladder, heart, ileum, kidney, pancreas, parathyroid, spleen, glandular stomach, testis, and thyroid. (Frith and Chandra 1991) Occasionally or rarely affected tissues include rectum, larynx, skeletal muscle, vagina, tongue, skin, eye, lacrimal gland, pituitary, preputial gland, mammary gland, prostate, seminal vesicle, sciatic nerve, cervical lymph node, pancreas, thymus, gall bladder, urinary bladder, uterus, renal lymph node, lumbar lymph node, and adipose tissue. (Frith and Chandra 1991; Majeed 1993) Other nonneoplastic conditions that have been considered to cause or contribute significantly to death on chronic studies include urinary tract obstruction in male mice with distended bladders and urethral plugs, sometimes called dysuria and similar to descriptions of mouse urologic syndrome (MUS) (Maita et al. 1988; Son 2003); renal disease including nephropathy, glomerulonephritis, glomerulosclerosis, or hydronephrosis, especially in female mice (Maita et al. 1988; Son 2003); skin lesions, including ulcers, abscesses, chronic dermatitis, especially in male mice (Maita et al. 1988; Son 2003) and sometimes attributed to fighting and self-trauma (Engelhardt et al. 1993); polyarteritis most commonly in thymus, ovary, uterus, kidney, and heart (Homburger et al. 1975; Maita et al. 1988; Son 2003); cardiomyopathy (Homburger et al. 1975; Son 2003), and left auricular thrombi especially in males (Maita et al. 1988). Cystic endometrial hyperplasia can be a common finding. (Maita et al. 1988; Engelhardt et al. 1993; Son 2003) Subcapsular spindle cell hyperplasia in the adrenal gland may be found in more than 80% of aged females and in fewer males. (Faccini et al. 1990). Spinal cord and sciatic nerve degeneration (Engelhardt et al. 1993), and ovarian cysts (Son 2003) are reported in some studies. Spontaneous Neoplasms
When Dr. Lynch’s Swiss mice were used for cancer research, necropsies were performed on all mice that died, and of those living 18 months or more the lung tumor incidence was 70-80%. Of mice living 6 months or more, 44% had lung tumors, 19% had mammary gland tumors, and about 1% had leukemia (lymphoma). (Lynch 1969) Hematopoietic, pulmonary, and hepatic neoplasms usually are the most common tumors and neoplastic causes of death in more recent chronic studies of CD-1® mice. Lymphoma incidences vary from 1-50% in females, 2-22% in males, and histiocytic sarcoma incidences from 2-18% in females and 1-8% in males, and there are occasional myeloid leukemias. Lung tumor incidences vary from 0-39% in females and 0-43% in males. Benign and malignant lung tumors are not distinguished in all studies, but when they are, benign exceed malignant, and malignant tumors may be implicated as a cause of death. Liver tumor incidences vary from 0-18% in females and 0-45% in males. Benign usually exceed malignant liver tumors, and malignant tumors may be implicated as a cause of
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death, sometimes with pulmonary metastasis. (Homburger et al. 1975; Maita et al. 1988; Engelhardt et al. 1993; Giknis and Clifford 2000; Son 2003) CFW Swiss mice have had a low reported incidence of spontaneous lymphomas and leukemias (Homburger et al. 1975; Fam and Mikhail 1996), but in a recent study of Mycobacterium leprae infection, mice of both control and infected groups developed splenomegaly and lymphadenopathy attributed to lymphoma, as early as 5 months of age. Over a two-year period, nearly 60% developed tumors, 85% of which were lymphomas, the majority of B cell origin. All tumors tested expressed ecotropic MuLVs. (Taddesse-Heath et al. 2000) Mammary tumors in female Swiss mice are less common in recent studies than in earlier reports by Lynch and Hauschka before the mice were cesarean derived. Incidences vary from 0 to 13% in female mice and usually are not recorded for male mice. The tumors usually are adenocarcinomas and may be implicated as a cause or contributor to death. Adenoma, adenoacanthoma, carcinosarcoma, and fibrosarcoma of the mammary gland also are reported occasionally. (Engelhardt et al. 1993; Giknis et al. 2000; Sher 1974; Homburger et al. 1975; Maita et al. 1988; Son 2003) Pituitary tumors are noted with incidences of up to 14% in female mice and up to 3% in male mice in some studies. (Engelhardt et al. 1993; Giknis et al. 2000; Homburger et al. 1975; Maita et al. 1988) Harderian gland tumors, usually adenomas in males, are noted in some studies with incidences of up to 14%. (Giknis et al. 2000; Maita et al. 1988) Hemangiomas or hemangiosarcomas of various tissues including spleen, skin, ovary, liver, heart, lymph nodes and bone marrow are common in some studies, with incidence of up to 18%. (Maita et al. 1988; Engelhardt et al. 1993; Giknis et al. 2000) Other neoplasms noted occasionally or at low incidences include gastric papilloma, carcinoma or squamous cell carcinoma; intestinal adenoma, carcinoma; gallbladder adenoma or carcinoma; pancreas acinar adenoma; nasal adenocarcinoma; renal adenoma or carcinoma; urinary bladder leiomyoma, leiomyoblastoma, or leiomyosarcoma; skin papilloma, squamous cell carcinoma, trichoepithelioma, basal cell tumor, fibroma, fibrosarcoma, hemangioma, hemangiosarcoma, nerve sheath tumors, neurofibroma, liposarcoma, rhabdomyosarcoma, or sarcoma; adrenal cortical adenoma, carcinoma, pheochromocytoma, or spindle cell tumor; thyroid C cell adenoma; follicular cell adenoma or carcinoma; pancreas islet cell tumor; parathyroid adenoma; pituitary adenoma or carcinoma; pituitary pars intermedia adenoma; brain ependymoma, oligodendroglioma, meningioma, or meningeal sarcoma; osteoma, osteosarcoma; ovary granulosa cell tumor, tubular, adenoma, luteal cell tumor, sertoliform adenoma, theca cell tumor; uterine sarcoma, leiomyoma, leiomyosarcoma, stromal cell sarcoma, stromal polyp, granular cell tumor; cervix squamous cell carcinoma, fibrosarcoma, hemangiopericytoma, nerve sheath tumor, myxoma, leiomyoma, leiomyosarcoma; vaginal papilloma,
660 polyp, adenocarcinoma, fibrosarcoma, leiomyoma, leiomyosarcoma; mast cell tumor; and mesothelioma. (Giknis et al. 2000) Related Stocks and Strains
While many of today’s important inbred strains derive from a relatively small group of non-Swiss mice developed by early “mousers,” including William Castle, Clarence Little, Leonell Strong, Jacob Furth, George Snell, Margaret and Earl Green, Howard Andervont, and others, several important inbred strains derive from the Swiss mice. (Morse 1978; Festing 1999; Beck et al. 2000; Paigen 2003; Rader 2005) Swiss-derived inbred strains include FVB/N, NOD, NON, SJL/J, and SWR. The FVB/N strain was developed in 1975 at NIH and has become important in the production of transgenic mice (more details above). NOD (nonobese diabetic) mice derive from Japanese ICR outbred stock that were selected for high fasting blood glucose. (Festing 1999) Diabetes development in NOD mice depends on environmental as well as on genetic factors (Leiter 1993). NON (nonobese nondiabetic) was separated from the main NOD diabetic colony at F13 and is one of several strains or stocks that has been used as control animals in studies of NOD mice. SJL/J mice were developed by James Lambert from three different sources of Swiss Webster mice (more details above). (Festing 1999; Jax 2004) SWR mice also derive from Clara Lynch’s inbred mice at the Rockefeller Institute that were transferred to Raymond Parker at the University of Toronto. (Festing 1999; Jax 2004) SENCAR outbred mouse stock, named for SENsitivity to CARcinogens, derive from crossing Charles River CD-1® mice with STS (skin-tumor-sensitive) mice (from Rockland Farm derived mice selected for sensitivity to skin tumor induction and maintained by non-inbred mating systems). SENCAR mice have a large database for sensitivity to carcinogens and promoters. The order of susceptibility within several mouse stocks and strains to epidermal two-stage carcinogenesis, using DMBA-initiation and TPA-promotion, has been summarized as follows: SENCAR > DBA/2 > CD-1® > C3H > BALB/c > C57BL/6 (Slaga 1986; NTP 1996; Coghlan et al. 2000). Compared to BALB/c mice, SENCAR mice are more susceptible to chemically induced skin tumors, and less susceptible to chemically induced lung, vascular, and uterine tumors. (Ward et al. 1986) While they are susceptible to chemically induced skin tumors, SENCAR mice do not seem to be unusually susceptible to spontaneous tumors. (Conti et al. 1985) In a chronic study in which 50% of control mice survived past 96 weeks of age, glomerulonephritis was the most common nonneoplastic condition, and inflammatory changes also were common. The most common neoplasms were histiocytic sarcoma, pulmonary adenoma or adenocarcinoma, mammary tumors, follicular center
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cell lymphoma, and hepatocellular adenoma. (Ward, Quander, et al. 1986; Ward, Rehm, et al. 1986) Several inbred lines or strains have been developed from outbred SENCAR mice, including SencarA/PtCr, SENCARB/PtJ, and SENCARC/PtJ, developed by Dr. Michael Potter (Pt) of the National Cancer Institute and currently maintained at NCI, Frederick (Cr), or by the Jackson Laboratory (J) as cryopreserved lines in repository, as well as the SSIN strain that was developed by Dr. Claudio Conti. These inbred strains have increased sensitivities to various carcinogen protocols compared to outbred SENCAR mice. (Hennings et al. 1997; Stern et al. 1998, 2002; JAX® 2004) Non-Swiss CF-1® Non-Swiss CF-1® stock with overall tumor incidences of 39-84% has been reported to have lower overall tumor incidence in chronic studies than CD-1® and CFW® Swiss stocks (Sher 1974). IV.
CONCLUSION
Historical data presented here or elsewhere do not obviate concurrent relevant controls in well-designed studies. Results vary profoundly with the source, strain, or substrain, age and sex of the animals, the environment in which they are or were maintained, the methods of evaluation, and the interpretations of the findings (e.g., inflammation vs. lymphocytic infiltration; adenoma vs. carcinoma vs. tumor). “Spontaneous” lesions, and morphologic differences between mice are phenotypes. They are subject to interpretation or misinterpretation as the result of genetic manipulation or another experimental manipulation. They may be a direct result of such manipulations, or they may be enhanced or diminished by the manipulations. Awareness of the spectrum of pathologic and other phenotypes of the mice under study facilitates and improves experimental design and interpretation of findings. This effort to summarize many years of findings from diverse studies necessarily excludes many details of the original studies. The original references may (or may not) include specifics regarding mouse strain, housing, methods of evaluation, or descriptions of pathologic findings. Examination of the original references for such details is recommended.
ACKNOWLEDGMENTS Many people have provided helpful information, editorial assistance, and advice. I apologize for any errors and offer sincere thanks to Stephen Barthold; Charles Frith; Wanda Haschek-Hock; Emily Bailey; Angela Brice; Nancy Everds; Nadine Forbes; Robert Hackman; Vida Hortenstine; Carol Linder; Peter Mann; Pat Mirley; Chuck Montgomery; Sandy Morse; Harshan Pisharath; Dawn Ruben; John Sharp; Brian Simons; Theresa Southard; John Sundberg; Paul Szauter; Gale Taylor; Ann Thompson; Jerrold Ward; Roxane Walden; and to the editors especially for their patience.
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V. KEY TO TABLES Table 25-1 Attributes of some common mouse strains and stocks. This table summarizes some important features of the mice discussed in this chapter. Table 25-2 Sources and origins of some Swiss derived mice and non Swiss ‘outbred’ mice available in North America. This table summarizes some of the history and characteristics of these mice. Table 25-3 Spontaneous non neoplastic conditions in some commonly used mouse strains or stocks. This table details
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incidences of nonneoplastic conditions the strains and stocks for which recent studies were available. Table 25-4 Spontaneous neoplasms in some commonly used mouse stains or stocks. This table details incidences of neoplasms in the strains and stocks for which recent studies were available. Table 25-5 Glossary by system: Non-neoplastic conditions. This table briefly defines or describes most of the nonneoplastic conditions discussed in this chapter. Table 25-6 Glossary by system: Neoplasms. This table briefly defines or describes most of the neoplastic conditions discussed in this chapter.
TABLE 25-1
ATTRIBUTES OF SOME COMMON STRAINS AND STOCKS Strain Name (abbreviation) 1
Color
Color Genotype
H2 Haplotype
129 (129) 2 A (A)
Agouti, etc.
Variable; often Aw, some p, d, etc. a/a Tyrp1b/Tyrp1b Tyrc/ Tyrc
Variable
AKR (AK) BALB/c (C) C3H (C3)
Albino
a d
hypocallosity; cardiac calcinosis; myoepitheliomas; induced plasmacytomas
Agouti
a/a Tyrc/ Tyrc a+/a+ Tyrp1b/Tyrp1b Tyrc/ Tyrc a+/a+
ES cell source; substrain variable features: +/- ahl 3, hypocallosity/acallosity, teratoma, hyalinosis, acidophilic macrophage pneumonia, etc. ahl; Apoa2c; 4 cleft lip/ palate; lung tumors; teratogen susceptibility; Helicobacter susceptibility; Dysfprmd 5 muscular dystrophy in A/J Apoa2a 6; thymic precursor T cell lymphoma
k
C57BL/6 (B6)
Black
a/a
b
DBA/2 (D2) FVB/N SJL/J (SJL) B6;129
Dilute brown
Myo5ad/Myo5ad Tyrp1b/Tyrp1b a/a a+/a+ Tyrc/ Tyrc p/p Tyrc/Tyrc Variable
d
rd1 7; cardiac calcinosis; alopecia areata; mammary tumors in F8; liver tumors M>F Lps-d 9 in C3H/HeJ Ahl; Apoa2a; microphthalmia; hydrocephalus; barbering/dermatitis F>M; amyloidosis; acidophilic macrophage pneumonia; lymphoma F>M; histiocytic sarcoma; pituitary tumors F>M Ahl; Apoa2a; cardiac calcinosis; glaucoma
q s
rd1; Fv1b 10 large pronuclei; seizures; mammary hyperplasia rd1; Apoa2c; Dysfim 11 muscular dystrophy; lymphoma; aggressive males
Variable
a+/a
b/k
Tyrc/Tyrc etc.
Variable
Variable genetic contributions of B6, 129; phenotype variability should be expected, e.g., hyalinosis, acidophilic macrophage pneumonia; lymphoma Robust hybrid; liver tumors M>F; lymphoma F>M, lung tumors, pituitary tumors F>M ; Harderian tumors Robust non-inbred; stock/source variable features e.g. +/-rd1; amyloid; lymphoma; lung tumor; liver tumor
B6C3F1 Swiss stocks
1From
Albino
Albino
Albino Albino Variable Agouti (dark) Albino
a
Eppig, 2004; Eppig, 2007; for additional details and abbreviations see these sources and subsequent updates. possible, abbreviations should indicate the substrain according to current nomenclature guidelines (Festing et al. 1999; Eppig 2004, 2007): 129P1, 129P2, 129P3, 129S1, 129S2, 129S4, 129S5, 129S6, 129S7, 129S8, 129T1, 129T2, 129X1. 3Ahl, age-related hearing loss 1 allele of Cdh23 (cadherin 23) gene. 4c (highly amyloidogenic) allele of Apoa2 (apolipoprotein A-II ) gene. 5prmd (progressive muscular dystrophy) allele of Dysf (dysferlin) gene. 6a allele of Apoa2 (apolipoprotein A-II ) gene. 7rd1 (retinal degeneration) allele in Pde6b (phosphodiesterase 6B, cGMP, rod receptor, beta polypeptide) gene. 8F, female; M, male. 9Lps-d (defective lipopolysaccharide response) allele in Tlr4 (Toll-like receptor 4) gene. 10b allele of fv1 (Friend virus susceptibility 1) gene. 11im (inflammatory myopathy) allele of Dysf (dysferlin) gene. 2Whenever
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TABLE 25-2
SOURCES AND ORIGINS OF SOME SWISS-DERIVED MICE AND NON-SWISS OUTBRED MICE AVAILABLE IN NORTH AMERICA Name [abbreviations, common names]1
Source or Vendor
Crl:CD-1®(ICR) [CD-1®14]
Rd2
Inbred/Non-inbred
History
Charles River Laboratories
0/60
Non-inbred
Crl:CF-1® [CF-1®14]
Charles River Laboratories
1/60
Non-inbred
Crl:CFW®(SW) [CFW®14]
Charles River Laboratories
59/60
Non-inbred
Hsd:ICR(CD-1®) [ICR]
Harlan
27/60
Non-inbred
Hsd:ND4 [ND4, Swiss Webster]
Harlan
Hsd:NIHS [NIH Swiss] Hsd:NSA™(CF-1®)15 [Non-Swiss Albino] SJL/J
Harlan
rd1/rd1
Non-inbred
Harlan
0/60
Non-inbred
The Jackson Laboratory
rd1/rd1
Inbred
SWR/J
The Jackson Laboratory
rd1/rd1
Inbred
FVB/N
NIH/NCI
rd1/rd1
Inbred
Cr:NGP(S) (NIH general purpose) Cr:NIH(S) (NIH Swiss) NIH Black Swiss
NIH/NCI
Non-inbred
NIH/NCI
Non-inbred
NIH/NCI
Non-inbred
Bom: [NMRI]
Taconic EU
Non-inbred
IcrTac:ICR [ICR]
Taconic
Swiss mice, from Lynch (at Rockefeller) to various sources, to Landis, to Hauschka, ICR (Ha/ICR) in 1948; to Mirand at RPMI (HaM/ICR); to CRL in 1959; Cesarean derived (CD). Charles River Laboratories, 2004, 2006. (Hauschka et al. 1973; Lang et al. 1996; Mirley 2004) Non-Swiss mice, probably of wild albino origin, from a Missouri laboratory; inbred by Carworth Farms (CF) >20 generations, outbred from a single pair as CF-1 stock; cesarean derived in 1974 by CRL “from a representative cross section of the Carworth CF-1 colony”. (Charles River Laboratories 2004, 2006) Swiss mice, from Lynch, inbred by Webster, at Rockefeller; outbred from a single pair by Carworth Farm as CFW; CD in 1974 by CRL “from a representative cross section of the Carworth CFW colony”. (Charles River Laboratories 2004, 2006) Swiss mice descended from Crl:CD1(ICR) stock obtained from CRL before 1980. (Harlan 2004, 2006) Swiss mice descended from Swiss Webster stock rederived by the University of Notre Dame, Notre Dame, Indiana; obtained by Harlan before 1980. (Harlan 2004, 2006) Swiss mice derived from NIH Swiss N:NIH(S) nucleus colony (Harlan 2004, 2006) Non-Swiss mice descended from Crl:CF1® stock obtained from CRL before 1980. (Harlan 2004, 2006) Descended from three sources of Swiss Webster mice brought to TJL between 1938 and 1943; pen bred until 1955; then inbred by J Lambert at TJL. (Festing 1999) Swiss mice inbred by Lynch at Rockefeller; to Parker, University of Toronto 1926; to TJL 1947 at F28+. (Festing 1999) Swiss mice derived from NIH Swiss N:NIH(S) 1936; selectively bred for HSFS/N ~1966; then selected for sensitivity to Friend leukemia virus B strain; inbred from 1975 at NIH; transferred to various suppliers (NIH 1982; Beck et al. 2000) Swiss mice from Lynch or Webster at Rockefeller to NIH; outbred as N:GP(S) 1935 (NIH 1982; Beck et al. 2000) Swiss mice derived from N:GP(S) as N:NIH(S) in 1936 (NIH 1982; Beck et al. 2000; NCI-APP 2006) Developed at NIH by Dr. Carl Hansen. N:NIH Swiss outbred mice were crossed to C57BL/6N mice to generate hybrid black mouse heterozygous for (non)agouti loci; ‘agouti gene was eliminated’ via test matings and backcrosses (N10) to N:NIH(S). (NCI-APP 2006) Swiss mice from Lynch or Webster at Rockefeller to Poiley NIH 1937; inbred by Poiley as NIH/Pl; NMRI in 1951 (NIH 1982; Beck et al. 2000). Random bred at Zentralinstitut für Versuchstierzucht, Hannover; M&B A/S. (Taconic’s Bomholt, Denmark facility, Laboratory code Bom) in 1961, 1985 (Taconic 2004, 2006) Swiss mice from Lynch, Rockefeller; transferred to Hauschka, ICR (Ha/ICR) in 1948; reconstituted from RPMI Ha/ICR stock ~1959; Fox Chase (ICR) to Taconic 1993 (Hauschka et al. 1973; Taconic 2004, 2006)
Non-inbred
48/60
Non-inbred
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TABLE 25-2
SOURCES AND ORIGINS OF SOME SWISS-DERIVED MICE AND NON-SWISS OUTBRED MICE AVAILABLE IN NORTH AMERICA—cont’d Name (common names, abbreviations) NTac:NIHBS [Black Swiss] [Swiss Webster]
Source or Vendor
Rd2
Inbred/Non-inbred
History
Taconic
rd1/rd1
Non-inbred
Taconic
47/60
Non-inbred
From NIH Black Swiss stock (source of Cr: NIH-BL(S)) transferred to Taconic in 1991, CD in 1992 (Taconic 2004) Swiss mice descended from Swiss Webster original stock from the Rockefeller Institute through Rockland Farms, Inc. to Taconic in 1940 (Taconic 2004)
1For non-inbred stocks, abbreviations are those listed by the primary breeder or source. Similarities among designations are substantial. CD-1®, CF-1®, CFW® are registered trademarks of Charles River Laboratories, Inc. NSA™ is a registered trademark of Harlan. 2Ratios in this column refer to numbers of mice evaluated that had retinal degeneration compatible with homozygosity for Pde6brd1, per Serfilippi et al. 2004. rd1/rd1 indicates that the inbred strain is known to be homozygous for Pde6brd1, or that the stocks (Hsd: NIHS and NTac: NIHBS) have been shown to be homozygous for Pde6brd1 (Clapcote et al. 2005).
TABLE 25-3
SPONTANEOUS NONNEOPLASTIC CONDITIONS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS SYSTEM Organ Condition GENERALIZED Multisystem condition Amyloidosis (systemic) Arteritis (systemic), polyarteritis ALIMENTARY Esophagus, dilatation; megaesophagus Gallbladder, inflammation Gallbladder, hyalinosis
129S4/ SvJae1
BALB/c2
C3H3
C57BL/6
FVB/N4
SJL
B6;1291
Listed by site
F 2%, M0
F 33% M 27% F 33% M 18% F 11% M 3%
F 6%, M 10%
F 15%, M7%
Liver, angiectasis Liver, centrilobular hypertrophy Liver, extramedullary hematopoiesis Liver, fatty change; vacuolization, hepatocellular
B6C3F15
F 55% M 5%
F 29%, M 22% VF 9% BF 7% VM 26% BM 73%
VF 1% BF 1% VM 22% BM 63%
F 2%, M 2% F 2%, M 6% F 6%, M 2% F 6%, M 2%
1Unless indicated, values in this column are adapted from Ward et al. 2000; Ward et al. 2001, study of 89 male and female SPF 129S4/SvJae, maintained through 27months of age. F, female; M, male. 2Unless indicated, values in this column are adapted from Frith et al. 1983, study of 3972 virgin and breeder BALB/cStrlfC3Hf/NCtr and C57BL/6JfC3Hf/NCtr (BALB/cStrl and C57BL/6J fostered onto gnotobiotic C3H/He) mice and barrier maintained up to 689 days. Data from animals over 500d are presented here. VF, virgin female; BF, breeder female; VM, virgin male; BM, breeder male. 3Unless indicated, values in this column are adapted from Hall et al. 1992, study of 298 male and 68 female C3H breeding mice sacrificed at 6–8 months of age. Mice were seronegative for viruses and Mycoplasma pulmonis. Helicobacter status unknown. 4Unless indicated, values in this column are adapted from Mahler et al. 1996, study of 29 male and 116 female SPF FVB/NTac mice sacrificed at 24 months of age. Information derived from the text of the report is indicated Y for yes, to indicate that the lesion occurred but the incidence was not given. 5Unless indicated, values in this column are adapted from NTP 2000, study of 50 male and 50 female naïve control B6C3F1 mice in a two-year study. Continued
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TABLE 25-3
SPONTANEOUS NONNEOPLASTIC CONDITIONS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS—cont’d SYSTEM Organ Condition
129S4/ SvJae1
BALB/c2
Liver, foci of cellular alteration Liver, inflammation or mononuclear infiltrates
F 36% M 33%
Liver, necrosis random or unspecified Pancreas, exocrine atrophy
VF 21% BF 39% VM 11% BM 9%
VF <1% BF 2% VM 0 BM 0
Pancreas, inflammation, chronic Salivary gland, inflammation
Teeth, dysplasia CARDIOVASCULAR Heart, cardiomyopathy Heart, coronary arteritis
F0 M <1% F 11% M 2%
F0 M 5% F0 M0
C57BL/6
FVB/N4
VF 7% BF 3% VM 6% BM 5%
Adrenal gland, pigment ceroid
6This
B6C3F15 F 22%6 M 54%
F 74% M 56%
F 8%, M 12% F 4%, M%
VF 1% BF 0 VM 1% BM 0
F 2% M0
F 56% M 42% VF 0 BF 2% VM 4% BM 2%
VF 5% BF 1% VM 1% BM 1% F 8% M 16%
F 48% M 31% F 46%, M 21%
F 17% M 16% F 6% M 8% F0 M <1%
F 19% M 49%
F 2% M 12%
F 40% M 75% F 6% M 35%
F 18%, M 20%
F 46% M 33% F 33% M12%
VF 1% BF 2% VM 11% BM 5% VF <1% BF 2% VM <1% BM 5%
F 33% M0
VF 11% BF 3% VM 16% BM 2% VF 3% BF 4% VM 0 BM 0
Heart, thrombus
ENDOCRINE Adrenal cortex, subcapsular cell hyperplasia
B6;1291
F 3% M 10%
Heart valves, melanosis Heart, mineralization, cardiac calcinosis
SJL
F 3% M <1%
Salivary gland (submandibular/submaxillary), duct hyperplasia Stomach, forestomach; squamous epithelial hyperplasia focal or diffuse Stomach, glandular; epithelial hyperplasia Stomach, glandular; epithelial hyalinosis Teeth, alveolitis
C3H3
F 28% M 6% F20/207 M1/10
VF 0 BF 0 VM 0 BM 0 VF 0 BF 0 VM 0 BM 0 VF 0 BF 0 VM 0 BM 0 VF 1% BF <1% VM 0 BM 0
F 83% M 14%
F 86% M 12%
F 4%, 0 M 6%
F 65% M 26%
is the sum of the incidence for basophilic focus, clear cell focus, eosinophilic cell focus, mixed cell focus. et al. 1996 study of 20 female and 10 male SPF C3H/HeN mice at approximately 6 months of age.
7Vargas
Continued
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TABLE 25-3
SPONTANEOUS NONNEOPLASTIC CONDITIONS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS—cont’d SYSTEM Organ Condition Pancreas, islet hyperplasia Parathyroid gland, melanosis
129S4/ SvJae1
VF 4% BF 6% VM 4% BM 4%
Pituitary pars distalis, hyperplasia
F 5% M 3%
Pituitary pars intermedia, hyperplasia
F 71% M 50%
Thyroid gland, arteritis
F 2% M 7%
Thyroid gland, follicular cell hyperplasia Thyroid gland, inflammation; lymphocytic infiltrate GENITAL, FEMALE Ovary, atrophy
F 29% M 13%
Ovary, cyst
F 42%
Ovary, hemorrhage + angiectasis or hematocyst Ovary, hyaline material Ovary, pigment, ceroid
F 15%
C57BL/6
FVB/N4
SJL
B6;1291
B6C3F15
F 29% M 45% F 93% M 88%
F 30% M 71%
F 11% M 4%
F 27% M0
VF 1% BF 0 VM 17% BM 4% F 7% M0 VF 52%8
F 26% M 16%
F 80%
F 56% F 51% F 25% F 60%
Uterus, hydrometra Uterus, mineralization GENITAL, MALE Epididymis, granulomatous inflammation or sperm granuloma Preputial gland, cystic ducts Preputial gland, inflammation Prostate gland, atypical epithelial hyperplasia Testes, atrophy or degeneration tubular Testes, interstitial (Leydig) cell hyperplasia Testes, mineralization
C3H3
F 28%, M 46% F 33% M 8%
Pituitary gland, cyst
Uterus, angiopathy Uterus, cystic endometrial hyperplasia Uterus, hemosiderosis
BALB/c2
F 60% M 4% VF 2% BF 0 VF 17% BF 8% VF 0 BF 0
VF 18% BF 0 VF 35% BF 16% VF 0 BF 2%
VF 12% BF 3%
VF 67% BF 40%
VF 4% BF 2% VF <1% BF 36% VF 8% BF 4% VF <1% BF 67%
VF 27% BF 30% VF 0 BF 16% VF 3% BF 13% VF 0 BF 7%
F 76% F 31%
F 24%
F 15%
F 12%
F 4% F 47%
F 72%
F 56%
M 31%
M 2%
M 46% M 2%
M 6/79 M 29% M 64%
M 8% VM 2% BM 1%
M 2% M 3%
M 56%
8Wakefield et al. 2003. VF, virgin female (n = 21); BF, breeder (multiparous) female (n = 6), incidence in FVB/N mice at 18 months old, see text for additional details. 9Six of seven glands examined had inflammatory changes. Continued
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TABLE 25-3
SPONTANEOUS NONNEOPLASTIC CONDITIONS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS—cont’d SYSTEM Organ Condition
129S4/ SvJae1
HEMATOPOIETIC Bone marrow, hyperplasia granulocytic Bone marrow, hyperplasia myeloid
C57BL/6
Bone marrow, myelofibrosis vertebra Lymph node, lumbar, infiltration histiocyte / macrophage Lymph node, lumbar, pigment
F 25% M 7%
SJL
B6;1291
F 2% M 49% F 67% M 31% F 67% M 31%10
Spleen, hyperplasia erythropoietic
Spleen, hyperplasia granulopoietic
F 54% M 74%
F 19% M0 F 6% M0
VF 8% BF 11% VM 11% BM 5% VF 5% BF 2% VM 4% BM 2% VF 3% BF 4% VM 2% BM 7% VF 7% BF 3% VM 5% BM 0
VF 8% BF 4% VM 7% BM 3% VF 7% BF 2% VM 2% BM 1% VF 8% BF 4% VM 4% BM 6% VF 9% BF 1% VM 4% BM 1%
Spleen melanosis F 83% M 62% VF 2% BF 4% VM 7% BM 5%
F 11% M0
F 44% M 46% F 10% M 6%
F 28% M 29% F 2% M0
F 6% M 4% F 21% M 16%
F 67% M 93%
VF 6% BF 3% VM 6% BM 2%
Thymus, hyperplasia lymphoid
F 28% M0
INTEGUMENT Skin, chronic active inflammation Skin, ulcer, inflammation
F 20% M 25%
MUSCULOSKELETAL Degenerative joint disease Bone hyperostosis endosteal
10Marginal
B6C3F15
F 17% M0 F 17% M% F 10% M%
Spleen, hyperplasia reticular
Thymus, atrophy cortex or lymphoid depletion Thymus, cysts
FVB/N4
F 2% M 4% F 37%, M0
Spleen, extramedullary hematopoiesis Spleen, hyperplasia lymphoid
C3H3
F 54% M 51%
Bone marrow, myelofibrosis (site unspecified) Bone marrow, myelofibrosis femur
Lymph node, mesenteric, hyperplasia lymphoid Spleen arteritis
BALB/c2
F 2% M0 F 2% M0
F 22%, M 10% F 40% M 39%
zone hyperplasia specified. Continued
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TABLE 25-3
SPONTANEOUS NONNEOPLASTIC CONDITIONS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS—cont’d SYSTEM Organ Condition NERVOUS Brain, melanosis meninges
129S4/ SvJae1
VF 3% BF 0 VM 7% BM 0 F 87% M 67% F 52% M 87%
Lung, alveolar epithelial hyperplasia Lung, lymphoid infiltrate perivascular peribronchiolar
Lung, mineralization Nose, inflammation Nose, olfactory epithelium hyalinosis Nose, respiratory epithelium hyalinosis Nose, septal amyloid like material Trachea, inflammation Trachea, hyalinosis Trachea, mucosal gland; acidophilic crystals SPECIAL SENSES Ear, otitis media Eye, cataract
C3H3
C57BL/6
F 32%, M 23%
Brain, thalamus mineralization
RESPIRATORY Lung, acidophilic macrophage pneumonia Lung, acidophilic crystals
BALB/c2
F0 M 1%
VF <1% BF 0 VM <1% BM 0
VF 4% BF 8% VM 3% BM 1%
VF 20% BF 0 VM 6% BM 0
VF 6% BF 0 VM 9% BM 8%
F 1% M <1% F 12% M 4%
F 65% M 68% F 4%, M 6%
F 33% M 18% F 65% M 36%
F0 M 2% F 51% M 42% F <1% M0
F 79% M 84% F 6% M0 F 9% M0
F 13% M 13%
F 54% M 15%
B6C3F15
F 78% M 32% F 94% M 58% F 100% M 100%
Eye, keratitis neovascularization
Harderian gland, inflammation
B6;1291
VF 10% BF 0 VM 6% BM 0
F 29% M 13% F 25% M 19% F 19% M 3%
Harderian gland, ectopic
SJL
F 2%, M 8%
F 25% M 20% F 17% M 20% F 19% M 3% F 77% M 31%
Eye, retinal atrophy
FVB/N4
VF 1% BF 6% VM 1% BM 5% VF 4% BF 0 VM 0 BM 0 VF 14% BF 8% VM 9% BM 5%
F0 M <1%
VF 0 BF <1% VM 0 BM 0 VF 12% BF 3% VM 10% BM 3% VF 29% BF 4% VM 48% BM 10% Continued
TABLE 25-3
SPONTANEOUS NONNEOPLASTIC CONDITIONS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS—cont’d SYSTEM Organ Condition
129S4/ SvJae1
URINARY Kidney, amyloidosis
Kidney, chronic nephropathy Kidney, chronic inflammation (lymphocyte infiltration) Kidney, glomerulonephritis
BALB/c2
C3H3
VF <1% BF 3% VM 0 BM 1% F 81% M 95% F 28% M 54%
C57BL/6
FVB/N4
SJL
B6;1291
VF 10% BF 0 VM 6% BM 0 F0 M0 F 3% M 1%
F 14% M 68% F 2%, M0 VF <1% BF <1% VM <1% BM 1%
VF 1% BF 0 VM <1% BM 0
F 71% M 52%
Kidney, “glomerulosclerosis” Kidney, hydronephrosis
F0 M 2%
Kidney tubule ,hyaline droplets Kidney tubule, mineralization
F 77% M 97%
Urinary bladder, inflammation (cystitis)
Urinary obstruction “dysuria,” MUS
F 2% M% F 1% M 3% F 6% M 32% F 63% M 8%
Eye, corneal opacity, mineralization17 11MUS
VF 1% BF 2% VM <1% BM 3%
VF 1% BF <1% VM <1% BM 1%
yes11
Studies that compare inbred strains Dental malocclusion12
Uterus, mucometra, imperforate vagina13 Brain, corpus callosum absent or small Brain, hydrocephalus16
F 6% M0 F 40% M 54%
F 15% M 1%
Kidney tubule, pigment (hemosiderin) Kidney tubule, regeneration
Urinary bladder, arteritis
B6C3F15
0.0018% c 0.0015% By
2 –80%14
0.0047%
12/44 4/35 ≥50%15
0.046% (6) 0.089% (Ks) 6/44 10/35
0.00023% c
0.0000%
0.029% 6 0.36% Ks
0.00075% By 10%
16%
4%
0.0019%
4/44
0.0000%
was listed as a cause of death in 5% of male mice on this study, but incidence of the condition is not indicated. from JaxNotes 2003 #489, incidence of malocclusion over 1 year for BALB/cJ (c), BALB/cByJ (By), C3H/HeJ, C57BL/6J, C57BLKS/J (Ks), DBA/2J, SJL/J are given here and discussed in text. 13Results in this row are from Jax notes 1990 #441, and Sundberg and Brown 1994. They represent the number of cases diagnosed in that strain/total number of cases diagnosed over two different periods (1987–1989 and 1990–1991), at the Jackson Laboratory. Strains are BALB/cJ, C57BL/6J, DBA/2J, SJL/J. 14Various strains, substrains of 129sv Balogh et al. 1999; Magara et al. 1999; Schalomon and Wahlsten 2002; Wahlsten 1982; Wahlsten et al. 2001; Wahlsten et al. 2003 re various strains, substrains of 129sv. 15Livy et al. 1997. 16Adapted from JaxNotes 2003 #490, incidence of hydrocephalic mice culled at weaning, over 1 year for BALB/cJ (c), BALB/cByJ (By), C3H/HeJ, C57BL/6J, C57BLKS/J (Ks), DBA/2J, SJL/J are given here and discussed in text. 17DBA/2 mice had 29% incidence in this study. (Van Winkel and Balk 1986) 12Adapted
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TABLE 25-4
SPONTANEOUS NEOPLASMS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS SYSTEM Organ, Neoplasm
129S4/ SvJae1
ALIMENTARY Intestine, polyp4
Intestine, adenoma Intestine, adenocarcinoma
BALB/c2
C3H
VF 1% BF 1% VM 2% BM 9%
C57BL/62
FVB3
B6C3F121
VF 1% BF 2% VM 1% BM 0
F 2% M0 F0 M 3%
F 4% M 4%
Intestine, cecum, carcinoid/fibroma
F 1% M0
Intestine, fibrous histiocytoma
F0 M 2%
Liver, tumor
Liver, adenoma(s)
VF 1% BF 2% VM 2% BM 12% F 6% M 15%
VF 2% BF 0 VM 5% BM 5% F0 M 1%
F0 M 21%
F 30% M 44% F 6% M 24%
Liver, carcinoma(s) Liver, hemangioma Liver, hemangiosarcoma Liver, hemangioma hemangiosarcoma Liver, hepatoblastoma
F0 M 2%
F 2%, M0 F0 M 2%
Salivary gland, myoepithelioma
Heart, angioma
F 10% M 12% F0 M 2% F 2% M0 F0 M 2%
F 2% M 8%
Pancreas, hemangioma
Stomach forestomach, squamous papilloma Stomach forestomach, squamous cell carcinoma CARDIOVASCULAR Heart, angiosarcoma
B6;129
VF 0 BF 1% VM 0 BM 0
VF 0 BF 0 VM 0 BM 0
VF 2% BF 6% VM 2% BM 2% VF 3% BF 0 VM 0 BM 0
VF 3% BF 0 VM 3% BM 14%
F 2% M0 F 2% M0
1Adapted
from Ward et al. 2000; Ward et al. 2001, study of 89 male and female SPF 129S4/SvJae, maintained through 27months of age. F, female; M, male. from Frith et al. 1983, study of 3972 virgin and breeder BALB/cStrlfC3Hf/NCtr and C57BL/6JfC3Hf/NCtr (BALB/cStrl and C57BL/6J fostered onto gnotobiotic C3Hf/NCtr) mice and barrier maintained up to 689 days. Data from animals surviving >500 d are listed here. VF, virgin female; BF, breeder female; VM, virgin male; BM, breeder male. 3Adapted from Mahler et al. 1996 study of 29 male and 116 female SPF FVB/NTac sacrificed at 24 months of age. 4Polyp was listed as a neoplasm in this study, and the term may refer to polypoid adenomas here. 2Adapted
Continued
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TABLE 25-4
SPONTANEOUS NEOPLASMS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS—cont’d SYSTEM Organ, Neoplasm ENDOCRINE Adrenal cortex, adenoma
Adrenal cortex, adenocarcinoma Adrenal medulla, pheochromocytoma Pancreas islet, adenoma
129S4/ SvJae1
F 3% M0
Thyroid follicular cells, adenoma
VF 8% BF <1% VM 6% BM 2%
C3H
C57BL/62
VF 0 BF 0 VM 0 BM 0
VF 11% BF 8% VM <1% BM 2%
F 13%
VF <1% BF 0
VF 4% BF <1%
Ovary, cystadenoma Ovary, granulosa cell tumor
F 10%
VF 1% BF 2%
VF 0 BF 0
Ovary, hemangiosarcoma Ovary, luteoma
F 2% F 2%
VF <1% BF <1%
VF 0 BF 0
B6;129
F 6% M 4% F 3% M0 F 14% M0 VF 19%5 BF 83%5
F 1% F 3%
F 2% M 2% F 6% M0
F 2% M0 F0 M 4% F 2% M 2%
F 2% M0
F 6% M 2% F 2%, M0
F 6%
F 17%
F 1% F 1%
F 2% F 27% F 2% F 15% F 8%
B6C3F121
F0 M 2%
F 2% M0 F 2% M0
Thyroid follicular cells, carcinoma GENITAL, FEMALE Ovary, adenoma
Ovary, teratoma Ovary, theca Sertoli cell tumor Ovary, tumor of any type Uterus, adenoma endometrial Uterus, hemangioma Uterus, hemangiosarcoma Uterus, polyp stromal
FVB3
VF 0 BF 0 VM 0 BM 0
F 2% M 11% F 2% M0
Pituitary gland, adenoma
Thyroid C cells, carcinoma
BALB/c2
F 2% F 2% VF 21% BF 36%
VF 1% BF 0
F 2%
F 4%
Uterus, leiomyoma Uterus, leiomyosarcoma Uterus, sarcoma stromal GENITAL MALE Testes, interstitial (Leydig) cell tumor Testes, teratoma Prostate, adenocarcinoma Seminal vesicle or coagulating gland, adenoma or carcinoma HEMATOPOIETIC Histiocytic sarcoma
F 4%
F 2% VM 2% BM 2%
VM <1% BM 0
M 1<%
VF <1% BF 0 VM 0 BM 2%
VF 5% BF 1% VM 2% BM 1%
F 6% M0
M 3%
F 4% M0
F0 M 2%
F 6% M 6%
5Wakefield et al. 2003. VF, virgin female (n = 21); BF, breeder (multiparous) female (n = 6), incidence in FVB/N mice at 18 months old, see text for additional details.
