Giardia and Cryptosporidium from Molecules to Disease

GIARDIA AND CRYPTOSPORIDIUM From Molecules to Disease Acknowledgement We are most grateful to Arturo Pérez-Taylor for...

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GIARDIA AND CRYPTOSPORIDIUM From Molecules to Disease

Acknowledgement We are most grateful to Arturo Pérez-Taylor for his excellent informatics assistance during the preparation of this book.

GIARDIA AND CRYPTOSPORIDIUM From Molecules to Disease

Edited by

Guadalupe Ortega-Pierres Department of Genetics and Molecular Biology, CINVESTAV-IPN, México City, Mexico

Simone M. Cacciò Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy

Ronald Fayer Environmental Microbial Safety Laboratory, Animal and Natural Resources Institute, ARS-USDA, Beltsville, MD, USA

Theo G. Mank Department of Parasitology, Public Health Laboratory, Haarlem, the Netherlands

Huw V. Smith Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow, UK and

R.C. Andrew Thompson WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, School of Veterinary and Biomedical Sciences, Murdoch University, WA, Australia

CABI is a trading name of CAB International CABI Head Office Nosworthy Way Wallingford Oxfordshire OX10 8DE UK Tel: +44 (0)1491 832111 Fax: +44 (0)1491 833508 E-mail: [email protected] Website: www.cabi.org

CABI North American Office 875 Massachusetts Avenue 7th Floor Cambridge, MA 02139 USA Tel: +1 617 395 4056 Fax: +1 617 354 6875 E-mail: [email protected]

© CAB International 2009. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. Library of Congress Cataloging-in-Publication Data Giardia and cryptosporidium : from molecules to disease / edited by Guadalupe Ortega-Pierres . . . [et al.]. p. cm. Includes bibliographical references and index. ISBN 978-1-84593-391-3 (alk. paper) 1. Giardia. 2. Cryptosporidium. 3. Giardiasis–Prevention. I. Ortega-Pierres, Guadalupe. II. C.A.B. International. [DNLM: 1. Giardia. 2. Cryptosporidiosis–prevention & control. 3. Cryptosporidium. 4. Giardiasis–prevention & control. 5. Water Pollution–prevention & control. QX 70 G4346 2008] QL368.D65G53 2008 616.9′3601–dc22 2008025358 ISBN-13: 978 1 84593 391 3 Typeset by AMA Dataset, Preston, UK. Printed and bound in the UK by the MPG Books Group, Bodmin. The paper used for the text pages in this book is FSC certified. The FSC (Forest Stewardship Council) is an international network to promote responsible management of the world's forests.

Contents

Contributors 1

The Impact of Giardia on Science and Society R.C. Andrew Thompson

2

Cryptosporidium in Cattle: From Observing to Understanding Ronald Fayer, Monica Santín and James M. Trout

ix 1

12

TAXONOMY, NOMENCLATURE AND EVOLUTION 3

Names Do Matter Dwight D. Bowman

4

Centenary of the Genus Cryptosporidium: From Morphological to Molecular Species Identification Jan Šlapeta

25

31

MOLECULAR EPIDEMIOLOGY AND TYPING 5

6

7

Molecular Epidemiology of Human Cryptosporidiosis in Developing Countries Lihua Xiao

51

Molecular Epidemiology and Typing of Non-human Isolates of Cryptosporidium Una M. Ryan and Lihua Xiao

65

Insights Into the Molecular Detection of Giardia duodenalis: Implications for Epidemiology Simone M. Cacciò, Marco Lalle, Relja Beck and Edoardo Pozio

81

v

vi

Contents

ZOONOTIC, HUMAN AND ANIMAL HEALTH ISSUES 8

Wildlife with Giardia: Villain, or Victim and Vector? Susan J. Kutz, R.C. Andrew Thompson and Lydden Polley

9

The Role of Livestock in the Foodborne Transmission of Giardia duodenalis and Cryptosporidium spp. to Humans Brent R. Dixon

10 The Risk of Zoonotic Genotypes of Cryptosporidium spp. in Watersheds Hussni O. Mohammed and Susan E. Wade

94

107

123

CLINICAL AND MOLECULAR EPIDEMIOLOGY 11 Clinical Presentation in Cryptosporidium-infected Patients Laetitia M. Kortbeek 12 Molecular Epidemiology of Cryptosporidium and Giardia Infections Paul R. Hunter

131

138

ADVANCES IN DIAGNOSIS 13 Advances in Diagnosis: is Microscopy Still the Benchmark? 147 Rachel M. Chalmers TREATMENT OF DRINKING WATER 14 Control of Cryptosporidium and Giardia in Surface Water by Disinfection Thomas M. Hargy, Jennifer L. Clancy and Lynn P. Landry

158

15 Towards Methods for Detecting UV-induced Damage in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy Huw V. Smith, B.H. Al-Adhami, Rosely A.B. Nichols, John R. Kusel and J. O’Grady

179

CONTROL IN WATER 16 Effect of Environmental and Conventional Water Treatment Processes on Waterborne Cryptosporidium Oocysts 198 Brendon King, Alexandra Keegan, Chris Saint and Paul Monis 17 Methods for Genotyping and Subgenotyping Cryptosporidium spp. Oocysts Isolated During Water and Food Monitoring Huw V. Smith, Rosely A.B. Nichols, Lisa Connelly and Christopher B. Sullivan

210

Contents

vii

18 Intervention in Waterborne Disease 227 Gordon Nichols, Iain R. Lake, Rachel M. Chalmers, Graham Bentham, Florence C.D. Harrison, Paul R. Hunter, Sari Kovats, Chris Grundy, Steve Anthony, Hester Lyons, Maureen Agnew and Chris Proctor OTHER WATERBORNE PROTOZOA 19 Occurrence and Control of Naegleria fowleri in Drinking Water Wells Charles P. Gerba, Barbara L. Blair, Payal Sarkar, Kelly R. Bright, R.C. Maclean and Francine Marciano-Cabral 20 Environmental Factors Influencing the Survival of Cyclospora cayetanensis Ynes R. Ortega

238

248

BASIC BIOLOGY 21 Recent Advances in the Developmental Biology and Life Cycle of Cryptosporidium 255 Nawal Samih Hijjawi, Annika C. Boxell and R.C. Andrew Thompson 22 Basic Biology of Giardia lamblia: Further Studies on Median Body and Funis 266 Marlene Benchimol 23 Giardia intestinalis: a Microaerophilic Parasite with Mitochondrial Ancestry Gloria León-Avila, José Manuel Hernández and Jorge Tovar

284

METABOLOMICS AND TRANSCRIPTOME 24 Cytoskeleton-based Lipid Transport in a Parasitic Protozoan, Giardia lamblia Cynthia Castillo, Yunuen Hernandez, Sukla Roychowdhury and Siddhartha Das 25 Signalling During Giardia Differentiation Tineke Lauwaet and Frances D. Gillin

292

309

GENOMICS 26 Preliminary Analysis of the Cryptosporidium muris Genome 320 Giovanni Widmer, Eric London, Linghui Zhang, Guangtao Ge, Saul Tzipori, Jane M. Carlton and Joana C. da Silva PROTEOMICS 27 Proteomic Analyses in Giardia Daniel Palm and Staffan G. Svärd

328

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Contents

28 Proteomic and Genomic Approaches to Understanding the ‘Power Plant’ of Cryptosporidium Lorenza Putignani, Sanya J. Sanderson, Cristina Russo, Jessica Kissinger, Donato Menichella and Jonathan M. Wastling

344

BIOCHEMISTRY AND PHYSIOLOGY 29 Energy Metabolism and Carbon Flow in Cryptosporidium parvum Guan Zhu

360

30 The Surface Protein Repertoires of Cryptosporidium spp. and Other Apicomplexans Thomas J. Templeton

369

31 Giardan: Structure, Synthesis, Regulation and Inhibition Keriman S ¸ener, Harry van Keulen and Edward L. Jarroll

382

CELL BIOLOGY AND SIGNALLING 32 Protein Kinase C in Giardia duodenalis: a Family Affair M. Luisa Bazán-Tejeda, Raúl Argüello-García, Rosa M. BermúdezCruz, Martha Robles-Flores and Guadalupe Ortega-Pierres 33 Secretory Granule Biogenesis and the Organization of Membrane Compartments via SNARE Proteins in Giardia lamblia Eliana V. Elías, Natalia Gottig, Rodrigo Quiroga and Hugo D. Luján 34 Molecular Mechanisms of Cryptosporidium-induced Host Actin Cytoskeleton Dynamics Steven P. O’Hara, Xian-Ming Chen and Nicholas F. LaRusso

398

409

418

PATHOGENESIS AND HOST–PARASITE RELATIONSHIP 35 Pathogenic Mechanisms in Giardiasis and Cryptosporidiosis Andre G. Buret

428

36 Interferon-gamma (IFN-γ) in Immunological Control of Cryptosporidial Infection Naheed Choudhry, Mona Bajaj-Elliott and Vincent McDonald

442

37 Immune Response to Giardia Infection: Lessons from Animal Models Steven M. Singer and Joel Kamda

451

DRUG TREATMENT AND NOVEL DRUG TARGETS 38 Drug Treatment and Novel Drug Targets Against Giardia and Cryptosporidium Jean-François Rossignol

463

Index

483

Contributors

Maureen Agnew, School of Environmental Sciences, University of East Anglia, Norwich, UK. B.H. Al-Adhami, CFIA, Centre for Animal Parasitology, Saskatoon Laboratory, 116 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 2R3. Steve Anthony, ADAS Consulting, Woodthorne, Wergs Road, Wolverhampton WV6 8TQ, UK. Raúl Argüello-García, Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados del IPN, México City, Av. Instituto Politécnico Nacional No. 2508, Col. San Pedro Zacatenco, México, D.F. 07360, Mexico. Mona Bajaj-Elliott, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. M. Luisa Bazán-Tejeda, Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados del IPN, México City, Av. Instituto Politécnico Nacional No. 2508, Col. San Pedro Zacatenco, México, D.F. 07360, Mexico. Relja Beck, Department for Parasitology and Parasitic Diseases, University of Zagreb, Heinzelova 55, 10000, Zagreb, Croatia. Marlene Benchimol, Universidade Santa Úrsula, Laboratório de Ultraestrutura Celular, Rua Jornalista Orlando Dantas 59, Botafogo, Rio de Janeiro, R.J., Brazil, CEP 222-31-010. Email: [email protected] Graham Bentham, School of Environmental Sciences, University of East Anglia, Norwich, UK. Rosa M. Bermúdez-Cruz, Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados del IPN, México City, Av. Instituto Politécnico Nacional No. 2508, Col. San Pedro Zacatenco, México, D.F. 07360, Mexico. Barbara L. Blair, Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, AZ 85721, USA. ix

x

Contributors

Dwight D. Bowman, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850, USA. Email: [email protected] Annika C. Boxell, Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia. Kelly R. Bright, Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, AZ 85721, USA. Andre G. Buret, Department of Biological Sciences, Inflammation Research Network, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N 1N4. Email: [email protected] Simone M. Cacciò, Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy. Email: [email protected] Jane M. Carlton, New York University, Department of Medical Parasitology, 550 First Avenue, New York 10016, USA. Cynthia Castillo, Naturopathic Medicine, Bastyr University, 14500 Juanita Drive NE, Kenmore, WA 98028, USA. Rachel M. Chalmers, UK Cryptosporidium Reference Unit (CRU), National Public Health Service for Wales, Microbiology Swansea, Singleton Hospital, Sketty, Swansea SA2 8QA, UK. Email: [email protected] Xian-Ming Chen, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA. Naheed Choudhry, Barts and the London School of Medicine and Dentistry, Centre for Gastroenterology, Queen Mary College, University of London, Turner Street, London E1 2AD, UK. Jennifer L. Clancy, Clancy Environmental Consultants, Inc., 20 Mapleville Depot, PO Box 314, Saint Albans, VT 05478, USA. Lisa Connelly, Scottish Parasite Diagnostic Laboratory (SPDL), Stobhill Hospital, 133 Balornock Road, Glasgow G21 3UW, UK. Siddhartha Das, Infectious Diseases and Immunology Unit, The Border Biomedical Research Center, Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968, USA. Email: [email protected] Brent R. Dixon, Microbiology Research Division, Banting Research Centre, 251 Sir Frederick Banting Driveway, PL 2204A2, Ottawa, Ontario, Canada K1A 0K9. Email: [email protected] Eliana V. Elías, Mercedes & Martin Ferreyra Institute for Medical Research, INIMEC-CONICET and School of Medicine, Catholic University of Cordoba, Parque Velez Sarsfield, CP 5000, Cordoba, Argentina. Ronald Fayer, Environmental Microbial Safety Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 173, BARC-East, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. Email: [email protected] Guangtao Ge, Department of Computer Sciences, School of Engineering, Tufts University, 200 Westboro Road, North Grafton, MA 01536, USA.

Contributors

xi

Charles P. Gerba, Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, AZ 85721, USA. Email: [email protected]. edu Frances D. Gillin, Department of Pathology, Division of Infectious Diseases, University of California at San Diego, San Diego, CA, USA. Email: [email protected] Natalia Gottig, Mercedes & Martin Ferreyra Institute for Medical Research, INIMEC-CONICET and School of Medicine, Catholic University of Cordoba, Parque Velez Sarsfield, CP 5000, Cordoba, Argentina. Chris Grundy, Public and Environmental Health Research Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK. Thomas M. Hargy, Clancy Environmental Consultants, Inc., 20 Mapleville Depot, PO Box 314, Saint Albans, VT 05478, USA. Florence C.D. Harrison, School of Environmental Sciences, University of East Anglia, Norwich, UK. José Manuel Hernández, Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, San Pedro, Zacatenco, CP 07300, México D.F., Mexico. Yunuen Hernandez, Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, Bldg 4/Rm16, 4 Center Drive, Bethesda, MD 20892, USA. Nawal Samih Hijjawi, Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, The Hashemite University, PO Box 150459, Zarqa 13115, Jordan. Paul R. Hunter, School of Medicine, Health Policy and Practice, University of East Anglia, Earlham Road, Norwich NR4 7TJ, UK. Email: Paul.Hunter@ uea.ac.uk Edward L. Jarroll, Department of Biology, Northeastern University, Boston, MA 02115, USA. Email: [email protected] Joel Kamda, Department of Biology and Center for Infectious Diseases, Georgetown University, Washington, DC 20057, USA. Alexandra Keegan, The Co-operative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Salisbury, South Australia 5108, Australia. Brendon King, The Co-operative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Salisbury, South Australia 5108, Australia. Jessica Kissinger, Center for Tropical and Emerging Global Diseases and Department of Genetics, University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA. Laetitia M. Kortbeek, Centre for Infectious Disease Control Netherlands, Laboratory for Infectious Diseases and Perinatal Screening (LIS), National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, the Netherlands. Email: [email protected] Sari Kovats, Public and Environmental Health Research Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.

xii

Contributors

John R. Kusel, Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. Susan J. Kutz, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. Email: [email protected] Iain R. Lake, School of Environmental Sciences, University of East Anglia, Norwich, UK. Marco Lalle, Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy. Lynn P. Landry, Utility Analysis and Environmental Management, Policy and Planning Department, Metro Vancouver, Greater Vancouver Regional District, 4330 Kingsway, Burnaby, BC, Canada V5H 4G8. Nicholas F. LaRusso, Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA. Email: larusso. [email protected] Tineke Lauwaet, Department of Pathology, Division of Infectious Diseases, University of California at San Diego, San Diego, CA, USA. Gloria León-Avila, Department of Parasitology, Escuela Nacional de Ciencias Biológicas, IPN, Carpio y Plan de Ayala, Casco de Santo Tomás, CP 11340, México, D.F., Mexico. Email: [email protected] Eric London, Tufts Cummings School of Veterinary Medicine, Division of Infectious Diseases, Tufts University, 200 Westboro Road, North Grafton, MA 01536, USA. Hugo D. Luján, Mercedes & Martin Ferreyra Institute for Medical Research, INIMEC-CONICET and School of Medicine, Catholic University of Cordoba, Parque Velez Sarsfield, CP 5000, Cordoba, Argentina. Email:hlujan@ immf.uncor.edu Hester Lyons, ADAS Consulting, Woodthorne, Wergs Road, Wolverhampton WV6 8TQ, UK. R.C. Maclean, Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA. Francine Marciano-Cabral, Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA. Vincent McDonald, Barts and the London School of Medicine and Dentistry, Centre for Gastroenterology, Queen Mary College, University of London, Turner Street, London E1 2AD, UK. Email: [email protected] Donato Menichella, Unit of Microbiology and Virology, Bambino Gesù Hospital, Scientific Institute, Piazza Sant’Onofrio 4, 00146, Rome, Italy. Email: [email protected] Hussni O. Mohammed, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. Email: [email protected] Paul Monis, The Co-operative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Salisbury, South Australia 5108, Australia. Email:[email protected]

Contributors

xiii

Gordon Nichols, Environmental and Enteric Diseases Department, Communicable Disease Surveillance Centre, Health Protection Agency (HPA) Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK. Email: [email protected] Rosely A.B. Nichols, Scottish Parasite Diagnostic Laboratory (SPDL), Stobhill Hospital, 133 Balornock Road, Glasgow G21 3UW, UK. J. O’Grady, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK. Steven P. O’Hara, Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA. Ynes R. Ortega, Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, GA 30223, USA. Email: [email protected] Guadalupe Ortega-Pierres, Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados del IPN, México City, Av. Instituto Politécnico Nacional No. 2508, Col. San Pedro Zacatenco, México, D.F. 07360, Mexico. Email: [email protected] Daniel Palm, Centre for Microbial Preparedness, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden. Lydden Polley, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, Canada S7N 5B4. Email:lydden. [email protected] Edoardo Pozio, Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy. Chris Proctor, ADAS Consulting, Woodthorne, Wergs Road, Wolverhampton WV6 8TQ, UK. Lorenza Putignani, Unit of Microbiology and Virology, Bambino Gesù Hospital, Scientific Institute, Piazza Sant’Onofrio 4, 00146, Rome, Italy. Email: [email protected] Rodrigo Quiroga, Mercedes & Martin Ferreyra Institute for Medical Research, INIMEC-CONICET and School of Medicine, Catholic University of Cordoba, Parque Velez Sarsfield, CP 5000, Cordoba, Argentina. Martha Robles-Flores, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apdo. Postal 70-159, México City, D.F. 04510, Mexico. Jean-François Rossignol, Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA. Email: [email protected] Sukla Roychowdhury, Infectious Diseases and Immunology Unit, The Border Biomedical Research Center, Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968, USA. Cristina Russo, Unit of Microbiology and Virology, Bambino Gesù Hospital, Scientific Institute, Piazza Sant’Onofrio 4, 00146, Rome, Italy. Email:russocri@ opbg.net Una M. Ryan, School of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch, WA 6150, Australia. Email: Una.Ryan@ murdoch.edu.au

xiv

Contributors

Chris Saint, The Co-operative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Salisbury, South Australia 5108, Australia. Sanya J. Sanderson, Department of Pre-Clinical Veterinary Science and Veterinary Pathology, Faculty of Veterinary Science, University of Liverpool, Crown Street, Liverpool L69 7ZJ, UK. Email: [email protected] Monica Santín, Environmental Microbial Safety Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 173, BARC-East, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. Payal Sarkar, Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, AZ 85721, USA. Keriman S¸ener, Department of Biology, Northeastern University, Boston, MA 02115, USA. Email:[email protected] Joana C. da Silva, Institute for Genome Sciences, University of Maryland School of Medicine, 20 Penn Street, Baltimore, MD 21201, USA. Steven M. Singer, Department of Biology and Center for Infectious Diseases, Georgetown University, Washington, DC 20057, USA. Email: sms3@ georgetown.edu Jan Šlapeta, Faculty of Veterinary Science, McMaster Building B14, University of Sydney, NSW 2006, Australia. Email: [email protected] Huw V. Smith, Scottish Parasite Diagnostic Laboratory (SPDL), Stobhill Hospital, 133 Balornock Road, Glasgow G21 3UW, UK. Email: huw.smith@ northglasgow.scot.nhs.uk Christopher B. Sullivan, Scottish Parasite Diagnostic Laboratory (SPDL), Stobhill Hospital, 133 Balornock Road, Glasgow G21 3UW, UK. Staffan G. Svärd, Department of Cell and Molecular Biology, BMC, Uppsala University, Box 596, SE-751 24 Uppsala, Sweden. Email: staffan.svard@ icm.uu.se Thomas J. Templeton, Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, Box 62, NY 10021 USA. Email: [email protected] R.C. Andrew Thompson, WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia. Email: a.thompson@ murdoch.edu.au Jorge Tovar, School of Biological Sciences, Royal Holloway University of London, Egham TW20 0EX, UK. James M. Trout, Environmental Microbial Safety Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 173, BARC-East, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. Saul Tzipori, Tufts Cummings School of Veterinary Medicine, Division of Infectious Diseases, Tufts University, 200 Westboro Road, North Grafton, MA 01536, USA. Harry van Keulen, Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA.

Contributors

xv

Susan E. Wade, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. Jonathan M. Wastling, Department of Pre-Clinical Veterinary Science and Veterinary Pathology, Faculty of Veterinary Science, University of Liverpool, Crown Street, Liverpool L69 7ZJ, UK. Email: [email protected] Giovanni Widmer, Tufts Cummings School of Veterinary Medicine, Division of Infectious Diseases, Tufts University, 200 Westboro Road, North Grafton, MA 01536, USA. Email: [email protected] Lihua Xiao, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Building 22, Mail Stop F-12, 4770 Buford Highway, Atlanta, GA 30341, USA. Email: [email protected] Linghui Zhang, Tufts Cummings School of Veterinary Medicine, Division of Infectious Diseases, Tufts University, 200 Westboro Road, North Grafton, MA 01536, USA. Guan Zhu, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX 77843, USA. Email: [email protected]

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1

The Impact of Giardia on Science and Society R.C.A. THOMPSON WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, Murdoch University, WA, Australia

Abstract Although Giardia has a long history, it is only recently that the clinical impact of Giardia in children has been recognized. Similarly, the emergence of Giardia as a frequent parasite of companion animals, livestock and wildlife raises questions about the clinical and zoonotic significance of such infections. Transmission patterns have already been described but the frequency of interaction between cycles of transmission is only just beginning to be addressed in molecular epidemiological studies. The application of molecular tools has provided information which sets the scene for revising the taxonomy of Giardia. With the completion of the Giardia genome sequencing project, it is hoped that it will soon be possible to compare genome and phenome and provide information of practical value for the control of Giardia, as well as increasing our understanding of the evolution and phylogenetic relationships of Giardia.

Introduction The protozoa that collectively comprise the genus Giardia have intrigued biologists and clinicians for over 300 years, ever since Antony van Leeuwenhoek first discovered the organism (Meyer, 1994). This enigmatic protozoan possesses a number of unusual characteristics including the presence of two similar, transcriptionally active diploid nuclei, the absence of mitochondria and peroxisomes, and a unique attachment organelle – the ventral sucking disc (Thompson and Monis, 2004; Morrison et al., 2007). Phylogenetic relationships are controversial, with one school of thought suggesting that Giardia is a basal eukaryote and the other that Giardia comprises one of many divergent eukaryotic lineages that adapted to a microaerophilic lifestyle rather than diverging before the endosymbiosis of the mitochondrial ancestor (Thompson and Monis, 2004; Morrison et al., 2007). Despite its long history, our understanding of the pathogenesis of Giardia infections and its relationship with its host is limited, and we do not know why clinical disease occurs in some individuals but may not be apparent in others © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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R.C.A. Thompson

(Savioli et al., 2006). There are no known virulence factors or toxins, and variable expression of surface proteins may allow evasion of host immune responses and adaptation to different environments (Morrison et al., 2007). For many years after van Leeuwenhoek first described Giardia, there was controversy as to whether it was a parasite that caused disease or a commensal. It was not until the 1920s that influential proponents such as Dobell and Miller gave support to the link between the presence of Giardia and disease and, more importantly, a link with malabsorption syndromes (Cox, 1998). Thirty years later, Rendtorff established infections with Giardia in humans following the oral inoculation of cysts (Rendtorff, 1954). Today, there is no doubt about the clinical significance of Giardia infections in humans, and particularly the impact of giardiasis in children and its association with failure to thrive and wasting syndromes (Thompson, 2008).

Taxonomy The recent application of molecular, PCR-based tools has enabled the genetic relationships of a range of morphologically identical ‘strains’ of Giardia to be determined (Thompson and Monis, 2004; Cacciò et al., 2005; Traub et al., 2005; Smith et al., 2006). As a consequence, a large number of species and genotypes of Giardia are now recognized that differ principally in their host range. The current taxonomy of Giardia is summarized in Table 1.1 and has been extensively reviewed (Thompson and Monis, 2004; Cacciò et al., 2005; Thompson et al., 2007). The nomenclature most widely accepted at the present time for the genotypes that have been characterized is ‘assemblage’, although a revised taxonomy has been proposed (Thompson and Monis, 2004; Cacciò et al., 2005). Some species and genotypes/assemblages appear to be restricted to particular species or types of hosts (e.g. Giardia assemblages C/D (G. canis) and E (G. bovis) in dogs and livestock, respectively; see Table 1.1) whereas others have broad host ranges, including humans, (e.g. G. duodenalis assemblages A and B; see Table 1.1) and are therefore of zoonotic significance. Giardia duodenalis (syn. G. intestinalis, G. lamblia) is the only species found in humans. Table 1.1 summarizes the proposed revised taxonomy for assemblages of Giardia. Such a formal nomenclature will avoid confusion and enhance communication.

Clinical Impact Humans In developed countries, infections with Giardia are most common in children, especially in daycare centres and among travellers, and a rising incidence in such settings has led to the designation of giardiasis as a re-emerging infectious disease in the developed world (Thompson, 2000, 2004; Eckmann, 2003; Thompson and Monis, 2004). The World Health Organization (WHO) has given consideration to intestinal protozoa for many years, but because of their very different disease dynamics they did not initially form part of the ‘Neglected Diseases Initiative’.

The Impact of Giardia on Science and Society Table 1.1.

3

Established and proposeda species/assemblages of Giardia.

Species/assemblage

Host

G. duodenalis/assemblage A

Humans and other primates, dogs, cats, livestock, rodents and other wild mammals Humans and other primates, dogs

G. duodenalis/assemblage B (G. enterica)a G. agilis G. muris G. psittaci G. ardeae G. duodenalis/assemblage C/D (G. canis)a G. duodenalis/assemblage F (G. cati)a G. duodenalis/assemblage E (G. bovis)a G. duodenalis/assemblage G (G. simondi )a aSee

Amphibians Rodents Birds Birds Dogs Cats Cattle and other hoofed livestock Rats

Thompson and Monis (2004), Cacciò et al. (2005).

However, since they all have a common link with poverty, the current view is to take a comprehensive approach to all these diseases. In September 2004, Giardia was included in WHO’s Neglected Diseases Initiative (Savioli et al., 2006). In developing countries, particularly in Asia, Africa and Latin America, about 200 million people have symptomatic giardiasis with some 500,000 new cases being reported each year (WHO, 1996). Children living in communities are most commonly infected in developing countries, particularly among disadvantaged groups living in isolated communities, such as Australian Aborigines (Thompson, 2000; Hesham et al., 2005; Savioli et al., 2006). These children are most at risk from the chronic consequences of Giardia infection. Although animals may serve as reservoirs of Giardia infection that under certain circumstances may spill over to humans; from a clinical viewpoint, direct human-tohuman transmission is of most significance, particularly in situations where the frequency of transmission is high. Human-to-human transmission of Giardia can occur indirectly through the accidental ingestion of cysts in contaminated water or food, or directly in environments where hygiene levels may be compromised, such as daycare centres or disadvantaged community settings, where the frequency of transmission is high and/or conditions are conducive to direct person-to-person transfer (Thompson, 2000; Hesham et al., 2005). Under such circumstances, children may be at constant risk of infection, even though chemotherapeutic interventions may be instituted (Thompson et al., 2001; Savioli et al., 2006). If children are constantly exposed they will be re-infected rapidly, since anti-giardial agents have no residual activity. The fact that children in such endemic settings do not appear to develop resistance to Giardia infection may be due to suboptimal immunological competence and/or infection with different ‘strains’/subgenotypes of Giardia (Hopkins et al.,

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1999). It might be expected that competitive interactions would result in the predominance of particular genotypes of Giardia and the exclusion of others (Thompson et al., 2001), but this does not appear to be the case, perhaps due to the suboptimal nutritional and immunological environments to which the parasites are exposed in such communities. Symptomatic infection in humans may not be evident in a significant proportion of infected individuals (Troeger et al., 2007), and represents only a fraction (20–80%) of all stool-positive Giardia infections (Nash et al., 1987; Flanagan, 1992; Rodriguez-Hernandez et al., 1996). Symptoms are highly variable but include persistent, usually short-term, diarrhoea, epigastric pain, nausea, vomiting and weight loss (Thompson et al., 1993; Eckmann, 2003). Symptoms typically occur 6–15 days after infection and last for 2–4 days. As such, infection is assumed to be self-limiting in more than 85% of cases (indicating that effective host defences exist), although chronic cases occur occasionally in the absence of apparent immunodeficiencies (Nash et al., 1987; Flanagan, 1992). The risk factors for clinical giardiasis, particularly in humans, have yet to be resolved, but clearly involve host and environmental factors as well as the ‘strain’/ genotype/assemblage of the parasite (Buret, 2007). However, a distinction needs to be made between the effects of a single infection, which may give rise to the ‘classical’ short-term episode of diarrhoea, and the long-term effects of persistent Giardia infection, particularly in children, in environments where the frequency of transmission is high. Here the picture is very different. In endemic foci where the frequency of transmission is high and often enhanced by poor hygiene and environmental contamination, children are at particular risk from the more serious and long-term consequences of Giardia infection that are associated with malnutrition, micronutrient deficiency and failure to thrive, iron deficiency anaemia and poor cognitive function (Hesham et al., 2004; Savioli et al., 2006; Gbakima et al., 2007; Gonen et al., 2007). Clearly, the impact of Giardia in such circumstances will be exacerbated by poor/suboptimal nutrition and concurrent infections with other enteric parasites such as Hymenolepis nana, Entamoeba coli and Blastocystis. Longitudinal studies on the impact of enteric parasites on childhood growth and mental development in such endemic areas is urgently required (Savioli et al., 2006). The children who are infected in such environments, particularly in developing countries and among disadvantaged groups, represent the most important group in terms of the clinical impact of Giardia (Thompson, 2000; Hesham et al., 2005; Savioli et al., 2006). In such circumstances, it is debatable whether the regular use of drugs is of any benefit. This is in contrast to the situation with gastrointestinal helminths such as hookworm, where regular mass chemotherapy has been shown to have great benefit in control (Reynoldson et al., 1998; Thompson et al., 2001).

Domestic animals Livestock, wildlife, and companion animals are frequently infected with Giardia and are susceptible to host-adapted and zoonotic species of Giardia. Although infections with Giardia are common in dogs and cats, most infected animals

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remain asymptomatic. If clinical disease occurs, it is usually associated with young animals and those in kennel or cattery situations (Robertson et al., 2000), where the effects of overcrowding, weaning and nutritional deficiency, may cause stress and exacerbate the effects of an infection (Thompson, 2004). The most consistent clinical sign of giardiasis in dogs and cats is small-bowel diarrhoea, which may be acute or chronic, and self-limiting, intermittent or continuous in nature. Giardia infection in ruminants is often asymptomatic but may also be associated with the occurrence of diarrhoea and ill-thrift in calves (O’Handley et al., 1999; Geurden et al., 2006). The importance of giardiasis as a cause of diarrhoea in ruminants is unclear, especially given that diarrhoea in ruminants is often multifactorial, with more than one pathogen detected (O’Handley and Olson, 2006). Nevertheless, the significance of Giardia infection in ruminants warrants further investigation, particularly with regard to production loss. In cattle, Giardia is commonly found alone or in combination with other pathogens as a cause of calf diarrhoea, which can have economic significance (O’Handley et al., 1999; Olson et al., 2004). In two studies in sheep, Giardia reduced the rate of weight gain, impaired feed efficiency and decreased carcass weight (Olson et al., 1995; Aloisio et al., 2006).

Wildlife It is often a common ‘knee-jerk’ reaction when parasites with zoonotic potential are found in wildlife that they represent a threat to public health as a reservoir and potential source of infection for humans (Thompson, 2004). Indeed, this was the case when WHO initially listed the common enteric protozoan parasite Giardia as a zoonosis over 25 years ago as a result of epidemiological observations suggesting that giardiasis in campers in Canada was caused by drinking stream water contaminated with Giardia from beavers (Thompson, 2004). No one thought to ask the question of where the beavers got their Giardia infections from until only beavers living downstream from a sewage works were found to be infected. With the subsequent application of molecular tools, it has been confirmed that beavers are susceptible to zoonotic strains of Giardia (see Thompson, 2004). The question now is: are they victim or villain with respect to human giardiasis? A similar situation has been reported in non-human primates for which there is a growing literature of the invasion of human pathogens into wild populations (Sleeman et al., 2000; Graczyk et al., 2001, 2002). For example, it was suggested that the discovery of Giardia and the cohabiting enteric protozoan Cryptosporidium in mountain gorillas in the Bwindi Impenetrable National Park, Uganda, was thought to indicate enhanced contact with humans and/or domestic livestock. This was confirmed when rangers and their cattle were found to be infected with Giardia and that the genotype was the same as that recovered from the gorillas (Graczyk et al., 2002). Muskoxen (Ovibos moschatus) are indigenous to the arctic tundra of Canada and Greenland and have been translocated to areas in Alaska, the USA, Russia, Norway and Sweden. These animals are well adapted to their northern environment, and tend to have a relatively simple parasite fauna. Recent surveys on the

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biodiversity and impacts of parasites in Arctic ungulates described Giardia duodenalis, assemblage A, the zoonotic genotype, in muskoxen (Kutz et al., 2008). This unexpected finding (a novel strain, or the livestock strain, was predicted) raises many interesting questions regarding the origin and epidemiology of this parasite in humans and wildlife in this Arctic ecosystem. In particular, is this a pathogen initially introduced to muskoxen by humans? Is Giardia now maintained as a sylvatic cycle in muskoxen (or other wildlife species in the locality) independent of humans? Does the Giardia from muskoxen spill back into humans? The impact of Giardia on the health and production (body condition, fecundity and pelage) of free-ranging ungulates, including muskoxen, remains unknown. In Australia, marsupials are commonly infected with Giardia (Kettlewell et al., 1998) but until recently it was not known to what species or strain(s) of Giardia they were susceptible. Studies on the quenda (Isoodon obesulus), a common widespread species of bandicoot in southern Australia, demonstrated that they were infected with a novel, genetically distinct, form of Giardia, so different from what has been described from humans and other animals that it probably represents a distinct species (Adams et al., 2004). The Giardia isolates genotyped from quenda in their natural habitats have all proved to be the novel strain. However, when quenda were trapped and examined on a farm, they were found to be infected with ‘domestic’ strains of Giardia normally found in livestock and humans (from assemblages A and E; see Table 1.1). Presumably, this reflects the susceptibility of the quenda to other strains of Giardia, as with the case of beavers in North America. This case study raises questions regarding the pathogenicity of non-host-adapted strains of Giardia in naïve wildlife hosts. Additionally, it also raises the question of competition between cohabiting ‘strains’ of Giardia (Thompson and Monis, 2004) and whether, in this case, and perhaps in other species of wildlife, zoonotic strains of Giardia can out-compete the host-specific wildlife strains.

Molecular Epidemiology Humans may be infected with Giardia genotypes belonging to assemblage A or assemblage B (Thompson and Monis, 2004; Cacciò et al., 2005). There is considerable evidence of phenotypic differences between these two assemblages in characters such as metabolism and growth rate (Thompson and Monis, 2004). It has therefore been proposed that there may be differences in the nature of infection with these two assemblages in humans which may be reflected in duration of infection, drug sensitivity and virulence (Thompson and Monis, 2004). There is growing evidence to support these suggestions but there is a need for more focused molecular epidemiological studies. For example, in tea-growing communities in Assam, India, the proportion of assemblage B and A infections in 18 infected people was 61% and 39%, respectively (Traub et al., 2004). Another study in the UK that examined 35 human clinical samples found that 64% were assemblage B, 27% were assemblage A genetic subgroup II, and the remainder were a mixture of assemblage B and assemblage A genetic group II (Amar et al., 2002).

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There is also increasing evidence supporting differences in virulence between genetic groups of G. duodenalis. Several studies have examined the relationship between clinical symptoms and the genetic assemblage of G. duodenalis infecting human patients. Three of these, undertaken in Australia, Bangladesh and Spain, all found a statistically significant correlation between the presence of symptoms, as defined by diarrhoea, and infection with Giardia belonging to assemblage A (Read et al., 2002; Haque et al., 2005; Sahagún et al., 2007). In contrast, it was found, in all studies, that infections with assemblage B were usually asymptomatic. A study in the Netherlands of individuals presenting general practitioners with diarrhoeal complaints found that those infected with assemblage A isolates had intermittent diarrhoea, whereas those infected with assemblage B had acute or persistent diarrhoea (Homan and Mank, 2001). However, this study only sampled patients presenting with diarrhoeal complaints. There is a need for additional large-scale molecular epidemiological surveys of Giardia infections in humans. With the limited data currently available it is not possible to determine the geographical distribution and prevalence of human-infective genotypes. With such data it may be possible to determine the significance of any ‘strain’-related differences in virulence. Although studies on the occurrence of the different genotypes of Giardia serve to emphasize the potential public health risk from domestic dogs and cats, data on the frequency of zoonotic Giardia transmission are lacking (Thompson, 2004; Leonhard et al., 2007). Such information can be obtained from molecular epidemiological studies that genotype isolates of the parasites from susceptible hosts in localized endemic foci of transmission or as a result of longitudinal surveillance and genotyping of positive cases. In the former, recent research in localized endemic foci of transmission has provided evidence in support of the role of dogs in cycles of zoonotic Giardia transmission involving humans and domestic dogs from communities in tea-growing areas of Assam, India, and in temple communities in Bangkok, Thailand (Traub et al., 2004; Inpankaew et al., 2007). In both these studies, some dogs and their owners sharing the same living area were shown to harbour isolates of G. duodenalia from the same assemblage. Other studies have shown that zoonotic genotypes of Giardia may occur frequently in individual pet dogs living in urban areas (for a review, see Leonhard et al., 2007).

Future Perspectives Sex Population genetic studies of Giardia in communities where the frequency of transmission is very high, have found evidence of occasional bouts of genetic exchange in Giardia (Meloni et al., 1995). These authors demonstrated multiple banding patterns in a number of isolates of Giardia by allozyme electrophoresis which, if a true reflection of the underlying genotypes of the isolates, would seem to indicate that G. duodenalis is functionally diploid, and that recombination or sexual reproduction must have occurred at some stage to produce the apparent

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heterozygotes (Tait, 1983; Meloni et al., 1995). These observations have been supported by subsequent population genetic studies (Cooper et al., 2007) and recent genomic studies (Ramesh et al., 2005; Morrison et al., 2007; Teodorovic et al., 2007). The evolutionary advantage this gives to Giardia is the capacity to respond to adversity, for example selection pressures imposed by regular exposure to anti-giardial drugs or competition with cohabiting ‘strains’ in circumstances where the likelihood of mixed infections is common (Hopkins et al., 1999).

Correlating phenotype and genotype Genetic studies and sequencing of the Giardia genome have laid an important foundation for understanding this parasite. As a consequence of delineating genotypic groupings, a clearer picture of transmission patterns and host specificity has been obtained. However, phenotypic differences in virulence, drug sensitivity and infectivity have been reported in isolates of G. duodenalis and there is a need to correlate these observations with genetic differences (Thompson and Monis, 2004; Cacciò et al., 2005). The correlation of phenotype and genotype will provide important information about the host–parasite relationship in Giardia infections. The complexity of any biological system, including that of Giardia, lies at the protein level and genomics alone cannot be used to understand these complexities. Using a proteomic approach to examine differences between the human infective genotypes of Giardia, we have found differences in a number of proteins between the two human infective genetic groups – assemblages A and B. These proteins of difference appear to be associated with virulence and pathogenicity but at what level of functionality remains unclear at this stage (Steuart et al., in press). We have also identified proteins, some of which are novel and Giardia-specific, that appear to be key to its survival and transmission, including growth factors and developmental triggers. Giardia is also of evolutionary and biological significance in terms of understanding the origin of higher animals from bacteria as well as fundamental questions about the parasitic way of life. Thus some of the proteins we have identified may contribute to a better understanding of Giardia’s pivotal position in our understanding of eukaryote biology and evolution.

References Adams, P.J., Monis, P.T., Elliot, A.D. and Thompson, R.C. (2004) Cyst morphology and sequence analysis of the small subunit rDNA and ef1 alpha identifies a novel Giardia genotype in a quenda (Isoodon obesulus) from Western Australia. Infection, Genetics and Evolution 4, 365–370. Aloisio, F., Filippini, G., Antenucci, P., Lepri, E., Pezzotti, G., Cacciò, S.M. and Pozio, E. (2006) Severe weight loss in lambs infected with Giardia duodenalis assemblage B. Veterinary Parasitology 142, 154–158. Amar, C.F.L., Dear, P.H., Pedraza-Díaz, S., Looker, N., Linnane, E. and McLauchlin, J. (2002) Sensitive PCR-restriction fragment length polymorphism assay for detection

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and genotyping of Giardia duodenalis in human feces. Journal of Clinical Microbiology 40, 446–452. Buret, A.G. (2007) Mechanisms of epithelial dysfunction in giardiasis. Gut 56, 316–317. Cacciò, S.M., Thompson, R.C.A., McLauchlin, J. and Smith, H.V. (2005) Unravelling Cryptosporidium and Giardia epidemiology. Trends in Parasitology 21, 430–437. Cooper, M.A., Adam, R.D., Worobey, M. and Sterling, C.R. (2007) Population genetics provides evidence for recombination in Giardia. Current Biology 17, 1984–1988. Cox, F. (1998) History of human parasitology. In: Cox, F.E.G., Kreier, J.P. and Wakelin, D. (eds) Microbiology and Microbial Infections. Vol. 5: Parasitology. Arnold, London, pp. 3–18. Eckmann, L. (2003) Mucosal defences against Giardia. Parasite Immunology 25, 259–270. Flanagan, P.A. (1992) Giardia – diagnosis, clinical course and epidemiology: a review. Epidemiology and Infection 109, 1–22. Gbakima, A.A., Konteh, R., Kallon, M., Mansaray, H., Sahr, F., Bah, Z.J., Spencer, A. and Luckay, A. (2007) Intestinal protozoa and intestinal helminthic infections in displacement camps in Sierra Leone. African Journal of Medical Science 36, 1–9. Gonen, C., Yilmaz, N., Yalcin, M., Simsek, I. and Gonen, O. (2007) Diagnostic yield of routine duodenal biopsies in iron deficiency anaemia: a study from Western Australia. European Journal of Gastroenterology and Hepatology 19, 37–41. Geurden, T., Claerebout, E., Dursin, L., Deflandre, A., Bernay, F., Kaltsatos, V. and Vercruysse, J. (2006) The efficacy of an oral treatment with paromomycin against an experimental infection with Giardia in calves. Veterinary Parasitology 135, 241–247. Graczyk, T.K., DaSilva, A.J., Cranfield, M.R., Nizeyi, J.B., Kalema, G.R.N.N. and Pieniazek, N.J. (2001) Cryptosporidium parvum genotype 2 infections in free-ranging mountain gorillas (Gorilla gorilla beringei) of the Bwindi Impenetrable National Park, Uganda. Parasitology Research 87, 368–370. Graczyk, T.K., Bosco-Nizeyi, J., Ssebide, B., Thompson, R.C., Read, C. and Cranfield, M.R. (2002) Anthropozoonotic Giardia duodenalis genotype (assemblage) A infections in habitats of free-ranging human-habituated gorillas, Uganda. Journal of Parasitology 88, 905–909. Haque, R., Roy, S., Kabir, M., Stroup, S.E., Mondal, D. and Houpt, E.R. (2005) Giardia assemblage A infection and diarrhoea in Bangladesh. Journal of Infectious Diseases 192, 2171–2173. Hesham, M.S., Edariah, A.B. and Norhavat, M. (2004) Intestinal parasitic infections and micronutrient deficiency: a review. Medical Journal of Malaysia 59, 284–293. Hesham, M.S., Azlin, M., Nor Aini, U.N., Shaik, A., Sa’iah, A., Fatmah, M.S., Ismail, M.G., Firdaus, M.S.A., Aisah, M.Y., Rozlida, A.R. and Norhayati, M. (2005) Giardiasis as a predictor of childhood malnutrition in Orang Asli children in Malaysia. Transactions of the Royal Society for Tropical Medicine and Hygiene 99, 686–691. Homan, W.L. and Mank, T.G. (2001) Human giardiasis: genotype linked differences in clinical symptomatology. International Journal for Parasitology 31, 822–826. Hopkins, R.M., Constantine, C.C., Groth, D.A., Wetherall, J.D., Reynoldson, J.A. and Thompson, R.C.A. (1999) PCR-based DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer. Parasitology 118, 531–539. Inpankaew, T., Traub, R., Thompson, R.C.A. and Sukthana, Y. (2007) Canine parasitic zoonoses and temple communities in Thailand. Southeast Asian Journal of Tropical Medicine and Public Health 38, 247–255. Kettlewell, J.S., Bettiol, S.S., Davies, N., Milstein, T. and Goldsmid, J.M. (1998) Epidemiology of giardiasis in Tasmania: a potential risk to residents and visitors. Journal of Travel Medicine 5, 127–130.

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R.C.A. Thompson Kutz, S.J., Thompson, R.C.A., Kandola, K., Nagy, J., Wielinga, C., Polley, L. and Elkin, B. (2008) Giardia assemblage A: human genotype in muskoxen in the Canadian Arctic. Parasites and Vectors 1, 32 pp. Leonhard, S., Pfister, K., Beelitz, P., Wielinga, C. and Thompson, R.C.A. (2007) The molecular characterisation of Giardia from dogs in Southern Germany. Veterinary Parasitology 150, 33–38. Meloni, B.P., Lymbery, A.J. and Thompson, R.C.A. (1995) Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: implications for reproductive biology, population structure, taxonomy, and epidemiology. Journal of Parasitology 81, 368–383. Meyer, E.A. (1994) Giardia as an organism. In: Thompson, R.C.A., Reynoldson., J.A. and Lymbery, A.J. (eds) Giardia: From Molecules to Disease. CAB International, Wallingford, UK, pp. 3–15. Morrison, H.G., McArthur, A.G., Gillin, F.D., Aley, S.B., Adam, R.D., Olsen, G.J., Best, A.A., Cande, W.Z., Chen, F., Cipriano, M.J., Davids, B.J., Dawson, S.C., Elmendorf, H.G., Hehl, A.B., Holder, M.E., Huse, S.M., Kim, U.U., Lasek-Nesselquist, E., Manning, G., Nigam, A., Nixon, J.E. J., Palm, D., Passamaneck, N.E., Prabhu, A., Reich, C.I., Reiner, D.S., Samuelson, J., Svard, S. and Sogin, M.L. (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317, 1921–1926. Nash, T.E., Herrington, D.A., Losonsky, G.A. and Levine, M.M. (1987) Experimental human infections with Giardia lamblia. Journal of Infectious Diseases 156, 974–984. O’Handley, R.M. and Olson, M.E. (2006) Giardiasis and cryptosporidiosis in ruminants. Veterinary Clinics of North America: Food Animal Practice 22, 623–643. O’Handley, R.M., Cockwill, C., McAllister, T.A., Jelinski, M., Morck, D.W. and Olson, M.E. (1999) Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea. Journal of the American Veterinary Medical Association 214, 391–396. Olson, M.E., McAllister, T.A., Deselliers, L., Morck, D.W., Cheng, K.J., Buret, A.G. and Ceri, H. (1995) Effects of giardiasis on production in a domestic ruminant (lamb) model. American Journal of Veterinary Research 56, 1470–1474. Olson, M.E., O’Handley, R.M., Ralston, B.J., McAllister, T.A. and Thompson, R.C.A. (2004) Update on Cryptosporidium and Giardia infections in cattle. Trends in Parasitology 20, 185–191. Ramesh, M.A., Malik, S.B. and Logsdon, J.M., Jr (2005) A phylogenomic inventory of meiotic genes: evidence for sex in Giardia and an early eukaryotic origin of meiosis. Current Biology 15, 185–191. Read, C., Walters, J., Robertson, I.D. and Thompson, R.C.A. (2002) Correlation between genotype of Giardia duodenalis and diarrhoea. International Journal for Parasitology 32, 229–231. Rendtorff, R.C. (1954) The experimental transmission of human intestinal protozoan parasites. II. Giardia lamblia cysts given in capsules. American Journal of Hygiene 59, 209–220. Reynoldson, J.A., Behnke, J.M., Gracey, M., Horton, R.J., Spargo, R., Hopkins, R.M., Constantine, C.C., Gilbert, F., Stead, C., Hobbs, R.P. and Thompson, R.C.A. (1998) Efficacy of albendazole against Giardia and hookworm in a remote Aboriginal community in the north of Western Australia. Acta Tropica 71, 27–44. Robertson, I.D., Irwin, P.J., Lymbery, A.J. and Thompson, R.C.A. (2000) The role of companion animals in the emergence of parasitic zoonoses. International Journal for Parasitology 30, 1369–1377. Rodriguez-Hernandez, J., Canut-Blasco, A. and Martin-Sanchez, A.M. (1996) Seasonal prevalences of Cryptosporidium and Giardia infections in children attending day

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care centres in Salamanca (Spain) studied for a period of 15 months. European Journal of Epidemiology 12, 291–295. Sahagún, J., Clavel, A., Goñi, P., Seral, C., Llorente, M.T., Castillo, F.J., Capilla, S., Arias, A. and Gómez-Lus, R. (2007) Correlation between the presence of symptoms and the Giardia duodenalis genotype. European Journal of Clinical Microbiology and Infectious Diseases 27, 81–83. Savioli, L., Smith, H. and Thompson, A. (2006) Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends in Parasitology 22, 203–208. Sleeman, J.M., Meader, L.L., Mudakikwa, A.B., Foster, J.W. and Patton, S. (2000) Gastrointestinal parasites of mountain gorillas (Gorilla gorilla beringei) in the Parc National des Volcans, Rwanda. Journal of Zoo and Wildlife Medicine 31, 322–328. Smith, H.V., Cacciò, S.M., Tait, A., McLauchlin, J. and Thompson, R.C.A. (2006) Tools for investigating the environmental transmission of Cryptosporidium and Giardia infections in humans. Trends in Parasitology 22, 160–167. Steuart, R.F.L., O’Handley, R., Lipscombe, R.J. and Thompson, R.C.A. (2008) Alpha 2 Giardin is an assemblage A specific protein of human infective Giardia duodenalsis. Parasitology (in press). Tait, A. (1983) Sexual processes in the Kinetoplastida. Parasitology 86, 29–57. Teodorovic, S., Braverman, J.M. and Elmendorf, H.G. (2007) Unusually low levels of genetic variation among Giardia Lamblia isolates. Eukaryotic Cell 6, 1421–1430. Thompson, R.C.A. (2000) Giardiasis as a re-emerging infectious disease and its zoonotic potential. International Journal for Parasitology 30, 1259–1267. Thompson, R.C.A. (2004) The zoonotic significance and molecular epidemiology of Giardia and giardiasis. Veterinary Parasitology 126, 15–35. Thompson, R.C.A. (2008) Giardiasis: modern concepts in control and management. Annales Nestlé 66, 23–29. Thompson, R.C.A. and Monis, P.T. (2004) Variation in Giardia: implications for taxonomy and epidemiology. Advances in Parasitology 58, 69–137. Thompson, R.C.A., Reynoldson, J.A. and Mendis, A.H.W. (1993) Giardia and giardiasis. Advances in Parasitology 32, 71–160. Thompson, R.C.A., Reynoldson, J.A., Garrow, S.J., McCarthy, J.S. and Behnke J.M. (2001) Towards the eradication of hookworm in an isolated Australian community. Lancet 357, 770–771. Thompson, R.C.A., Traub, R.J. and Parameswaran, N. (2007) Molecular epidemiology of foodborne parasitic zoonoses. In: Murrell, K.D. and Fried, B (eds) Food-Borne Parasitic Zoonoses: Fish and Plant-Borne Parasites. Springer, New York, pp. 383–415. Traub, R.J., Monis, P.T., Robertson, I., Irwin, P., Mencke, N. and Thompson, R.C.A. (2004) Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology 128, 253–262. Traub, R.J., Monis, P.T. and Robertson, I.D. (2005) Molecular epidemiology: a multidisciplinary approach to understanding parasitic zoonoses. International Journal for Parasitology 35, 1295–1308. Troeger, H., Epple, H.-J., Schneider, T., Wahnschaffe, U., Ullrich, R., Burchard, G.-D., Jelinek, T., Zeitz, M., Fromm, M. and Schulzke, J.-D. (2007) Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum. Gut 56, 316–317. WHO (1996) Fighting Disease, Fostering Development: The World Health Report 1996. World Health Organization, Geneva.