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TABLE 25-4
SPONTANEOUS NEOPLASMS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS—cont’d SYSTEM Organ, Neoplasm
129S4/ SvJae1
Lymphoma (any type)6
F 6% M 2%
BALB/c2
C3H
C57BL/62
FVB3
B6C3F121
B6;129
VF 34% BF 11% VM 15% BM 12%
F0 M0
VF 32% BF 11% VM 21% BM 9%
F 6% M0
F 16% M 14%
F 67% M 42%
Marrow, hemangioma
F 2% M% F0 M 2%
Marrow, mast cell tumor malignant Spleen, hemangioma
F 1% M0
Spleen, hemangiosarcoma
F 2% M 2% F0 M 2%
Spleen, mast cell tumor INTEGUMENT Mammary gland; tumor
F0 M0
VF 1% BF 3%
VF 0 BF 0 Y7
Mammary gland, adenocarcinoma Skin, fibrosarcoma Skin, fibrous histiocytoma Skin, hemangioma or hemangiosarcoma Skin, liposarcoma
F0 M 7%
F 1% M0 F 1% M0
Skin, mast cell tumor
F0 M 2%
Skin, neural crest tumor Skin, squamous papilloma
F 3% M 10% F 1% M0
F 2% M0
MUSCULOSKELETAL Bone, metastatic tumor NERVOUS RESPIRATORY Lung tumors
F 2% M0
F 31% M 62%
Lung tumors, metastatic Lung, adenoma(s) Lung, carcinoma(s) Nasal cavity, hemangioma Nasal cavity, schwannoma
6Because
F 2% M0 F 6% M% F0 M 2% F 2% M0
F 25% M 40% F 8% M 30% F0 M 3% F0 M 3%
VF 28% BF 30% VM 42% BM 41%
F0 M <1%
VF 3% BF 0 VM 6% BM 1%
F8 14 14% F 24 37% M 14 13% M 24 41%
F 65% M 36%
F 4% M 10% F 8% M 14% F 8% M 8%
F 18% M 30% F 4% M 2%
of variations in classification of lymphomas, leukemias, these values represent the sum of lymphomas reported. Nieto et al. 2003. Six carcinomas diagnosed in almost 500 FVB/N submissions included squamous carcinoma, adenosquamous carcinoma, adenocarcinoma. 8F24, M24 are data from mice sacrificed at 24 months. F14, M14 are from additional 98 females (F14) and 45 males (M14) sacrificed at 14 months of age. Continued 7
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TABLE 25-4
SPONTANEOUS NEOPLASMS IN SOME COMMONLY USED MOUSE STRAINS OR STOCKS—cont’d SYSTEM Organ, Neoplasm
129S4/ SvJae1
SPECIAL SENSES Harderian gland, tumor
Harderian gland, adenoma Harderian gland, adenocarcinoma URINARY Kidney, adenoma
Kidney, adenocarcinoma
BALB/c2
C3H
VF 14% BF 9% VM 9% BM 8%
C57BL/62
B6C3F121
B6;129
F 6% M 7%
F 3/58 M 2/28 F 2/58
F 4% M 4%
VF 5% BF 1% VM 3% BM 2%
F 30%, M 51% F0 M 3% F 2% M 3%
FVB3
VF 0 BF 0 VM 1% BM 0 VF 0 BF 0 VM <1% BM 1%
VF 0 BF 0 VM 0 BM 0 VF 0 BF 0 VM 0 BM 0
Kidney, tumor metastatic
F0 M 2%
8Harderian glands in this study were collected and examined histologically only if gross lesions were identified at necropsy. Only 5 female and 2 male Harderian glands were examined, so these data are expressed as number of tumors/number of glands examined.
TABLE 25-5
GLOSSARY BY SYSTEM:NONNEOPLASTIC CONDITIONS; Brief Definitions or Descriptions of Some Conditions (Phenotypes) That May Be Encountered in Mice. This Is Not a Complete List and Glossary of All Nonneoplastic Conditions That May Occur Spontaneously or Be Induced in Mice. The Information Is Derived Primarily from Resources Used in the Chapter. It Is Provided to Facilitate Understanding of Terminology in the Chapter, and Is Not an Officially Sanctioned Glossary, Dictionary, or Ontology. Terminology and Definitions Will Continue to Change as Conditions Are Characterized Further and as New Conditions Are Induced SYSTEM Organ, Condition; Synonyms (historical terms) MULTISYSTEM Amyloidosis
i.e., generalized condition that involves multiple systems simultaneously, or condition that may occur in various systems or at various sites Amyloid deposition in mice can occur in many tissues including liver, spleen, kidney, lung, heart, parotid, gland, adrenal gland, thyroid, esophagus, skin, stomach, and small and large intestines. With H&E, amyloid is homogeneous eosinophilic amorphous extracellular material. It stains positively with crystal violet, Congo red, and thioflavine T stains. In Congo red–stained section exposed to polarized light, amyloid should demonstrate green birefringence. Intestinal amyloid deposition is in lamina propria and submucosa, with ileum usually affected most severely. Renal amyloid occurs primarily in glomerular mesangium, but may be interstitial especially around collecting tubules in papilla. Splenic amyloid occurs primarily in marginal zones around follicles. Lymph node involvement is primarily of the mesenteric lymph node with amyloid deposits at the periphery of the node in the subcapsular sinuses. Hepatic amyloid occurs first around portal veins and then in perisinusoidal spaces of Disse. Cardiac amyloid spreads from around capillaries. Pulmonary amyloid deposition is in septa. Adrenal amyloid deposits are primarily in the inner cortex surrounding the medulla. Parotid gland interstitial amyloid deposition may separate acini. Thyroid and parathyroid glands can have interstitial amyloid deposition (Frith et al. 1991; Higuchi et al. 1991; Hogen-Esch et al. 1996).
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TABLE 25-5—cont’d SYSTEM Organ, Condition; Synonyms (historical terms) Amyloidosis, reactive AA Amyloidosis, senile AApoAII Amyloidosis, systemic Arteritis; polyarteritis; systemic arteritis (periarteritis)
Angiectasis
Choristoma, lipomatous hamartoma
Hyalinosis
Inflammation; lymphocytic infiltrates
Melanosis
Mineralization
ALIMENTARY Esophagus, dilatation; megaesophagus
Gallbladder; inflammation Gallbladder; hyalinosis
Intestine, amyloidosis
AA-amyloid has a predilection for spleen, liver, gut and kidney, and often is associated with chronic inflammatory conditions (Frith et al. 1991; Higuchi et al. 1991; Hogen-Esch et al. 1996). Masses of amyloid in lung, heart, and ileum suggest AApoAII. Senile amyloid exclusively is likely to be found in gonads, papillary dermis, epineurium, and lung (Frith et al. 1991; Higuchi et al. 1991; Hogen-Esch et al. 1996). Maita et al. 1998 defined the term for their study as amyloidosis involving 3 or more different organs or tissues. Thyroid, adrenal, kidney, small intestine, and ovary were predilection sites in this study. Inflammation of arteries. Small-medium muscular arteries, usually in multiple sites, have medial thickening with variable deposition of eosinophilic material, and have mild-marked perivascular fibrosis and predominantly lymphocytic, mononuclear infiltration. There may be early fibrinoid necrosis or thrombi. Common sites include the heart, thymus, tongue, uterus, testes, mesentery, kidney, and urinary bladder (Frith et al. 1988; Faccini et al. 1990; Plendl et al. 1996; Elwell et al. 1999); Maita et al. 1988 defined systemic arteritis as arteritis involving 3 or more different organs or tissues. Thymus, ovary, uterus, kidney, and heart were predilection sites in this study, and thrombosis frequently was associated with the lesions. Marked dilatation of vascular channels (veins, sinusoids or lymphatics). The lesion can occur in any organ but most commonly involves the spleen, ovary, liver or lymph nodes. It may be difficult to distinguish from hemangioma with angiectatic areas, but the lining endothelial cells should be normal in size and morphology. (Frith et al. 1988; Plendl et al. 1996; Elwell et al. 1999; Harada et al. 1999) Similar to hamartoma, including the mass lesion requirement, but unlike hamartoma, includes heterotopic tissue of an adult or embryonic nature (topographical and developmental anomaly) Pathbase 2004. They are soft, raised masses on the dorsal midline, primarily above the sutures of the skull. They may be noticed because of abnormally long hair, change in direction of the hairs, or change in hair color compared. Microscopically, the masses consist of normal adipose tissue in the reticular dermis and subcutis that sometimes extends through the cranial sutures, entering the brain, or expanding into the ventricles. Large masses may contain normal appearing thyroid, intestine, respiratory epithelium lined cysts, squamous epithelial cysts, bone and marrow, cartilage, glands, and angiomatous anomalies. Overlying epidermis is intact. This condition resembles “lipomatous” hamartomas, a congenital defect in human beings. (Adkison et al. 1991) Epithelial cytoplasmic eosinophilic degenerative change. It may be especially common in various tissues in 129-related mice including B6;129 mice, but similar changes have been recognized in multiple strains. The material, which may be intracytoplasmic or extracellular as hyaline acidophilic material or as needle shaped, rectangular, or square crystals, has been identified as a chitinase. Affected tissues can include the lung, in which the condition is known as acidophilic macrophage or acidophilic crystalline pneumonia, nasal mucosa, trachea, lung, stomach, gallbladder, bile ducts. (Ward et al. 2000; Ward et al 2001) See additional details in discussion above or listed by organ below. Inflammatory lesions especially lymphocytic foci, may occur in various tissues in aging mice, in the absence of discernible inciting agents or factors. Frequently these are interstitial, perivascular or periductal depending on the tissue. Affected tissues may include: Harderian glands, salivary glands, nasal cavity, trachea, lung, gall bladder, hepatic portal areas, glandular stomach, thyroid, kidney, urinary bladder, prostate gland. (Radovsky et al. 1999; Ward et al. 2000; Haines et al. 2001) Melanin pigment can be found in various tissues, other than skin, hair follicles, and retinal pigmented epithelium. Because it is considered to be a normal finding, it is not reported in some studies. Commonly affected tissues include; spleen, heart valves, meninges, choroid plexus, parathyroid. (Ward et al. 2000; Haines et al. 2001) Especially in DBA-related mice, dystrophic calcification or mineralization is likely to occur in various tissues especially in the heart but also in aorta, testes, tongue, skeletal muscle, cornea, kidney, stomach, small intestine, ovary with incidence and severity increasing with age and without apparent sex differences (in non-gonadal tissues). (Rings et al. 1972; Maeda et al. 1986; Yamate et al. 1987; Yamate et al. 1990). In BALB and C3H-related mice mineralization is most common in the heart and cornea.(Van Winkle et al. 1986 Frith et al. 1983; Everitt et al. 1988; Vargas et al. 1996. Extracardiac soft tissue mineralization also may occur in C3H mice. (Highman et al. 1951). In most other strains, soft tissue mineralization is unusual (Elwell et al. 1999). Dilatation of the esophagus. Dilatation usually is of the thoracic esophagus, visible at necropsy, with apparent impaction by ingested material. The condition may be a contributory cause of death in some case (Ward et al. 2000; Haines et al. 2001). In some strains (ICRC) megaesophagus has been associated with the presence of smooth muscle instead of (normal) skeletal muscle in the distal esophagus. (Randelia et al. 1988; Randelia et al. 1990) See above, MULTISYSTEM. See above, MULTISYSTEM. Gallbladders with hyalinosis may be grossly enlarged with thickened opaque walls. Extracellular crystals (identified as chitinase), when present, tend to be large and rectangular to square. Affected bile ducts had associated mucoid metaplasia and fibrosis. The acidophilic hyaline or crystalline material has been identified as a chitinase (Ward et al. 2000, 2001). See above, MULTISYSTEM. Amyloid deposition is in the lamina propria of the small intestine, especially in the ileum (Frith et al. 1991; Higuchi et al. 1991; Hogen-Esch et al. 1996) Continued
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TABLE 25-5—cont’d SYSTEM Organ, Condition; Synonyms (historical terms) Liver, amyloidosis Liver, angiectasis; telangiectasis, peliosis hepatis Liver, centrilobular hypertrophy Liver, centrilobular necrosis hepatocyte Liver, extramedullary hematopoiesis (EMH)
Liver, foci of cellular alteration; altered hepatocellular foci
Liver inflammation
Liver, necrosis hepatocytes
Liver, hepatocellular inclusions
Liver, hepatocellular vacuolization, steatosis, (fatty change, fatty metamorphosis) Liver, hepatocyte karyomegaly, cytomegaly; polyploidy Pancreas, exocrine atrophy Pancreas, inflammation, chronic Salivary gland, amyloidosis
See above, MULTISYSTEM. Amyloid deposition is in sinusoids beneath the sinusoidal-lining cells, is distinctly homogeneous and eosinophilic. (Frith et al. 1991; Higuchi et al. 1991; Hogen-Esch et al. 1996) See above, MULTISYSTEM. Hepatic angiectasis involves dilatation of the vascular sinusoids and may be either focal or diffuse. Angiectasis may be difficult to distinguish from hemangioma. In angiectasis, the vascular spaces are dilated and prominent and their lining endothelial cells are normal in appearance, number, and size. (Frith et al. 1988; Harada et al. 1999) Enlargement of centrilobular hepatocytes, with increased amount and variable staining characteristics of cytoplasm, with gradual decrease in cell size closer to portal areas. It is especially likely in studies of toxicants that induce proliferation of peroxisomes or of smooth endoplasmic reticulum. (Harada et al. 1999) Centrilobular hepatocellular necrosis can occur with ischemia or chronic passive congestion, and is seen in FVB mice believed to have died after severe or prolonged seizures. (Goelz et al. 1998) EMH occurs normally in fetal and neonatal mouse liver. In the adult mouse it may be secondary to infectious disease or neoplasia. Small foci or nests of immature granulocytes, nucleated erythrocytes, or megakaryocytes are scattered in sinusoids. Granulopoietic foci may be primarily periportal. Megakaryocytes and nucleated erythrocytes help to distinguish EMH from inflammation and leukemia or lymphoma. (Frith et al. 1988; Harada et al. 1999) Altered staining qualities and textural appearance of the cytoplasm and size of hepatocytes, compared to adjacent normal hepatocytes. There is no obvious disruption of the liver architecture, nor compression of adjacent normal parenchyma. They may be classified as eosinophilic, basophilic, clear cell, or mixed. They are much more common in carcinogen treated than in control mice, and are more common in male than in female mice. Clear cell foci consist of cells with pale or sometimes lacy cytoplasm that stain with PAS stain prior to but not after diastase digestion, suggesting the presence of glycogen. Their nuclei tend to be central rather than flattened against the cell membrane as in lipid-vacuolated cells. Eosinophilic foci consist of cells that tend to be larger than adjacent normal hepatocytes with eosinophilia due to increased cytoplasmic mitochondria and/or smooth endoplasmic reticulum. Basophilic foci consist of cells that tend to be smaller than adjacent normal hepatocytes with basophilia due to relatively increased cytoplasmic free ribosomes and rough endoplasmic reticulum. Mixed cell foci contain varying proportions of 2 or more of any of the cell types. (Frith et al. 1979; Harada et al. 1996; Harada et al. 1999) See above, MULTISYSTEM. Foci of acute and/or chronic inflammatory cells (primarily lymphocytes) distributed randomly and sporadically. Mild chronic inflammation characterized by random and/or portal or perivascular primarily lymphocytic infiltrates. Some of these cases may be attributable to helicobacter or other infections. (Harada et al. 1996; Harada et al. 1999; Ward et al. 2000) Focal necrosis may be an incidental finding of unknown etiology, or may be related to infections (MHV, Clostridium piliforme, helicobacters), toxicants. It may involve single cells, single or multiple lobules, and it may vary in distribution. Coagulation necrosis with hypereosinophilic cytoplasm and pyknotic or absent nuclei is the typical morphologic feature, and can be common in some studies. (Harada et al. 1996; Harada et al. 1999; Ward et al. 2000) Distinctive round eosinophilic intranuclear inclusions in hepatocytes may nearly fill the nucleus. Their incidence increases with age and they are considered to be invaginations of the cytoplasm into the nucleus. Intracytoplasmic inclusions are less common and may occur near neoplasms, and may consist of condensed secretory proteins in dilated cisternae of rough endoplasmic reticulum. (Frith et al. 1979; Harada et al. 1996; Harada et al. 1999) Hepatocellular vacuolization due to fatty change is especially common in old obese controls and is more common in male than in female mice. It also may occur in response to a toxicant. Initially there is usually a centrilobular distribution. The empty clear vacuoles peripherally compress nuclei and represent lipid that was removed during tissue processing. The lipid nature can be confirmed by staining frozen sections with Oil Red O or Sudan Black B. (Frith and Ward 1988; Harada et al. 1996) Hepatocellular anisocytosis and anisokaryosis, with enlarged cells and nuclei increase with age, and in response to various agents. There may be binucleate or multinucleate hepatocytes. The increase in nucleus size or number is associated with polyploidy. (Frith et al. 1988; Lu et al. 1993; Styles 1993) Premature polyploidy occurs in some mutant mice with defective DNA repair mechanisms. (Chipchase et al. 2003) Focal or lobular change resulting in complete absence of acini, or only few small acini with pale exocrine cells due to reduced zymogen granules, and normal islets of Langerhans suspended in fatty stroma. Its occurrence only in aged mice suggests that it is a true atrophy and not a hypoplasia. (Frith et al. 1988; Faccini et al. 1990; Boorman et al. 1991) See above, MULTISYSTEM. Mild chronic lymphocytic interstitial infiltrates in the exocrine pancreas. See above, MULTISYSTEM. Salivary glands may develop amyloidosis. The parotid glands are serous glands that extend from the base of the ears ventrally and posteriorly. With severe amyloidosis acini may be widely separated by amorphous acidophilic material, with histochemical (staining) properties of amyloid. (West et al. 1965; Sashima et al. 1990)
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TABLE 25-5—cont’d SYSTEM Organ, Condition; Synonyms (historical terms) Salivary gland, ductal hyperplasia
Salivary, gland inflammation Stomach, forestomach; squamous epithelial hyperplasia Stomach, fundic mucosal hyperplasia, gastric hyperplasia; adenomatous hyperplasia Stomach, glandular hyperplasia, plaque
Stomach, gastric hyalinosis
Teeth, alveolitis, periodontitis, periodontal disease Teeth, dental dysplasia
Teeth, malocclusion
CARDIOVASCULAR Heart, cardiomyopathy
Heart, coronary arteritis Heart, melanosis valves Heart, mineralization (dystrophic cardiac calcinosis, epicardial mineralization)
Heart, thrombi
Ductal hyperplasia usually is associated with lobular acinar atrophy and is more common in the submaxillary and parotid salivary glands than in the sublingual gland. The lesion typically involves a single lobule in which some acini are atrophied and replaced by hyperplastic ducts, and there may be associated inflammatory infiltrates. (Frith et al. 1988; Botts et al. 1999; Seely 1999; Ward et al. 2000) See above, MULTISYSTEM. This typically is chronic lymphocytic or lymphoplasmacytic interstitial, perivascular or periductular inflammation, and may be more common in male than in female mice. (Frith et al. 1988; Faccini et al. 1990; Botts et al. 1999) Increased thickness of squamous mucosa due to hyperplasia of stratified squamous epithelium, usually with hyperkeratosis, and likely to occur after administration of irritants. (Frith et al. 1988; Leininger et al. 1999) Increased thickness of glandular mucosa due to hyperplasia with increased pit and gland length. Pits become elongated and more basophilic. There may be mucosal folding but gland architecture is retained. Epithelial cells may be enlarged, hypertrophied. Severe cases may have herniation of glands into muscularis mucosae, but basement membrane is not penetrated. (Betton et al. 2001) Gastric plaque has been used to refer to foci of glandular hyperplasia associated with gastric hyalinosis in 129S4/SvJae and related mice. They are most common in the cardiac glandular stomach at or near the limiting ridge. Grossly discernible areas of plaque like thickening, sometimes with hemorrhage, are periesophageal. Histologically glands are elongated, hyperplastic, and may be focally disorganized with loss of normal differentiation patterns. The plaques contain foci of hyalinized epithelial cells that seemed to arise from chief cells in the mid-region of the glands. Some epithelial cells contain only a few intracytoplasmic droplets, or cells may be distended with nuclei displaced peripherally by brightly eosinophilic material. Glandular lumina may contain abundant extracellular rectangular eosinophilic crystals, as opposed to needle-shaped or square crystals. They are metachromatic with Dominici stain as are the hyaline granules within epithelial cells. The eosinophilic hyaline and crystalline material has been identified as a chitinase. (Haines et al. 2001; Ward et al. 2001) Eosinophilic cytoplasmic degenerative change, termed hyalinosis, is most common in the cardiac glandular stomach at or near the limiting ridge, and frequently is associated with plaque-like thickening. See above, MULTISYSTEM. (Haines, Chattopadhyay, et al. 2001; Ward, Yoon, et al. 2001) Inflammation in the dental alveolus (tooth socket) or around the tooth. In mice the disease is most severe in the maxillary arch. It can begin as a gingivitis initiated by hair impaction, plaque, or calculus, and can progress to severe inflammatory changes with resorption of tooth and alveolar bone (Losco 1995; Long et al. 1999) Abnormal development or malformations of injured and/or displaced odontogenic tissues; may be used to refer to the spectrum of nonneoplastic incisor malformations involving the tooth and periodontal structures. Histologically there can be mild cystic changes in the developing portions of the incisors or the tooth may be replaced and socket filled and expanded irregular masses of dentin-like material with fragments of tooth and bone. (Losco 1995; Long et al. 1999; Ward et al. 2000) Malocclusion in mice ultimately manifests as long maxillary incisors that grow out from and curl back into the maxilla, and long mandibular incisors that tend to grow upwards from the mandible. Mice with this condition cannot eat hard pelleted chow. Trauma and genetics have been implicated in this condition. (Losco 1995; Long et al. 1999; Jax Notes 2003) Foci of myocardial necrosis or myocyte degeneration (myocytolysis) with minimal to mild mononuclear infiltration by, with or without fibrosis, which may be more common in the left ventricle. (Plendl et al. 1996; Price et al. 1996) Some authors include mineralization in this definition, e.g. in BALB/c mice. Some authors may use ‘myocardial necrosis, inflammation, fibrosis’. (Frith et al. 1988; Faccini et al. 1990) Arteritis/polyarteritis involving coronary arteries. See above, MULTISYSTEM. See above, MULTISYSTEM. See above, MULTISYSTEM. In BALB/c and related mice mineralization is epicardial, on the right ventricular free wall (Frith et al. 1983) Mineralization, may be associated with degenerative changes especially in female C3H mice, and involves myocardium. (Frith et al. 1983; Everitt et al. 1988; Vargas et al. 1996) In DBA mice mineralization is myocardial and epicardial and there may be mineralization of other soft tissues, e.g. tongue, testes, aorta. (Yamate et al. 1987; Brunnert et al. 1999) Mineralized foci are basophilic with hematoxylin and eosin (H&E), black with von Kossa, and red with Alizarin Red staining. (Frith et al. 1988; Maita et al. 1988) Thrombi are usually in the left atrium, which may be enlarged and red. The degree of organization depends on the age of the thrombus. Some thrombi may contain foci of cartilaginous metaplasia. With large thrombi there may be secondary chronic passive congestion. Thrombosis may be associated with uremia and kidney disease, including amyloidosis, may not be associated with arteritis, and may be more common in males. (Frith et al. 1983; Frith et al. 1988; Maita et al. 1988; Faccini et al. 1990; Elwell et al. 1999). In some cases both atria may be involved or there may be ventricular thrombi (Hagiwara et al. 1996) Continued
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TABLE 25-5—cont’d SYSTEM Organ, Condition; Synonyms (historical terms) Vessels, angiectasis Vessels, arteritis; polyarteritis; systemic arteritis (periarteritis) ENDOCRINE Adrenal cortex, accessory cortical nodule Adrenal cortex, atrophy
Adrenal cortex, hyperplasia subcapsular cell
Adrenal cortex, hypertrophy Adrenal gland, amyloidosis Adrenal gland, pigment ceroid (lipogenic pigmentation; lipoid pigment; brown degeneration)
Pancreatic islet hyperplasia
Parathyroid melanosis Pituitary cysts
Pituitary pars distalis; hyperplasia
Pituitary pars intermedia hyperplasia Thyroid gland, ectopic tissue
Thyroid gland, cysts Thyroid gland, inflammation
Marked dilatation of small vessels. See above, MULTISYSTEM. Inflammation of arteries. See above, MULTISYSTEM.