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Cryptosporidium in Cattle: From Observing to Understanding R. FAYER, M. SANTÍN AND J.M. TROUT United States Department of Agriculture, Beltsville, MD, USA

Abstract Cryptosporidium parvum is a zoonotic pathogen transmissible from a variety of animals to humans and is a considerable public health concern. Dairy cattle have been identified in numerous reports as a major source of environmental contamination with this pathogen. However, the vast majority of these reports have been based on microscopic examination of the organism in faeces from cattle. This chapter traces the progress of research on bovine cryptosporidiosis from the first observations of infection to the present understanding of susceptibility on the part of the bovine host and pathogenicity on the part of the parasites that constitute the taxa under the umbrella of the genus Cryptosporidium. It includes information based on molecular typing with the SSU rRNA gene and subtyping with the GP60 gene, which enables epidemiologists and others to trace the sources of Cryptosporidium-related outbreaks.

Introduction Recent archaeological evidence from Egypt suggests that cattle were herded in prehistoric times, possibly from around 12,500 BC. It is not clear whether these cattle originally came from the ‘Fertile Crescent’. Initially, it is presumed, cattle were killed for food, later they were used as draught animals, and for thousands of years cows’ milk was consumed only by calves. As farming methods evolved, cattle became a major source of protein, milk and milk products, and leather. Over time, livestock production intensified to support growing human populations. Larger and larger herds became concentrated in increasingly smaller areas to maximize the efficiency of production. In industrialized countries, as well as less affluent areas of the world, the consequence of this evolution in animal husbandry was seen through the impact of enteric diseases on neonates, of which cryptosporidiosis is but one. Through the millennia of this close association between humans and cattle, enteric pathogens had billions upon billions of opportunities to adapt and to be readily transmitted between these hosts. Those that have are 12

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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of concern to public health and among them is the species Cryptosporidium parvum. Cryptosporidium infections have been reported in cattle worldwide. Before the development and application of molecular methods to aid in species determination, numerous publications simply documented the presence of Cryptosporidium oocysts in cattle faeces. Subsequently, many other publications have identified Cryptosporidium species in cattle but with limited information on prevalence and herd management. Cryptosporidiosis, especially in calves, has been associated with a wide range of clinical signs from no apparent ill effects to severe morbidity, resulting in poor performance and production losses and in some instances mortality. From the perspective of human health, cattle have often been implicated as a source of zoonotic Cryptosporidium species. Risk of human infection has been based on physical contact with cattle, contamination of fresh fruits and vegetables with manure, and manure runoff from farms into drinking water supplies. With the goal of producing healthy cattle while protecting food and water supplies, studies have been undertaken to obtain a clear understanding of the species of Cryptosporidium that infect cattle, the prevalence of infection, and the relationship of these species to the age of the animals. There has been much progress in defining the Cryptosporidium species, genotypes, and some subtypes present in cattle. This chapter traces the progress of research on bovine cryptosporidiosis from the first observations of infection in cattle to our present understanding of susceptibility and pathogenicity. It includes those parasites that constitute the taxa under the umbrella of the genus Cryptosporidium. Application of this information can benefit both animal and human health.

Early Reports of Cryptosporidiosis in Cattle The first report of bovine cryptosporidiosis appeared in 1971 and described stages of the parasite in tissue sections of the jejunum from an 8-month-old heifer with chronic diarrhoea (Panciera et al., 1971). This observation was soon followed by others in which diarrhoeic dairy and beef calves, 2 weeks old and younger, had similar infections (Barker and Carbonell, 1974; Meuten et al., 1974; Morin et al., 1976). The association between the parasite and illness became stronger when it was reported that cryptosporidia were probably common enteropathogens of calves (Pohlenz et al., 1978) and that Cryptosporidium was a pathogen in experimentally infected calves (Tzipori et al., 1983). A survey of neonatal dairy calves in Maryland, USA, found Cryptosporidium in healthy as well as in diarrhoeic calves (Leek and Fayer, 1984). This finding led to a study of factors contributing to clinical illness in calves experimentally infected with Cryptosporidium obtained from pooled calves’ faeces (Fayer et al., 1985). In that study, clinical illness was not consistently found in neonatal dairy calves experimentally infected with 3.2–30 × 106 oocysts, except when rotavirus and/or Clostridium perfringens was also present. It appeared that Cryptosporidium required the presence of other pathogens to produce illness. These findings gave rise to the following questions: 1. What was different in this study from reports in which cryptosporidiosis in calves was strongly associated with morbidity and mortality?

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2. Were other isolates of the parasite more pathogenic or did other studies simply lack viral and bacterial assessments? 3. Why were some naturally infected calves healthy and others severely ill? At that time there appeared to be a single species of Cryptosporidium that lacked host specificity, and answers to those questions would, in part, require the development of better taxonomic data. In the next few years, cattle were recognized as hosts for two species, C. parvum and C. muris (Anderson, 1987), infecting the intestine and the abomasum, respectively, and represented by small and large oocysts (C. parvum, ~4.5 × 5.5 mm and C. muris, ~5.5 × 7.4 mm). Molecular techniques and cross-species transmission studies eventually identified the abomasal form with the large oocysts as a new species, C. andersoni (Lindsay et al., 2000). Cryptosporidium parvum was then associated with diarrhoea in young calves and C. andersoni with asymptomatic adults. As reports accumulated it became apparent that under farm and field conditions calves acquired infections shortly after birth. The highest prevalence of cryptosporidiosis in cattle was found at 1–3 weeks of age, with oocysts excreted for an average of 12 days and diarrhoea, when present, lasting an average of 8 days (reviewed by Santín and Trout, 2008).

What is the Immune Status of Calves and How Does it Affect Susceptibility to Infection? Cell-mediated immunity had been shown to play an important role in control of C. parvum infections in mouse models, and by the early 1990s it was clear that HIV AIDS patients with low levels of T cells were extremely susceptible to cryptosporidiosis and other opportunistic pathogens, although little was known regarding the immune status of calves. To determine why young calves were so susceptible to infection, studies were designed to identify lymphoid cell populations at the site of infection in the ileum where lymphocytes could respond directly to the parasite (Pasquali et al., 1997; Canals et al., 1998). Intra-epithelial lymphocytes (IEL), and lamina propria lymphocytes (LPL) were collected from C. parvum-infected calves and uninfected control calves; the infected group was inoculated with oocysts at 1–2 days of age. Cells were collected from both groups at 7–9 days of age and analysed for phenotype and cytokine mRNA production. Significant increases in CD2+, CD3+, CD4+ and CD8+ T cells were observed in the IELs of infected versus uninfected calves. These findings were supported by reports of elevated IEL CD8+ T cells in infected versus uninfected calves (Wyatt et al., 1997) and of elevated LPL CD4+, CD8+ T, and g/d T cells in infected versus uninfected calves (Abrahamsen et al., 1997). IELs and LPLs from uninfected calves contained much lower percentages of CD4+ and CD8+ T cells than found in adult cattle (Pasquali et al., 1997) which could explain the age-related susceptibility of neonatal calves while supporting early observations of others that T cell subsets protect against cryptosporidiosis in mice (Ungar et al., 1991; Chen et al., 1993; McDonald et al., 1994) and humans (Flanigan et al., 1992). Correlated with

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the increases in CD4+ and CD8+ IELs was the finding of elevated IFN-g and IL-12 mRNA in IELs and LPLs from the ileum of infected calves. However, no significant increases were detected in mRNA levels for IL-2, IL-4 or IL-10 (Canals et al., 1998). These cytokine and phenotypic cell differences in primary infected versus uninfected neonatal calves indicated that the susceptibility of these young calves to enteric infections had an immunological basis, the absence or extremely low levels of T cells.

What Accounts for the Variability in Severity of Infections? Although pre-weaned calves are highly susceptible, infection with different isolates of C. parvum appears to result in variation in the severity of diarrhoea and number of oocysts produced. Such variation with different isolates has been demonstrated in human infectivity studies and in calves (Pozio et al., 1992; Okhuysen et al., 1999). Clinical cryptosporidiosis could not always be induced in experimental calf infections at inoculating doses of 3.2–30 × 106 C. parvum oocysts per calf (Fayer et al., 1985). Subsequently, inoculation of calves with 1 × 104 oocysts of a bovine C. parvum isolate from Alabama, USA, consistently induced severe clinical disease in experimentally infected calves (Fayer and Ungar, 1986). Another isolate of C. parvum from Auburn, Alabama (AUCP-1) was transmitted from one Cryptosporidium-naïve calf to another (26 calves) over a period of 3 years; these infections consistently resulted in oocyst production rates of 0.3–41.5 × 106/g of faeces for 1 day or more during the patent period, accompanied by profuse diarrhoea (Fayer et al., 1997). Then, unexpectedly, over a period of the next six serial passages through calves, the severity of clinical signs and oocyst output steadily decreased until inoculation with 1.5 × 106 oocysts resulted in no diarrhoea and the recovery of fewer than 1 × 106 oocysts per calf. A similar decrease in productivity and pathogenicity with the same isolate occurred at Colorado State University. The same isolate was also serially transmitted, over a 3-year period, through 40 groups of C57BL/6 mice (four mice per group) immunosuppressed with dexamethasone. The mice showed no clinical signs but oocyst output remained consistently high. When oocysts from the mice were transmitted to a calf, the AUCP-1 isolate once again exhibited the earlier pathogenicity and oocyst production. It was not determined what factors led to the loss of pathogenicity in progressive serial passage through calves or why pathogenicity was unaffected by passage through mice; however, one might speculate that the original isolate contained multiple genetically distinct subpopulations that were selected for or against during passage through a particular host.

Selection In an attempt to use selection to obtain a non-pathogenic precocious strain of C. parvum, an experiment was designed in much the same way as the two live commercial vaccines Paracox and Livacox were selected for in poultry (Fayer, 1994). Ten calves were serially infected with oocysts collected from the previous

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calf on the first day of the patent period. Instead of the expected decrease in pathogenicity, as seen with coccidia, the duration and severity of diarrhoea steadily increased from calf 1 through to calf 9, although no pattern was seen in number of oocysts shed per calf. Perhaps it was the ability of C. parvum to autoinfect, unlike the Eimeria species in the coccidiosis vaccines, that invalidated the concept of a precocious non-pathogenic immunizing strain of C. parvum. These observations of different isolates and of serial passage of specific isolates of C. parvum demonstrate that not only do differences in pathogenicity exist among field isolates but also that both the pathogenicity and fecundity of an isolate can change over time.

Double-stranded RNA Virus-like Particles Double-stranded RNA (dsRNA) virus-like particles have been found in Babesia, Trichomonas, Giardia, Leishmania and Eimeria (Hotzel et al., 1995). Two sizes of extrachromosomal dsRNAs were found in the cytoplasm of sporozoites of C. parvum and C. hominis but not in seven other species of Cryptosporidium (Khramtsov et al., 1997; Khramtsov and Upton, 2000). Sequence analysis showed distinctly different dsRNA sequences in isolates (species) from calves versus those from humans (Khramtsov et al., 2000). Small dsRNA sequences of isolates from 23 calves and 38 humans (Xiao et al., 2001) showed that isolates from the same outbreak had identical sequences; eight distinct nucleotide sequences were from cattle (C. parvum) and ten from humans (C. hominis). If any dsRNAs in Cryptosporidium are associated with pathogenicity this has not been demonstrated, but a recent study with our colleagues (M. Jenkins and J. Higgins, USDA, personal communication) has shown an association with fecundity. Calves infected with C. parvum-Beltsville oocysts produced substantially more oocysts than calves infected with C. parvum-Iowa oocysts. Increased fecundity was correlated with levels of C. parvum virus (CPV) as measured by real-time RT-PCR using CPV RNA-specific primers. The CPV signal in C. parvum-Beltsville sporozoites relative to C. parvum-Iowa was 3–4 times greater as measured by RT-PCR. The greater fluorescence intensity of C. parvum-Beltsville sporozoites labelled with antibodies to CPV 40 kDa capsid protein supported this observation. These findings suggest that CPV affects fecundity, which might in turn affect the severity of infection. What other factors affect severity of infection is not known.

Prevalence of Severe Morbidity and Mortality There is no formal reporting system for cryptosporidiosis in cattle. Reports of cases, surveys and outbreaks provide information, but not on the same basis as the public health reports of ProMed, FoodNet and MMWR. At the US Department of Agriculture in Beltsville, Maryland, telephone calls from farmers and veterinarians attributing high morbidity and mortality to cryptosporidiosis in dairy and beef calves were frequent, peaking in the late 1980s through to the

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early 1990s, but steadily decreased in number until in 2006 no such calls were received. Whether the actual number of severe outbreaks declined or whether the association of severe diarrhoea and death of calves with cryptosporidiosis was no longer a ‘new’ phenomenon, and therefore consultation was no longer sought, is not known. Nevertheless, this informal barometer of activity suggests a change in the frequency of severe and economically important outbreaks perhaps due to a time when an exceptionally pathogenic strain of C. parvum was present.

Species Other than C. parvum Cryptosporidium andersoni, which colonizes the gastric glands of the abomasum, has been reviewed by Santín and Trout (2008). Infections, primarily in calves older than 4 weeks of age, produce no diarrhoea or visible clinical signs. Infections are more chronic (sometimes lasting over a year) and oocyst production is less than that of C. parvum. Elevated plasma pepsinogen and decreased milk production have been attributed to infection. For other species and genotypes infecting cattle (Table 2.1), there are no reports of clinical signs, histological data or subclinical pathology.

Molecular Identification of Species in Cattle For decades, microscopy was the sole method for detecting oocysts, first by direct faecal smears routinely stained, and later stained by IFA techniques. Microscopy Table 2.1. Species and genotypes of Cryptosporidium found in cattle and locations where they have been detected (adapted from Santín and Trout, 2008). Cryptosporidium species/genotypes C. parvum C. bovis C. andersoni C. ryanae C. hominis C. suis C. suis-like Pig genotype II C. felis C. canis

Prevalence

Geographical location

Frequent Frequent Frequent Frequent Rare Rare (2 calves) Rare (3 cattle) Rare (1 cow) Rare (1 cow) Experimental infection only

Worldwide Worldwide Worldwide Worldwide Scotland, India, Korea USA and Zambia Denmark Denmark Poland USA

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was time-consuming and required highly trained personnel. Unless oocysts differed significantly in size, species identification was impossible. PCR was found to be more sensitive than IFA and, when combined with gene sequencing, could differentiate species and genotypes of Cryptosporidium. The most frequently used gene for identification has been the SSU rRNA (small subunit ribosomal RNA) gene (the 18S gene). Molecular analysis of Cryptosporidium from cattle has identified seven species and three genotypes: C. parvum, C. andersoni, C. bovis, C. canis, C. felis, C. hominis, C. suis, C. suis-like genotype, C. ryanae, and Cryptosporidium pig genotype II (Bornay-Llinares et al., 1999; Fayer et al., 2001, 2006; Santín et al., 2004; Smith et al., 2005; Geurden et al., 2006; Park et al., 2006; Starkey et al., 2006; Feng et al., 2007; Langkjær et al., 2007) (Table 2.1).

Prevalence of Species in Cattle In large-scale studies of cattle, C. parvum, C. andersoni, C. bovis and C. ryanae were found most frequently (Santín et al., 2004; Xiao et al., 2004; Fayer et al., 2006; Geurden et al., 2006; Feng et al., 2007; Langkjær et al., 2007). Other species and genotypes were found infrequently or rarely. Crytosporidium hominis was found in a few cattle in Scotland, India and Korea (Smith et al., 2005; Park et al., 2006; Feng et al., 2007), C. suis was found in one calf in the USA and another in Zambia (Fayer et al., 2006; Geurden et al., 2006), C. suis-like genotype and Cryptosporidium pig genotype II were found in three cattle and one cow in Denmark, respectively (Langkjær et al., 2007), C. felis was found in a cow in Poland (Bornay-Llinares et al., 1999), and C. canis infected calves experimentally but natural infections have not been detected (Fayer et al., 2001) (Table 2.1).

Large-scale Cross-sectional Study and Longitudinal Study of Species Prevalence Related to Age of Cattle A large-scale study involving 1615 cattle was conducted over a period of 4 consecutive years (Santín et al., 2004; Fayer et al., 2006, 2007). Each year 15 commercial dairy farms were included, two or three from each of seven states, ranging from Vermont to Florida along the east coast of the USA. Each year an attempt was made to collect faeces from 30 or more cattle from each farm. During the first 2 years, calves from 5 days to 11 months of age were examined. During the third year, dairy heifers 1–2 years of age were examined. During the fourth year, cows over 2 years of age were examined. Most of the farms visited in the first year were visited again in the following years, but in a few cases, because of management practices, either the required age or the required number of cattle were not available and substitute farms were introduced into the study. Based on SSU rRNA gene sequencing from PCR-positive specimens, virtually all infections in pre-weaned calves (<8 weeks of age) were C. parvum, with low levels of C. bovis, C. ryanae and C. andersoni in some calves. The number

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of C. parvum infections decreased markedly in post-weaned calves (2–11 months of age), while the number of calves with C. bovis, C. ryanae and C. andersoni increased; C. bovis was the predominant species in this age group. In cattle older than 12 months, C. andersoni was the most prevalent species. In Denmark, a similar pattern was observed: C. parvum was the predominant species in calves younger than 1 month of age, and C. bovis became the predominant species in calves 3–12 months of age (Langkjær et al., 2007). Both C. bovis and C. ryanae have been found to be geographically widespread in cattle (Santín et al., 2004; Fayer et al., 2006, 2007; Starkey et al., 2006; Feng et al., 2007; Langkjær et al., 2007). Cryptosporidium andersoni constitutes the majority of infections in mature cattle in Denmark, the Czech Republic, Japan and the USA (Enemark et al., 2002; Kvac and Vitovec, 2003; Sakai et al., 2003; Fayer et al., 2006, 2007; Kvac et al., 2006) with the exception that Langkjær et al. (2007) in Denmark, did not detect this species in cattle of any age. The prevalence of Cryptosporidium in cattle declines significantly with age (Santín et al., 2004; Fayer et al., 2006, 2007; Kvac et al., 2006; Langkjær et al., 2007). Therefore, species and genotypes found in mature cattle are actually present in relatively few animals. A longitudinal study involving 30 Holstein cattle on a single dairy farm was conducted over a 2-year period (M. Santín et al., 2008). Faeces were examined consecutively at weekly, two-weekly and then at monthly intervals from 1 week to 24 months of age for the presence of Cryptosporidium oocysts using a twostep nested PCR protocol to amplify the SSU rRNA gene. Every PCR-positive product was purified and sequenced. Cryptosporidium parvum, C. bovis, C. andersoni and C. ryanae were detected in every animal and their presence was strongly associated with the age of the animal. This longitudinal study strongly supports the findings of the cross-sectional studies. In the longitudinal study all pre-weaned calves were infected with C. parvum, followed in the post-weaned cattle by C. bovis and C. ryanae infections, and then in the older cattle C. andersoni infections.

Subtyping of C. parvum and C. hominis Because C. parvum is the zoonotic species most commonly identified in cattle, it became the focus of subtyping to better determine its genetic diversity and to provide a more accurate tool for source tracking. Most subtyping protocols have been based on the 60 kDa glycoprotein gene (GP60). Sequence analysis of GP60 has revealed several subtype families (alleles) and subtypes within those families for C. parvum and C. hominis (Cacciò et al., 2000; Strong et al., 2000; Peng et al., 2001, 2003; Alves et al., 2003; Tanriverdi et al., 2003; Wu et al., 2003; Xiao and Ryan, 2004). Most cattle isolates belong to the C. parvum subtype family IIa, which is the zoonotic subtype family. Within this family the most common subtype found worldwide was IIA15G2R1. For example, in Portugal 61 of 72 isolates were positive for this subtype (Alves et al., 2003, 2006). Likewise, in India 5 of 9 were positive (Feng et al., 2007), and in the USA 135 of 175 were positive for this subtype (Xiao et al., 2007). Greater diversity was found within allele IIa in

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Northern Ireland (Thompson et al., 2007). Subtypes IIaA18G3R1 and IIA15G2R1 were found in 120 and 28 of 214 isolates, respectively. The identity of IIA15G2R1 as the subtype most prevalent in calves and frequently detected in humans worldwide (Strong et al., 2000; Glaberman et al., 2002; Alves et al., 2003, 2006; Peng et al., 2003; Stantic-Pavlinic et al., 2003; Wu et al., 2003; Chalmers et al., 2005; Sulaiman et al., 2005; Abe et al., 2006; Trotz-Williams et al., 2006), as well as other subtypes identified by analysis of the GP60 gene, provides an important tool to accurately identify and track Cryptosporidium of veterinary and public health concern. The recent finding that C. parvum subtypes IIaA15G2R1 and IIaA17G2R1 were detected in stools from 12 laboratory-confirmed case patients who drank apple cider at a school outing in Ohio, and that subtype IIaA17G2R1 was also detected in the remains of some of the cider (Blackburn et al., 2006) illustrates the usefulness of subtyping. It is interesting to note that the same two subtypes were detected in a high percentage of faeces from cattle in Ohio (Xiao et al., 2007). Although these initial findings based on subtyping with GP60 have already served to provide a greater understanding of the transmission dynamics of C. parvum and C. hominis, the number of isolates examined is relatively few, so much more can still be learned. For other species and genotypes of Cryptosporidium affecting cattle and humans, additional typing tools are needed to reach a similar level of identification and understanding.

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Pohlenz, J., Moon, H.W., Cheville, N.F. and Bemrick, W.J. (1978) Cryptosporidiosis as a probable factor in neonatal diarrhea of calves. Journal of the American Veterinary Medical Association 172, 452–457. Pozio, E., Gomez Morales, M.A., Barbieri, F.M. and LaRosa, G. (1992) Cryptosporidium: different behaviour in calves of isolates of human origin. Transactions of the Royal Society of Tropical Medicine and Hygiene 86, 636–638. Sakai, H., Tsushima, Y., Nagasawa, H., Ducusin, R.J., Tanabe, S., Uzuka, Y. and Sarashina, T. (2003) Cryptosporidium infection of cattle in Tokachi District, Hokkaido. Journal of Veterinary Medical Science 65, 125–127. Santín, M. and Trout, J.M. (2008) Cryptosporidiosis of livestock. In: Fayer, R. and Xiao, L. (eds) Cryptosporidium and Cryptosporidiosis, 2nd edn. CRC Press, Boca Raton, FL, pp. 451–483. Santín, M., Trout, J.M., Xiao, L., Zhou, L., Greiner, E. and Fayer, R. (2004) Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Veterinary Parasitology 122, 103–117. Santín, M., Trout, J.M. and Fayer, R. (2008) A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age. Veterinary Parasitology 155, 15–23. Smith, H.V., Nichols, R.A., Mallon, M., Macleod, A., Tait, A., Reilly, W.J., Browning, L.M., Gray, D., Reid, S.W. and Wastling, J.M. (2005) Natural Cryptosporidium hominis infections in Scottish cattle. Veterinary Record 156, 710–711. Stantic-Pavlinic, M., Xiao, L., Glaberman, S., Lal, A.A., Orazen, T., Rataj-Verglez, A., Logar, J. and Berce, I. (2003) Cryptosporidiosis associated with animal contacts. Wiener Klinische Wochenschrift 115, 125–127. Starkey, S.R., Zeigler, P.E., Wade, S.E., Schaaf, S.L. and Mohammed, H.O. (2006) Factors associated with shedding of Cryptosporidium parvum versus Cryptosporidium bovis among dairy cattle in New York State. Journal of the American Veterinary Medical Association 229, 1623–1626. Strong, W.B., Gut, J. and Nelson, R.G. (2000) Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products. Infection and Immunity 68, 4117–4134. Sulaiman, I.M., Hira, P.R., Zhou, L., Al-Ali, F.A., Al-Shelahi, F.A., Shweiki, H.M., Iqbal, J., Khalid, N. and Xiao, L. (2005) Unique endemicity of cryptosporidiosis in children in Kuwait. Journal of Clinical Microbiology 43, 2805–2809. Tanriverdi, S., Arslan, M.O., Akiyoshi, D.E., Tzipori, S. and Widmer, G. (2003) Identification of genotypically mixed Cryptosporidium parvum populations in humans and calves. Molecular and Biochemical Parasitology 130, 13–22. Thompson, H.P., Dooley, J.S.G., Kenny, J., McCoy, M., Lowery, C.J., Moore, J.E. and Xiao, L. (2007) Genotypes and subtypes of Cryptosporidium in neonatal calves in Northern Ireland. Parasitology Research 100, 619–624. Trotz-Williams, L.A., Martin, D.S., Gatei, W., Cama, V., Peregrine, A.S., Martin, S.W., Nydam, D.V., Jamieson, F. and Xiao, L. (2006) Genotype and subtype analyses of Cryptosporidium isolates from dairy calves and humans in Ontario. Parasitology Research 99, 346–352. Tzipori, S., Smith, M., Halpin, C., Angus, K.W., Sherwood, D. and Campbell, I. (1983) Experimental cryptosporidiosis in calves: clinical manifestations and pathological findings. Veterinary Record 112, 116–120. Ungar, B.L., Kao, T.C., Burris, J.A. and Finkelman, F.D. (1991) Cryptosporidium infection in an adult mouse model: independent roles for IFN-gamma and CD4+ T lymphocytes in protective immunity. Journal of Immunology 147, 1014–1022. Wu, Z., Nagano, I., Boonmars, T., Nakada, T. and Takahashi, Y. (2003) Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length

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R. Fayer et al. polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses. Applied and Environmental Microbiology 69, 4720–4726. Wyatt, C.R., Brackett, E.J., Perryman, L.E., Rice-Ficht, A.C., Brown, W.C. and O’Rourke, K.I. (1997) Activation of intestinal intraepithelial T lymphocytes in calves infected with Cryptosporidium parvum. Infection and Immunity 65, 185–190. Xiao, L. and Ryan, U.M. (2004) Cryptosporidiosis: an update in molecular epidemiology. Current Opinion in Infectious Diseases 17, 483–490. Xiao, L., Limor, J., Bern, C. and Lal, A.A. (2001) Tracking Cryptosporidium parvum by sequence analysis of small double-stranded RNA. Emerging Infectious Diseases 7, 141–145. Xiao, L., Fayer, R., Ryan, U. and Upton, S.J. (2004) Cryptosporidium taxonomy: recent advances and implications for public health. Clinical Microbiological Reviews 17, 72–97. Xiao, L., Zhou, L., Santín, M., Yang, W. and Fayer, R. (2007) Distribution of Cryptosporidium parvum subtypes in calves in eastern United States. Parasitology Research 100, 701–706.

3

Names Do Matter D.D. BOWMAN Cornell University, Ithaca, NY, USA

Abstract In zoology, each species is assigned a name by an expert or group of experts on the basis of characters that define an organism or groups of organisms that are designated as type specimens. As more is learned about a genus, the importance of certain characters for species designation may wax and wane, but the type materials are expected to remain constant and are usually held within museum collections for the purpose of later examination by additional experts as the need requires. Historically, in forms that have mating as part of the life cycle, the biological verification of a species has often been done, if possible, by crossing to prove that the offspring are viable and are capable of producing sustainable offspring themselves. Thus, in the case of Cryptosporidium species, crosses are potentially possible for species determination, but are not possible in the case of species within the genus Giardia. In both cases, however, it is expected that type specimens that correlate with the originally defined species will remain available for examination. Specific names sometimes have major impacts and ramifications that extend beyond the simple designations assigned to specimens by the experts naming a species. Thus, drug labels, if at all possible, contain the names of certain species of organisms, unless they can be shown to inactivate or kill more than one member of a species within a genus. Thus, a drug that kills the hookworm, Ancylostoma caninum, may have no effect on the related species, Ancylostoma braziliense; both names will not appear on the label unless the drug is shown to kill both species, and some drugs may kill only one species and not the other. It is possible that similar rationale could be applied to Cryptosporidium parvum in cattle; where a drug would have an effect on this species but would be without effect on the related Cryptosporidium bovis; this is typically what currently occurs with bovine coccidia. If a species of Giardia or Cryptosporidium is designated as zoonotic, regulations may be created narrowly for the control of one species and not another if the means exist for the ready designation of species. In the world of ecology and species protection, species or even subspecies are often protected against environmental encroachment or extinction, and this can have major implications for species protection by public and federal agencies. Names can be very important both scientifically and in the formation of policy.

Introduction The question as to the importance of names is not new. In 1920, Kofoid asked the same major questions regarding the taxonomy of Giardia (and also Cryptosporidium) that are still being asked today: © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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D.D. Bowman The several species which have been described, especially by Benson (1908), are based on slight differences in form and proportions, and Noe (1908) raises the question as to the validity of these distinctions…We are inclined to regard Noe’s scepticism as not well grounded. This matter [of separate species] is one of more than restricted interest for it impinges on the one hand on the general biological question as to how far specificity of parasites to particular hosts obtains, and on the other hand on the very practical question as to whether or not rodents, especially mice, rats, and Belgian hares, are sources of infection to man, for these flagellate parasites, in mice and men at least, are often associated with chronic intestinal disturbances. (Kofoid, 1920)

These questions remain as the major concern of biologists today relative to these genera of organisms. Are different groups of organisms in different hosts restricted to those hosts? What is the zoonotic potential of the different groups of organisms relative to each other? Will the addition of names to the different species and assemblages bring clarity to the problem relative to the public and the medical community, or would they just serve to continue the confusion as to what to say when a diagnosis of cysts and oocysts is made? Would the clarification of species assist in the establishment of public policy for assessing the zoonotic risk of oocysts in drinking water or sewage effluents?

The International Code of Zoological Nomenclature The rules for names are set in the International Code of Zoological Nomenclature, published by the International Commission on Zoological Nomenclature (ICZN, 2006). This organization sets the rules for how names are made, defines what a taxon is and sets rules for how taxa are associated with a given name, decides what constitutes the publication of a name, and even has the power to suppress names for the purpose of maintaining stability. Because protozoa fall under the realm of eukaryotes in the world of zoology, they are covered by the code of zoological nomenclature rather than the codes that exist for fungi and bacteria. The code sets the rules for taxonomy from the names at the family level to the names used at the species level. The name of a species is a binomen (genus and species). The code also allows for the creation of subgenera (species aggregates within a genus) and subspecies within a species; this usage can actually form names consisting of three or four words. The use of species aggregates and subspecies may turn out to be very useful in the naming and categorization of organisms that appear on the many different cladograms and phenograms that have been created from the molecular identification of different clusters or taxa. Again, the code only specifically deals with the family through the species; higher taxonomic classifications are not bound by the code. The code also sets forth what the requirements are for describing a new species. Every new name published after 1999 must be explicitly indicated as intentionally new, the description must be associated with the explicit fixation of a holotype, or syntypes, for the nominal taxon and, where the holotype or syntypes are extant specimens, by a statement of intent that they will be (or have

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been) deposited in a collection along with a statement indicating the name and location of that collection. The code states the rules for publication, and paper publication is required, descriptions solely on the web are not recognized at this time. Authors are also cautioned that they should avoid confusing the literature by citing new names in abstracts or proceedings in a manner that does not really constitute publication, but will cause confusion as to priority. The code also establishes the principle of priority. The first name is the true name. However, it is possible that a name with priority may be discovered that has not been used for a very long time. This can be very confusing if another name is already entrenched in the literature. Thus, it is possible to suppress a name with priority under certain circumstances: Prevailing usage must be maintained when the following conditions are both met: the senior synonym or homonym has not been used as a valid name after 1899, and the junior synonym or homonym has been used for a particular taxon, as its presumed valid name, in at least 25 works, published by at least 10 authors in the immediately preceding 50 years and encompassing a span of not less than 10 years.

The goal here, as stated above, is the maintenance of stability amongst taxa. The standard of the code is the ‘type specimen’. The code goes to great lengths to describe different types and how to define a type: Each nominal taxon in the family, genus or species groups has actually or potentially a name-bearing type. The fixation of the name-bearing type of a nominal taxon provides the objective standard of reference for the application of the name it bears.

And: No matter how the boundaries of a taxonomic taxon may vary in the opinion of zoologists, the valid name of such a taxon is determined from the name-bearing type(s) considered to belong within those boundaries.

The neotype is an important concept describing species for which types may not be in existence or which have been destroyed. This may be used, for example, in the redescription of species of Giardia: Neotype is the name-bearing type of a nominal species-group taxon when no name-bearing type specimen (i.e. holotype, lectotype, syntype or prior neotype) is believed to be extant and an author considers that a name-bearing type is necessary to define the nominal taxon objectively.

And: A neotype is validly designated when there is an exceptional need and only when that need is stated expressly and when the designation is published with the following particulars: a statement that it is designated with the express purpose of clarifying the taxonomic status or the type locality of a nominal taxon; a statement of the characters that the author regards as differentiating from other taxa the nominal species-group taxon for which the neotype is designated; data and description sufficient to ensure recognition of the specimen designated; the author’s reasons for believing the name-bearing type specimen(s) (i.e. holotype, or lectotype, or all syntypes, or prior neotype) to be lost or destroyed, and the steps that

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D.D. Bowman had been taken to trace it or them; evidence that the neotype is consistent with what is known of the former name-bearing type from the original description and from other sources; however, a neotype may be based on a different sex or life stage, if necessary or desirable to secure stability of nomenclature; evidence that the neotype came as nearly as practicable from the original type locality and, where relevant, from the same geological horizon or host species as the original namebearing type; a statement that the neotype is, or immediately upon publication has become, the property of a recognized scientific or educational institution, cited by name, that maintains a research collection, with proper facilities for preserving name-bearing types, and that makes them accessible for study.

The code specifically states that the word ‘genotype’ should not be used in matters taxonomic. The reason is simply for clarity (from the Glossary of the Code: ‘Genotype: A term not recognized by the Code, formerly used for type species, but that should not now be used in zoological nomenclature’). The reasoning seems straightforward enough, and having access to words like ‘assemblage’ and ‘clade’, there probably are other highly useful alternatives to the use of the word ‘genotype’.

Ramifications In 1922, Hegner described Giardia canis very carefully according to the standards of the day. Also, he deposited type material: ‘The types and cotypes are deposited in the collections of the Department of Medical Zoology, School of Hygiene and Public Health, Johns Hopkins University’ (Hegner, 1922). The work on assemblages has shown that dogs may have their own Giardia in the form of assemblages C&D. So, what is to be done with Hegner’s description? Can the types be found? If so, what good are they for modern study? They were probably fixed with mercuric chloride and stained with haematoxylin after treatment with ferric ammonium sulphate; the chances seem good, therefore, that there will be no DNA available for molecular analysis. So will neotypes have to be selected? Will assemblages C&D turn into subspecies, or will the assemblages be recognized instead as subgenera? At the same time, for a redescription of Giardia canis and neotype selection, care would have to be taken to try and represent a collection of organisms similar to those that Hegner had in mind in his original description.

Splitters Splitters are currently holding sway in the taxonomy of Giardia and Cryptosporidium. The dissection of Cryptosporidium parvum has moved along relatively easily because there were almost no species described before the development of modern molecular methods. However, in the case of Giardia, there were many species described previously, so the creation of new names is much more complicated. This leaves open the question as to what is required to name a species, and instead of talking species, we discuss assemblages.

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Splitting can have major ramifications, especially in the field of regulatory affairs and liability. Classic cases include the environmental ramifications caused by the description of the snail darter, the spotted owl, and various subspecies of gorillas (Etnier, 1976; Noon and McKelvey, 1996; Jensen-Seaman et al., 2004). These have all had major fiscal, ecological and social implications for large geographical areas, with huge amounts being spent on litigation and management expenses. The same thing has happened with Giardia and Cryptosporidium. During the period when they were each considered to consist of a single species infecting all mammals other than rodents, the great fear was zoonosis, mainly fuelled by the devastating effects of cryptosporidiosis in AIDS patients. Giardia and Cryptosporidium received NIH funding, there were all sorts of funds spent for improved diagnostic methods for cysts and oocysts in drinking water, regulations were passed by the EPA with regard to allowable levels in water, and there was significant discussion by the EPA and USDA about the regulation of cysts and oocysts leaving confined animal feeding operations (CAFOs). Very large amounts of money and effort were expended. So, now the splitters are in charge. The discovery that the only major zoonotic species of Cryptosporidium in cattle is Cryptosporidium parvum, which is found only in young calves, means that it is going to be easier to manage the relatively small quantity of calf waste for pathogen control. Similarly, the recognition of Cryptosporidium canis and Cryptosporidium felis as parasites of dogs and cats, respectively, that only very, very rarely occur in people, can put pet owners’ and veterinarians’ minds at ease when dealing with a diagnosis in the family pet. Concerns about liability are real. If a pet is shedding Giardia cysts, is it zoonotic? Dogs without any clinical signs have been treated repeatedly with the goal of removing all cysts from their faeces. In some cases, this has resulted in very large bills for the client for an infection that did not seem to be bothering the dog and very probably was not putting the family at serious risk. The use of a separate name for the species found in the dog would make it easier for the veterinarian to tell the owners that they did not have to worry, and the veterinarian would not have to worry about the possibility of having to appear in court to defend his decision not to treat the asymptomatic animal.

Conclusion Names matter greatly, both for scientists and the public. Names should bring together information that adds to clarity as to what a species really is, should assist in understanding basic biology and assumed phylogenetic relationships, and allow discussion as to what risks a given species may pose to the health of humans and animals. The system does seem to be working in the realm of these two pathogens, and the taxonomy is sorting itself out. The work and methods being applied are giving us new insights on a regular basis. The problem is that the discoveries resulting in new taxonomy and systematics of the two different groups are being made too quickly for some and too slowly for others. However, it does appear that the community is learning and that it is working towards a sense of consensus that, of course, will probably never reach perfect agreement

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among gatherings of hundreds of very interested scientists intimately familiar with many different aspects of the organisms under discussion.

References Etnier, D.A. (1976) A new percid fish from the Little Tennessee River, Tennessee. Proceedings of the Biological Society of Washington 88, 469–488. Hegner, R.W. (1922) A comparative study of the Giardias living in man, rabbit, and dog. American Journal of Hygiene 2, 442–454. ICZN (2006) International Commission on Zoological Nomenclature. Available at http:// www.iczn.org/index.htm Jensen-Seaman, M.I., Sarmiento, E.E., Deinard, A.S. and Kidd, K.K. (2004) Nuclear integrations of mitochondrial DNA in gorillas. American Journal of Primatology 63, 139–147. Kofoid, C.A. (1920) A critical review of the nomenclature of human intestinal flagellates Cercomonas, Chilomastix, Trichomonas, Tetratrcihomonas, and Giardia. University of California Publications in Zoology 20, 145–168. Noon, B.R. and McKelvey, K.S. (1996) Management of the spotted owl: a case history of conservation biology. Annual Reviews in Ecology and Systematics 27, 135–162.

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Centenary of the Genus Cryptosporidium: From Morphological to Molecular Species Identification J. ŠLAPETA University of Sydney, NSW, Australia

Abstract The biology and species diversity of the genus Cryptosporidium is mystifying for many protozoologists and even more so for non-specialists. Historically, two morphologically distinct parasites of the gastrointestinal tract were originally described by Ernest E. Tyzzer from mice (i.e. Cryptosporidium muris and Cryptosporidium parvum). Later, these two parasites were thought to parasitize all mammals, including cattle. However, such a scheme became insupportable and human-to-human transmission is now associated with Cryptosporidium hominis. The causative parasite of the zoonotic animal-to-human transmission is Cryptosporidium pestis. In this chapter, all the species names applied to the genus Cryptosporidium are reviewed in the light of historical records and molecular taxonomy initiatives. A DNA approach to taxonomy stands on the implicit assumption that the reference databases used for comparison are sufficiently complete and feature-rich, with annotated entries. However, the uncertain taxonomic reliability of annotations in public DNA repositories forms a major obstacle to sequence-based species identification. Finally, but importantly, a huge gap exists between the number of described names and the number of identified genotypes. The closure of this gap represents a prime challenge for the decades to come.

Introduction Species of the genus Cryptosporidium have attracted a great deal of attention since the late 1970s as an object for epidemiological and molecular biological studies. However, while going through the published records, one is quickly confronted with the masked identity of the organism. There is a dramatic difference between the features of C. parvum as reported in 1980, 2000 and nowadays. The huge number of names and the jargon used to describe some isolates, along with the inability to continuously culture these parasites in vitro, has become a major obstacle to the comparison of experimental results from different laboratories over the century of existence of the genus Cryptosporidium, which was first described by Ernest E. Tyzzer (Fig. 4.1). © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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Fig. 4.1. Portrait of Ernest E. Tyzzer, who described Cryptosporidium muris from a mouse stomach in 1907. Courtesy of the National Library of Medicine, negative no. 85-204.

Traditionally, taxonomy is based on morphology. However, for microbes such as Cryptosporidium species, morphological differences are minute or do not exist at all. This fact has been repeatedly emphasized, and additional characteristics, including site of infection and host specificity, proved to be useful for species identification. But while these biological characteristics were useful, they are largely impractical for routine identification. Thus, an accurate identification technique using molecular methods has been particularly welcome. Using molecular techniques, formerly neglected organisms have been rediscovered and others have become topical. The following is an account of advances in the taxonomy of the genus Cryptosporidium. The aim was to consider old reports based on morphology in the context of more recent work on DNA sequencing, which is generating ‘barcodes’ of these life forms. A consensus is presented within the rules of the International Commission of Zoological Nomenclature (1999) along with an annotated checklist of each species. Additionally, as part of the description, available DNA accession numbers of small subunit rDNA (SSU rDNA), Cryptosporidium oocyst wall protein 1 (COWP1), actin, and cytosolic 70 kDa heat shock protein (HSP70)

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gene sequences from reference specimens are tabulated as molecular identifiers of the respective species.

Mounting Complexity: the Value of Host Specificity Cryptosporidium was an obscure genus and very few reports were published prior to the 1980s. The genus Cryptosporidium Tyzzer 1907 was established for C. muris parasitizing mouse gastric glands (Tyzzer, 1907, 1910). A related sporozoan from the mouse intestine was named C. parvum (Tyzzer, 1912). Tyzzer correctly identified and experimentally verified the life cycle and, based on his histological findings, he correctly speculated about autoinfection within the host. It all seemed relatively simple, and common wisdom dictated that each host would have its own species. Providing the proof for this concept was very illfated. Vetterling et al. (1971) published a revision of the taxonomy and amended the description of the genus Cryptosporidium. Ironically, in contrast to Tyzzer’s original precise work, Vetterling et al. (1971) failed to identify oocysts as a source of infection. At the same time, Vetterling et al. (1971) described host-specific C. wrairi from the guinea pig. Unfortunately, C. wrairi has a peculiar nature, since it is a species never recovered from the host in the wild but only reported on a handful of occasions in guinea pig laboratory colonies. Nevertheless, host specificity was established as the sole character for species identification within the genus Cryptosporidium. Follow-up papers started to introduce new names haphazardly, solely on the basis of different host species. The life cycle and transmission were deemed elusive until the end of the 1970s (Bird and Smith, 1980) despite accumulating information on the significance of the parasite in immunocompromised hosts. Oocysts, the exogenous stage, were re-demonstrated as the source of the infection, and the complete life cycle was experimentally confirmed and validated on C. felis (Iseki, 1979). The genus description was amended to include the oocyst with four sporozoites in accordance with Tyzzer’s (1910) early description. Using the putative species host specificity, many new species were quickly described and named (i.e. Barker and Carbonell, 1974). Other evidence, however, suggested the existence of only a single species in the genus (Tzipori et al., 1980). Increasing medical interest due to the recognition of acquired immune deficiency syndrome (AIDS) caused an avalanche of publications describing numerous aspects of Cryptosporidium from the early 1980s onwards. Early studies supported the animal-to-human transmission of Cryptosporidium spp., but some data already suggested person-to-person transmission or human infections not acquired directly from infected animals (Casemore et al., 1985). Host specificity studies and morphological diagnostic techniques definitely delimitated the life cycle and identified oocysts as a primary cause of infection (Current and Reese, 1986; Fayer and Ungar, 1986). Experimental infections and ultrastructural studies provided evidence for the thin-walled, autoinfective oocysts and recycling type I meronts, thereby explaining overwhelming host infections after exposure to inocula containing small numbers of oocysts (Current and Reese, 1986).

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To simplify the mounting complexity of the taxonomy, Levine (1984) reviewed existing cross-transmission experiments and hosts of Cryptosporidium spp. and suggested the existence of only a single species in each of the animal groups: mammals, birds, reptiles and fish. This idea did not agree with the original description for the localization of gastric C. muris and intestinal C. parvum in mice (Tyzzer, 1912). A response to Levine’s paper was published by Upton and Current (1985), clearly identifying two morphologically distinct Cryptosporidium spp. in cattle. Both C. parvum and C. muris were deemed to be prototype species for all mammalian species until further evidence suggested otherwise (Upton and Current, 1985). Early isoenzyme analysis and later polymerase chain reaction (PCR)-based diagnostic techniques provided new evidence for the existence of human-tohuman, animal-to-human and waterborne transmission (Awad-el-Kariem et al., 1995; Morgan et al., 1995). Introduction of DNA sequencing provided the data to identify genetic variants affecting different or even the same host species. It seemed logical to believe that individual hosts each have their own unique hostspecific Cryptosporidium spp., as well as species that are less host-specific affecting a wider spectrum of hosts (Xiao et al., 2004). Molecular tools provided the techniques for accurate diagnosis and genesequencing-generated data which allowed the construction of molecular phylogenies mapping the evolutionary relationships between individual species and isolates. All this effort led to the reopening of the question: ‘Is the number of species currently recognized sufficient, or do we need to define more?’