Small nodules of adrenal cortical cells surrounded by a connective tissue capsule are sometimes associated with the adrenal capsule, and may contain cell of zona glomerulosa and/or zona fasciculata. (Yarrington 1996; Nyska et al. 1999) Especially in male mice, small adrenals may be difficult to find. Histologically the capsular surface is irregular, cortical thickness is reduced and/or variable, and there may be 1 or more foci of cortical cell hypertrophy or hyperplasia (Hall et al. 1992; Yarrington 1996; Nyska et al. 1999) Hyperplasia of adrenal subcapsular cells that may represent adrenal subcapsular reserve cells. They may be spindled type A cells or polygonal lipid-laden type B cells that more closely resemble normal cortical cells. Aged mice, especially females in some strains, commonly have subcapsular accumulations or proliferations of basophilic spindle (type A) cells, with sparse cytoplasm and indistinct cytoplasmic borders, which may be associated with mast cell infiltration. The lesion may be focal or diffuse, involving the entire subcapsular cortex. Hyperplastic foci may slightly bulge the capsule but exhibit nominimal compression, and should not be larger than the regular width of the cortex in a young mouse, but the distinction between hyperplasia and adenoma may be arbitrary and difficult (Frith et al. 1988; Faccini et al. 1990; Yarrington 1996; Kim et al. 1997; Nyska et al. 1999) Enlargement of adrenal cortical cells due to increased cytoplasm with zona fasciculata principally affected. (Yarrington 1996; Nyska et al. 1999; NTP 2000) See above, MULTISYSTEM. The innermost cortex is the first predilection site for amyloid deposition, and deposits can progress peripherally to involve zona fasciculate, but usually spare zona glomerulosa. (Hogen-Esch et al. 1996; Yarrington 1996; Nyska et al. 1999) Aged mice may develop deposition of ceroid (lipogenic) pigment initially in cortical cells and macrophages near the corticomedullary junction that can progress to surround the medulla. Small amounts of the cytoplasmic pigment are granular to amorphous yellow-brown. Affected cells become enlarged and distended with brown material. Their nuclei may become pyknotic and there may be multinucleated cells. Ceroid deposits in the ovary are mainly in interstitial cells. Ceroid pigments are yellow brown lipid-derived pigments that autofluoresce, are PAS positive, acid fast, stain blue with the Schmoll reaction, are positive with Sudan black, and are negative for iron. (Yarrington 1996; Nyska et al. 1999) Increased size of islets of Langerhan to hyperplasia (increased number) of cells, which morphologically are similar to those in smaller normal islets. Hyperplasia may be difficult to distinguish from the normal size variation of islets and hyperplasia of beta cells occurs normally during pregnancy. Hyperplasia usually involves more than one islet (multifocal), and may involve all islets in a section. (Sass et al. 1978; Frith et al. 1988; Faccini et al. 1990; Boorman et al. 1999) See above, MULTISYSTEM. Single or multiple small cysts occur primarily in the pars distalis or pars tuberalis. They may be lined by ciliated epithelium and sometimes contain an eosinophilic colloid-like secretion. Some may represent craniopharyngeal duct (Rathke’s pouch) remnants. Cystic or cyst-like degeneration of the pars distalis occurs occasionally in aging untreated control mice, but occurs earlier and with higher incidence with increasing doses of estrogenic compounds. Focal degeneration and loss of cells in the pars distalis results in small irregular spaces often containing some eosinophilic material and cell debris. (Frith et al. 1988; Mahler et al. 1999) Hyperplastic foci have indistinct edges and are not compressive, they are distinguished from adjacent tissue by slightly different staining properties (usually paler) with increased number or density of cells. They may be composed of chromophobe or acidophil cells. Chromophobe cells may secrete prolactin. (Frith et al. 1988; Mahler et al. 1999; Capen et al. 2001) Diffuse or nodular thickening of the pars intermedia due to hyperplasia. The cells are large and pale compared to pars distalis cells, and may be larger and more basophilic than typical pars intermedia cells. Tumors of the pars intermedia are rare. (Frith et al. 1988; Mahler et al. 1999; Ward et al. 2000; Capen et al. 2001) Ectopic thyroid tissue, typically consisting of a few colloid containing follicles may occur in the mediastinum or thymus. Small cysts in or around the thyroid gland, lined by ciliated cuboidal cells or squamous epithelial cells are considered to be remnants of the pharyngobrachialis duct or of the ultimobranchial duct respectively. Thymic rests (small accumulations of thymic tissue) may occur in or near the thyroid glands. Ultimobranchial cysts, lined by ciliated, cuboidal, or squamous epithelial cells occasionally occur in or near the thyroid gland. (Faccini et al. 1990; Hardisty et al. 1999) See above, MULTISYSTEM. Inflammation in the thyroid gland is uncommon in untreated mice. There may be occasional lymphoid infiltrates or arteritis. (Hardisty et al. 1999)
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TABLE 25-5—cont’d SYSTEM Organ, Condition; Synonyms (historical terms) Parathyroid gland, cysts Parathyroid gland, ectopic tissue GENITAL, FEMALE Ovary, atrophy
Ovary cyst
Ovary pigment
Uterus, adenomyosis
Uterus, angiopathy Uterus, cystic endometrial hyperplasia
Uterus, hemosiderosis Uterus, hydrometra, mucometra
Uterus, mineralization GENITAL, MALE Epididymis, karyomegaly Epididymis, sperm granuloma
Preputial gland, cystic ducts, ectasia
Parathyroid cysts may be multilocular, are lined by a monolayer of cuboidal to columnar, often ciliated, epithelium, and usually contain eosinophilic proteinaceous material. (Faccini et al. 1990; Hardisty et al. 1999) Ectopic parathyroid tissue may occur in the mediastinum or thymus. Thymic rests (small accumulations of thymic tissue) may occur in or near the parathyroid glands. (Faccini et al. 1990; Hardisty et al. 1999) The incidence and severity of ovarian atrophy increases with age in female mice, and also may be induced with estrogenic compounds. Atrophic ovaries are smaller than normal ovaries with reduced numbers of follicles and especially of corpora lutea, and relatively increased interstitial tissue. Clusters of large yellow-brown (ceroid) pigmented interstitial cells are common. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999) Ovarian cysts may be single or multiple, may vary in size and most are age-related. (Davis et al. 1999). Follicular and luteal cysts derive from anovulatory graafian follicles and are the most common ovarian lesion in mice on some studies. They may be single or multiple and may essentially replace the ovary. They are lined by 1-4 layers of cuboidal granulosa cells. They should not be confused with cystic corpora lutea that derive from ovulatory follicles and produce progesterone. These have more than 6 layers of hypertrophied luteal cells surrounding a central cavity that may be blood filled. Epithelial inclusion cysts are lined by 1/ several layers of columnar epithelial cells that form papillary structures that project into the central lumen. They may be precursors of cystadenomas or cystadenocarcinomas. Epidermoid cysts are lined by squamous epithelial cells, may be filled with laminated keratin debris, and are often found with teratomas. Paraovarian cysts arise from the mesovarium, are lined by ciliated columnar epithelial cells, and the wall contains smooth muscle. Rete cysts derive from dilated tubules of the rete ovarii. They are lined by columnar epithelial cells with apical nuclei. Bursal cysts can be common. The ovary may be compressed within the cystically distended ovarian bursa. Ceroid is the most common pigment in mouse ovaries, and there may be a high incidence of deposition of this age-related pigment after 1 year of age. Ceroid deposits in the ovary are mainly in interstitial cells. They are yellow-brown lipid-derived pigments that autofluoresce, are PAS positive, acid fast, stain blue with the Schmoll reaction, are positive with Sudan black, and are negative for iron. Hemosiderin laden macrophages are likely in sites of earlier follicular hemorrhage. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999) The presence of endometrial glands in the myometrium is seen with cystic endometrial hyperplasia and can be induced by administration of estrogenic compounds. Glands sometimes may extend to the serosa. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001) Angiectasis in the uterus usually occurs in the myometrium, and arteritis also can occur in the uterus. See above, MULTISYSTEM. Cystic endometrial hyperplasia is the most common uterine change in some studies of aged female mice and the incidence may approach 100%. The condition also can be induced by hormones and agents with estrogenic properties. The uterus may be markedly enlarged by increased numbers of glands plus cystic dilatation of many glands. Endometrial glands are cystic and increased in number. There may be adenomyosis (endometrial glands within the myometrium) in severe cases. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001). Hemosiderin deposition is especially likely in aged multiparous females, likely related to involution of placental sites. (Frith et al. 1983; Maekawa et al. 1996) Marked dilatation of the horns or body of the uterus. One or both horns and the corpus may be involved. The lumen contains serous proteinaceous or mucoid material, and the uterine wall may be thin and atrophic due to prolonged distention. The cause often is not determined, imperforate vagina is a likely cause. (Sheldon et al. 1980; Frith et al. 1988; Sundberg et al. 1994) Mineralization is especially likely in aged multiparous females, likely related to involution of placental sites. (Frith et al. 1983; Maekawa et al. 1996) Karyomegaly frequently with associated cytoplasmic vacuolation in the epithelium of the cauda epididymis causing the enlarged cell to bulge into the lumen, has been a common finding in recent evaluations of 129 and related mice. (Ward et al. 2000; Haines et al. 2001) Granuloma, or pyogranulomatous inflammation, resulting from rupture of a duct with release of sperm and duct contents to interstitium. The lesion may include multinucleated giant cells and cholesterol clefts. It may be secondary to ruptured spermatocele, which is a dilated duct segment filled with spermatozoa. (Frith et al. 1988; Radovsky et al. 1999) Ductal ectasia and gland atrophy can be common in preputial and clitoral glands, which are composed of modified sebaceous acini and squamous ducts. Associated suppurative and chronic inflammation is common. (Frith et al. 1988; Seely et al. 1999) Continued
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TABLE 25-5—cont’d SYSTEM Organ, Condition; Synonyms (historical terms) Preputial gland, inflammation Prostate gland, atypical epithelial hyperplasia Prostate gland, inflammation Testes, atrophy, degeneration tubular
Testes, interstitial (Leydig) cell hyperplasia
Testes, mineralization
HEMATOPOIETIC Bone marrow, fibroosseous change (myelofibrosis)
Bone marrow, hyperplasia myeloid granulocytic Lymph node, hyperplasia, lymphoid
Lymph node, infiltration, macrophages; sinus histiocytosis Lymph node, infiltration, plasma cells Spleen, amyloidosis Spleen, arteritis Spleen, melanosis
Spleen, extramedullary hematopoiesis, EMH (myeloid metaplasia) Spleen, hyperplasia erythroid Spleen, hyperplasia myeloid granulocytic
Spleen, hyperplasia reticular
Suppurative and chronic inflammation in preputial and clitoral glands can be common, with development of abscesses. (Frith et al. 1988; Seely et al. 1999) Proliferation of prostate epithelium, without disturbance of acinar architecture or compression of adjacent tissue. (Radovsky et al. 1999; Ward et al. 2000) See above, MULTISYSTEM. Lymphocytic foci and arteritis can occur in the prostate. (Radovsky et al. 1999; Haines et al. 2001) Degeneration in seminiferous tubules manifests initially as scattered vacuolated or necrotic spermatogenic cells often with multinucleated giant cells and reduced numbers of germinal epithelial cells. More advanced degeneration manifests as severely depleted germ cells, depleted and flattened Sertoli cells, and few remaining spermatogonia. Lipofuscin or ceroid pigment in the testes increases with age and may be associated with degenerative changes. (Radovsky et al. 1999) Increased numbers (hyperplasia) of interstitial (Leydig) cells between seminiferous tubules. These noncompressive collections of typical interstitial cells may be focal, multifocal, or diffuse, and range in diameter from about 25% of the diameter of a seminiferous tubule to 3% seminiferous tubules diameter. Interstitial (Leydig) cells have abundant eosinophilic, sometimes vacuolated, cytoplasm, and usually central nucleus with prominent nucleolus. (Gordon et al. 1996; Radovsky et al. 1999; Rehm et al. 2001; Pathbase 2004) Focal dystrophic mineralization of the seminiferous tubules may occur occasionally in some strains and commonly in DBA/2 mice. It may represent previous areas of injury. Histologically the basophilic concretions may be amorphous or concentrically laminated. (Rings et al. 1972; Frith et al. 1988; Yamate et al. 1990; Radovsky et al. 1999) Foci of replacement of marrow by fibrous connective tissue, not associated with renal or (Wijnands et al. 1996) parathyroid lesions, can occur in any bones but are commonly reported in sternum, femur and vertebrae especially in aging female mice. In advanced lesions the marrow cavity may be almost replaced by fibrous connective tissue or bony trabeculae. (Sass et al. 1980; Frith et al. 1988; Wijnands et al. 1996; Long et al. 1999; Ward et al. 2000) Intramedullary bone proliferation may be referred to as hyperostosis. See below, MUSCULOSKELETAL. Relative or absolute increase in normal (usually granulopoietic) myeloid elements in the bone marrow usually is a response to infection or necrosis, e.g. tumoral necrosis. Severe granulopoietic hyperplasia may be difficult to distinguish from granulocytic leukemia. (Frith et al. 1985; Kogan et al. 2002) Lymphoid hyperplasia of the lymph nodes can be common, especially in females, in some studies of aging mice. Expansion due to hyperplasia occurs in B cell areas (follicles, germinal centers) and T cell thymic-dependent areas (paracortex). Marginal sinus is often filled with lymphocytes and medullary cords expanded by plasma cells. The lymphocytes are mature, usually there are few mitotic figures. Lymphoid hyperplasia and plasmacytosis van be a reaction to chronic inflammatory lesions or tumor antigens. (Frith et al. 1985; Frith et al. 1988) Accumulations of macrophages or histiocytes in the subcapsular and medullary sinuses of lymph nodes. The plump macrophages may have abundant distinctly eosinophilic cytoplasm and may contain hemosiderin, other pigments, erythrocytes, and other phagocytosed material. (Frith et al. 1985; Frith et al. 1988) Accumulations of plasma cells in the subcapsular and medullary sinuses of lymph nodes. See above, MULTISYSTEM. Early lesions occur in the white pulp and spread to the red pulp. See above, MULTISYSTEM. See above, MULTISYSTEM. Both hemosiderin and melanin pigment occur in the spleens of mice. Hemosiderin is golden brown and usually in cytoplasm of macrophages or reticular cells. It stains positively with iron stains such as Prussian blue. Melanin also occurs in the spleen of mice with pigmented skin. It is slightly darker than hemosiderin, maybe in elongate strands rather than clumps (like hemosiderin), is not associated with macrophages and is iron negative. (Frith et al. 1985; Frith et al. 1988) The spleen is an important hematopoietic organ, and the red pulp expands due to increased, proliferative, normal hematopoietic elements. The increase may be primarily in erythroid or granulopoietic elements. Megakaryocytes also are common in the red pulp and may increase as well. (Frith et al. 1985; Kogan et al. 2002) Increased erythropoiesis is characterized by foci of immature erythrocytic precursors with small darkly staining nuclei in the red pulp. Increased erythropoiesis may or may not be associated with an increase in granulopoiesis. (Frith et al. 1985; Frith et al. 1988) Increased granulopoiesis (e.g., due to infection or abscess) usually occurs in bone marrow as well as in spleen and there may be granulopoietic elements in liver, adrenals, and lymph nodes as well. Normal maturation including mature neutrophils should be discerned in granulopoiesis. A primary (inciting) cause such as abscess, ulcerative dermatitis, or tumoral necrosis should be sought in cases of marked granulopoiesis. (Frith et al. 1985; Kogan et al. 2002). This term may be used to refer to apparent increase in monocyte/macrophage or reticuloendothelial cells. (Frith et al. 1985; Wijnands et al. 1996)
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TABLE 25-5—cont’d SYSTEM Organ, Condition; Synonyms (historical terms) Thymus, atrophy cortex or lymphoid depletion or involution
Thymus, cysts
Thymus, hyperplasia, lymphoid INTEGUMENT Mammary hyperplasia, functional without atypia
Skin ulcer, inflammation, ulcerative dermatitis
Alopecia areata MUSCULOSKELETAL Skeletal, degenerative joint disease; osteoarthritis Bone hyperostosis endosteal NERVOUS Brain, hypocallosity Brain, lipofuscin Brain, melanosis meninges Brain, mineralization thalamus
RESPIRATORY Lung, acidophilic Macrophage pneumonia
Lung, acidophilic crystals Lung, alveolar epithelial hyperplasia bronchoalveolar hyperplasia (Type II cell hyperplasia)
The thymus reaches maximal size at sexual maturity and then undergoes gradual involution. Thymus size and rate of involution varies with mouse strain. (Peleg et al. 1984; Hsu et al. 2003) During involution thymus size decreases due to gradual reduction in cortex and lymphocyte content, and the medulla becomes more prominent, with apparent increase in connective tissue, and in epithelial elements, which may form cysts, cords, or tubules. Temporary and reversible involution or atrophy occurs during pregnancy, infection, malnutrition, after surgery or other stressors, and can be caused by toxic insults. Early atrophy may manifest as a starry sky appearance due to phagocytosis of necrotic/apoptotic lymphocytes by phagocytes. (Wijnands et al. 1996; Ward et al. 1999) Epithelial cysts in the thymus may be come more prominent during involutions. They may be lined by squamous to columnar cells with some ciliated cells. Some cysts may contain cell debris and PAS positive glycoprotein material and may be involved in cell disposal. Focal or diffuse hyperplastic changes can occur in the cortex or medulla, in 1 or both lobes. There may be focal lymphocytic accumulations in the cortex or medullary, or follicles with germinal center. (Wijnands et al. 1996; Ward et al. 1999) Mammary glands may be grossly enlarged and have lobuloalveolar hyperplasia with distended secretory alveoli and ducts that that contain eosinophilic colloid-like secretory material. They resemble glands during pregnancy or delayed involution. The condition may be common in older virgin and multiparous FVB/N mice with or without associated pituitary lesions, and has been associated with mouse mammary tumor virus infection. In FVB/N mice there may be squamous nodules within the hyperplastic gland. (Medina 1982; Frith et al. 1988; Nieto et al. 2003; Wakefield et al. 2003) The condition can occur in many strains of mice and acariasis should be ruled out. It has been reported most commonly in C7BL related strains. It may begin as a dorsal papular dermatitis, progressing to foci of alopecia and small ulcers, then expansion of the ulcers, with deep necrosis, intense suppurative and chronic inflammatory responses, and secondary changes such as lymphadenopathy, myeloid hyperplasia and amyloidosis. (Sundberg 1996) In outbred Swiss mice the ears and neck may be affected most commonly and severely and has been called progressive necrosing dermatitis of the pinna. (Slattum et al. 1998) Histologically, all species have dystrophic anagen stage hair follicles associated with a peri- and intrafollicular inflammatory cell infiltrate. (McElwee et al. 1998; McElwee et al. 1999) Non-inflammatory, progressive loss or destruction of articular cartilage, with thickening of underlying bone, and sometimes subchondral cysts and osteophytes in various strains. Knee and elbow joints may be most severely affected, and vertebrae, especially thoracic vertebrae, may be affected also. (Long et al. 1999) Endosteal bone proliferation. Estrogens may induce this condition in mice. In advanced stages of spontaneous fibroosseous lesions, the medullary cavities may be nearly filled by bone (see Bone Marrow, above). (Highman et al. 1981; Ward et al. 1999) Small or absent corpus callosum connecting cerebral hemispheres occurs especially in 129 and BALB/c strains. (Wahlsten 1982; Livy et al. 1991; Livy et al. 1997) Brown-tinged cytoplasm of neuronal cell bodies, due to lipofuscin accumulation, increases with age, and may be most prominent in granule cells of the dentate gyrus. (Moore et al. 1995; Moore et al. 1995; Radovsky et al. 1999) See above, MULTISYSTEM. Small foci (up to 100 µm diameter) of basophilic mineralized material occurs usually bilaterally in the thalamus of old mice, with an incidence of about 5% in CD-1® and B6C3F1 mice. (Faccini et al. 1990) They usually are associated with blood vessels and concentric lamination may be evident with H&E. They stain negative for amyloid and iron, and are weakly positive with Alcian blue, primarily at the periphery of the deposits. They are dark brown or black with distinct lamination by Verhoeff’s method, and have a red core and dark periphery with Alizarin red. (Morgan et al. 1982; Frith et al. 1988; Radovsky et al. 1999) Acidophilic macrophage pneumonia, also referred to as acidophilic crystalline pneumonia, is characterized by abundant macrophages distended with eosinophilic (acidophilic) granular or crystalline material in airways. Intracellular or extracellular eosinophilic needle-like crystals in airways may or may not be conspicuous and may stain blue with Perl’s reaction (for iron). (Ward et al. 2000, 2001) Intracellular or extracellular, non-birefringent, acidophilic crystals, identified as a chitinase, can be striking or dominant feature of acidophilic macrophage pneumonia (acidophilic crystalline pneumonia) in some cases. (Ward et al. 2000; 2001) Hyperplasia (increased numbers) of alveolar type II cells or bronchiolar secretory cells. These typically are poorly demarcated foci of hypercellularity with septal thickening due to increased numbers of plump type II cells or bronchiolar-type cells with preservation of alveolar septal architecture. Atypia and mitoses are unusual. These may be precursors of adenoma or carcinoma. (Dixon et al. 1999; Dungworth et al. 2001) Continued
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TABLE 25-5—cont’d SYSTEM Organ, Condition; Synonyms (historical terms) Lung, inflammation, perivascular peribronchiolar Nose, amyloidosis, nasal amyloidosis
Nose; olfactory epithelium hyalinosis Nose; respiratory epithelium hyalinosis
Trachea inflammation Trachea hyalinosis Trachea, mucosal gland acidophilic crystals SPECIAL SENSES Ear, otitis media
Eye, Blepharoconjunctivitis
Eye, cataract gross Eye, cataract microscopic
Eye, keratitis, neovascularization Eye, corneal opacity, corneal dystrophy, corneal mineralization, band keratopathy Eye, retinal degeneration
Eye, retinal atrophy
Harderian gland, chronic inflammation URINARY Kidney, amyloidosis
See above, MULTISYSTEM. These usually consist of small perivascular lymphocyte aggregates, or mild increase bronchiole associated lymphoid tissue (BALT). They should be minimal in untreated mice not exposed to respiratory pathogens. (Ernst et al. 1996) Expansion of the nasal septum by submucosal deposition of amorphous acellular eosinophilic material also has been referred to as septal amyloidosis. The submucosal amorphous eosinophilic material in the ventral nasal septa of aged mice does not stain with Congo red as amyloid should. Trichrome staining is compatible with collagen. (Monticello et al. 1990; Hogen-Esch et al. 1996; Haines et al. 2001) Nasal olfactory epithelial hyalinosis is primarily near the olfactory/respiratory transition areas, with foci of hyalinosis characterized by eosinophilic intracytoplasmic inclusions, identified as a chitinase, originating at the basal aspects of lining cells. (Ward et al. 2000, 2001) Nasal respiratory epithelium tends to be more affected than olfactory epithelium, particularly in the regions of the nasal glands. Affected respiratory epithelial cells may be distended with peripheral displacement of nuclei by eosinophilic hyaline material. Inflammation usually is not associated with epithelial changes. Extracellular crystals were variably needle-like, rectangular, or square. The acidophilic hyaline material and crystals have been identified as a chitinase. Inflammation usually is not associated with epithelial changes. (Ward et al. 2000; 2001) See above, MULTISYSTEM. These usually consist of small submucosal lymphocytic aggregates. Tracheal epithelial cytoplasm contains and may be distended by amorphous or spicular hyaline eosinophilic material that has been identified as a YM1 chitinase. (Ward et al. 2000, 2001) Epithelial cytoplasm contains and may be distended by amorphous or spicular hyaline eosinophilic material that has been identified as a YM1 chitinase. (Ward et al. 2000; Ward et al. 2001) Inflammation of the middle ear. There may be intense suppurative exudates in the lumen, with inflammatory and proliferative changes in the lining epithelium, or the chamber may be filled with eosinophilic serous material with few inflammatory cells and little change in the lining epithelium. Some lesions have fibrosis and lipid-like material with cholesterol clefts resembling cholesteatoma or cholesterolinic granuloma. Organisms may be difficult to discern. (Haines et al. 2001) Inflammation of the eyelid and conjunctiva. This condition may be quite common in several pigmented and non-pigmented mouse strains. Initially there is suppurative conjunctivitis and/or ulceration at the mucocutaneous junction, progressing to suppurative inflammation involving meibomian ducts, with conjunctival ulceration. Various bacterial species can be isolated, but their role as pathogens or opportunists is unclear (Smith et al. 1996) Opacification of the lens, resulting in a white or gray lens. (Smith et al. 1994; Hubert et al. 1999) Changes in liens fibers include fiber swelling, vacuolation, liquefaction, and formation of morgagnian globules. Damage to lens epithelium can result in epithelial flattening, proliferation, layering, and formation of bladder cells. Later changes may include mineralization, cholesterol/lipid deposition, or complete liquefactive necrosis. With hypermature cataracts, leakage of lens material may provoke an inflammatory response. (Frame et al. 1996; Smith, Roderick et al. 1994; Hubert, Gerin et al. 1999) Inflammation of the cornea with vascularization of the normally avascular cornea. There may be ulceration, corneal thickening due to edema, erosion or ulceration of the anterior surface. (Frame et al. 1996; Hubert et al. 1999) The grossly visible white area on the anterior cornea is attributable to mineralization. Histologically there is mineralization of the basement membrane and stroma with varying degrees of edema, inflammation, vascularization of the stroma, and erosion or ulceration. (Van Winkle et al. 1986; Frame et al. 1996) When associated with the rd1 mutation in Pde6b gene there is bilateral complete loss of the outer nuclear layer (rod and cone nuclei) and of the outer granular layer (inner and outer segments of the rod and cone photoreceptors), so that the inner nuclear layer appears to abut the retina pigmented epithelium (RPE). As the disease progresses there is loss of retinal vasculature and of pigment in the RPE (in pigmented mice), foci of thinning of the inner nuclear layer, and loss of ganglion cells and nerve fibers. (Smith et al. 1996; Chang et al. 2002; Serfilippi et al. 2004) Age-related or light-related degenerative changes in the retina with reduction of the outer layers due to loss of photoreceptor cells. Normally the outer nuclear layer is 10–12 nuclei thick. (Smith et al. 1996) Some studies may use the term for rd1-associated retinal degeneration. (Hall et al. 1992) See above, MULTISYSTEM. These usually consist of discrete interstitial mononuclear infiltrates. (Frith et al. 1988; Faccini et al. 1990; Botts et al. 1999) See above, MULTISYSTEM. This lesion can morphologically resemble glomerulonephritis and should be distinguished by special stains. Glomeruli are enlarged with mesangium expanded by nodular or diffuse accumulations of acellular eosinophilic homogeneous material. Renal papillary necrosis is usually associated with renal amyloidosis or toxins. (Frith et al. 1988; Wolf et al. 1996)
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TABLE 25-5—cont’d SYSTEM Organ, Condition; Synonyms (historical terms) Kidney, glomerulonephritis
Kidney, glomerulosclerosis
Kidney, hydronephrosis
Kidney inflammation Kidney, interstitial nephritis
Kidney, nephropathy
Kidney tubule, hyaline droplets Kidney tubule, mineralization (nephrocalcinosis) Urinary obstruction, ‘dysuria’
Urinary obstruction, MUS
Urinary bladder, arteritis Urinary bladder, inflammation
Deposition of eosinophilic hyaline material in glomerular basement membranes with proliferation of mesangial cells and inflammatory cell infiltration. (Son 2003) It may be characterized further as membranoproliferative glomerulonephritis. A chronic glomerulonephritis characterized by mesangial cell proliferation, increased lobular separation of glomeruli, thickening of glomerular capillary walls, and increased mesangial matrix. Early changes are focal mesangial thickening with increased numbers of epithelial cells. Special stains or techniques are required to identify amyloid, PAS positive thickening of glomerular basement membranes, and other material such as collagen. When diagnoses are based on H&E sections only, these distinctions are not made, and the term may be used to include various glomerular changes including glomerular amyloidosis. (Faccini et al. 1990; Montgomery 1998; Son 2003; Pathbase 2004) The term implies scarring or fibrosis and related late changes in the glomerular tuft in progressive glomerular disease, including small, fibrotic tufts and adhesions to the glomerular capsule (synechiae). (Faccini et al. 1990) The term also may be used to include a wide range of glomerular changes (including very little sclerosis) with various tubular changes and interstitial inflammation. (Maita et al. 1988) Distention of the renal pelvis may be mild with little change in kidney size, or the dilated pelvis and kidney may cause abdominal distention with compression atrophy of kidney remnants. Possible causes include urinary obstruction due to calculi, tumors, or inflammation. It may be unilateral or bilateral. There may be hydroureter if obstruction is chronic and distal in the urinary tract. (Frith et al. 1988; Seely 1999) The condition should not be confused with genetically determined polycystic kidney syndromes that usually involve bilateral progressive development of multiple cysts of tubules and/or collecting ducts, culminating in renal failure, and sometimes associated with extrarenal cysts and other abnormalities. (Werder et al. 1984; Trudel et al. 1991; Nakayama et al. 1994; Ricker et al. 2000) See interstitial nephritis. In diffuse chronic interstitial nephritis, kidneys are reduced in size with granular or nodular surfaces. In the subacute stage cellular infiltrate includes lymphocytes, plasma cells, and fewer neutrophils or macrophages. Tubules are dilated with varying degrees of degeneration and regeneration. The chronic stage is characterized by focal or diffuse fibrosis, with lymphocytic or lymphohistiocytic infiltration; tubules may be cystic with proteinaceous casts or be atrophic, there may be areas of tubular regeneration or hyperplasia. Glomeruli usually are spared if primary insult is interstitial, or the condition may be secondary to glomerulonephritis. (Faccini et al. 1990; Montgomery 1998) The term usually includes tubular changes: tubular regeneration, occasionally with tubular casts, thickened basement membrane, crowding of nuclei, and inflammatory cell infiltration. (Son 2003) Some authors may include glomerulonephritis in this term. Some authors refer to the condition as chronic progressive nephropathy, similar to the condition in rats (Wolf et al. 1996). Interstitial nephritis and nephropathy have been used to refer to similar or identical conditions. (Faccini et al. 1990) Hyaline droplets that contain lysozyme from tumor cells can be found in proximal tubules in some mice that have histiocytic sarcoma. The brightly eosinophilic cytoplasmic droplets can be striking in affected kidneys. (Hard et al. 1991; Lacroix-Triki et al. 2003) Foci of mineralization are granular, faintly basophilic, and fill tubular lumina, usually at the corticomedullary junction, or in the loops of Henle in the medulla. It may occur as part of the spectrum of changes in nephropathy or interstitial nephritis, and not diagnosed as a separate entity. (Frith et al. 1988; Faccini et al. 1990; Seely 1999) Maita et al. (1988) and Son (2003). uses the term dysuria to refer to urinary obstruction, with urinary bladder severely dilated and urethra near isthmus plugged by gelatinous plug with sperm, or the obstruction may not be identified, and there may be hydroureter or hydronephrosis. Other authors refer to this condition as obstructive uropathy. (Faccini et al. 1990) or MUS (Mouse urologic syndrome) (Faccini et al. 1990; Bendele 1998) Mouse urologic syndrome (MUS) has one or more of the following features: bladder distension; peripreputial urine staining, alopecia, and edema; paraphimosis; urethral blockage; ulcerative balanoposthitis; hydronephrosis; pyelonephritis; rectal prolapse; and perineal ulcerative dermatitis. (Everitt et al. 1988) In acute MUS mice are found dead with no prior clinical signs. In the chronic form, there may be ventral wetting, dermatitis, paraphimosis, penile trauma, urinary calculi, pyelonephritis, or hydronephrosis. (Bendele 1998) See above, MULTISYSTEM. See above, MULTISYSTEM.
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TABLE 25-6
GLOSSARY BY SYSTEM: NEOPLASMS, Brief Definitions, or Descriptions of Some Neoplasms that may be Encountered in Mice. This Is Not a Complete List and Glossary of All Neoplasms That May Occur Spontaneously or Be Induced in Mice. The Information Is Derived Primarily from Resources Used in the Chapter. It Is Provided to Facilitate Understanding of terminology in the Chapter, and Is Not an Officially Sanctioned Glossary, Dictionary, or Ontology. Terminology and Definitions Will Continue to Change as Neoplasms Are Characterized Further and as New Neoplasms Are Induced. SYSTEM Organ, Neoplasm (historical or obsolete terms) ALIMENTARY Intestine, polyp
Intestine, adenoma
Intestine, adenocarcinoma
Intestine, cecum carcinoid
Intestine, fibrous histiocytoma
Liver tumor
Liver, adenoma hepatocellular (type A nodule hepatoma, hyperplastic nodule, carcinoma) Liver, carcinoma hepatocellular (type B nodule, trabecular carcinoma, malignant hepatoma)
A mass of tissue which projects outward or upward from the normal surface level being macroscopically visible as a hemispheroidal, spheroidal, or irregular mound-like structure growing from a relatively broad base or a slender stalk. These may be adenomas but are not specified in some reports. (Maekawa et al. 1996; Shackelford et al. 1999; Pathbase 2004) Benign neoplasm of enterocytes of intestinal mucosa. These are uncommon spontaneous neoplasms in mice, and are more likely to occur in the small intestine (especially in the duodenum) than in the large intestine. They are frequently small and may not be detected in the unopened intestine. Typically they are polypoid and project into the lumen. Mucosal architecture may be distorted and there may be branching villi or tubular crypt proliferation. There may be crypt herniation but without penetration of basement membrane. The epithelium is relatively well differentiated, but may be more basophilic than adjacent normal epithelium. There may be associated inflammatory changes especially if there is ulceration, and the neoplasms may arise near a Peyer’s patch. (Frith et al. 1988; Maekawa et al. 1996; Shackelford et al. 1999; Betton et al. 2001) Malignant neoplasm of enterocytes of intestinal mucosa. Large tumors may be recognized grossly when they expand the intestine. There may be single or multiple nodules or polypoid masses that project into the lumen or endophytic, sessile neoplasms may result in a thickened wall with irregular mucosal surface, and present grossly as a diverticulum or bulge of the serosal surface. Distinction between adenoma and adenocarcinoma may be difficult when there is no metastasis or obvious invasion through the basement membrane into the intestine wall. Invasive glands frequently are associated with a marked inflammatory and scirrhous response. Normal architecture is lost and there may be cystic and solid areas. The neoplastic cells usually are basophilic, more anaplastic and pleomorphic than in adenomas, cuboidal to columnar, and there may be goblet or ‘signet ring,’ or Paneth cell components. There are increased mitotic figures and nuclei may be pleomorphic. (Frith et al. 1988; Maekawa et al. 1996; Shackelford et al. 1999; Betton et al. 2001) Neoplasm of neuroendocrine (enterochromaffin) cells of the intestinal mucosa. Gastrointestinal carcinoid or neuroendocrine tumors are very rare neoplasms in mice. They may be induced in the stomach by antisecretory agents that cause hypergastrinemia. Malignancy is determined by invasion and/or distant metastasis. Neuroendocrine tumors typically feature a packeted pattern of clusters of polygonal cells supported delicate fibrovascular stroma. Cytologic features may be similar to other endocrine cells with moderate to ample granular cytoplasm, a round nucleus with prominent nucleus/i, and the bulk of the cytoplasm, rather than the nucleus, may appose the vasculature. The cells may be argyrophilic, and stain with chromogranin A or neuron specific enolase. (Maekawa et al. 1996; Betton et al. 2001) These neoplasms derive from pluripotential mesenchymal stem cells and also exhibit histiocytoid features. In mice these are rare outside of the subcutis/skin. Histiocytic sarcoma (see below, HEMATOPOIETIC) should be considered especially when there is involvement of liver and other organs. Schwannoma (nerve sheath tumor) and fibroma or fibrosarcoma also should be ruled out. (See below, INTEGUMENT.) (Ernst et al. 2001) Hepatocellular neoplasm, adenoma vs. adenocarcinoma not distinguished. Usually other types of tumors in the liver (cholangioma, cholangiocarcinoma, hemangioma, hemangiosarcoma, hepatoblastoma, histiocytic sarcoma, metastatic neoplasm) are not included in this category, unless diagnoses were made by gross examination only. Benign neoplasm derived from hepatocytes. These usually are distinctly demarcated or circumscribed nodules, 1–10 mm diameter, that lack lobular organization, compress adjacent parenchyma, and may bulge from the liver surface. They do not invade adjacent parenchyma or vessels and do not metastasize. They consist of a uniform population of welldifferentiated cells that resemble normal hepatocytes but may be larger or smaller than adjacent normal hepatocytes, and can have more basophilic, eosinophilic, or vacuolated cytoplasm. Hepatocellular carcinomas may arise within adenomas. (Frith et al. 1994; Harada et al. 1999; Deschl et al. 2001) Malignant neoplasm derived from hepatocytes. Carcinomas in mice often have distinct trabecular or adenoid patterns. Moderately-well differentiated hepatocellular carcinomas are composed of larger hepatocytes that vary in size and shape in trabecular or solid patterns. The poorly differentiated tumors are composed of cells with less cytoplasm and more immature nuclei and some have extremely large anaplastic cells. Metastases are typically to the lung, and careful examination may reveal pulmonary metastases in up to 40% of male B6C3F1 or C3H mice with hepatocellular carcinoma that are allowed to live out their lifespan. Metastases usually occur only when tumors are large (>10 mm). (Frith et al. 1994; Harada et al. 1999; Deschl et al. 2001)
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TABLE 25-6—cont’d SYSTEM Organ, Neoplasm (historical or obsolete terms) Liver, foci of cellular alteration Liver, hemangioma Liver, hemangiosarcoma Liver, hepatoblastoma
Salivary gland, myoepithelioma
Stomach, forestomach, squamous papilloma
Stomach, forestomach, squamous cell carcinoma
CARDIOVASCULAR Heart, hemangioma (angioma) Heart, hemangiosarcoma (angiosarcoma) Vessels, hemangioma
Vessels, hemangiosarcoma
Heart, mesothelioma
See Table 25-5. They may be reported as neoplastic or nonneoplastic findings, depending on the study. Hemangioma in liver, see hemangioma below. These neoplasms may be difficult to distinguish from angiectasis (Table 25-5). Hemangiosarcoma in liver, see hemangiosarcoma below. These rare spontaneous or induced liver tumors may be an undifferentiated variant of hepatocellular carcinoma, and fetal or ductular origins have been proposed. Histologically similar neoplasms occur in children, but hepatoblastomas only occur in aged mice. They are almost always found within or adjacent to hepatocellular carcinomas and are distinct because of their basophilia relative to adjacent parenchyma. The tumors frequently have distinctive patterns including rosettes, rows or ribbons, organoid, or nest-like structures lined by distinct but delicate vascular channels. The channels are surrounded by one/several layers of radially or concentrically arranged neoplastic cells. (Frith et al. 1994; Harada et al. 1999; Deschl et al. 2001) Neoplasm of myoepithelial cells of glandular structures including salivary gland; mammary gland and preputial/clitoral glands. They are rare in mice, and are most likely to occur in the submaxillary or parotid salivary glands. The tumors can become large and appear to be cystic. Histologically, they are biphasic tumors, composed of large pleomorphic cells including elongated or spindle-shaped mesenchymal-type cells, mixed with areas of polyhedral epithelioid cells. Areas of degeneration and necrosis can result in pseudocysts filled with mucus and necrotic cell debris. The neoplastic cells may palisade around blood vessels. Larger tumors may metastasize to the lung. (Sundberg 1992; Botts et al. 1999; Pathbase 2004) Benign neoplasm of stratified squamous epithelium of the nonglandular stomach (forestomach). These are usually exophytic villous or arborescent outgrowths of fibrovascular stroma covered by neoplastic stratified. There may be acanthosis and hyperkeratosis but maturation is normal and mitotic figures are rare. The incidence in control B6C3F1 mice, Swiss mice, and other strains usually is <2%. (Maekawa et al. 1996; Betton et al. 2001; Pathbase 2004) Squamous cell carcinoma arising from stratified squamous epithelium of the forestomach. In the stomach these malignant neoplasms tend to be polypoid, with marked cellular pleomorphism, keratin pearls, and many mitotic figures. They exhibit invasion, and ulceration, inflammation, and fibrosis of submucosa are common features. The incidence in control B6C3F1 mice, Swiss mice, and other strains usually is <2%. See below, Hemangioma. See below, Hemangiosarcoma. Benign neoplasm of endothelial cells can be found at any site in the body. The most common sites are the spleen and liver. The subcutis, skeletal muscle, and female reproductive tract are other common sites. Cavernous hemangiomas are dilated cavernous spaces that are lined by endothelial cells and filled with red blood cells. Capillary hemangiomas are circumscribed accumulations of small cleft-like spaces lined by typically plump endothelial cells and containing red blood cells, or the spaces may be vacant or flattened. Mitotic figures are rare in hemangiomas. (Frith et al. 1982; Booth et al. 1995; Peckham et al. 1999; Ernst et al. 2001) Malignant neoplasms of endothelial cells. They consist of dilated vascular spaces of varying sizes which may or may not be filled with red blood cells. They may be very pleomorphic and vessel-like structures may be difficult to appreciate in predominantly solid neoplasms. The cells lining the vascular spaces are plump with oval basophilic nuclei and with indistinct cell borders, and may have bizarre mitotic figures. There is often piling-up of the lining cells. In areas, there may be solid sheets of cells. Particularly in the spleen, the tumors may be predominantly solid. Necrosis, hemorrhage, and thrombosis are frequent. They are non-circumscribed and are locally invasive. (Frith et al. 1982; Booth et al. 1995; Peckham et al. 1999; Ernst et al. 2001 Neoplasm of mesothelial cells that line pleural and peritoneal cavities. These are rare spontaneous tumors in mice but may be induced by intrapleural or aerosol exposure to asbestos or other mineral fibers. They have epithelial, mesenchymal, or mixed patterns, and exhibit nodular or diffuse involvement of pleural or peritoneal surfaces. Nodular mesotheliomas are characterized by 1 to several nodules on pleural or peritoneal surfaces, and may be benign or malignant. Diffuse mesotheliomas have many contiguous nodules or thick confluent, creeping, growth over surfaces and tend to be malignant with deep invasion. Epithelial-type neoplasms may be papillary with exophytic fronds of pleomorphic cells supported on fibrous stalks, or tubular with atypical cells forming glandular patterns, or solid with many bizarre atypical cells with karyomegaly or several nuclei. Mesenchymal-type areas consist of interlacing bundles of spindle cells with nuclear pleomorphism. (Dixon et al. 1999; Dungworth et al. 2001) Continued
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TABLE 25-6—cont’d SYSTEM Organ, Neoplasm (historical or obsolete terms) ENDOCRINE Adrenal cortex, subcapsular cell hyperplasia Adrenal cortex, adenoma (adrenal adenoma; adrenal cortical adenoma)
Adrenal medulla, pheochromocytoma (adrenal medullary (chromaffin) cell tumor)
Pancreas isle (adenoma insulinoma)
Pituitary gland, adenoma
Pituitary gland, hyperplasia, pars distalis/intermedia Thyroid gland, C cell, adenoma
Thyroid gland, C cell, carcinoma Thyroid gland, follicular cell, adenoma
Thyroid gland, follicular cell, carcinoma
See Table 25-5. Benign neoplasm of adrenal cortical tissue. These are expansile accumulations of neoplastic cells with low mitotic activity that are well delineated from adjacent normal cortex, typically bulge the surface of the gland, compress underlying cortex, and exceed the regular width of the cortex in a young mouse. Type A adenomas are composed of spindled cells. Type B adenomas (solid adenomas) are composed of more polygonal, small, or larger lipid-laden cells that resemble normal adrenal cortical cells. In both types neoplastic cells appear to be packeted by fine vascular stroma. The tumors frequently have both cell types, and usually may be named for the predominant cell type. Mast cells intermingled with the tumor cells often are associated with the type A tumors. Adenomas are relatively uncommon, usually <1% incidence even in old populations, but are more common than carcinomas. (Yarrington 1996; Nyska et al. 1999; Capen et al. 2001) Neoplasm of the adrenal medulla. These tumors are much less common in mice than they are in rats, and like human pheochromocytomas, spontaneous tumors in mice produce catecholamines. The neoplastic cells are relatively uniform polyhedral cells that resemble normal medullary secretory cells with central nuclei and finely stippled cytoplasm, and are supported or packeted in delicate fibrovascular stroma. As tumors enlarge, the stroma tends to be less conspicuous and the capillaries are distended with blood. Cytoplasm of neoplastic cells may be more basophilic with H&E than that of normal medullary cells. Lesions that involve <50% of the medulla and do not compress adjacent tissue may be diagnosed as hyperplasia. Lesions that invade the adrenal capsule or spread beyond the adrenal gland are classified as malignant pheochromocytoma. (Tischler et al. 1996; Nyska et al. 1999; Capen et al. 2001) Islet cell adenomas in mice typically involve a single islet within a histologic section, in contrast to hyperplasia which typically affects multiple islets or all islets in a lobule. Islet adenomas are larger than hyperplastic islets and compress adjacent normal pancreas. Adenomas may be more vascular than normal or hyperplastic islets and cells tend to be well differentiated, with few mitotic figures. Usually these are functional beta cell tumors that produce insulin without causing hypoglycemia. Carcinomas are even less common and tend to be larger than adenomas but also composed of well-differentiated cells, but demonstrate invasion or distant metastasis usually to the liver. (Boorman et al. 1999; Capen et al. 2001) Benign neoplasm of the pituitary gland. Adenomas of the pars distalis are more common than adenomas of the pars intermedia, and pars distalis adenomas can be common in aged female mice. Adenomas are distinguished from pituitary hyperplasia by being well-delineated and causing compression of adjacent normal cells, but the distinction may be subtle and difficult. They are typically composed of large cells with abundant eosinophilic cytoplasm, and angiectatic or cyst-like space are common. Mammotroph or prolactin secreting adenomas of the pars distalis may be especially common in female C57BL6/J mice. Carcinomas of the pars distalis are rare and are diagnosed when there is unequivocal invasion into surrounding tissues or distant metastases. (Schechter et al. 1981; Frith et al. 1988; Mahler et al. 1999; Capen et al. 2001) See Table 25-5. Benign neoplasm derived from calcitonin producing C-cells (parafollicular cells) of the thyroid gland. These are much less common than thyroid follicular adenomas. They occur as solid nests (as opposed to papillary or follicular structures typical of follicular adenomas) of polygonal pale polyhedral cells with indistinct cytoplasmic borders. They are (arbitrarily) distinguished from hyperplastic foci by occupying an area larger than 5 average follicles. (Hardisty et al. 1999; Capen et al. 2001) Malignant neoplasm derived from the calcitonin producing C-cells (parafollicular cells) of the thyroid gland. The distinction between adenoma and carcinoma is not clearly defined. There may be central necrosis. Invasion of extrathyroidal tissue may be the only useful criterion for malignancy. (Hardisty et al. 1999; Capen et al. 2001) Benign neoplasm of thyroid follicular cells. Thyroid tumors are uncommon in mice, <1% in most studies, and follicular cell adenoma is the most commonly reported spontaneous tumor. These usually are single well-delineated lesions within the normal thyroid gland. The most common type is a small papillary adenoma, in which a papillary projection of follicular epithelium extends into a cystic lumen. Follicular patterns consisting of small colloid-containing follicles mixed with normal sized follicles, and solid patterns with cells in sheets and densely packed nodules are less common. The follicular epithelium is cuboidal, the cytoplasm stains slightly more basophilic than adjacent normal thyroid, and there may be colloid in the follicle lumens. Solid pattern adenomas may resemble C-cell tumors. (Thomas et al. 1996; Hardisty et al. 1999; Capen et al. 2001) Malignant neoplasm of thyroid follicular cells. These are much less common than follicular cell adenomas, and may be difficult to distinguish from them. The primary pattern is solid, but they may have follicular, papillary, or mixed patterns. They are usually larger than adenomas, with more atypia, and evidence of invasion or distant metastases. (Thomas et al. 1996; Hardisty et al. 1999; Capen et al. 2001)
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TABLE 25-6—cont’d SYSTEM Organ, Neoplasm (historical or obsolete terms) GENITAL, FEMALE Ovary, adenoma Ovary, choriocarcinoma
Ovary, cyst adenoma
Ovary, granulosa cell tumor
Ovary, hemangioma Ovary, hemangiosarcoma Ovary, sex cord stromal tumors
Ovary, luteoma
Ovary, Sertoli cell tumor
Ovary, Sertoli–Leydig cell tumor Ovary, teratoma
Ovary, theca Sertoli cell tumor Ovary, thecoma
Benign tumor of the ovary. The type of adenoma is not specified in some reports. Tubulostromal adenoma or cystadenoma are likely, but with gross examination, the term may refer to any ovarian neoplasm. Malignant neoplasm derived from trophoblasts, and considered to be germ cell neoplasms. Trophoblasts are giant cells with nuclei up to 50 µ diameter that are found in normal embryonic membranes and produce chorionic gonadotropins. Choriocarcinomas are rare neoplasms in mice even in carcinogen studies, and usually occur in the uterus. They are composed of sheets of large anaplastic multinucleated syncytioblasts, and smaller basophilic cells that resemble placental cytotophoblasts pleomorphic, as well as more typical giant uninucleated trophoblasts and highly anaplastic trophoblastlike cells. Hemorrhage is a prominent feature. (Davis et al. 1999; Davis et al. 2001) Benign neoplasm derived from the surface epithelium of the ovary. It is a common spontaneous ovarian neoplasm in some strains of mice. Anastomosing papillary fronds, lined by non-ciliated cuboidal-columnar cells project into the cyst lumen. The cyst lining typically is cuboidal-columnar epithelium, which may include ciliated cells. The cyst lumen may contain eosinophilic serous fluid or blood. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001) Ovarian neoplasm derived from sex cord stromal cells. Granulosa cell tumors are usually unilateral, but may be bilateral or occur in conjunction with another ovarian tumor. They are characterized by a diversity of patterns including solid, tubular, follicular, and trabecular. The neoplastic cells resemble normal granulosa cells and have scant to moderate amphophilic vacuolated cytoplasm depending on their degree of luteinization. They tend to have small oval nuclei with stippled chromatin, and there may be few to many mitotic figures. Some tumors have distinctive areas of fusiform theca-like cells. Some large tumors have areas of necrosis and hemorrhage and prominent lipofuscin-laden cells. Mitoses vary in number from few to numerous. Call-Exner-like rosettes are rare but may be found in the follicular types. Malignant granulosa cell tumors exhibit more cellular pleomorphism, high mitotic rates, local invasion, frequent necrosis and hemorrhage, and may metastasize to the lungs, kidneys, and lymph nodes. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001) Hemangioma in the ovary, see above, CARDIOVASCULAR. Hemangiosarcoma in the ovary, see above, CARDIOVASCULAR. Neoplasms derived from ovarian sex cord stromal cells. They are composed of endocrine-stromal cells of the ovary and include neoplasms of granulosa, theca and lutea cell origin, Sertoli cell tumors, and fibroma or fibrosarcoma of stromal cell origin. Neoplasms may include more than one sex cord stromal cell type and usually are named for the predominant cell type, but some reports may use names that indicate more than one cell type. (Davis et al. 1999; Davis et al. 2001) Benign ovarian neoplasm derived from sex cord stromal cells. They may be the most common ovarian tumor in some strains, e.g., BALB/c. Luteomas are generally well circumscribed but not encapsulated, and may involve the entire ovary. They consist of large polygonal cells that closely resemble the cells of a normal corpus luteum. They should exceed the size of 3 normal corpora lutea. The neoplastic cells have abundant pale granular eosinophilic, sometimes vacuolated, cytoplasm, and a single round nucleus, usually with few mitotic figures. In some luteomas the cells may be brown tinged due to ceroid, which is PAS-positive and acid fast. Mast cells, scattered or in clusters, may be common in luteomas of BALB/c and C57BL/6 but are less common in those of the C3H strains. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001) Ovarian neoplasm derived from sex cord stromal cells. Sertoli cell tumor is a rare ovarian neoplasm, and histologically resembles testicular Sertoli cell tumors, with a distinctive tubular pattern of elongated epithelial cells with homogeneous faintly eosinophilic cytoplasm and basal, round nuclei, palisading on thin strands of fibrovascular stroma. Mitotic activity is usually low. This term probably refers to a mixed sex cord stromal tumor. Benign or malignant germ cell derived neoplasm containing derivatives from all 3 germ layers (endoderm, mesoderm, ectoderm). Ovarian teratomas are rare but usually seen in young mice, and are frequently cystic with a mixture of epithelial cell types, including prominent areas of cornifying, stratified squamous epithelium, ciliated tall columnar epithelium and intestinal epithelium, sometimes thyroid or pancreas, along with well-differentiated cartilage, bone, skeletal or smooth muscle, and variable amounts of well-differentiated nervous tissue resembling cerebral cortex. Benign tumors tend to appear more differentiated with easily recognizable mature tissues. Malignant tumors tend to be less differentiated, with large areas of necrosis and hemorrhage, and are highly metastatic. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999, Davis et al. 2001) Ovarian neoplasm derived from sex cord stromal cells, with theca cell and Sertoli cell-like areas. Benign ovarian neoplasm derived from sex cord stromal cells. The nodules are composed of densely packed distinctive fusiform theca cells typically in bundles or whorling patterns, supported on delicate fibrovascular stroma. Paler luteinized cell clusters may be interspersed between strands of fusiform thecal cells. The principle cell type is fusiform, with an oval nucleus and sparse basophilic cytoplasm. Luteinization is characterized by increased cell size, faintly eosinophilic, brownish, foamy cytoplasm and a round nucleus with a delicate chromatin pattern. Large tumors may have extensive necrosis with only perivascular persistence of viable tissue. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001) Continued
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TABLE 25-6—cont’d SYSTEM Organ, Neoplasm (historical or obsolete terms) Ovary, tubulostromal adenomas; tubular adenoma (tubular mesothelioma)
Ovary, tubulostromal adenocarcinoma Uterus, adenoma endometrial
Uterus, adenocarcinoma endometrial
Uterus, hemangioma Uterus, hemangiosarcoma Uterus, leiomyoma
Uterus, leiomyosarcoma
Uterus, polyp stromal
Uterus, stromal sarcoma
GENITAL, MALE Testes, interstitial cell tumor, Leydig cell tumor
Testes, teratoma; testicular germ cell tumor (TGCT)
Benign neoplasm derived from the surface epithelium of the ovary. It is the most common spontaneous ovarian neoplasm in some strains of mice. Tubulostromal adenomas are at least 2–3 mm diameter and compress adjacent ovarian tissue or efface the ovary. They are composed of cords or tubules of non-ciliated cuboidal or columnar cells separated by large round-polygonal cells that resemble stromal interstitial cells and have eosinophilic foamy or vacuolated cytoplasm, sometimes with golden-brown pigment. They have been interpreted as invaginations of mesothelial (or germinal) epithelium into the ovarian stroma, sometimes dividing the ovary into multiple lobules. These invaginations usually present as variably sized tubular structures, lined by simple columnar, cuboidal, or occasionally flattened epithelium. Tubulostromal hyperplasia does not form discrete masses, is not compressive, nor invasive, but differentiation of these conditions may be arbitrary. (Frith et al. 1988; Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001) Malignant neoplasm derived from the surface epithelium of the ovary. Compared to tubulostromal adenoma, these neoplasms are much less common, and feature increased cellular atypia, increased mitotic index, hemorrhage metastasis, and invasion beyond the ovarian bursa. (Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001). Benign neoplasm of uterine mucosal epithelium. These may be on a broad base or on a stalk (when they may be called polyps). They form well-differentiated glandular or tubular papillary structures lined by cuboidal epithelium. Stromal proliferation should not be a feature (see stromal polyp). (Maekawa, Maita, et al. 1996; Davis, Dixon, et al. 1999; Davis, Harleman, et al. 2001) Malignant neoplasm of uterine mucosal epithelium. These are uncommon in mice. They are poorly circumscribed masses that extend into and occlude the uterine lumen, invade deeply into myometrium and beyond, and may metastasize to lungs. The neoplastic epithelial cells may be well differentiated or very pleomorphic. Necrosis and hemorrhage are common. (Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001) Hemangioma in the uterus, see above, CARDIOVASCULAR. Hemangiosarcoma in the uterus, see above, CARDIOVASCULAR. Benign neoplasm of smooth muscle cells of the myometrium. They may be reported in up to 3% of CD-1® or B6C3F1 mice in some studies and are more common than leiomyosarcomas. They are solitary well-circumscribed masses that may compress adjacent tissues. They are composed of densely cellular sheets of interlacing bundles and whorls of spindle cells, with eosinophilic cytoplasm similar to adjacent myometrial smooth muscle cells. (Maekawa et al. 1996; Davis et al. 1999) Malignant neoplasm of smooth muscle cells of the myometrium. They are poorly delineated masses with disorganized and invasive growth patterns, and may have areas of necrosis and hemorrhage. The spindled cells may be more pleomorphic than in leiomyoma. (Maekawa et al. 1996; Davis et al. 1999) Polypoid mass of uterine stromal tissue that projects into the uterine lumen. It is covered by cuboidal-columnar epithelium that is continuous with and similar to endometrial lining epithelium. There may be endometrial glandular elements as well. Most endometrial polyps have abundant loose stroma composed of stellate or spindle cells and numerous small blood vessels, and may have a few large dilated or pleomorphic endometrial glands. When the polyp is composed primarily of endometrial glandular tissue with little stroma, it may be referred to as a glandular polyp. (Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001) Malignant neoplasm of uterine fibrovascular stroma. These are uncommon tumors, found in <1% of CD-1® or B6C3F1 mice on chronic studies. Some may arise in the stroma of uterine polyps. They are composed of sheets of poorly differentiated spindle cells, there may be pleomorphism and some giant cell and many mitoses. There are variable amounts of fibrillar or collagenous matrix, and endothelium-lined vascular spaces. There may be necrosis, hemorrhage and infiltration of myometrium, cervix, and adjacent abdominal structures. Metastasis is rare. Differential diagnoses include histiocytic sarcoma, leiomyosarcoma, fibrosarcoma, and schwannoma, and they may be difficult to distinguish from the more common histiocytic sarcoma involving the uterus. (Maekawa et al. 1996; Davis et al. 1999; Davis et al. 2001) Tumor derived from testicular Leydig cells (interstitial cells). Both benign and malignant spontaneous tumors of the interstitial cells of Leydig are rare in mice, but can be induced with synthetic or natural estrogens in certain strains, especially in BALB/c mice. They are unilateral, with no right or left predilection. ‘Tumors’ may be difficult to distinguish from interstitial hyperplasia. Hyperplastic foci may be distinguished by size (<3 seminiferous tubules diameter) and non-compression, compared to adenomas which are larger (>3 seminiferous tubules diameter), compress surrounding tissue, and exhibit some cellular pleomorphism. Spontaneous Leydig cell tumors tend to be well differentiated and of the solid, diffuse type, composed of round homogeneous cells with eosinophilic granular cytoplasm. Small, well-circumscribed tumors are adenomas, and large tumors which are invasive or metastasize are referred to as carcinomas. The larger carcinomas may metastasize to the lungs. (Frith et al. 1988; Prahalada et al. 1994; Gordon et al. 1996; Mahler et al. 1997; Radovsky et al. 1999; Pathbase 2004) Benign or malignant germ cell derived tumors of the testes containing tissues from all 3 germ cell layers (endoderm, mesoderm, ectoderm). Testicular teratomas are rare but usually seen in young mice, especially of 129 strains. They typically contain a variety of epithelial types, including cornifying, stratified squamous epithelium, ciliated tall columnar epithelium and intestinal epithelium, sometimes thyroid or pancreas, along with well-differentiated cartilage, bone, skeletal or smooth muscle, and variable amounts of well-differentiated nervous tissue resembling cerebral cortex. Benign tumors tend to appear more differentiated with easily recognizable mature tissues. Malignant tumors tend to be less differentiated, with large areas of necrosis and hemorrhage, and may metastasize. (Gordon et al. 1996; Rehm et al. 2001; Pathbase 2004)
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TABLE 25-6—cont’d SYSTEM Organ, Neoplasm (historical or obsolete terms) Testes, seminoma
Testes, Sertoli cell tumor
HEMATOPOIETIC Histiocytic sarcoma (reticulum cell sarcoma or reticulum cell neoplasm, type A; endometrial sarcoma)
Leukemia, lymphoid Leukemia, nonlymphoid
Lymphoma (any type)
Lymphoma, diffuse large B cell lymphoma; follicular center cell lymphoma Lymphoma, follicular B cell lymphoma; follicular center cell lymphoma; reticulum cell sarcoma or reticulum cell neoplasm, type B Lymphoma, precursor T cell lymphoblastic lymphoma (thymic leukemia)
Plasmacytoma
Testicular germ cell tumor derived from spermatogenic cells resembling spermatogonia or spermatocytes. These are very rare in mice, composed of fairly uniform large cells with clear (glycogen-containing) cytoplasm and well-defined cell borders, resembling primitive germ cells. (Gordon et al. 1996; Rehm et al. 2001; Pathbase 2004) Testicular germ cell tumor derived from sex cord/stromal cells (Sertoli cells). These are very rare in mice, composed of elongate cells typically arranged in distinctive palisades on delicate fibrovascular stroma (picket fence pattern), forming tortuous tubular structures without distinct lumina. A poorly organized diffuse pattern with few or indistinct tubular structures is less common. Neoplastic cells tend to have indistinct borders, pale vacuolated cytoplasm, and many mitotic figures. (Rehm et al. 2001; Pathbase 2004) Solid tumor mass composed predominantly of histiocytic cells. (Kogan et al. 2002) These can be common hematopoietic neoplasms in some strains, and usually are more common in female mice. They usually are diagnosed after 12 months of age. The liver and uterus or vagina are commonly involved. Spleen, lymph node, bone marrow, lung, kidney, and ovaries also may be involved. There are noncircumscribed, highly infiltrative accumulations of usually large histiocytoid cells with ample eosinophilic cytoplasm. There may be areas where cells are primarily elongate in sarcomatous patterns, and there may be multinucleated cells, and Erythrophagocytosis. There may be areas of necrosis surrounded by palisading cells. In the liver sinusoids may be filled and expanded by neoplastic cells, and (metastatic) neoplastic cells may be prominent in pulmonary vasculature. The cells should be immunocytochemically positive for histiocytic markers including lysozyme. Eosinophilic hyaline droplets (of lysozyme) have been reported in kidney tubules in animals with this condition. Increased hematopoiesis in the liver also is reported in animals with this condition. (Ward et al. 1999; Frith et al. 2001; Lacroix-Triki et al. 2003) The majority of lymphoid leukemias in mice, as defined by involvement of the blood, represents “spillover” of lymphoma cells into the blood. These are not primary leukemias but rather lymphomas with leukemic phases. (Morse et al. 2002) Nonlymphoid leukemias, myeloid dysplasias, and myeloid proliferations (nonreactive) include diseases that arise primarily as increased numbers of nonlymphoid hematopoietic cells in the spleen and/or bone marrow. Leukemias are disseminated diseases that are rapidly fatal. Many leukemias are characterized by impaired differentiation, but myeloproliferativedisease–like (MPD-like) myeloid leukemias retain differentiation to mature forms. Myeloid dysplasias are characterized by cytopenias and abnormal differentiation. Nonlymphoid hematopoietic sarcomas are cellular proliferations that arise primarily as solid tumors, e.g., histiocytic sarcoma. Myelogenous leukemias or erythroleukemias are uncommon in mice and can be induced by some murine leukemia viruses and by genetic manipulations. (Kogan et al. 2002) Malignant neoplasm of lymphoid cells. This term usually designates solid neoplasms. Some studies do not distinguish hematopoietic neoplasms, or it is not clear what the correct current nomenclature should be based on the nomenclature used. Immunohistochemistry and/or molecular techniques should be used to determine accurate diagnoses and subclassifications. Only a few of the more common spontaneous lymphomas are listed here. (Ward et al. 1999; Frith et al. 2001; Morse et al. 2002) Neoplasm of mature B lymphocytes. This has been reported as a spontaneous tumor in some congenic and recombinant inbred mice. There usually is splenic involvement with little or late lymph node involvement, sometimes liver and blood involvement. The cells are uniformly small B cells. (Morse et al. 2002) Neoplasm of mature B lymphocytes, although infiltrating (polyclonal) T cells may be numerous. These are the most common spontaneous hematopoietic neoplasm in many mouse strains. Usually they are diagnosed after 12 months of age. There is progressive enlargement of the spleen with mottling on cut section due to neoplastic enlargement of follicles in white pulp. Advanced cases have massively enlarged mesenteric lymph nodes and spleen, and prominent GALT. Histologically there is diffuse involvement of splenic white pulp and cells are small or large, with cleaved or noncleaved nuclei with clumped or vesicular chromatin of apparently mixed-cell types, although early tumors may be more plasmacytoid. (Ward et al. 1999; Frith et al. 2001; Morse et al. 2002) Neoplasm of immature T cells that arises in the thymus. Lymphomas of T cell origin usually arise in the thymus. Clinically mice with thymic lymphoma may be dyspneic due to massive enlargement of the thymus. Necropsy findings commonly also include enlarged spleen and lymph nodes. Involvement of liver, kidneys, and bone marrow may occur in advanced stages, or may represent an additional neoplastic process. Histologically neoplastic cells are monomorphic and medium sized with scant cytoplasm, and the starry sky pattern is attributed to scattered larger paler (tingible body) macrophages, which are engulfing apoptotic or necrotic cells and debris, in sheets of homogeneous neoplastic lymphoid cells. This is the most common lymphoma affecting the thymus, is very common in AKR mice, and is the most commonly induced tumor by viruses and carcinogens. (Ward et al. 1999; Frith et al. 2001; Karpova et al. 2002; Morse et al. 2002) Neoplasm of mature secretory B cells. It is an uncommon spontaneous neoplasms in mice but can be induced by pristane and mineral oil, especially in BALB/c mice. The spontaneous tumor presents with splenomegaly and lymphadenopathy. The pristane-induced tumor arises in peritoneal granulomas. Histologically it is characterized by mature plasmacytoid cells with ample amphophilic cytoplasm, frequently with paranuclear pallor due to golgi material, and round clock-face nuclei with central nucleoli. They should be immunocytochemically positive for cytoplasmic immunoglobulins. (Ward et al. 1999; Frith et al. 2001; Morse et al. 2002) Continued
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TABLE 25-6—cont’d SYSTEM Organ, Neoplasm (historical or obsolete terms) Spleen, hemangioma INTEGUMENT Mammary gland, hyperplasia functional without atypia Mammary gland, hyperplastic alveolar nodule, HAN
Mammary gland, plaques
Mammary gland, tumor Mammary gland, adenoma
Mammary gland, adenocarcinoma
Mammary gland, carcinoma with squamous differentiation, adenoacanthoma, adenosquamous carcinoma Mammary gland, fibroadenoma Skin subcutis, fibroma
Skin subcutis, fibrosarcoma
Skin subcutis, fibrous histiocytoma
Skin subcutis, hemangioma (angioma) Skin subcutis, lipoma, liposarcoma
See above, CARDIOVASCULAR. See Table 25-5.
HAN are common preneoplastic findings in MMTV-infected and MMTV-free mice, as well as in carcinogen-treated mice. Grossly HAN are 1–5 mm nodules, frequently outlined by yellow pigment. Histologically they are foci of lobuloalveolar hyperplasia, characterized by closely crowded acini that are lined by a single layer of epithelium, and lack significant dysplasia, within a background of normal fatty stroma. This hyperplastic mammary tissue is immortal and can be serially transplanted, with development into focal proliferations and neoplastic lesions. (Medina 1982; Rehm et al. 1996; Cardiff et al. 1999; Cardiff et al. 2000) Mammary plaques are epithelial proliferations that occur in mouse mammary glands during pregnancy or after hormone induction, but regress after withdrawal of the stimulus. These were formerly known as type P or pregnancy dependent tumors. Histologically they consist of radiating ducts surrounded by dense connective tissue. (Medina 1982; Rehm et al. 1996; Cardiff et al. 1999) Mammary neoplasm, adenoma vs. adenocarcinoma vs. other not specified Benign neoplasm of the mammary gland. These are rare in mice, but well-differentiated acinar pattern carcinomas may be referred to as adenomas in some studies. These are well-differentiated, circumscribed, or encapsulated nodules composed of closely packed, small, uniform, acinar structures. (Seely et al. 1999; Bruner et al. 2001) Malignant neoplasm of the mammary gland. Most spontaneous or MMTV-induced mammary tumors in mice have been classified as type A, B, or C adenocarcinomas according to Thelma Dunn’s original 1959 classification. Type A (acinar) or microacinar adenocarcinomas are composed of small acini lined by a single layer of cuboidal cells. These also have been referred to as adenoma and tubular carcinoma. Type B (bizarre) or ductal tumors are most common, have more variable histologic features, with well- and poorly differentiated regions of neoplastic cells in cords or sheets or papilloma-like configurations. They can rise from carcinogen-induced ductal hyperplasias. Type C (cystic) tumors are less common than A or B tumors, and feature cystic epithelial structures in more abundant stroma. (Medina 1982; Rehm et al. 1996; Cardiff et al. 1999; Cardiff et al. 2000) The term adenoacanthoma has been used to refer to benign and malignant adenomatous epithelial tumors with some squamous differentiation. In mice mammary adenoacanthoma usually is used to refer to a malignant mammary epithelial neoplasm with squamous cell areas covering >25% of the lesion. When the squamous component is <25%, it is an adenocarcinoma. (Bruner et al. 2001) Adenoacanthomas are uncommon in high tumor strains, are more likely to occur in old retired breeders than in young animals, are more common in BALB/c (and maybe FVB/N mice) than in other common strains, and they can have a high incidence some carcinogenesis protocols. (Rehm 1990; Rehm et al. 1996; Cardiff et al. 1999; Cardiff et al. 2000; Nieto et al. 2003) Benign neoplasm of mammary epithelium plus fibrocollagenous connective tissue. While this is a common neoplasm in rats, it is unusual in mice. It is composed of proliferating ducts within a dense fibrous stroma. (Seely et al. 1999; Bruner et al. 2001) Benign neoplasm of fibroblasts or fibrocytes, characterized by production of collagen. They present as firm rounded masses or elevations of the skin. Histologically they are circumscribed masses of collagenous connective tissue that can compress or distort surrounding tissues. Spindle cells are enmeshed in the collagen fibers in interlacing bundles. (Sundberg 1996; Peckham et al. 1999; Ernst et al. 2001) Malignant neoplasm of fibroblasts or fibrocytes. These tend to have more irregular patterns, higher cellularity, more cellular pleomorphism or atypia, and may have less collagen than the benign counterpart. They may have herring bone patterns, necrosis, hemorrhage, inflammation, and alopecia and ulceration of overlying skin. Local invasion can be extensive but metastasis late and infrequent. (Peckham et al. 1999; Ernst et al. 2001) Neoplasm of pluripotential mesenchymal stem cells. They should exhibit storiform patterns and have histiocytoid features to be diagnosed. They may be positive by immunohistochemistry for histiocytic enzymes such as lysozyme, cathepsin B, alpha 1 antitrypsin, and alpha1 antichymotrypsin as well as mesenchymal markers. They have been reported as the most common skin/subcutaneous neoplasm in CD-1® mice. Histologically benign fibrous histiocytoma are composed of primarily well-differentiated spindled fibroblast-like cells in storiform and cartwheel patterns, and may have abundant collagen, but also have a histiocytoid component of plumper cells that may exhibit phagocytosis. There may be inflammatory cells scattered in and around the neoplasm. Malignant neoplasms have more variable patterns, fibrous, myxoid, pleomorphic, and mixed. These neoplasms may be difficult to distinguish from poorly differentiated fibrosarcomas, and may be diagnosed as fibrosarcoma or undifferentiated sarcoma in some report. (Peckham et al. 1999; Ernst et al. 2001) Hemangiomas in skin or subcutis are dark red or purple raised lesions that bleed profusely when cut. See above, Hemangioma. (Booth et al. 1995; Booth et al. 1996). Also see above, CARDIOVASCULAR. Lipoma and liposarcoma are rare in mice. Lipomas are well-circumscribed accumulations of mature unilocular adipocytes with peripherally compressed nuclei. Liposarcomas tend to be firmer, poorly circumscribed, more cellular with more poorly differentiated spindle cells, and fewer typical adipocytes. (Peckham et al. 1999; Ernst et al. 2001)
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TABLE 25-6—cont’d SYSTEM Organ, Neoplasm (historical or obsolete terms) Skin subcutis, neural crest tumor Skin subcutis, neurofibroma, neurofibrosarcoma Skin subcutis, Schwannoma, nerve sheath tumor Skin, papilloma;
MUSCULOSKELETAL Rhabdomyosarcoma
NERVOUS Brain, astrocytoma
Brain, meningioma
Brain, oligodendroglioma
Neurofibroma, neurofibrosarcoma Schwannoma, nerve sheath tumor
RESPIRATORY Nasal cavity, hemangioma Nasal cavity, schwannoma, neurilemmoma Lung, tumors
Subcutaneous spindle cell tumors occurring on the pinna and tail of FVB/N mice were diagnosed as neural crest tumors. They resemble the amelanotic melanomas of the pinnae of Fischer 344 rats. (Mahler et al. 1996) See below, NERVOUS. See below, NERVOUS.
A benign neoplasm consisting of villous or arborescent outgrowths of fibrovascular stroma covered by squamous papilloma neoplastic squamous epithelial cells. These are uncommon spontaneous neoplasms, but are induceed in various carcinogen protocols. They are usually exophytic or papillary, but may be pedunculated (on a thin stalk) or sessile (flat). The basal cell layer or borderline should be distinct. Acanthosis and hyperkeratosis or parakeratosis are typical. (Bruner et al. 2001; Pathbase 2004) Malignant neoplasm of striated skeletal muscle. These neoplasms arise as nodules in skeletal muscle. Neoplastic cells are pleomorphic, with elongate, strap-like cells with multiple nuclei in tandem array, and smaller plump to spindled haphazardly oriented cells. Cross striations in neoplastic cells distinguish this from other sarcomas, but can be very difficult to discern. They may be enhanced by phosphotungstic acid hematoxylin (PTAH) stains. (Sundberg et al. 1991; Sundberg et al. 1996 Neoplasm of astrocytic glial cells. Astrocytomas are even less common than oligodendrogliomas in mice. The lesion should be confined to one area of the brain but tumor margins are indistinct and there may be areas of edema, hemorrhage. The cells tend to be monomorphic but with an indistinct cytoplasmic border, and large, oval, or slightly folded nuclei. Hemorrhage and necrosis may be more typical of astrocytomas than of oligodendrogliomas. Malignant neoplasms may be multicentric with invasion of perivascular spaces and meninges. (Morgan, Frith, et al. 1984; Frith and Ward 1988; Radovsky and Mahler 1999; Krinke, Fix, et al. 2001) Neoplasm of meningeal cells. These usually present as discrete nodules on the surface of the brain or spinal cord. They are expansile and compress adjacent soft tissue, and rarely exhibit invasion (malignancy). The fibrous type has a regular pattern of loosely interwoven bundles of delicate spindle cells, with single small hyperchromatic oval nuclei. The neoplasms may appear myxomatous when there is abundant faintly basophilic finely granular ground substance. Meningothelial types are less common and have larger, more epithelioid cells with abundant eosinophilic cytoplasm forming sheets or lobules. (Morgan et al. 1984; Frith et al. 1988; Radovsky et al. 1999; Krinke et al. 2001) Neoplasm of oligodendrocytes that normally form myelin sheaths in the CNS. Brain tumors are uncommon in spontaneous or induced tumors in mice but these are the most commonly diagnosed CNS neoplasms. It occurs in the cerebrum and/or diencephalon, usually is ventro-lateral and involves much of the thalamus, hypothalamus, and amygdaloid. It is a poorly demarcated mass but expansile and distorts surrounding tissues. It is distinct from adjacent neuropil due to distinctive monomorphic cell population of small cell with small dark round nuclei, and a typical perinuclear clear halo that may result in a honeycomb pattern. Blood vessels within the neoplasm may have hyperplastic endothelium. There may be necrosis and hemorrhage. (Morgan et al. 1984; Frith et al. 1988; Radovsky et al. 1999; Krinke et al. 2001) Neoplasm derived from fibroblasts of perineural connective tissue (perineurium), distinct from schwannoma. Histologically they resemble fibroma or fibrosarcoma, with neoplastic spindle cells arranged in bundles of eosinophilic fibers. (Peckham et al. 1999) Neoplasm derived from nerve sheath cells (Schwann cells), which normally produce the neurilemma and myelin layers surrounding axons in the peripheral nervous system (PNS), and are of ectodermal, neural crest origin. These are uncommon neoplasms in mice but most likely to be diagnosed in the subcutis and heart. Benign neoplasms are discrete, expansile, compressive. Malignant neoplasms are invasive, usually with more atypia and mitotic figures. These spindle cell tumors have 2 typical growth patterns. Antoni type A pattern features elongate cells in whirls and bundles with their nuclei palisading in parallel array. These areas may be called Verocay bodies. Antoni B pattern is a looser pattern with sparser cells more haphazardly arranged in a clear or edematous matrix. Ultrastructurally these cells should have basal laminae, and immunohistochemical staining for S 100 indicating neural crest origin. (Peckham et al. 1999; Ernst et al. 2001) Hemangioma in the nasal cavity, see above, CARDIOVASCULAR. Schwannoma of the nasal cavity. See above, NERVOUS.