Systematics and Initial Nomenclatural Considerations Systematics is a fundamental discipline in biology, consisting of three ideally inseparable processes: (i) the identification of a species with its characteristics and the description of a type specimen; (ii) applying taxonomy to name a species or taxon; and (iii) phylogenetics, which describes the relationships between species and other taxa. The major product of systematics is a classification system of species. The system, an anthropocentric view on the species history, is a flexible structure of arbitrary taxa. Understandably, there is controversy about the boundaries of each taxon, due to the existence of several opinions on what constitutes a species, i.e. species concepts. Before one starts to talk about species in the genus Cryptosporidium, it is important to distinguish between two distinct elementary concepts. 1. The species is a biological entity that is represented by a distinct population of individuals. The definition of a species is a controversial issue and many theories exist, each concept favouring some aspects over others (e.g. biological species concept, phylogenetic species concept, etc.). Importantly, the species is based on the preferences of the group of researchers and their own local judgement, experience and consensus applying the ‘whatever species concept’ in favour at the time. With no strict rules preset, the biologists in each field tend to be either those who prefer to group several possible species into a single species, thus

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representing the ‘lumpers’ category; or those who tend to divide existing species into new species (who are therefore called ‘splitters’). In the case of parasites, clinicians often seem to represent the first category, aligning with the conservative, less name-rich scenario, unlike zoologists, who constantly modify and adjust the systematic scheme. 2. The name is the binominal name of a species consisting of Latin genus and species names, and is an arbitrary but very useful identifier of the species. The name must be unique, to make communication easier between researchers. In contrast to the species definition, the rules for naming species are set very strictly within an international code. In the case of the genus Cryptosporidium, as well as for the majority of the parasitic protozoa, this is the International Code of Zoological Nomenclature (ICZN), specifically the 4th edition (International Commission on Zoological Nomenclature, 1999), which supersedes all previous editions as from 1 January 2000. It stands on the holotype, the name-bearing specimen of the species that the author has used for the original description. The holotype is deposited in a museum and is the reference material for the species. However, in the sense of protistan terminology, the term holotype (= type) is substituted with hapantotype, i.e. one or more preparations consisting of directly related individuals representing distinct stages of the life cycle (Article 72.5.4, 73.3). Importantly for Cryptosporidium spp., the type (= hapantotype) is the specimen or specimens illustrated and/or photographed or described, but not the illustration/photograph itself or its description (Article 72.5.6). Thus, in fact, for the majority of protozoa there is no original museum material for subsequent investigators to use for comparative purposes, apart from the original printed publication. On the other hand, the fact that the specimens illustrated no longer exist or cannot be traced does not invalidate the type designation (Article 73.1.4). The genus Cryptosporidium was traditionally classified within coccidia, where the oocysts contain, in general, sufficient morphological differences to allow identification. Such a model worked quite well for the scenario described by Upton and Current (1985) with respect to distinguishing between the stomach and the intestinal species in cattle. The morphological detail of the oocyst proved quite practical for species such as Eimeria, and high-quality micrographs of sporulated oocysts along with validated data were considered to form an acceptable substitute for the type specimens (Duszynski, 1999). However, taking into consideration the 4th edition of the ICZN (International Commission on Zoological Nomenclature, 1999), such a practice had two drawbacks: (i) illustration is not a syntype, i.e. name-bearing type (Article 72.5); and (ii) the type must be fixed (Article 72.3). Even so, Cryptosporidium oocysts possess very few morphological characteristics and consequently the identity of Cryptosporidium species cannot usually be assigned using oocyst morphology alone (Fall et al., 2003); therefore such illustrations are of limited diagnostic and descriptive value. Taking into account all of the above, all existing names in the genus Cryptosporidium were re-evaluated in accordance with the ICZN and categorized as follows: 1. The names are reported as nomen dubium (s.)/nomina dubia (pl.) for those species that are species inquirenda (s.)/species inquirendae (pl.), that are impossible

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to recognize and identify again but where the name was based on correct rules of the code. These species are placed to one side until a detailed review is initiated. 2. The names incorrectly defined, i.e. without respecting ICZN, are assigned as nomen nudum (s.)/nomina nuda (pl.). The name is unavailable, but upon new description can be made available for the same or a different concept. For species where there is evidence of an original misidentification of a different organism, the names and species are removed from Cryptosporidium. 3. Otherwise, the name is valid. So much for the technicalities of taxonomy; now let’s apply the above to the Cryptosporidium species names (Table 4.1). In total, throughout the 100 years that the genus Cryptosporidium has been in existence, 37 names have been introduced. Of these, four names are nomina nuda and five have been removed from the genus as being different organisms (e.g. Sarcocystis crotali – syn. Cryptosporidium crotali). The remaining 28 names are treated here as available names; nevertheless, five of these currently need redescription (i.e. C. anserinum, C. agni) or remain aside due to insufficient information (i.e. C. rhesi, C. garnhami). This leaves us with 23 names to be associated with individual species. Here, I recognize in total 21 species; thus two names are reported as synonyms (C. tyzzeri, C. saurophilum). On one hand, the majority of these have a dominant host, but on the other hand, they are accidentally found in possibly aberrant hosts; as is evidenced by rare findings of diverse Cryptosporidium isolates in human and calf faeces. The exception to this rule is the zoonotic C. pestis, which has the potential to temporarily affect the majority of mammalian hosts, and C. meleagridis, which is known to be the only species affecting birds as well as mammals (Table 4.1).

Nucleotide Sequence Data: Reference Material – ‘Barcode’ Approach Nucleotide data have played a dominant role in revealing the epidemiology and Cryptosporidium population structure. While Cryptosporidium isolates remain difficult to grow in vitro, the use of PCR and animal models have provided muchneeded characterization of many former morphologically defined species. Several single-locus analyses provided a secure foundation for multiple previously uncharacterized species. The leading role was given to SSU rDNA as the most universal marker, soon followed by additional conservative markers (actin, HSP70). Supportive evidence has also come from COWP1. Characterization of the sequence of these four genes from a diverse selection of isolates has provided the groundwork for follow-up studies that mapped new isolates within the existing diversity of each of these genes. These four markers are the universal and dominant markers used to characterize individual isolates, and the availability of existing sequences makes them a suitable entry point for any survey. Since the late 1990s there has been a steady increase in the number of individual sequences in public databases (Fig. 4.2). The SSU rDNA gene remains the preferred

Update on Cryptosporidium spp. names and species status.

Name

Available/ Unavailable

1 2 3 4 5 6 7

C. muris Tyzzer 1907 C. parvum Tyzzer 1912 C. crotali Triffit 1925 C. vulpis Wetzel 1938 C. baikalika Matschoulsky 1947 C. meleagridis Slavin 1955 C. tyzzeri Levine 1961

Available Available Removed* Removed* Removed* Available Available

8

Removed*

12

C. lampropeltis Anderson, Duszynski & Marquardt 1968 C. ameivae Arcay de Peraza & Bastardo de San Jose 1969 C. ctenosauris Duszynski 1969 C. wrairi Vetterling, Jervis, Merill & Sprinz 1971 C. agni Barker & Carbonell 1974

13

C. anserinum Proctor & Kemp 1974

Available

14

C. bovis Barker & Carbonell 1974

Available

15

C. cuniculus Inman & Takeuchi 1979

Available

NN

9 10 11

Host range NS

*

Homo

Valid – redescribed by Tyzzer (1910) Valid

1 2

M M

Yes

Valid Subjectively invalid (junior subjective synonym with C. meleagridis)

3

MB

Yes

4

M

5

M

6

M

Name status/Species status

Unavailable

Nomen nudum/incomplete description

Removed* Available

Valid

Available

Subjectively invalid (nomen dubium/species inquirenda) Subjectively invalid (nomen dubium/species inquirenda) Valid – redescribed by Fayer, Santín and Xiao (2005) Valid

Bos

Centenary of the Genus Cryptosporidium

Table 4.1.

Yes

37

(Continued)

38

Table 4.1.

Continued Host range

Name

16

C. felis Iseki 1979

Available

Valid

17

C. rhesi Levine 1980

Available

18 19

C. serpentis Levine 1980 C. garnhami Bird 1981

Available Available

20

Available Unavailable

Nomen nudum/incomplete description

Available

Valid

Available Unavailable

Subjectively invalid (nomen dubium/species inquirenda) Nomen nudum/incomplete description

Available

Valid

11

Available Available

Valid Valid

12 13

F F

28

C. nasoris Hoover, Hoerr, Carlton, Hinsman & Ferguson 1981 C. enteritidis Payne, Lancaster, Heinzman & McCutchan 1983 C. baileyi Current, Upton & Haynes 1986 C. curyi Ogassawara, Benassi, Larsson & Hagiwara 1986 C. villithecum Paperna, Landsberg & Ostrovska 1986 C. varanii Pavlásek, Lávicˇková, Horák, Král & Král 1995 C. cichlidis Paperna & Vilenkin 1996 C. reichenbachklinkei Paperna & Vilenkin 1996 C. saurophilum Koudela & Modrý 1998

Subjectively invalid (nomen dubium/species inquirenda) Valid – redescribed by Tilley et al. (1990) Subjectively invalid (nomen dubium/species inquirenda) Valid

Available

29

C. galli Pavlásek 1999

Subjectively invalid (junior subjective synonym with C. varanii) Valid – redescribed by Ryan et al. (2003)

14

B

21 22 23 24 25 26 27

Available

Name status/Species status

NS

*

Homo

Bos

7

M

Yes

Yes

8

R

9

F

10

B

J. Šlapeta

NN

Available/ Unavailable

31 32

33

34 35

36

37

C. andersoni Lindsay, Upton, Owens, Morgan, Mead & Blagburn 2000 C. canis Fayer, Trout, Xiao, Morgan, Lal & Dubey 2001 C. blagburni Morgan, Monis, Xiao, Limor, Sulaiman, Raidal, O’Donoghue, Gasser, Murray, Fayer, Blagburn, Lal & Thompson 2001 C. hominis Morgan-Ryan, Fall, Ward, Hijjawi, Sulaiman, Fayer, Thompson, Olson, Lal & Xiao 2002 C. molnari Alvarez-Pellitero and Sitja-Bobadilla 2002 C. suis Ryan, Monis, Enemark, Sulaiman, Samarasinghe, Read, Buddle, Robertson, Zhou, Thompson & Xiao 2004 C. scophthalmi Alvarez-Pellitero, Quiroga, Sitjà-Bobadilla, Redondo, Palenzuela, Padrós, Vázquez & Nieto 2004 C. pestis Šlapeta 2006

Available

Valid

15

M

Yes

Yes

Available

Valid

16

M

Yes

Unavailable

Nomen nudum/incomplete description

Available

Valid

17

M

Yes

Yes

Available

Valid

18

F

Available

Valid

19

M

Yes

Yes

Available

Valid

20

F

Available

Valid

21

M

Yes

Yes

Centenary of the Genus Cryptosporidium

30

* (from Cryptosporidium) Notes: NN – name number; NS – currently recognized species number; M, mammal; B, bird; R, reptile; F, fish.

39

40

J. Šlapeta

Number of sequences in nucleotide databases

500 small subunit rDNA oocyst wall protein 1 400

actin 70 kDa heat shock protein 60 kDa glycoprotein precursor

300

200

100

0 1997

1998

1999

2000

2001 2002 Year

2003

2004

2005

2006

Fig. 4.2. The increasing numbers of individual SSU rDNA, COWP1, actin, HSP70 and GP60 gene sequences of Cryptosporidium spp. in nucleotide databases over the 10 years from 1997 to 2006.

marker in recent studies, with over 430 available sequences to compare, followed by COWP1 with some 130 available sequences. The sequence numbers of actin and HSP70 are appreciably lower, due to difficulties with the amplification of these genes from some materials. While the above genes remain extremely useful in primary characterization of the individual isolates, especially from animal hosts, it soon became apparent that for fine epidemiological studies these markers are too conservative. A 60-kDa glycoprotein precursor (GP60) proved to be the ideal marker for the elucidation of the predominant human-derived isolates. Nowadays, besides SSU rDNA, human epidemiological studies require characterization of GP60, with the comparison of over 260 available sequences being possible (Fig. 4.2). These numbers underestimate the real number of genotypes made, due to the absence of published genotyping results in the nucleotide databases. The current practice of submitting only the unique sequences, while referencing the identity to known sequences should be avoided. Curators of the primary nucleotide databases (NCBI, EMBL, DDBJ) recommend the submission of complete datasets of all sequences regardless of the identity match with already available sequences. Such a practice, with thorough annotation, will lay the foundations for quantitative meta-analyses with direct reference to features associated with these sequences, i.e. host species. The available sequences in public databases are an extremely valuable source of information; nevertheless, they represent secondary material and thus, in general, their reliability may be questionable (Harris, 2003; Morrison, 2006).

Centenary of the Genus Cryptosporidium

41

As well as the quality of the nucleotides reported, the accuracy and the depth of annotation remains a serious issue (Morrison, 2006; Nilsson et al., 2006). First, individual submitters provide their preferred species name, which is then retained in the database. However, the definition of a species often differs between researchers and over time. Since the late 1990s, ten new names have been introduced (Table 4.1). For many of these species, DNA has been the primary source of evidence for their distinct status. First these individuals acquired type/isolate/ genotype status within known species or only as a Cryptosporidium sp. For example, C. canis sequence accession numbers may be found in three distinct organism ranks despite representing a single valid species; i.e. SSU rDNA is AF112576, but this accession number retains its definition as ‘Cryptosporidium parvum strain CPD1’, COWP1 is AF266274, defined as ‘Cryptosporidium sp. 715’, HSP70 AF221529 is defined as ‘Cryptosporidium parvum isolate 244’, and finally actin AF382340 is correctly defined as ‘Cryptosporidium canis isolate 715’. Second, the annotation of many sequences does not provide sufficient information to associate the sequence with its host, isolate number, country of origin, etc. The use of common names for hosts is insufficient, i.e. monkey or snake. It may be argued that such information appears in the paper; however, in many cases, this information is absent there as well. It is now imperative to take advantage of the ‘features’ entry currently available during the process of sequence submission including: ‘country’, ‘specific_host’, ‘isolate’ and ‘dev_stage’. Unfortunately, the popular genotype status is not a recognized feature, but generally it is acceptable to provide this information in the ‘note’ field. To reduce the ambiguity present in the database derived from a diverse array of submitters, I provide a table with reference sequence accession numbers (Table 4.2). This table aims to list all available full-length sequences, complete where possible, for each named species of the genus Cryptosporidium (Table 4.1). I have taken all precautions to choose the most representative and complete sequence, if multiple sequences exist in databases. These sequences can be viewed as the ‘barcodes’ of individual species; moreover, as more data become available they will be improved and extended. Where possible, I chose to use only single sequences, to avoid future misinterpretations resulting from the construction of a consensus sequence. One exception is the SSU rDNA of C. galli, which is a consensus of two overlapping partial sequences. The four genes SSU rDNA, COWP1, actin and HSP70 are the most commonly used markers in genotyping analyses and species delimitation studies. The hypervariability of the GP60 marker prohibits its use in species identification; moreover, it is only available for the study of dominant populations which infect humans. The 21 recognized species are relatively well represented within the four genes (Table 4.2). The most problematic species are the fish species (five species), with only the assumed C. molnari species represented by SSU rDNA and actin. The COWP1 sequence is missing for C. bovis, C. cuniculus and C. galli. The list and completeness of the sequences may seem very strong, but multiple sequence alignments reveal the hidden incompleteness of many sequences in public databases. The partial sequence representation is further reflected in Cryptosporidium surveys, where only a diagnostic hypervariable region (400–800 bp) is amplified and sequenced, i.e. the use of SSU rDNA primers targeting the hypervariable

42

Table 4.2.

Reference sequences for named Cryptosporidium spp. SSU rDNA

COWP1

actin

HSP70

C. andersoni C. baileyi C. bovis C. canis C. cichlidis C. cf. cuniculus C. felis C. galli C. hominis C. hominis draft genome TU502 strain&&

AF093496 L19068 AY741305 AF112576 n.a. AY120901 AF108862 AF316624+AY168847*** AF108865 chromosome 2: Chro.rrn013 (6112–7866) chromosome 2: Chro.rrn021 (334–2082) chromosome 2: Chro.rrn022 (21763–23515) chromosome 7: Chro.rrn016 (23535–25283) chromosome 8: Chro.rrn005 (599–1001) chromosome 8: Chro.rrn006 (1064–1516) AF112574

AF266262 AF266276 n.a. AF266274 n.a. n.a. AF266263 n.a. AF266265 chromosome 6: Chro.60244

AF382352 AF382346 AY741307 AF382340 n.a. AY120924 AF382347 AY163901 AF382337 chromosome 5: Chro.50056

AF221542 AJ310880 AY741306 AF221529 n.a. AY273775 AF221538 AY168849 AF221535 chromosome 2: Chro.20010

AF266266

AF382351

AF221537

AY524773 AB089284

n.a. AF161579

AY524772 AF382350

n.a. AF221543

C. meleagridis (syn. C. tyzzeri) C. cf. molnari C. muris

J. Šlapeta

Species (alphabetically)

n.a. AF112571 AF108864 chromosome 1: cgd1_5 (46–743) chromosome 2*: cgd2_? (200603–202360) chromosome 7: cgd7_5535 (1278301–1278425**) chromosome 7: cgd7_7 (1582–3330) chromosome 8: cgd8_5425 (1155551–1156729)

n.a. AF266268 BX538351.1:61269..66140 chromosome 6: cgd6_2090

n.a. AF382343 M86241# chromosome 5: cgd5_3160

n.a. AF221530 U11761## chromosome 2: cgd2_20

C. reichenbachklinkei C. scophthalmi C. serpentis C. suis C. varanii (syn. C. saurophilum) C. wrairi

n.a. n.a. AF151376 AF115377 AF112573

n.a. n.a. AF266275 AF266270 AF266277

n.a. n.a. AF382353 AF382344 AF382349

n.a. n.a. AF221541 AF221533 AF221540

AF115378

U35027+U35028

AF382348

AF221536

Centenary of the Genus Cryptosporidium

C. nasoris C. parvum C. pestis C. pestis draft genome IOWA strain&

Notes: n.a. – sequences are not available; SSU rDNA – small subunit ribosomal DNA, >1500 bp (complete or almost complete) is desirable for comparative purposes; COWP1 – oocyst wall protein with type I and type II cysteine-rich repeats, oocyst EB module wall protein (Templeton et al., 2004); HSP70 – 70 kDa heat shock protein, cytosolic form; & – draft genome sequence of C. pestis IOWA (Abrahamsen et al. 2004, AAEE01000000) has five SSU rDNA (some are partial); for the purpose of this table chromosome 7: cgd7_7 sequence serves as reference sequence; LeBlancq et al. (1997) identified the total of five copies and two slightly different types of rDNA units per genome of C. pestis KSU-1: referred to as Type A (AF015772) and Type B (AF308600); && – draft genome sequence of C. hominis TU502 (Xu et al. 2004, AAEL01000000) has six SSU rDNAs (some partial); for the purpose of this table chromosome 7: Chro.rrn016 and chromosome 2: Chro.rrn022 as reference sequences; * – on chromosome 2, cgd2_1375 (299599–301356) is currently erroneously annotated as SSU rDNA, a different non-annotated region on the same contig AAEE01000005 is the true SSU rDNA; ** – cgd8_5425 (1155551–1156706); here several additional nucleotides are added as of the contig; *** – the sequence is concatenated from two overlapping partial sequences; # – the sequence from chromosome 3 (Kim et al., 1992) but genome projects of C. pestis Iowa and C. hominis TU502 both identified this gene on chromosome 5 (Abrahamsen et al., 2004; Xu et al., 2004); ## – the sequence reported by Khramtsov et al. (1995); see also mRNA sequence U69698 – unpublished, GenBank.

43

small subunit rDNA C. pestis IOWA (1749 nt) C. hominis TU502 (1753 nt)

200

150

100

50

0 0

5 -2

0

40

70 60 50 40 30 20 10 0

actin C. pestis IOWA (1131 nt) C. hominis TU502 (1131 nt)

70 kDa heat shock protein C. pestis IOWA (2022 nt) C. hominis TU502 (2034 nt)

1 50

0

51

0 10

0

0

5 12

01

0 15

51

0

01

5 17

0

0 20

51

30 25 20 15 10 5 0 0

0

80

7 12 15 10 17 Sequence length category (nt)

0

5 -7

35

5 -2

oocyst wall protein 1 C. pestis IOWA (4872 nt) C. hominis TU502 (4863 nt)

0 0 0 0 0 0 0 0 0 25 -50 -75 100 125 150 175 200 500 1 01 1- 1- 1- 1- 1- 15 5 75 00 25 50 75 00 2 1 1 2 1 1 Sequence length category (nt)

0

0 -5

1 25

Number of sequences in the length category

J. Šlapeta

0

0

0 -5

1 25

1 50

5 -7

0

0

51

0 10

01

5 12

7 10 Sequence length category (nt)

Number of sequences in the length category

Number of sequences in the length category

Number of sequences in the length category

44

0-

40 35 30 25 20 15 10 5 0

0 0 0 0 0 0 0 0 0 25 -50 -75 100 125 150 175 200 225 1 01 1- 1- 1- 1- 1- 15 2 5 75 00 25 50 75 00 2 1 1 1 1 Sequence length category (nt)

0-

Fig. 4.3. Representation SSU rDNA, COWP1, actin and HSP70 sequences available in primary nucleotide databases (NCBI/EMBL, DDBJ). Individual sequences were categorized according to their length. The full length of individual genes from C. pestis and C. hominis draft genomes is given above the graph.

region by Johnson et al. (1995), CPB-DIAGF and CPB-DIAGR primers or COWP1 by Spano et al. (1997), and Cry-15 and Cry-9 primers (Fig. 4.3). Many of the sequence entries for HSP70 and COWP1 are short in sequence length, in contrast to actin, where many species are almost fully sequenced. Moreover, many of the currently recognized genotypes are difficult to examine further due to the generation of only partial sequences by investigators.

Centenary of the Genus Cryptosporidium

45

It should be stressed that every effort should be made to generate full-length sequences for already existing species, genotypes or new genotypes being described. These sequences represent a permanent record upon which an investigation for biological characters can be based. To avoid the confusion that arises from the rapidly expanding complexity of species names and number of sequences generated in different laboratories since the late 1990s, a website has been developed (‘Taxonomy of the genus Cryptosporidium’ at http://www.vetsci.usyd.edu.au/staff/JanSlapeta/). This website aims to provide a common gateway for analysing Cryptosporidium species and to standardize the terminology of individual isolates. Alignments of the reference sequences from the named species are provided, with further detailed annotation of individual species. Furthermore, currently under development is a webbased analysis (BLAST, multiple sequence alignment, tree building) as well as an in-house annotation database of sequences to streamline and standardize the process for the identification of Cryptosporidium isolates.

Will the Real Cryptosporidium parvum Please Stand Up? When reviewing the large amount of information on the human and bovine derived isolates, it became apparent that the mouse species, C. parvum Tyzzer 1912, described and illustrated by Tyzzer (1912), is different from the commonly encountered species affecting humans and cattle (Šlapeta, 2006). Ernest E. Tyzzer described the parasite from the intestine of tame mice in his Harvard laboratory (Tyzzer, 1912). The species was easily transmissible to mice and was present in great numbers in the intestines of affected mice. In the absence of the type material and with a lack of data about the host specificity of Tyzzer’s original material, we need to subjectively align the available data with the current information. Two studies provided noteworthy details on Cryptosporidium spp. in mice (Mus musculus). The first study, by Klesius et al. (1986), determined up to 30% incidence of cryptosporidiosis in mice at a calving site and susceptibility of calves to some of these oocysts from mice faeces. In the second study, Morgan et al. (1999) genotyped many samples of Cryptosporidium spp. from mice obtained worldwide and genetically characterized a specific ‘mouse genotype’ using several independent nuclear loci distinct from known genotypes. This ‘mouse genotype’ was genetically different from the ‘bovine’ and ‘human’ genotypes previously identified in Australia and Maryland, USA, and lumped under the umbrella name C. parvum (Xiao et al., 2004). Thus, the genotyping of mice isolates provided additional evidence for the existence of the ‘bovine genotype’ in wild mice (Morgan et al., 1999). Consequently one can speculate that it was this ‘bovine genotype’ that was infectious for calves in the study of Klesius et al. (1986). This ‘bovine genotype’ essentially infects only neonatal mice and only affects adult mice transiently (Korich et al., 2000), unlike authentic rodent C. parvum isolates that produce heavy infections in adult laboratory mice (Bednarska et al., 2003). No experimental calf infections were conducted using the true C. parvum ‘mouse genotype’. Despite this fact, cattle and human isolates of Cryptosporidium

46

J. Šlapeta

spp. have attracted considerable attention in recent years, but no ‘mouse genotype’ (C. parvum) has been identified (Xiao et al., 2002; Santín et al., 2004). Therefore, C. parvum (C. parvum ‘mouse genotype’) appears to be host-specific, because either there are no natural calf or human infections or they represent an extremely rare case. It is generally agreed that the species that Tyzzer used for his description is indeed the ‘mouse genotype’ (Šlapeta, 2007; Xiao et al., 2007). An urgent step towards the stability of historical information is to apply Tyzzer’s name to the species he used for the description and to use appropriate names for the two dominant human species. The above findings led to the recent taxonomic treatment and support for the identity of the ‘mouse genotype’ with the original description, type host, experimental transmission and illustrations of C. parvum (Šlapeta, 2006). Cryptosporidium parvum sensu Tyzzer, 1912 is a host-adapted species of Mus musculus with no documented capacity to infect either humans or domestic cattle.

Consensus, Reversibility and Universality Two species which can by supported by morphological characteristics and by localization and development in the host are C. muris and C. parvum. The larger C. muris is found in the stomach, whereas the intestine is the location of the smaller C. parvum. The status quo of only these two species affecting mammals is retained by Upton and Current (1985), and is suggested to be practical unless further evidence shows otherwise. However, a diverse spectrum of hosts and distinct DNA has provided further evidence of multiple lineages with different evolutionary history, and thus provides only preliminary evidence for a species. Older names synonymized under the umbrella names C. parvum and C. muris have been re-erected as full species (i.e. C. felis, C. wrairi). Those for which no name was thought to be available and whose identification was based on DNA sequencing have acquired ‘genotype’ status or have been named as new species. However, mixing the latter with the former has introduced inconsistency to what is C. parvum. Indeed, the name C. parvum has lost its purpose; it is no longer a unique identifier. Genotypes now seem to be much more useful descriptors, i.e. human genotype, mouse genotype, canine genotype, snake genotype, bovine genotype, etc. Nevertheless, this is all work in progress. To reinstate some stability to some of the major and clinically important genotypes, backed up by epidemiological data and experimental information, some were logically named as new species. Thus, the status quo of Upton and Current (1985) was abandoned and the following consensus was practically applied to cattle species. The abomasum is parasitized by C. andersoni and at least two named species are recognized to affect the intestine of cattle, the hostspecific C. bovis and the zoonotic C. pestis (Šlapeta, 2006). This approach logically aligns with the recognition of C. hominis which, along with C. pestis, were formerly classified as human and bovine genotypes of C. parvum.

Centenary of the Genus Cryptosporidium

47

Nevertheless this opinion is capable of reverting to the former conservative opinion, thus justifying the continuity of the names according to the ICZN, i.e. grouping C. hominis, C. parvum and C. pestis under the oldest available name, which is C. parvum, thereby reinstating the terms ‘human genotype’, ‘mouse genotype’ and ‘bovine genotype’, respectively. Underlying genetic differences have the potential to be used to distinguish between multiple taxa. While morphologically it is hard to find any differences, and host specificity provides only a partial picture, the use of DNA provides a virtual but very specific and repeatable label for each individual. If even a minor genetic difference is detected then it may reflect the niche adaptation, and so should be viewed as a practical tag for identification. Hence, those populations causing significant disease will stand out from the crowd and ultimately acquire a unique name. Indeed, the name C. hominis is now used for the dominant species transmitted from human to human, unequivocally typified by the draft genome of TU502 (Xu et al., 2004). Similarly, C. pestis is the name for the already known zoonotic species for which the draft genome of the Iowa strain is available (Abrahamsen et al., 2004). With the acceptance of both C. pestis and C. hominis we are clarifying the identity of the medically and veterinary important species. The synopsis for the three species is as follows: Cryptosporidium parvum Tyzzer, 1912 Syn. Cryptosporidium parvum ‘mouse genotype’ Xiao et al. (2002, 2004) Type host: Laboratory mouse, Mus musculus Type locality: Harvard Laboratory, Massachusetts, USA Cryptosporidium hominis Morgan-Ryan, Fall, Ward, Hijjawi, Sulaiman, Fayer, Thompson, Olson, Lal & Xiao, 2002 Syn. Cryptosporidium parvum ‘human genotype’ Xiao et al. (2002) Type host: Human, Homo sapiens Type locality: Perth, Western Australia, Australia Cryptosporidium pestis Šlapeta, 2006 Syn. Cryptosporidium parvum ‘bovine genotype’ Xiao et al. (2002, 2004) Type host: Domestic cattle, Bos taurus Type locality: Iowa, USA

Future Challenges Historically, two morphologically distinct populations of parasites of the gastrointestinal tract were originally described by Ernest E. Tyzzer from mice (i.e. Cryptosporidium muris and C. parvum). Nowadays, DNA sequences are increasingly seen as primary information sources for species identification in many organism groups, including Cryptosporidium. Such approaches stand on the implicit assumption that the reference databases used for comparison are sufficiently complete and feature-rich, with annotated entries. However, the uncertain taxonomic reliability and lack of annotations in public DNA repositories form a major obstacle to sequence-based species identification. The current taxonomic

48

J. Šlapeta

expansion of the genus Cryptosporidium is important relative to the dominant host, pathogenicity and genetic diversity. Unfortunately only few taxonomically available and named species have so far been molecularly characterized which, however, does not invalidate them from scientific usage. On the other hand, data from molecular epidemiological studies constantly reveal new genetic variants (= genotypes), but the lack of sufficient annotation and additional biological characteristics prohibits their elevation to species and ultimately taxonomic recognition. Nevertheless, descriptions of new genotypes remain an essential part of our understanding of the host specificity, the diversity and the epidemiology of the genus Cryptosporidium. In addition to the poor annotation of sequences in public DNA repositories, a huge gap exists between the number of described names and the number of identified genotypes. The closure of this gap represents a prime challenge for the decades to come.

Acknowledgements I would like to thank the organizers of the Second International Giardia and Cryptosporidium Conference (II IGCC) for their invitation to present the paper which forms the basis for this chapter. Financial support for the preparation of this article and participating in the II IGCC was partly provided by the Faculty of Veterinary Science, University of Sydney. I also thank Professor John Ellis (University of Technology Sydney) for editorial suggestions. I apologize to those authors whose work could not be cited owing to space limitations.

References Abrahamsen, M.S., Templeton, T.J., Enomoto, S., Abrahante, J.E., Zhu, G., Lancto, C.A., Deng, M., Liu, C., Widmer, G., Tzipori, S., Buck, G.A., Xu, P., Bankier, A.T., Dear, P.H., Konfortov, B.A., Spriggs, H.F., Iyer, L., Anantharaman, V., Aravind, L. and Kapur, V. (2004) Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304, 441–445. Awad-el-Kariem, F.M., Robinson, H.A., Dyson, D.A., Evans, D., Wright, S., Fox, M.T. and McDonald, V. (1995) Differentiation between human and animal strains of Cryptosporidium parvum using isoenzyme typing. Parasitology 110, 129–132. Barker, I.K. and Carbonell, P.L. (1974) Cryptosporidium agni sp.n. from lambs, and Cryptosporidium bovis sp.n. from a calf, with observations on the oocyst. Zeitschrift für Parasitenkunde 44, 289–298. Bednarska, M., Bajer, A., Kulis, K. and Sinski, E. (2003) Biological characterisation of Cryptosporidium parvum isolates of wildlife rodents in Poland. Annals of Agricultural and Environmental Medicine 10, 163–169. Bird, R.G. and Smith, M.D. (1980) Cryptosporidiosis in man: parasite life cycle and fine structural pathology. Journal of Pathology 132, 217–233. Casemore, D.P., Sands, R.L. and Curry, A. (1985) Cryptosporidium species a 'new' human pathogen. Journal of Clinical Pathology 38, 1321–1336. Current, W.L. and Reese, N.C. (1986) A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice. Journal of Protozoology 33, 98–108.

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Duszynski, D.W. (1999) Revisiting the code: clarifying name-bearing types for photomicrographs of protozoa. Journal of Parasitology 85, 769–770. Fall, A., Thompson, R.C.A., Hobbs, R.P. and Morgan-Ryan, U.M. (2003) Morphology is not a reliable tool for delineating species within Cryptosporidium. Journal of Parasitology 89, 399–402. Fayer, R. and Ungar, B.L. (1986) Cryptosporidium and cryptosporidiosis. Microbiological Reviews 50, 458–483. Fayer, R., Santín, M. and Xiao, L. (2005) Cryptosporidium bovis n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus). Journal of Parasitology 91, 624–629. Harris, D. (2003) Can you bank on GenBank? Trends in Ecology and Evolution 18, 317–319. International Commission on Zoological Nomenclature (1999) International Code of Zoological Nomenclature (ICZN), London, 306 pp. Available at: http://www.iczn.org/ iczn/index.jsp Iseki, M. (1979) Cryptosporidium felis sp. n. (Protozoa: Eimeriorina) from the domestic cat. Japanese Journal of Parasitology 28, 285–307. Johnson, D.W., Pieniazek, N.J., Griffin, D.W., Misener, L. and Rose, J.B. (1995) Development of a PCR protocol for sensitive detection of Cryptosporidium in water samples. Applied and Environmental Microbiology 61, 3849–3855. Khramtsov, N.V., Tilley, M., Blunt, D.S., Montelone, B.A. and Upton, S.J. (1995) Cloning and analysis of a Cryptosporidium parvum gene encoding a protein with homology to cytoplasmic form Hsp70. Journal of Eukaryotic Microbiology 42, 416–422. Kim, K., Gooze, L., Petersen, C., Gut, J. and Nelson, R.G. (1992) Isolation, sequence and molecular karyotype analysis of the actin gene of Cryptosporidium parvum. Molecular and Biochemical Parasitology 50, 105–113. Klesius, P.H., Haynes, T.B. and Malo, L.K. (1986) Infectivity of Cryptosporidium sp. isolated from wild mice for calves and mice. Journal of the American Veterinary Medical Association 189, 192–193. Korich, D.G., Marshall, M.M., Smith, H.V., O’Grady, J., Bukhari, Z., Fricker, C.R., Rosen, J.P. and Clancy, J.L. (2000) Inter-laboratory comparison of the CD-1 neonatal mouse logistic dose-response model for Cryptosporidium parvum oocysts. Journal of Eukaryotic Microbiology 47, 294–298. LeBlancq, S.M., Khramtsov, N.V., Zamani, F., Upton, S.J. and Wu, T.U. (1997) Ribosomal RNA gene organization in Cryptosporidium parvum. Molecular and Biochemical Parasitology 90, 463–478. Levine, N.D. (1984) Taxonomy and review of the coccidian genus Cryptosporidium (Protozoa, Apicomplexa). Journal of Protozoology 31, 94–98. Morgan, U.M., Constantine, C.C., O’Donoghue, P., Meloni, B.P., O’Brien, P.A. and Thompson, R.C. (1995) Molecular characterization of Cryptosporidium isolates from humans and other animals using random amplified polymorphic DNA analysis. American Journal of Tropical Medicine and Hygiene 52, 559–564. Morgan, U.M., Sturdee, A.P., Singleton, G., Gomez, M.S., Gracenea, M., Torres, J., Hamilton, S.G., Woodside, D.P. and Thompson, R.C. (1999) The Cryptosporidium “mouse” genotype is conserved across geographic areas. Journal of Clinical Microbiology 37, 1302–1305. Morrison, D.A. (2006) Phylogenetic analyses of parasites in the new millennium. Advances in Parasitology 63, 1–124. Nilsson, R., Ryberg, M., Kristiansson, E., Abarenkov, K., Larsson, K. and Koljalg, U. (2006) Taxonomic reliability of DNA sequences in public sequence databases: a fungal perspective. PLoS ONE 1, e59.

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J. Šlapeta Ryan, U.M., Xiao, L., Read, C., Zhou, L., Lal, A.A. and Pavlasek, I. (2003) Identification of novel Cryptosporidium genotypes from the Czech Republic. Applied and Environmental Microbiology 69, 4302–4307. Santín, M., Trout, J.M., Xiao, L., Zhou, L., Greiner, E. and Fayer, R. (2004) Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Veterinary Parasitology 122, 103–117. Šlapeta, J. (2006) Cryptosporidium species found in cattle: a proposal for a new species. Trends in Parasitology 22, 469–474. Šlapeta, J. (2007) Response to Xiao et al.: Further debate on the description of Cryptosporidium pestis. Trends in Parasitology 23, 42–43. Spano, F., Putignani, L., McLauchlin, J., Casemore, D.P. and Crisanti, A. (1997) PCRRFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin. FEMS Microbiology Letters 150, 209–217. Templeton, T.J., Lancto, C.A., Vigdorovich, V., Liu, C., London, N.R., Hadsall, K.Z. and Abrahamsen, M.S. (2004) The Cryptosporidium oocyst wall protein is a member of a multigene family and has a homolog in Toxoplasma. Infection and Immunity 72, 980–987. Tilley, M., Upton, S.J. and Freed, P.S. (1990) A comparative study on the biology of Cryptosporidium serpentis and Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae). Journal of Zoo and Wildlife Medicine 21, 463–467. Tyzzer, E.E. (1907) A sporozoan found in the peptic glands of the common mouse. Proceedings of the Society for Experimental Biology and Medicine 5, 12–13. Tyzzer, E.E. (1910) An extracellular coccidium, Cryptosporidium muris (gen. et sp. nov.) of the gastric glands of the common mouse. Journal of Medical Research 23, 487–509. Tyzzer, E.E. (1912) Cryptosporidium parvum (sp. nov.), a coccidium found in the small intestine of the common mouse. Archiv fur Protistenkunde 26, 394–412. Tzipori, S., Angus, K.W., Campbell, I. and Gray, E.W. (1980) Cryptosporidium: evidence for a single-species genus. Infection and Immunity 30, 884–886. Upton, S.J. and Current, W.L. (1985) The species of Cryptosporidium (Apicomplexa: Cryptosporidiidae) infecting mammals. Journal of Parasitology 71, 625–629. Vetterling, J.M., Jervis, H.R., Merrill, T.G. and Sprinz, H. (1971) Cryptosporidium wrairi sp. n. from the guinea pig Cavia porcellus, with an emendation of the genus. Journal of Protozoology 18, 243–247. Xiao, L., Sulaiman, I.M., Ryan, U.M., Zhou, L., Atwill, E.R., Tischler, M.L., Zhang, X., Fayer, R., Lal, A.A. (2002) Host adaptation and host–parasite co-evolution in Cryptosporidium: implications for taxonomy and public health. International Journal for Parasitology 32, 1773–1785. Xiao, L., Fayer, R., Ryan, U. and Upton, S.J. (2004) Cryptosporidium taxonomy: recent advances and implications for public health. Clinical Microbiological Reviews 17, 72–97. Xiao, L., Fayer, R., Ryan, U. and Upton, S.J. (2007) Response to the newly proposed species Cryptosporidium pestis. Trends in Parasitology 23, 41–42. Xu, P., Widmer, G., Wang, Y., Ozaki, L.S., Alves, J.M., Serrano, M.G., Puiu, D., Manque, P., Akiyoshi, D., Mackey, A.J., Pearson, W.R., Dear, P.H., Bankier, A.T., Peterson, D.L., Abrahamsen, M.S., Kapur, V., Tzipori, S. and Buck, G.A. (2004) The genome of Cryptosporidium hominis. Nature 431, 1107–1112.

5

Molecular Epidemiology of Human Cryptosporidiosis in Developing Countries L. XIAO Centers for Disease Control and Prevention, Atlanta, GA, USA

Abstract Genotyping and subtyping tools have been used to characterize the transmission of human cryptosporidiosis in developing countries. Thus far, five Cryptosporidium spp. – C. hominis, C. parvum, C. meleagridis, C. canis and C. felis – are responsible for most Cryptosporidium infections in both immunocompetent and immunocompromised individuals. In most areas, C. hominis is responsible for over 70% of human cryptosporidiosis cases, with C. parvum accounting for 10–20% of infections. Differences have been observed among endemic areas in the proportion of infections due to each species, and in some areas C. meleagridis is also endemic. Results of subtyping suggest that there is high genetic heterogeneity in C. hominis in developing countries, and in these areas, human infections with C. parvum and other species are mostly the result of anthroponotic rather than zoonotic transmission. There is geographical segregation in C. hominis or C. parvum subtypes. Mixed and sequential infections with different Cryptosporidium species/genotypes and subtypes are common. Differences in oocyst shedding and clinical presentation have been observed among Cryptosporidium species and C. hominis subtypes. These findings reveal the diversity of cryptosporidiosis transmission in endemic areas and highlight the need for more extensive studies of cryptosporidiosis epidemiology in diverse areas with a wide spectrum of socioeconomic and environmental conditions.

Introduction Cryptosporidiosis is prevalent in developing countries, perhaps due to the high intensity of environmental contamination and poor hygiene conditions. Children in these highly endemic areas develop cryptosporidiosis very early in life, with peak infections usually occurring before 2 years of age. There is usually a strong association between the occurrence of cryptosporidiosis and the rainy season in tropical areas, or the cooler months in dry areas. These countries often have less intensive animal husbandry. Thus, the transmission of cryptosporidiosis in developing countries is probably different from that in the industrialized nations. Even though most molecular epidemiological studies of cryptosporidiosis have © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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been conducted in the industrialized nations, an increasing number of studies are being done in developing countries. This has significantly improved our understanding of cryptosporidiosis transmission in these areas. This includes a better knowledge of parasite diversity, the roles of various transmission routes in cryptosporidiosis epidemiology, and the significance of parasite genetics in pathogenesis and clinical presentations. These developments have enabled health officials to better educate the public about the risk factors involved in the acquisition of cryptosporidiosis in vulnerable populations.

Molecular Epidemiological Tools Genotyping tools Many genotyping tools have been used to differentiate between Cryptosporidium species in humans. The PCR primers of earlier tools were mostly based on antigenic, structural, housekeeping genes and unknown genomic fragments of C. parvum, and included various formats of detection and differentiation (Xiao and Ryan, 2004; Cacciò, 2005). With few exceptions, most of these techniques can efficiently differentiate C. parvum, C. hominis and perhaps C. meleagridis, but are unlikely to amplify some of the more distant species (such as C. canis, C. felis, C. muris and C. andersoni). Therefore, these genotyping tools are mostly replaced by the genus-specific PCR-RFLP techniques based on the SSU rRNA gene, which have higher sensitivity and allow broad species detection.

Subtyping tools Subtyping tools are increasingly used in epidemiological studies of C. parvum and C. hominis. Several types of genetic targets are used in the development of subtyping tools, including microsatellites and minisatellites, double-stranded (ds) RNA elements, and ITS-2. In developing countries, the most widely used subtyping tool is DNA sequencing of the 60 kDa glycoprotein (GP60) gene. The GP60 gene is similar to a microsatellite sequence by having tandem repeats of the serine-coding trinucleotide TCA/TCG/TCT at the 5′ end of the gene. However, in addition to variations in the number of trinucleotide repeats, there are extensive sequence differences in the non-repeat regions, which categorize C. parvum and C. hominis each to several subtype families (alleles). Some of the common subtype families are Ia, Ib, Id, Ie and If for C. hominis and IIa, IIc, IId and IIe for C. parvum (Fig. 5.1). Members of different subtype families differ from each other extensively in the primary sequences. Within each subtype family, subtypes differ from each other mostly in the number of trinucleotide repeats TCA, TCG and TCT (mostly seen in Ie). It should be kept in mind that GP60 and other subtyping tools (including the MST and MLST tools described below) do not amplify the DNA of C. felis, C. canis and other species distant from C. parvum and C. hominis.

Molecular Epidemiology of Human Cryptosporidiosis in Developing Countries 100

IIi-Akiyoshi-154

100

0.1 substitutions/site

53

Id-AF164497 I-Akiyoshi-90

100

70

II-Kenya-9589R IIe-7870

82

II-Alpaca-13987 IIj-NI-8934 IIf-7490 I-Peru9911

100 98

Ia-AF164502 Ig-NI3986

99

IId-4833 IIb-4736

99 100

IIg-Akiyoshi-66

100

FelisAY700394.1| 99

IIa-4804: Zoonotic C. parvum Ib-4746

100

Ie-5632 67

If-4755

55 IIh-Akiyoshi-112

52

II-kenya-9408R

67

IIc-4742: Anthroponotic C. parvum III-DQ067570

100 98

III-4500 III-295

Fig. 5.1. Phylogenetic relationships between the known subtype families of C. hominis (Ia, Ib, Id, Ie, If, etc.) and C. parvum (IIa, IIc, IId, IIe, etc.) based on a neighbour-joining analysis of the GP60 gene sequences. Sequences starting with III were derived from C. meleagridis. Two major anthroponotic and zoonotic C. parvum subtype families (shown in boxes) are identified.

MLT and MLST The recent genome sequencing of C. parvum and C. hominis has allowed the identification of microsatellite and minisatellite sequences in C. parvum and C. hominis genomes and other targets that are highly polymorphic between C. parvum and C. hominis. They are frequently used in multilocus analysis to increase the typing resolution. Two types of techniques are used in typing. In multilocus typing (MLT), variation in microsatellites and minisatellites is assessed on the basis of length variations, using polyacrylamide gel electrophoresis or the GeneScan technology (Ngouanesavanh et al., 2006). This allows the use of

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many targets in the MLT techniques economically. The second kind of typing technique, multilocus sequence typing (MLST), relies on the detection of genetic heterogeneity by DNA sequencing of the amplified PCR products (Gatei et al., 2006a, 2007). Compared to MST, MLST allows the detection of nucleotide substitution and the inclusion of markers with single nucleotide polymorphisms.

Proper use of molecular epidemiological tools Caution should be exercised in interpreting typing results, as accurate diagnosis of species and subtypes has important implications for understanding infection sources. In two studies in India, Cryptosporidium mouse genotype was identified in one child and one HIV-positive adult, based on results of RFLP analysis of the SSU rRNA PCR products (Muthusamy et al., 2006; Ajjampur et al., 2007). The true identity of the parasite was probably C. meleagridis, which has the same RFLP pattern as the mouse genotype in the genotyping technique used. The detection of C. andersoni in a Malawian child was also largely based on RFLP analysis (Morse et al., 2007), which needs to be confirmed by DNA sequencing, as DraI and AseI RFLP was mainly used in species differentiation and C. muris may also share the same pattern. The use of RFLP instead of DNA sequencing in differentiating GP60 subtypes should be avoided, as some subtype families share high sequence homology in one region but not in other regions of the gene, and many new subtype families have been found since the initial development of the RFLP differentiation tool. The sequence mosaic in the gene is also responsible for the polyphyletic nature of both C. parvum and C. hominis in the gene (Fig. 5.1). Thus, the identification of IIa and If subtype families in humans in southern India (Muthusamy et al., 2006) needs to be confirmed by the results of DNA sequencing. The subtype family name Ic is still occasionally used by some researchers to describe C. parvum (Muthusamy et al., 2006; Ajjampur et al., 2007), even though this was the result of an initial misidentification of species at the SSU rRNA locus (Strong et al., 2000), and the subtype family was renamed as IIc some time ago (Alves et al., 2003). IIc should be used to avoid unnecessary confusion. In one study (Ajjampur et al., 2007), the so-called Cryptosporidium mouse genotype GP60 sequence was probably from C. meleagridis, and the C. felis GP60 sequences were probably not from C. felis.

Cryptosporidium Species and Genotypes in Humans In a similar way as for people living in the industrialized nations, five Cryptosporidium spp. are responsible for most human Cryptosporidium infections in developing countries; including C. hominis, C. parvum, C. meleagridis, C. canis and C. felis. They were initially found in otherwise healthy children in Peru in a longitudinal cohort study using a SSU rRNA-based genotyping tool (Xiao et al., 2001), but have also recently been found in diarrhoeic children in Kenya (Gatei et al., 2006b). One large-scale study of Ugandan children reported only

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Table 5.1. Distribution of common human-pathogenic Cryptosporidium species in children in developing countries. No. of isolate C. hominis C. parvum C. meleagridis C. felis C. canis Reference

Location China India India Kenya Malawi Malawi Uganda Iran Guatemala Lima, Peru Chile

5 50 58

5 47 47

0 0 7

175 37 43 444

153 35 25 326

15 2 10 85

7

3

4

15 85 4

14 67 2

1 8 2

1?

1 3

1

2

3

1

2

2 5

7

Peng et al. (2001) Gatei et al. (2007) Ajjampur et al. (2007) Gatei et al. (2006b) Peng et al. (2003) Morse et al. (2007) Tumwine et al. (2003) Meamar et al. (2007) Xiao et al. (2004) Xiao et al. (2001) Neira-Otero et al. (2005)

C. hominis, C. parvum and C. meleagridis, but the genotyping tool used is not capable of detecting C. canis, C. felis and other species genetically distant from C. parvum and C. hominis (Tumwine et al., 2003). Other studies examined only small numbers of specimens, which was probably responsible for the low species diversity detected (Table 5.1). These five Cryptosporidium spp. have also been found in immunocompromised people in developing countries (Table 5.2). Studies conducted in Thailand, India and Peru showed the presence of these five species in HIV-positive adults (Gatei et al., 2002b; Cama et al., 2003). Small-scale studies in other areas have also revealed the presence of some of the five species in HIV-positive adults or children (Table 5.2). In Lima, Peru, there is no significant difference in the distribution of the five species between children and HIV-positive adults (Xiao et al., 2001; Cama et al., 2003). It is likely that other Cryptosporidium species can infect humans under certain circumstances. Cryptosporidium muris has been found in human cases in Indonesia (Katsumata et al., 1998), Kenya (Gatei et al., 2002a, 2006b), Peru (Palmer et al., 2003) and India (Muthusamy et al., 2006). Other Cryptosporidium species found in humans in developing countries include C. suis in one HIVpositive adult and Cryptosporidium cervine genotype in a child, both in Lima, Peru (Xiao et al., 2002; V. Cama et al., unpublished). New Cryptosporidium genotypes will probably be found in humans in future, even though these parasites account for a very small proportion of Cryptosporidium infections in humans.