Pulmonary neoplasm, adenoma vs. adenocarcinoma not distinguished. Usually other types of tumors or metastases in the lung are not included in this category, unless diagnoses were made by gross examination only. Continued
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TABLE 25-6—cont’d SYSTEM Organ, Neoplasm (historical or obsolete terms) Lung, bronchoalveolar hyperplasia Type II cell hyperplasia Lung, adenoma (BAA, bronchioloalveolar adenoma)
Lung, carcinoma (bronchioloalveolar carcinoma)
SPECIAL SENSES Harderian gland, tumor Harderian gland, adenoma
Harderian gland, carcinoma or adenocarcinoma
URINARY Kidney, renal tubular hyperplasia
Kidney, adenoma (renal tubular cell adenoma)
Kidney adenocarcinoma (renal tubular cell carcinoma, renal cell carcinoma)
See Table 25-5.
Benign neoplasm of airway epithelium supported on fibrovascular stroma in acinar or papillary patterns. Right lobes are involved more frequently than the left. The most common tumor type, previously called bronchoalveolar or bronchioloalveolar adenoma, currently is classified simply as adenoma of the lung, and they may have acinar (solid) papillary or mixed patterns. (Nikitin 2004) Grossly, these tumors are yellow-white, discrete nodules ranging in size from 1.0–10 mm. Adenomas are usually less than 4 mm in diameter with solid > papillary > mixed patterns. (Dixon et al. 1991; Dixon et al. 1999; Festing et al. 1994; Dungworth et al. 2001) Malignant neoplasm of airway epithelium. Carcinomas usually are irregular nodules larger than 4 mm in diameter with papillary > mixed patterns. Carcinomas are less common than adenomas and may metastasize to liver. They may be difficult to distinguish from large adenomas when there is not obvious destruction of parenchyma, invasion of bronchiolar walls, interstitial tissue or pleura, lymphatic dissemination, or distant metastasis. (Dixon et al. 1991, 1999; Festing et al. 1994; Dixon et al. 1999; Dungworth et al. 2001) Neoplasm of the Harderian gland adenoma vs. carcinoma not specified. Benign neoplasm of acinar epithelium of Harderian gland. These can be quite common in some studies, and more common in males than in females. Adenomas are more common than carcinomas. They may be under-reported because the gland may not be examined unless there is a grossly obvious lesion. These usually are well-demarcated or encapsulated nodules, with compression of surrounding gland. Patterns may be papillary, cystic, cystic-papillary, or acinar, and the neoplastic cells usually are well differentiated and in a single layer. (Botts et al. 1999; Krinke et al. 2001) Malignant tumor of acinar epithelium of the Harderian gland. These usually are larger than adenomas and may cause facial selling and/or exophthalmos. They are highly cellular, and disorganized compared to adenomas, with piling up of pleomorphic cells. Areas of medullary or solid growth patterns are common. There may be necrosis and hemorrhage, invasion beyond the orbit and distant metastasis to lungs, local lymph nodes, thymus, or liver. (Botts et al. 1999; Krinke et al. 2001) Hyperplasia or proliferation of renal tubule epithelium. By convention foci of hyperplasia are <3 times the diameter of a normal tubule. They may be precursors of adenomas or carcinomas. The tubule may be expanded by several layers of slightly pleomorphic epithelial cells, or the tubule may be dilated with an expanded lumen and lined by irregular and crowded pleomorphic epithelial cells. Crowded cells with nuclear crowding usually apparent. Tubule structure is essentially maintained and there is no compression of adjacent parenchyma. (Seely 1999; Hard et al. 2001) Benign neoplasms of renal tubule epithelium. These are uncommon spontaneous neoplasms in mice, with adenoma and carcinoma incidence usually <1%. They usually are solitary, and classified morphologically as cystic, papillary, or solid, with papillary being the most common type. The neoplastic cells are uniformly cuboidal with eosinophilic cytoplasm and relatively small nuclei. Mitotic figures are rare. Cystic and papillary adenomas usually are encapsulated. Solid adenomas usually are well demarcated from adjacent parenchyma, but not encapsulated. (Frith et al. 1988; Seely 1999; Hard et al. 2001) Malignant neoplasm of renal tubule epithelium. These are uncommon spontaneous neoplasms in mice, with adenoma and carcinoma incidence usually <1%. They may have solid, papillary or tubular, or anaplastic patterns. They are compressive and may have necrosis or hemorrhage, or cystic areas with intraluminal pale eosinophilic material. Neoplastic cells vary from small and uniform to large and pleomorphic, with granular eosinophilic, clear, or basophilic cytoplasm. Their nuclei may be uniformly small and round or oval, or large and pleomorphic, and the mitotic index is variable. Metastasis is rare and usually to the lungs. (Frith et al. 1988; Seely 1999; Hard et al. 2001)
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Fig. 25-1 A, Six-week-old mouse, malocclusion, incisor overgrowth. B, Mouse; megaesophagus; dorsal view of stomach, esophagus, lungs; black arrow points to right lung lobes. Thoracic esophagus ((between white arrows) is distended by ingesta. C, Mouse, ventral view, hydronephrosis, right kidney. D, Mouse, decalcified head, transverse section. Hydrocephalus.
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Fig. 25-2 A, Small intestine (ileum), amyloidosis. Villi are mildly distended and thickened by accumulation of congophilic, acellular, extracellular, amorphous eosinophilic material in the lamina propria. B, Kidney, mild glomerular amyloidosis in a male mouse. Glomerular mesangium is mildly expanded by congophilic, acellur, extracellular, amorphous eosinophilic material. With H&E staining, glomerular amyloidoses resembles other glomerulopathies. Special stains of other techniques can be used to further characterize glomerulopathies. Note cuboidal parietal epithelium surrounding one of the two glomeruli. C, Parotid and submandibular salivary glands with amyloidosis of the parotid gland. In contrast to the underlying submandibular salivary gland, parotid gland interstitium is widely expanded by congophilic, acellular, extracellur, amorphous eosinophilic material (arrow), with apparent reduction in size (atrophy) of acinar elements. D, Thyroid gland, mild amyloidosis is characterized by mild perifollicular or interstitial deposition of congophilic, acellular, extracellular, amorphous eosinophilic material.
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Fig. 25-3 A, Heart, amyloidosis. Cardiac myofibers are variably separated by congophilic, acellular, amorphous eosinophilic material, which also is perivascular (arrow). B, Nasal septum. Submucosal glands are separated by acellular, extracellular, amorphous eosinophilic material (arrow) that may not be congophilic, and may have staining properties compatible with collagen.
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Fig. 25-4 A, Lung, mouse, acidophilic macrophage pneumonia characterized by abundant plump macrophages, distended by cytoplasmic acidophilic (eosinophilic) material, in alveoli and bronchioles, with few scattered acidophilic spicules or crystals. Note the central spicular acidophilic crystal surrounded by plump macrophages. B, Lung, mouse, acidophilic macrophage pneumonia characterized by prominent extracellular acidophilic (eosinophilic) elongate spicular crystals in a bronchiole, with fewer, sparser plump macrophages compared to. C, Nasal cavity, mouse, decalcified head section. At this level, the nasal cavity is lined by olfactory epithelium (arrow) as well as ciliated respiratory epithelium (rectangle), within which are small intracellular slender acidophilic cytoplasmic crystals. Such crystals have been identified as a chitinase in nasal, gastric, and biliary epithelium as well as in acidophilic macrophage pneumonia. D, Higher magnification of respiratory epithelium with intracytoplasmic crystals from E.
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Fig. 25-5 A, Adrenal gland, 8-month-old male mouse, subcapsular cell hyperplasia. B, Higher magnification of A, with hyperplasia of spindled and plump subcapsular cells in the adrenal cortex. C, Adrenal gland, 11-month-old female mouse. Accessory adrenal cortical nodule (lower left). Large cells near the corticomedullary junction contain golden brown ceroid lipofuscin pigment and may be multinucleated. D, Higher magnification of corticomedullary junction of C with large pigmented cells near the corticomedullary junction. E, Adrenal gland of 6-month-old virgin female mouse with vacuolation of cells of the X zone.
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Fig. 25-6 A, Heart, right ventricular free wall, BALB/c mouse, epicardial mineralization. B, Kidney medulla, female mouse, collecting ducts, mild focal mineralization. C, Brain, thalamus, 14-month-old male mouse, foci of mineralization in the thalamus of old mice frequently are bilateral and were in this case.
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Fig. 25-7 A, Brain, hippocampus, dentate gyrus, FVB/N mouse (post-seizure), segmental necrosis, and loss of neurons, with dark pyknotic neuronal nuclei to the left of the arrow, and larger (relatively normal) neuronal nuclei right of the arrow. B, Liver, FVB/N mouse (post-seizure), centrilobular necrosis of hepatocytes. C, Mammary gland, nonpregnant female FVB/N mouse, mammary gland hyperplasia. D, Mammary gland, nursing female mouse, diffuse (physiologic) mammary gland hyperplasia.
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Fig. 25-8 A, Heart, left auricle, mouse, thrombi with mineralization. B, Higher magnification of A with dark areas of mineralization fibroplasia (spindle cells). C, Heart, right ventricle, mouse, thrombus. While cardiac thrombi are reported more commonly in the left auricle or atrium of mice, they may occur in other sites. D, Heart, mouse, trichrome stain reveals acellular areas of collagen deposition as well as mild chronic inflammation. Cardiac degeneration, inflammation, and fibrosis may be listed as separate diagnoses or may be included in the term cardiomyopathy.
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Fig. 25-9 A, Heart, mouse, coronary arteritis with marked periarterial fibrosis and inflammation. B, Decalcified head, meninges, mouse, arteritis with periarterial inflammation and mild fibrosis. C, Spleen, mouse, angiectasis, dilatation of vascular spaces, characterized by cavernous blood-filled vascular spaces, lined by flattened endothelial cells. D, Liver, mouse, angiectasis (telangiectasis, peliosis), dilatation of vascular spaces, characterized by cavernous and variably sized blood-filled vascular spaces, lined by flattened endothelial cells, without evidence of proliferation or atypia.
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Fig. 25-10 A, Brain, mouse, normal corpus callosum connecting the hemispheres (arrow). B, Brain, mouse, B6;129 origin; complete absence of corpus callosum.
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Fig. 25-11 A, Decalcified head section, maxillary incisor, decalcified head, 14-month-old mouse, infoldings of enamel layer (arrow) are compatible with dental dysplasia. B, Decalcified head section, maxillary incisor, decalcified head, 14-month-old mouse, cystic structure, and abnormal architecture (arrow) of the dentin layer are compatible with dental dysplasia. C, Decalcified head section, maxillary molar, decalcified head, 14-month-old mouse; there is distention of periodontal sulci by accumulations of debris, including hairs, bacteria, and inflammatory cells. D, Decalcified head section, maxillary molar, decalcified head, 14-month-old mouse; there is marked distention of periodontal sulci by accumulations of debris, including hairs, bacteria, and inflammatory cells, loss of most of the tooth structure, including enamel and much dentin, colonization of the surface by bacteria.
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Fig. 25-12 A, Decalcified head, 14-month-old mouse, “fibroosseous lesion,” with replacement of hematopoietic marrow by B, Decalcified head, 14-month-old mouse, “fibroosseous lesion,” with replacement of hematopoietic marrow by fibrovascular connective tissue. C, Decalcified femur, 14-month-old mouse, hyperostosis, endosteal bone proliferaion, or fibroosseous lesion with replacement of marrow by osseous connective tissue, bone.
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Chapter 26 Zoonoses and Other Human Health Hazards Christian E. Newcomer and James G. Fox
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Viral Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Lymphocytic Choriomeningitis Virus (LCMV) . . . . . . . . . . . . . . . . . . . 1. Reservoir and Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mode of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Clinical Signs, Susceptibility, and Resistance in Humans . . . . . . . . 4. Diagnosis, Treatment, and Control . . . . . . . . . . . . . . . . . . . . . . . . . . B. Rabies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Reservoir and Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Diagnosis, Treatment, and Control . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Rickettsial Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Rickettsialpox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Murine Typhus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Bacterial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Leptospirosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Reservoir and Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mode of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Epidemiology and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Rat Bite Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Reservoir and Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mode of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Reservoir and Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mode of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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D. Other Potential Bacterial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Borrelia Species (Tick-borne Relapsing Fever) . . . . . . . . . . . . . . . . 2. Helicobacteriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Dermatophytosis (Ringworm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Reservoir and Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mode of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Helminth Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Tapeworms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Hymenolepis diminuta, the Rat Tapeworm . . . . . . . . . . . . . . . . . . . . 2. Rodentolepsis (formerly Hymenolepis) nana, the Dwarf Tapeworm of Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Rodentolepsis microstoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Roundworms (Syphacia obvelata) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Reservoir and Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mode of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Arthropod Infestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Ornithonyssus bacoti—Tropical Rat Mite . . . . . . . . . . . . . . . . . . . . B. Fleas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Bites IX. Allergic Sensitivities—Laboratory Animal–Associated Allergy (LAA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Incidence and Clinical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Treatment and Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
Zoonoses is derived from the Greek words zoon (meaning animals), and noses (meaning disease), and refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. The biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. The extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modern animal care and use environment. However, when these essential animal program quality measures fail or are not incorporated into the animal facility operations, zoonotic pathogens may be unwittingly introduced and perpetuated, placing personnel at increased risk of exposure. Wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated
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hazards. In addition to the zoonoses, mice are capable of inflicting bites on inadequately trained personnel and are a rich source of allergens for a substantial number of persons predisposed to develop mouse-associated allergic sensitivities. This chapter discusses the mouse-associated zoonotic diseases and other health hazards and explains the strategies that are helpful for reducing or eliminating the risk of personnel exposure to these conditions.
II. A.
VIRAL DISEASES
Lymphocytic Choriomeningitis Virus (LCMV)
Lymphocytic choriomeningitis virus (LCMV) is an enveloped single-stranded RNA virus in the Old World serocomplex group of the family Arenaviridae. It is the only representative of the group of Old World arenaviruses that is present in the United States and survives in the house mouse, Mus musculus, as its principal host through which it has achieved a worldwide distribution (Buchmeier et al. 2001) (see also Chapter 7 of this volume). The arenaviruses have a predilection for rodent reservoirs, and several with zoonotic
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implications in the New World serocomplex group are present among the wild rodents endemic to the United States such as Neotoma spp. and Peromyscus spp. (2000; Buchmeier et al. 2001; Fulhorst et al. 2002). Research animal programs that import wild rodents for laboratory studies should stay abreast of the developments on the identification and host-range characteristics of the New World arenaviruses of emerging importance. This section will focus only on lymphocytic choriomeningitis (LCM), a naturally occurring viral zoonosis of the laboratory mouse. 1.
Reservoir and Incidence
Many published reports of human LCM infection are associated with laboratory animal and pet contact, particularly mice and hamsters, and these studies now span many decades (Armstrong and Lillie 1934; Bowen et al. 1975; Dykewicz et al. 1992; Jahrling and Peters 1992; Lehmann-Grube et al. 1979; Rousseau et al. 1997). There seems to be a resurgent awareness among physicians that LCM should be sought as an etiology in human neurological disease and in pediatric congenital brain disorders (Barton and Hyndman 2000; Barton et al. 1995, 2002; Romero and Newland 2003). LCMV is widely distributed among the wild mouse population throughout most of the world presenting a zoonotic hazard (Childs et al. 1992; Childs and Peters 1993; Morita et al. 1996; Smith et al. 1993). Surveys conducted within the urban environment of Baltimore, Maryland, reported that 9% of house mice and 4.7% of persons tested had measurable LCMV antibody titers (Childs et al. 1991, 1992). A similar serological survey conducted in Spain across urban and rural ecological settings also found a 9% prevalence in mice and a 1.7% prevalence among persons by immunofluorescence assay (Lledo et al. 2003). The recent serological detection of LCMV in five mice on the treeless, sub-Antarctic, Macquarie Island of Australia, indicates the extent of distribution of this agent to the remotest areas of our planet (Moro et al. 2003). The apparent ease with which LCMV is transmitted to humans also occurs in a variety of other laboratory animal species; hamsters, guinea pigs, swine, dogs, and nonhuman primates, especially callitrichids, which readily sustain natural infections. In the case of the callitrichids, there have been numerous reports of epizootic infectious hepatitis (callitrichid hepatitis) due to LCMV, with a high mortality rate in zoological parks in both the United States and England over the past two decades (Lucke and Bennett 1982; Montali et al. 1989; Stephensen et al. 1990, 1991, 1995). Rodent (mouse) infestations of these zoos and/or the supplementation of the diets of tamarins and marmosets with suckling mice, a common practice (Richter et al. 1984), are potentially rich sources for LCMV. In the research animal facility environment, the laboratory mouse continues to merit attention as the species of primary concern as a reservoir for cases of human LCM (Dykewicz et al. 1992). In the laboratory mouse, and to a lesser degree the hamster, breeding colonies can become endemically infected when the virus is transmitted to pups in utero or early in the
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neonatal period, producing a tolerant subclinical infection characterized by chronic viremia and viruria. When infected, athymic and other immunodeficient mouse strains may be predisposed to harboring silent, persistent infections and present a higher risk to personnel (Dykewicz et al. 1992). Under some circumstances LCMV also produces a pantropic infection and may be copiously present in blood, cerebrospinal fluid, urine, nasopharyngeal secretions, feces, and tissues of infected natural hosts and possibly humans. Bedding material and other fomites contaminated by LCMV-infected animals can also be important sources of infection for humans, as demonstrated in a recent outbreak among laboratory animal technicians and on many previous occasions (Biggar et al. 1975; Dykewicz et al. 1992). The experimental passage of tumors and cell lines contaminated with LCMV has long been recognized (Haas and Stewart 1956) and represents one of the biggest threats for the introduction of LCMV into animal facilities at the present time (Bhatt et al. 1986; Dykewicz et al. 1992; Nicklas et al. 1993). Reported that 17 of 63 rodent transplantable tumors screened were positive for LCMV and identified contamination in 4 of 14 hamster tumors and 2 of 81 mouse tumors that had been propagated in animals (Nicklas et al. 1993). The growth of LCMV in insect cell lines has also been demonstrated (Rahacek 1965), and the article by Hotchin summarized the work of others indicating that numerous experimentally infected, bloodsucking ectoparasites are capable of transmitting the disease to laboratory rodents (Hotchin 1971). LCM virus also has been recovered from cockroaches (Armstrong and Lillie 1934). 2.
Mode of Transmission
The diagnosis and control of LCMV infection in mouse colonies has been reviewed in Chapter 7 of this volume. Tumor and cell-line screening before animal passage, the control of wild rodent infestations in areas where animals are housed or used, and the early detection of colony infections through sound colony health surveillance practices are of critical importance to the prevention of infection in mouse colonies. Once established in mouse breeding colonies, the high viral load characteristically shed by mice infected congenitally or neonatally represents a very serious hazard to personnel. Most of the cases of human infection, whether involving exposure in the home, agricultural, or laboratory setting, have involved contact with live mice and their excreta or mouse carcasses (Dykewicz et al. 1992; Havens 1948; Morbidity and Mortality Weekly Report 1984). Several authors have emphasized the association between the actual handling of infected mice and the contraction of the disease by humans (Dykewicz et al. 1992; Havens 1948; Smithard and Macrae 1951). Several cases of human infection have suggested the possibility that infected rodent tissues can serve as a source of infection for laboratory personnel (Baum et al. 1966; Dykewicz et al. 1992; Tobin 1968). Humans may be infected by inhalation or by the contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals. The transmission
722 of LCMV by the bite of an infected mouse can also occur (Scheid et al. 1964). Also, Hotchin reported the findings of other researchers that LCMV was transmissible experimentally through the intact skin of the guinea pig, but this finding has not been reported in humans (Hotchin 1971). Airborne transmission is well documented and plays a very important role in human infections, especially through the ready dispersion and inhalation of viral-contaminated dust from the animal cage or room (Biggar et al. 1975; Hinman et al. 1975). 3.
Clinical Signs, Susceptibility, and Resistance in Humans
Following an incubation period of 1 to 3 weeks, humans may experience asymptomatic or a mild febrile disease ranging to a serious flu-like illness characterized by anorexia, malaise, diffuse myalgia and arthralgia, fever, chills, vomiting, headache, stiff neck, and photophobia. Some patients enter a second stage of the disease several days after the resolution of early mild symptoms, developing meningoencephalitis and exhibiting additional signs such as drowsiness, confusion, sensory disturbances, and motor abnormalities. Patients can also develop more serious nonneurological manifestations of the disease such as maculopapular rash, lymphadenopathy, parotitis, orchitis, arthritis, and epicarditis (Peters 1997). Central nervous system involvement has resulted in death in several cases. Infections during pregnancy pose a risk of infection for the human fetus (Wright et al. 1997). Wright et al. reported 26 cases in human infants, with LCMV confirmed serologically over a two-year period in a major U.S. medical center (Wright et al. 1997). These infants presented with ocular abnormalities, macrocephaly, and hydrocephalus with microcephaly. Fifty percent of the mothers reported having had illnesses compatible with LCMV infection, and over half reported exposures to rodents during their pregnancies. 4.
Diagnosis, Treatment, and Control
The diagnosis of LCM infection in humans is currently made by serological testing using either the immunofluorescent antibody (IFA) test or the enzyme-linked immunosorbent assay (ELISA) (Barton et al. 2002). Both of these tests are available through the Centers for Disease Control and Prevention and are superior to the complement fixation test that is widely available commercially. Although there are no proven effective antiviral therapies for LCM infection, intravenous ribavirin therapy reduces mortality in patients infected with Lassa fever virus (a member of the Old World Arenavirus serocomplex) and may be of some benefit in patients with severe LCMV infections (Andrei and De Clercq 1993; McCormick et al. 1986). This disease can be prevented in the laboratory through periodic serological surveillance using ELISA and IFA tests of newly introduced animals with inadequate disease profiles and of resident animal colonies at risk. Thorough screening of all tumors and cell lines intended for animal passage using the highly sensitive mouse antibody production assay or newer
CHRISTIAN E. NEWCOMER AND JAMES G. FOX
PCR-based laboratory tests for the presence of LCMV is another crucial element in the program to prevent the introduction of LCMV into established animal colonies (Besselsen et al. 2003 and Chapter 7 of this volume). Sound animal facility sanitation practices and the use of microbarrier caging systems with proper infection-control techniques should prevent or suppress the spread of LCMV if present in the environment. The elimination of wild rodent infestations in animal facilities is very important to prevent the introduction of LCMV into the animal facility environment. Also, facilities with wild rodent infestations may encounter the relatively common, free-living, bloodsucking mite of the rodent, Ornithonyssus bacoti, in abundance (personal communication). Although the natural LCMV transmission to humans from bloodsucking ectoparasites is unproven, the control of potential ectoparasitic vectors of this type would be a prudent measure.
B.
Rabies
Rabies is an acute, almost invariably fatal disease that occurs worldwide with the exception of a few countries, generally island nations, and other regions that have excluded the disease through animal importation and control programs and the aid of geographic barriers. Neither the laboratory mouse nor other small wild rodent hosts appear to be important as reservoirs of natural rabies infection. Hence, the principal reason for our discussion of rabies as a zoonotic disease of the laboratory mouse is to provide information that should quickly allay the fears of uninformed research and animal facility staff who suffer the bite of a mouse. On the other hand, the experimental use of mice in the study and characterization of rabies virus and in rabies vaccine development is an important component of some animal care and use programs that deserves special attention by the institution during all phases of research planning and implementation. 1.
Reservoir and Incidence
There are no known cases of human rabies from rodents in the United States (2002). The incidence in larger wild rodent species within the United States has increased in recent years, however. During the interval 1971–1984, a total of 97 cases of rabies in rodents were recorded, but from 1995 to 2000 approximately 52 cases were reported in large rodents annually (2002). The rodents involved were woodchucks and beavers, both species presumably large enough to survive the chance encounter and attack by a wild rabid carnivore such as the raccoon, skunk, fox, or feral cat. Earlier literature on the Federal Republic of Germany summarized findings from 1961 to 1967, which indicated that three mice, one rat (species not given), nine Norway rats, and three muskrats were infected with rabies and had bitten humans (Scholz and Weinhold 1969). Despite rare reports of this nature, rodents are not a proven source of rabies transmission to humans (Johnson 1989).
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2.
Clinical Signs
All mammals are generally regarded as susceptible to rabies. In humans, the course of the disease proceeds through several phases: incubation, prodromal, acute neurological, coma, and rarely, recovery (Johnson 1989). The incubation period varies from 9 days to over 8 months. During the prodromal stage lasting 2 to 4 days, patients experience a period of apprehension and develop headache, malaise, and fever. An abnormal, indefinite sensation at the site of a prior animal bite wound is the first specific symptom. Patients also may develop intermittent periods of excitation, nervousness, or anxiety interspersed with quiet periods when the mental state appears normal. Further progression of the disease involves paresis or paralysis, inability to swallow, and the related hydrophobia, delirium, convulsions, and coma. Rabies produces an almost invariably fatal acute viral encephalomyelitis, with death due to respiratory paralysis. Adult mice used experimentally in rabies studies usually exhibit clinical signs between 5 and 15 days following inoculation and die within 5 days of the onset of clinical signs coinciding with the period of viral shedding. Clinical signs in the mouse consist of muscular tremors, incoordination, excitation, or paralysis. Certain rabies virus isolates from skunks produce a spastic paralysis in adult mice followed by recovery in a high percentage of infected mice. Also, infant mice inoculated with certain strains of street rabies virus are capable of full recovery (Johnson 1989). 3.
Diagnosis, Treatment, and Control
Personnel working with mice experimentally infected with rabies virus should adhere to the well-established and detailed procedures that have been described in other sources for animal inoculation, husbandry, and tissue harvest procedures (Johnson 1989). Vaccination of personnel involved in rabies studies with laboratory animals also is clearly indicated, regardless of the animal species involved. C.
Other Viruses
Four other viruses that produce natural infections in the mouse have been implicated previously or are known to be infectious for humans. These include hantavirus, Sendai virus, Reovirus 3, and mouse hepatitis virus. For none of these viral agents is there documented evidence of zoonotic transmission of the agent from naturally infected laboratory mice to personnel in the laboratory. Hantaviruses are zoonotic viruses comprising at least 22 species that are maintained among natural rodent reservoirs, despite the presence of neutralizing antibody in the rodent host (Elliott et al. 2000; Meyer and Schmaljohn 2000). Approximately half of the hantaviruses are known human pathogens producing virus-specific patterns of disease that include hantavirus pulmonary syndrome, hemorrhagic fever with renal syndrome, and its benign form,
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nephropathia endemica (Mills and Childs 1998). Although Mus musculus can exhibit the pattern of persistent hantavirus infection in the presence of neutralizing antibody when induced experimentally (Araki et al. 2003), this does not occur in natural infections of the mouse (Meyer and Schmaljohn 2000). This is the likely reason that the wild mouse apparently is not important as a natural reservoir for the hantaviruses. This interpretation is supported by serological studies of wild Mus musculus that detected either no or a very low incidence of serological evidence to hantavirus exposure even when other wild rodent species in the vicinity had a high level of endemic infection (Kantakamalakul et al. 2003; Meyer and Schmaljohn 2000; Pacsa et al. 2002; Zuo et al. 2004). Mus musculus is used in the laboratory as an animal model to study various aspects of hantavirus infection ranging from vaccine development (Choi et al. 2003), viral pathogenicity (Ebihara et al. 2000; Kim and McKee 1985), and immunological aspects of persistence (Araki et al. 2003), and it is primarily in this context that hantavirus deserves mention as a zoonotic infection in the mouse. The control of wild rodent infestations in animal facilities, quarantine and testing of wild caught rodent species during importation, and proper observance of animal biosafety guidelines for hantavirus-infected animals would be expected to virtually eliminate the risk of this zoonosis in laboratory maintained mice. Mouse hepatitis virus (MHV) remains a prevalent infection in many mouse colonies where it potentially impacts colony health and disrupts experimental studies (see Chapter 6). Earlier studies have demonstrated that human sera contained complement-fixing and neutralizing antibodies to MHV (Hartley et al. 1964). Later studies suggested that this was most likely due to cross-reactive antibodies from human cold virus infections (Bradburne 1970; McIntosh et al. 1967). Mouse hepatitis virus and the two prototype human cold viruses (OC43 and 229E) are members of the antigenic group 2 coronaviruses, and it is now known that members of this group share four, and in some cases five, structural genes that could account for this crossreactivity (Navas-Martin and Weiss 2003). The coronaviruses have a very narrow host range and generally replicate only in the cells of the host species (Navas-Martin and Weiss 2003). However, under unique laboratory conditions involving persistent cell culture infection, the use of mixed cell cultures of murine and of a nonpermissive species, or the use of cells possessing modified receptors, MHV has been adapted to grow in human, nonhuman primate, and hamster cells. Also, the many strains of MHV in combination with use of targeted RNA recombinant system have also been very useful for experimental study of the molecular basis of coronavirus pathogenicity (Masters 1999). Application of the targeted RNA recombinant system to MHV for exploring of emerging coronavirus infections such as SARS may delineate the molecular basis for expanding of host range. These studies may warrant the reader’s future attention, but at the present, MHV can be reasonably dismissed as a zoonotic infection.
724 Sendai virus was once a prevalent agent in mouse colonies but has become a rarity in most institutions. This is due to its ease of eradication through the use of temporary cessation of breeding to eliminate a naïve population that is susceptible to infection and through the use of caging systems that prevent transmission (see Chapter 11). Sendai virus was originally isolated and described in the 1950s during the investigation of cases of human respiratory illness (Gerngoss 1957; Kuroya et al. 1953a, 1953b; Sano et al. 1953; Zhandoff et al. 1957). In the original report involving the isolation of Sendai virus from Japanese newborn human infants suffering from fatal pneumonitis, lung suspensions from the newborns were inoculated intranasally into mice, producing lung consolidation and death in several cases (Kuroya et al. 1953b). In later studies, Sendai virus isolates were reported to produce disease in human volunteers (Kuroya et al. 1953a; Yamada 1956), and reports from many countries indicated that serological evidence of Sendai virus infection was associated with outbreaks of human respiratory illness (Demeio and Walker 1958; Gardner 1957; Jensen et al. 1955). Tennant et al. demonstrated that personnel working with laboratory animals had antibody titers to Sendai virus, but personnel with no known exposure to laboratory animals also had significant titers to the agent (Tennant et al. 1967). Recombinant Sendai virus is widely used for gene transfer experiments, and these vectors can readily infect human airway epithelium and a variety of other human tissues under experimental conditions (Nagai 1999; Pinkenburg et al. 2004). Although these recent studies have clearly demonstrated that Sendai virus is capable of infecting human tissues, the initial evidence for its role as a human pathogen remains doubtful. The mice used for the early isolations of the agent may have already been endemically infected with Sendai and served as the source, or it may be that other serologically cross-reactive parainfluenza viruses, which were not characterized during this era, were responsible for producing false positive reactions to Sendai virus (Ishida and Homma 1978). Reoviruses are generally regarded as the cause of childhood infections producing asymptomatic or very mild disease, and there are few reports linking these infections with a particular disease (Tsai 2000). Reovirus 3 was originally isolated from the feces of a clinically ill child (Stanley et al. 1953), and it continues to receive attention as a possible etiology for neonatal hepatitis and extrahepatic biliary atresia in infants (Richardson et al. 1994; Steele et al. 1995). Reovirus 3 is highly infectious for infant laboratory mice and still receives some attention in the health-screening programs for the mouse and laboratory rodent species. Jacoby and Lindsey (1997) reported that mouse colonies in the United States continue to have a 5 to 20% prevalence of reovirus 3 infection. Although there are no confirmed reports of reovirus 3 transmission from mice (or other laboratory animal species) to humans, the laboratory mouse should be considered a possible source for this infectious agent for humans and other susceptible species.
CHRISTIAN E. NEWCOMER AND JAMES G. FOX
III.
RICKETTSIAL DISEASE A.
Rickettsialpox
The Rickettsiae are fastidious, small pleomorphic coccobacilliary organisms maintained in nature in a cycle of infection involving mammalian hosts and arthropod vectors as reservoirs (Saah 2000). In most rickettsial infections, humans serve only as an incidental host and do not contribute to the propagation of the organism in nature. Such is the case for rickettsialpox, a nonfatal, self-limiting zoonotic disease caused by Rickettsia akari, which is perpetuated in a cycle of infection involving Mus musculus as the primary host and a mite vector (Liponyssoides sanguineus). Isolation of the organisms from rats (Rattus) and voles (Microtus) has also been reported. The first cases of human rickettsialpox were described in patients in New York City (Huebner et al. 1946a, 1946b), and outbreaks of the disease have generally remained clustered within large urban areas of the United States and in rural North Carolina, Utah, South Africa, Korea, and the former Soviet Union (Koss et al. 2003). According to Koss et al. approximately 800 cases of rickettsialpox have been reported in the literature, with nearly 500 of these within the three years following the original description of the disease (Koss et al. 2003). Prior to the report by Koss et al. the largest case study included 13 patients accumulated over a 10-year period. The recent report by Koss et al., however, included 18 patients in New York City reporting over only a 20-month period in the wake of the September 11, 2001 attacks, suggesting that perhaps the heightened sensitivities to possible bioterrorism events stimulated an upsurge in the reporting of cases as a byproduct of increased patient concerns (Koss et al. 2003). Authors have commented on a variety of other social and demographic factors that may also be contributing to the noticeable increase in the incidence of rickettsialpox and murine typhus in the urban environment (Comer et al. 1999; Paddock et al. 2003). There are no reported cases of rickettsialpox in personnel related to exposure to naturally infected laboratory mice. The mite vector L. sanguineus has not been reported in laboratory mouse colonies either historically or contemporarily. However, the tropical rat mite (Ornithonyssus bacoti) can be experimentally infected with R. akari but has not been shown to play a role in the natural cycle of infection. In the authors’ experience of Ornithonyssus bacoti infestations of laboratory mouse or rat colonies are still seen with some frequency in facilities that have resident wild or feral mouse populations and should be addressed in the institution’s pest control and infection control programs.
1.
Clinical Signs
Rickettsialpox has an incubation period of 7 to 21 days following the bite of the infected mite (Saah 2000). The disease is
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mild to severe with an abrupt onset, and it typically presents with a classic triad of an eschar, fever, and a papulovesicular rash. The papule develops at the site of the bite and later ulcerates and progresses to an eschar, 0.5 to 3 cm in diameter, as R. akari proliferates locally in the skin and vasculitis develops (Koss et al. 2003; Saah 2000). The rash begins with firm, generally nonpruritic, erythematous papules, 2 to 10 mm in diameter, that develop into vesicles and heal by crusting. In addition to fever, patients may experience chills and headache, and less commonly, backache, myalgia, and photophobia. The disease is mild and self-limiting within 6 to 10 days, and serious complications or death have rarely been reported (Saah 2000). Patients typically respond quickly after the initiation of antirickettsial therapy with tetracycline, doxycycline, or other appropriate agent (Koss et al. 2003; Saah 2000). However, following resolution of the infection, headache and lassitude can persist for 1 to 2 weeks. The reader should refer to Koss et al. for information on other rash-producing or eschar-related diseases that should be considered in the differential diagnosis to rickettsialpox (Koss et al. 2003). Rickettsia akari are diagnosed by complement fixation tests or the more sensitive indirect immunofluorescent antibody test. Serum antibody to R. akari generally takes 2 weeks to develop, and paired sera are needed to confirm a four-fold rise in antibody titer (Saah 2000). During the acute phase of the infection, immunohistochemistry or PCR analysis can be used for a rapid diagnosis on biopsy material obtained from the papulovesicular rash or eschar (Koss et al. 2003; Paddock et al. 2003). Laboratory mice infected with R. akari develop fatal pneumonia with intranasal inoculation and severe illness and death with intraperitoneal inoculation. Mice develop anorexia, depression, and dyspnea. Peritonitis, splenomegaly, and lymphadenitis are found upon necropsy examination. Subcutaneous inoculation produces an active infection for 1 month, with organisms being recovered from the spleen but not the feces or urine (Bell 1970). The control and eradicaton of R. akari infections depend on the prevention of wild mice and the mite vector from entering laboratory animal facilities and human dwellings.
B.
Murine Typhus
Murine typhus is a rickettsial disease caused by Rickettsia typhi (previously R. mooseri) that occurs worldwide with epidemics or with high prevalence in particular geographic areas (Dumler and Walker 2000). In the United States, most human cases of the disease are concentrated in Texas and Southern California. The disease is now predominantly associated with the rat as the primary host species for the Oriental rat flea (Xenopsylla cheopis), which serves as the principal ectoparasitic vector transmitting the disease to humans. However, the mouse can also serve as a host for this flea, as well as for the northern rat flea (Nasopsyllus fasciatus) and the mouse flea
(Leptosylla segnis), which also bite man and can be involved in the transmission of R. typhi (Flynn 1973; Pratt and Wiseman 1962; Yunker 1964). An early report in the literature indicated that X. cheopis was easily established in an animal facility inhabiting rooms used for housing laboratory mice (Yunker 1964). It is now also known that the cat and opossum and the cat flea (Ctenocephalides felis) can be involved in sustaining the cycle in some geographic localities (Azad et al. 1997). Clark and Will (1994) reported on use of the laboratory mouse as an experimental host for rearing X. cheopis, but there have been no reports of natural infestations of mouse colonies with any of the flea vectors of R. typhi for several decades. Also, R. typhi has not been isolated from natural infections in laboratory mice. Murine typhus is a more serious disease than rickettsialpox and presents with fever, headache, chills, nausea, and vomiting. Splenomegaly, hepatomegaly, central nervous system involvement, and multiorgan failure can occur as severe and potentially fatal complications. A skin rash, which is typically maculopapular in the case of murine typhus, occurs much less commonly than in rickettsialpox, and its absence should not dissuade the clinician from making a diagnosis of murine typhus and from promptly instituting therapy due to the potential severity of the disease (Dumler and Walker 2000). The methods used for the laboratory diagnosis and treatment of the disease in humans and the principles of preventing the introduction of R. typhi into laboratory animal colonies are similar to those for R. akari.
IV.
BACTERIAL DISEASES A.
Leptospirosis
Leptospirosis microorganisms were discovered in 1914 when they were isolated from jaundiced patients (Inada et al. 1916); after further study they were named in 1917 (Noguchi 1918). Leptospirosis is solely a zoonotic disease of livestock, pet and stray dogs, and wildlife, including wild rodents. Rodent reservoir hosts of leptospirosis include, in addition to rats, mice, field moles, hedgehogs, gerbils, squirrels, rabbits, and hamsters (Fox and Lipman 1991; Torten 1979). Human to human transmission is extremely rare. Leptospira interrogans (comprising more than 200 serovars) have been isolated worldwide (Tappero et al. 2000). L. interrogans contains 23 serogroups with strains pathogenic for amphibians, reptiles, and mammals including humans. Serovars australis, ballum, bataviae, hardjo, grippotyphosa, icterohemorrgagiae, javanica, and pomona are associated with rodent infections. Leptospira serovars, including L. australis, bataviae, grippotyphosa, hebdomidis, icterohaemorrhagiae, pomona, and pyrogenes, are found in the house mouse (Torten 1979). Leptospira ballum has also been reported from mice and is
726 most commonly associated with zoonotic outbreaks (Borst et al. 1948; Friedmann et al. 1973; Stoenner and Maclean 1958). Although particular serovars usually have distinct host species, most serovars can be carried by several hosts. Leptospira are well adapted to a variety of mammals, particularly wild animals and rodents. 1.
Reservoir and Incidence
In the chronic form, the organism chronically infects the host and is shed in the urine inconspicuously for long periods of time. Rodents are the only major animal species that can shed leptospires throughout their lifespan without clinical manifestations (Fox and Lipman 1991; Torten 1979). Active shedding of leptospires by rodents can go unrecognized until personnel handling the animals become clinically infected or are infected by exposure to water or food contaminated by urine. Rats and mice are common animal hosts for serotype, L. ballum, although it has been found in other wildlife as well. Water can often be contaminated with infected rodent urine. The infection can persist unnoticed in laboratory rodents, though their carrier rates for laboratory-maintained rodents in the United States are unknown, but it is probably low. The organism is not routinely screened on health surveillance protocols for mouse colonies; however, there was a report of leptospirosis in 1984 in a research colony of mice in the United States being housed in a large research institution (Alexander 1984). 2.
Mode of Transmission
Because leptospirosis in humans is often difficult to diagnose, the low incidence of reported infection in humans may be misleading. From 1974 to 1979, only 498 cases were reported, for an incidence of 0.05 per 100,000 people per year (Sanger and Thiermann 1988). Leptospirosis was removed from the reportable disease category in the United States in 1995 because of the small number of cases reported. Outbreaks have been documented in the United States from personnel working with laboratory mice (Barkin et al. 1974; Stoenner and Maclean 1958). In one study, 8 of 58 employees handling the infected laboratory mice (80% of breeding females were excreting L. ballum in their urine) contracted leptospirosis (Stoenner and Maclean 1958). Infection with leptospira most frequently results from handling infected animals (contaminating the hands with urine) or from aerosol exposure during cage cleaning (Barkin et al. 1974; Friedmann et al. 1973; Stoenner and Maclean 1958). Skin abrasions or exposure to mucous membranes may serve as the portal of entry. All secretions and excretions from infected animals should be considered infective. In one instance, a father apparently was infected after his daughter used his toothbrush to clean a contaminated pet mouse cage (Boak et al. 1960). Rodent bites can also transmit the disease (Looke 1986). In Detroit, children from the inner city had a significantly higher
CHRISTIAN E. NEWCOMER AND JAMES G. FOX
L. icterohaemorrhagiaes antibody when compared to children living in the Detroit suburbs. Therefore, children living in rodent-infested tenements may be at increased risk of infection (Demers et al. 1983). In Europe and more recently in North America, rodents including house mice have provided a source of leptospira infection for swine and by extension could also infect personnel working in swine production units (Galton 1966; Smith et al. 1992). Leptospira interrogans serovar bratislava is commonly reported in these mice. 3.
Clinical Signs
The disease may vary from unapparent infection to severe infection and death. A self-limited systemic illness is seen in approximately 90% of infected humans. The incubation period is usually 5 to 14 days. Individuals infected with leptospira experience a biphasic disease (Faine 1991; Sanger and Thiermann 1988; Stoenner and Maclean 1958). They become suddenly ill with weakness, headache, myalgia, malaise, chills, and fever and usually exhibit leukocytosis. During the second phase of the disease, conjunctival suffusion and a rash may occur. Upon examination, renal, hepatic, pulmonary, and gastrointestinal findings may be abnormal. Intravenous penicillin is the drug of choice in treating early-onset and late-stage leptospirosis infection (Faine 1991; Taber and Feigin 1979; Watt et al. 1988). Ampicillin and doxycycline also have been effective in treating people with mild to moderate forms of leptospirosis. 4.
Diagnosis
Because of the variability in clinical symptoms and the lack of pathognomonic pathologic findings in humans and animals, serologic diagnosis or actual isolation of leptospires is imperative (Faine 1991). As an aid to diagnosis, leptospires can sometimes be observed by examination or direct staining of body fluids or fresh tissue suspensions (Sulzer et al. 1968). The definitive diagnosis in humans or animals is made by culturing the organisms from tissue or fluid samples, or by animal inoculation (particularly in 3- to 4-week-old hamsters) and subsequent culture and isolation. Culture media with long-chain fatty acids with 1% bovine serum albumin are routinely used as a detoxicant (Faine 1991). Serologic assessment is accomplished by indirect hemagglutination, agglutination analysis, complement fixation, microscopic agglutination, and fluorescent antibody techniques (Faine 1991). The serologic test most frequently used is the modified microtiter agglutination test. Titers of 1:100 or greater are considered significant. Molecular techniques including PCR and randomly amplified polymorphic DNA fingerprinting are used for identification of serovars (Tappero et al. 2000). 5.
Epidemiology and Control
In mouse colonies infected with L. ballum, antibodies against L. ballum were detected in sera of mice of all ages, but
2 6 . Z O O N O S E S A N D O T H E R H U M A N H E A LT H H A Z A R D S
leptospires could be recovered only from mature mice. Progeny of seropositive females had detectable serum antibodies at 51 days of age but not at 65 days. It was also reported that progeny of seropositive female mice, which possessed antibody at birth and acquired additional antibody from colostrums, remained free of leptospires if isolated from their mothers at 21 days of age, despite exposure during the nursing period (Stoenner 1957). Studies in mice experimentally infected with L. grippotyphosa demonstrated that maternal antibodies, whether passed through milk or placental transfer, conferred protection of long duration against the carrier state and shedding of leptospires. Thus, serologically positive immune mothers do not transmit the disease to their offspring. However, mice born to nonimmune mothers, if infected at 1 day postpartum, become carriers with no trace of antibodies. Thus a population of carrier pregnant mice without antibody could serve as a precipitator in outbreaks among susceptible mouse populations (Birnbaum et al. 1972). Field surveys have supported this data in that a significant percentage of carrier mice do not have antibodies. This led to the diagnostic approach, which specifies that both serologic and isolation methods must be utilized to determine the rate of leptospiral infection in rodents (Galton et al. 1962). Leptospira ballum is frequently found in the common house mouse (M. musculus) (Brown and Gorman 1960; Yager et al. 1953). Therefore, eradication of infected colonies, use of surgically derived and barrier-maintained mice or of conventional laboratory mice free of leptospira infection, coupled with the prevention of ingress of wild rodents, should effectively preclude introducing of the organism into research and commercial laboratories (Loosli 1967). Leptospira ballum has been eliminated from a mouse colony by administration of feed containing 1000 gm chlorotetracycline hydrochloride per ton for 10 days. After 7 days of antibiotic therapy, mice were transferred to clean containers and administered clean water, both having been sterilized by steam. Mouse traps and rodenticides were used to destroy escaped mice and to prevent reintroduction of L. ballum by the common house mouse (Stoenner et al. 1958). Commercial animal colonies maintained in research vivarium today are not routinely screened for leptospirosis, assuming that the organism has been effectively eliminated from commercial and research-maintained mice.
B. 1.
Rat Bite Fever
Reservoir and Incidence
Rat bite fever can be caused by either of two microorganisms: Streptobacillus moniliformis or Spirillum minus. Streptobacillus moniliformis causes the diseases designated as streptobacillary fever, streptobacillary rat bite fever, or streptobacillosis. Haverhill fever and epidemic arthritic erythemia are diseases associated with ingestion of water, food, or raw milk contaminated with
727
Str. moniliformis. Sodoku, derived from the Japanese words for rat (so) and poison (doku), spirilosis, and spirillary rat bite fever are caused by another bacterium, Spirillum minus. The bite of an infected rat is the usual source of infection. In some cases, other animal bites, including mice, gerbils, squirrels, weasels, ferrets, dogs, and cats, or rare traumatic injuries unassociated with animal contact, cause the infection. In a retrospective analysis covering three decades (1970–1998) of 45 S. moniliformis isolates (91% from humans) from the Department of Public Health in Berkeley, California, 50% of the isolates were from children ≤ 9 years of age (Graves and Janda 2001). In 75% of the human infections where a diagnosis was made, rat bite fever (RBF) was suspected; 83% of those suspected cases involved either known rat bite or exposure to rodents. Two cases of RBF were attributed to exposure— in one case a squirrel, and in the second a mouse (Graves and Janda 2001). 2.
Mode of Transmission
Interestingly, ≥ 9% of the cases could not be attributed to a rat bite or scratch, indicating that close contact with infected rodents can be sufficient to become infected (Graves and Janda 2001). Other reports have indicated that the disease can occur in individuals who have no history of rat bites, but reside or work in rat-infested areas or have pet rats with whom they have close contact (Fordham et al. 1992; Holroyd et al. 1988; Rumley et al. 1987). Rat scratches can also be the source of the organism (Edwards and Finch 1986; Shanson et al. 1985). Exposure to cats and dogs that prey on wild rodents may also be the source of the organisms. These organisms are present in the oral cavity and upper respiratory passages of asymptomatic rodents, usually rats (Wilkins et al. 1988). Mice can be infected with resulting morbidity and mortality due to arthritis and pneumonia. In one study, Streptobacillus moniliformis was isolated as the predominant microorganism from the upper trachea of laboratory rats (Paegle et al. 1976). Presumably the incidence of Str. moniliformis is now lower in high-quality, commercially reared specific pathogen-free rats. Surveys in wild mice indicate 0 to 25% infection with Spirillum minus (Hull 1955). 3.
Clinical Signs
Rat bite fever is not a reportable disease, which makes it difficult to assess its prevalence, geographic location, racial data, and source of infection in humans. The disease, though uncommon in humans, has nonetheless appeared among researchers or students working with laboratory rodents, particularly rats (Anderson et al. 1983). Historically, wild rat bites and subsequent illness (usually small children) relate to poor sanitation and overcrowding (Hull 1955). Acute febrile diseases, especially if associated with animal bites, are routinely treated with penicillin or other antibiotics. Therefore, accurate data regarding prevalence is usually not provided.
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CHRISTIAN E. NEWCOMER AND JAMES G. FOX
TABLE 26-1
CLINICAL SIGNS OF RAT BITE FEVER
Clinical Features Incubation period Fever Chills Myalgia Rash Lymphadenitis Arthralgia, arthritis Indurated bite wound Recurrent fever/ constitutional signs (untreated)
Streptobacilliary fever (Streptobacillus moniliformis)
Spirillosis (Spirillum minus)
2–10 days +++ +++ +++ ++ Morbilliform, petechial + ++ − Irregular periodicity
1–6 weeks +++ +++ +++ ++ Maculopapular ++ +− +++ Regular periodicity
From Fox (2004).
Streptobacillus moniliformis incubation varies from a few hours to 2 to 10 days, whereas Spirillum minus incubation ranges from 1 to 6 weeks (Table 26-1). Fever is present in either form. Inflammation associated with the bite and lymphadenopathy are frequently accompanied by headache, general malaise, myalgia, and chills (Arkless 1970; Cole et al. 1969; Gilbert et al. 1971; McGill et al. 1966). The discrete macular rash that often appears on the extremities may generalize into pustular or petechial sequelae. Arthritis occurs in 50% of all cases of S. moniliformis but is less common in Spirillum minus. Streptobacillus moniliformis may be cultured from serous to purulent effusion, which is recovered from affected larger joints. A total of 18 cases of endocarditis due to S. moniliformis were reported from 1915 to 2000 (Shvartsblat et al. 2004). Death has occurred in cases of S. moniliformis involving preexistent valvular disease or as a result of endocarditits in a previously healthy individual. Infants can also die of the infection (Sens et al. 1989). If antibiotic treatment––usually penicillin at doses of 400,000 to 600,000 daily for 7 days––is not instituted early, complications such as pneumonia, hepatitis, pyelonephritis, enteritis, and endocarditis may develop (Richter 1954). If endocarditis is present, the penicillin should be given parenterally at doses of 15 to 20 million units daily for 4 for 6 weeks. Streptomycin and tetracyclines are also effective antibiotics for those individuals with penicillin-associated allergies. Addition of streptomycin to standard therapy is also advised in cases where isolates of Str.. moniliformis are cell wall deficient (Rupp 1992). 4.
Diagnosis
Spirillum minus does not grow in vitro and requires inoculation of culture specimens into laboratory animals, with subsequent identification of the bacteria by dark-field microscopy. Streptobacillus moniliformis grows slowly on
artificial media but only in the presence of 15% blood and sera, usually 10% to 20% rabbit or horse serum incubated at reduced partial pressures of oxygen. Sodium polyanethol sulfonate sometimes found in blood-based media because of its properties as a bacterial growth promoter should not be used due to its inhibitory effects on Str. moniliformis (Lambe et al. 1973; Shanson et al. 1985). Growth on agar consists of 1 to 2 mm gray, glistening colonies. The API ZYM diagnostic system can be used for rapid biochemical analysis and diagnosis. A PCR-based assay has also been described to diagnose Str. moniliformis (Berger et al. 2001).
C. 1.
Salmonella
Reservoir and Incidence
The genus Salmonella are gram-negative bacteria with approximately 2000 serotypes. Nontyphoidal salmonellosis is caused by any of these serotypes. Other than Salmonella typhi, the causative agent of typhoid fever, salmonellosis occurs worldwide and is important in humans and animals. S. typhi and Salmonella choleraesuis have only one serotype, whereas the remaining 2000 serotypes are within the species Salmonella enteritidis. References to the Salmonella enteritidis serotypes are abbreviated such that “enteritidis” is dropped; for example, S. enteritidis serotype typhimurium is called Salmonella typhimurium. Salmonella typhimurium is the serotype most commonly associated with disease in both animals and humans. Other serotypes most commonly reported from humans and animals are Salmonella heidelberg, Salmonella agona, Salmonella montevideo, and Salmonella newport. Salmonellae are pathogenic to a variety of animals.
2.
Mode of Transmission
Salmonella are ubiquitous in nature and are routinely found in water or food contaminated with animal or human excreta. Fecal-oral transmission is the primary mode for spreading infection from animal to animal or to humans. Transmission is enhanced by crowding and poor sanitation. During the early 1900s, rodenticides containing live cultures of S. enteritidis were distributed on a large-scale basis by commercial and public health organizations in an attempt to eliminate feral rats. These cultures were known as “rat viruses” and were widely used in Europe, England, and the United States as “rat poisons” (Weisbroth 1979). However, enthusiasm for their use waned when it was discovered that the spread of the organisms couldn’t be limited; predictably, the baiting program was implicated in several epidemics among exposed human populations (Weisbroth 1979). Surprisingly, as late as the 1950s in England, S. enteritidis (serovar danzy) was isolated from adults living four miles apart. The source of infection was traced to contaminated cakes from a local bakery. Mice that had acquired the
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infection from living S. danzy cultures in rodenticide baits had infected food in the bakery (Brown and Parker 1957). As with other fecal-oral transmitted diseases, control depends on eliminating contact with feces, food, or water contaminated with Salmonella or animal reservoirs excreting the organism. Salmonella survive for months in feces and are readily cultured from sediments in ponds and streams previously contaminated with sewage or animal feces. Fat and moisture in food promote survival of salmonella. Pasteurization of milk and proper cooking of food (56°C for 10 to 20 minutes) effectively destroys salmonella. Municipal water supplies should be routinely monitored for coliform contamination (Pavia and Tauxe 1991). 3.
Clinical Signs
Clinical signs of salmonellosis in humans include acute sudden gastroenteritis, abdominal pain, diarrhea, nausea, and fever. Diarrhea and anorexia may persist for several days. Organisms invading the intestine may create septicemia without severe intestinal involvement; most clinical signs are attributed to hematogenous spread of the organisms. As with other microbial infections, the severity of the disease relates to the organism’s serotype, the number of bacteria ingested, and the host’s susceptibility. In experimental studies with volunteers, several serovars induced a spectrum of clinical disease ranging from brief enteritis to serious debilitation. Incubation varied from 7 to 72 hours. Cases of asymptomatic carriers, persisting for several weeks, were common (Hull 1955). Salmonella are flagellated, nonsporulating, aerobic gramnegative bacilli that can be readily isolated from feces on selective media designed to suppress bacterial growth of other enteric bacteria. Salmonella serotyping requires antigenic analysis (Fox 1991). Salmonella gastroenteritis is usually mild and self-limiting. With careful management of fluid and electrolyte balance, antimicrobial therapy is not necessary. In humans, antimicrobial
therapy may prolong rather than shorten the period that salmonella are shed in the feces (Nelson et al. 1980; Pavia and Tauxe 1991). In one double-blind placebo study of infants, oral antibiotics did not significantly affect the duration of salmonella carriage. Bacteriologic relapse after antibiotic treatment occurred in 53% of the patients, and 33% of these suffered a recurrence of diarrhea, whereas none of the placebo group relapsed (Nelson et al. 1980). D. 1.
Other Potential Bacterial Diseases
Borrelia Species (Tick borne Relapsing Fever)
Tick-borne relapsing fever occurs primarily in foci in the western part of the United States, as well as other parts of the world. The disease is caused by at least 15 Borrelia species and is transmitted to humans from a variety of rodents (chipmunks, squirrels, rats, mice, prairie dogs, hedgehogs) via soft ticks of the genus Ornithodorus. 2.
Helicobacteriosis
Of recent interest are the increasingly recognized enterohepatic Helicobacter spp., which cause both hepatic and intestinal disease in mice (Whary and Fox 2004). One of these, H. bilis, isolated routinely from mice, has been found using PCR-based assays in bile and gallbladder of Chilean patients with chronic cholecystitis and in biliary cancers in Japanese patients (Fox et al. 1998; Matsukura et al. 2002). Whether these new helicobacters will be linked to zoonotic transmission from wild or laboratory rodents will require further studies. 3.
Staphylococcus aureus
Pathogenic Staphylococcus aureus of human phage type can cause clinical disease in mice and rats. This organism has been
TABLE 26-2
TRICHOPHYTON MENTAGROPHYTES INFECTION ASSOCIATED WITH LABORATORY MICE, RATS, OR PET MICE Probable Source of Infection
Number of Persons Infected
Lesions Appearing On Infected Mice Or Rats
References
Pet white mice; inbred albino laboratory mice (VSBS, A2G) Laboratory mice Laboratory mice
7 children; 2 lab technicians
2 or 104, diffuse alopecia
(Mackenzie 1961)
6 lab technicians 2 lab technicians
0 f 96 (222 cultured), survey of commercial stock
(Alteras 1965) (Dolan et al. 1958)
BALB/c C3H/BI mice
6 lab technicians
<1% of all mice, carrier rate 90%
(Davies and Shewell 1964)
White mice
1 lab worker
(Booth 1952)
White mice
1 bacteriologist
Wistar rats Rats
1 technician 1 technician
% not determined, alopecia, increased scaling on head and back, 10 mice 60 of 400, crusted or crustless plaques, circular with prominent periphery; general alopecia; mortality in some mice 20% colony with alopecia and scaly skin Alopecia with crusting an erythema
(Cetin et al. 1965)
(Dolan et al. 1958) (Povar 1965)
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introduced into SPF barrier-maintained mouse colonies and SPF rats and guinea pigs; the same phage type was isolated from their animal caretakers (Davey 1962; Shults et al. 1973). Colonization by normal S. aureus strains in the nasopharyngeal area of humans presumably minimizes the zoonotic potential of animal-originated S. aureus.
V. DERMATOPHYTOSIS (RINGWORM) As reviewed comprehensively in Blank, reports of ringworm (favus) in the mouse began to appear in the European literature in the mid-nineteenth century and in the North American literature during the early twentieth century (Blank 1957). Several of the early authors noted the similarities between the lesions of favus in the mouse and in humans. Quincke, who is generally credited with isolating the causative agent which he named γ-Pilz (now Trichophyton mentagrophytes), suggested that the infection in the mouse was also a source of infection of the cat, and thereby, of humans. Earlier reports of murine ringworm referred to the causative agent as T. quinckeanum, but the successful crossing of T. quinckeanum with the perfect state of T. mentagrophytes, Arthroderma behamiae, indicates that T. quinckeanum is not a distinct species (Ajello et al. 1968). A later study of the two varieties, T. mentagrophytes var. mentagrophytes and T. m. var. quinckeanum, noted that the conidia from both produced two morphological variants on cultivation (granular and fluffy), and these variants were A. behamiae type + and pathogenic (Hejtmanek and Hejtmankova 1989). In addition to T. mentagrophytes, Epidermophyton floccosum, Mircrosporum gallinae, M. gypseum, M. canis, T. erinacai, T. schoenleini, and T. (keratinomyces) ajello have been reported as zoophilic dermatophytes that can infect mice and cause ringworm in humans (Dvorak 1964; Krempl-Lamprecht and Bosse 1964; Marples 1967; Papini et al. 1997; Refai and Ali 1970). 1.
Reservoir and Incidence
The dermatophytes are distributed worldwide and can involve a variety of small animal host species in addition to the mouse. Chmel et al. (1975) conducted field studies in a wooded farm setting in Czechoslovakia and detected an overall prevalence rate of 4.4% (57 positive of 1288) for T. mentagrophytes infection in 6 of 13 species sampled; the prevalence in Mus musculus was 3.4%, with mice comprising 15.8% of the infections detected. Of the species that harbored the infection, all frequented the barn or granary area; the seasonal incidence was highest during the winter months when the rodent carriers were more likely to seek harborage indoors. Chmel et al. (1975) also analyzed patient data and demonstrated that T. mentagrophytes was the predominant isolate from those who did agricultural work, while T. verrucosum was the main isolate
from individuals who worked with farm animals. Also, human T. mentagrophytes infections were most common on the hands, wrist, forearm, face, and neck, unprotected skin sites readily contaminated by fodder, litter, or other materials while working in the barns. Ringworm infections associated with the handling of bags of grain in which mice had been living have also been reported (Alteras 1965; Blank 1957). Ringworm infection in laboratory mice is often asymptomatic, remaining unrecognized until laboratory personnel become infected. Early reports in the literature indicated that the prevalence of T. mentagrophytes was 80 to 90% among some laboratory mouse stocks (Davies and Shewell 1964; Dolan et al. 1958). However, these reports predated the era of modern laboratory animal colony management marked by the commercial availability of cesarean-derived, barrier-maintained rodents. Moreover, the modern production practices that have been universally adopted by the industry for decades and the use of microbarrier cages with appropriate technique have further reduced the opportunity for ringworm to become a significant problem in contemporary colonies. In recognition of this fact, none of the major commercial vendors in the United States survey their colonies for dermatophyte infections as part of routine health monitoring. Sporadic cases of ringworm infections in rodents have been reported in the past three decades (Hironaga et al. 1981; Mizoguchi et al. 1986; Papini et al. 1997). Programs involved in importing mice from sources that fail to meet contemporary rodent production and husbandry practices should consider screening mice for dermatophytes during the quarantine period. 2.
Mode of Transmission
The ease of transmission of dermatophytes from animals to humans is well known and is a significant health hazard. Laboratory mice, as well as other laboratory animal species, can harbor dermatophyte infection, with few or no visible skin lesions transmitting the infection to unsuspecting personnel (Dolan et al. 1958). Transmission can occur through direct contact with the infected animal or through indirect contact with animal bedding or other materials in the environment of the contaminated animal room. Rigorous facility and equipment sanitation has long been recognized as an essential element of an effective control program and should be undertaken in conjunction with efforts to treat valuable animals or to repopulate previously contaminated areas of a facility (Davies and Shewell 1964; Dolan et al. 1958; Mizoguchi et al. 1986). The importance of barrier protections by donning appropriate clothing, using gloves and other personal protective equipment, and modifying work practices to minimize skin exposure to dermatophytes has also been acknowledged for the prevention of transmission (Dolan et al. 1958). When prevention methods fail, allowing the introduction of dermatophyte infection into a mouse colony, and when transmission to humans occurs, clinical cases of dermatophytosis routinely respond well either to topical or systemic antifungal therapy.
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TABLE 26-3
SELECTED ECTOPARASITES OF RODENTS WITH ZOONOTIC POTENTIAL* Species Mites Obligate skin mites Sarcoptes scabiei subspecies Nest-inhabiting parasites Ornithonyssus bacoti
Liponyssoides sanguineus Haemogamasus pontiger Ixodids (ticks) Dermacentor variabilis Amblyomma americanum Ixodes scapularis Ixodes dammini Fleas Xenopsylla cheopis Nasopsyllus fasciatus Leptopsylla segnis
Disease in humans
Host
Agent transmitted
Scabies
Mammals
Dermatitis, murine typhus, rickettsialpox
Rodents and other vertebrates
Dermatitis, rickettsialpox Dermatitis
Rodents, particularly Mus musculus Rodents, insectivores, straw bedding
Irritation, RMSF,++ tularemia, tick paralysis, other diseases Irritation, RMSF,++ tularemia Irritation, possible tularemia Human babesiosis, Lyme disease
Wild rodents, cottontail rabbits, dogs from endemic areas Wild rodents, dogs Dogs, wild rodents Wild rodents, especially Peromyscus sp.
Dermatitis, plague, murine typhus, R. nana, R. diminuta Dermatitis, plague, R. nana, R. diminuta, murine typhus R. diminuta, R. nana, murine typhus
Rat, mouse, wild rodents Rat, mouse, wild rodents Rat
Coxsackie, WEE,** SLE+ virus, Rickettsia typhi, Rickettsia akari, Francisella tularensis Rickettsia akari
Rickettsia rickettsia, F. tularensis
Borrelia burgdorferi, Babesia microti Rodent tapeworms, Yersinia pestis, Rickettsia typhi Rodent tapeworms, Yersinia pestis, Rickettsia typhi Rodent tapeworms, harbors salmonella, Rickettsia typhi
*Found
in laboratory animals that cause allergic dermatitis or from which zoonotic agents have been recovered in nature (see Yunker 1964). western equine encephalitis. +SLE, St. Louis equine encephalitis. ++RMSF, Rocky Mountain spotted fever. **WEE,
3.
Clinical Signs
Dermatophytosis or ringworm in humans can be asymptomatic and minor, often self-limiting and drawing little attention from the affected individual. The infection usually causes an expanding, scaly and erythematous inflammatory plaque on the skin that occasionally contains fissures or vesicles when severely eczematous. On the trunk and extremities, the lesion may consist of one or more circular lesions with a central clearing and sharply defined margins, forming a ring, and hence the name “ringworm” (Fig. 26-1)(Merlin et al. 1994). Other dermatophytes are named according to the sites of involvement on the body (e.g., tinea pedis for foot infections, tinea capitis for scalp infections). The dermatophyte infections of humans associated with direct or indirect contact from mice usually involve the body or extremities, especially the arms and hands. Zoophilic T. mentagrophytes infection usually produces a highly inflammatory lesion and often undergoes rapid resolution. However, it can also produce furunculosis––deep infection of the hair follicles or widespread tinea corporis–– which is also seen in infections of E. floccosum. In a laboratory-acquired infection with T. (keratinomyces) ajelloi, mice
were the source of infection for a laboratory technican who developed small, grayish-white, scaly lesions on both hands. Hand lesions yielded the organism, as did 2 of 250 apparently health mice (Refai and Ali 1970).
Fig. 26-1 Tropical rat mite bites in human.
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CHRISTIAN E. NEWCOMER AND JAMES G. FOX
VI.
HELMINTH DISEASES A.
1.
Tapeworms
Hymenolepis diminuta, the Rat Tapeworm
A. RESERVOIR AND INCIDENCE Although this parasite occurs in the mouse intestine, it is more commonly associated with rats and is especially common in wild Norway (Rattus norvegicus) and black (Rattus rattus) rats throughout the world (Faust and Russell 1970; Stone and Manwell 1966; Wardle and McLeod 1952). It has been reported in humans worldwide, including several areas in the United States. B. MODE OF TRANSMISSION Like other tapeworms, H. diminute requires an intermediate host, usually a flour beetle (Tribolium sp.), moth, or flea (Voge and Heyneman 1957). Larval development in Tribolium sp. at 30°C requires 8 days. Therefore, humans become infected only through ingestion of infected insects, such as flour beetles, which may contaminate rodent food or cereal marketed for human consumption. C. CLINICAL SIGNS The infection in humans is usually asymptomatic, but in moderate to heavy infections it may cause headaches, dizziness, abdominal discomfort, and diarrhea. The greatest length of an adult parasite removed from a patient was 1 meter. Usually, adult parasites are 20 to 50 cm long and as much as 4 mm wide (Markell et al. 1999).
2.
Rodentolepsis nana (formerly Hymenolepis) the Dwarf Tapeworm of Humans
A. RESERVOIR AND INCIDENCE The dwarf tapeworm is a common parasite of both the wild house mouse and the laboratory mouse. As indicated earlier in the text, in most well- managed mouse colonies, R. nana incidence is low compared to earlier reports of its high incidence in rodent colonies (Wescott 1982). The estimate that 20 million humans in the world are infected was made many years ago but probably is an underestimate (Markell et al. 1999). Surveys conducted in Central Europe report that this tapeworm in humans is more prevalent in warm than in temperate regions. An incidence of 10% has been noted in some South American countries (Jelliffe and Stanfield 1978). It is most commonly diagnosed in children. Diagnosis is made by observing characteristic eggs in the feces. B. MODE OF TRANSMISSION R. nana is unique among tapeworms in that the adult worm develops after the egg is ingested. The hooked oncosphere then invades the intestinal mucosa and develops into a cysticeroid larva. Rodentolepsis nana eggs can contaminate hands, be trapped on particulate matter, or be aerosolized, and then accidentally ingested. Since no intermediate host is required, the eggs are readily infective for the
reciprocal hosts (Faust and Russell 1970). Precautions against infection include strict personal hygiene, appropriate laboratory uniforms, and use of disposable gloves and face masks when handling contaminated bedding and feces. C. CLINICAL SIGNS The clinical picture of R. nana infection is quite cosmopolitan. In well-nourished persons, essentially no symptoms occur; the infection is noted when the proglottids or ova are seen in the stool. In other persons, the symptoms include headaches, dizziness, anorexia, inanition, pruritis of the nose and anus, periodic diarrhea, and abdominal distress. A tapeworm identified as R. nana was found in a tumor removed from the chest wall (Jelliffe and Stanfield 1978). The diagnosis is based on identification of the characteristic eggs or proglottids in the stool. D. TREATMENT Praziquantfel, given orally in a single dose of 25 mg/kg body weight is the drug of choice. Alternatively, niclosamide is given daily for 5 days because of the tissue phase of the parasite. The dose is 2 gm for adults and 1.5 gm for children > 34 kg, and 1.0 gm for children between 11 and 34 kg (Markell et al. 1999). 3.