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Table 5.2.

Distribution of common human-pathogenic Cryptosporidium species in HIV-positive people in developing countries.

Location

Age

Vellore, India Bangkok, Thailand

Adults 4 children, 25 adults Adults Adults Adults Children Children Children Adults Adults Mostly adults Adults Adults

Bangkok, Thailand Taiwan Kenya Malawi Ugandaa South Africa Iran Venezuela Colombia Haiti Lima, Peru a Including

No. of isolate C. hominis C. parvum C. meleagridis C. felis C. canis Reference 48 29

31 24

9

1 + 1 (?) 3

5 1

34 4 24 6 76 21 8 10 6 49 302

17 2 14 6 56 16 1 8 3 31 204

5

7 1 1

3 1

8 14 5 7 1 2 16 34

Muthusamy et al. (2006) Tiangtip and Jongwutiwes (2002) 2

3

1 1? 38

1 1? 10

12

Gatei et al. (2002b) Hung et al. (2007) Gatei et al. (2003) Peng et al. (2003) Tumwine et al. (2005) Leav et al. (2002) Meamar et al. (2007) Certad et al. (2006) Navarro-i-Martinez et al. (2006) Ngouanesavanh et al. (2006) Cama et al. (2003)

nine HIV-negative children.

L. Xiao

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Cryptosporidium hominis Infection In developing countries, more than 70% of Cryptosporidium infections in children are caused by C. hominis in most of the areas studied (Table 5.1). In these areas, children acquire cryptosporidiosis very early in life, with peak incidence of infections occurring between 1 and 2 years of age. Under such conditions, the transmission of cryptosporidiosis is probably mostly through a direct person-to-person route rather than an indirect waterborne or foodborne route. This is probably responsible for the high proportion of infections attributable to C. hominis. This predominance of C. hominis in humans in developing countries applies also to HIV-infected adults (Table 5.2). A diarrhoea outbreak caused by C. hominis also occurred in a daycare centre in São Paulo, Brazil (Goncalves et al., 2006). Molecular characterizations of C. hominis isolates have revealed the complexity of cryptosporidiosis epidemiology. Despite the seemingly lower diversity of Cryptosporidium in humans at the species/genotype level in developing countries, the results of GP60 subtyping have revealed the complexity of Cryptosporidium transmission in endemic areas. This is evident from the existence of many C. hominis subtype families in each endemic area. Thus, 3–4 C. hominis subtype families were seen in humans in India, Peru, Kenya, Malawi and South Africa (Leav et al., 2002; Peng et al., 2003; Xiao et al., 2004; Ajjampur et al., 2007; Cama et al., 2007; Gatei et al., 2007). In these areas, the complexity of transmission is frequently reflected by the existence of many subtypes within C. hominis subtype families Ia and Id in one endemic area (Leav et al., 2002; Peng et al., 2003; Cama et al., 2007; Gatei et al., 2007). The high C. hominis heterogeneity in developing countries is probably an indicator of intensive and stable cryptosporidiosis transmission in the area. Four common C. hominis subtype families – Ia, Ib, Id and Ie – have been found in humans in many developing countries (Xiao et al., 2004) (Fig. 5.1). Nevertheless, there are geographical differences in their distribution. For example, Ia, Ib and Id are common in Malawi, South Africa, India and Peru (Leav et al., 2002; Peng et al., 2003; Ajjampur et al., 2007; Cama et al., 2007; Gatei et al., 2007). Children in South Africa were commonly infected with If (erroneously named as Ie in the publication), a subtype family not seen in most other studies (Leav et al., 2002). In contrast, the common subtype family Ie was not seen in cryptosporidiosis in South African children (Leav et al., 2002). Two space–time clusters of Ia infections were identified in children in southern India (Ajjampur et al., 2007), but it is unclear whether each cluster was caused by one Ia subtype, as many subtypes of the Ia subtype family are usually present in an area (see above). Within each subtype family, one subtype is frequently seen in certain areas but not in others. For example, there are only two common subtypes within C. hominis subtype family Ib: IbA9G3 and IbA10G2. The former is commonly seen in Malawi, Kenya and India, whereas the latter is commonly seen in South Africa, Botswana, Jamaica and Peru (Leav et al., 2002; Cama et al., 2007; Gatei et al., 2006a, 2007; L. Xiao et al., unpublished). This geographical segregation of C. hominis subtypes becomes much more obvious when specimens from different countries are genetically compared using a MLST tool (Gatei et al., 2006a).

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Cryptosporidium parvum Infection Most developing countries have very low frequency of C. parvum infections, although the burden attributable to this species seems to vary by area. In these countries, C. parvum transmission in humans is frequently anthroponotic. This is supported by results of GP60 subtyping in several regions. Two major GP60 subtype families, the zoonotic IIa and the anthroponotic IIc, are responsible for most C. parvum infections in humans (Fig. 5.1). IIa subtypes are rarely seen in humans in developing countries. Instead, the subtype family IIc is responsible for most human C. parvum infections in these areas (Leav et al., 2002; Peng et al., 2003; Xiao et al., 2004; Akiyoshi et al., 2006; Ajjampur et al., 2007). In some places, such as Lima, Peru and southern India, the IIc subtype family is the only C. parvum in humans, whereas in other developing countries such as Malawi and Kenya, IIe (another anthroponotic C. parvum subtype family) is seen in humans in addition to IIc (Peng et al., 2003; Xiao et al., 2004; Ajjampur et al., 2007; Cama et al., 2007). In Uganda, even though IIc subtypes are the dominant C. parvum in children, several new subtype families have been reported (Akiyoshi et al., 2006). All these are likely indicators of the reduced role of zoonotic transmission of C. parvum in these areas. Nevertheless, a recent study of asymptomatic cryptosporidiosis in Zambian dairy farm workers and their household members has identified C. parvum as being more common than C. hominis (Siwila et al., 2007). Zoonotic infection was also suspected to be one cause of the seemingly wider species diversity in Malawian children living in rural areas (Morse et al., 2007). No subtyping of C. parvum was done in these studies to identify possible infection sources. One major outbreak of diarrhoea largely attributed to cryptosporidiosis occurred in Botswana in early 2006 after heavy rain, causing thousands of infections and several hundreds of deaths in a number of districts. Both C. parvum and C. hominis were identified in infected people, with the former responsible for more cases. As expected, five C. hominis subtype families and six subtypes were identified in a small number of specimens analysed. However, the C. parvum specimens analysed had only IIcA5G3a or IIcA5G3b subtypes of the IIc subtype family (L. Xiao et al., unpublished). Because C. parvum was identified as the major species and some patients in the outbreak had enteropathogenic E. coli, cattle waste was initially suspected as the major source of contamination. The finding of only the C. parvum subtype IIc family and the presence of C. hominis clearly suggests that the major initial source of contamination was human sewage.

Infections with Other Species/Genotypes In most developing countries studied, C. parvum and C. hominis are responsible for more than 90% of human cases of cryptosporidiosis, with the remainder attributable to C. meleagridis, C. canis and C. felis. Some areas, however, have a high prevalence of these unusual species. In Lima (Peru) and Bangkok (Thailand), C. meleagridis is as prevalent in humans as C. parvum, being responsible for

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10–20% of human cryptosporidiosis cases (Xiao et al., 2001; Gatei et al., 2002b; Cama et al., 2003). Subtyping tools are largely unavailable for these rare species because most C. parvum- and C. hominis-based subtyping primers are not capable of PCR amplification in these species. GP60 and HSP70-based typing tools, however, are available to subtype C. meleagridis. Multiple C. meleagridis subtypes were seen in the small number of human specimens from several different areas (Glaberman et al., 2001). A recent MLST analysis in a small community in Lima, Peru, has shown the presence of multiple subtypes in children, AIDS patients, and birds, with no apparent host segregation of the subtypes found (L. Xiao et al., unpublished). Like C. parvum, most human infections with the usual species are probably the result of anthroponotic transmission, judged by the concurrent presence of C. hominis in some of the C. meleagridis-, C. canis- or C. felis-infected individuals (see below). Possible transmission of C. canis between two siblings and a dog occurred in a household in a slum in Lima, Peru, but the direction of the transmission was not clear (Xiao et al., 2007).

Mixed Infections and Sequential Infections No matter which PCR tool is used in genotyping Cryptosporidium, all broadly specific tools have the problem that they detect only the dominant genotype in the specimen because of the inherent nature of exponential amplification by PCR and the requirement of a substantial amount of PCR product for them to be visible on an agarose gel. Thus, concurrent infection with mixed Cryptosporidium genotypes is more challenging to diagnose and minor populations of species or genotypes in specimens are probably under-detected by these tools (Reed et al., 2002). A low prevalence of mixed infections of multiple Cryptosporidium genotypes has been reported in children in Uganda (19/444), Malawi (1/43) and India (2/50) (Tumwine et al., 2003; Gatei et al., 2007; Morse et al., 2007). Using genotype-specific primers in combination with other genus-specific primers, Cama et al. (2006) identified concurrent C. hominis, C. parvum or C. meleagridis infections in seven out of 21 C. canis- or C. felis-infected HIV-positive people. Seven out of 55 C. meleagridis-infected children and HIV-positive people in Lima, Peru, had concurrent C. hominis infection in a MLST analysis (L. Xiao et al., unpublished). Thus, the accurate identification of infections with mixed genotypes has important implications for understanding the transmission of so-called zoonotic species and genotypes in humans. In developing countries, children frequently have multiple episode of cryptosporidiosis, although the likelihood of clinical illness decreases with increased infection episodes (Bern et al., 2002). Molecular analysis of longitudinal samples from Peruvian children with multiple cryptosporidiosis episodes indicated that immunity against both homologous and heterologous Cryptosporidium species/ genotypes was short-lived, with time intervals between infections of about 1 year. As might be expected, sequential infections with heterologous Cryptosporidium species were more common than sequential infections with homologous

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L. Xiao

Cryptosporidium species (Xiao et al., 2001, Cama et al., 2008). Even though some children had the same genotype (C. hominis) in sequential infections, results of subtyping suggested that these were in fact due to heterogeneous subtypes of the parasite (Cama et al., 2008).

Cryptosporidium Species/Genotypes, Subtypes, and Virulence The clinical and epidemiological significance of various Cryptosporidium species and subtypes in humans is not yet clear. Results of recent genotyping studies nevertheless support the theory that C. hominis and C. parvum behave differently in humans. Studies in slums in Peru and Brazil have shown that children infected with C. hominis have higher oocyst shedding intensity and longer duration than those infected with C. parvum and other genotypes (Xiao et al., 2001; Bushen et al., 2007). Children infected with C. hominis had a significantly greater severity of diarrhoea than those infected with other species in southern India (Ajjampur et al., 2007). In AIDS patients in Lima, Peru, only infections with C. canis, C. felis or subtype family Id of C. hominis were significantly associated with diarrhoea, and infections with C. parvum were significantly associated with chronic diarrhoea and vomiting. Infections with C. hominis Ib subtype family were also marginally associated with diarrhoea and vomiting. In contrast, infections with C. meleagridis and Ia and Ie subtype families of C. hominis were usually asymptomatic (Cama et al., 2007). These results demonstrate that different Cryptosporidium genotypes and subtype families are linked to different clinical manifestations. Because of the differences in pathogenicity, C. hominis and C. parvum seemingly have different nutritional effects on infected children. In Brazil, heightfor-age (HAZ) Z-scores showed significant declines within 3 months of infection for children infected with either C. hominis or C. parvum. However, in the 3–6 month period following infection, only C. hominis-infected children continued to demonstrate declining HAZ score and those with asymptomatic infection showed even greater decline (P = 0.01). Thus, C. hominis is associated with greater growth shortfalls, even in the absence of symptoms (Bushen et al., 2007).

Conclusions Molecular epidemiological studies of cryptosporidiosis in developing countries are still in their infancy, but significant progress has already been made towards a better understanding of the transmission of the infection. We are beginning to use second-generation molecular tools to answer epidemiological questions that are difficult to address by traditional methods, such as maintenance of immunity and cross-protection, transmission dynamics in different settings, temporal and geographical variations in Cryptosporidium transmission, and the role of parasite factors in the variability in transmission and clinical spectrum of cryptosporidiosis. More importantly, we are starting to see better collaboration between epidemiologists, clinicians, molecular biologists and parasitologists in well-designed

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epidemiological studies. Such an integrated approach will undoubtedly lead to better utilization of the available molecular diagnostic tools and a better understanding of the epidemiology of cryptosporidiosis.

Acknowledgements The findings and conclusions reported in this chapter are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention.

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Molecular Epidemiology and Typing of Non-human Isolates of Cryptosporidium U.M. RYAN1 AND L. XIAO2 1Murdoch

University, WA, Australia; 2Center for Disease Control and Prevention, Atlanta, GA, USA

Abstract Cryptosporidium has been reported in a wide variety of hosts, with C. parvum and C. hominis being responsible for most human infections. Until recently, it has been assumed that farm animals and wild animals are important zoonotic reservoirs for human cryptosporidiosis. However, recent molecular analysis has revealed a wide range of Cryptosporidium species and genotypes infecting both domestic and wild animals, and the epidemiology of cryptosporidiosis is clearly more complicated than was previously thought.

Introduction Cryptosporidium has been reported in a wide variety of vertebrate hosts (Fayer et al., 2000) and, at present, 16 species of Cryptosporidium are regarded as valid on the basis of differences in oocyst morphology, site of infection, vertebrate class specificity and genetic differences: C. muris in rodents; C. andersoni and C. bovis in cattle and sheep; C. suis in pigs; C. parvum in cattle, humans and other mammals; C. meleagridis in birds and humans; C. hominis in humans; C. baileyi and C. galli in birds; C. serpentis and C. saurophilum in snakes and lizards; C. molnari and C. scophthalmi in fish; C. wrairi from guinea pigs; C. felis in cats; and C. canis in dogs (Fayer et al., 2000, 2001; Alvarez-Pellitero and Sitjà-Bobadilla, 2002; Morgan-Ryan et al., 2002; Ryan et al., 2003a, 2004a; Alvarez-Pellitero et al., 2004). Cryptosporidium parvum and C. hominis are responsible for most human infections (Morgan-Ryan et al., 2002), and it has been assumed that the majority of Cryptosporidium infections in farmed animals that had oocysts in the size range of 4–6 mm were due to C. parvum (cattle genotype) and that farm animals and wild animals represented an important zoonotic reservoir for human cryptosporidiosis. However, recent molecular analysis has revealed a wide range of Cryptosporidium species and genotypes infecting both domestic and wild animals, © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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and the epidemiology of cryptosporidiosis is clearly more complicated than was previously thought.

Cryptosporidium Species and Genotypes in Sheep Cryptosporidium has been reported in sheep worldwide; however, most studies on Cryptosporidium in sheep have been based on microscopy, with the reported prevalence ranging from 2.6% to 82% (see Ryan et al., 2005). Recent molecular characterization of Cryptosporidium in sheep suggests that the majority of sheep are not infected with zoonotic genotypes. In Australia, a survey of 1647 sheep faecal samples reported a prevalence of 26.2%. The most common Cryptosporidium genotypes identified were the cervid genotype (55%) and C. bovis (23%). Low levels of C. andersoni, C. suis, marsupial genotype, pig II genotype, C. hominis and a novel previously unidentified genotype were also detected (Ryan et al., 2005). Cryptosporidium parvum was not detected in any of the samples. More recently, in the USA, sequence analysis of 57 specimens corresponding to 8 ewes and 24 lambs that were positive for Cryptosporidium identified the cervid genotype (84%), C. bovis-like genotype (~12%) and C. parvum (3.5%) (Santín et al., 2007). In the UK, C. parvum and a novel genotype were reported in sheep (Chalmers et al., 2002); however, RFLP analysis of the COWP locus was used (Chalmers et al., 2002) and recent studies have shown that this analysis can erroneously identify isolates as C. parvum due to the extent of conservation at the COWP locus (Hamnes et al., 2007).

Cryptosporidium Species and Genotypes in Pigs Cryptosporidium was first reported in pigs in the USA in 1977 (Kennedy et al., 1977). Subsequent studies have been undertaken worldwide, including Australia, Japan, Germany, Spain and Ireland, with prevalences ranging from 1.4% to 100% in pigs aged 1 week to adulthood (Quílez et al., 1996; Izumiyama et al., 2001; Wieler et al., 2001; Maddox-Hyttel et al., 2006; Xiao et al., 2006). The prevalence in piglets under 2 months of age has been reported to range between 0% and 59.2%. The age at which Cryptosporidium is most prevalent is in pigs between 6 and 12 weeks old (see Hamnes et al., 2007). The majority of nursing piglets and weanlings that have been positive for Cryptosporidium have been shown to be infected with two Cryptosporidium spp. – C. suis and pig genotype II (Guselle et al., 2003; Ryan et al., 2003a, 2004a; Vitovec et al., 2006; Xiao et al., 2006; Hamnes et al., 2007; Langkjær et al., 2007) (see Table 6.1). Cryptosporidium suis is adapted to porcine hosts, but poorly infective for cattle (Enemark et al., 2003) and not infective for nude mice and neonatal BALB/c mice (Morgan et al., 1999a; Ryan et al., 2003a; Vitovec et al., 2006). Cryptosporidium suis has been identified in humans (Xiao et al., 2002a) but is not a common human pathogen. Little is known about the pathogenicity or zoonotic potential of pig genotype II but it has not been reported in humans to date and is unlikely to be a major threat to public health. Phylogenetic analysis

Molecular Epidemiology and Typing of Non-human Isolates Table 6.1.

67

Cryptosporidium genotypes infecting pigs.

Country Denmark Weaners Piglets Norway Australia

No. genotyped

C. suis

Pig genotype II

170 13 9 12

24% 71% ~66% 60%

76% 29% ~33%

Other species

Reference Langkjær et al. (2007)

C. parvum (40%)

Australia Weaners Piglets Australia Weaners Piglets Ireland

14 14

50% 50%

50% 50%

27 18 25

24% 100% 57%

76% 0% 39%

Czech Republic

13

100%

Hamnes et al. (2007) Morgan et al. (1999a) Ryan et al. (2003a)

Johnson et al. (2008)

C. muris (3.5%)

Xiao et al. (2006) Vitovec et al. (2006)

at multiple loci has confirmed the species status of C. suis and provides strong evidence that pig genotype II is also a valid species (see Ryan et al., 2003a; Xiao et al., 2004a). In Denmark, sequence analysis of 183 Cryptosporidium-positive pig isolates from sows, weaners and piglets from various herds identified C. suis in 24% of weaners and 71% of piglets, and pig genotype II in 76% of weaners and 29% of piglets (Langkjær et al., 2007). Higher oocyst concentrations were observed in samples genotyped as C. suis than in samples genotyped as pig genotype II. There also appeared to be an age-related change in the species/genotypes infecting pigs, as pig genotype II was more prevalent in weaners whereas the majority of piglets were infected with C. suis (Langkjær et al., 2007). This is in contrast with a previous Australian study in which 28 Cryptosporidium isolates from pigs were genotyped, revealing equal numbers of C. suis and pig genotype II (Ryan et al., 2003a). No correlation between genotype and host age was found, but some pigs infected with pig genotype II seemed to have a high excretion of oocysts (Ryan et al., 2003a). However, a more recent study in Australia reported that pig genotype II was more prevalent in weaners (71%) than C. suis (17%) (Johnson et al., 2008). One of the first genotyping studies on pigs identified C. suis but also C. parvum (cattle genotype) by sequence analysis of the 18S rRNA gene in outdoor pigs suffering from diarrhoea in Western Australia (Morgan et al., 1999a). Interestingly, C. parvum has not been detected in pig isolates since then and it is likely that C. parvum is responsible for only occasional infections in pigs. In the Czech Republic, 0/135 sows, 193/3368 (5.7%) pre-weaned and 201/835 (24.1%) post-weaned piglets were positive for Cryptosporidium infection. Only C. suis was identified; however, only a few isolates were genotyped

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(Vitovec et al., 2006). The authors reported that the C. suis strains obtained were larger (6.2 × 5.5 mm) than the oocyst sizes given in the original C. suis description (4.6 × 4.2 mm) (Ryan et al., 2004a). However, studies have shown that there is substantial morphological variation in oocyst size within individual species and that morphology alone is not a reliable indicator for delimiting species (Fall et al., 2003). In Norway, 31 (31%) herds and 57 (8.3%) litters from 684 litters of suckling piglets from 100 indoor swine herds from all regions tested positive for Cryptosporidium. Molecular characterization of nine Cryptosporidium isolates demonstrated both C. suis and Cryptosporidium pig genotype II (Hamnes et al., 2007). A study in Ireland reported that the spreading of pig slurry onto pasture or crops may lead to increases in the occurrence of Cryptosporidium in water, as C. suis, Cryptosporidium pig genotype II, and C. muris were identified in 25 of 56 pig slurry samples from 33 Irish farms (Xiao et al., 2006). The finding of C. muris in pig slurry is unusual and could have originated from either infected pigs or rodents. Even though C. muris infection has never been reported in pigs, this species does have the widest host range of all Cryptosporidium species, and this range includes humans (Iseki et al., 1989).

Cryptosporidium Species and Genotypes in Cattle Since the late 1980s, cattle have been identified as being one of the main reservoir host for the zoonotic C. parvum; however, recent studies in the USA suggest that cattle are infected with at least four Cryptosporidium parasites: C. parvum, C. bovis, C. andersoni and the Cryptosporidium deer-like genotype (Santín et al., 2004; Fayer et al., 2006, 2007; Feng et al., 2007; Xiao et al., 2007). Recent studies in the USA indicated that the occurrence of these Cryptosporidium spp. in cattle is age-related (Santín et al., 2004), as the zoonotic C. parvum was responsible for about 85% of the Cryptosporidium infections in pre-weaned calves but only 1% of the Cryptosporidium infections in post-weaned calves and heifers (Santín et al., 2004). Post-weaned calves were mostly infected with C. bovis, C. andersoni and the deer-like genotype (Santín et al., 2004) (see Table 6.2). These findings clearly demonstrate that neonatal calves are an important source of zoonotic cryptosporidiosis in humans. Neonatal calves are also the age group of cattle mostly affected by cryptosporidiosis in terms of prevalence of infection and the associated morbidity and mortality (Fayer et al., 1997). Studies on heifers aged 12–24 months of age on dairy farms in Pennsylvania, Vermont, New York, Maryland, Virginia, North Carolina and Florida reported a much lower prevalence of C. parvum and reported that C. suis, C. parvum, the deer-like genotype, C. bovis and C. andersoni accounted for 1%, 6%, 15%, 35% and 43%, respectively (Fayer et al., 2006). A study on mature dairy cattle identified an even lower prevalence of Cryptosporidium and reported that C. parvum, C. bovis and C. andersoni were found infecting 0.4%, 1.7% and 3.7% of the 541 cows, respectively (Fayer et al., 2007). The overall lower prevalence of Cryptosporidium in these cows was very highly significant (P < 0.0001) compared

Cryptosporidium genotypes infecting cattle.

Country USA Pre-weaned Post-weaned USA Heifers USA Milking cows New York state Georgia, USA Pre-weaned Post-weaned Milking cows China India Denmark Cows Older calves Young calves Hungary Calves Portugal Calves Adults aC.

No. Genotyped

C. parvum

C. bovis

C. andersoni

Deer-like genotype

278

85% 1%

9% 55%

1% 13%

5% 31%

68

6%

35%

43%

15%

31 115

6.5% 61% (70/115)

29% 37% (42/115)

23 6 3 6 12

26% 0% 0%

3 61 90

100% 4% 82%

22

95%

63 7

100% 71.5%

Other species

Reference Santín et al. (2004)

Fayer et al. (2006) 1%a Fayer et al. (2007) 64.5% 3% (3/115)

Starkey et al. (2006) Feng et al. (2007)

8.4%

39% 66% 66% 83% 91.6% – 73% 14%

22% 16% 0%

33%b

16%

Feng et al. (2007) Feng et al. (2007) Langkjær et al. (2007)

– 14% 3%

– 4%c Plutzer and Karanis (2007) Mendonça et al. (2007)

5% 14.25%

Molecular Epidemiology and Typing of Non-human Isolates

Table 6.2.

14.25%d

suis; b1 mixed C. parvum + C. andersoni isolate; cC. suis-like genotype + <1% pig genotype II; dC. meleagridis. 69

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with younger cattle. The very low level of infection with zoonotic C. parvum, suggests that mature dairy cattle are a relatively low-risk source of infection for humans (Fayer et al., 2007). A study on 115 faecal samples positive for Cryptosporidium from dairy cattle in New York state, identified 70 of the 115 (61%) as C. parvum, 42 (37%) as C. bovis, and 3 (3%) as the deer-like genotype (Starkey et al., 2006). The authors also suggested that C. bovis may be more host-adapted and thus less pathogenic to dairy cattle than C. parvum (Starkey et al., 2006). The prevalence of Cryptosporidium species in cattle in other countries is less well characterized but recent studies have shown that C. bovis and the deer-like genotype are present in cattle studied in China and India (Feng et al., 2007) (see Table 6.2). In that study, the prevalence of Cryptosporidium species in cattle in Georgia was compared with that in China and India. Analysis revealed that the prevalence of C. bovis and the deer-like genotype was much higher than that of C. parvum, even in pre-weaned calves, in Georgia. This is in contrast to the studies by Fayer et al. (2006) and Santín et al. (2004), which reported that that C. bovis and the deer-like genotype were mostly found in post-weaned calves and older animals. This may have been due to the fact that the pre-weaned calves examined in the Feng study were mostly 1 month or older and only a small number of animals were sampled. Alternatively, cattle of all ages may be susceptible to infections with both species and there may not be an age-associated occurrence of the two parasites in cattle. It is also possible that calves may acquire infection with C. bovis or the deer-like genotype early in life, but the infection may be concealed by the overwhelming C. parvum infection. The pre-patent period for C. bovis is 10–12 days, which is much longer than for C. parvum (about 4 days) (Fayer et al., 2005). Further studies are required to elaborate on this. A study in Denmark also showed age structuring, with C. bovis causing 14% and 73% of Cryptosporidium infections in young and older calves, respectively (Langkjær et al., 2007). Cryptosporidium parvum and the deer-like genotype were responsible for 82% and 4% in young calves and 3% and 14% of Cryptosporidium infections in older calves (Langkjær et al., 2007). A novel C. suislike genotype which exhibited 99% and 98% nucleotide identity to C. suis at the 18S rDNA and HSP70 loci, respectively, was identified in three calves originating from two different herds (Langkjær et al., 2007). Pig genotype II, which until now has appeared to be pig-specific, was also identified in a young calf (Langkjær et al., 2007). Cryptosporidium andersoni, which was found previously in Danish cattle (Enemark et al., 2002), was not detected. This is probably explained by the fact that C. andersoni is primarily found in older animals and that only three isolates from cows could be successfully genotyped. In Portugal, a study of 467 dairy and beef cattle, of which 291 were calves and 176 were adults, identified 74/291 calves (25.4%) and 8/176 adults (4.5%) as positive for Cryptosporidium by microscopy (Mendonça et al., 2007). Molecular characterization of 63 isolates from calves and 7 isolates from adults, identified all of the isolates as C. parvum using sequence analysis of the HSP70 locus. However, sequence analysis of the 18S rRNA locus identified two of the C. parvum isolates as C. meleagridis and C. andersoni (Mendonça et al., 2007). This is the first report of C. meleagridis in cattle.

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In Hungary, 79 faecal samples from calves with diarrhoea were collected on 52 farms from different counties (Plutzer and Karanis, 2007). Immunofluorescence microscopy identified 39 samples as positive for Cryptosporidium and 18S rRNA RFLP analysis of 22 isolates identified C. parvum in 21 samples and the Cryptosporidium deer-like genotype in 1 sample (Plutzer and Karanis, 2007).

Cryptosporidium in Wild Animals Little is known of the prevalence and distribution of Cryptosporidium spp. infecting wild animals, but studies to date suggest that the majority of wild animals do not shed oocysts that are of public health significance. A recent study detected 36 Cryptosporidium positives from 471 faecal samples from foxes, raccoons, muskrats, otters and beavers living in wetlands adjacent to Chesapeake Bay, Maryland, USA. Genotyping analysis identified five types of Cryptosporidium, including the C. canis dog and fox genotypes, Cryptosporidium muskrat genotypes I and II, and Cryptosporidium skunk genotype (Zhou et al., 2004a). The Cryptosporidium ferret genotype has been identified in ferrets, river otters and American mink (Xiao et al., 2002b; Gaydos et al., 2007; Gomez-Couso et al., 2007). Another study examined faeces and duodenal scrapings from 22 coyotes killed in managed hunts in north-eastern Pennsylvania. Six coyotes (27%) were positive for Cryptosporidium spp.; one isolate shared 99.7% homology with C. muris, whereas five others (23%) shared 100% homology with C. canis coyote genotype (Trout et al., 2006). In marsupials, multiple species and genotypes have been identified, including marsupial genotype I and II, opossum genotype I (which is genetically very closely related to marsupial type I), opossum genotype II and C. muris (Morgan et al., 1997; Xiao et al., 1999a, 1999b, 2002b; Warren et al., 2003; Power et al., 2004, 2005). A recent study examined the occurrence of Cryptosporidium oocysts in faeces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia (Power et al., 2005). Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3557 (6.7%) eastern grey kangaroo faecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Partial 18S rRNA gene sequencing of 51 isolates representing 10 of the 11 faecal sampling periods identified marsupial genotypes I and II in 61% and 39% of samples, respectively (Power et al., 2005). As marsupial genotypes I and II have not been identified in humans to date, it is unlikely that marsupials in water catchment areas are a serious public health threat.

Cryptosporidium in Rodents There have been few studies conducted on the epidemiology of Cryptosporidium in wild rodents; however, it is important to establish whether they are a

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reservoir of infection for humans and animals. Studies in different geographical areas have reported a prevalence ranging from 5.0% to 39.2% in wild rodents, but most have relied on morphology for identification (Chalmers et al., 1997; Sturdee et al., 1999, 2003; Torres et al., 2000; Bednarska et al., 2003). The limited molecular characterization studies that have been conducted on rodents have identified eight species/genotypes to date: 1. Mouse genotype I, which has been identified in mice and rats in Spain, the UK, Australia and the USA (Morgan et al., 1999b, L. Xiao et al., unpublished) and to date has not been identified in humans. 2. The zoonotic C. parvum, which was detected in mice trapped near sheepgrazing pastures in Victoria, Australia (Morgan et al., 1999b). 3. Cryptosporidium muris, which infects a range of rodents and is generally not zoonotic but which can infect other hosts including dogs, guinea pigs, chipmunks, rabbits, lambs, cats and humans (Iseki et al., 1989; Chalmers et al., 1997; Torres et al., 2000; Hurkova et al., 2003; Hikoska and Nakai, 2005). 4. A novel genotype detected in one wood-mouse (Apodemus sylvaticus) sample from the Czech Republic (Hajdušek et al., 2004). 5. Cryptosporidium meleagridis from a brown rat (Rattus norvegicus) in Japan (Kimura et al., 2007). 6. Eight isolates from brown rats in Japan clustered with sequences denoting W19 and W19 variants found in New York storm water (Jiang et al., 2005; Kimura et al., 2007). 7. Three isolates from brown rats in Japan were identical to a recently described snake isolate 2162 (AY268584) (Xiao et al., 2004b; Kimura et al., 2007). 8. Another isolate from brown rats in Japan showed 100% identity with isolates from non-human primates at Polonnaruwa, Sri Lanka (EF446679) (Ekanayake et al., 2007; Kimura et al., 2007). A recent study in Australia identified mouse genotype I and a novel genotype (mouse genotype II), which is genetically distinct and appears to be hostspecific, as it has not been identified in any other hosts to date (Foo et al., 2007). Unfortunately, it is not possible to determine whether mouse genotype II was the same as the novel mouse genotype described from the Czech Republic, as only the COWP and not the 18S or actin loci were sequenced as part of the Czech study. A previous study, which examined mice from different geographical loci including Australia, the UK and Spain, revealed that the majority of Cryptosporidium-positive mice were infected with mouse genotype I (Morgan et al., 1999b). In that study, five of the Australian mouse samples, which were infected with the zoonotic C. parvum, came from mice which had been trapped on farms in Victoria where large numbers of sheep were grazing. This indicates that sheep may transmit C. parvum to mice, which may in turn transmit Cryptosporidium to other domestic animals. A more recent study by Foo et al. (2007) did not detect the zoonotic C. parvum. This may be because all the positive samples were from arable farms where the mice were unlikely to encounter sheep and cattle. It is possible that mice are only occasionally infected with the C. parvum during periods of heavy environmental contamination.

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Cryptosporidium in Birds Cryptosporidiosis is one of the most prevalent parasitic infections in domesticated, caged and wild birds (Lindsay and Blagburn, 1990; O’Donoghue, 1995), and the parasite has been reported in more than 30 avian species worldwide belonging to the orders Anseriformes, Charadriiformes, Columbiformes, Galliformes, Passeriformes, Psittaciformes and Struthiniformes (Lindsay and Blagburn, 1990; O’Donoghue, 1995; Ng et al., 2006). However, few studies have examined the genetic diversity of Cryptosporidium spp. amongst avian hosts. Currently only three avian Cryptosporidium spp. are recognized: C. meleagridis, C. baileyi and C. galli. These three Cryptosporidium species can each infect a broad range of birds, but they differ in preferred sites. Even though both C. meleagridis and C. baileyi are found in the small and large intestine and bursa, they differ significantly in oocyst size and only C. baileyi is also found in the respiratory tissues such as the conjunctiva, sinuses and trachea. In contrast, C. galli infects only the proventriculus (see Xiao et al., 2004a). Naturally occurring cryptosporidiosis in birds manifests itself in three clinical forms: respiratory disease, enteritis and renal disease. Usually only one form of the disease is present in an outbreak (Lindsay and Blagburn, 1990). Although three Cryptosporidium species are considered to be valid in birds, and a number of avian genotypes have been identified that are likely to be re-classified as species in the future, most reports of natural infections have not provided enough information to conclusively determine the species/genotype of Cryptosporidium involved. In addition to the three currently recognized avian species of Cryptosporidium, ten genetically distinct avian genotypes have been described: avian genotypes I–IV from various avian hosts (Meireles et al., 2006; Ng et al., 2006); a novel genotype from a Eurasian woodcock (Scolopax rusticola) (isolate B2-1) from the Czech Republic (Ryan et al., 2003b); a duck genotype from a black duck (Anas rubripes) (Morgan et al., 2001); and goose genotypes I and II, an additional two novel genotypes (geese 3b and 7) from Canada geese (Jellison et al., 2004; Zhou et al., 2004b). Morphometric analysis has not been conducted on most avian genotypes; however, limited morphometric analysis (<10 oocysts per genotype) on avian genotypes II and III suggests that they are morphologically distinct. However, larger numbers of oocysts from each genotype (~50) would need to be analysed in order to produce statistically meaningful results. The results of these recent studies indicate that Cryptosporidium is able to infect a large range of avian species and that, potentially, there are more avian Cryptosporidium species yet to be identified. The potential of these newly identified genotypes to cause disease in birds or humans is unknown, and further studies are required to understand the extent of host adaptation for avian-derived Cryptosporidium genotypes, their species status, transmission dynamics and host range, and the public health implications, if any, of these novel avian genotypes. Four additional species have also been identified in birds: C. hominis, C. parvum, C. muris and C. andersoni (Zhou et al., 2004b; Ng et al., 2006). However, the presence of these oocysts in avian faeces is more likely to be due to mechanical transport rather than an actual infection, as previous studies have shown that, for C. parvum at least, the oocysts were merely passing through the

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digestive tracts of foraging Canada geese without establishing infection (Graczyk et al., 1997).

Cryptosporidium in Fish Little is known about the prevalence or geographical distribution of isolates of Cryptosporidium infecting fish. The first report of Cryptosporidium spp. in fish was in a tropical marine fish (Naso lituratus) in 1981 (Hoover et al., 1981). Cryptosporidium nasorum was subsequently proposed as a species by Levine in 1984 (Levine, 1984). However, only developmental stages of the parasites on the microvillous surface of intestinal epithelial cells were described by light and electron microscopy. No measurements of viable oocysts were provided, and no taxonomically useful diagnostic features were presented. The species was named solely on the basis of the presumed host specificity of Cryptosporidium spp. and is now considered a nomen nudem (Xiao et al., 2004a). Recently, two new piscine Cryptosporidium species have been described: Cryptosporidium molnari from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.), and Cryptosporidium scophthalmi from cultured turbot (Scophthalmus maximus) (Alvarez-Pellitero and Sitjà-Bobadilla, 2002; Alvarez-Pellitero et al., 2004). Cryptosporidium molnari was found mainly in the stomach epithelium, whereas C. scophthalmi was found mainly in the intestinal epithelium. Oocysts for both species were similar in size to C. parvum and averaged 4.72 × 4.47 mm and 4.44 × 3.91 mm for C. molnari and C. scophthalmi, respectively. In both species, parasite stages were observed deep within the epithelium (Alvarez-Pellitero and Sitjà-Bobadilla, 2002; Alvarez-Pellitero et al., 2004). Unfortunately no molecular characterization of C. molnari or C. scophthalmi has been conducted thus far, and its relationship to other species of Cryptosporidium remains unknown. A recent study conducted histological, genetic and phylogenetic analysis of a C. molnari-like isolate of Cryptosporidium from a guppy (Poecilia reticulata) (Ryan et al., 2004b). The size of the oocysts, the location of the oocysts in the stomach, and the presence of oogonial and sporogonial stages deep within in epithelium, was consistent with C. molnari (Ryan et al., 2004b). Phylogenetic analysis of the 18S rRNA and actin loci revealed that the C. molnari-like isolate formed a distinct group that was basal to all other Cryptosporidium species, suggesting that C. molnari may be the most primitive of the Cryptosporidium spp. However, until genetic sequences are made available for C. molnari and C. scophthalmi, it is impossible to determine the identity of the guppy isolate. Indeed it could be argued that the naming of C. molnari and C. scophthalmi did not follow the criteria for naming species in the genus Cryptosporidium (Xiao et al., 2004a) and therefore should be considered invalid species until genetic and phylogenetic analysis is provided.

Cryptosporidium in Reptiles Cryptosporidium infections are common in reptiles and have been reported in at least 57 reptilian species (O’Donoghue, 1995). Unlike in other animals in which

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Cryptosporidium infection is usually self-limiting in immunocompetent individuals, cryptosporidiosis in reptiles is frequently chronic and sometimes lethal in snakes. Two Cryptosporidium spp. are recognized in reptiles: Cryptosporidium serpentis and C. saurophilum in snakes and lizards, which differ from each other in morphology (oocysts of C. serpentis are bigger than those of C. saurophilum) and predilection sites (C. serpentis is a gastric parasite, whereas C. saurophilum is an intestinal parasite) (Morgan et al., 1999c; Fayer et al., 2000; Xiao et al., 2000, 2004b; Hajdušek et al., 2004). In addition to the two recognized species, a total of seven genotypes have been identified in reptiles including a tortoise genotype, two new snake genotypes, and a lizard genotype, which was genetically distinct but was related to C. serpentis (Xiao et al., 2004b; Alves et al., 2005). Cryptosporidium muris, C. parvum and mouse genotype I have also been identified in reptiles (Morgan et al., 1999c; Xiao et al., 2004b). However, it is likely that that latter three probably do not represent true parasites of these animals but are instead oocysts from rodents ingested by these carnivorous reptiles. This possibility is supported by the fact that the snakes are fed mice and that none of the animals with these oocysts had clinical symptoms. It is also likely that one of the snake genotypes identified by Xiao et al. (2004b) may also be derived from mice, as snake isolate 2162 (AY268584) was identical to Cryptosporidium sequences obtained from brown rats in Japan (Kimura et al., 2007). Currently, there are no effective control strategies against cryptosporidiosis in reptiles. In a small-scale study, it was demonstrated that snakes with clinical and subclinical cryptosporidiosis could be effectively treated with hyperimmune bovine colostrum raised against C. parvum (Graczyk et al., 1998). A common control practice is to euthanize Cryptosporidium-infected snakes, which would prevent the spread of infection to other animals. However one problem with this control measure is the frequent presence of oocysts of C. muris and mouse genotype I in snakes because of the use of feeder mice as part of the diet, which would lead to the killing of uninfected animals.

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Graczyk, T.K., Cranfield, M.R., Helmer, P., Fayer, R. and Bostwick, E.F. (1998) Therapeutic efficacy of hyperimmune bovine colostrum treatment against clinical and subclinical Cryptosporidium serpentis infections in captive snakes. Veterinary Parasitology 74, 123–132. Guselle, N.J., Appelbee, A.J. and Olson, M.E. (2003) Biology of Cryptosporidium parvum in pigs: from weaning to market. Veterinary Parasitology 113, 7–18. Hajdušek, O., Ditrich, O. and Šlapeta, J. (2004) Molecular identification of Cryptosporidium spp. in animal and human hosts from the Czech Republic. Veterinary Parasitology 122, 183–192. Hamnes, I.S., Gjerde, B.K., Forberg, T. and Robertson, L.J. (2007) Occurrence of Cryptosporidium and Giardia in suckling piglets in Norway. Veterinary Parasitology 144, 222–233. Hikoska, K. and Nakai, Y. (2005) A novel genotype of Cryptosporidium muris from large Japanese field mice, Apodemus speciosus. Parasitology Research 97, 373–379. Hoover, D.M., Hoerr, F.J., Carlton, W.W., Hinsman, E.J. and Ferguson, H.W. (1981) Enteric cryptosporidiosis in a naso tang, Naso lituratus Bloch and Schneider. Journal of Fish Diseases 4, 425–428. Hurkova, L., Hajdušek, O. and Modry, D. (2003) Natural infection of Cryptosporidium muris (Apicomplexa: Cryptosporiidae) in Siberian chipmunks. Journal of Wildlife Diseases 39, 441–444. Iseki, M., Maekawa, T., Moriya, K., Uni, S. and Takada, S. (1989) Infectivity of Cryptosporidium muris (strain RN 66) in various laboratory animals. Parasitology Research 75, 218–222. Izumiyama, S., Furukawa, I., Kuroki, T., Yamai, S., Sugiyama, H., Yagita, K. and Endo, T. (2001) Prevalence of Cryptosporidium parvum infections in weaned piglets and fattening porkers in Kanagawa Prefecture, Japan. Japanese Journal of Infectious Diseases 54, 23–26. Jellison, K.L., Distel, D.L., Hemond, H.F. and Schauer, D.B. (2004) Phylogenetic analysis of the hypervariable region of the 18S rRNA gene of Cryptosporidium oocysts in feces of Canada geese (Branta canadensis): evidence for five novel genotypes. Applied and Environmental Microbiology 70, 452–458. Jiang J., Alderisio, K.A. and Xiao, L. (2005) Distribution of Cryptosporidium genotypes in storm event water samples from three watersheds in New York. Applied and Environmental Microbiology 71, 4446–4454. Johnson, J., Buddle, R., Reid, S., Armson, A. and Ryan, U. (2008) Prevalence of Cryptosporidium genotypes in pre- and post-weaned pigs in Australia. Experimental Parasitology 119, 418–421. Kennedy, G.A., Kreitner, G.L. and Strafuss, A.C. (1977) Cryptosporidiosis in three pigs. Journal of the American Veterinary Medical Association 170, 348–350. Kimura, A., Edagawa, A., Okada, K., Takimoto, A., Yonesho, S. and Karanis, P. (2007) Detection and genotyping of Cryptosporidium from brown rats (Rattus norvegicus) captured in an urban area of Japan. Parasitology Research 100, 1417–1420. Langkjær, R.B., Vigre, H., Enemark, H.L. and Maddox-Hyttel, C. (2007) Molecular and phylogenetic characterization of Cryptosporidium and Giardia from pigs and cattle in Denmark. Parasitology 134, 339–350. Levine, N.D. (1984) Taxonomy and review of the coccidian genus Cryptosporidium (Protozoa, Apicomplexa). Journal of Protozoology 31, 94–98. Lindsay, D.S. and Blagburn, B.L. (1990) Cryptosporidiosis in birds. In: Dubey, J.P., Speer C.A. and Fayer, R. (eds) Cryptosporidiosis in Man and Animals. CRC Press, Boca Raton, FL, pp. 133–148. Maddox-Hyttel, C., Langkjær, R.B., Enemark, H.L. and Vigre, H. (2006) Cryptosporidium and Giardia in different age groups of Danish cattle and pigs: occurrence and management associated risk factors. Veterinary Parasitology 141, 48–55.