Rodentolepsis (formerly Hymenolepis) microstoma
Recently, a parasite known to naturally colonize mice, R. microstoma, has been identified in the feces of humans living in the northwest of Western Australia (Macnish et al. 2003). Although R. nana was the most common enteric parasite based on microscopic examination of feces, R. microstoma was identified as a mixed infection in 4 of 11 individuals by using a molecular-based assay consisting of restriction fragment length polymorphism of tapeworm DNA as well as a sequencing of the PCR product of the internal transcribed spacer 1 region of ribosomal DNA (Macnish et al. 2002). Given that R. microstoma requires an intermediate host, Tribolium confusum for its life cycle, it is understandable why it was not as common as R. nana in this study. However, given the morphological similarities of the eggs of R. nana and R. microstoma, the true prevalence of R. microstoma in humans won’t be known until molecular techniques to differentiate the two species are utilized in future studies.
B. 1.
Roundworms (Syphacia obvelata)
Reservoir and Incidence
Syphacia obvelata is an ubiquitous parasite in both wild and laboratory mice. Although parasitology texts report that Syphacia is infectious to humans, this citation originates from a publication in 1919, in which two S. obvelata adult worms and eggs reportedly were found in the formalin preserved feces of a Filipino child whose entire family of five was infected with
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H. nana (Riley 1919). No mention is made of the method of collecting the feces, nor is it known whether the feces could have been contaminated with murine feces or with the parasite and/or eggs. The only other report is an unpublished finding of S. muris eggs in the feces of two children and two rhesus monkeys, cited in a personal letter from Dr. E. E. Faust of Tulane University, dated January 6, 1965 (Stone and Manwell 1966). Both of these cases may therefore be examples of spurious parasitism, but definitive information for that conclusion is lacking. Regardless, no published information indicates that laboratory personnel have been infected by working with Syphacia-infected mice. 2.
Mode of Transmission
Contamination of food or utensils or accidental ingestion of Syphacia ova (e.g., via contaminated hands) could result in infection of humans. People working with infected mice probably ingest ova occasionally, but there is no evidence that this exposure results in active infection. 3.
Clinical Signs
Because Syphacia infection in humans has not been described, clinical signs have not been noted. 4.
Diagnosis
There are striking differences in size between specimens of female S. obvelata and those of Enterobius vermicularis, the pinworm, in humans (Markell and Voge 1965). Syphacia is 3.5 to 5.8 mm long, whereas the Enterobius sp. female reaches a length of 8 to 13 mm. The male Syphacia sp. measures 1.1 to 1.5 mm compared to 2.5 mm for Enterobius. The size difference between the eggs of the two species is also marked: Syphacia eggs are more than twice as long (125 µm versus 52 µm as those of Enterobius). It is unlikely therefore that Syphacia sp. would be misdiagnosed as Enterobius sp., assuming, of course, that the observer was aware of the size difference and measured the eggs.
VII.
ARTHROPOD INFESTATIONS A.
Mites
Although many species of mites are found on laboratory mice, only Ornithonyssus bacoti, the tropical rat mite, and Liponysoides sanguineus, the house mouse mite, are vectors of human disease. Ornithonyssus bacoti is seen in laboratory mice (Fox 1982); L. sanguineus has been identified only on wild mice. Bites from these mites, as well as from another mouse mite, Haemalaelaps casalis, are responsible for allergic dermatitis, or local inflammation, in humans.
1.
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Ornithonyssus bacoti––Tropical Rat Mite
Ornithonyssus bacoti can be found on many rodents; the brown Norway rat and the black roof rat are probably the primary host species (Beaver and Jung 1985). Since the time of the first report of human Ornithonyssus bacoti–associated dermatitis in Australia in 1913, and a 1923 report in humans in the United States, many other cases have continued to be described throughout the world (see Table 26-4) (Charlesworth and Clegern 1977; Chung et al. 1998; Dove and Shelmire 1931; Dowlati and Maguire 1970; Engel et al. 1998; Fox 1982; Haggard 1955; Hetherington et al. 1971; Riley 1940; Theis et al. 1981; Wainschel 1971; Weber 1940). Ornithonyssus bacoti is an obligate bloodsucking parasite, usually tan but red when engorged with blood. Both the male and female feed on a rodent as their preferred host. The female is 700 µm to 1 mm in length; the male is smaller (Fig. 26-2). Eggs are laid in bedding or wall crevices by the female, which survives for about 70 days and feeds about every two days during this period. The mite has five developmental stages: adult, egg, nonfeeding larva, bloodsucking protonymph, and nonfeeding deutonymph. After feeding, the adults and protonymphs leave their host and seek refuge in cracks and crevices. The life cycle from adult to egg requires 7 to 16 days at room temperature. Unfed protonymphs have survived for 43 days (Brettman et al. 1981). The mite often gains access to the premises on wild rodents and lives in crevices. If wild rodents are not readily available or are captured, the mite will seek blood elsewhere, either from the wild or laboratory rodent (if in an animal research facility) and/or humans. In some infestations, the rodent shows no clinical signs. However, in more chronic cases, dermatitis and anemia may develop. Historically, this mite has been a troublesome parasite in certain laboratory animals, especially rats, mice, and hamsters (Fox 1982; Keefe et al. 1964). a. CLINICAL SIGNS Tropical rat mites produce painful, pruritic lesions in humans. Examination of patients often discloses papular lesions on the wrists, arms, abdomen, and chest. Raised erythematous papules and nodules several millimeters to greater than 1 cm in size occur singly or in linear configuration (Fig. 26-3). Epidemiologically, cases usually occur in clusters that involve a common source of exposure to the mite. Experimentally, cases have been shown to be a vector of pathogens. In the laboratory, mite transmission of various rickettsial species, Pasteurella tularensis, and Coxsackie virus between different laboratory animals has been shown (Hopla 1951; Petrov 1971; Philip and Hughes 1948; Schwab et al. 1952). Affected individuals may be treated with topical lindane or treated symptomatically, given that the mite does not reside on humans for any extended periods. Papular dermatitis will regress after a period of 7 to 10 days post-therapy. Recurrence of Ornithonyssus bacoti infestations is common unless the
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TABLE 26-4
REPORTS OF ORNITHONYSSUS BACOTI–INDUCED DERMATITIS IN HUMANS: UNITED STATES, 1931–1998 Host
Person(s) afflicted
Environment
Lesions
Anatomical location
References
Adults: urticarial wheals, papules Children: papules, urticarial wheals, vesicles Wheals, papules, few wheals with central puncture
Adults: ankles, trunk, back, neck Children: beltline, upper part of shoulders Women: arms, forearms Man: hands, ankles, legs, beltline, shoulders, neck —
(Dove and Shelmire 1931)
Rat
200 adults and children
Residence, theater
Rat
4 women, 1 man
Department store
Rat Mice Rat
Employees
Department store
Macular skin eruptions
Infants, adult occupants 8-yr.-old boy; 5 siblings, and both parents affected with milder symptoms 60 yr. old
Foundling home
Papular urticaria, grouping of bites Excoriated urticarial papules
—
(Haggard 1955)
Trunk, upper part of arms, buttocks
(Dowlati and Maguire 1970)
Residence
1- to 4-mm papules, excoriated macules
Neck, shoulders, back, scalp, forearm, arms, abdomen
(Hetherington et al. 1971)
56-yr.-old father and 2 sons; 73-yr.-old woman 69-yr.-old woman
Residence (apartment over food store)
“Insect bites,” papular excoriated dermatitis
Thorax, extremities, buttocks, genitalia, entire body
(Wainschel 1971)
Residence
Papules with erythema
Breast, shoulders, arm
Residence
Papular urticaria, erythematous Several millimeters to >1 cm raises erythematous papules and nodules Erythematous papules
Neck, shoulders, arms, legs, abdomen, back Wrists, arm, abdomen, chest
(Charlesworth and Clegern 1977) (Theis et al. 1981)
Rat
Norway rat Rat
Rat Rat Mice
Rodents Rodents
3 female adults, 3 children 5 research personnel, 2 animal care technicians 10 medical students (9 males, 1 female) 6 medical students
Residence
Animal research laboratory Library Residence in centuries old house
Red papules and seropapules
Neck axilla, abdomen, both extremities Legs, arms, waist, laterally on the trunk
(Weber 1940)
(Riley 1940)
(Fox 1982)
(Chung et al. 1998) (Engel et al. 1998)
Fig. 26-2 Left, Adult Ornithonyssus recovered from mouse cage (×10). Right, Enlarged view of mite mouth parts (×100).
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Fig. 26-3 A circular ringworm lesion on the arm of a man; contracted from a rodent infected with Trichophyton mentagrophytes. (Courtesy of Dr. William Kaplan.)
premises have been treated with an appropriate insecticide, and any feral rodents eradicated (Engel et al. 1998).
B.
Fleas
Fleas are seldom found in laboratory mice but are common parasites of feral rodents. The Oriental rat flea, Xenopsylla cheopis, and another flea, Nasopsyllus fasciatus, both naturally infest mice and rats; they are vectors for murine typhus. Apparently, X. cheopis is easily established in animal facilities. At a midwestern U.S. university, it inhabited a room housing laboratory mice where, on two separate occasions, the flea caused distress by biting students (Yunker 1964). Leptopsylla segnis, the mouse flea, bites humans and is a vector for plague and typhus, serious diseases in humans. Also, L. segnis can serve as an intermediate host for the rodent tapeworms R. nana and R. diminuta, which can infect people. The flea’s bite can also be irritating and cause allergic dermatitis.
epidemiological perspective on the transmission of infectious diseases, principally rabies. The bite of the rat is far more powerful and more likely to be disfiguring than that of the mouse, and rat bites are known to be associated with the transmission of bacterial zoonoses such as rat bite fever and leptospirosis (see elsewhere in this chapter). One should assume that the mouse is also capable of transmitting these agents via bite. Rabies transmission from small rodents in the wild occurs but is exceedingly rare (Gdalevich et al. 2000); therefore, rabies is of concern only if experimental studies with the virus are being conducted in mice. Mice can also transmit hantavirus infection and lymphocytic choriomeningitis virus infection via bites. Anecdotally, most animal care and use programs report that rodent bites among personnel are a reasonably common occurrence that often are unreported to an institution’s occupational medical service, despite the fact that bites inflict pain, produce anxiety, and may have significant health consequences. In addition to the hazard of zoonotic disease or local wound infection with pyogenic or toxin producing bacteria such as Clostridium tetani, Staphylococcus spp., Streptococcus spp., Escherichia coli, and Bacillus subtilis, rodent bites, including those of the mouse, can induce a severe local allergic reaction or anaphylaxis in individuals previously sensitized to allergen (Hesford et al. 1995; Teasdale et al. 1983; Thewes et al. 1999). Thus, bite wounds from mice should be immediately cleaned thoroughly and reported to the institutional occupational medical service to permit evaluation of the person’s tetanus immunization status and need for additional local wound or other medical care. The need for additional training of bitten persons in animal handling may also be indicated.
IX.
ALLERGIC SENSITIVITIES––LABORATORY ANIMAL–ASSOCIATED ALLERGY (LAA) A.
VIII.
BITES
Authoritative information on the incidence and impact of animal bites in the general population over the past several decades is scant, and reliable data on the incidence of mouse bites among personnel who work in laboratory animal facilities or among the general populace is lacking. There have been occasional studies on the occurrence and clinical characteristics of rat bites within urban populations, including a recent investigation of 622 bites over the period 1974–1996 that associated this phenomenon with urban blight, poverty, and unemployed populations (Hirschhorn and Hodge 1999). Traditionally, animal bites have received attention from the clinical perspective of wound management and complications and from the
Incidence and Clinical Signs
Allergic skin and respiratory reactions to laboratory mice are very common in laboratory animal caretakers and technicians who work with these animals. A survey by Lutsky (1987) demonstrated that three-fourths of all institutions with laboratory animals had animal care personnel with allergic symptoms. The prevalence of symptoms of laboratory animal–associated allergy (LAA) among personnel working with laboratory animals has been estimated as between 10 and 46% in numerous recent studies, and among these individuals, approximately 10% are estimated to eventually proceed to the development of asthma (Chan-Yeung and Malo 1994; Eggleston and Wood 1992; Hollander et al. 1996; Hunskaar and Fosse 1990; Knysak 1989; Renstrom et al. 1994). Furthermore, other sources have suggested that among atopic individuals with preexisting allergic disease, up to 73% of persons exposed to lab animal allergens
736 may eventually develop LAA (Committee on Occupational Health and Safety in Research Animal Facilities/National Research Council Allergens 1997). The population at risk for work-related exposure to rodents was estimated at 90,000 (Newill et al. 1986); this population has likely grown in the intervening years to the expanding populations of genetically modified mice that are used in contemporary biomedical research programs and require care. Moreover, a recent study would seem to suggest that the risk of exposure to mouse allergens is not confined to those working in the laboratory animal facility environment. Data analyzed from the first National Survey of Lead and Allergens in Housing in the United States demonstrated that 82% of homes of diverse types and income levels across geographic locations had evidence of mouse allergen; 57% had detectable levels on the kitchen floors specifically; and 22% had allergen concentrations greater that 1.6 µg/g of dust collected, a level previously correlated with the significantly increased rate of sensitization to mouse allergen (Cohn et al. 2004). The large number of staff at risk of exposure in the workplace or already presensitized, in combination with the substantial added costs to employers for the medical management, operational disruptions, and retraining efforts related to employees who develop LAA and later proceed to asthma, should provide the impetus for many institutions to pay grater attention to this element of the occupational health and safety program (Schweitzer et al. 2003). The major allergen of the laboratory mouse is the Mus m 1 protein, a member of the mouse major urinary proteins encoded by a multigene family consisting of approximately 35 genes (Clark et al. 1984a, 1984b). The earlier literature on the subject of mouse allergy referred to the mouse urinary proteins (MUPs), whereas recent literature cites the specific protein (Mus m 1) that is now known to be the primary offending allergen in the MUP multigene family. The Mus m 1 protein is in the lipocalin family of proteins that are produced in the saliva and liver and are excreted in the urine at levels 100 times higher than are present in mouse serum. Lipocalins serve to bind small hydrophobic molecules and function biologically to transport vitamins, small volatile odorants, and pheromones conferring the characteristic odor to mouse urine (Cavaggioni et al. 1999; Flower et al. 1993; Konieczny et al. 1997; Santa et al. 1998; Virtanen et al. 1999). Several studies have indicated that production of Mus m 1 is under hormonal control and that the urine of male mice contains four-fold higher levels than the urine of female mice (Hastie et al. 1979; Lorusso et al. 1986; Price and Longbottom 1987). In addition to being present in the saliva and urine, Mus m 1 in the serum becomes incorporated into the pelt, conferring the allergenic property to mouse dander. The main allergens of many furred animals are structurally similar proteins within the lipocalin family, including those of the cow (Bos d 2), horse (Equ c 1), dog (Can f 1), and rat (Rat n 1) (Virtanen et al. 1999). The Mus m 1 and Rat n 1 lipocalin allergens, to which 90% of mouse and rat allergic individuals react, respectively, are closely related, sharing a 66% homology (Clark et al. 1984a).
CHRISTIAN E. NEWCOMER AND JAMES G. FOX
Some have proposed that personnel exposed to laboratory animal allergens can be categorized into four basic risk groups based on their history of allergic disease and sensitization to animal proteins (Committee on Occupational Health and Safety in Research Animal Facilities/National Research Council Allergens 1997). These risk groups are (1) normal individuals, (2) atopic individuals with preexisting allergic disease, (3) asymptomatic individuals with IgE antibodies to allergic animal proteins, and (4) symptomatic individuals with clinical symptoms upon exposure to animal allergens. Individuals in the normal risk group do not have a history of allergic disease, and 90% will never develop symptoms of LAA. If LAA develops in individuals in the normal risk group, it usually appears during the first three years of exposure. However, infrequently individuals in this risk group who have remained free of LAA for 10 or more years of exposure have developed a delayed onset of the condition (Department of Health and Human Services, National Institute of Occupational Safety and Health 1997). Atopic individuals have a genetic predisposition for an exaggerated tendency to mount IgE responses to common environmental allergens. Atopic individuals have higher total levels of IgE in the circulation and higher blood eosinophil counts compared to normal individuals, possibly as a result of the activation of cytokines involved in IgE isotype switching, eosinophil survival, and mast cell proliferation (Janeway et al. 2001). Among atopic individuals, up to 73% of those exposed to allergenic animal proteins eventually develop symptoms (Agrup et al. 1986). In asymptomatic individuals with elevated circulating IgE antibodies to animal allergens, up to 100% are at risk of developing allergic symptoms. Of the individuals in risk groups that are already symptomatic for LAA, approximately 33% will develop chest symptoms and 10% are likely to develop occupational asthma and face the prospect that continued exposure will result in permanent impairment. Allergic rhinitis, allergic conjunctivitis, and contact urticaria are the most common disorders seen in LAA (Committee on Occupational Health and Safety in Research Animal Facilities/ National Research Council Allergens 1997). Clinically, allergic rhinitis and conjunctivitis present with the symptoms of sneezing, clear nasal discharge, nasal congestion, itchiness, and watery eyes. Contact urticaria presenting as raised, circumscribed, erythematous lesions may also be present in LAA patients who report an intense itchiness to the skin in the area of contact with the allergen. Figs. 26-4 and 26-5 (Fox and Brayton 1982) illustrate the typical wheal and flare reaction on the skin provoked in an individual who had developed hypersensitivity to mouse urine over a period of several years and who was exposed by having a mouse with urine-contaminated feet walk over his arm (Ohman 1978). One large survey of laboratory animal workers summarized in the NIOSH Alert (Department of Health and Human Services, National Institute of Occupational Safety and Health 1997) reported that of 5641 animal workers from 137 animal
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In the most severe cases, the cascading events of angioedema, stridor, respiratory obstruction, hypotension, and shock can be life threatening (Committee on Occupational Health and Safety in Research Animal Facilities/National Research Council Allergens 1997).
B.
Fig. 26-4 Small wheals on the mid-forearm (arrows) and a large wheal adjacent to the tip of the tail of the mouse in the skin of a patient sensitive to mice.
facilities, 23% developed allergic symptoms related to laboratory animals. Of the workers reporting symptoms, 82% had nasal or eye symptoms, 46% had skin complaints, and 33% had asthma. Patients who develop asthma as a more serious complication of LAA manifest symptoms of wheezing, intermittent dyspnea or shortness of breath, cough, often nocturnal or in the early morning, and tightness of the chest. The key clinical sign in these patients is wheezing on auscultation, and physiological abnormalities include airflow obstruction, which may vary over time, bronchodilator responsiveness, and increased airway responsiveness (airway hyperreactivity) (Tang et al. 2003). Though quite rare, generalized anaphylactic reactions that are potentially life threatening can occur in individuals highly sensitized to animal allergens. Anaphylaxis may manifest as diffuse itching, hives, and swelling of the face, lips, and tongue. In some individuals, breathing becomes difficult owing to laryngeal edema, and others develop asthma and wheezing.
Laboratory animal-associated allergy is an example of the Type I, IgE antibody-mediated, immune reaction, and the reader should refer to other sources for a detailed discussion of the molecular mechanisms involved in developing this reaction (Janeway et al. 2001). In the case of animal allergens, the usual route of initial exposure is airborne, although bite exposures (saliva) and direct contact with the skin can also become important in later clinical symptoms. In the Type I reaction, upon exposure to antigen, which is often a protein or glycoprotein, the allergen is taken up and processed by cells of the innate and adaptive immune systems and by dendritic cells located in the mucosal-associated lymphoid cells, gut-associated lymphoid cells, and/or the dermis. The cytokine profiles of these cells favor the development of naïve CD4 T cells into TH2 cells that induce B cells to produce IgE specific for the allergen. Once the IgE response is initiated, it can be further enhanced by basophils, mast cells, and eosinophils that also drive allergen-specific IgE production (Janeway et al. 2001). IgE is normally found only in low levels in the circulation because it binds to tissue mast cells and circulating basophils. In the sensitized individual, restimulation with the sensitizing allergen results in allergen binding to IgE and the release of histamine and other chemical mediators from the mast cells and basophils. These mediators produce the array of clinical signs and symptoms that are characteristic of the allergy: itchiness, nasal congestion, sneezing, nasal and ocular drainage, coughing, wheezing, and shortness of breath.
C.
Fig. 26-5 Large wheal and flare in the skin (arrow) of a patient sensitive to mice.
Pathogenesis
Diagnosis
To establish the diagnosis of LAA related to mouse exposure, the physician should begin by considering the strength of the history, physical examination findings, the temporal relationship between the patient symptoms and the environmental exposure to mice, and possibilities of alternative explanations for the patient’s problems such as exposure to other potential allergens in the workplace or allergens of a nonoccupational nature. The development of clinical symptoms concomitant with or following exposure to an environment containing mice or Mus m 1 laden mouse products should help in narrowing the number of allergens tested. The patient’s family history of allergy is also very important to consider because atopy is a proven risk factor in developing LAA (Botham et al. 1995; Meijer et al. 2002; Venables et al. 1988).
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Physical examination of the patient and clinical monitoring for the progression of allergic disease incorporate a number of approaches. Pulmonary function tests such as bronchial hyperresponsiveness (in response to pharmacologic challenge with methacholine and not the specific allergen) and the forced expiratory volume in one second (FEV1) are commonly used to evaluate the degree of airway impairment and the response to bronchodilators, glucocorticoids, and other therapeutic agents. Radiographs may also be useful in patients with pulmonary involvement. Routine laboratory tests may also aid in the characterization and management of the patient’s condition, such as complete blood count and nasal smears for eosinophilia which is common in allergic individuals but also can be seen in those with perennial nonallergic rhinitis (Dykewicz et al. 1998). Measurement of total serum IgE has little value to the physician as an aid in distinguishing whether a particular patient has allergic disease, but it may offer some potential for the identifying of populations at risk for developing of LAA as indicated in both prospective and cross-sectional studies of laboratory animal workers (Hollander et al. 1996; Renstrom et al. 1995). Use of the radioallergosorbent test (RAST) for the detection of human IgE antibodies of defined allergen specificity is also available for patient evaluation. However, the quality of the laboratory performing the in vitro RAST assays, the specificity of the allergens used, and the potential for cross-reactivity are important considerations in adopting the RAST as a diagnostic tool (Hamilton 2003). Even when properly conducted, in vitro tests usually fail to detect a modest number of skin test– positive individuals, and on a per-test basis, skin tests have lower time and reagent costs (Hamilton 2003). Clinicians agree that when properly performed, prick-puncture skin tests are generally considered the most convenient and least expensive screening method for detecting allergic reactions in most patients (Demoly et al. 2003). The valid interpretation of these tests relies on standardized allergens and methods, and negative prick-puncture tests may be confirmed by more sensitive intradermal techniques. Even after falsepositive and false-negative tests have been eliminated, the proper interpretation of results requires thorough knowledge of the patient’s history and physical findings. A positive skin test alone does not confirm a definite clinical sensitivity to an allergen in the asymptomatic patient but possibly predicts the onset of allergic symptoms. A positive skin test in conjunction with a history suggestive of clinical sensitivity strongly indicates the allergen as the cause of the disease (Horak 1985). Strong positive skin tests along with a suggestive clinical history also correlate well with results of bronchial or nasal challenges with the antigen.
D.
Treatment and Prevention
The animal facility conditions and practices that contribute to mouse-associated LAA as a serious and prevalent workplace
hazard have received considerable study over the past several decades, enabling effective strategies for achieving control of exposures in most research animal care and use settings. In summary, these strategies involve exposure reduction through source reduction, containment of hazard through the use of modern equipment and engineering controls, and barrier protection with personal protective equipment. The Mus m 1 allergen load in the environment is markedly increased when male mice are used in studies due to the fact that they excrete 4-fold higher levels of allergen in the urine than do female mice (Lorusso et al. 1986). Therefore, sources have recommended, that whenever scientifically possible, use of only female mice would be a means of reducing allergen load in the environment and minimizing the exposure of personnel (Department of Health and Human Services, National Institute of Occupational Safety and Health 1997; Renstrom et al. 2001). Furthermore Renstrom et al. (2001) reported a three-fold higher rate of allergic sensitization in technicians who worked with male rodents. Although this approach may be useful in a few studies, this method of source reduction would appear to have only very limited applicability across the broad scope of contemporary studies using mouse models. Source reduction of mouse allergen is also achieved through reduction of animal density within an animal room (the number of animals per room volume) and through use of frequent, effective facility sanitation practices (Department of Health and Human Services, National Institute of Occupational Safety and Health 1997). The risk of exposure to mouse allergen varies by the type of animal-related activities conducted by personnel and by the type of animal housing systems and equipment containment devices employed in the use and maintenance of laboratory mice (Gordon et al. 1997, 2001; Schweitzer et al. 2003; Thulin et al. 2002). Many studies have examined the different caging systems used for mouse housing, and the ability of each cage system design to reduce environmental allergen is well known (Gordon et al. 2001; Schweitzer et al. 2003). The application of just a simple filter sheet top or fitted filter bonnet to an open cage is effective in reducing ambient allergen levels (ReebWhitaker et al. 1999). However, studies indicate the clear superiority of individually ventilated caging (IVC) systems run under negative pressure for the purpose of controlling room allergen levels (Gordon et al. 2001; Reeb-Whitaker et al. 1999; Schweitzer et al. 2003). Gordon et al. (2001) suggested that the use of efficient negative IVC in combination with other engineering controls for allergen containment during procedures and waste processing would potentially produce a virtually allergen-free work environment. When negative pressure IVC housing is not available, the placement of cages in a HEPAfiltered, ventilated cabinet is effective at reducing room allergen loads (Thulin et al. 2002). Gordon et al. (1997) reported that individuals who have direct contact with mice (animal technicians) have the highest exposure, followed by those who have intermittent contact with
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anesthetized animals or mouse tissues (scientists and necropsy technicians), followed by those with indirect contact (office workers or histology technicians). The specific animal husbandry activities that are known to result in high exposure of personnel to mouse allergen are cage-changing activities, including animal transfer, stacking dirty cages, and manual emptying of cages; handling animals directly (particularly males); and room sweeping (Gordon et al. 1997). For each of these activities, use of improved containment equipment and changes in equipment handling procedures are effective in the controlling the allergen hazard and should be encouraged. For example, use of ventilated cabinets or biological safety cabinets during cage changing and animal handling is effective in conjunction with the use of microisolation cages (Gordon et al. 2001; Schweitzer et al. 2003; Thulin et al. 2002). Containment equipment has also been designed for the capture of airborne allergens generated when the bottom of one dirty cage is placed into the opening of another to stack the cages for transport to the cage wash area or when dirty bedding is removed prior to cage washing (Gordon et al. 1997; Kacergis et al. 1996). Room cleaning with a vacuum equipped with HEPA filtration followed by mopping with a damp mop also aids in reducing the environmental allergen load and personnel exposure (Kacergis et al. 1996). Use of personal protective equipment and dedicated work clothing for personnel involved in high-exposure activities is an important asset in reducing allergen exposure. It is important for the work clothing to remain at work, as evidenced by the finding that children of laboratory animal workers had a higher incidence of clinical signs during provocative testing, positive skin tests, and IgE specific to laboratory rodents than did the children of parents who worked in other occupations (Krakowiak et al. 1999). Full sleeve protection and gloves should be worn to prevent the urticarial reactions in persons who are highly sensitive to the Mus m 1 allergen. Personnel should also be provided with respiratory protection and eye or face protection when warranted. Either filtering facepiece particulate respirators (N95 equivalent) or powered air purified respirators are effective in reducing exposure and alleviating clinical symptoms (Schweitzer et al. 2003; Thulin et al. 2002). Special attention must be paid to the selection and fitting of N95 filtering facepiece particulate respirators to ensure proper function (Morbidity and Mortality Weekly Report 1998). When the elimination of mouse allergen exposure in the workplace is not achieved through the use of engineering controls, work practices, and personal protective equipment, allergic reactions in persons sensitive to Mus m 1 can be managed with pharmacological agents that have a long history of use for this condition. These include antihistamines, topical α-adrenergic agents (bronchodilators), cromolyn sodium as a nasal spray, and intranasal potent glucocorticoids (Austen 2004). Prophylaxis in patients with mild symptoms is often provided by topical cromolyn sodium on a continuous basis, supplemented with the intermittent use of antihistamines often at bedtime. The selection
of the antihistamine has been an area of considerable discussion, and the reader should refer to Casale et al. (2003) for further insights into this matter. In more serious cases, potent topical glucocorticoids may be necessary for alleviating clinical signs. Immunotherapy, or hyposensitization, is typically reserved for patients who are unable or unwilling to escape the allergen provoking the response. Although allergy to the dog or the cat can be ameliorated by immunotherapy (Norman 1998, 2004), the infrequent reports in the literature of immunotherapy for allergy to mice and other small laboratory animals have failed to establish the usefulness of this approach for the control of allergy to these species (Sorrell and Gottesman 1957; Wahn and Siraganian 1980).
X.