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U.M. Ryan and L. Xiao Meireles, M.V., Soares, R.M., dos Santos, M.M. and Gennari, S.M. (2006) Biological studies and molecular characterization of a Cryptosporidium isolate from ostriches (Struthio camelus). Journal of Parasitology 92, 623–626. Mendonça, C., Almeida, A., Castro, A., Delgado, M. de L., Soares, S., da Costa, J.M.C. and Canada, N. (2007) Molecular characterization of Cryptosporidium and Giardia isolates from cattle from Portugal. Veterinary Parasitology 147, 47–50. Morgan, U.M., Constantine, C.C., Forbes, D.A. and Thompson, R.C. (1997) Differentiation between human and animal isolates of Cryptosporidium parvum using rDNA sequencing and direct PCR analysis. Journal of Parasitology 83, 825–830. Morgan, U.M., Buddle, R., Armson, A. Elliot, A. and Thompson, R.C.A. (1999a) Molecular and biological characterisation of Cryptosporidium in pigs. Australian Veterinary Journal 77, 44–47. Morgan, U.M., Sturdee, A.P., Singleton, G., Gomez, M.S., Gracenea, M., Torres, J., Hamilton, S.G., Woodside, D.P. and Thompson, R.C. (1999b) The Cryptosporidium “mouse” genotype is conserved across geographic areas. Journal of Clinical Microbiology 37, 1302–1305. Morgan, U.M., Xiao, L., Fayer, R., Graczyk, T.K., Lal, A.A., Deplazes, P. and Thompson, R.C. (1999c) Phylogenetic analysis of Cryptosporidium isolates from captive reptiles using 18S rDNA sequence data and random amplified polymorphic DNA analysis. Journal of Parasitology 85, 525–530. Morgan, U.M., Monis, P.T., Xiao, L., Limor, J., Sulaiman, I., Raidal, S., O’Donoghue, P., Gasser, R., Murray, A., Fayer, R., Blagburn, B.L., Lal, A.A. and Thompson, R.C. (2001) Molecular and phylogenetic characterisation of Cryptosporidium from birds. International Journal for Parasitology 31, 289–296. Morgan-Ryan, U.M., Fall, A., Ward, L.A., Hijjawi, N., Sulaiman, I., Fayer, R., Thompson, R.C.A., Olson, M., Lal, A. and Xiao, L.H. (2002) Cryptosporidium hominis n. sp (Apicomplexa: Cryptosporidiidae) from Homo sapiens. Journal of Eukaryotic Microbiology 49, 433–440. Ng, J., Pavlasek, I. and Ryan, U. (2006) Identification of novel Cryptosporidium genotypes from avian hosts. Applied and Environmental Microbiology 72, 7548–7553. O’Donoghue, P.J. (1995) Cryptosporidium and cryptosporidiosis in man and animals. International Journal for Parasitology 25, 139–195. Plutzer, J. and Karanis, P. (2007) Genotype and subtype analyses of Cryptosporidium isolates from cattle in Hungary. Veterinary Parasitology 146, 357–362. Power, M.L., Slade, M.B., Sangster, N.C. and Veal, D.A. (2004) Genetic characterisation of Cryptosporidium from a wild population of eastern grey kangaroos Macropus giganteus inhabiting a water catchment. Infection Genetics and Evolution 4, 59–66. Power, M.L., Sangster, N.C., Slade, M.B. and Veal, D.A. (2005) Patterns of Cryptosporidium oocyst shedding by eastern grey kangaroos inhabiting an Australian watershed. Applied and Environmental Microbiology 71, 6159–6164. Quílez, J., Sánchez-Acedo, C., Clavel, A., del Cacho, E. and López-Bernad, F. (1996) Prevalence of Cryptosporidium infections in pigs in Aragón (northeastern Spain). Veterinary Parasitology 56, 345–348. Ryan, U.M., Samarasinghe, B., Read, C., Buddle, J.R., Robertson, I.D. and Thompson, R.C.A. (2003a) Identification of a novel Cryptosporidium genotype in pigs. Applied and Environmental Microbiology 69, 3970–3974. Ryan, U.M., Xiao, L., Read, C., Zhou, L., Lal, A.A. and Pavlasek, I. (2003b) Identification of novel Cryptosporidium genotypes from the Czech Republic. Applied and Environmental Microbiology 69, 4302–4307. Ryan, U.M., Monis, P., Enemark, H.L., Sulaiman, I., Samarasinghe, B., Read, C., Buddle, R., Robertson, I., Zhou, L., Thompson, R.C.A. and Xiao, L. (2004a) Cryp-

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tosporidium suis n. sp. (Apicomplexa: Cryptosporidiidae) in pigs (Sus scrofa). Journal of Parasitology 90, 769–773. Ryan, U.M., O’Hara, A. and Xiao, L. (2004b) Molecular and biological characterization of a Cryptosporidium molnari-like isolate from a guppy (Poecilia reticulata). Applied and Environmental Microbiology 70, 3761–3765. Ryan, U.M., Bath, C., Robertson, I., Read, C., Elliot, A., Mcinnes, L., Traub, R. and Besier, B. (2005) Sheep may not be an important zoonotic reservoir for Cryptosporidium and Giardia parasites. Applied and Environmental Microbiology 71, 4992–4997. Santín, M., Trout, J.M., Xiao, L., Zhou, L., Greiner, E. and Fayer, R. (2004) Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Veterinary Parasitology 122, 103–117. Santín, M., Trout, J.M. and Fayer, R. (2007) Prevalence and molecular characterization of Cryptosporidium and Giardia species and genotypes in sheep in Maryland. Veterinary Parasitology 146, 17–24. Starkey, S.R., Zeigler, P.E., Wade, S.E., Schaaf, S.L. and Mohammed, H.O. (2006) Factors associated with shedding of Cryptosporidium parvum versus Cryptosporidium bovis among dairy cattle in New York State. Journal of the American Veterinary Medical Association 229, 1623–1626. Sturdee, A.P., Chalmers, R.M. and Bull, S.A. (1999) Detection of Cryptosporidium oocysts in wild mammals of mainland Britain. Veterinary Parasitology 80, 273–280. Sturdee, A.P., Bodley-Tickell, A.T., Archer, A. and Chalmers, R.M. (2003) Long-term study of Cryptosporidium prevalence on a lowland farm in the United Kingdom. Veterinary Parasitology 116, 97–113. Torres, J., Gracenea, M., Gomez, M.S., Arrizabalaga, A. and Gonzalez-Moreno, O. (2000) The occurrence of Cryptosporidium parvum and C. muris in wild rodents and insectivores in Spain. Veterinary Parasitology 92, 253–260. Trout, J.M., Santín, M. and Fayer, R. (2006) Giardia and Cryptosporidium species and genotypes in coyotes (Canis latrans). Journal of Zoo and Wildlife Medicine 37, 141–144. Vitovec, J., Hamadejova, K., Landova, L., Kvac, M., Kvetonova, D. and Sak, B. (2006) Prevalence and pathogenicity of Cryptosporidium suis in pre- and post-weaned pigs. Journal of Veterinary Medicine Series B 53, 239–243. Warren, K.S., Swan, R.A., Morgan-Ryan, U.M., Friend, J.A. and Elliot, A. (2003) Cryptosporidium muris infection in bilbies (Macrotis lagotis). Australian Veterinary Journal 81, 739–741. Wieler, L.H., Ilieff, A., Herbst, W., Bauer, C., Vieler, E., Bauerfeind, R., Failing, K., Klos, H., Wengert, D., Baljer, G. and Zahner, H. (2001) Prevalence of enteropathogens in suckling and weaned piglets with diarrhoea in southern Germany. Journal of Veterinary Medicine Series B 48, 151–159. Xiao, L., Escalante, L., Yang, C., Sulaiman, I., Escalante, A.A., Montali, R.J., Fayer, R. and Lal, A.A. (1999a) Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Applied and Environmental Microbiology 65, 1578–1583. Xiao, L., Morgan, U.M., Limor, J., Escalante, A., Arrowood, M., Shulaw, W., Thompson, R.C., Fayer, R. and Lal, A.A. (1999b) Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Applied and Environmental Microbiology 65, 3386–3391. Xiao, L., Morgan, U.M., Fayer, R., Thompson, R.C. and Lal, A.A. (2000) Cryptosporidium systematics and implications for public health. Parasitology Today 16, 287–292. Xiao, L., Bern, C., Arrowood, M., Sulaiman, I., Zhou, L., Kawai, V., Vivar, A., Lal, A.A. and Gilman, R.H. (2002a) Identification of the Cryptosporidium pig genotype in a human patient. Journal of Infectious Diseases 185, 1846–1848.

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Insights Into the Molecular Detection of Giardia duodenalis: Implications for Epidemiology S.M. CACCIÒ1, M. LALLE1, R. BECK2 AND E. POZIO1 1Istituto

Superiore di Sanità, Rome, Italy; 2University of Zagreb, Croatia

Abstract Giardia duodenalis is a widespread parasite of mammalian species, including humans. Due to its invariant morphology, investigation of aspects such as host specificity and transmission patterns requires a direct genetic characterization of cysts/trophozoites from host samples. A number of molecular assays have been developed to help in unravelling the complex epidemiology of this infection. Recently, however, molecular approaches have been complicated by the recognition of intra-isolate sequence heterogeneity (i.e. ‘mixed templates’), which affects subtype identification and the assignment of isolates to specific G. duodenalis assemblages. This raises concerns about the previous interpretation of genotyping data, and indicates the need to understand the mechanisms that are responsible for these unexpected findings. In this chapter, we critically review those different mechanisms and discuss some possible experimental strategies that can be used in future studies.

Introduction Giardia is a genus of intestinal flagellates that infect a wide range of vertebrate hosts. The genus currently comprises six species; namely Giardia agilis detected in amphibians, Giardia ardeae and Giardia psittaci detected in birds, Giardia microti and Giardia muris detected in rodents, and Giardia duodenalis detected in a wide spectrum of mammals, which are distinguished on the basis of the morphology and ultrastructure of their trophozoites (Adam, 2000). Giardia duodenalis (syn. G. intestinalis, G. lamblia) is the only species found in humans, although it is also found in other mammals, including domestic pets and livestock (Thompson, 2004). A considerable amount of data have shown that G. duodenalis should be considered as a species complex whose members, while showing little variation in their morphology, can be assigned to at least seven distinct assemblages (A to G) based on genetic analyses (Monis et al., 2003). Only assemblages A and B have been detected in humans and in a wide © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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Giardia duodenalis assemblages and their distribution in mammalian

Assemblages

Hosts

A

Humans and other primates, livestock, horses, dogs, cats, guinea pigs, fallow deer, white-tailed deer, ferrets Humans and other primates, livestock, horses, dogs, coyotes, muskrats, beavers Dogs, cats, coyotes, wolves Cattle, sheep, goats, water buffaloes, muflons Cats Rats

B C, D E F G

range of other mammalian hosts, whereas the remaining assemblages (C to G) are likely to be host-specific (Table 7.1). Due to the lack of variation in cyst and trophozoite morphology, the reliable discrimination of G. duodenalis isolates is necessarily based on their genetic characterization. Over the last decade or so, specially after the introduction of DNA amplification techniques, this approach has been extensively used, in particular to understand the role of animals in the epidemiology of human infection, i.e. to estimate the extent of zoonotic transmission (Cacciò et al., 2005), as well as to detect and genotype Giardia spp. cysts from water and food samples (Smith et al., 2006). Most of these studies, however, were based on the analysis of single markers, and no systematic evaluation of the genetic variability and usefulness of the different genetic loci has been performed. Indeed, the recognition of subgroups within assemblages A and B, as well as the extent of genetic variability among G. duodenalis isolates, are mainly based on isoenzyme analysis (Monis et al., 2003), an approach which is not routinely applicable to clinical and environmental samples, as it requires the in vitro propagation of Giardia spp. cysts.

Current Genotyping Approach Molecular typing: genetic loci, their variability, and the issue of nomenclature Compared to other protozoan pathogens, the genotyping technique for Giardia spp. is not particularly advanced, and the vast majority of studies rely on the analysis of the small subunit ribosomal RNA (ssu-rRNA), the β-giardin (bg), glutamate dehydrogenase (gdh), elongation factor 1-alpha (ef-1), triose phosphate isomerase (tpi), and the GLORF-C4 (C4) genes (reviewed in Cacciò et al., 2005). Some of these genes have been mapped, using hybridization on chromosomes separated by pulsed field gel electrophoresis (Adam, 2000), on to chromosome 5 (tpi and bg genes), chromosome 4 (gdh gene) and chromosome 1 (the majority of the ssu-rRNA gene copies). A search of the latest (April 2007) version of the G. duodenalis database (see http://gmod.mbl.edu/) showed that the tpi and bg genes are located in supercontig 577 (2 mega-bases (Mb) in size), and are

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about 150 kb apart. Similarly, the ef-1 gene is located on supercontig 608 (64 kb), and the C4 gene is located on supercontig 573 (1.5 Mb). Therefore, the genes can be considered unlinked in the G. duodenalis genome. This is an important and desirable feature for genotyping studies. A large number of sequences have been obtained from each of these genes and from isolates of human and animal origin collected worldwide (120 for ssu-rRNA, 170 for bg, 134 for gdh, 10 for ef-1, 70 for tpi, and 3 for C4, according to a search of the April 2007 GenBank release). Unfortunately, most sequences are partial, particularly in the case of the ssu-rRNA gene and, in general there is a tendency to reduce the amplicon size, either to increase PCR sensitivity or as a result of the design of new primers when more sequences become available. In terms of their polymorphism, these genes are quite different, with tpi and gdh genes being the more variable, followed by bg and C4 genes, then by the more conserved ef-1 and ssu-rRNA genes (Monis et al., 1999). This is also reflected by differences in substitution patterns, with the bg and ef-1 genes showing few, if any, non-synonymous changes, whereas the tpi and gdh genes appear to also tolerate amino acid replacements. Previous analyses, mainly using G. duodenalis isolates adapted to in vitro growth, and based on isoenzymes (Meloni et al., 1995) and DNA sequence analyses (Monis et al., 1999), have shown the existence of two subgroups in assemblage A (AI and AII) and in assemblage B (BIII and BIV). It is clear, however, that more variability is present in these as well as in isolates from the other G. duodenalis assemblages (Monis et al., 2003). Substructuring was evident within this assemblage, with isolates from the same host species forming distinct subclusters, which may correspond to yet undefined subgroups (Monis et al., 2003). More recent DNA-based genotyping studies have indeed identified a number of subtypes from both human and animal isolates (e.g. Sulaiman et al., 2003; Lalle et al., 2005a) and have confirmed substructuring within major G. duodenalis assemblages (Fig. 7.1). In these studies, a different terminology was used to describe subtypes, such as A3, A4, A5, or B1, B2, B3 at the bg locus (Lalle et al., 2005b), BS1, BS2, BS3, or CS1, CS2, CS3 at the tpi locus, or even B0, B1, B2 at the gdh locus. This has generated a certain degree of confusion, particularly when different subtypes were found by sequencing different genetic markers, i.e. when there was a lack of concurrence between the results for these genes (Robertson et al., 2006).

Epidemiology of Giardiasis Several characteristics of G. duodenalis influence the epidemiology of infection: ● ●

●

●

The infective dose is low (about 10–100 cysts; Rendtorff, 1954). Cysts are immediately infectious when excreted in faeces, and can be transmitted by person-to person or animal-to-animal contact (Thompson, 2004). Cysts are remarkably stable and can survive for weeks to months in the environment (Smith et al., 2006). Environmental contamination can lead to the contamination of drinking water and food (Karanis et al., 2007).

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98 99

AI A II A

100

F (cat) 100

E (hoofed animals)

91 100

B III

G. duodenalis

100 B IV

84

100 100

D (dog)

C (dog)

G (rat) 100 G. microti

G. ardeae G. muris

0.05

Fig. 7.1. Phylogenetic relationships of Giardia spp. inferred by the neighbour-joining analysis of the triose phosphate isomerase (tpi) nucleotide sequences. Only bootstrap values >70 are indicated. Modified from Lalle et al. (2007).

Direct and indirect transmission between infected hosts and susceptible individuals is favoured by high population densities (e.g. during lambing or calving), and by close contact (e.g. during recreational bathing or consumption of contaminated water) (Cacciò et al., 2005). Human isolates from different geographical locations, examined by PCR amplification of DNA extracted directly from faeces, demonstrate that only G. duodenalis assemblages A and B are associated with human infections. The prevalence of each assemblage varies considerably from country to country; assemblage B seems more common overall, but no strong conclusions can be drawn from current data (Cacciò et al., 2005). One important and still debated aspect in the epidemiology of human giardiasis is the extent of zoonotic transmission (Thompson, 2004). A number of animals have been considered to act as reservoirs, including livestock, pets and aquatic animals, but although the World Health Organization has considered G. duodenalis to have zoonotic potential for over 20 years (WHO, 1979), direct evidence is still lacking. As an example, beavers are often indicated as a source

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of environmental contamination and potential waterborne infection in humans (giardiasis is known as ‘beaver fever’ in Canada), but application of molecular techniques to samples collected in British Columbia (from humans, beavers and drinking water) could not support a causative link (McIntyre et al., 2000). Furthermore, the isoenzyme data of Monis et al. (2003) have shown that human and animal-derived isolates of G. duodenalis from assemblages A and B are rarely identical, and tend to form separate clusters in phylogenetic analyses. Finally, typing of wildlife isolates worldwide increasingly shows a predominance of hostadapted genotypes. Isolates from wild hoofed animals are often typed as assemblage A, but they are so genetically distinct from human isolates that zoonotic transmission seems unlikely (Lalle et al., 2007). Therefore, the available data, albeit still fragmentary, indicate a minor role of animals in the epidemiology of human giardiasis. However, zoonotic transmission may occur in endemic foci, where humans and animals live in close association with each other. In such cases, molecular characterization of cysts and detailed epidemiological investigations can provide insights into transmission patterns, as recently shown by a study of giardiasis in humans and dogs from a remote tea-growing community of north-east India (Traub et al., 2004). Certainly, we need more sensitive molecular methods to assess the zoonotic potential of Giardia spp. cysts shed by animals. In this respect, an important lesson can be learned from studies on cryptosporidiosis. Here, human infection with Cryptosporidium parvum has been traditionally considered zoonotic, with cattle being the main reservoir (Cacciò, 2005). However, the application of fingerprinting techniques, based on a combination of highly polymorphic micro- and minisatellite markers, has shown that a number of C. parvum types appear restricted to humans and may be maintained largely, if not exclusively, by an anthroponotic cycle (Mallon et al., 2003). Whether a similar situation exists for giardiasis is not known, but deserves to be considered. Transmission of G. duodenalis through drinking and recreational water is well documented, as are outbreaks of giardiasis following the consumption of contaminated water. Indeed, of the 325 water-associated outbreaks of parasitic protozoan disease reported so far (Karanis et al., 2007), G. duodenalis was the causative agent in about 40%. Despite this fact, little is known about the Giardia spp. or G. duodenalis assemblages present in the aquatic environment (Smith et al., 2006). In all wastewater samples examined, only G. duodenalis cysts were identified, and, more importantly, only human pathogens (assemblages A and B) were found (Cacciò et al., 2003; Sulaiman et al., 2004). This is in sharp contrast with results obtained for Cryptosporidium spp., but probably reflects the nature of the samples tested (raw urban wastewater) and the very limited number of surveys conducted. Most foodborne outbreaks of giardiasis were ascribed to direct contamination by a food handler, but in two instances a role for zoonotic transmission was suggested, namely the consumption of a Christmas pudding contaminated with rodent faeces and tripe soup made from the offal of an infected sheep (Smith et al., 2006). Notably, no information is available on the G. duodenalis assemblage(s) associated with these outbreaks.

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Current Problems in Molecular Genotyping Mixed templates: impact and possible origin The presence of mixed templates, characterized by two overlapping nucleotide peaks at specific positions in the electropherograms, was already reported 10 years ago (Hopkins et al., 1997). As a matter of fact, a number of sequences submitted to GenBank (for example, U47632) over the last few years contained undetermined positions (R, Y, etc.) and are likely to represent cases of mixed templates. The impact of this phenomenon is quite evident, as it hampers the precise assignment of isolates at the assemblage or subtype level. This is particularly relevant for assemblage B isolates, where a considerable fraction of isolates are characterized by mixed templates at different genetic loci (unpublished data from our laboratory). Two principal mechanisms can explain the occurrence of mixed templates: (i) ‘true’ mixed infections; and (ii) allelic sequence heterozygosity (ASH).

Mixed infections The occurrence of mixed infections has been reported in molecular-based surveys performed in Australia, the UK, India, Italy and Ethiopia (Hopkins et al., 1997; Amar et al., 2002; Traub et al., 2004; Lalle et al., 2005a, 2005b; Gelanew et al., 2007). The percentage of mixed infections ranged from 2.0% to 21.0% and was higher in less economically developed countries. Mixed infections are known to occur both at the inter- and intra-assemblage levels (for example, A plus B, or AI plus AII). In such cases, co-amplification of genetic material from genetically different cysts, followed by direct sequencing of PCR products, may result in heterogeneous sequencing profiles.

Allelic sequence heterozygosity Allelic sequence heterozygosity (ASH) has long been considered to be an explanation for mixed templates. Baruch et al. (1996) first reported ASH by sequence analysis of the tpi gene. They sequenced multiple cloned PCR products to demonstrate that the ambiguity in sequencing gels was due to ASH and not to technical artefacts. Interestingly, ASH was found between two human isolates from the AII subgroup, as well as between assemblage B isolates of human and animal origin. The level of ASH, however, was extremely low, less than 0.1%, a finding that seems to be confirmed by the genome sequencing project. However, the assembled portion of the genome does not include sub-telomeric regions, where chromosome size heterogeneity (heteromorphy) is known to occur (Adam, 2000). These regions carry copies of the rDNA of variable surface protein genes, and also of retroposons (Prabhu et al., 2007). Therefore, G. duodenalis

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chromosomes appear to be organized into conserved, central domains and polymorphic chromosome ends, resembling other parasitic genomes (Lanzer et al., 1995). A low ASH level is considered surprising for a polyploid, asexually replicating organism such as G. duodenalis (Adam, 2000). Indeed, substantial differences are expected to accumulate between the chromosome homologues in asexual organisms with a ploidy of two or higher, as has been shown for bdelloid rotifers (Welch and Meselson, 2000). In this context, it is important to note that in other asexual organisms, such as Daphnia spp., the rate of loss of nucleotide heterozygosity by ameiotic recombination is substantially greater than the rate of introduction of new variation by mutation. This suggests that the evolutionary potential of asexual diploid species is not only a matter of mutation accumulation and reduced efficiency of selection, but it underscores the limited utility of using neutral allelic divergence as an indicator of ancient asexuality (Omilian et al., 2006). Recent studies have shown that each nucleus has at least one copy of the genome, and that the two nuclei are partitioned equationally at cytokinesis (Yu et al., 2002), therefore ruling out two possible explanations for low ASH, namely that each nucleus contains a different genome, or that nuclear segregation is reductional (Yu et al., 2002). This result, however, was not confirmed by another study (Tumova et al., 2006), which reported differences in the number and size of chromosomes between the nuclei of single cells and, notably, suggested that each nucleus behaves as a clonal lineage, and should therefore accumulate mutations independently. Clearly, this complex issue deserves future investigation.

Assemblage swapping: impact and possible origin Recent publications (Read et al., 2004; Traub et al., 2004; Gelanew et al., 2007) have reported that the assignment of isolates to specific G. duodenalis assemblages is not always reliable, as different markers can give different results. This has been found both in human and animal isolates, appears to be rather frequent (up to 25% of dog isolates; Read et al., 2004), and has been found using different combinations of gene markers. It is of particular relevance that animal isolates can be typed as ‘potentially zoonotic’ with one marker, but as ‘host-specific’ with another. For molecular epidemiological studies, therefore, this has very important implications, as totally different conclusions may be drawn, depending on the way that genotyping data are obtained and interpreted. A number of mechanisms have been proposed to account for assemblage swapping, including preferential amplification of one assemblage over another (in cases of mixed infection), introgression, retention of ancestral polymorphisms and, more generally, recombinational (sexual) events. The occurrence of mixed infections has been summarized above. The possibility that a high percentage of infections are sustained by two genetically different parasites cannot be disregarded, particularly in regions of high endemicity.

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Introgression Introgression requires hybridization between closely related species and backcrossing between hybrids and one of the parental populations to produce gene flow (Anderson, 2001). This mechanism can result in segregation of divergent alleles in the same population. Conversely, identical alleles can occur in genetically distinct populations due to the retention of ancestral polymorphism, if the time of divergence of sister taxa from a common ancestor has been insufficient to allow fixation of the alleles produced by mutation and genetic drift (Anderson, 2001). If the organisms under study are morphologically identical, as is the case for G. duodenalis, the use of single-locus markers in molecular epidemiological studies may lead to ambiguous or even wrong conclusions, as demonstrated by Anderson (2001) using Ascaris spp. as a case study.

Meiosis and sex The evidence for the presence of sexuality in flagellates of the genus Giardia is, at present, only indirect. Using a phylogenomic approach, Ramesh et al. (2005) identified a core group of meiotic genes in the G. duodenalis genome (that are true orthologues of their counterparts in sexual organisms) and concluded that the parasite has the potential to perform meiosis, and thus, sexual reproduction. In a study of genome ploidy in different life cycle stages (Bernander et al., 2001), it has been shown that, during excystation, the recently excysted cell divides twice without DNA replication. The authors commented that: differentiation in G. lamblia is therefore reminiscent of meiosis, in which the genome is first replicated and then divided twice without DNA replication. It is possible that differentiation of primitive eukaryotes into cystic forms is an ancestral form of sexual processes.

If meiosis is present in G. duodenalis, what we need to know is how the diploidy is restored (Birky, 2005), and if this happens between nuclei from two different individuals (outcrossing) or from the same individual (selfing), as the net result of these two alternatives is crucially different.

Clonality As mentioned above, other studies have challenged this view, and suggested a ‘clonal’ mode of reproduction (Tumova et al., 2006). The (apparent) stable transmission of an aneuploid pattern is possible only if meiosis is absent, as the presence of homologous copies of each chromosome is a prerequisite for the first meiotic division (Tumova et al., 2006). In the early 1990s, Tibayrenc et al. (1990) proposed a clonal theory of population structure where sexual recombination is not frequent enough to break the prevalent pattern of clonal population structure. In this study, allozyme data were used to propose that G. duodenalis is a clonally propagating organism.

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The concept of clonality was then challenged by Maynard-Smith et al. (1993), who proposed an epidemic population structure where there is a background level of frequent sexual recombination with occasional clonal expansion of a few particular genotypes. Whether the population structure of G. duodenalis is clonal, panmictic or epidemic remains an open question, mainly because an insufficient number of isolates have been tested, and statistical tests could not be applied (Monis et al., 2003). Considering the conceptual and practical implications of clonality, this is clearly an important area for future investigation (Tibayrenc, 2005).

Mobile Elements and the Giardia-specific Virus Other mechanisms that could enhance genetic exchanges within and between trophozoites should also be considered. In particular, genetic mobile elements have been identified in the G. duodenalis genome, and a Giardia-specific virus has been widely studied. Giardia duodenalis harbours non-LTR (non-long terminal repeats) retroelements of the LINEs family (long interspersed nuclear elements), that are transposed by reverse transcription of mRNA directly into the site of integration. Most G. duodenalis sub-telomeres consist of tandem copies of active LINE retroposons (either GilM or GilT elements), which directly abut the telomeric repeats and are oriented such that reverse transcription would have run towards the chromosome end (Wickstead et al., 2003). Repeats play an integral part in ongoing genomic evolution and can perform diverse roles at different times, imparting a greater changeability to genomes. When an organism faces a changeable environment, the advantages of genomic flexibility (particularly if it can be contained at specific loci) may outweigh the extra cost of replication and of mutagenic effect exerted on other genomic regions. It is currently unknown what role the retroposons are playing (or have played) in the evolution of the G. duodenalis genome. Giardia-specific virus (GLV) is a double-stranded (ds) RNA virus of the Totiviridae family, constituted by a 36 nm non-enveloped icosahedron comprising one dsRNA of about 7 kb (Wang and Wang, 1991). GLV infects many G. duodenalis from assemblages A, B, C/D and E, although no correlation between the presence or absence of the virus and the specific assemblage has been found (Sedinova et al., 2003; Chen et al., 2007), and cohabitation of multiple GLV species in the same parasite has also been demonstrated (Tai et al., 1996). In G. duodenalis Portland I strain, which is chronically infected by this virus, viral RNA was detected in the cytoplasm as well as in the twin nuclei (Tai et al., 1991). Recombinant GLV cDNA has been successfully introduced into GLV-infected trophozoites to express a heterologous gene in G. duodenalis. Moreover, the chimeric RNA could be replicated as double-stranded RNA and packaged into virus-like particles, and the recombinant virions, by themselves, can superinfect G. duodenalis trophozoites and start new rounds of expression (Yu et al., 1996). All these observations are compatible with fragment(s) of the G. duodenalis genome being inserted in the GLV and then shuttled between trophozoites of different G. duodenalis assemblages.

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Intriguingly, plasmid DNA transfected into trophozoites of assemblage A is maintained as a multimeric episome, whereas in assemblage B it is always integrated into the genome by homologous recombination, with insertion of multiple copies (Singer et al., 1998). This contrasting behaviour could reflect isolate-specific differences in the factors determining whether exogenous DNA is integrated or maintained episomally. If plasmid- or virus-mediated lateral exchange of DNA between two trophozoites can occur, homologous recombination could, at least partially, explain the higher rate of ASH observed in assemblage B compared with assemblage A.

Future Experimental Strategies To understand the origin of mixed templates, different approaches can be envisaged, including the use of assemblage-specific primers (to estimate more precisely the occurrence of mixed infections and its contribution to both phenomena), the cloning of PCR products from single isolates followed by sequencing of multiple clones (to evaluate ASH), and the direct typing of single nuclei isolated from cultured trophozoites (to prove genetic heterogeneity between the two nuclei). For a more comprehensive analysis, however, it is still invaluable to obtain and maintain cloned isolates that are representative of each assemblage. This research will also benefit from international collaboration and from the use of validated sets of markers and methods that allow direct comparisons between studies.

References Adam, R.D. (2000) The Giardia lamblia genome. International Journal for Parasitology 30, 475–484. Amar, C.F.L., Dear, P.H., Pedraza-Díaz, S., Looker, N., Linnane, E. and McLauchlin, J. (2002) Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces. Journal of Clinical Microbiology 40, 446–452. Anderson, T.J.C. (2001) The dangers of using single locus markers in parasite epidemiology: Ascaris as a case study. Trends in Parasitology 17, 183–188. Baruch, A.C., Isaac-Renton, J. and Adam, R.D. (1996) The molecular epidemiology of Giardia lamblia: a sequence-based approach. Journal of Infectious Diseases 174, 233–236. Bernander, R., Palm, J.E. and Svard, S.G. (2001) Genome ploidy in different stages of the Giardia lamblia life cycle. Cellular Microbiology 3, 55–62. Birky, C.W., Jr (2005) Sex: is Giardia doing it in the dark? Current Biology 15, 56–58. Cacciò, S.M. (2005) Molecular epidemiology of human cryptosporidiosis. Parassitologia 47, 185–192. Cacciò, S.M., De Giacomo, M., Aulicino, F. and Pozio, E. (2003) Giardia cysts in wastewater treatment plants in Italy. Applied and Environmental Microbiology 69, 3393–3398. Cacciò, S.M., Thompson, R.C.A., McLauchlin, J. and Smith, H.V. (2005) Unravelling Cryptosporidium and Giardia epidemiology. Trends in Parasitology 21, 430–437.

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Chen, L., Li, J., Zhang, X., Liu, Q., Yin, J., Yao, L., Zhao, Y. and Cao, L. (2007) Inhibition of krr1 gene expression in Giardia canis by a virus-mediated hammerhead ribozyme. Veterinary Parasitology 143, 14–20. Gelanew, T., Lalle, M., Hailu, A., Pozio, E. and Cacciò, S.M. (2007) Molecular characterization of human isolates of Giardia duodenalis from Ethiopia. Acta Tropica 102, 92–99. Hopkins, R.M., Meloni, B.P., Groth, D.M., Wetherall, J.D., Reynoldson, J.A. and Thompson, R.C. (1997) Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. Journal of Parasitology 83, 44–51. Karanis, P., Kourenti, C. and Smith, H. (2007) Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. Journal of Water Health 5, 1–38. Lalle, M., Jimenez, E., Cacciò, S.M. and Pozio, E. (2005a) Genotyping of Giardia duodenalis from humans and dogs from Mexico using β-giardin nested PCR assay. Journal of Parasitology 91, 203–205. Lalle, M., Pozio, E., Capelli, G., Bruschi, F., Crotti, D. and Cacciò, S.M. (2005b) Genetic heterogeneity at the b-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. International Journal for Parasitology 35, 207–213. Lalle, M., Frangipane di Regalbono, A., Poppi, L., Nobili, G., Tonanzi, D., Pozio, E. and Cacciò, S.M. (2007) A novel Giardia duodenalis assemblage A subtype in fallow deer. Journal of Parasitology 93, 426–428. Lanzer, M., Fischer, K. and Le Blancq, S.M. (1995) Parasitism and chromosome dynamics in protozoan parasites: is there a connection? Molecular and Biochemical Parasitology 70, 1–8. Mallon, M.E., MacLeod, A., Wastling, J.M., Smith, H. and Tait, A. (2003) Multilocus genotyping of Cryptosporidium parvum Type 2: population genetics and sub-structuring. Infection Genetics and Evolution 3, 207–218. Maynard-Smith, J., Smith, N.H., O’Rourke, M. and Spratt, B.G. (1993) How clonal are bacteria? Proceedings of the National Academy of Sciences of the USA 90, 4384–4388. McIntyre, L., Hoang, L., Ong, C.S., Lee, P. and Isaac-Renton, J.L. (2000) Evaluation of molecular techniques to biotype Giardia duodenalis collected during an outbreak. Journal of Parasitology 86, 172–177. Meloni, B.P., Lymbery, A.J. and Thompson, R.C.A. (1995) Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: implications for reproductive biology, population structure, taxonomy, and epidemiology. Journal of Parasitology 81, 368–383. Monis, P.T., Andrews, R.H., Mayrhofer, G. and Ey, P.L. (1999) Molecular systematics of the parasitic protozoan Giardia intestinalis. Molecular Biology and Evolution 16, 1135–1144. Monis, P.T., Andrews, R.H., Mayrhofer, G. and Ey, P.L. (2003) Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infection Genetics and Evolution 3, 29–38. Omilian, A.R., Cristescu, M.E., Dudycha, J.L. and Lynch, M. (2006) Ameiotic recombination in asexual lineages of Daphnia. Proceedings of the National Academy of Sciences of the USA 103, 18638–18643. Prabhu, A., Morrison, H.G., Martinez, C.R., III and Adam, R.D. (2007) Characterisation of the subtelomeric regions of Giardia lamblia genome isolate WBC6. International Journal for Parasitology 37, 503–513.

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Wildlife with Giardia: Villain, or Victim and Vector? S.J. KUTZ1, R.C.A. THOMPSON2 AND L. POLLEY3 1Faculty

of Veterinary Medicine, University of Calgary, Alberta, Canada; Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, Murdoch University, WA, Australia; 3Western College of Veterinary Medicine, University of Saskatchewan, Canada 2WHO

Abstract Among mammalian wildlife, Giardia has been reported on every continent and from a wide variety of species, including marsupials, rodents, insectivores, ungulates, marine mammals, felids, canids and ursids. Increasing efforts have been made to determine the strain and zoonotic potential of Giardia and this has resulted in new host and geographical records for Giardia duodenalis. According to many peer-reviewed publications, the mere presence of Giardia in wildlife is sufficient to implicate wildlife as source of infection for humans. However, the bulk of the evidence to support this claim demonstrates correlation, not causation. In fact, in many cases wildlife are more likely to be the victims, with ‘spillover’ of zoonotic strains from people and/or domestic animals. A critical re-evaluation of zoonotic strains of Giardia in wildlife is needed. This should include a return to classical parasitology and epidemiology in order to develop a quantitative understanding of the temporal and spatial patterns and impacts of the various Giardia strains in wildlife and the potential interactions with domestic systems. It has become abundantly clear that the mere detection of Giardia cysts in wildlife is no longer sufficient evidence of zoonotic transmission and that there remains considerable uncertainty about the structure of the parasite web and the direction and intensity of parasite flow for ‘zoonotic’ strains of Giardia among domestic and wild animal, and human hosts.

Introduction Since its discovery in 1681, Giardia has been recovered from a wide range of mammalian, avian, reptilian, and amphibian hosts, many of which have been considered to be important reservoirs of infection for people (Thompson, 2002). At one time over 50 species of Giardia had been described. The recent application of molecular tools has led to a rapid evolution in our understanding of the parasite’s taxonomy and host specificity, confirming some previously named species while refuting others. Importantly, recognition of these host specificities has generated doubt concerning the ubiquitous zoonotic threat once thought 94

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to be associated with Giardia. Today there are seven recognized species assemblages defined on the basis of molecular and phylogenetic analyses. Based on our current understanding, only Giardia duodenalis assemblages A and B, described from a variety of mammalian species, have demonstrated zoonotic potential, although the zoonotic potential of novel subtypes is unclear.

Are Wildlife Species Significant Reservoirs for Zoonotic Transmission of Giardia? Parasites of the genus Giardia are widespread in mammalian, avian, reptilian and amphibian wildlife. Mammalian hosts, in particular, have long been considered important sources of infection for people. In fact, the beaver is considered such a common source of human infection that in North America the disease in people is often referred to as ‘beaver fever’. The link to beavers is so ingrained among the public and healthcare professionals that physicians have been known to advise that there is no risk of Giardia in areas where beavers are absent, and one peer-reviewed paper suggested that the movement of beavers northwards will enhance the emergence of giardiasis in people in northern regions (Parkinson and Butler, 2005). The belief in the integral role of beavers in the propagation of giardiasis is also reflected in the active education campaigns in parks and protected areas across Canada and the USA, in the culture of campers and backcountry hikers, and is a tremendous stimulus for the purchase of water filters. While several wildlife species are, to varying degrees, suitable hosts for the human Giardia duodenalis assemblages A and B, beavers appear to be particularly susceptible to these strains, are able to maintain them for several months and, once infected, may serve as suitable reservoirs, and perhaps amplifiers, for spill-back to people (Davies and Hibler, 1979). Most of the evidence implicating beavers and other wildlife hosts as significant sources of Giardia for people is, however, anecdotal and correlational – direct links have rarely, if ever, been established (Appelbee et al., 2002; Sulaiman et al., 2003; Cacciò et al., 2005). One of the most extensive epidemiological studies examining the links between wildlife, domestic animals and people, is that by Davies and Hibler from 1975 to 1977 (Davies and Hibler, 1979). These authors investigated Giardia in multiple species (26 wildlife, 5 domestic – dogs, cats, cattle, horses, sheep – and people) in multiple watersheds in Colorado, USA. They also completed a series of experimental cross-infections using Giardia from different host species to determine host specificity. This landmark study demonstrated several important aspects of the spatial, temporal, and host distribution of Giardia: ● ●

●

Giardia was already present in people and dogs entering wilderness areas. Of 26 wildlife species sampled, only beavers and coyotes were infected with Giardia in the wild. There was a temporal pattern of Giardia infection for beavers, coyotes and cattle, with none of these species infected in the spring but all infected by autumn, suggesting that the parasite did not overwinter in these species, but that they became infected during the summer.

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●

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Prevalence in beavers was higher at or downstream from outbreak locations or human settlements, campgrounds, or sewage plants, compared with upstream, and prevalence in beavers increased with distance downstream. Giardia from people, beavers or (captive) deer varied in their ability to infect other host species.

Through this survey, Davies and Hibler established host, temporal and spatial distributions of Giardia spp. in areas of Colorado, and through their experimental work they established pre-patent and patent periods and reported any clinical signs associated with the parasite in various host species, including people (Davies and Hibler, 1979). They also observed that ‘beaver may be an excellent sentry animal’ for watershed contamination, and that once infected ‘beaver are an important reservoir … they carry the infection for at least 3 months and defecate into the water where Giardia cysts survive very well’. Domestic livestock were considered as part of the transmission cycle in that they congregate around waterbodies and defecate in and around them and thus may become important reservoirs. Dogs were also considered as important sources of infection and disseminators of Giardia because they accompany people everywhere, defecate at will, are coprophagic, and often roll in faeces. Ultimately Davies and Hibler (1979) stated that ‘Humans are the most important component in the epidemiology of giardiasis’. They commented that: people use watersheds heavily and frequently defecate indiscriminately in or near waterbodies (the ‘wilderness experience’); ‘human feces and toilet paper were found in several locations’ along waterbodies; used diapers have been found in lakes and streams, and; there are several reports of people emptying recreational vehicle toilets into rivers. They concluded that ‘giardiasis is a growing problem and will remain so for many years’ because there will be increased use of wilderness areas by people and their pets (some of which bring Giardia with them), and there is a lack of sanitary facilities and a reluctance of people to use them (Davies and Hibler 1979). Subsequent advances in molecular biology have allowed us to unravel some of the issues associated with host-specificity, and the zoonotic potential of some strains of Giardia has been confirmed. Despite these developments, no studies have demonstrated unequivocally that wildlife serve as the origin or main reservoir for human infections with the parasite. In many waterborne outbreaks affecting people, sewage contamination of the water supply has been implicated as the source of Giardia (Craun et al., 2005). In reports where wildlife has been identified as the source of the parasite, supporting evidence is lacking and wildlife appear to be blamed by default (Charron et al., 2004). Additionally, Giardia cysts are invariably much more abundant in sewage outflow (treated and untreated) than in the raw water intake in most communities, suggesting that contamination of waterbodies is of human (or domestic animal) origin. It seems that beavers, and other wildlife using water downstream from these outflows, are unfortunate victims of this environmental contamination (Roach et al., 1993; Craun et al., 2005). The uncertainty of the relationship between Giardia in wildlife and people was eloquently captured by Cacciò et al. (2005):

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The occurrence of Giardia in wildlife … has been the single most important factor implicating Giardia as a zoonotic agent. It is therefore surprising that there is little evidence to support the role of wildlife as a source of disease in humans. Although wildlife, particularly aquatic mammals, is commonly infected with Giardia, there is little evidence to implicate such infections as the original contaminating source in waterborne outbreaks. It would seem that such animals are more likely to have become infected from water contaminated with faecal material of human or, less likely, domestic animal origin, thus serving to amplify the numbers of the originally contaminating isolate. The few studies that have genotyped Giardia of beaver origin have confirmed previous suggestions that the source of Giardia infection in beavers was likely to be of human origin.

It has become abundantly clear that the mere detection of Giardia cysts in wildlife is no longer sufficient evidence of zoonotic transmission and that there remains considerable uncertainty about the structure of the parasite web and the direction and intensity of parasite flow for ‘zoonotic’ strains of Giardia among domestic and wild animal, and human hosts.

Emergence of Giardia in Wildlife In mammalian wildlife, Giardia has been reported on every continent and from a wide variety of species, including marsupials, rodents, insectivores, ungulates, marine mammals, felids, canids and ursids (Appelbee et al., 2005; Hamnes et al., 2006; S.J. Kutz et al., unpublished). Increasingly, efforts have been made to determine the strain and zoonotic potential of Giardia, and this has resulted in new host and geographical records for Giardia duodenalis assemblage A in muskoxen (Kutz et al., 2008), bowhead whale (Hughes-Hanks et al., 2005), harp seals (M. Olson, 2007, Calgary, personal communication) and gorillas (Graczyk et al., 2002); assemblage B in beavers (Fayer et al., 2006) and coyotes (Trout et al., 2006); and assemblages A and B from red foxes (Hamnes et al., 2007), ringed seals (B. Dixon and M. Olson, 2007, Morelia, personal communication) and house mice ((Moro et al., 2003). Additional reports of Giardia spp. or Giardia duodenalis in wildlife are common (Hamnes et al., 2006; Gaydos et al., 2007), but unfortunately, without genotyping, the interpretation of these findings is limited with respect to understanding parasite flow and impacts.

Some Case Studies: ‘Zoonotic’ Giardia in Free-ranging Ungulates In general, ungulates typically appear to be accidental hosts for Giardia assemblage A. This is supported by the studies on domestic cattle that invariably report assemblage E as the dominant strain. Giardia is rare in free-ranging ungulate populations (see Hamnes et al., 2006), but zoonotic strains have been recovered from white-tailed deer, elk and muskoxen (Deng and Cliver, 1999; Trout et al., 2003; Hamnes et al., 2006) (S.J. Kutz et al., unpublished). Two studies illustrate some of the key features of the ecology of Giardia that may have significance in the transmission of the parasite to people, and are worthy of further consideration.

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Giardia in Norwegian cervids Giardia is emerging as a parasite of concern in people and animals in Norway (Robertson and Gjerde, 2006). In a study of the parasite in cervids, Giardia was found in red deer (prevalence 1.7%), reindeer (7.1%), moose (12.3%), and roe deer (15.5%) (Hamnes et al., 2006). The difference in prevalence among species was hypothesized to be related to host behaviour and distribution. The higher prevalence in moose was linked to the aquatic habitat that this species prefer and the higher prevalence in roe deer to the fact that the deer are relatively sedentary and commonly found in close proximity to people and agricultural lands (Hamnes et al., 2006). In contrast, reindeer were found in higher, drier and less populated areas, and both red deer and reindeer are migratory species (in contrast to moose and roe deer) and are less likely to re-infect themselves from contaminated soil and water. Thus, this study begins to unravel the potential role of host ecology in the structure of a parasite web for Giardia and in the rates of parasite flow.

Giardia in muskoxen in an arctic ecosystem Giardia was first found in wild muskoxen in 1994 on Banks Island, in arctic Canada. It was found that 4% of muskoxen tested from 1994 to 1998 were positive for the parasite, but the strain was not determined (Nagy et al., 1998). In 2004, muskoxen were sampled again and Giardia assemblage A (probably A1) was reported at a prevalence of 21% (Kutz et al., 2008). These results were interesting for two reasons. First, in this remote region it was anticipated that any Giardia in muskoxen would be either a novel strain or a livestock strain; and secondly, the prevalence of infection appeared to have increased from the mid 1990s to 2004. It was hypothesized that people first introduced Giardia to muskoxen and that the parasite may now be cycling in the muskox population (Kutz et al., 2008), roe deer, moose and reindeer (van der Giessen et al., 2006; Robertson et al., 2007). The time frame for this introduction is unknown. Muskoxen were rare (perhaps absent) on the island in the early 1900s and were unlikely to maintain a population density sufficient to maintain the parasite. Thus the introduction of Giardia may more likely have occurred within the last century. Giardia has also been reported in sympatric Peary caribou (prevalence 3%) as well as in residents of the island. Chronic ‘stomach flu’ has often been reported by people in the community of Sachs Harbour on Banks Island and, since the discovery of Giardia in muskoxen, faecal samples from symptomatic people have been examined for Giardia with some positive results. Ringed seals in the marine environment surrounding the island have also tested positive for Giardia (Olson et al., 1997) and assemblage A has been isolated from some of these animals (M. Olson, 2007, Calgary, personal communication), suggesting a possible terrestrial–marine cycle. The isolates of Giardia from people on Banks Island have not yet been typed and this hinders our understanding of the flow of the parasite among wildlife and people. Several aspects of human and animal behaviour may influence parasite

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transmission in this ecosystem. First, the residents of Banks Island still maintain close ties with the land and spend time ‘on-the-land’ engaged in subsistence hunting and fishing. They visit and camp in areas rich in wildlife, such as river valleys, and obtain water directly from the rivers and streams. The limited summer tourism on the island is also concentrated around waterbodies. Typically there are no latrines in these remote regions and environmental contamination with human waste is common. Additionally, muskoxen on the island are relatively sedentary and are concentrated in the lush river valleys. Thus spatial overlap of habitat use between people and muskoxen is common and is usually centred on waterbodies – ideal locations for the persistence and transmission of Giardia (Kutz et al., 2008). Second, annually during the winter there is an organized, inspected commercial slaughter of muskoxen for sale of meat and qiviut (undercoat). Muskoxen are herded into a central location, held for up to 72 h, and then shot. Historically, the offal from the slaughter was removed to a nearby hillside, but practices have changed and it is now deposited on the sea ice. Depending on survival of the Giardia cysts in the sub-zero environment, the concentration of muskoxen at the slaughter site and the disposal of offal on the land and sea ice may enhance transmission and dispersal of Giardia among the marine and terrestrial wildlife as well as people (Kutz et al., 2008). Preliminary work suggests that the muskox is a good host for Giardia assemblage A, not unlike the beaver. The high prevalence in muskoxen in an area of such low human density is perhaps surprising and suggests that the parasite may be maintained in this host in the absence of people. Possible reduced immunocompetence of muskoxen (this species seems to be exquisitely sensitive to many pathogens) together with enhanced survival and transmission of parasite cysts in an arctic environment may in part explain this pattern of infection. Many questions remain as to how Giardia is maintained in this arctic environment: is it amplified in the muskox population, did it originate from people (spillover) and is there spill-back to people, what other wildlife species are infected, and is there a terrestrial/marine cycle? Giardia may be emerging in muskoxen (increased prevalence since the mid-1990s), but more long-term data on seasonal, temporal, age- and sex-related patterns of infection are needed to confirm this. Further work is planned in order to better understand the flow, effects, patterns of survival and transmission of Giardia in terrestrial and marine environments at these high latitudes (Kutz et al., 2008).

‘Zoonotic’ Giardia in Other Wildlife Hosts Marine ecosystems Reports of Giardia in marine environments are becoming more common. The parasite has been reported in a variety of pinnipeds, and more recently in cetaceans (Olson et al., 1997; Measures and Olson, 1999, 2002; Deng et al., 2000; Hughes-Hanks et al., 2005). Few of these isolates have been typed; however, both assemblage A and B have been recovered from marine mammals

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(Appelbee et al., 2005; B. Dixon, 2007, Morelia, personal communication). In some cases the source of Giardia may be of anthropogenic origin (runoff from human activities) or domestic livestock sources, and the finding of Giardia assemblage A in Macoma and Asian freshwater clams in fresh water and estuaries along the east coast of the USA (Graczyk et al., 1999a, 1999b) supports this hypothesis. Non-human primates Zoonotic transmission of pathogens between human and non-human primates has long been of great concern for human health. With increased ecotourism, the risk of transmission of pathogens from people to non-human primates, and the impact on the conservation of these species, has become a major concern (Nizeyi et al., 2001). Recently, Giardia assemblage A was reported in sympatric gorillas (prevalence of 2%), cattle (10%) and people (5%) in the Bwindi Impenetrable National Park, Uganda. It is hypothesized that the parasite was introduced by people and now circulates among these three host species, and that transmission may be maintained and enhanced by poor husbandry and hygiene practices together with ingestion of untreated stream water (Graczyk et al., 2002).

Drivers of Emergence of Giardia in Wildlife In recent years there has been an apparent increase in the frequency of reports of Giardia in wildlife. This may represent apparent emergence attributable in part to increased sampling efforts related to an interest in the potential role of wildlife as reservoirs for zoonoses, and to improvements in molecular tools for diagnosis. There is, however, strong evidence to suggest that there is a real emergence of human and domestic animal strains of Giardia in wildlife as a result of environmental changes and the breakdown of ecological barriers separating host species. For example, new host assemblages resulting from agricultural encroachment on to wild lands, suburbanization of people and urbanization of wildlife, and pathogen pollution from increased sewage discharge and runoff from agricultural lands, may be linked to a real emergence of Giardia in at least some wildlife species (roe deer and agriculture; gorillas, ecotourism and cattle; and terrestrial runoff to marine systems). Directional climate change, which will continue to alter habitats, habitat use, and water availability and distribution, is likely to result in new host distributions and associations and the subsequent emergence of Giardia strains and other pathogens in new host species and in new locations (Kutz et al., 2004, 2005).

Impacts on Wildlife Although Giardia is recognized as an important pathogen in people and domestic animals, causing gastrointestinal disease and adversely affecting several

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aspects of childhood development (Savioli et al., 2006), effects of Giardia in wildlife have been only superficially investigated. In people and domestic animals Giardia can be treated, and for domestic livestock the impacts of parasitism can often be mitigated through abundant high-quality feed and shelter. For freeranging wildlife, however, this is not the case. Wildlife are often ‘living on the edge’, exposed to the elements, predators, and often with limited food sources. Any additional drain on their resources, such as parasitism, may have significant impacts on the survival and lifetime reproductive success of a free-ranging species.

Host infections with multiple strains It is important to recognize that different strains (historical versus recently acquired) may have different impacts on wildlife hosts. In particular, assemblages A and B may have evolved primarily in a domestic cycle (people/livestock/dogs), and a host switch to wildlife may have more significant impacts on these new hosts. Recent molecular typing has demonstrated that a single host species can be infected with multiple strains of Giardia concurrently. For example, the small Australian marsupial, the quenda, is host to a ‘new’ strain of Giardia which is the only strain present when quenda are in their native habitat (Adams et al., 2004). Quenda in agricultural areas, however, also harbour the livestock strain of Giardia (Thompson, Chapter 1, this volume) indicating host-switching by Giardia and a lack of strong cross-immunity between the two strains. Similarly, coyotes and foxes have recently been reported to harbour several different strains of Giardia in a single host population, and possibly within a single host (Trout et al., 2006; Hamnes et al., 2007). These results raise questions about whether infection with multiple strains of Giardia at population and individual animal levels reflect the historical situation, or whether it is a result of relatively recent increases in contact between previously separated host species. Apparent host switching by Giardia draws attention to the need for an improved understanding of host specificity, the health effects of the parasite in historical and in naïve hosts, how Giardia species and strains are maintained in a variety of ecosystems, and the potential for spillover and spill-back among host species and populations.

Implications for People of ‘Zoonotic Strains’ in Wildlife Although the evidence for wildlife as a significant, ongoing source and reservoir of human infection with Giardia is equivocal, it is conceivable that, once infected, these hosts may serve as a source of ‘spill-back’ of the parasite to people. Beavers and muskoxen, with high reported prevalences and intensities of these strains, and typically an association with waterbodies, stand out as possible wildlife species that may be able to amplify zoonotic Giardia (Fig. 8.1). There may be other, as yet uninvestigated, wildlife hosts with a similar role in the epidemiology of zoonotic Giardia. Despite this potential, there is, at present, no clear evidence that these hosts are a source of human infection, and wildlife may more probably

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Beavers Muskoxen

People

Other wildlife

Domestic animals

Fig. 8.1. A proposed parasite web for Giardia duodenalis assemblages A or B within and among species and between domestic and sylvatic cycles. The thickness of arrows reflects the rate of parasite flow in the various parts of the web, with dashed arrows indicating that parasite flow is unlikely. Note that for beavers and muskoxen a strong ‘within species’ transmission cycle is hypothesized.

be victims of environmental contamination with Giardia from anthropogenic sources as opposed to the primary villains responsible for infections in people. Proposed relative rates of flow between people, domestic animals and wildlife for Giardia assemblages A and B are shown in Fig. 8.1.

Moving Forward Giardia, the ‘cosmopolitan parasite’, is recognized as an important component of the World Health Organization ‘Neglected Disease Initiative’ (Savioli et al., 2006). We propose that, because of the current lack of clarity surrounding the role of wildlife in parasite webs for Giardia, and especially the role of these hosts as sources of infection for people, wildlife should be considered the ‘neglected hosts’ of the ‘Neglected Disease Initiative’. Good datasets on the prevalence, intensity, distribution, and host range of Giardia are rare, and the temporal, spatial and quantitative patterns of parasite flow within and between species (wild, domestic and human) remain poorly described. It is now time to apply our rapidly advancing molecular tools, together with classical epidemiology and parasitology, to better understand the ecology, impacts and flow of Giardia among wild and domestic hosts. This can be done by focusing on several key approaches. Survey and inventory Equipped with molecular techniques to better define species and strain diversity, we now need to revisit host species and geographical regions to (re)define the

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host range and geographical distribution of the various strains of Giardia. Studies that report the presence of ‘Giardia’ with no data on strain identification provide little insight into parasite flow or the epidemiology of the parasite among its various host species. Strain-level identification of Giardia is essential to generate important new knowledge on host–parasite associations and establish baselines for future comparisons. Sampling wildlife poses several logistic challenges, and often there is limited or substandard material available; thus sensitive and efficient diagnostic techniques are essential in order to encourage strain identification (Fayer et al., 2006). Further taxonomic and phylogenetic analyses are also needed to evaluate host–parasite associations and historical biogeographical relationships which should shed light on epidemiology and impacts of the various strains in wildlife and domestic systems.

Classical parasitology Moving beyond basic survey and inventory, there is a need to return to the examples of Davies and Hibler (1979) and others to describe and understand the temporal, spatial, demographic and quantitative patterns of occurrence, survival and transmission of Giardia in wildlife and among wildlife, domestic species and people. Our advantage today is that we can identify strains, greatly enhancing our understanding of observed patterns in wild and experimental settings. Experimental infections of various host species are needed in order to determine the host susceptibility, pre-patent and patent periods, clinical signs and temporal patterns of parasite production. The viability of various Giardia strains from different host species and under different environmental conditions needs to be quantified. Together, these data will provide tremendous insight into the flow of zoonotic strains of Giardia between domestic and wild systems, moving us towards an understanding of causal, as opposed to correlational, relationships.

Giardia archives Finally, there is a need for a concerted effort to archive Giardia isolates from various species and geographical locations. If research in recent years has taught us nothing else, advances in molecular techniques invariably result in the need to go back and re-evaluate previous isolates and species and strain designations. Methodologies and standards for preserving and archiving specimens should be developed and widely promoted to ensure that these valuable materials are available for these retrospective analyses.

Conclusions In the research reported in many peer-reviewed publications, the mere presence of Giardia in wildlife has been sufficient to implicate wildlife as source of infection

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for people. However, the bulk of the evidence to support this claim demonstrates correlation, not causation. In fact, in many cases, wildlife species are more probably the victims, with ‘spillover’ of zoonotic strains from people and/or domestic animals. A critical re-evaluation of zoonotic strains of Giardia in wildlife is needed. This should include a return to classical parasitology and epidemiology to develop a quantitative understanding of the temporal and spatial patterns and impacts of the various Giardia strains in wildlife and the potential interactions with domestic systems. These efforts should lead to an understanding of the structure of parasite webs and parasite flow for Giardia among domestic and sylvatic hosts and people, and should ultimately provide an evidence-based assessment of the critical control points and management activities required to reduce environmental contamination and cross-species transmission. We should not be surprised if we discover that the key management steps needed to reduce Giardia in people may be centred on the appropriate containment and disposal of our own waste!

References Adams, P.J., Monis, P.T., Elliot, A.D. and Thompson, R.C. (2004) Cyst morphology and sequence analysis of the small subunit rDNA and ef1 alpha identifies a novel Giardia genotype in a quenda (Isoodon obesulus) from Western Australia. Infection, Genetics and Evolution 4, 365–370. Appelbee, A., Thorlakson, C. and Olson, M. (2002) Genotypic characterization of Giardia cysts isolated from wild beaver in southern Alberta, Canada. In: Olsen, B.E., Olsen, M.E. and Wallis, P.M. (eds) Giardia: The Cosmopolitan Parasite. CABI Publishing, Wallingford, UK and New York, pp. 299–300. Appelbee, A.J., Thompson, R. and Olson, M.E. (2005) Giardia and Cryptosporidium in mammalian wildlife: current status and future needs. Trends in Parasitology 21, 370–376. Cacciò, S.M., Thompson, R.C.A., McLauchlin, J. and Smith, H.V. (2005) Unravelling Cryptosporidium and Giardia epidemiology. Trends in Parasitology 21, 430–437. Charron, D.F., Thomas, M.K., Waltner-Toews, D., Aramini, J.J., Edge, T., Kent, R.A., Maarouf, A.R. and Wilson, J. (2004) Vulnerability of waterborne diseases to climate change in Canada: a review. Journal of Toxicology and Environmental Health – Part A 67, 1667–1677. Craun, G.F., Calderon, R.L. and Craun, M.F. (2005) Outbreaks associated with recreational water in the United States. International Journal of Environmental Health Research 15, 243–262. Davies, R.B. and Hibler, C.P. (1979) Animal reservoirs and cross-species transmission of Giardia. In: Jakubowski, W. and Hoff, J.C. (eds) Waterborne Transmission of Giardiasis. US Environmental Protection Agency, Office of Research and Development, Environmental Research Centre, Cincinnati, OH, EPA-600/7-79-001, pp. 104–126. Deng, M. and Cliver, D. (1999) Improved immunofluorescence assay for detection of Giardia and Cryptosporidium from asymptomatic adult cervine animals. Parasitology Research 85, 733–736. Deng, M.Q., Peterson, R.P. and Cliver, D.O. (2000) First findings of Cryptosporidium and Giardia in California sea lions (Zalophus californianus). Journal of Parasitology 86, 490–494.

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Fayer, R., Santín, M., Trout, J.M. and Greiner, E. (2006) Prevalence of species and genotypes of Cryptosporidium found in 1–2-year-old dairy cattle in the eastern United States. Veterinary Parasitology 135, 105–112. Gaydos, J.K., Miller, W.A., Gilardi, K.V.K., Melli, A., Schwantje, H., Engelstoft, C., Fritz, H. and Conrad, P.A. (2007) Cryptosporidium and Giardia in marine-foraging river otters (Lontra canadensis) from the Puget Sound Georgia Basin ecosystem. Journal of Parasitology 93, 198–202. Graczyk, T.K., Fayer, R., Conn, D.B. and Lewis, E.J. (1999a) Evaluation of the recovery of waterborne Giardia cysts by freshwater clams and cyst detection in clam tissue. Parasitology Research 85, 30–34. Graczyk, T.K., Thompson, R.C., Fayer, R., Adams, P., Morgan, U.M. and Lewis, E.J. (1999b) Giardia duodenalis cysts of genotype A recovered from clams in the Chesapeake Bay subestuary, Rhode River. American Journal of Tropical Medicine and Hygiene 61, 526–529. Graczyk, T.K., Bosco-Nizeyi, J., Ssebide, B., Thompson, R.C., Read, C. and Cranfield, M.R. (2002) Anthropozoonotic Giardia duodenalis genotype (assemblage) A infections in habitats of free-ranging human-habituated gorillas, Uganda. Journal of Parasitology 88, 905–909. Hamnes, I.S., Gjerde, B., Robertson, L., Vikoren, T. and Handeland, K. (2006) Prevalence of Cryptosporidium and Giardia in free-ranging wild cervids in Norway. Veterinary Parasitology 141, 30–41. Hamnes, I.S., Gjerde, B.K., Forberg, T. and Robertson, L.J. (2007) Occurrence of Giardia and Cryptosporidium in Norwegian red foxes (Vulpes vulpes). Veterinary Parasitology 143, 347–353. Hughes-Hanks, J.M., Rickard, L.G., Panuska, C., Saucier, J.R., O’Hara, T.M., Dehn, L. and Rolland, R.M. (2005) Prevalence of Cryptosporidium spp. and Giardia spp. in five marine mammal species. Journal of Parasitology 91, 1225–1228. Kutz, S.J., Hoberg, E.P., Nagy, J., Polley, L. and Elkin, B. (2004) “Emerging” parasitic infections in arctic ungulates. Integrative and Comparative Biology 44, 109–118. Kutz, S.J., Hoberg, E.P., Polley, L. and Jenkins, E. (2005) Global warming is changing the dynamics of arctic host–parasite systems. Proceedings of the Royal Society B: Biological Sciences 272(1581), 2571–2576. Kutz, S.J., Thompson, R.C.A., Polley, L., Kandola, K., Nagy, J., Wielinga, C.M. and Elkin, B.T. (2008) Giardia assemblage A: human genotype in muskoxen in the Canadian Arctic. Parasites and Vectors. Available at http://www.biomedcentral.com/ (Doi: 10.1186/1756-3305-1-32.) Measures, L.N. and Olson, M. (1999) Giardiasis in pinnipeds from eastern Canada. Journal of Wildlife Diseases 35, 779–782. Measures, L.N. and Olson, M.E. (2002) Giardiasis in Canadian phocid seals. In: Olsen, B.E., Olsen, M.E. and Wallis, P.M. (eds) Giardia: The Cosmopolitan Parasite. CAB International, Wallingford, UK and New York, p. 316. Moro, D., Lawson, M.A., Hobbs, R.P. and Thompson, R.C.A. (2003) Pathogens of house mice on arid Boullanger Island and subantarctic Macquarie Island, Australia. Journal of Wildlife Diseases 39, 762–771. Nagy, J.A., Larter, N., Branigan, M., McLean, E. and Hines, J. (1998) Co-management Plan for Caribou Muskoxen, Arctic Wolves, Snow Geese, and Small Herbivores on Banks Island, Inuvialuit Settlement Region, Northwest Territories. Department of Resources, Wildlife, and Economic Development for the Wildlife Management Advisory Council, Inuvik, Northwest Territories, Canada.

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S.J. Kutz et al. Nizeyi, J.B., Innocent, R.B., Erume, J., Kalema, G.R., Cranfield, M.R. and Graczyk, T.K. (2001) Campylobacteriosis, salmonellosis, and shigellosis in free-ranging humanhabituated mountain gorillas of Uganda. Journal of Wildlife Diseases 37, 239–244. Olson, M.E., Roach, P.D., Stabler, M. and Chan, W. (1997) Giardiasis in ringed seals from the Western Arctic. Journal of Wildlife Diseases 33, 646–648. Parkinson, A.J. and Butler, J.C. (2005) Potential impacts of climate change on infectious diseases in the Arctic. International Journal of Circumpolar Health 64, 478–486. Roach, P.D., Olson, M.E., Whitley, G. and Wallis, P.M. (1993) Waterborne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada. Applied and Environmental Microbiology 59, 67–73. Robertson, L.J. and Gjerde, B.K. (2006) Fate of Cryptosporidium oocysts and Giardia cysts in the Norwegian aquatic environment over winter. Microbial Ecology 52, 597–602. Robertson, L.J., Forberg, T., Hermansen, L., Hamnes, I.S. and Gjerde, B. (2007) Giardia duodenalis cysts isolated from wild moose and reindeer in Norway: genetic characterization by PCR-rflp and sequence analysis at two genes. Journal of Wildlife Disease 43, 576–585. Savioli, L., Smith, H. and Thompson, A. (2006) Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends in Parasitology 22, 203–208. Sulaiman, I.M., Fayer, R., Bern, C., Gilman, R.H., Trout, J.M., Schantz, P.M., Das, P., Lal, A.A. and Xiao, L. (2003) Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerging Infectious Diseases 9, 1444–1452. Thompson, R.C.A. (2002) Towards a better understanding of host specificity and the transmission of Giardia: the impact of molecular epidemiology. In: Olsen, B.E., Olsen, M.E. and Wallis, P.M. (eds) Giardia: The Cosmopolitan Parasite. CAB International, Wallingford, UK and New York, pp. 55–69. Trout, J., Santín, M. and Fayer, R. (2003) Identification of assemblage A Giardia in whitetailed deer. Journal of Parasitology 89, 1254–1255. Trout, J.M., Santín, M. and Fayer, R. (2006) Giardia and Cryptosporidium species and genotypes in coyotes (Canis latrans). Journal of Zoo and Wildlife Medicine 37, 141–144. van der Giessen, J.W.B., de Vries, A., Roos, M., Wielinga, P., Korteek, L.M. and Mank, T.G. (2006) Genotyping Giardia in Dutch patients and animals: a phylogenic analysis of human and animal isolates. International Journal for Parasitology 36, 849– 858.

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The Role of Livestock in the Foodborne Transmission of Giardia duodenalis and Cryptosporidium spp. to Humans B.R. DIXON Health Canada, Ottawa, Canada

Abstract While person-to-person and waterborne transmission probably account for most human infections with Giardia duodenalis and Cryptosporidium spp., zoonotic transmission, particularly from livestock, has generated a great deal of interest in recent years. Both G. duodenalis and Cryptosporidium spp. are highly prevalent in cattle and other livestock, and zoonotic genotypes and species of these parasites have been identified in numerous studies. There is limited evidence, however, supporting the role of livestock as reservoirs for human infection. The association of livestock with the foodborne transmission of these parasites is also poorly recognized. Nevertheless, these animals have been associated with the presence of Giardia cysts and Cryptosporidium oocysts in a variety of foods, as well as with some foodborne outbreaks. The possible role of livestock in the contamination of produce, meats and other foods with G. duodenalis and Cryptosporidium spp., and its public health significance will be discussed.

Introduction Giardia and Cryptosporidium are common protozoan parasites of the intestinal tract of humans and a wide variety of animals worldwide. They represent the most common causes of enteric illness associated with protozoan parasites in humans. Giardia and Cryptosporidium are also responsible for illness and production losses in domestic animals (Olson et al., 2003; Olson et al., 2004, O’Handley and Olson, 2006). Transmission of Giardia and Cryptosporidium occurs by means of the faecal–oral route. Giardia cysts and Cryptosporidium oocysts are either ingested directly, as in person-to-person transmission, or indirectly by means of a vehicle such as contaminated water or food. Numerous waterborne outbreaks have been documented worldwide and this has, in fact, been recognized as one of the most common means of transmission for these © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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pathogens. A number of foodborne illness outbreaks associated with Giardia and Cryptosporidium, and involving a variety of foods, have also been reported. In recent years, there has been considerable interest in the high prevalence of both Giardia and Cryptosporidium infections in livestock, and the possible role these animals may play in the zoonotic transmission of these parasites.

Taxonomy Giardia There are currently six recognized species of Giardia (Olson et al., 2004, Thompson and Monis, 2004; Cacciò et al., 2005). Three of these species, G. duodenalis, G. muris and G. microti, are infectious to mammals, but only G. duodenalis has a broad host range, infecting a wide range of mammals including humans, livestock and companion animals, and is the only species of public health concern. Numerous genotypes, or assemblages, as well as subgroups or subgenotypes within these assemblages, have been demonstrated for G. duodenalis (Thompson, 2004; Thompson and Monis, 2004; Lalle et al., 2005). Thompson (2004) suggested that the genetic distance separating some of these assemblages, including assemblages A and B, is high enough to warrant separating them into different species. Humans are only susceptible to infection with two of these genotypes, assemblages A and B (Cacciò et al., 2005). Cattle and other livestock were thought to be susceptible to infection only with the so-called zoonotic genotype, assemblage A, and with the hoofed livestock genotype, assemblage E (Olson et al., 2004). A recent study, however, has demonstrated that cattle can also be infected with assemblage B (Lalle et al., 2005).

Cryptosporidium The taxonomy of Cryptosporidium is in a constant state of flux. Recently, Fayer (2004) recognized a total of 15 species of Cryptosporidium infecting fish, amphibians, reptiles, birds and mammals. These species are discussed in depth by Ryan (2003) and by Xiao et al. (2004). At least two other species, C. suis (Ryan et al., 2004) and C. bovis (Fayer et al., 2005), have been described since then. There have also been numerous host-specific genotypes of the ‘C. parvum group’ which have been reported in recent years, some of which may represent distinct species (Ryan, 2003). For many years, C. parvum was thought to be the only species infective to humans. However, many additional Cryptosporidium species have recently been identified as causing infection and illness in both immunocompromised and immunocompetent humans (Xiao et al., 2003, 2004; Cacciò et al., 2005). Currently, at least seven species and two genotypes of Cryptosporidium are associated with human disease (Cacciò et al., 2005). The two species of most significance in terms of public health, however, are C. parvum and C. hominis. C. parvum, previously known as the bovine genotype, has a very wide host range, including humans and livestock, and is thought to be the most important

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species in terms of zoonotic transmission. C. hominis is predominantly found in humans; however, there have been some rare natural infections reported in cattle and sheep, as well as some experimental infections (Ryan et al., 2005; Smith et al., 2005).

Transmission Routes The direct faecal–oral route (person-to-person) and the indirect transmission of Giardia cysts and Cryptosporidium oocysts by means of contaminated water probably account for the majority of human cases worldwide. Person-to-person transmission has been identified as the cause of many outbreaks, and is largely associated with poor hygiene. Outbreaks at daycare facilities, in particular, have been the focus of much attention. A large number of surveys have been done on surface and groundwater worldwide, and have demonstrated a relatively high prevalence of both Cryptosporidium and Giardia. As a result, numerous drinking-water-related outbreaks of Cryptosporidium (Fayer, 2004) and Giardia (Jakubowski and Craun, 2002) have also been reported. In recent years, outbreaks, particularly of Cryptosporidium infections, have also been associated with recreational water, including swimming pools, splash pads and water parks (Fayer, 2004; Smith et al., 2006). A few recent review articles have addressed the issue of foodborne transmission of protozoan parasites, including Giardia and Cryptosporidium (Nichols and Smith, 2002; Dixon, 2003; Dawson, 2005). While the foodborne route of transmission is reported much less frequently, and accounts for fewer outbreaks and cases, than the waterborne route or the direct faecal–oral route, it is becoming more important due to factors such as global trade, international travel, and changes in consumer habits, such as the consumption of more raw and undercooked foods (Dixon, 2003). Zoonotic transmission of Giardia and Cryptosporidium remains a controversial issue, and has become the focus of considerable research in recent years. While the zoonotic significance of companion animals and wildlife remains somewhat questionable (Thompson, 2004; Cacciò et al., 2005), livestock, and cattle in particular, have been identified as a potential significant source of environmental contamination with Giardia cysts and Cryptosporidium oocysts, and subsequent human infections. This is largely based on the high infection rates, and the presence of zoonotic genotypes and species of Giardia and Cryptosporidium in these animals. Zoonotic transmission may occur through direct contact with cysts or oocysts in animal faeces, as in the case of animal handlers, veterinarians, farmers and their families, and farm or petting zoo visitors. Zoonotic transmission, however, may also occur by means of a vehicle such as water or food. Surface or groundwater, for example, may become contaminated through the application of fresh manure to fertilize agricultural land or through agricultural runoff (Heitman et al., 2002; Olson et al., 2004). Similarly, produce may become contaminated directly through the use of manure application to crop lands, or indirectly through irrigation or processing with contaminated water. There is also some evidence for the presence of Giardia cysts and Cryptosporidium oocysts in products of animal origin, such as milk and meat.

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Prevalence of Giardia and Cryptosporidium in Livestock A high prevalence of both Giardia and Cryptosporidium has been reported worldwide in both dairy and beef cattle (Olson et al., 2003). O’Handley (2002), however, indicated that prevalence rates can vary among different studies and that this may result from differences in climate, farm management, study design and diagnostic test used. A large number of prevalence and molecular characterization studies have been done worldwide in recent years on Giardia and Cryptosporidium infections in dairy calves. The prevalence of both parasites has generally been reported to be high, with some studies reporting a 100% cumulative prevalence of both Giardia and Cryptosporidium in dairy calves (Xiao and Herd, 1994; O’Handley et al., 1999; O’Handley, 2002). Studies in Canada have reported a prevalence in dairy calves of between 45.7% and 73% for G. duodenalis, and between 40.6% and 88.7% for Cryptosporidium spp. (Olson et al., 1997b; Ruest et al., 1998; O’Handley et al., 2000; Trotz-Williams et al., 2005). In the USA, Cryptosporidium spp. prevalences of between 7.5% and 49% have been reported in dairy calves (Garber et al., 1994; Nydam et al., 2005; Starkey et al., 2005). Trout et al. (2004, 2005) examined the prevalence and genotypes of G. duodenalis in pre- and post-weaned dairy calves from several states in the USA. These authors reported a widely varying prevalence of 9–93%, with an average of 40% in preweaned calves and an overall prevalence of 52% in post-weaned calves. Numerous prevalence studies in dairy calves have also been undertaken in Europe, primarily on Cryptosporidium, and have demonstrated a Cryptosporidium prevalence ranging from 2.4% to 47.8%, while Giardia prevalence ranged from 0.8% to 49% (Lefay et al., 2000; Huetink et al., 2001; Castro-Hermida et al., 2002; Joachim et al., 2003; Hamnes et al., 2006; Thompson et al., 2007). In India, Singh et al. (2006) reported a Cryptosporidium prevalence in dairy calves of 50% in diarrhoeic calves and 25.7% in non-diarrhoeic calves. Becher et al. (2004) reported Cryptosporidium and Giardia in 48% and 89% of sampled dairy calves, respectively, in Western Australia. Several studies have examined Cryptosporidium and Giardia infections in older dairy cattle, and have reported a generally lower prevalence than in calves (Fayer et al., 2000; Santín et al., 2004; Fayer et al., 2006, 2007). Conversely, Trout et al. (2006) reported a relatively high and varied prevalence of Giardia in 1–2-year-old dairy cattle. Uehlinger et al. (2006) also reported a high prevalence of Giardia in adult dairy cattle in a veterinary college teaching herd, along with a complete absence of Cryptosporidium spp. There have been generally fewer studies on the prevalence of Giardia and Cryptosporidium in beef cattle. Ralston et al. (2003) reported a Cryptosporidium prevalence of only 5% in beef calves in Alberta, Canada. A low prevalence of Cryptosporidium in beef cows and calves was also reported in western Canada by Gow and Waldner (2006). C. parvum has been reported to be considerably less prevalent in beef calves than in dairy calves (Kvac et al., 2006; O’Handley and Olson, 2006). Conversely, some studies have demonstrated a relatively high Cryptosporidium prevalence in beef cattle (Fayer et al., 2000; McAllister et al., 2005). For Giardia, Ralston et al. (2002, 2003) reported a 100% cumulative

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prevalence in beef calves in Alberta, Canada, while more moderate Giardia infection rates have been reported for beef cattle in other studies (Fayer et al., 2000; Appelbee et al., 2003; McAllister et al., 2005; Gow and Waldner, 2006). A relatively high prevalence of both Giardia and Cryptosporidium has also been reported worldwide in swine (Olson et al., 1997a; Ryan et al., 2003; Xiao et al., 2006). As with dairy and beef calves, a 100% cumulative prevalence of Cryptosporidium in pigs has also been reported (Guselle et al., 2003). A number of studies have been done worldwide on the prevalence of Giardia and Cryptosporidium in sheep and have reported prevalences of between 6.2% and 68.6% for Giardia, and between 10.1% and 68.3% for Cryptosporidium (Olson et al., 1997a; Ryan et al., 2005). Santín et al. (2007) reported a much higher prevalence of Cryptosporidium in lambs (77.4%) than in ewes (25%) on a sheep farm in Maryland, USA, and a relatively low prevalence of Giardia in both lambs (4%) and ewes (12%). A few recent studies have also reported the presence of Giardia and Cryptosporidium in other livestock including horses (Olson et al., 1997a; Atwill et al., 2000), and goats (Bomfim et al., 2005; Castro-Hermida et al., 2005).

Zoonotic Genotypes and Species of Giardia and Cryptosporidium in Livestock Giardia duodenalis Cattle may be infected with either the zoonotic G. duodenalis assemblage A or the host-adapted, non-zoonotic, assemblage E. Numerous studies have reported a higher prevalence of G. duodenalis assemblage E than assemblage A in cattle (O’Handley et al., 2000; Appelbee et al., 2003; Trout et al., 2004, 2005, 2006; O’Handley and Olson, 2006), and Olson et al. (2004) concluded that this probably limits their role as reservoirs for human infection. However, a similar prevalence of both assemblages was reported by Uehlinger et al. (2006) in adult dairy cattle in a veterinary college teaching herd, and is suggestive of a potential zoonotic risk of infection to humans. Common genotypes or species in different hosts is not necessarily evidence for zoonotic transmission, however (Thompson, 2002). O’Handley (2002) suggested that in order to understand the importance of livestock in the zoonotic transmission of Giardia, it must be determined whether the genotypes infectious to humans are naturally transmitted among livestock, or whether livestock become infected with these genotypes through contact with a human or other animal source. Recently, O’Handley and Olson (2006) indicated that the presence of assemblage A in ruminants may, in fact, be suggestive of a human source of infection. It seems feasible, therefore, that the relatively high proportion of assemblage A in cattle from a teaching herd reported by Uehlinger et al. (2006) may be evidence for human-to-animal transmission, particularly given the frequent human contact with these animals. Recently, cattle have also been reported to be infected with G. duodenalis assemblage B (Lalle et al., 2005), greatly increasing the potential reservoir of infection for humans, and raising questions about the actual source of infection

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in these animals. Recent unpublished results from our laboratory on the molecular characterization of Giardia in livestock support these findings and have, in fact, demonstrated a predominance of assemblage B in pooled dairy cattle manure and in pooled swine manure in eastern Ontario, Canada. Specifically, we found a predominance of G. duodenalis assemblage B in pooled swine manure, with the remainder being assemblage E. In dairy cattle, we identified both G. duodenalis assemblages B and E, as well as a small number of samples which typed as assemblage A. This predominance of assemblage B is very suggestive of humanto-animal transmission, but it is not clear whether this genotype may, in fact, be naturally transmitted among livestock in this region. Traub et al. (2005) examined the genotypes of G. duodenalis in horses from New York and from Western Australia, and reported the presence of assemblage AI in Australia and AI, AII and BIV in New York. The authors concluded that horses may constitute a risk of zoonotic transmission either through direct contact with oocysts or via watersheds.

Cryptosporidium spp. Cattle are susceptible to infection with at least four species of Cryptosporidium (Xiao et al. (2007). These include the zoonotic species, C. parvum, as well as the non-zoonotic host-specific species and genotypes C. andersoni, Cryptosporidium deer-like genotype and the recently described C. bovis (Fayer et al., 2005). Santín et al. (2004) demonstrated that infections with these species in cattle are age-related, with C. parvum predominating in pre-weaned calves, and being replaced with other Cryptosporidium species, including C. andersoni, C. bovis and the deer-like genotype, in post-weaned calves and older animals. Similar findings have been reported by Fayer et al. (2006) and Thompson et al. (2007). Recently, however, Feng et al. (2007) reported that both C. bovis and the Cryptosporidium deer-like genotype were found in all age groups of cattle in different parts of the world. Based on the high prevalence of C. parvum in young animals, Xiao et al. (2007) concluded that only neonatal calves are an important source of zoonotic transmission. Similarly, due to the very low prevalence of C. parvum in mature dairy cattle, Fayer et al. (2007) suggested that these animals pose a low risk of infection to humans. In pigs, two distinct Cryptosporidium genotypes have been reported, pig genotype I and pig genotype II (Ryan et al., 2003). Ryan et al. (2004) determined that pig genotype I actually constituted a new species, and named it C. suis. Cryptosporidium suis has subsequently been included in the list of Cryptosporidium species infective to humans. Guselle et al. (2003) reported the presence of C. suis in all pigs tested in Alberta, Canada. Xiao et al. (2006) identified C. suis, Cryptosporidium pig genotype II, and another potentially zoonotic species, C. muris, in pig slurry samples in Ireland, and concluded that spreading pig slurry onto agricultural land could result in contamination of water sources with zoonotic isolates of Cryptosporidium. Recent unpublished results from our laboratory demonstrated a predominance of C. parvum and Cryptosporidium pig genotype II in pooled swine manure, while only one isolate was identified as C. suis.

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Ryan et al. (2005) noted that, with the exception of one C. hominis isolate, most sheep tested in Australia were infected with species and genotypes rarely or never reported in humans, and these authors concluded that sheep may not represent an important zoonotic reservoir for Giardia and Cryptosporidium infections in humans. More recently, Santín et al. (2007) found that species and genotypes in sheep included mainly a novel C. bovis-like genotype, and Cryptosporidium cervine genotype, with only two lambs exhibiting C. parvum. These authors further reported that most Giardia infections were assemblage E, with only one ewe having an infection with assemblage A. They concluded that although there was a low prevalence of C. parvum and G. duodenalis assemblage A, the high prevalence of the zoonotic Cryptosporidium cervine genotype indicated that sheep may play a role in transmission to humans through environmental contamination.

The Significance of Zoonotic Transmission from Livestock While a high prevalence of both Giardia and Cryptosporidium has been reported worldwide in cattle and other livestock, and common genotypes and species exist in humans and livestock, direct evidence for zoonotic transmission remains rather scant (Olson et al., 2004; Thompson, 2004). For example, a waterborne outbreak of cryptosporidiosis in British Columbia, Canada in 1996 is the only waterborne outbreak in North America in which oocysts of the bovine genotype have been identified (Cacciò et al., 2005). Waterborne outbreaks associated with livestock are, in fact, largely based on epidemiological or circumstantial evidence (Olson et al., 2004, Thompson, 2004). Based on the absence of C. hominis infection in dairy calves from farms near New York City, USA, Nydam et al. (2005) concluded that cattle are probably not an important source of infection in waterborne outbreaks, in which C. hominis is generally identified in North America. However, in a recent study, Feltus et al. (2006) identified C. parvum as the predominant cause of sporadic human cases in Wisconsin, USA, and suggested that this was indicative of zoonotic transmission, with cattle and other ruminants being the likely sources based on subtyping data. Further evidence for zoonotic transmission include a foodborne outbreak in which Cryptosporidium-infected calves were thought to be directly associated with oocyst-contaminated apple cider in Maine, USA (Millard et al., 1994). Outbreaks of infection with the bovine genotype (C. parvum) have occasionally been epidemiologically linked to direct or indirect contact with animals (Ryan, 2003, Olson et al., 2004). These included an outbreak among a rural family in Pennsylvania, USA, in 1997, and an outbreak involving a water fountain at the Minnesota Zoo in 1997. Despite this evidence in support of zoonotic transmission, recent studies involving molecular epidemiology have, in fact, suggested that the role of cattle and other livestock in human infections with Giardia and Cryptosporidium may not be as important as was originally thought (O’Handley, 2002; Olson et al., 2004; O’Handley and Olson, 2006). O’Handley and Olson (2006) suggested that only young ruminants pose a risk for zoonotic transmission of Cryptosporidium, and that, overall, human-to-human transmission may be more important than zoonotic transmission.

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The Role of Livestock in Foodborne Transmission of Giardia and Cryptosporidium While a number of different foods have been epidemiologically associated with outbreaks of infection with Giardia and Cryptosporidium, including fresh produce, apple cider, salads and other foods (Dixon, 2003), the actual source of contamination of the food is often not clear. Contamination by infected farm workers, irrigation and/or wash water containing cysts or oocysts, or food handlers, has been suspected in some outbreaks. Another potential source of contamination of food, however, is by means of direct or indirect contact with livestock. Laberge and Griffiths (1996) suggested that the high prevalence of Cryptosporidium spp. in domestic animals directly or indirectly increases the risk for contamination of foods including produce, milk and meat. Foodborne outbreaks associated with livestock are rarely reported but warrant further investigation as to how the foods actually become contaminated, and what can be done to reduce the risk of contamination. Poor farm management practices, for example, may allow livestock access to crops (direct contamination) or surface water (indirect contamination). The irrigation of crops, or the washing or processing of produce, with Giardia- or Cryptosporidium-contaminated raw water represents a significant risk for foodborne transmission. The application of manure onto crop lands is another important source of contamination of fresh produce (Olson et al., 2004; Moore et al., 2007). Giardia cysts and Cryptosporidium oocysts are environmentally resistant and can remain viable in water, soil or manure for extended periods (Olson et al., 1999). Giardia cysts and, particularly, Cryptosporidium oocysts are also very resistant to the environmental conditions found in the field, as well as those during storage or transportation of produce (Dixon, 2003). Further, the moist surface of some fruits and vegetables may, in fact, further protect any contaminating cysts or oocysts from desiccation and unfavourable temperatures. As such, it cannot be assumed that cysts and oocysts contaminating produce in the field, or during storage, will become inactivated before reaching the consumer. In light of the high prevalence of both Giardia and Cryptosporidium in livestock, it is also feasible that carcasses or cuts of meat may become contaminated in the slaughterhouse, either directly by faecal contact or by means of washing or processing with contaminated water.

Prevalence of Giardia and Cryptosporidium in Foods Surveys have reported a relatively high prevalence of Cryptosporidium oocysts on market produce in Costa Rica and Peru (Monge and Chinchilla, 1996; Monge et al., 1996; Ortega et al., 1997; Calvo et al., 2004). Ortega et al. (1997) reported that 14.5% of the total vegetables examined from markets in Peru contained oocysts, and suggested that the consumption of raw or undercooked produce purchased at these markets could be a source of transmission to humans. Giardia cysts have been detected in a variety of foods, including Christmas pudding, lettuce and strawberries (Barnard and Jackson, 1984; Boreham, 1987;

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Boreham et al., 1990). Giardia cysts were reported in 1% of vegetables obtained from supermarkets and a public market in the Philippines (de Leon et al., 1992). The authors speculated that the vegetables may have been contaminated through the application of human and animal manure on agricultural lands, among other sources. Giardia cysts were also reported in market vegetables tested in Costa Rica (Monge et al., 1996). A recent survey done in Norway showed a relatively high prevalence of both Cryptosporidium oocysts and Giardia cysts in a variety of fruits and vegetables, as well as in water samples related to irrigation and production (Robertson and Gjerde, 2001). There is very little information regarding the prevalence of Giardia or Cryptosporidium in or on raw meats. Moriarty et al. (2005) investigated the prevalence of Cryptosporidium in the faeces and on the carcasses of individual beef cattle at slaughter. While Cryptosporidium spp. oocysts were detected in 7.3% of the faecal samples, no Cryptosporidium oocysts were detected in any of the carcass tissue. Similarly, Cryptosporidium oocysts have been detected in adult cattle in slaughterhouses in Japan (Kaneta and Nakai, 1998; Koyama et al., 2005). Recent unpublished results from our laboratory demonstrated the presence of both G. duodenalis and Cryptosporidium spp. on raw meats purchased from retail outlets. Chicken breasts, minced beef and pork chops were all positive for both parasites by nested PCR, while one minced beef sample was positive for Cryptosporidium by immunofluorescence microscopy and one pork chop sample was positive for Giardia using this method. All Giardia isolates in meats were determined by DNA sequence alignment to be G. duodenalis assemblage B, with the exception of one minced beef isolate which belonged to assemblage E. The identification of assemblage B in meats is indicative of a human source of contamination rather than an animal source. In addition, C. parvum was identified in all meats by PCR sequencing positive samples. These results represent the first report of Giardia and Cryptosporidium on naturally contaminated raw meats. A number of recent studies have also reported on the presence and survival of Cryptosporidium oocysts in tissues of oysters and other molluscan shellfish, and the potential for transmission to humans through consumption (Graczyk, 2003; Fayer et al., 2004). Giardia cysts have also been isolated from clams in a sub-estuary of Chesapeake Bay, USA (Graczyk et al., 1999). Along with sewage discharge, agricultural runoff probably contributes significantly to the levels of cysts and oocysts detected in seawater and subsequently in shellfish.

Foodborne Outbreaks Associated with Livestock Approximately 10% of all cases of cryptosporidiosis in the USA have been attributed to foodborne transmission (Mead et al., 1999). Many of these outbreaks have been associated directly or indirectly with livestock. For example, earlier foodborne outbreaks of cryptosporidiosis reported in the UK and Australia, and in travellers to Mexico, were associated with salad, raw milk (cow and goat), raw sausages and raw tripe prepared from the stomach of a cow (Laberge and Griffiths, 1996). More recently, a Cryptosporidium outbreak in Maine, USA, in 1993 was epidemiologically associated with drinking unpasteurized, fresh-pressed apple

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cider (Millard et al., 1994). Oocysts were detected in leftover cider and on swabs from the cider press. It is worth noting that the apples used in making the cider were probably contaminated after falling to the ground near the edge of a pasture from which Cryptosporidium-infected calves were identified. An outbreak of cryptosporidiosis in 1995, involving 50 cases, was reported in the UK and was linked to drinking school milk which had been inadequately pasteurized (Gelletlie et al., 1997). These authors suggested that the milk may have become contaminated through poor udder hygiene. Other recent outbreaks of cryptosporidiosis have been associated with foods such as chicken salad, raw produce and apple cider, but were considered to have been the result of infected food handlers, contaminated water or other unknown sources (Besser-Wiek et al., 1996; Mshar et al., 1997; Quinn et al., 1998; Quiroz et al., 2000). As with cryptosporidiosis, an estimated 10% of all cases of giardiasis in the USA are attributable to foodborne transmission (Mead et al., 1999). There have been numerous foodborne outbreaks of giardiasis reported in North America (Dixon, 2003). The majority of these outbreaks were attributed to foods contaminated by food handlers who are themselves infected, or those who have been in close contact with other infected individuals, particularly young children in diapers. One example of an outbreak of giardiasis with a possible animal origin occurred in Turkey, in which a small number of people became ill following the consumption of tripe soup (Karabiber and Aktas, 1991). The tripe was prepared from sheep, and the authors suggested that the animal could have been infected and that cysts could have remained viable in the crevices of the intestine during cooking.

Conclusions Both Giardia duodenalis and Cryptosporidium spp. are highly prevalent in cattle and other livestock, especially in young animals. Further, molecular characterization has demonstrated that zoonotic genotypes and species of these parasites are common in these animals. However, while zoonotic transmission from livestock to humans has generated considerable interest among researchers in recent years, there remains limited evidence for this form of transmission. Zoonotic transmission by means of the direct or indirect contamination of foods by livestock is less well recognized than other forms, including livestock-contaminated surface water, or direct animal contact. Nevertheless, livestock have been associated with foodborne outbreaks of giardiasis and cryptosporidiosis, and with the presence of cysts and oocysts in a variety of foods. Further work is required to further clarify the role of livestock in the contamination of foods with zoonotic isolates of Giardia and Cryptosporidium, and the control measures that can be taken to minimize this risk.

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Laberge, I. and Griffiths, M.W. (1996) Prevalence, detection and control of Cryptosporidium parvum in food. International Journal of Food Microbiology 31, 1–26. Lalle, M., Pozio, E., Capelli, G., Bruschi, F., Crotti, D. and Cacciò, S.M. (2005) Genetic heterogeneity at the b-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. International Journal for Parasitology 35, 207–213. Lefay, D., Naciri, M., Poirier, P. and Chermette, R. (2000) Prevalence of Cryptosporidium infection in calves in France. Veterinary Parasitology 89, 1–9. McAllister, T.A., Olson, M.E., Fletch, A., Wetzstein, M. and Entz, T. (2005) Prevalence of Giardia and Cryptosporidium in beef cows in southern Ontario and in beef calves in southern British Columbia. Canadian Veterinary Journal 46, 47–55. Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., Griffin, P.M. and Tauxe, R.V. (1999) Food-related illness and death in the United States. Emerging Infectious Diseases 5, 607–625. Millard, P.S., Gensheimer, K.F., Addiss, D.G., Sosin, D.M., Beckett, G.A., Houck-Jankoski, A. and Hudson, A. (1994) An outbreak of cryptosporidiosis from fresh-pressed apple cider. Journal of the American Medical Association 272, 1592–1596. Monge, R. and Chinchilla, M. (1996) Presence of Cryptosporidium oocysts in fresh vegetables. Journal of Food Protection 59, 202–203. Monge, R., Chinchilla, M. and Reyes, L. (1996) Occurrence of parasites and intestinal bacteria in vegetables that are consumed raw in Costa Rica. Revista de Biologia Tropical 44, 369–375. Moore, J.E., Millar, B.C., Kenny, F., Lowery, C.J., Xiao, L., Rao, J.R., Nicholson, V., Watabe, M., Heaney, N., Sunnotel, O., McCorry, K., Rooney, P.J., Snelling, W.J. and Dooley, J.S.G. (2007) Detection of Cryptosporidium parvum in lettuce. International Journal of Food Science and Technology 42, 385–393. Moriarty, E.M., McEvoy, J.M., Lowery, C.J., Thompson, H.P., Finn, M., Sheridan, J.J., Blair, I.S., McDowell, D.A. and Duffy, G. (2005) Prevalence and characterization of Cryptosporidium species in cattle faeces and on beef carcases at slaughter. Veterinary Record 156, 165–168. Mshar, P.A., Dembek, Z.F., Cartter, M.L., Hadler, J.L., Fiorentino, T.R., Marcus, R.A., Mcguire, J., Shiffrin, M.A., Lewis, A., Feuss, J., Vandyke, J., Toly, M., Cambridge, M., Guzewich, J., Keithly, J., Dziewulski, D., Braunhowland, E., Ackman, D., Smith, P., Coates, J. and Ferrara, J. (1997) Outbreaks of Escherichia coli O157H7 infection and cryptosporidiosis associated with drinking unpasteurized apple cider: Connecticut and New York, October 1996. Journal of the American Medical Association 277, 781–782. Nichols, R.A.B. and Smith, H.V. (2002) Parasites: Cryptosporidium, Giardia and Cyclospora as foodborne pathogens, In: Blackburn, C. de W. and McClure, P.J. (eds) Foodborne Pathogens: Hazards, Risk Analysis and Control. Part III: Non-bacterial and Emerging Foodborne Pathogens. Woodhead Publishing, Cambridge, UK, pp. 453–478. Nydam, D.V., Lindergard, G., Santucci, F., Schaaf, S.L., Wade, S.E. and Mohammed, H.O. (2005) Risk of infection with Cryptosporidium parvum and Cryptosporidium hominis in dairy cattle in the New York City watershed. American Journal of Veterinary Research 66, 413–417. O’Handley, R.M. (2002) Giardia in farm animals. In: Olsen, B.E., Olsen, M.E. and Wallis, P.M. (eds) Giardia: The Cosmopolitan Parasite. CAB International, Wallingford, UK, pp. 97–105. O’Handley, R.M. and Olson, M.E. (2006) Giardiasis and cryptosporidiosis in ruminants. Veterinary Clinics of North America: Food Animal Practice 22, 623–643. O’Handley, R.M., Cockwill, C., McAllister, T.A., Jelinski, M., Morck, D.W. and Olson, M.E. (1999) Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves

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B.R. Dixon and their association with diarrhea. Journal of the American Veterinary Medical Association 214, 391–396. O’Handley, R.M., Olson, M.E., Fraser, D., Adams, P. and Thompson, R.C. (2000) Prevalence and genotypic characterization of Giardia in dairy calves from Western Australia and western Canada. Veterinary Parasitology 90, 193–200. Olson, M.E., Thorlakson, C.L., Deselliers, L., Morck, D.W. and McAllister, T.A. (1997a) Giardia and Cryptosporidium in Canadian farm animals. Veterinary Parasitology 68, 375–381. Olson, M.E., Guselle, N.J., O’Handley, R.M., Swift, M.L., McAllister, T.A., Jelinski, M.D. and Morck, D.W. (1997b) Giardia and Cryptosporidium in dairy calves in British Columbia. Canadian Veterinary Journal 38, 703–706. Olson, M.E., Goh, J., Phillips, M., Guselle, N. and McAllister, T.A. (1999) Giardia cyst and Cryptosporidium oocyst survival in water, soil, and cattle faeces. Journal of Environmental Quality 28, 1991–1996. Olson, M.E., Ralston, B.J., O’Handley, R., Guselle, N.J. and Appelbee, A.J. (2003) What is the clinical and zoonotic significance of cryptosporidiosis in domestic animals and wildlife. In: Thompson, R.C.A., Armson, A. and Ryan, U.M. (eds) Cryptosporidium: From Molecules to Disease. Elsevier, New York, pp. 51–68. Olson, M.E., O’Handley, R.M., Ralston, B.J., McAllister, T.A. and Thompson, R.C.A. (2004) Update on Cryptosporidium and Giardia infections in cattle. Trends in Parasitology 20, 185–191. Ortega, Y.R., Roxas, C.R., Gilman, R.H., Miller, N.J., Cabrera, L., Taquiri, C. and Sterling, C.R. (1997) Isolation of Cryptosporidium parvum and Cyclospora cayetanensis from vegetables collected in markets of an endemic region in Peru. American Journal of Tropical Medicine and Hygiene 57, 683–686. Quinn, K., Baldwin, G., Stepak, P., Thorburn, K., Bartleson, C., Goldoft, M., Kobayashi, J. and Stehr-Green, P. (1998) Foodborne outbreak of cryptosporidiosis: Spokane, Washington, 1997. Morbidity and Mortality Weekly Report 47, 565–567. Quiroz, E.S., Bern, C., MacArthur, J.R., Xiao, L., Fletcher, M., Arrowood, M.J., Shay, D.K., Levy, M.E., Glass, R.I. and Lal, A. (2000) An outbreak of cryptosporidiosis linked to a foodhandler. Journal of Infectious Diseases 181, 695–700. Ralston, B.J., McAllister, T.A. and Olson, M.E. (2002) Prevalence and infection pattern of naturally acquired giardiasis in beef calves and their dams from birth to weaning. In: Olsen, B.E., Olsen, M.E. and Wallis, P.M. (eds) Giardia: The Cosmopolitan Parasite. CAB International, Wallingford, UK, pp. 47–52. Ralston, B.J., McAllister, T.A. and Olson, M.E. (2003) Prevalence and infection pattern of naturally acquired giardiasis and cryptosporidiosis in range beef calves and their dams. Veterinary Parasitology 113, 113–122. Robertson, L.J. and Gjerde, B. (2001) Occurrence of parasites on fruits and vegetables in Norway. Journal of Food Protection 64, 1793–1798. Ruest, N., Faubert, G.M. and Couture, Y. (1998) Prevalence and geographical distribution of Giardia spp. and Cryptosporidium spp. in dairy farms in Quebec. Canadian Veterinary Journal 39, 697–700. Ryan, U.M. (2003) Molecular characterization and taxonomy of Cryptosporidium. In: Thompson, R.C.A., Armson, A. and Ryan, U.M. (eds) Cryptosporidium: From Molecules to Disease. Elsevier, New York, pp. 147–160. Ryan, U.M., Samarasinghe, B., Read, C., Buddle, J.R., Robertson, I.D. and Thompson, R.C.A. (2003) Identification of a novel Cryptosporidium genotype in pigs. Applied and Environmental Microbiology 69, 3970–3974. Ryan, U.M., Monis, P., Enemark, H.L., Sulaiman, I., Samarasinghe, B., Read, C., Buddle, R., Robertson, I., Zhou, L., Thompson, R.C.A. and Xiao, L. (2004) Cryptosporidium

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suis n. sp. (Apicomplexa: Cryptosporidiidae) in pigs (Sus scrofa). Journal of Parasitology 90, 769–773. Ryan, U.M., Bath, C., Robertson, I., Read, C., Elliot, A., Mcinnes, L., Traub, R. and Besier, B. (2005) Sheep may not be an important zoonotic reservoir for Cryptosporidium and Giardia parasites. Applied and Environmental Microbiology 71, 4992–4997. Santín, M., Trout, J.M., Xiao, L., Zhou, L., Greiner, E. and Fayer, R. (2004) Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Veterinary Parasitology 122, 103–117. Santín, M., Trout, J.M. and Fayer, R. (2007) Prevalence and molecular characterization of Cryptosporidium and Giardia species and genotypes in sheep in Maryland. Veterinary Parasitology 146, 17–24. Singh, B.B., Sharma, R., Kumar, H., Banga, H.S., Aulakh, R.S., Gill, J.P. and Sharma, J.K. (2006) Prevalence of Cryptosporidium parvum infection in Punjab (India) and its association with diarrhea in neonatal dairy calves. Veterinary Parasitology 140, 162–165. Smith, H.V., Nichols, R.A., Mallon, M., Macleod, A., Tait, A., Reilly, W.J., Browning, L.M., Gray, D., Reid, S.W. and Wastling, J.M. (2005) Natural Cryptosporidium hominis infections in Scottish cattle. Veterinary Record 156, 710–711. Smith, H.V., Cacciò, S.M., Tait, A., McLauchlin, J. and Thompson, R.C.A. (2006) Tools for investigating the environmental transmission of Cryptosporidium and Giardia infections in humans. Trends in Parasitology 22, 160–167. Starkey, S.R., Wade, S.E., Schaaf, S. and Mohammed, H.O. (2005) Incidence of Cryptosporidium parvum in the dairy cattle population in a New York City watershed. Veterinary Parasitology 131, 197–205. Thompson, H.P., Dooley, J.S.G., Kenny, J., McCoy, M., Lowery, C.J., Moore, J.E. and Xiao, L. (2007) Genotypes and subtypes of Cryptosporidium in neonatal calves in Northern Ireland. Parasitology Research 100, 619–624. Thompson, R.C.A. (2002) Towards a better understanding of host specificity and the transmission of Giardia: the impact of molecular epidemiology. In: Olsen, B.E., Olsen, M.E. and Wallis, P.M. (eds) Giardia: The Cosmopolitan Parasite. CAB International, Wallingford, UK, pp. 55–69. Thompson, R.C.A. (2004) The zoonotic significance and molecular epidemiology of Giardia and giardiasis. Veterinary Parasitology 126, 15–35. Thompson, R.C.A. and Monis, P.T. (2004) Variation in Giardia: implications for taxonomy and epidemiology. Advances in Parasitology 58, 69–137. Traub, R., Wade, S., Read, C., Thompson, A. and Mohammed, H. (2005) Molecular characterization of potentially zoonotic isolates of Giardia duodenalis in horses. Veterinary Parasitology 130, 317–321. Trotz-Williams, L.A., Jarvie, B.D., Martin, S.W., Leslie, K.E. and Peregrine, A.S. (2005) Prevalence of Cryptosporidium parvum infection in southwestern Ontario and its association with diarrhea in neonatal dairy calves. Canadian Veterinary Journal 46, 349–351. Trout, J.M., Santín, M., Greiner, E. and Fayer, R. (2004) Prevalence of Giardia duodenalis genotypes in pre-weaned dairy calves. Veterinary Parasitology 124, 179–186. Trout, J.M., Santín, M., Greiner, E. and Fayer, R. (2005) Prevalence and genotypes of Giardia duodenalis in post-weaned dairy calves. Veterinary Parasitology 130, 177–183. Trout, J.M., Santín, M., Greiner, E.C. and Fayer, R. (2006) Prevalence and genotypes of Giardia duodenalis in 1–2 year old dairy cattle. Veterinary Parasitology 140, 217–222. Uehlinger, F.D., Barkema, H.W., Dixon, B.R., Coklin, T. and O’Handley, R.M. (2006) Giardia duodenalis and Cryptosporidium spp. in a veterinary college bovine teaching herd. Veterinary Parasitology 142, 231–237.