CONCLUSION
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Index A Actinobacillus muris, see Pasteurellaceae Adrenalitis, murine cytomegalovirus myocarditis model, 24 Age-related pathology, see also specific organisms amyloidosis, 632–633 hematopoietic neoplasms, 633 AKR strain development, 638 phenotype, 638 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 639 nonneoplastic conditions, 638–639 pathogen susceptibility, 639 related strains, 639 Akt, prostate cancer signaling, 599–600 Albendazole, Giardia muris management, 521 Alopecia, C3H mice, 644 Amoxicillin Clostridium perfringens infection management, 357 Helicobacter infection management, 428–429 Ampicillin Clostridium perfringens management, 357 Corynebacterium bovis management, 401 Amyloidosis A strain mice, 637 Balb/cJ mice, 641 C57BL mice, 649 definition, 672–673 features, 692–694 SJL/J mice, 654 Swiss mice, 658–659 Animal bite, management, 735 Animal husbandry, phenotypic effects, 626 Antibiotic therapy, see specific antibiotics Antibodies, see B cell Apicomplexa, general features, 528–529 Apoptosis, mouse polyoma virus antiapoptotic responses, 111–112
Arteritis features, 699 strain 129 mice, 635 Aspiculuris tetraptera diagnosis, 556 differentiation from Syphacia obvelata, 553 life cycle, 556 morphology, 555 research-confounding effects, 556 treatment, 556–559 A strain development, 636–637 phenotype, 637 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 637–638 nonneoplastic conditions, 637 pathogen susceptibility, 638 Atherosclerosis, murine cytomegalovirus myocarditis model, 24
B B6;129 mouse phenotype, 655 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 655 nonneoplastic conditions, 655 B6C3F1 mouse phenotype, 655–656 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 656–657 nonneoplastic conditions, 656 pathogen susceptibility, 657
Bacitracin Clostridium difficile infection management, 360 Clostridium perfringens infection management, 357 Balb/c phenotype, 639–640 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 641–642 nonneoplastic conditions, 640–641 pathogen susceptibility, 642–643 related strains, 643 Balb/cBy phenotype, 639–640 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 641–642 nonneoplastic conditions, 640–641 pathogen susceptibility, 642–643 related strains, 643 B cell lactate dehydrogenase-elevating virus response, 224 lymphocytic choriomeningitis virus persistence role, 200–201 mouse adenovirus type 1 response, 60 mouse hepatitis virus response, 155, 157 mousepox response, 82 murine cytomegalovirus immune response, 28–29 myocarditis role, 23 Sendai virus response, 296 Bite, see Animal bite Borrelia, epidemiology, 729
C C3H development, 643 phenotype, 643 spontaneous diseases
747
748 C3H (continued) comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 645–646 nonneoplastic conditions, 643–645 pathogen susceptibility, 646 related strains, 646–647 C57BL development, 647 phenotype, 647 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 649–650 nonneoplastic conditions, 647–649 pathogen susceptibility, 650 related strains, 650–651 C57L, phenotype, 651 C58, phenotype, 651 Cancer, see Tumor CAR bacillus, see Cilia-associated respiratory bacillus CAR, see Cocksackie-adenovirus receptor Cardioviruses, see Encephalomyocarditis virus; Theiler’s murine encephalomyelitis virus Catnb, transgenic mice, 599, 602–603 CBA, phenotype, 646–647 Chilomastix bettencourti, features, 525 Chlamydia muridarum culture, 333 developmental cycle, 331–332 genital infection course, 339–340 immune response, 341–342 pathologic response, 340–341 genome, 331 history of study, 326–328 metabolism, 332–333 morphology, 330–331 pathogenesis, 334–335 respiratory infection course, 335–336 immune response, 337–339 pathologic response, 336–337 strains, 333–334 structure, 331 taxonomy, 333 Chlamydia pneumoniae history of study, 342–343 mouse infection studies, 330, 343–344 taxonomy, 333 Chlamydia psittaci, mouse infection studies, 330, 344 Chlamydia trachomatis history of study, 327–328 mouse infection studies, 329–330, 339–342 taxonomy, 333
INDEX
Cilia-associated respiratory bacillus clinical features, 455–456 control and prevention, 459 culture, 455 diagnosis culture, 458 electron microscopy, 459 enzyme-linked immunosorbent assay, 458 histopathology, 458–459 immunohistochemistry, 459 polymerase chain reaction, 459 geographic distribution, 457 history of study, 454–455 host range, 457–458 pathogenesis, 456–457 pathology, 456 prevalence of infection, 458 properties, 455 strains, 455 transmission, 458 treatment, 459 Citrobacter rodentium classification, 373 clinical features, 375–376 diagnosis, 376 epizootiology, 376 history of study, 373–374 pathogenesis, 374–375 properties, 374 treatment and control, 377 Clindamycin Clostridium difficile infection management, 359 Clostridium perfringens infection management, 357 Clostridium difficile clinical features, 358–359 control and prevention, 360 culture, 358 diagnosis, 359 epizootology, 359 history of study, 357–358 pathogenesis, 359 properties, 358 strains and antigenic relationships, 358 treatment, 359–360 Clostridium perfringens clinical features, 356 control and prevention, 357 culture, 356 diagnosis, 357 epizootology, 357 history of study, 355 pathogenesis, 356–357 properties, 356 strains and antigenic relationships, 356 treatment, 357 Clostridium piliforme clinical features, 351–353 control and prevention, 355 culture, 351 diagnosis, 353–355 epizootology, 353 history of study, 350 pathogenesis, 353
properties, 350 strains and antigenic relationships, 350 treatment, 355 Coat color, genetics and nomenclature, 628–630 Cocksackie-adenovirus receptor, human function and murine homolog, 54 Comparative tumor biology, see Tumor pathology, genetically engineered mice Congenic strain, definition, 628 Conjunctivitis, Pasteurellaceae, 484–485 Corneal dystrophy, Balb/c mice, 640 Corynebacterium bovis clinical features, 400 culture, 399 diagnosis, 401 epizootiology, 400–401 properties, 399 strains, 399 treatment and control, 401–402 Corynebacterium kutscheri clinical features, 402–403 culture, 402 diagnosis, 403–404 epizootiology, 403 properties, 402 strains, 402 treatment and control, 404 Cryptosporidium muris cell biology, 539 clinical features, 539 diagnosis, 539 history of study, 538 life cycle, 538–539 prevention, 539–540 research implications, 540 treatment and control, 539–540 Cytomegalovirus, see Murine cytomegalovirus
D DBA development, 651 phenotype, 651 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 651–652 nonneoplastic conditions, 651–652 pathogen susceptibility, 651–653 DC, see Dendritic cell Demodex musculi clinical features, 568 diagnosis, 568 host range, 568 life cycle, 568 morphology, 568 pathobiology, 568 prevention and control, 568 treatment, 568
749
INDEX
Dendritic cell lymphocytic choriomeningitis virus persistence role, 199 murine cytomegalovirus immune response, 27 Dermatophytosis fungal diagnosis, 511 epidemiology, 510–511 history of study, 510 pathology, 511 taxonomy, 510 treatment and control, 511 ringworm in humans clinical signs, 731 reservoir and incidence, 730 transmission, 730
E Early transposon-related elements features, 276 insertional mutagenesis, 272 Ectromelia virus, see Mousepox EDIM, see Rotavirus Eimeria cell biology, 531 clinical features, 531 diagnosis, 531 life cycle, 529, 531 prevention, 531 research implications, 531–532 taxonomy, 529 treatment and control, 531 Electron microscopy cilia-associated respiratory bacillus diagnostics, 459 Mycoplasma pulmonis diagnostics, 451 ELISA, see Enzyme-linked immunosorbent assay EMCV, see Encephalomyocarditis virus Encephalitozoon cuniculi cell biology, 541 clinical features, 541–541 diagnosis, 542 life cycle, 541 prevention, 542–543 research implications, 543 taxonomy, 540–541 treatment and control, 542–543 Encephalomyocarditis virus antigenic properties, 315 biophysical properties, 312 clinical features, 315–316 control and prevention, 320 diagnosis, 319–320 epizootiology, 318–319 genome, 312–313 history of study, 311–312 propagation, 315 receptors, 314–315 structure, 313–314 Enrofloxacin Citrobacter rodentium management, 377 Corynebacterium bovis management, 401
Entamoeba muris cell biology, 527 clinical features, 527–528 diagnosis, 528 life cycle, 527 morphology, 527 prevention, 528 research implications, 528 taxonomy, 527 treatment and control, 528 Enterbacteriaceae, see also specific organisms culture media, 373 genera, 366–367 growth characteristics, 367 virulence factors and pathogenicity islands, 367–369 Enzyme-linked immunosorbent assay cardiovirus diagnostics, 319 cilia-associated respiratory bacillus diagnostics, 458 Clostridium piliforme diagnostics, 354 Corynebacterium kutscheri diagnostics, 404 Helicobacter diagnostics, 427–428 lactate dehydrogenase-elevating virus diagnostics, 227–228 lymphocytic choriomeningitis virus diagnostics, 203–204, 722 mammalian reovirus diagnostics, 257 minute virus of mice diagnostics, 100 mouse adenovirus diagnostics, 61 mouse parvovirus diagnostics, 100 mouse thymic virus diagnostics, 34 mousepox virus diagnostics, 87, 160–161 murine cytomegalovirus diagnostics, 32 Mycoplasma pulmonis diagnostics, 450 Pasteurellaceae diagnostics, 495–496 pneumonia virus of mice diagnostics, 304 rotavirus diagnostics, 245 Sendai virus diagnostics, 297–298 Erbb2, transgenic mice, 598, 599–602 Escherichia coli clinical features, 377 diagnosis, 377 properties, 377
F F1 hybrid, definition, 628 Fenbendazole, pinworm management, 558–559 Fipronil, Polyplax serrata management, 568 Flea human interaction, 735 rodent species, 735 Fungal infection, see also Dermatophytosis; Pneumocystis murina animal models, 513–514 systemic and opportunistic infection diagnosis, 513 epidemiology, 511–512 history of study, 511 pathology, 512–513 taxonomy, 511 treatment and control, 513
FVB/N development, 653 phenotype, 653 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 653 nonneoplastic conditions, 653 pathogen susceptibility, 654
G Gastric cancer, Helicobacter models, 425–426 Gastritis, Helicobacter models, 424–425 Genetically engineered mice, see Tumor pathology, genetically engineered mice Giardia muris cell biology, 520–521 clinical features, 521 diagnosis, 521 life cycle, 520 morphology, 520 prevention, 521 research implications, 521, 523 taxonomy, 520 treatment and control, 521 Grey lung agent, features and infection, 454 Griseofulvin, dermatophytosis management, 511
H H antigen, serotyping, 367–369 Haemophilus influenzaemurium, see Pasteurellaceae Helicobacter classification, 409–410 colony management in prevention, 429–430 diagnosis culture, 427 enzyme-linked immunosorbent assay, 427–428 histology, 428 polymerase chain reaction, 427 epizootiology, 426–427 genomic diversity analysis, 428 Helicobacter bilis, 417–418 Helicobacter felis, 424 Helicobacter ganmani, 419 Helicobacter hepaticus history of study, 426 oxidative stress and cytotoxicity biomarkers, 416–417 pathogenesis, 410, 412 susceptibility of host, 412–415 tumorigenesis mechanisms, 415–416 Helicobacter mastomyrinus, 420–421 Helicobacter muricola, 420 Helicobacter muridarum, 419–420 Helicobacter pylori, 408, 424–425
750
INDEX
Helicobacter (continued) Helicobacter rappini, 421 Helicobacter rodentium, 419 Helicobacter trogontum, 424 Helicobacter typhlonius, 419 history of study, 409–410 host range and tissue tropism, 409 human disease, 408 human infection, 729 mouse models gastric cancer, 425–426 gastritis, 424–425 hepatitis and hepatic cancer, 421–422 inflammatory bowel disease, 422–424 overview, 410 phylogenetic relationships, 408 properties of mouse isolates, 410–411 research-confounding effects, 426 sentinel mice in monitoring, 430 treatment, 428 Heligmosomoides polygyrus, features and management, 559 Helminths, see also specific organisms cestodes, 559–561 human infection, 732–733 nematodes, 559 oxyurids, 552–556 Hemolytic uremic syndrome, mouse model development, 366 Hepatitis Helicobacter hepaticus model, 422 lymphocytic choriomeningitis virus model, 194–196 murine cytomegalovirus myocarditis model, 21–22 Hepatitis virus, see Mouse hepatitis virus Hermaphroditism, chimeric mice, 634 Herpesviruses, see Mouse thymic virus; Murine cytomegalovirus Humoral immunity, see B cell HUS, see Hemolytic uremic syndrome Hydrocephalus, C57BL mice, 647 Hymenolepis diminuta features and management, 560–561 human infection, 732
I IAP, see Intracisternal A particles IBD, see Inflammatory bowel disease IFA, see Immunofluorescence assay IL-1, see Interleukin-1 IL-6, see Interleukin-6 Immunofluorescence assay Clostridium piliforme diagnostics, 354 lymphocytic choriomeningitis virus diagnostics, 203–204, 722 mouse thymic virus diagnostics, 34 mousepox virus diagnostics, 160–161 Inbred strain, see also specific strains attributes, 661 definition, 628 Incipient inbred, definition, 628 Infectious ectromelia, see Mousepox
Inflammatory bowel disease, Helicobacter models, 422–424 Interferons lactate dehydrogenase-elevating virus response, 222–223 lymphocytic choriomeningitis virus persistence role, 201–202 mammalian reovirus response, 257 mouse adenovirus type 1 response, 59 mouse hepatitis virus response, 155 murine cytomegalovirus response, 27–28 Sendai virus response, 292–295 Interleukin-1, murine cytomegalovirus response, 28 Interleukin-6, murine cytomegalovirus response, 28 Interstitial pneumonitis, murine cytomegalovirus myocarditis model, 20–21 Intracisternal A particles classification, 276 insertional mutagenesis, 272 Itraconazole, dermatophytosis management, 511 Ivermectin pinworm management, 557–558 Heligmosomoides polygyrus management, 559 Polyplax serrata management, 568 Myobia musculi management, 571–572 Myocoptes musculinus management, 574
K Klebsiella history of study, 379–380 properties, 380 epizootiology, 380 clinical features, 380 diagnosis, 380 Klossiella muris history of study, 533 life cycle, 533 cell biology, 533 clinical features, 533 diagnosis, 533–534 prevention, 534 treatment and control, 534 research implications, 534
L LAA, see Laboratory animal-associated allergy Laboratory animal-associated allergy allergens, 736 clinical signs, 738–739 diagnosis, 737–738 incidence, 735–737 pathogenesis, 737 treatment and prevention, 738–739 Lactate dehydrogenase-elevating virus classification, 216 control and prevention, 228 diagnosis
enzyme-linked immunosorbent assay, 227–228 lactate dehydrogenase activity, 227 polymerase chain reaction, 227 history of study, 216 host range, 226 immune response autoimmunity, 225 B cells, 224 cytokines, 222–223 immunosuppression, 224–225 macrophages, 223 natural killer cells, 223 T cells, 223–224 lactate dehydrogenase clearance impairment, 221–222 morphology, 216 pathology, 222 persistence, 222 physicochemical properties and structure, 216–217 polioencephalomyelitis, 225–226 receptor, 221 replication cell culture studies, 220–221 kinetics, 220 RNA synthesis, 218 sites, 219–220 stability, 218–219 strains, 219 transmission, 226–227 tumor studies, 226 LCMV, see Lymphocytic choriomeningitis virus LDV, see Lactate dehydrogenase-elevating virus Leptospirosis clinical signs, 726 control, 726–727 diagnosis, 726 host range, 725 humans, 726 reservoir and incidence, 726 serovars, 725–726 transmission, 726 Lice, see Polyplax serrata Lipopolysaccharide, virulence factors, 367–369 LPS, see Lipopolysaccharide Lymphocytic choriomeningitis virus autoimmune disease susceptibility, 203 behavioral effects, 203 classification, 180–181 contamination of biological material, 202 control and prevention, 204 diagnosis molecular detection, 204 serology, 203–204 disease syndromes in laboratory mice autoimmune disease in transgenic mice, 197 hematopoietic disorders, 196–197 hepatitis, 194–196 immunopathogenesis, 187–188
751
INDEX
lymphocytic choriomeningitis, 192–194 prenatal and neonatal infection endocrine disorders, 191 immune complex disease, 189–191 wasting disease, 194 history of study, 180 host range, 186 human infection clinical signs, 722 diagnosis, 722 overview, 186–187, 720–721 reservoir and incidence, 721 susceptibility, 722 transmission mode, 721–722 treatment, 722 immunosuppression, 202–203 major histocompatibility complex and genetic susceptibility, 197–198 natural history, 184–185 persistence roles B cells, 200–201 dendritic cells, 199 interferons, 201–202 natural killer cells, 201 overview, 198–199 T cells CD4+ cells, 200 CD8+ cells, 199–200 propagation cells, 183 mouse bioassays, 184 replication, 181–182 safety, 204–205 strains antigenic and genetic relationships, 182 biologic differences, 183 transmission horizontal, 185 vertical, 185–186 virion structure, 181
M Macrophage lactate dehydrogenase-elevating virus response, 223 murine cytomegalovirus immune response, 27 pneumonia, see Pneumonia Malocclusion features, 691 C57BL mice, 648 MaLR, see Mammalian apparent LTR-retrotransposons Mammalian apparent LTR-retrotransposons features, 276 insertional mutagenesis, 272 Mammalian reovirus cardiorespiratory system infection, 254 cell culture growth studies, 251–252 central nervous system infection, 253–254 control and prevention, 258
diagnosis, 257–258 endocrine system effects, 255–256 genome, 247 hepatobiliary system infection, 254–255 history of study, 245–246 host entry, 252–253 host range, 257 human infection, 724 immune response, 256–257 stability, 249 strains mutants, 250–251 reassortants, 250 serotypes, 249–250 structure, 247–249 transmission, 257 vaccination, 248 MAP, see Mouse antibody production Mastitis, Pasteurellaceae, 485 MAV-1, see Mouse adenovirus type 1 MAV-2, see Mouse adenovirus type 2 MCMV, see Murine cytomegalovirus Mebendazole, Trichuris muris management, 559 Megaesophagus features, 691 strain 129 mice, 635 Metastasis, mouse tumor models, 611–612 Metronidazole Clostridium perfringens management, 357 Giardia muris management, 521 MHV, see Mouse hepatitis virus Minute virus of mice clinical signs of infection, 96 control and prevention, 101 diagnosis, 100 epizootiology, 96–97 genome features, 94–95 history of study, 93–94 pathology and pathogenesis, 98–100 physicochemical properties, 95–96 replication, 95 research applications, 101 Mites, see Demodex musculi; Myobia musculi; Myocoptes musculinus; Ornithonyssus bacoti; Psorergates simplex; Radfordia; Trichoecius romboutsi MMTV, see Mouse mammary tumor virus MoPn, see Chlamydia muridarum Mouse adenovirus type 1 control and prevention, 61–62 diagnosis, 61 genetic susceptibility, 61 genome E1, 51–52 E3, 52–53 E4, 53 major late promoter, 53 structure, 51 history of study, 50–51 host range and prevalence, 61 immune response
cell-mediated immunity, 59 humoral immunity, 60 innate immunity, 58–59 model, 60 infection in mouse age effects, 56 cell tropism, 57–58 E1A mutants, 58 E3 mutants, 58 inoculation route effects, 57 persistence, 56 infection in vitro E1A mutants, 55 E3 mutants, 55–56 kinetics of replication, 53–54 receptor, 54 physical properties, 51 Mouse adenovirus type 2 history of study, 50–51 infection in mouse, 60–61 physical properties, 51 Mouse antibody production mouse hepatitis virus diagnostics, 161 mousepox virus prevention, 89 Sendai virus prevention, 298 Mouse hepatitis virus cell interactions, 148–149 classification, 146 control, 169–170 diagnosis enzyme-linked immunosorbent assay, 160–161 immunofluorescence assay, 160–161 immunohistochemistry, 167 mouse antibody production, 161 pathology, 162–165, 167 polymerase chain reaction, 161–162 serology, 161 duration of infection, 144–145 genome mutation and recombination, 148 history of study, 142–143 host range, 145–146, 149 human infection, 723 immune response B cells, 155, 157 experimental brain disease, 155–156 immunomodulation, 157–158 interferons, 155 passively acquired maternal immunity, 156–157 reinfection immunity, 157 T cells, 154–155, 157 vaccination immunity, 157 isolates, 142–143 isolation and propagation, 158–160 natural history, 143–144 pathogenesis enterotropic infection, 150 experimental encephalitis and demyelination, 151–154 experimental hepatitis, 151 respiratory infection, 150 replication, 148 research-confounding effects, 625
752 Mouse hepatitis virus (continued) surveillance, 168–169 transmission, 145 virion structure, 146–148 Mouse mammary tumor virus host range, 271 loci, 275 receptors, 271 research-confounding effects, 625 tumor pathogenesis, 595 Mouse minute virus, see Minute virus of mice Mouse parvovirus clinical signs of infection, 96 control and prevention, 101 diagnosis, 100 epizootiology, 96–97 genome features, 94–95 history of study, 94 pathology and pathogenesis, 97–98 physicochemical properties, 95–96 replication, 95 research applications, 101 Mouse Phenome Project, overview, 627 Mouse polyoma virus cell interactions in culture cell transformation, 108 productive and nonproductive infections, 107–108 history of study, 106–107 natural history, 108–109 prospects for study, 129–130 receptors and uptake, 122–123 regulatory sequences, 121–122 structure, 113 susceptibility genetics in inbred strains, 123–126 transgenic mouse studies JC virus T antigen, 127 polyoma T antigens, 127–128 polyoma virus regulatory sequences in transgene expression, 128 SV40 large T antigen, 126–127 tumor antigens genetic interactions, 119–120 molecular interactions, 118–119 pathogenicity determinants, 120–121 structures and functions, 116–118 tumor induction cancer modeling anti-apoptotic responses, 111–112 early progression, 110–111 genomic instability and progression, 111 immune response, 112 initiation, 110 invasion and metastasis, 112–113 profile, 109 sites, 107 tissue interactions, 109–110 VP1 pathogenicity determinants, 113–116 recombinant proteins, 113 self-assembly, 113
INDEX
VP2, 116 VP3, 116 Mousepox clinical disease age effects, 73 patterns, 73 sexual dimorphism, 73 control depopulation and disinfection, 87 rederivation of mouse strains, 88 serological screening, 88 vaccination, 87–88 diagnosis clinical signs, 86–87 enzyme-linked immunosorbent assay, 87 pathology, 87 polymerase chain reaction, 87 serology, 87 virus isolation, 87 ectromelia virus species distribution, 69 enzootic mousepox, 86 epidemiology, 84–85 epizootic mousepox, 85–86 history of study, 68–69 host range species, 83–84 strain susceptibility, 84 immune response adaptive immunity, 81–82 innate immunity, 80–81 resistance genetics, 82–83 pathogenesis footpad infection and viral spread, 75–76 inoculation routes and mechanisms arthropod vectors, 74 feeding, 74 intracerebral inoculation, 75 intradermal inoculation and scarification, 73–74 intranasal inoculation, 74–75 intraperitoneal inoculation, 74 intrauterine infection, 75 lower respiratory tract inoculation, 75 pathology intestine, 79–80 liver, 78 lymphoid tissue, 79 resistant mouse strains, 80 skin, 77–78 spleen, 78–79 prevention mouse antibody production, 89 quarantine, 88–89 sentinel surveillance, 88 propagation chick embryo, 72 replication cycle, 71–72 tissue culture, 72–73 strains Hampstead strain, 70–71 Ishibashi strain, 71 Moscow strain, 71 NAV strain, 71 NIH-79 strain, 71
taxonomy, 68–69 virion properties composition, 70 morphology, 69–70 stability, 70 structure, 70 Mouse thymic virus diagnosis, 34–35 history of study, 33 pathogenesis, 34 properties, 33–34 Mouse urinary syndrome, AKR strain, 639 MptV, see Murine pneumotropic virus MPV, see Mouse parvovirus MTV, see Mouse thymic virus MuLV, see Murine leukemia virus Murine cytomegalovirus classification, 3–4 control, 32 diagnosis enzyme-linked immunosorbent assay, 32 polymerase chain reaction, 32 serology, 32 history of study, 2 host range, 12–13 human cytomegalovirus similarities adrenalitis, 24 atherosclerosis, 24 central nervous system infection, 24–25 clinical significance, 2–3, 17 genome, 4–5 hemopoiesis studies, 26 hepatitis, 21–22 interstitial pneumonitis, 20–21 intrauterine infection and congenital disease embryonic development effects, 19 epidemiology, 18 gonadal tissue infection, 19–20 hearing loss, 25–26 reproduction effects, 20 myocarditis, 22–24 retinitis, 25 immune response B cell response, 28–29 cytokines, 27 dendritic cell, 27 evasion, 30–32 immunosuppression induction, 26–27 macrophage, 27 natural killer cells, 28 T cell CD4+, 30 CD8+, 29–30 laboratory mouse infection, 11–12 life cycle morphogenesis, 6–7 replication, 5–6 viral entry, 5 natural history, 11 pathogenesis age effects, 13 dose effects, 13
753
INDEX
inoculation route effects, 13 latency and reactivation, 16–17 resistance mechanisms acute infection, 14–15 cell culture studies, 15–16 chronic resistance, 15 propagation in mice, 10–11 propagation in vitro cell growth cycle effects, 9–10 centrifugal enhancement, 9 kinetics of replication, 9 multicapsid virion production, 10 non-murine cells, 9 nonpermissive murine cells, 9 permissive murine cells, 8–9 species distribution of cytomegalovirus, 3–4 strains, 7–8 transmission mode, 12 vaccines, 32–33 virion structure, 4–5 Murine leukemia virus common integration site, 274 Fv1 in susceptibility, 273–274 host range, 271 inbred mouse strain distribution, 271–272 insertional mutagenesis, 272–273 receptors, 271 tumor pathogenesis, 595 Murine pneumotropic virus features, 128 propagation, 128–129 prospects for study, 129–130 tumor antigens, 129 Murine typhus features, 725 human risks, 725 MVM, see Minute virus of mice Myc, transgenic mice, 597, 599, 602–603 Mycoplasma arthritides, features and infection, 453–454 Mycoplasma collis, features and infection, 454 Mycoplasma muris, features and infection, 454 Mycoplasma neurolyticum, features and infection, 454 Mycoplasma pulmonis clinical signs, 441 culture, 440–441 diagnosis culture, 450–451 electron microscopy, 451 enzyme-linked immunosorbent assay, 450 general considerations, 449–450 histopathology, 451 immunohistochemistry, 451 polymerase chain reaction, 452 geographic distribution, 448 history of study, 438–439 host range, 448 pathogenesis disease expression factors, 444–445 host injury, 445–446 immmune response, 446–448 pathology
genital disease, 443 polyarthritis, 443–444 respiratory disease, 441–443 prevalence of infection, 448–449 properties, 439–440 strains, 440 transmission, 449 Myobia musculi clinical features, 571 diagnosis, 571 host range, 571 life cycle, 569, 571 morphology, 569 pathobiology, 571 prevention and control, 572 treatment, 571–572 Myocarditis, murine cytomegalovirus myocarditis model, 22–24 Myocoptes musculinus clinical features, 573 diagnosis, 573 host range, 573 life cycle, 573 morphology, 572–573 pathobiology, 573 prevention and control, 573–574 treatment, 573
N Natural killer cell lactate dehydrogenase-elevating virus response, 223 lymphocytic choriomeningitis virus persistence role, 201 murine cytomegalovirus immune response, 28 murine cytomegalovirus myocarditis role, 23 Sendai virus response, 295 Neomycin, Citrobacter rodentium infection management, 377 Neu, transgenic mice, 597 Niclosamide, Rodentolepis nana management in humans, 732
O O antigen, serotyping, 367–369 Octomitus pulcher, features, 525 Ornithonyssus bacoti features, 733 host range, 733 human infestation, 733–735 Osteoporosis, C57BL mice, 648 Otitis, Pasteurellaceae, 485 Outbred stock definition, 628 sources and origins, 662–663 Oxantel, Trichuris muris management, 559
P p53, mutant transgenic mice, 601 PAI, see Pathogenicity island Parvoviruses, see Minute virus of mice; Mouse parvovirus
Pasteurellaceae classification, 471, 480 clinical disease co-pathogens and opportunism, 483 latency, 484 primary pathogenicity, 482–483 target tissues conjunctivitis, 484–485 mastitis, 485 otitis, 485 overview, 481 respiratory tract, 484 urogenital tract, 485–486 culture, 480–481 diagnosis culture chemotaxonomic criteria, 494–495 growth conditions, 492–493 organs, 493 phenotypic identification, 493–494 enzyme-linked immunosorbent assay, 495–496 polymerase chain reaction, 495 environmental sensitivity, 474–475 eradication in control, 497 geographic distribution, 491 history of study Actinobacillus, 472–473 Haemophilus, 473–474 overview, 470–471 Pasteurella, 471–472 host range, 488–489 human infection potential, 489–491 morbidity and mortality, 481–482 pathogenesis, 482 pathology, 486–487 phenotypic characteristics Actinobacillus muris, 477 growth factor-dependent bacteria, 479–480 Haemophilus influenzaemurium, 477–479 overview, 474–475 Pasteurella pneumotropica, 475–477 prevalence of infection, 491 prevention, 497–499 research-confounding effects, 487–488 storage, 475 transmission, 491–492 treatment, 496 Pathogenicity island, features, 369 Pathology cilia-associated respiratory bacillus, 456 control and prevention, 453 lactate dehydrogenase-elevating virus, 222 minute virus of mice, 98–100 mouse hepatitis virus, 162–165, 167 mouse parvovirus, 97–98 mousepox, 77–80 Mycoplasma pulmonis genital disease, 443 polyarthritis, 443–444 respiratory disease, 441–443 Pasteurellaceae, 486–487
754 Pathology (continued) Pneumocystis murina, 509 pneumonia virus of mice, 301–302 Sendai virus, 290–292 treatment, 452–453 tumors, see Tumor pathology, genetically engineered mice PCR, see Polymerase chain reaction Permethrin Myobia musculi management, 572 Polyplax serrata management, 568 PFGE, see Pulsed-field gel electrophoresis Pinworm, see Aspiculuris tetraptera; Syphacia obvelata Pneumocystis murina animal models of infection, 513 diagnosis, 509 epidemiology, 508–509 history of study, 508 morphology, 508 pathology, 509 taxonomy, 508 treatment and control, 509–510 Pneumonia, acidophilic macrophage pneumonia C57BL mice, 649 features, 694 strain 129 mice, 634–635 Pneumonia virus of mice age effects, 301 classification, 298 clinical features, 300 control and prevention, 304 diagnosis, 303–304 genome, 299 history of study, 298 host range, 302 immune response, 302 pathology gross changes, 301 microscopic changes, 302 propagation cell culture, 300 eggs, 300 mice, 300 sex differences, 301 strains, 299 structure, 299 susceptibility of inbred strains, 301 target sites, 300–301 transmission, 303 Pneumotropic virus, see Murine pneumotropic virus Polymerase chain reaction cardiovirus diagnostics, 320 cilia-associated respiratory bacillus diagnostics, 459 Clostridium piliforme diagnostics, 354 Corynebacterium bovis diagnostics, 401 Helicobacter diagnostics, 427 lactate dehydrogenase-elevating virus diagnostics, 227 lymphocytic choriomeningitis virus diagnostics, 204
INDEX
mammalian reovirus diagnostics, 258 minute virus of mice diagnostics, 100 mouse parvovirus diagnostics, 100 mousepox diagnostics, 87 mousepox virus diagnostics, 161–162 murine cytomegalovirus diagnostics, 32 Mycoplasma pulmonis diagnostics, 452 Pasteurellaceae diagnostics, 495 Sendai virus diagnostics, 297 Polyoma virus, see Mouse polyoma virus; Murine pneumotropic virus Polyplax serrata clinical features, 567 diagnosis, 567–568 host range, 567 life cycle, 566 morphology, 566 pathobiology, 567 prevention and control, 568 treatment, 568 Poxviruses, see Mousepox Praziquantfel, Rodentolepis nana management in humans, 732 Proteus mirabilis classification, 378 clinical features, 378 diagnosis, 379 epizootiology, 378–379 history of study, 377–378 pathogenesis, 378 properties, 378 treatment and control, 379 Protozoa, see also specific organisms comparison of murine pathogens, 519 taxonomy, 518–519 Pseudomonas aeruginosa clinical features, 382 control, 382–383 diagnosis, 382 epizootiology, 382 history of study, 380–381 pathogenesis, 381–382 properties, 381 Psorergates simplex clinical features, 575 diagnosis, 575 host range, 574 life cycle, 574 morphology, 574 pathobiology, 575 prevention and control, 575 treatment, 575 PTEN knockout mouse, 599603 prostate cancer signaling, 599–601 Pulsed-field gel electrophoresis, Helicobacter genomic diversity analysis, 428 PVM, see Pneumonia virus of mice Pyrimethamine Sarcocystis muris management, 533 Toxoplasma gondii management, 536
R Rabies clinical signs, 723 diagnosis, treatment and control, 723 reservoir and incidence, 722 Radfordia clinical features, 575 diagnosis, 575 host range, 575 life cycle, 575 morphology, 575 pathobiology, 575 prevention and control, 575–576 treatment, 575–576 Radioallergosorbent test, laboratory animal-associated allergy diagnosis, 738 Ras, transgenic mice, 597, 599–600, 603 RAST, see Radioallergosorbent test Rat bite fever clinical signs, 727–728 diagnosis, 728 pathogens, 727 reservoir and incidence, 727 transmission, 727 Recombinant inbred strain, definition, 628 Related inbred strain, definition, 628 Reoviridae, see Mammalian reovirus; Rotavirus Retinitis, murine cytomegalovirus myocarditis model, 25 Retroelement, features, 269–270 Retrotransposon insertional mutagenesis, 272 types, 270 Retrovirus, see specific viruses Rickettsialpox clinical signs, 724–725 control and prevention, 725 geographic distribution, 724 Ringworm, see Dermatophytosis Rodentolepis microstoma, features and management, 561 Rodentolepis nana features and management, 559–560 human infection, 732 Rotavirus age effects, 243–244 cell culture growth studies, 240–241 control and prevention, 245 diagnosis, 246 diarrhea mechanisms, 242 genome, 238 history of study, 236–237 host range, 244–245 immune response, 242–243 pathogenesis, 241–242 stability, 238–239 strains electropherotypes, 239 groups, 239 murine strains, 240 serotypes, 239–240 structure, 237–238
755
INDEX
S Salmonella classification, 369–370 clinical features humans, 729 mice, 371–372 diagnosis, 372–373 epizootiology, 371 history of study, 370 pathogenesis, 371 properties, 370–371 reservoir and incidence, 728–729 Sarcocystis muris cell biology, 532 clinical features, 532–533 diagnosis, 533 history of study, 532 life cycle, 532 prevention, 533 research implications, 533 treatment and control, 533 Selamectin, pinworm management, 558 Sendai virus age effects, 288 classification, 282 clinical features, 287 control and prevention, 298 diagnosis, 297–298 genome, 282–283 geographic distribution, 296 history of study, 282 host range, 296 human infection, 724 immune response adaptive immunity, 295–296 innate immunity, 292–295 pathology gross changes, 290 microscopic changes, 290–292 propagation cell culture, 286–287 eggs, 286 mice, 285–286 receptors, 283 sex differences, 288 strains, 284–285 structure, 283–284 susceptibility of inbred strains, 288–290 target sites, 287–288 transmission, 297 Senescence-accelerated mice, features, 639 Sexual dimorphism adrenal gland, 631 definition, 630 kidney, 631 parotid gland, 632 pathology, see specific organisms submandibular salivary gland, 631–632 SJL/J development, 654 phenotype, 654 spontaneous diseases comparison between strains and stocks, 663–672
glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 655 nonneoplastic conditions, 654 pathogen susceptibility, 654–655 Spindle cell tumor, features, 596 Spironucleus muris cell biology, 524 clinical features, 524 diagnosis, 524 life cycle, 523 morphology, 523 prevention, 524 research implications, 524 taxonomy, 523 treatment and control, 524 Spontaneous disease, see specific strains Staphylococcus aureus, human infection, 729–730 Staphylococcus clinical features, 391–394 culture, 390 diagnosis, 394–395 epizootiology, 394 properties, 390 strains, 390–391 treatment and control, 382–383 Strain 129 historical perspective, 633 nomenclature, 633–634 spontaneous diseases comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 636 nonneoplastic conditions, 634–636 related strains, 636 Streptobacillus moniliformis classification, 383 clinical features, 384 control, 384 diagnosis, 384 epizootiology, 384 history of study, 383 properties, 383 Streptococcus clinical features, 396–398 control and prevention, 399 culture, 396 diagnosis, 398–399 epizootiology, 398 properties, 395–396 strains, 396 Substrain, definition, 628 Sulfaquinoxaline, Sarcocystis muris management, 533 SV40, transgenic mice, 126–127, 597–599 Swiss mice development, 657 phenotype, 657–658 sources and origins, 662–663 spontaneous diseases
comparison between strains and stocks, 663–672 glossaries neoplasms, 682–690 nonneoplastic conditions, 672–681 neoplasia, 659–660 nonneoplastic conditions, 658–659 related strains, 660 Syphacia muris, features and management, 559 Syphacia obvelata diagnosis, 555 differentiation from Aspiculuris tetraptera, 553 human infection, 732–733 life cycle, 554 morphology, 553–554 research-confounding effects, 556 treatment, 556–559
T Taenia taeniaeformis, features and management, 561 T cell lactate dehydrogenase-elevating virus response, 223–224 lymphocytic choriomeningitis virus persistence role CD4+ cells, 200 CD8+ cells, 199–200 lymphocytic choriomeningitis virus response, 187–188 mammalian reovirus response, 256 mouse adenovirus type 1 response, 59 mouse hepatitis virus response, 154–155, 157 mouse polyoma virus response, 112 mousepox response, 81–82 murine cytomegalovirus immune response CD4+ cells, 30 CD8+ cells, 29–30 myocarditis role, 23 Sendai virus response, 295–296 Tetracycline Citrobacter rodentium infection management, 377 Clostridium piliforme infection management, 355 Corynebacterium bovis management, 401 Mycoplasma pulmonis infection management, 452 Theiler’s murine encephalomyelitis virus antigenic properties, 315 biophysical properties, 312 clinical course intracerebral inoculation, 316–318 oral inoculation, 316 control and prevention, 320 diagnosis, 319–320 epizootiology, 318–319 genome, 312–313 history of study, 311–312 immune response, 318
756 Theiler’s murine encephalomyelitis virus (continued) persistence, 317–318 propagation, 315 receptors, 314–315 structure, 313–314 TLRs, see Toll-like receptors TMEV, see Theiler’s murine encephalomyelitis virus Toll-like receptors mutations, 625 Salmonella susceptibility role, 371 Toltrazuril, Eimeria management, 531 Toxoplasma gondii cell biology, 535–536 clinical features, 536 diagnosis, 533 history of study, 534–535 life cycle, 535 prevention, 536 research implications, 536, 538 treatment and control, 536 Trichoecius romboutsi clinical features, 576–577 diagnosis, 577 host range, 576 life cycle, 576 morphology, 576 pathobiology, 576 prevention and control, 577 treatment, 577 Trichomonas muris cell biology, 525 clinical features, 525 diagnosis, 525 life cycle, 524–525 morphology, 524–525 prevention, 525 research implications, 525 taxonomy, 524 treatment and control, 525 Trichuris muris, features and management, 559 Trimethoprim, Pneumocystis murina management, 509–510 Trypanosoma musculi cell biology, 526 clinical features, 526–527 diagnosis, 527 life cycle, 526
INDEX
morphology, 526 prevention, 527 taxonomy, 525–526 treatment and control, 527 Tumor antigens, see Mouse polyoma virus Helicobacter hepaticus mechanisms, 415–416, 422 spindle cell tumor features, 596 susceptibility of mouse strains A strain, 637–638 AKR strain, 639 Balb/c, 641–642 C3H, 645–646 C57BL, 649–650 DBA, 651–652 FVB/N, 653 glossary of neoplasms, 682–689 SJL/J, 654 strain 129, 636 Swiss mice, 659–660 Tumor pathology, genetically engineered mice comparative human pathology accuracy, 582, 584, 612 digital imaging, 614 morphometrics, 615 prospects, 616–617 reporting of results, 613–614 spontaneous tumor surveillance, 615–616 validation, 612–613 experimental design, 587, 592, 594 gene targets, 591 historical perspective, 584 Internet resources, 591–592 nomenclature conventions, 594–595 oncogenic event considerations molecular alterations and microscopic structure, 601–603 spontaneous and carcinogen-induced tumors, 595–596 uniqueness of genetically engineered mouse tumors, 596–601 progression metastasis versus microinvasion, 611–612 sequential microscopic changes, 605–606, 608–611
promoters for tissue-specific expression, 592, 601–602 signature phenotypes, 586 tissue context effects microscopic structure, 604–605 strain effects, 604–605 tumor biology, 603–604 weak oncogenes, 603 Tylosin Clostridium perfringens infection management, 357 Mycoplasma pulmonis infection management, 452 Tyzzer’s disease, see Clostridium piliforme
U Ulcerative dermatitis, C57BL mice, 648–649
V Vaccination mousepox, 87–88, 157 murine cytomegalovirus, 32–33 Mycoplasma pulmonis, 453 Vancomycin, Clostridium difficile infection management, 360 VL30 elements, features, 276
W Wasting disease, lymphocytic choriomeningitis virus model, 194 Wnt, transgenic mice, 598–599, 603–604
Z Zoonoses, see also specific diseases allergy, see Laboratory animal-associated allergy arthropod infestation, 733–735 bacterial disease, 725–730 bites, 735 definition, 720 dermatophytosis, 730–731 helminth disease, 732–744 rickettsial disease, 724–725 viruses, 720–724