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10

The Risk of Zoonotic Genotypes of Cryptosporidium spp. in Watersheds

H.O. MOHAMMED AND S.E. WADE College of Veterinary Medicine, Cornell University, USA

Abstract Cryptosporidium parvum is a coccidian protozoan that has zoonotic significance. Genotypes of this protozoan are known to contribute significantly to calf morbidity and mortality and hence have become an economic liability for many dairy and beef herds. In addition to its negative impact on the efficiency of animal production, Cryptosporidium has emerged as one of the most significant waterborne pathogens causing enterocolitis in humans worldwide. Because of the broad host spectrum of this pathogen, inefficiency of the common drinking water treatment methods, and the lack of reliable therapy in humans, zoonotic genotypes of Cryptosporidium spp. are considered to be among the major threats found in water supply systems. We have been carrying out studies to evaluate the risk associated with environmental degradation, and hence the contamination of drinking water supply systems with zoonotic Cryptosporidium spp. in a watershed in New York state. There are many agricultural sources that could contribute to the contamination of water supply systems including dairy cattle operations and associated farming activities, wildlife, and sewage treatment plants. The risk assessment approach, based on data collected from this watershed ecosystem, was employed to evaluate the potential environmental degradation. A fault-tree pathway scenario approach was used to assess the likelihood of contamination of the water supply in the watershed. Estimates of the parameters used in the risk assessment model were obtained from our studies and from the literature. The analysis demonstrated the importance of farming activities in mitigating the risk of this protozoan associated with dairy cattle, and highlighted the role of sewage treatment plants.

Introduction Cryptosporidium is a coccidian that can infect most mammals, including humans. Infection is contacted by ingestion of viable oocysts that are shed in the faeces of infected hosts (Fayer et al., 1997). Cryptosporidium has been found to be prevalent in mammals (Atwill et al., 1997; Olson et al., 1997; O’Handley and Olson, © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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2006; Shukla et al., 2006; Ziegler et al., 2007) including cattle (Garber et al., 1994; Olson et al., 1997; Mohammed et al., 1999; Wade et al., 2000). Most of the dairy herds and wildlife that have been investigated in these studies are inhabitants of the watershed ecosystem. By virtue of their residency in the watershed ecosystem and the likelihood of harbouring genotypes of Cryptosporidium, cattle are likely to pose a risk to human health. This risk can be inferred from the fact that some of these zoonotic genotypes are likely to enter the water supply systems that collect water from the respective watershed and, hence, infect humans. There are several genotypes of Cryptosporidium, some of which are known to be zoonotic. It is plausible that environmental and host factors may have modified the host–parasite ecology and led to the emergence of new pathogenic strains. As a consequence, the parasites have adapted to novel hosts, favouring the survival of ecological generalists (affecting multiple hosts) rather than specialists (affecting one host). At present, 15 distinct species are recognized (Fayer et al., 2005; Hunter and Thompson, 2005). The vast majority of outbreaks of cryptosporidiosis in humans are attributed either to the host-adapted C. hominis or the less fastidious C. parvum (Xiao and Ryan, 2004). C. parvum is known to be prevalent among dairy cattle populations and at a higher rate in calves (Nydam et al., 2001; Santín et al., 2004; Starkey et al., 2006). Cattle also harbour non-zoonotic strains of Cryptosporidium, e.g. C. bovis (Fayer et al., 2005). This organism is microscopically indistinguishable from C. parvum and, therefore, studies which investigated the public health risk associated with dairy animals in watersheds might have overestimated the contributory role of agricultural animals. The dynamics of Cryptosporidium infection in wildlife is not fully understood (Appelbee et al., 2005). Recent studies have incriminated some wildlife species as potential reservoirs of zoonotic genotypes of Cryptospordium (Daszak et al., 2000; Kruse et al., 2004). Several species that maintain high populations within a limited geographical area are reported to harbour C. parvum (Bednarska et al., 2003; Atwill et al., 2004; Ziegler et al., 2007). Environmental loading of Cryptosporidium oocysts depends upon a variety of factors, including the number of infected hosts within a geographical region (Slifko et al., 2000). As hosts these animals constitute an uncontrolled source of pathogen pollution contributing to the degradation of watershed quality. Contamination of the environment by these populations is possible through excretion and through the process of manure-spreading on farmland (Barwick et al., 2003). Runoff from the land contaminated by these sources may serve as a vehicle through which the Cryptosporidium oocysts can be transported into water sources. In this study we carried out a quantitative risk assessment analysis to evaluate the potential risk that dairy herds and wildlife, which resides in the watershed ecosystem, pose to the water supply system, with emphasis at the reservoir level.

Materials and Methods A quantitative risk assessment approach using a pathway model was used to address the stated objective. The risk assessment approach is a systematic process

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that involves the collection, collation and integration of information on a specific hazard to assess its adverse consequences on a particular population (Covello and Merkhofer, 1993). The emphasis in this study was on the release and exposure assessments of C. parvum from components of the watershed ecosystem. The study focused on the New York City and Upper Susquehanna watersheds. Information relating to the likelihood of shedding zoonotic C. parvum and the number of oocysts shed by dairy animals was collected using a couple of crosssectional studies and a longitudinal study (Wade et al., 2000; Starkey et al., 2005, 2006). In addition, data on risk factors hypothesized to associate with the presence of these genotypes in dairy populations in these two watersheds were also collected and analysed (Mohammed et al., 1999; Nydam et al., 2001; Starkey et al., 2005, 2006). In addition to studies on dairy animals, we carried out a cross-sectional study to determine the spectrum of wildlife species that shed C. parvum in the watershed ecosystem (Ziegler et al., 2007). Data on the amount of oocysts shed by each species and the factors that were associated with the presence of the protozoan in these animals were also collected. To complement our understanding of the dynamics of this hazard in the watershed ecosystem we carried out a study to determine the presence of C. parvum in the soil and associated factors (Barwick et al., 2003). We developed a scenario delineating the pathway by which the hazard would reach the stream edge in the hope of capturing the adverse effects associated with the presence of C. parvum in the watershed ecosystem. Figure 10.1 shows the conceptual framework for the fault-tree pathway model used in this study. The primary sources of oocysts were dairy farms, wildlife and agricultural land. These are represented by circles in the figure. Agricultural land was included in the pathway as a separate component because the land could be fertilized by sludge from other sources outside the watershed system, e.g. sludge from neighbouring cities. Since the oocysts are not motile they can either be transported mechanically from the immediate environment of the calf to different sites or moved with the manure as it is carried in waterbodies or runoffs. Only new manure was assumed to be stored near the barn area. Manure that was stored or spread on the agricultural fields had the potential to contaminate the reservoir with oocysts through runoff from the fields. A proportion of the manure produced daily by calves housed in barns, animal houses or the farmyard would also be washed off towards the reservoir. Our studies and others have shown that calves under 30 days of age are at high risk of infection with C. parvum (Wade et al., 2000; Starkey et al., 2005). Figure 10.2 shows the pattern of infection among calves enrolled in the longitudinal study as the data were analysed using the survival analysis method (Starkey et al., 2005). The median time to infection among the calves younger than 60 days of age was 15 days. The number of oocysts shed daily on each farm depends on the number of animals at risk, the infection rate within the farm, and the amount of faeces produced by each calf (Nydam et al., 2005). To capture the potential uncertainty in the estimates of these parameters at the watershed level, the respective distributions were assumed using the parameters obtained through our studies (Table 10.1). The quantity of faeces produced by each calf varies

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Top event

Risk of C. parvum

AND gate

Others

Agricultural fields

OR gate

Wildlife

Species

Sources

Other sources

Cattle

Calves

Soil

Fig. 10.1. The fault-tree scenario describing the pathway by which C. parvum could be transported from the sources to the reservoir.

Time of infection

Survivorship S(t)

1.00

0.75

0.50

0.25

0.00 0

20

40

60

Follow-up time (days)

Fig. 10.2. Time to infection among calves in the longitudinal study analysed using the Kaplan-Meier method.

daily and we assumed that this variability could be captured using a Poisson distribution (Wilkerson et al., 1997). Other parameters that were used in the model are described in Table 10.1. Isolates recovered from dairy cattle in these two watersheds were genotyped using the methods described in our previous study (Starkey et al., 2006). Only the C. parvum genotype, which represented

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Table 10.1. Description, distribution of input variables and the source of the estimates used in the analysis. Variable description Dairy animals Number of herds Number of stock per herd Proportion of calves in a herd Shedding rate per calf Amount of faeces per day Oocysts per gram per day Efficiency of composting pH of the soil Efficiency of soil neutralization Wildlife Occurrence in wildlife Amount of faeces per animal Oocysts per gram per day Cryptosporidium in agricultural field Occurrence in soil Oocysts per gram

Value/Parameter

Source/Reference

Pert (109, 258, 400) Normal (100, 20) Pert (0.01,0.03,0.05) Pert (0.02, 0.08, 0.12) Poisson (1) Exponent (5.6) Pert (0.2, 0.5, 0.7) Triangular (6, 7.4, 8.6) Binomial (0.5, 200)

Barwick et al. (2003) Starkey et al. (2006)

Beta (409, 2992) Lognormal (4, 1) Exponent (3)

Ziegler et al. (2007) Atwill et al. (2004)

Beta (133, 647) Pert (10, 100, 1000)

Barwick et al. (2003) Barwick et al. (2003)

Starkey et al. (2005) Wilkerson et al. (1997) Nydam et al. (2005) Kato et al. (2003) Barwick et al. (2003) Barwick et al. (2003)

61% of all of the isolates that were recovered from cattle in the watershed, was used in the analysis. Some of the practices for managing manure include composting, storage until spreading, or daily spreading on agricultural fields. We assumed that composting is 100% efficacious. During storage and spreading there was some loss due to desiccation of the oocysts as a result of high temperature or dryness (Robertson et al., 1992; Kato et al., 2001; Barwick et al., 2003). In addition to dairy calves we considered the potential contribution from wildlife and agricultural fields. An estimate of the prevalence among the wildlife population in the watershed ecosystem was obtained from our study (Ziegler et al., 2007). An average estimate of 12% for all wildlife in the study was used in the analysis (Table 10.1). To capture the potential variability in this estimate, we used the beta distribution. We observed that the average number of oocysts shed per animal was 1000/g of faeces. An estimate of the average amount of faeces produced by the wildlife was obtained from Atwill et al. (2004). Oocysts were recovered from agricultural land at a rate of 17%, and each gram contained on average 100 oocysts (Barwick et al., 2003). These estimates were used in the final analysis with the respective assumed distribution (Table 10.1). To capture the variability and uncertainty associated with some of the parameters used in the simulation, we assumed that these estimates were obtained from theoretical distributions. We ran the simulation with respective parameters using @risk software (Palisade Corporation, Ithaca, New York, USA).

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Results and Discussion The results of the simulation are shown in Fig. 10.3. Our analysis, using the assumptions about the number of affected calves, the size of the herd, the decay and the wash-off rates, and the presence of oocysts in wildlife and in agricultural fields, showed that on average 10 oocysts could be detected per litre of water per day in the reservoir. However, in the majority of cases it was unlikely that oocysts would be detected in the water (probability 0.078). In a few instances the analysis showed that it would be likely to find more than 24 oocysts per litre in the water samples collected from the reservoir. Although this value was within the range reported in the literature, the associated probability of finding such a concentration was less than 1% (Gibson et al., 1998). This observed number is consistent with what has been reported in the literature. The results reported in this study were similar to the findings of Okun et al. (1997). These authors reported on the average concentration of Cryptosporidium oocysts at the Kensico Reservoir which feeds into the New York City supply system. In our sensitivity analysis several factors influenced the likelihood of finding oocysts at the reservoir. These factors included the level of infection on the farm in general, the shedding rate by the calves, and the pH of the soil. Modification of these practices is likely to reduce the output or environmental loading from dairy farms. In our analysis we assumed that the recovery rate of the water sampling methods was 100%. Methods used in water sampling to date have a recovery rate much lower than that. Our analysis, despite its limitations, has demonstrated the potential contributory role of dairy farms to the risk of water degradation by zoonotic genotypes of Cryptosporidium, namely C. parvum. However, this risk is likely to be mitigated by the implementation of routine practices at the farm level, such as the use of milk replacer, improving the biosecurity measures, or the use of salt to neutralize the soil. 0.100 0.090

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Fig. 10.3. Results of the simulation of the average number of C. parvum oocysts per litre of water per day.

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References Appelbee, A.J., Thompson, R. and Olson, M.E. (2005) Giardia and Cryptosporidium in mammalian wildlife: current status and future needs. Trends in Parasitology 21, 370–376. Atwill, E.R., Phillips, R., Pereira, M.D., Li, X. and McCowan, B. (2004) Seasonal shedding of multiple Cryptosporidium genotypes in California ground squirrels (Spermophilus beecheyi). Applied and Environmental Microbiology 70, 6748–6752. Barwick, R.S., Mohammed, H.O., White, M.E. and Bryant, R.B. (2003) Factors associated with the likelihood of Giardia spp. and Cryptosporidium spp. in soil from dairy farms. Journal of Dairy Science 86, 784–791. Bednarska, M., Bajer, A., Kulis, K. and Sinski, E. (2003) Biological characterisation of Cryptosporidium parvum isolates of wildlife rodents in Poland. Annals of Agricultural and Environmental Medicine 10, 163–169. Daszak, P., Cunningham, A.A. and Hyatt, A.D. (2000) Emerging infectious diseases of wildlife: threats to biodiversity and human health. Science 287, 443–449. Fayer, R., Speer, C.A. and Dubey, J.P. (1997) The general biology of Cryptosporidium. In: Fayer, R. (ed.) Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, FL, pp. 1–42. Fayer, R., Santín, M. and Xiao, L. (2005) Cryptosporidium bovis n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus). Journal of Parasitology 91, 624–629. Garber, L.P., Salman, M.D., Hurd, H.S., Keefe, T. and Schlater, J.L. (1994) Potential risk factors for Cryptosporidium infection in dairy calves. Journal of the American Veterinary Medical Association 205, 86–91. Hunter, P.R. and Thompson, R.C.A. (2005) The zoonotic transmission of Giardia and Cryptosporidium. International Journal for Parasitology 35, 1181–1190. Kato, S., Lindergard, G. and Mohammed, H.O. (2003) Utility of the Cryptosporidium oocyst wall protein (COWP) gene in a nested PCR approach for detection infection in cattle. Veterinary Parasitology 11, 153–159. Kruse, H., Kirkemo, A.M. and Handeland, K. (2004) Wildlife as source of zoonotic infections. Emerging Infectious Diseases 10, 2067–2072. Nydam, D.V., Lindergard, G., Santucci, F., Schaaf, S.L., Wade, S.E. and Mohammed, H.O. (2005) Risk of infection with Cryptosporidium parvum and Cryptosporidium hominis in dairy cattle in the New York City watershed. American Journal of Veterinary Research 66, 413–417. O’Handley, R.M. and Olson, M.E. (2006) Giardiasis and cryptosporidiosis in ruminants. Veterinary Clinics of North America: Food Animal Practice 22, 623–643. Olson, M.E., Thorlakson, C.L., Deselliers, L., Morck, D.W. and McAllister, T.A. (1997) Giardia and Cryptosporidium in Canadian farm animals. Veterinary Parasitology 68, 375–381. Robertson, L.J., Campbell, A.T. and Smith, H.V. (1992) Survival of Cryptosporidium parvum oocysts under various environmental pressures. Applied and Environmental Microbiology 58, 3494–3500. Santín, M., Trout, J.M., Xiao, L., Zhou, L., Greiner, E. and Fayer, R. (2004) Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Veterinary Parasitology 122, 103–117. Shukla, R., Giraldo, P., Kraliz, A., Finnigan, M. and Sanchez, A.L. (2006) Cryptosporidium spp. and other zoonotic enteric parasites in a sample of domestic dogs and cats in the Niagara region of Ontario. Canadian Veterinary Journal 47, 1179–1184.

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11

Clinical Presentation in Cryptosporidium-infected Patients

L.M. KORTBEEK National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Abstract In immunocompetent patients Cryptosporidium can lead to a self-limited diarrhoea, sometimes recurrent. Other symptoms are vomiting, nausea, decreased appetite, weight loss, flatulence and abdominal pain and cramps. High levels of oocyst shedding have been associated with inflammation, villous atrophy and subsequent malabsorption. In immunocompromised patients CD4 T cell counts at <200 mm3 are associated with persistent (>30 days) diarrhoeal infection and can lead to severe illness. The knowledge of the clinical presentation of Cryptosporidium is changing since it is now possible to type the different strains. In C. hominis cases, non-gastrointestinal symptoms (e.g. joint pain, eye pain, headache, dizziness and fatigue) were seen more often than in cases of C. parvum. In young children, infections with C. hominis and, if symptomatic, C. parvum, are often heavy, associated with faecal lactoferrin and growth shortfalls. C. hominis appears to stimulate inflammation irrespective of age; this raises important questions regarding how it may be specifically inducing greater pro-inflammatory responses. The role of immunity was studied in healthy volunteers. The results indicate that prior exposure to C. parvum provides protection from infection and illness at low oocyst doses. Cryptosporidium can be a cause of serious disease, with a spectrum of symptoms that can vary between asymptomatic infection (non-diarrhoeal but not healthy) to serious infection causing death. The importance of extra-intestinal symptoms is not yet clear and needs to be studied. Typing and subtyping of Cryptosporidium in a standardized way will probably change our ideas and knowledge of clinical symptomatology of cryptosporidiosis in the near future.

Introduction Although Cryptosporidium has been known as an animal parasite since first being described by Tyzzer in 1907, it was not recognized as a human parasite until 1976. The first report of Cryptosporidium as the causative agent of diarrhoea in a 3-year-old child with self-limiting enterocolitis was by Nime et al. (1976). In the same year, Meisel et al. published a paper which reported that Cryptosporidium © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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had been found in an immunosuppressed patient (Meisel et al., 1976). It was, however, not until the start of the HIV epidemic in the 1980s that Cryptosporidium was recognized as an important human parasite (Petersen, 1992).

Clinical Symptoms The clinical symptoms of Cryptosporidium differ between non-immunocompromised and immunocompromised individuals.

Non-immunocompromised individuals In non-immunocompromised individuals, the disease is usually a self-limiting acute gastroenteritis, characterized by vomiting, weight loss, fever, watery diarrhoea, cramping, abdominal pains, flatulence, malaise and myalgia (Chin, 2000). Recurrent gastrointestinal symptoms may occur in 30–40% of the cases. The duration of the symptoms is about 13 days but this can vary between 1 day and more than 26 days. The severity of the infection is related to the condition and age of the patient. In young children, an important factor is dehydration, especially in children living in developing countries. In a study of 191 children with C. parvum in Uganda, 12.6% died, compared with 6.2% for children without C. parvum in Uganda (Tumwine et al., 2003). Immunocompetent patients do not usually require medical treatment. The number of drugs that are effective is very limited. Most anti-diarrhoeal drugs, such as paramomycin, have no proven effect. In a study in Egypt, Rossignol et al. (2006) demonstrated an effect of a 3-day course of 2 dd 500 mg nitazoxanide, which significantly improved the resolution of diarrhoea and parasitological eradication in non-immunodeficient patients aged 12 years and over. Nitazoxanide has been FDA approved and was marketed in the USA in 2003 (Hussar, 2004).

Immunocompromised individuals In immunosuppressed individuals, i.e. HIV-infected patients with low CD4 T cell counts (<200 mm3), patients undergoing chemotherapy, elderly people and transplant patients, among others, the disease can be severe and debilitating. Patients with idiopathic diarrhoea, biliary disease, primary T cell immune deficiency, acute leukaemia, lymphoma or bone marrow transplant cases, and to a lesser extent those with solid organ transplants and patients taking T cell immunosuppressants, are at high risk for cryptosporidiosis. These patients may develop severe chronic diarrhoeal disease or atypical gastrointestinal disease such as cholangitis, cholecystitis and hepatitis (R.M. Chalmers, 2007, Morelia, personal communication). Infection is associated with persistent diarrhoeal infection, lasting more than 30 days, and can also result in pulmonary or tracheal cryptosporidiosis, low-grade fever or severe intestinal symptoms.

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The most effective therapy in patients with AIDS is highly active antiretroviral treatment (HAART) of HIV. A Cochrane study to assess the efficacy of interventions for the treatment and prevention of cryptosporidiosis among immunocompromised individuals confirms the absence of evidence for effective agents in the management of cryptosporidiosis. The results of this study indicate that nitaxozanide reduces the load of parasites and may be useful in immunocompetent individuals. Due to the seriousness of the potential outcomes of cryptosporidiosis, the use of nitaxozanide should also be considered in immunocompromised patients (Abubakar et al., 2007). The natural history of cryptosporidiosis and its relationship with immune response has been described by Pozio et al. (1997) in their study of a waterborne outbreak in Italy in 1995–1997 involving members of a drug rehabilitation community, their children, and staff members. Of this community, 19.6% were HIVpositive. The attack rate of clinical cryptosporidiosis was 13.6% for HIV-negative members of the community and 30.7% for HIV-positive individuals. The CD4 cell count was indirectly proportional to the attack rate. In general terms, the severity of symptoms was greater among HIV-positive individuals and among those with lower CD4 cell counts. The effect was particularly clear for those with <100 CD4 cells/mm3. Other clinical symptoms, such as abdominal cramps, nausea, vomiting, flatulence and fever, were seen in both groups, but most of them more often in the HIV-positive group. None of the 190 HIV-negative people who complained of diarrhoea at the end of January or at the beginning of February had recurrent or chronic disease. Diarrhoea lasting longer than 14 days was observed only in 22 HIV-positive individuals who had ≤ 300 CD4 cells/mm3. For six of these, the diarrhoea lasted ≥ 30 days. Sera samples of 198 individuals, taken before and after the outbreak, were available. Twenty-eight (14.1%) of the 198 serum samples were already positive (titre 1/50) for specific IgG versus C. parvum antigen before the outbreak. Eightyseven of the 170 initially seronegative individuals (51.2%) developed specific antibodies during the outbreak, reaching titres up to 1/200. As suggested by the attack rates, only a low percentage (17.1%) of the individuals exposed to Cryptosporidium infection developed clinical symptoms, while antibodies against the parasite were detected in 58.0% of the systematic sample. According to these findings, since 17.1% of the participants developed diarrhoea, the authors roughly estimated that almost 40% of infected individuals developed clinical disease. This observation is supported by the rate of seroconversion (52.0%) in asymptomatic HIV-negative individuals, which certainly underestimates the ill-to-infected ratio, since not all infected individuals had detectable specific antibodies in their serum samples collected in March and April, shown by the percentage of seroconversion (90.5%) in symptomatic HIV-negative individuals (Pozio et al., 1997).

Molecular Typing Our knowledge of the clinical presentation of Cryptosporidium is changing, since it is now possible to type the different strains. In the UK, Hunter et al. (2004)

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showed a clear difference in the symptomatology of sporadic cases of cryptosporidiosis between C. hominis and C. parvum. They observed the frequent recurrence of gastrointestinal-related symptoms (e.g. loss of appetite, recurrent vomiting, abdominal pain and diarrhoea) reported by people after recovery from infections caused both by C. parvum and by C. hominis. There were, however, significant differences in the occurrence of non-gastrointestinal symptoms (e.g. joint pain, eye pain, headache, dizziness and fatigue) between the two groups. Following infections with C. hominis, 44.3% had at least one non-gastrointestinal symptom and 27.9% had two or more of these symptoms. This was lower in C. parvum infections, respectively 28.0% and 4.0%, and in a control group (15.1% and 5.2%, respectively) (Hunter et al., 2004). In a study of children in Peru, V. Cama showed an association of different symptoms with infection caused by different subtypes. Cryptosporidium-positive samples were subtyped using the GP60 gene. Subtype C. hominis Ib was associated with nausea, vomiting and general malaise, while subtypes Ia, Ib and Ie were associated with diarrhoea (V. Cama, 2007, Morelia, personal communication).

Asymptomatic infections Asymptomatic infections can now also be studied in more detail. In a study of dairy farm workers and their household contacts in Zimbabwe, 18 asymptomatic Cryptosporidium cases were detected. Cryptosporidium parvum was detected in 12 samples (66.7%; 75% of the positive farm workers and in 60% of the household contacts) and C. hominis in 3 samples (16.7%), while the dairy harboured C. parvum (66%) and C. bovis 33% (Siwila et al., 2007). Asymptomatic infections can also be seen in HIV-positive patients. This seems to occur more frequently with C. hominis than with C. parvum. In a study of HIV-positive adults in Tanzania, Houpt et al. (2005) showed that C. hominis was associated with a longer duration of symptoms, a higher rate of asymptomatic infection, and a lower CD4 cell count compared with C. parvum-infected patients (P < 0.05). This study suggests there may be important differences in the natural history of Cryptosporidium infection in HIV-infected people, depending on parasite species (Houpt et al., 2005). Using more sensitive methods to detect Cryptosporidium, such as PCR, more asymptomatic cases will probably be detected in future. However, it remains to be seen whether these cases are really asymptomatic, or only lacking diarrhoea. There is only limited information on growth and mental development in asymptomatic cases. In a study in Brazil, Bushen et al. (2007) showed that, even in asymptomatic children, C. hominis is associated with heavy infection, lactoferrin and growth shortfalls which are more significant than previously thought. Faecal lactoferrin is a sensitive marker for faecal leucocytes and hence an inflammatory process. It was found that 68% of the Cryptosporidium-infected children were lactoferrin-positive, with no relationship to Cryptosporidium genotype. High levels of oocyst shedding were associated with inflammation, villous atrophy and subsequent malabsorption. In symptomatic C. parvum cases, the numbers of oocysts shed were higher than in asymptomatic cases. Asymptomatic C.

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hominis cases had high numbers of oocysts and long-term growth shortfall; half were lactoferrin-positive (Bushen et al., 2007).

Immunity Another issue that needs attention is the role of immunity in the clinical presentation of Cryptosporidium infections. Chappell et al. (1999) showed, in a study in healthy volunteers, that there is a significant difference in the number of oocysts that are required to cause an infection: the ID50 was 1880 oocysts in healthy volunteers with pre-existing a-C. parvum serum IgG antibodies, while the ID50 was 83 oocysts in volunteers without pre-existing a-C. parvum serum IgG. These data indicate that prior exposure to C. parvum provides protection from infection and illness at low oocyst doses (Chappell et al., 1999). In another study in healthy volunteers it was shown that there is a difference in the serological response by the different Cryptosporidium types. In contrast to C. parvum (Iowa isolate), C. hominis resulted in a serological response in 8 (38.1%) of 21 challenged volunteers and in 8 (53.3%) of 15 who had evidence of infection. Interestingly, only volunteers receiving 30 oocysts or more had a serum IgG response, even though all had diarrhoea or faecal oocyst shedding. Furthermore, the degree of response was influenced by post-challenge outcome. Volunteers who had a diarrhoeal illness and who shed detectable levels of oocysts yielded the highest responses (Chappell et al., 2006).

Where Does the Science Go from Here? One of the questions that was asked at the II International Giardia and Cryptosporidium Conference held in May 2007 in Morelia, Mexico, was: ‘Where should the science go from here?’ A paper published after the first Giardia and Cryptosporidium meeting in Amsterdam (2003) by Savioli et al. put Giardia and Cryptosporidium on the list of neglected diseases. Most of the important questions that were raised then still need to be addressed (Savioli et al., 2006). In order to understand the clinical significance of cryptosporidiosis, it is important to study the sequelae of an infection and the mechanisms behind these sequelae. We should also study the frequency of the non-intestinal symptoms of the infection (arthritis, uveitis and urticaria) and determine their severity: the same applies to the following unanswered questions: ● ● ● ● ● ●

How often does malabsorption occur in cryptosporidiosis? What are the main risk groups? Which markers can be used to detect it? How often do deficiencies of micronutrients (vitamin A, minerals, etc.) occur? Is there a relation with post-infection IBS/IBD? If yes, how frequently does this occur and what are the risk groups?

To be able to compare studies in different parts of the world and in different settings, we need definitions for clinical cryptosporidiosis and giardiasis; and also

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a definition for diarrhoea (e.g. ≥ 3 stools/day and different consistency). In addition, we need to establish the clinical significance of laboratory-confirmed cryptosporidiosis and giardiasis: the significance of an isolated positive PCR without detectable cysts or oocysts; whether we need multiple sampling; and the value of serum antibodies, etc. We also need to be able to agree on the definition of acute and chronic diarrhoea. I suggest that acute diarrhoea lasts between 1 and 14 days and chronic diarrhoea persists for ≥ 30 days. We need good diagnostic routines so that clinicians know when to test for Giardia or Cryptosporidium. In a normal diagnostic setting it is possible to use available information (age, season, duration of symptoms) to reduce the requests to test for different microorganisms considerably. The methods chosen, or the combination of methods, can also assist in more sensitive and cost-effective diagnostics.

Conclusions Cryptosporidium can cause serious disease with a spectrum of symptoms that can vary between asymptomatic infection (non-diarrhoeal but not healthy) to serious infection causing death. The importance of extra-intestinal symptoms is not yet clear and needs to be studied. Typing and subtyping of Cryptosporidium in a standardized way will probably change our ideas and knowledge of clinical symptomatology of cryptosporidiosis in the near future.

References Abubakar, I., Aliyu, S.H., Arumugam, C., Hunter, P.R. and Usman, N.K. (2007) Prevention and treatment of cryptosporidiosis in immunocompromised patients. Cochrane Database Systematic Reviews 24, CD004932. Bushen, O.Y., Kohli, A., Pinkerton, R.C., Dupnik, K., Newman, R.D., Sears, C.L., Fayer, R., Lima, A.A. and Guerrant, R.L. (2007) Heavy cryptosporidial infections in children in northeast Brazil: comparison of Cryptosporidium hominis and Cryptosporidium parvum. Transactions of the Royal Society of Tropical Medicine and Hygiene 101, 378–384. Chappell, C.L., Okhuysen, P.C., Sterling, C.R., Wang, C., Jakubowski, W., Dupont, H.L. (1999) Infectivity of Cryptosporidium parvum in healthy adults with pre-existing anti-C. parvum serum immunoglobulin. American Journal of Tropical Medicine and Hygiene 60, 157–164. Chappell, C.L., Okhuysen, P.C., Langer-Curry, R., Widmer, G., Akiyoshi, D.E., Tanriverdi, S. and Tzipori, S. (2006) Cryptosporidium hominis: experimental challenge of healthy adults. American Journal of Tropical Medicine and Hygiene 75, 851–857. Chin, J. (2000) Cryptosporidiosis. In: Chin, J. (ed) Control of Communicable Diseases Manual, 17th edn. American Public Health Association, Washington, DC, pp. 134–137. Houpt, E.R., Bushen, O.Y., Sam, N.E., Kohli, A., Asgharpour, A., Ng, C.T., Calfee, D.P., Guerrant, R.L., Maro, V., Ole-Nguyaine, S. and Shao, J.F. (2005) Short report: asymptomatic Cryptosporidium hominis infection among human immunodeficiency virus-infected patients in Tanzania. American Journal of Tropical Medicine and Hygiene 73, 520–522.

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Hunter, P.R., Hughes, S., Woodhouse, S., Raj, N., Syed, Q., Chalmers, R.M., Verlander, N.Q. and Goodacre, J. (2004) Health sequelae of human cryptosporidiosis in immunocompetent patients. Clinical Infectious Diseases 39, 504–510. Hussar, D.A. (2004) New drugs of 2003. Journal of the American Pharmaceutical Association 44, 168–206. Meisel, J.L., Perera, D.R., Meligro, C. and Rubin, C.E. (1976) Overwhelming watery diarrhea associated with a cryptosporidium in an immunosuppressed patient. Gastroenterology 70, 1156–1160. Nime, F.A., Burek, J.D., Page, D.L., Holscher, M.A. and Yardley, J.H. (1976) Acute enterocolitis in a human being infected with the protozoan Cryptosporidium. Gastroenterology 70, 592–598. Petersen, C. (1992) Cryptosporidiosis in patients infected with the human immunodeficiency virus. Clinical Infectious Diseases 15, 903–909. Pozio, E., Rezza, G., Boschini, A., Pezzotti, P., Tamburrini, A., Rossi, P., Di Fine, M., Smacchia, C., Schiesari, A., Gattei, E., Zucconi, R. and Ballarini, P. (1997) Clinical cryptosporidiosis and human immunodeficiency virus (HIV)–Induced immunosuppression: findings from a longitudinal study of HIV-positive and HIV-negative former injection drug users. Journal of Infectious Diseases 176, 969–975. Rossignol, J.-F., Kabil, S.M., El-Gohary, Y. and Younis, A.M. (2006) Effect of nitazoxanide in diarrhea and enteritis caused by Cryptosporidium species. Clinical Gastroenterology and Hepatology 4, 320–324. Savioli, L., Smith, H. and Thompson, A. (2006) Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends in Parasitology 22, 203–208. Siwila, J., Phiri, I.G., Vercruysse, J., Goma, F., Gabriel, S., Claerebout, E. and Geurden, T. (2007) Asymptomatic cryptosporidiosis in Zambian dairy farm workers and their household members. Transactions of the Royal Society of Tropical Medicine and Hygiene 101, 733–734. Tumwine, J.K., Kekitiinwa, A., Nabukeera, N., Akiyoshi, D.E., Rich, S.M., Widmer, G., Feng, X. and Tzipori, S. (2003) Cryptosporidium parvum in children with diarrhea in Mulago Hospital, Kampala, Uganda. American Journal of Tropical Medicine and Hygiene 68, 710–715.

12

Molecular Epidemiology of Cryptosporidium and Giardia Infections

P.R. HUNTER University of East Anglia, Norwich, UK

Abstract For infectious diseases, molecular epidemiology can be defined as the use of molecular (predominantly genetic) methods to distinguish between strains of a microbial pathogen in order to identify markers of virulence or host range or to elucidate different transmission pathways. In this regard, molecular epidemiological methods have the same objectives as other typing methods that have been in use for several decades. The characteristics of a good typing method are typability, reproducibility and discriminatory power. There have been significant advances in typing methods for Cryptosporidium spp. in recent years. The first advances were for methods to distinguish the different species, predominantly C. parvum from C. hominis. More recently, microsatellite markers have been used to distinguish different strains within these two species. Microsatellite typing of C. parvum is sufficiently discriminatory to be useful, although more information is needed to determine reproducibility. Typing methods for C. hominis are not yet sufficiently discriminatory for general use. Molecular typing methods for Giardia are not yet well developed and have not been used in well-designed epidemiological studies.

Introduction The phrase ‘molecular epidemiology’ has become very popular in the title of papers and grant applications in the last few years (Fig. 12.1). So it is perhaps appropriate to discuss what we mean by this before moving on to discuss the molecular epidemiology of Cryptosporidium and Giardia infections. Molecular epidemiology is more generally used to refer to any study that uses molecular methods (and increasingly this means genetic methods) to identify markers of susceptibility in hosts or virulence in pathogens. The other main use of molecular epidemiology is to track the spread of pathogens in order to elucidate transmission pathways. In this regard molecular epidemiology does not differ in its objectives, or indeed its philosophy, from other work on microbial epidemiology that uses non-molecular typing methods. 138

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Any discussion of molecular epidemiology needs to focus on two key areas. The first concerns the methods used to characterize the strains, and the second concerns the epidemiological studies themselves. There seems to be a view in some quarters that because molecular methods have been used then the results are somehow more compelling than those obtained by traditional typing methods. However, the need for proper validation of molecular methods is just as great as that for non-molecular methods. It is somewhat disappointing that proper validation is often not done. This chapter will first discuss the validation of typing methods. Before taking the discussion any further we need to ask what microbiological typing methods are used for. The primary role of a typing method is to distinguish between two strains. No typing method can say that two strains are the same, only that they are different. For example, if serotyping of two strains of E. coli shows that both are E. coli O157 this does not necessarily imply that they are epidemiologically related. On the other hand, if one is O157 and the other is O135 we can be confident that the two are not the same. The other use of typing methods is in the identification of markers that may indicate particular virulence, host preferences, or routes of transmission. In the E. coli serotyping example, if a strain of E. coli O157 was isolated from a patient with bloody diarrhoea, then we would be fairly confident that this strain was an enterohaemorrhagic strain. However, one should not make the mistake of assuming that the marker = the virulence factor. Many strains of E. coli O157 are not particularly pathogenic and, if one was isolated from an environmental water sample, it would be dangerous to assume that it was enterohaemorrhagic without tests to identify the virulence factor directly. It is also important to understand the differences between typing methods and typing schemes (Hunter, 1991). A typing method is any method that is

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used to distinguish between different strains of the same species. A typing scheme, on the other hand, is a typing method that has been subjected to rigorous inter-laboratory comparisons and has been agreed on as an international standard. Examples include the phage typing of Staphylococcus aureus and serotyping of Salmonella or E. coli. There are six characteristics of a typing method that need to be considered when assessing its value (Hunter, 1991): 1. 2. 3. 4. 5. 6.

Typability. Reproducibility. Discriminatory power. Cost. Timeliness. Ease of use.

The latter three characteristics are fairly obvious. An ideal typing method would be cheap, give a quick result and be sufficiently easy to do that extensive training of staff would not be required. The other characteristics need more explanation. Typability is a measure of the proportion of strains that can be typed by the method. Some strains, for example, simply may not amplify with one of the primers and so cannot be allocated to a specific type (Hunter et al., 2007). Typability is represented as the percentage of strains that can be fully typed by the method. One should be careful not to assume that the non-typing strain is in any way different from a strain that is fully typed. A non-typed strain cannot be said to be different from one that is typed. There are two aspects of reproducibility: in vitro and in vivo reproducibility. In vitro reproducibility is the proportion of strains that, should they be retyped, would give exactly the same result. In vitro reproducibility is primarily a measure of the reproducibility of the typing method itself. Sources of poor in vitro reproducibility include poor operator experience, difficulties with the method itself, and sloppiness in recording results. In vivo reproducibility is a more difficult concept, but is concerned with the stability of the organism itself. Some pathogens have a habit of switching their phenotypes (see Jones et al., 1994). Of particular concern to genetic-based typing methods is the possibility of mutation or, for the case of microsatellites, slippage (e.g. Eckert et al., 2002; Tanriverdi and Widmer, 2006). Whatever the mechanism, if genetic change occurs, then the possibility of getting different type results increases when strains are, in fact, related. Discriminatory power is a measure of the ability of the typing method to distinguish between unrelated strains. Discriminatory power is a function of the number of different stain types identifiable and also the distribution of strains between these different types. Discriminatory power is greater where there are more types, and strains are evenly distributed between types. Poor discriminatory typing methods identify few types, and most strains belong to a single type. In 1988 we proposed an index of discriminatory power that has become the standard way of describing the discriminatory power (Hunter and Gaston 1988). A short while later we described a generalization of the index that is applicable to situations where reproducibility is less than 100% (Hunter, 1990). Other workers

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have described an approach to determine the confidence intervals of estimated discriminatory power. Clearly, discriminatory power will increase as more typing variables are used. One of the uses to which indices of discriminatory power can be put is the choice of parsimonious collections of typing characters or methods to give the most efficient discriminatory tests (Gaston and Hunter, 1989). If one starts with the most discriminatory test, then as each furthermost discriminatory parameter is included, the combined discriminatory power increases, but very soon any increase in discrimination will level off (Fig. 12.2). On the other hand, as the number of variables increases, reproducibility continues to decline. Consequently, increasing the number of variables beyond a certain point is counterproductive.

Initial Work on Cryptosporidium One of the early studies of genotyping and cryptosporidiosis to suggest two distinct genotype types with different host ranges was that of Peng et al. (1997). Based on typing 39 strains from human and animal sources, they established that C. parvum genotype 1 was isolated only from humans whilst genotype 2 was isolated from humans and animals. Furthermore, human cases with genotype 2 tended to report contact with cattle or cattle products. The author of this chapter usually has serious concerns about the strength of conclusions based on the examination of a relatively small number of strains. However, on this occasion the conclusions appear to be sound. McLaughlin et al. (2000) reported a much larger study from the UK. In this study the authors examined 1705 positive faecal samples from humans and 105 from livestock animals. Genotype 1 was detected in 37.8% of human samples, whilst genotype 2(B) was detected in 61.5%. C. meleagridis was detected in 0.3%. On the other hand, all the samples from livestock yielded genotype 2. 1 0.9 0.8

D or R

0.7 0.6

D

0.5

R

0.4 0.3 0.2 0.1 0 1

2

3

4

5

6

7

8

9

10

Number of typing variables

Fig. 12.2. Indication of the impact of increasing numbers of typing variables on discriminatory power (D) and reproducibility (R).

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Probably the first large-scale analytical epidemiological study done on sporadic cryptosporidiosis using these ‘genotyping’ methods was that of Hunter et al. (2004a). In this study the authors were able to identify personal risk factors between genotype 1 and genotype 2 strains. The authors found that the strongest risk factors for genotype 1 or C. hominis were travel abroad (OR = 6.841, 95% CI 2.622, 17.85) and changing nappies (OR = 3.991, 95% CI 1.848, 8.618). By contrast the strongest association for genotype 2 or C. parvum was direct contact with farm animals (OR = 2.653, 95% CI 1.113, 6.323). Of particular interest was the observation that the consumption of tomatoes (OR = 0.317, 95% CI 0.140, 0.719) or other raw vegetables (OR = 0.222, 95% CI 0.086, 0.572) were negatively associated with risk. Using the same population, the authors were also able to show that long-term sequelae were likely to follow infection with C. hominis but not with C. parvum (Hunter et al., 2004b). At one time, these studies would have counted as molecular epidemiological studies. However, since the different genotypes were deemed to be two separate species (Morgan-Ryan et al., 2002), the molecular methods used would be better classified as species identification. Consequently these studies would not now be classed as molecular epidemiology.

Subspecies Typing of Cryptosporidium Most of the currently published studies of strain typing methods for Cryptosporidium spp. have been based on microsatellite markers (Aeillo et al., 1999; Cacciò et al., 2000, 2001; Enemark et al., 2002; Mallon et al., 2003a, 2003b). These studies were generally focused on comparing polymorphisms in human and animal sources. Of particular interest is the work of Mallon et al. (2003a, 2003b), undertaken in Scotland, that identified a cluster of C. parvum strains that were identified in humans but not animals. Based on this finding, they suggested that there was a specific clone of C. parvum that was anthropogenic. This was a valuable insight. However, care must be taken when interpreting such data, especially from relatively small geographical locations and when not accompanied by specific exposure data. In this context the study design would not be able to distinguish between a strain that was truly anthropogenic and one that was zoonotic but the focus of animal infection was localized elsewhere. So, for example, based on just the evidence from this study, it is plausible that the type was a zoonotic infection in the Mediterranean, was acquired by people on holiday and brought back to the UK, and had not yet developed any animal cycle of infection. We have recently published a study that looked at strain typing of both C. parvum and C. hominis using three microsatellite markers: ML1, ML2 and GP60 (Hunter et al., 2007). In this study we typed strains that were part of our earlier case control study (Hunter et al., 2004a). Taken together, these three markers were highly discriminatory for C. parvum (D = 0.957) but very poorly discriminatory for C. hominis (D = 0.197). This means that for C. hominis the method would be able to distinguish between two unrelated strains on fewer than 20% of occasions, and so was not suitable for epidemiological studies of this species.

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The relatively poor typability of C. hominis should come as no surprise. It has been pointed out that, as would be predicted by the theory of adaptive polymorphism, that pathogens with a limited host range and no environmental replication are generally much less diverse than pathogens with a wide host range and/ or environmental life stages (Hunter and Fraser, 1990). For C. parvum, three main clusters were identifiable, one large cluster and two smaller clusters. This was in line with earlier findings of Mallon et al. (2003a, 2003b). All the members of the larger cluster had a single MLT1 fragment length 242 bp while all others were MLT1-227. Interestingly, we were able to show that all cases who reported farm animal contact had the MLT1-242 type, and furthermore the attack rate was more common in rural areas. All C. parvum strains from animals in a related study were also MLT1-242 type. Thus our findings would strengthen Mallon’s suggestion, although again our study would not be able to exclude the hypothesis that MLT1-227 strains were zoonotic in other parts of the world.

Application of Molecular Epidemiology to Outbreak Investigations Microbial typing has a long tradition of use in the investigation of outbreaks of infectious disease. Strain typing data can be helpful in one of several ways. First, typing can be an indicator of the source of infection. For example, as is the case in the investigation of waterborne outbreaks, strain typing may give important indications of the source of contamination. Knowing whether the infection is due to C. parvum or C. hominis will let the investigator know whether or not the source of pollution was human or cattle (e.g. Glaberman et al., 2002; Dalle et al., 2003; Goncalves et al., 2006). Microbial typing is also of value in identifying whether or not individual cases are part of an outbreak. This is important to enhance the strength of the epidemiological studies. If many unrelated cases are included in the analysis, important risk factors may be missed. To date, no good subspecies typing studies of strains from outbreaks of cryptosporidiosis have been reported in the literature. However, preliminary results from the investigation of a waterborne outbreak in the north-west of England have identified multiple MLFT types. The implications of this finding are currently unclear. It may be the case that the outbreaks are in fact due to multiple strains. Alternatively, it may be the case that the typing characters are not sufficiently stable for the investigation of large outbreaks. Whatever the explanation, if it is the case that waterborne outbreaks are usually due to multiple strain types, then the value of strain typing would be severely adversely affected.

Giardia In general, strain typing issues for Giardia are similar to those for Cryptosporidium. However, epidemiological studies for Giardia are not as advanced as those for Cryptosporidium (Hunter and Thompson, 2005). In part, this is because the

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characterization of different species/assemblages is not yet as well accepted as species differentiation in Cryptosporidium. Also it would appear that the methods available for further typing below the species/assemblage level are not as well characterized. One typing method that may be valuable is sequence heterogeneity in the β-giardin gene (Lalle et al., 2005).

Conclusions In recent years the development of molecular typing methods has contributed to our understanding of the epidemiology of cryptosporidiosis and, to a much lesser extent, giardiasis. However, few of the methods have yet to be adequately characterized in the published literature to enable their reproducibility and discriminatory power to be adequately assessed. Furthermore, relatively few of the studies that have used molecular typing methods have actually linked the strain typing data to basic epidemiological data. Many studies have been reported that have simply listed identified strain types in different hosts. Grinberg et al. (2007) have recently pointed out the dangers of relying solely on differences in detected strain types in different host populations to draw too strong a conclusion. There is always a strong desire amongst microbiologists to develop their own tests, and so it is likely that the microbiological community will spend considerable time and effort in developing new typing methods for both Giardia and Cryptosporidium. However, the increase in our knowledge of the epidemiology of these two pathogens will be best served if our community focuses on better characterizing the methods we currently have available. There is also an obvious need for microbiologists and parasitologists who can do strain typing to develop their project proposals in collaboration with epidemiologists. Only then will they really understand what the results of their strain typing are telling us about the epidemiology of these two pathogens.

References Aeillo, A.E., Xiao, L., Limor, J.R., Liu, C., Abrahmason, M.S. and Lal, A.A. (1999) Microsatellite analysis of the human and bovine genotypes of Cryptosporidium parvum. Journal of Eukaryotic Microbiology 46, 46S–47S. Cacciò, S., Homan, W., Camilli, R., Traldi, G., Kortbeek, T. and Pozio, E. (2000) A microsatellite marker reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum. Parasitology 120, 237–244. Cacciò, S., Spano, F. and Pozio, E. (2001) Large sequence variation at two microsatellite loci among zoonotic (genotype C) isolates of Cryptosporidium parvum. International Journal for Parasitology 31, 1082–1086. Dalle, F., Roz, P., Dautin, G., Di-Palma, M., Kohli, E., Sire-Bidault, C., Fleischmann, M.G., Gallay, A., Carbonel, S., Bon, F., Tillier, C., Beaudeau, P. and Bonnin, A. (2003) Molecular characterization of isolates of waterborne Cryptosporidium spp. collected during an outbreak of gastroenteritis in South Burgundy, France. Journal of Clinical Microbiology 41, 2690–2693.

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Eckert, K.A., Mowery, A. and Hile, S.E. (2002) Misalignment-mediated DNA polymerase beta mutations: comparison of microsatellite and frame-shift error rates using a forward mutation assay. Biochemistry 41, 10490–10498. Enemark, H.L., Ahrens, P., Juel, C.D., Petersen, E., Petersen, R.F., Andersen, J.S., Lind, P. and Thamsborg, S.M. (2002) Molecular characterization of Danish Cryptosporidium parvum isolates. Parasitology 125, 331–341. Gaston, M.A. and Hunter PR. (1989) Efficient selection of tests for bacteriological typing schemes. Journal of Clinical Pathology 42, 763–766. Glaberman, S., Moore, J.E., Lowery, C.J., Chalmers, R.M., Sulaiman, I., Elwin, K., Rooney, P.J., Millar, B.C., Dooley, J.S.G., Lal, A.A. and Xiao, L. (2002) Three drinkingwater-associated cryptosporidiosis outbreaks, Northern Ireland. Emerging Infectious Diseases 8, 631–633. Goncalves, E.M., da Silva, A.J., Eduardo, M.B., Uemura, I.H., Moura, I.N., Castilho, V.L. and Corbett, C.E. (2006) Multilocus genotyping of Cryptosporidium hominis associated with diarrhea outbreak in a day care unit in São Paulo. Clinics (São Paulo, Brazil) 61, 119–126. Grinberg, A., Lopez-Villalobos, N., Pomroy, W., Widmer, G., Smith, H. and Tait, A. (2007) Host-shaped segregation of the Cryptosporidium parvum multilocus genotype repertoire. Epidemiology and Infection 136, 273–278. Hunter, P.R. (1990) Reproducibility and indices of discriminatory power of microbial typing methods. Journal of Clinical Microbiology 28, 1903–1905. Hunter, P.R. (1991) A critical review of typing methods for Candida albicans. Critical Reviews in Microbiology 17, 417–434. Hunter, P.R. and Fraser, C.A.M. (1990) Application of the theory of adaptive polymorphism to the ecology and epidemiology of pathogenic yeasts. Applied and Environmental Microbiology 56, 2219–2222. Hunter, P.R. and Gaston, M.A. (1988) A numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. Journal of Clinical Microbiology 26, 2465–2566. Hunter, P.R. and Thompson, R.C.A. (2005) The zoonotic transmission of Giardia and Cryptosporidium. International Journal for Parasitology 35, 1181–1190. Hunter, P.R., Hughes, S., Woodhouse, S., Syed, Q., Verlander, N.Q., Chalmers, R.M., Morgan, K., Nichols, G., Beeching, N. and Osborn, K. (2004a) Sporadic cryptosporidiosis case-control study with genotyping. Emerging Infectious Diseases 10, 1241–1249. Hunter, P.R., Hughes, S., Woodhouse, S., Raj, N., Syed, Q., Chalmers, R.M., Verlander, N.Q. and Goodacre, J. (2004b) Health sequelae of human cryptosporidiosis in immunocompetent patients. Clinical Infectious Diseases 39, 504–510. Hunter, P.R., Hadfield, S.J., Wilkinson, D., Lake, I.R., Harrison, F.C.D. and Chalmers, R.M. (2007) Subtypes of Cryptosporidium parvum in humans and disease risk. Emerging Infectious Diseases 13, 82–88. Jones, S., White, G. and Hunter, P.R. (1994) Increased phenotypic switching in strains of Candida albicans associated with invasive infections. Journal of Clinical Microbiology 32, 2869–2870. Lalle, M., Pozio, E., Capelli, G., Bruschi, F., Crotti, D. and Cacciò, S.M. (2005) Genetic heterogeneity at the β-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. International Journal for Parasitology 35, 207–213. Mallon, M., MacLeod, A., Wastling, J., Smith, H., Reilly, B. and Tait., A. (2003a) Population structures and the role of genetic exchange in the zoonotic pathogen Cryptosporidium parvum. Journal of Molecular Evolution 56, 407–417.

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P.R. Hunter Mallon, M.E., MacLeod, A., Wastling, J.M., Smith, H. and Tait, A. (2003b) Multilocus genotyping of Cryptosporidium parvum Type 2: population genetics and substructuring. Infection Genetics and Evolution 3, 207–218. McLaughlin, J., Amar, C., Pedraza-Diaz, S. and Nichols, G.L. (2000) Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: Results of genotyping Cryptosporidium spp. in 1705 fecal samples from humans and 105 fecal samples from livestock animals. Journal of Clinical Microbiology 38, 3984–3990. Morgan-Ryan, U.M., Fall, A., Ward, L.A., Hijjawi, N., Sulaiman, I., Fayer, R., Thompson, R.C.A., Olson, M., Lal, A. and Xiao, L.H. (2002) Cryptosporidium hominis n. sp (Apicomplexa: Cryptosporidiidae) from Homo sapiens. Journal of Eukaryotic Microbiology 49, 433–440. Peng, M.M., Xiao, L., Freeman, A.R., Arrowood, M.J., Escalante, A.A., Weltman, A.C., Ong, C.S.L., Mackenzie, W.R., Lal, A.A. and Beard, C.B. (1997) Genetic polymorphism among Cryptosporidium parvum isolates: evidence of two distinct human transmission cycles. Emerging Infectious Diseases 3, 567–573. Tanriverdi, S. and Widmer, G. (2006) Differential evolution of repetitive sequences in Cryptosporidium parvum and Cryptosporidium hominis. Infection, Genetics and Evolution 6, 113–122.

13

Advances in Diagnosis: is Microscopy Still the Benchmark?

R.M. CHALMERS UK Cryptosporidium Reference Unit, NPHS Microbiology, Swansea, UK

Abstract Despite advances in the biomedical sciences, leading to developments in laboratory diagnostics, examination (usually of faeces) by microscopy remains the keystone of laboratory diagnosis of cryptosporidiosis and giardiasis. In Europe, standard methods include the direct examination of wet preparations (with and without concentration) for Giardia, and tinctorial staining of faecal smears (usually with strong carbol fuchsin) or fluorescent staining (e.g. using Auramine O) for Cryptosporidium. There is an increasing trend in the UK towards the routine use of Auramine O staining, and over 70% of laboratories use this stain, while 26% use a modified Ziehl-Neelsen stain. Immunoassays to detect copro-antigens are gradually being introduced into routine diagnostics, particularly in larger laboratories processing large numbers of samples. These methods are more commonly used in the USA, as is immunofluorescent microscopy, which offers increased sensitivity and specificity over conventional staining. However, cost constraints preclude their use in many countries. Advances in molecular diagnostics have shown that PCR-based methods have exquisite sensitivity in pure (oo)cyst suspensions, but amplification inhibitors and barriers to DNA extraction need to be overcome in faecal samples. Additional benefits are Cryptosporidium species/genotype or Giardia assemblage identification. However, few diagnostic laboratories are well placed to undertake PCR. The modernization and amalgamation of pathology services may be a driver for the increased use of immunoassays and PCR in diagnostic settings, most logically as multiplexed tests for panels of gastrointestinal pathogens. However, while these and other biomedical advances may aid diagnosis, any loss of microscopy skills would be to the detriment of parasitology services worldwide.

Introduction It is over 300 years since Antony van Leeuwenhoek’s microscopic examination of his own stools in 1681 facilitated his description of what we now recognize as Giardia cysts and trophozoites. The first formal description was published by Lambl in 1859, who proposed the name Cercomonas intestinalis. The following © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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50 years or so saw many proposed genus and species names, until Giardia lamblia and Giardia enterica were proposed for the parasite isolated from humans (Kofoid and Christensen, 1915; Kofoid, 1920). The currently used Giardia duodenalis and Giardia intestinalis are most accurate taxonomically, although some prefer the former since it conforms to Filice’s morphologically based nomenclature published in 1952 (Filice, 1952). Giardia cysts are readily visible in microscopic examination of a wet preparation from a faecal sample, but trophozoites, being more fragile and less likely to survive intact, are seen less frequently. Similarly, astonishingly accurate and detailed early descriptions of Cryptosporidium life cycle stages were made by Ernest E. Tyzzer in 1907, 1910, 1912 and 1929, establishing the life cycle of the parasite. The only element later amended by examination using electron microscopy was the added description of extracellular developmental stages (merozoites and microgametes). Cryptosporidium was initially recognized as an animal pathogen, and staining techniques were improved to detect oocysts in faeces for veterinary diagnosis. Transfer of these improvements to clinical laboratories enabled diagnosis by examination of stools instead of histological staining of gut biopsy. This facilitated important epidemiological studies during the early 1980s, which led to the recognition of the parasite of importance not only as a potentially fatal infection of immunocompromised patients but also as a cause of acute, self-limiting gastroenteritis in the general population, particularly children (Casemore et al., 1985). Widespread testing and reporting of microbiological results to disease surveillance schemes was thus enabled. In England and Wales, this has been ongoing since 1983. The impact of infection with Cryptosporidium and Giardia was recognized by WHO in 2004 by their inclusion in the ‘Neglected Pathogen Initiative’. Routine laboratory diagnosis of these pathogens is undertaken for a variety of reasons: ● ● ● ● ● ●

Routine clinical diagnosis for patient care. For surveillance and public health purposes. Contact screening. Outbreak/cluster investigation. Enhanced surveillance, e.g. incorporating specialist test results or patient data. Special studies, e.g. carriage rates; experimental studies (transmission/infectivity/treatment trials).

This chapter describes the current situation with regard to routine and specialist diagnostic processes for Cryptosporidium and Giardia.

Testing Algorithms and Diagnostic Procedures Testing algorithms describe the appropriate ordering and use of laboratory tests and are designed to provide guidance to ensure appropriate sample submission, reduce unnecessary testing and accelerate the diagnostic process. Expansion of the algorithm may include investigation and management of suspected and confirmed cases of illness and reporting systems. Algorithms may be produced for individual pathogens, symptoms (e.g. gastrointestinal illness), patient groups (e.g. high-risk patients, travellers returning from abroad) or settings (e.g. schools

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and other child care centres, hospitals) at the local, region, national or international level. National diagnostic test algorithms for Cryptosporidium and Giardia are currently produced in the form of guidance, such as the Investigation of Specimens other than Blood for Parasites produced by the Health Protection Agency (HPA) in England (see http://www.hpa-standardmethods.org.uk/documents/ bsop/pdf/bsop31.pdf) and its sister document Staining Procedures (http://www. hpa-standardmethods.org.uk/documents/bsopTP/pdf/bsoptp39.pdf). The equivalent in the USA is Laboratory Detection of Parasites of Public Health Concern produced by the Centers for Disease Control and Prevention (http://www.dpd. cdc.gov/dpdx/HTML/Cryptosporidiosis.htm). UK veterinary testing is guided by a working group for the Harmonization of Laboratory Diagnostic Methods of the National Surveillance Group on Diseases and Infections of Animals for the testing of samples from farmed animals in Veterinary Laboratories Agency (VLA) regional laboratories. The methods used are recognized by the Office International des Epizooties Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, published in 2004 and updated in 2008 (http://www.oie.int/eng/normes/ mmanual/A_summry.htm). However, this document does not include Giardia, the impact of which on livestock and companion animals may be underestimated because in some countries, including the UK, it is not routinely reported by veterinary laboratories. In many countries Cryptosporidium is only tested for on specific request or in HIV & AIDS patients, and Giardia only as part of an ova and parasites (O&P) request. Requesting primary care physicians and clinicians may not be aware that Cryptosporidium is not necessarily included in this. The UK guidance is for all samples from symptomatic patients to be tested for Cryptosporidium, based on the findings of a working group published in 1993 (Casemore and Roberts, 1993). Testing for Giardia often depends on the patient history (mainly recent foreign travel) and clinical details. Recent surveys by the UK Cryptosporidium Reference Unit have shown that there is an increasing trend for compliance in testing for Cryptosporidium (Chalmers et al., 2002; UK Cryptosporidium Reference Unit, unpublished data). In a survey of 187 laboratories carried out in 2003, 53% reported routinely testing all diagnostic stools from community cases for Cryptosporidium, a figure which had risen to 78% by 2006. The overall positivity rate is in the region of 1%. In the 2003 survey, 36% of laboratories tested all such stools by microscopic examination of a wet preparation, which would include examination for Giardia. It is common practice in the UK to treat hospital in-patients differently from community cases and a ‘3- or 5-day rule’ is often applied. This means that if a patient who had been in hospital develops diarrhoeal illness after this time period, they are not tested for the same suite of pathogens as community cases, and tests for Cryptosporidium and Giardia are not performed unless specifically requested. In VLA regional veterinary laboratories, Cryptosporidium is examined for only in neonatal animals with diarrhoea, unless pathological examination (for example post mortem) suggests the involvement of this parasite. Diagnosis of intestinal protozoa traditionally relies on the detection of life cycle stages in appropriate samples. For Cryptosporidium this is mainly the oocyst, and for Giardia the cyst, in faeces (Table 13.1). Giardia trophozoites may

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Table 13.1.

Routine diagnostic procedures for Cryptosporidium in faeces.

Procedure

Detection method

Microscopy

Modified acid-fast stains

Immunoassay

Microscopy

Diagnostic target

Format

Advantages

Disadvantages

Oocyst

Fixed microscopy slide

Low cost

Fluorescent stains

Oocyst

Fixed microscopy slide

Immunofluorescent stains

Oocyst

Fixed microscopy slide

Enzyme

Antigen

Microplate

Chromatographic

Antigen

Strip-based cartridge

Modified acid-fast stains

Oocyst

Fixed microscopy slide

Low cost Improved specificity over acid-fast stains Low power, more rapid screening Improved sensitivity over acid-fast and fluorescent stains Mass testing Lower skill base Lower skill base than microscopy Portable (field testing) See above

Skilled microscopist Detects other pathogens Poor sensitivity and specificity Skilled microscopist

Higher cost

High cost False positives High cost False positives and negatives See above R.M. Chalmers

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also occasionally be seen. Throughout Europe it is current practice for most diagnostic stools to be submitted fresh, without preservatives. While this has some advantages, the vegetative stages of many parasites may degrade if there are delays in processing. Testing of other sample types depends on the clinical history of the patient: for immunocompetent patients the main symptom is diarrhoea and stool is the most common and appropriate specimen type. Other specimen types for diagnosis of Cryptosporidium, such as tissue biopsy, sputum, bronchoalveolar lavage and bile are generally from severely immunocompromised patients who may suffer from extra-intestinal infection or infection of the biliary tree (Farthing, 2000; Hunter and Nichols, 2002). Diagnosis in cases of suspected biliary involvement may also be assisted by imaging techniques, including ultrasound, CT scans and, more specifically, endoscopic retrograde cholangio-pancreatography (the definitive test for disruption of biliary anatomy). Difficulty in detecting Giardia may be assisted by testing duodenal aspirate. Detection methods for Cryptosporidium oocysts are traditionally tinctorial or fluorescent staining of faecal smears examined by bright field or epifluorescence microscopy, respectively (Nichols and Thom, 1984; Casemore, 1991), while Giardia cysts and occasionally trophozoites can be seen in examination of a wet preparation (Table 13.1). Methods recommended in the HPA guidance are stool microscopy, specifically modified Ziehl-Neelsen or auramine phenol staining for Cryptosporidium. These methods are also specified for animal testing in VLA laboratories. Improved sensitivity is provided by immunofluorescence microscopy (IFM) using a fluorescein isothiocyanate (FITC)-labelled monoclonal antibody raised against oocyst wall antigens (Arrowood, 1997; Casemore, 1992), which is also available in combination for the simultaneous detection of these two protozoa. These stains can be applied to unconcentrated as well as concentrated faecal samples. Concentration by the formol-ether method is suitable and there are commercially available kits providing the plastic ware to facilitate this. Garcia (2001) provides an excellent laboratory manual for parasitological diagnosis. In a survey undertaken in 2006 of 170 primary diagnostic laboratories in the UK, 26% used modified Ziehl-Neesen staining and 72% used auramine phenol staining (UK Cryptosporidium Reference Unit, unpublished data). Just three laboratories used other methods: one used flotation and direct examination and two used antigen detection kits. None used IFM as a primary diagnostic test, for reasons of cost, although this is available as a specialist reference test at the UK Cryptosporidium Reference Unit (see below). The emergence of copro-antigen test kit use in the UK is recent: in a previous survey 3 years earlier no clinical laboratories were using this technology, although individual private veterinary practices sometimes use it. The use of IFM and copro-antigen detection kits in diagnosis is more common elsewhere, particularly in the USA. Two main types of antigen detection system are employed in the kits: enzyme immunoassay assay (EIA) and immunochromatography. The EIA kits are available in multiwell or microplate format, making them an attractive option for laboratories testing large numbers of samples. Indeed, this is the justification for the transfer from microscopy in clinical diagnostics in the UK: laboratories receiving

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large numbers of samples are finding that, particularly if they batch the tests to be performed three times each week, the cost differential with conventional microscopy is reduced. Immunochromatographic tests are available in capsule format, including the dip-stick style for individual sample testing, and offer rapid results within 1–2 hours. Parasites are often shed intermittently and multiple stool samples should be submitted for examination. Traditionally, diagnosis of Giardia in stool, duodenal or jejunal aspirate has been performed by examination of wet preparations (before and after concentration by the formol-ether method) of cysts and trophozoites. Observation is further improved by Lugol’s iodine staining, and examination of a stained permanent smear is also desirable. However, it is doubtful whether sufficient multiple stool samples are submitted for many patients. Vegetative stages in unpreserved stools often degrade, compromising the microscopic diagnosis, but these are still detectable in copro-antigen tests. It has been reported that testing a single stool for copro-antigens by EIA may be an acceptable alternative, since this offers sensitivity approaching that of two sequential stools examined by microscopy (Mank et al., 1997). Improved diagnosis is further enabled by the ‘Triple Faeces Test’ adopted in the Netherlands (van Gool et al., 2003). EIA and immunochromatographic kits are available for individual target pathogens or in combination for Cryptosporidium and Giardia or Cryptosporidium, Giardia and Entamoeba histolytica/dispar. Tests for Cryptosporidium and Giardia can be applied to fresh, frozen or formalin-preserved stools, while those incorporating Entamoeba histolytica/dispar require fresh, unpreserved stools. Sample preparation for antigen detection kits (in contrast to oocyst detection) should not include concentration, since antigens may be lost in the process.

Test Performance Monitoring Should Include Positive and Negative Controls Confirmation of positive test results should always be undertaken, which for microscopy includes accurate measurement of oocysts and observation of morphology. Confirmation of positive results from test kits is important because false positive results have been reported, sometimes driven by high numbers of other microorganisms in the sample. The confirmatory test for diagnostic samples should be of high diagnostic specificity. Most of the test kits were developed using Cryptosporidium parvum: all are useful in detecting Cryptosporidium hominis, but their affinity for other species/genotypes is subject to evaluation.

Test Choice, Evaluation and Quality Assurance The drivers for test choice are theoretically numerous and include cost, simplicity, ease of interpretation, equipment required, personnel/level of training and experience, number of tests ordered, speed, sensitivity, specificity, and the value of incidental findings in non-specific tests. The key attributes of routine test

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options are shown in Table 13.1. However, the main driver is often cost, particularly in publicly funded laboratories. The consumables cost of a modified ZiehlNeelsen test is less than US$1 per sample compared with over US$5 for IFM. However, increasingly, the number of tests ordered is becoming more important as there is a current trend for centralization of laboratory services and there are fewer but larger laboratories testing larger numbers of samples. Here, batch tests such as EIA become more cost-effective when staff costs are taken into account, particularly when the cost of a skilled microscopist’s time is included in the equation. The analytical sensitivity of acid-fast or fluorescence staining of unconcentrated stools is in excess of 104 oocysts per gram (opg), depending on the consistency of the sample, with liquid stools providing greater sensitivity (Anusz et al., 1990; Weber et al., 1991). EIA and immunohistochemical kits offer comparable sensitivity. Improved sensitivity is provided by IFM, but is still in excess of 103 opg. These tests all limit the volume of sample tested. This can be increased to over 2 g by the use of immunomagnetic separation (Robinson et al., 2008) but the cost of this is not justified in routine diagnosis. However, it has a place as a specialist test (see below). The evaluation of new tests should take all the above factors into account and it must also be made clear what is meant by sensitivity and specificity. This is because there are two meanings. One refers to diagnostic sensitivity and specificity and the other to the analytical sensitivity and specificity. Diagnostic parameters are important in studies investigating disease, enabling calculation of positive and negative predictive values for diagnostic tests. The relevant diagnostic definitions are: ●

●

Diagnostic sensitivity = the proportion of subjects with the disease who have a positive test result. Diagnostic specificity = the proportion of subjects with the disease who have a negative test result.

For calculation of these values, there clearly needs to be a clinical definition of disease. However, for Cryptosporidium and Giardia this does not exist in the absence of laboratory confirmation. This presents something of a problem in the above context, which would be only partially overcome by incorporating ‘gold standard’ laboratory confirmation in the definition. Unfortunately, these are not defined either. Nominal definitions may be chosen with justification for individual studies, and Bayesian techniques have also been applied to overcome this problem (Geurden et al., 2006). If a nominal analytical ‘gold standard’ is chosen for comparison of analytical tests, IFM is usually selected for Cryptosporidium and microscopy (wet preparation and stained smear) or IFM for Giardia. Results may be compared using the kappa statistic (k) or McNamar’s test without the necessity to define a gold standard. In studies investigating carriage rates, the analytical sensitivity and specificity are important and are defined thus: ●

Analytical sensitivity = the smallest detectable amount of the analyte in question.

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●

Analytical specificity = the degree to which analytes, other than that in question, react in a test.

Specialist and Reference Tests Routine local primary diagnostic services are often limited to testing stool samples for Cryptosporidium and Giardia, biopsy samples where histological services are available for Cryptosporidium, and duodenal aspirate or string test material for Giardia. Further specialist diagnostic tests may be provided for patients with appropriate indications, or where oocyst numbers are low, either locally or in reference laboratories. The UK Cryptosporidium Reference Unit algorithm recommends specialist testing for patients with idiopathic diarrhoea and/or biliary disease from the following high-risk groups: ● ● ●

HIV with CD4 < 200. Acute leukaemic/lymphoma/BMT. Primary T cell immune deficiency.

Sensitive testing is also available for patients with solid organ transplants or taking T cell immunosuppressants. In addition to stool, appropriate samples types depend on symptoms. For those with cholangitis or biliary disease, stool and bile from ERCP are appropriate and liver biopsy may also be tested. For those with unexplained chest symptoms, sputum or bronchioalveolar lavage may be tested, and for those with unexplained sinusitis, antral washout is appropriate. Test methods are IFM and PCR, which show fair agreement. Improved analytical sensitivity can be provided, where required, by use of immunomagnetic separation (Robinson et al., 2008) or PCR (Webster et al., 1996). Detection using PCR has an emerging place in clinical diagnostics, and is particularly cost-effective when performed as a multiplexed test for two or more pathogens. This approach is being introduced in the Netherlands (L. Kortbeck, personal communication) but the exact choice of which pathogens to co-test for needs careful consideration. None of the routinely used diagnostic tests identifies Cryptosporidium genotypes or Giardia assemblages. Genotyping isolates is currently a specialist test, and provides valuable epidemiological data to measure changes, improve risk assessment, and remove epidemiological noise by separating genotypes in the analysis (Chalmers and Elwin, 2000). Typing can be undertaken from faecal samples or from material fixed on microscope slides in routine diagnostic processing (Amar et al., 2002). It is clear from continuing long-term enhanced surveillance at the UK Cryptosporidium Reference Unit since 2000, involving the testing of over 16,000 isolates, that Cryptosporidium parvum and Cryptosporidium hominis have different epidemiologies (Anonymous, 2002; Nichols et al., 2006) and risk factors (Hunter et al., 2004). Detailed analysis of changes in epidemiology of Cryptosporidium infections is enhanced by genotyping data (Smerdon et al., 2003; Sopwith et al., 2005).

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Reporting and Disease Surveillance While testing for Cryptosporidium is generally voluntary and subject to individual laboratory testing algorithms, reporting for surveillance purposes depends on the notifiable status of the diagnosis in each country. In the UK, Cryptosporidium is not statutorily notifiable unless it is suspected to be foodborne (the definition of which includes waterborne) disease, and reporting is voluntary. Despite this, in 2006 over 80% of laboratories reported cases, indicating the recognized importance of testing and surveillance in the UK. In Ireland, Germany and Sweden, for example, Cryptosporidium is notifiable and reporting compulsory. However, testing is voluntary. Despite advances in the biomedical sciences leading to developments in laboratory diagnostics, examination (usually of faeces) by microscopy remains the keystone of laboratory diagnosis of cryptosporidiosis and giardiasis.

References Amar, C.F.L., Chalmers, R.M., Elwin, K., Tynan, P. and McLauchlin, J. (2002) Blinded evaluation of Cryptosporidium genotyping from DNA recovered from stained smear. Letters in Applied Microbiology 35, 486–488. Anonymous (2002) The Development of a National Collection for Oocysts of Cryptosporidium. Final Report to DEFRA: Drinking Water Inspectorate. Foundation for Water Research, Marlow, Bucks, UK. Available at: http://www.fwr.org/ Anusz, K.Z., Mason, P.H., Riggs, M.W. and Perryman, L.E. (1990) Detection of Cryptosporidium parvum oocysts in bovine feces by monoclonal antibody capture enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 28, 2770– 2774. Arrowood, M.J. (1997) Diagnosis of Cryptosporidium and cryptosporidiosis. In: Fayer, R. (ed.) Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, FL, pp. 43–46. Casemore, D.P. (1991) ACP Broadsheet 128, June 1991: Laboratory methods for diagnosing cryptosporidiosis. Journal of Clinical Pathology 44, 445–451. Casemore, D.P. (1992) Cryptosporidium and Giardia. In: Caul, E.O. (ed.) Immunofluorescent Antigen Detection Techniques in Diagnostic Microbiology. PHLS, London. Casemore, D.P. and Roberts, C. (1993) Guidelines for screening for Cryptosporidium in stools: Report of a Joint Working Group. Journal of Clinical Pathology 46, 2–4. Casemore, D.P., Sands, R.L. and Curry, A. (1985) Cryptosporidium species a “new” human pathogen. Journal of Clinical Pathology 38, 1321–1336. Chalmers, R. and Elwin, K. (2000) Implications and importance of genotyping cryptosporidium. Communicable Disease and Public Health 3, 155–158. Chalmers, R.M., Hughes, S., Thomas, A.L., Woodhouse, S., Thomas, P.D. and Hunter P. (2002) Laboratory ascertainment of Cryptosporidium and local authority public health policies for the investigation of sporadic cases of cryptosporidiosis in two regions of the United Kingdom. Communicable Disease and Public Health 5, 114–118. Farthing, M.J.G. (2000) Clinical aspects of human cryptosporidiosis. In: Petry, F. (ed.) Cryptosporidiosis and Microsporidiosis. Contrib Microbiolo, Karger, Basel, Switzerland, pp. 50–74. Filice, F.P. (1952) Studies on the cytology and life history of a Giardia from the laboratory rat. University of California Publications in Zoology 57, 53–146.

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R.M. Chalmers Garcia, L S. (2001) Diagnostic Medical Parasitology, 4th edn. American Society for Microbiology, Washington, DC. Geurden, T., Berkvens, D., Geldhof, P., Vercruysse, J. and Claerebout, E. (2006) A Bayesian approach for the evaluation of six diagnostic assays and the estimation of Cryptosporidium prevalence in dairy calves. Veterinary Research 37, 671–682. Hunter, P.R. and Nichols, G. (2002) Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clinical Microbiology Reviews 15, 145– 154. Hunter, P.R., Hughes, S., Woodhouse, S., Syed, Q., Verlander, N.Q., Chalmers, R.M., Morgan, K., Nichols, G., Beeching, N. and Osborn, K. (2004) Sporadic cryptosporidiosis case-control study with genotyping. Emerging Infectious Diseases 10, 1241– 1249. Kofoid, C.A. (1920) A critical review of the nomenclature of human intestinal flagellates Cercomonas, Chilomastix, Trichomonas, Tetratrcihomonas, and Giardia. University of California Publications in Zoology 20, 145–168. Kofoid, C.A. and Christensen, E.B. (1915) On binary and multiple fission in Giardia muris (Grassi). University of California Publications in Zoology 16, 30–54. Lambl, W. (1859) Mikroskopische untersuchungen der Darmexcrete. Vierteljahrsschr Prakst Heikunde 61, 1–58. Mank, T.G., Zaat, J.O.M., Deelder, A.M., van Eijk, J.T.M. and Polderman, A.M. (1997) Sensitivity of microscopy versus enzyme immunoassay in the laboratory diagnosis of giardiasis. European Journal of Clinical Microbiology and Infectious Diseases 16, 615–619. Nichols, G. and Thom, B.T. (1984) Screening for Cryptosporidium in stools. Lancet 1, 735. Nichols, G., Chalmers, R., Lake, I., Sopwith, W., Regan, M., Hunter, P., Grenfell, P., Harrison, F. and Lane, C. (2006) Cryptosporidiosis: A Report on the Surveillance and Epidemiology of Cryptosporidium Infection in England and Wales. Drinking Water Inspectorate Contract Number DWI 70/2/201. Drinking Water Inspectorate, London. Robinson, G., Watkins, J. and Chalmers, R.M. (2008) Evaluation of a modified semiautomated immunomagnetic separation technique for the detection of Cryptosporidium oocysts in human faeces. Journal of Microbiological Methods 75, 139–141. Smerdon, W.J., Nichols, T., Chalmers, R.M., Heine, H. and Reacher, M.H. (2003) Decrease in human cryptosporidiosis coincident with the Foot and Mouth Disease epidemic: a measure of the livestock attributable fraction in England and Wales? Emerging Infectious Diseases 9, 22–28. Sopwith, W., Osborn, K., Chalmers, R. and Regan, M. (2005) The changing epidemiology of cryptosporidiosis in north west England. Epidemiology and Infection 133, 785–793. Tyzzer, E.E. (1907) A sporozoan found in the peptic glands of the common mouse. Proceedings of the Society for Experimental Biology and Medicine 5, 12–13. Tyzzer, E.E. (1910) An extracellular coccidium, Cryptosporidium muris (gen. et sp. nov.) of the gastric glands of the common mouse. Journal of Medical Research 23, 487–509. Tyzzer, E.E. (1912) Cryptosporidium parvum (sp. nov.), a coccidium found in the small intestine of the common mouse. Archiv fur Protistenkunde 26, 394–412. Tyzzer, E.E. (1929) Coccidiosis in gallinaceous birds. American Journal of Hygiene 10, 269–383. van Gool, T., Weijts, R., Lommerse, E. and Mank, T.G. (2003) Triple faeces test: an effective tool for detection of intestinal parasites in routine clinical practice. European Journal of Clinical Microbiology and Infectious Diseases 22, 284–290.

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Weber, R., Bryan, R.T., Bishop, H.S., Walquist, S.P., Sullivan, J.J. and Juranek, D.D. (1991) Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current methods. Journal of Clinical Microbiology 29, 1323–1327. Webster, K.A., Smith, H.V., Giles, M., Dawson L. and Robertson, L.J. (1996) Detection of Cryptosporidium parvum oocysts in faeces: comparison of conventional coproscopical methods and the polymerase chain reaction. Veterinary Parasitology 61, 5–13.

14

Control of Cryptosporidium and Giardia in Surface Water by Disinfection

T.M. HARGY1, J.L. CLANCY1 AND L.P. LANDRY2 1Clancy

Environmental Consultants, Saint Albans, VT, USA; 2Greater Vancouver Regional District, Burnaby, Canada

Abstract The Greater Vancouver Regional District (GVRD) serves the Greater Vancouver, BC, area, delivering water to 18 Lower Mainland municipalities, which in turn deliver water to approximately 2.1 million people. Water is collected from three mountainous watersheds: Capilano, Coquitlam and Seymour. The GVRD adopted its Drinking Water Management Plan (DWMP) in August 2005 and its strategy is to use a risk-management multi-barrier approach from source to tap. The results of this project have confirmed that the GVRD has selected an effective multi-barrier approach for protecting the health of consumers of unfiltered drinking water. However, caution is recommended if any circumstance arise such that turbidity spikes due to some cause that enriches the organic, rather than the inorganic, fraction of the suspended particles. An algal bloom or terrestrial upset, such as the immediate fallout of a forest fire, might have an effect not tested in this study. The protected nature of the watershed makes a nutrient influx fuelling an algal bloom an unlikely scenario, but a natural or human-induced forest fire is possible. Ozone was shown to be a very effective pretreatment of Coquitlam water for UV, regardless of turbidity. The UV transmittance (UVT) of Coquitlam water, and thus its UV treatability, was uniformly improved by ozonation.

Introduction The Greater Vancouver Regional District (GVRD) serves the Greater Vancouver, BC, area, delivering water to 18 Lower Mainland municipalities, which in turn deliver water to approximately 2.1 million people. Water is collected from three mountainous watersheds: Capilano, Coquitlam and Seymour. The GVRD adopted its Drinking Water Management Plan (DWMP) in August 2005 and its strategy is to use a risk-management multi-barrier approach from source to tap. The Coquitlam watershed is closed to the public, industry and agriculture to minimize potential contamination of the source water. The Coquitlam source is currently using ozone for primary disinfection and chlorine for secondary disinfection. The GVRD plans to install UV for Cryptosporidium control downstream of the 158

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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ozone treatment for Giardia and viruses. While Coquitlam source water turbidity is low and filtration can be avoided in accordance with the Guidelines for Canadian Water Quality, there may be infrequent, but possible, events likely to cause high source-water turbidity. This study was undertaken to demonstrate the degree to which an elevated turbidity event would diminish the effectiveness of the disinfection system. Specifically, the question asked was whether elevated turbidity at the Coquitlam source could directly impair the effectiveness of either the UV disinfection of Cryptosporidium or the ozone disinfection of Giardia and viruses. A second concern was whether elevated turbidity might minimize the advantage gained when UV is applied downstream of ozone. In this study, bulk water samples and concentrated suspended material were collected and used to quantify and characterize the microbiological and physical characteristics of Coquitlam source water. Further efforts were directed toward re-suspending subsamples of the collected concentrated material into reactor volumes of the bulk water, and evaluating the efficacy of ozone and UV at disinfecting natural or seeded microorganisms across a range of turbidities experimentally prepared from native Coquitlam particles. Specifically, we examined the disinfection by ozone and UV of seeded coliphage MS2, Cryptosporidium oocysts and Giardia cysts. Finally, we investigated the effect of ozone on UV absorbance to determine whether the oxidant would continue to improve the UV treatability of Coquitlam water under high turbidity conditions, to the same degree as it has been shown to do at the lower turbidity levels generally present.

Enumeration of Indigenous Free- and Particle-associated Microorganisms in Coquitlam Water A combination of methods was used to assess the microbiological quality of the Coquitlam source water that supplies Greater Vancouver’s water needs. Knowing the types and concentrations of microorganisms present in a source water is important in assessing the type(s) of treatment necessary to comply with drinking water regulations or guidelines and to protect public health. Organisms of interest include pathogens such as Giardia and Cryptosporidium, relatively resistant organisms such as bacterial spores, algae – which are often the most abundant organisms in a waterbody, and macroorganisms that include a variety of invertebrates that are not pathogenic but contribute to the total biomass. Macro- and microorganisms are charged particles and tend to become associated with other particles, forming aggregates. To characterize the water quality, both the levels and types of biota present under various conditions were examined. The methods used to measure the microbiological quality of the Coquitlam water supply included direct microscopic observation using a technique known as microscopic particulate analysis or MPA; USEPA method 1623 (USEPA, 2005) for detection of Giardia cysts and Cryptosporidium oocysts; heterotrophic plate count (HPC), a measure of general microbial quality; total aerobic spores (TAS), which measures the population of aerobic resistant spore-forming bacteria; and male-specific and somatic coliphages, bacterial viruses that infect E. coli.

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Sample collection Samples for subsequent testing and analyses were collected by GVRD from the Coquitlam source immediately downstream of the utility intake at a sample port.

Giardia/Cryptosporidium and MPA Analyses Filter processing For each high-volume sample, a spiral wound filter and two Envirochek HV capsule filters were collected. The spiral wound filter was processed as described in the MPA consensus method (USEPA, 1992) and the HV filters were processed as described in USEPA method 1623 (USEPA, 2005), with the exception that, for the purpose of characterizing the microbial population of the captured particulate material, each filter was processed using an aseptic technique. MPA analysis A portion equivalent to 0.7 l of each sample collected was examined using bright field microscopy. Giardia/Cryptosporidium enumeration Low-volume samples were collected on Envirochek HV filter capsules. The retained particles were eluted from the filter as described above, with the final concentrate being resuspended to 10–20 ml. A portion equivalent to 10–15 l of the original sample volume was transferred for Cryptosporidium and Giardia analysis by immunomagnetic separation (IMS). Recovered organisms were enumerated using epifluorescence microscopy. Pellet preparation for microbial analysis Packed pellet material was gently resuspended and an aliquot (1% of this original suspension) was diluted in sterile PBW and mixed. This sample was split into three subsamples, one for each of the three microbial assays (HPC, TAS and coliphages). Each subsample was split in half: one half was analysed directly, and the other half was analysed following a disassociation procedure involving a series of vortexing and sonication steps. Enumeration of heterotrophic bacteria Heterotrophic bacteria were enumerated by the spread plate technique (Standard Method 9215C) on R2A agar to measure HPC. Plates were incubated inverted

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for 4–7 days at room temperature (25°C). Colony forming units (CFU) from dilutions were recorded and the concentration of HPC was calculated as CFU/ml.

Enumeration of total aerobic spores TAS were enumerated by the spread plate technique (Standard Method 9215C) on TAS medium. Following pasteurization, samples were subjected to serial tenfold dilution in PBW (if necessary). An appropriate volume of sample water was aseptically spread on TAS medium using a sterile spreader and allowed to absorb completely before incubating. Plates were incubated inverted for 24 ± 2 h at 35 ± 0.5°C. Counts were recorded and the TAS concentration calculated as CFU/ml.

Enumeration of male-specific and somatic coliphages Male-specific and somatic coliphage concentrations were determined by the double-agar-layer plaque assay (Standard Methods 9224B and 9224C) using host bacteria Escherichia coli HS(pFamp)R (ATCC No. 700891). Viral plaque counts from dilutions in the countable range were tallied and reported as plaque forming units per ml (PFU/ml).

Results Giardia and Cryptosporidium Data for pathogenic protozoa are shown in Table 14.1. No fluorescing objects resembling Giardia cysts or Cryptosporidium oocysts were observed in either of the samples analysed.

Microscopic particulate analysis Two MPA samples were collected and analysed using the USEPA consensus method (USEPA, 1992). Results are shown in Table 14.2. The increase in turbidity from Coquitlam 1 to Coquitlam 2 led to a large increase in packed pellet volume; the Coquitlam 1 sample had a packed pellet Table 14.1.

Giardia and Cryptosporidium sampling data.

Sampling round Sample matrix Coquitlam 1 Coquitlam 2

Envirochek HV capsule Envirochek HV capsule

Giardia cysts Cryptosporidium oocysts <1/10 l <1/15 l

<1/10 l <1/15 l

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Microscopic particulate analysis data.

Sample date Volume sampled (l) Start/end turbidity (NTU) Packed pellet volume (ml)

Coquitlam 1

Coquitlam 2

11/7/2006 400

11/16/2006 124

1.5/2.0

6.2/5.3

0.5

4

(No. per 100 l)

(No. per 100 l)

Algae

5.7 × 106

7.0 × 106

Amoebae

1.1 × 104

6.2 × 103

Crustaceans

MPA category

1.4 ×

102

ND

Diatoms

3.1 ×

104

3.4 × 104

Insects/parts

2.0 ×

103

2.8 × 102

Nematodes/eggs

2.4 × 103

7.1 × 102

Pollen

5.7 × 102

3.4 × 103

ND

ND

Free-living protozoa Rotifers/eggs Spores Vegetative debris

8.5 ×

102

4.3 × 102

ND

2.3 × 103

1.4 × 102

1.8 × 103

ND: none detected.

volume of 0.5 ml/400 l (0.1 ml/100 l) while Coquitlam 2 had a 4 ml pellet from a 124 l sample volume (3.3 ml/100 l). Coquitlam 1 contained biological particles typical of pristine lake water, with low levels of algae and diatoms. Inorganic debris in the sample was typical of lake water, with bits of diatom shells adding to the ‘inorganic’ or solids portion of the sample. The amount of inorganic debris present was small (<15 µm), colourless and fairly sparse. When a portion of the sample was concentrated by centrifugation, a layer of algae was observed on top of the inorganic portion of the sample. This observation is unusual; however, if algae are present that form gas vacuoles or are contained in colonial mucilage, this phenomenon can occur. The algae layer comprised approximately 10% of the volume of the pellet material present in this sample. Coquitlam 2 contained a much larger number of inorganic particles. The concentrations and types of biological particles in this sample were similar to the Coquitlam 1 sample. The biological portion of the sample comprised <0.1% of the particle concentration in this sample. While similar concentrations of algae and diatoms were noted in both MPA samples examined, the amounts of inorganic debris increased significantly in the higher-turbidity sample. The number of inorganic particles increased by a log, indicating that the increase in turbidity did not result in an increased load of biological particles, but rather in inorganic particles due to runoff. Concentrations of pollen and vegetative debris were

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greater in Coquitlam 2; while these are biological in nature, they are also likely to be increased due to runoff during the rainfall event. The photomicrographs in Fig. 14.1 show the difference observed in the two samples. The Coquitlam 1 sample (Photos 1A–1D) had much less inorganic debris than did Coquitlam 2 (Photos 2A–2D).

Heterotrophic bacteria HPC data are shown in Table 14.3. The HPC concentrations were enumerated from the 1-l bulk water samples collected aseptically immediately on receipt of the samples, and the counts were similar for both samples (1.5–1.8 × 103/ml). The pre- and post-disaggregation sample data were measured on the packed pellet material and represent hundreds of litres of filtered water concentrate; they do not relate to the bulk water data in the table. The pellet counts show that there was no increase in HPCs after the sample was subjected to vortexing and sonication, suggesting that particle absorption did not result in clumping of HPC bacteria, and thus cause an underestimation of their presence.

Total aerobic spores Table 14.4 shows the TAS data. The spore data show that TAS was very low or not detected in all of the samples tested. Even the packed pellet material, which is hundreds-fold concentrated, showed no spores before or after treatment to disrupt aggregates. Spores are highly resistant to inactivation but their low numbers in this source water would not allow their use as a natural indicator of disinfection.

Male-specific and somatic coliphage Coliphage data are shown in Table 14.5. Like TAS, the phage data showed very low counts in both samples of the bulk water. The packed pellet concentrates were very low and disaggregation did not increase counts. Like TAS, coliphages would not be a useful natural indicator of disinfection efficacy.

Disinfection of MS2, Cryptosporidium and Giardia Project approach In this phase of the project we characterized the dose response of coliphage MS2, Giardia and Cryptosporidium in baseline Coquitlam water to a range of disinfectant doses, with the purpose of identifying the dose necessary for 4-log (MS2) and 3-log (protozoa) inactivation. This was followed by testing the efficacy of that disinfectant dose at inactivating the microorganisms suspended in

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Coquitlam 1A - Minimal inorganic debris; centric diatom, girdle view; Cyanophyta; 200

Coquitlam 1B - Minimal inorganic debris, Pleurotaenium; 200

Coquitlam 1C - Aggregated inorganic debris, 400

Coquitlam 1D – Merismopedia and Chroococcus, 400

Coquitlam 2A - Inorganic debris with amoebae shell, 200

Coquitlam 2B - Pennate diatom and Chroococcus, 400

Coquitlam 2C - Merismopedia, 400

Coquitlam 2D- Inorganic debris, 400

Fig. 14.1. Photomicrographs of particles in Coquitlam waters 1 and 2, showing the differences noted in each sample.

Control of Cryptosporidium and Giardia in Surface Water Table 14.3.

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Heterotrophic plate count data.

Sampling round

Sample matrix

Average CFU/ml

Standard deviation

1 2 1 2 1 2

1 l bulk water 1 l bulk water Pellet pre-disaggregation Pellet pre-disaggregation Pellet post-disaggregation Pellet post-disaggregation

1.8E+03 1.5E+03 6.3E+05 7.2E+06 6.0E+05 7.3E+06

1.2E+02 1.0E+02 7.6E+04 1.2E+05 9.8E+04 1.2E+05

Average CFU/ml

Standard deviation

0.3 0.1 0 0 0 0

0.1 0 0 0 0 0

Table 14.4. Total aerobic spore data. Sampling round

Sample matrix

1 2 1 2 1 2

1 l bulk water 1 l bulk water Pellet pre-disaggregation Pellet pre-disaggregation Pellet post-disaggregation Pellet post-disaggregation

Table 14.5.

Coliphage data.

Sampling round

Sample matrix

1 2 1 2 1 2

1 l bulk water 1 l bulk water Pellet pre-disaggregation Pellet pre-disaggregation Pellet post-disaggregation Pellet post-disaggregation

Male-specific phage

Somatic phage

Average PFU/ml

Average PFU/ml

0.2 <0.3 <0.01 <0.003 <0.01 <0.003

0.2 <0.3 <0.01 <0.003 0.01 <0.003

Coquitlam water that had previously been adjusted to a range of turbidity levels in order to evaluate any degradation of disinfection with increase in turbidity. The turbidity levels selected were from baseline up to 5 NTU in 1-NTU increments, for evaluation of disinfection efficacy across the turbidity range permissible under the Guidelines for Canadian Drinking Water Quality (Health Canada, 2005); also selected were 10, 15 and 20 NTU for evaluation of disinfection in the face of currently potential operating conditions (up to the 99.995% percentile value of 20 NTU) wherein the Coquitlam source could not be shut down during turbidity events. Such a scenario will no longer present itself on completion of the Seymour Capilano filtration plant. Finally, the 50 NTU level was selected as an extraordinary turbidity level at which to observe any impacts to disinfection practices.

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Turbidity adjustment In all disinfection against turbidity tests, the test microbes were mixed with sufficient particulate material to achieve the target turbidity in the reactor volume, and allowed to stand overnight at 4°C to allow contact time for particle association. Sufficient MS2 were added to result in reactor concentrations of 1 × 107 PFU/ ml for UV tests and 2 × 106 PFU/ml for ozone tests. Protozoa were added in quantities producing a total of 1 × 106 microorganisms per reactor volume. At the time of testing, agitation of the microbe/particle mixture for resuspension was kept to a minimum to avoid breaking up any aggregates that may have formed.

UV disinfection methods Collimated beam dose response determination The UV dose delivery process, known as a collimated beam test, was carried out using the methods described by Bolton and Linden (2003), which adjusts exposure time based on UV irradiance, absorbance and distribution. UV dose was defined as the average irradiation so calculated, multiplied by the exposure time. Exposures providing UV doses of 0, 20, 40, 60, 80 and 100 mJ/cm2 for MS2, and 0, 1, 2, 3, 4 and 5 mJ/cm2 for Cryptosporidium were used. A control (0 mJ/ cm2 dose) was run simultaneously with the longest exposure for each microbe, but in the absence of UV. The control sample provided the base count for determination of log inactivation. UV dose selection and determination in the presence of turbidity The same collimated beam apparatus and methods employed for dose response determination were used to evaluate the effects of turbidity on UV disinfection of MS2 and Cryptosporidium, but with the following adjustments. Rather than present a range of exposure times, and thus UV doses, to suspended organisms, the UV dose determined from the above dose response characterization was applied to the test microbes suspended in a range of turbidities from baseline to as high as 50 NTU. As noted above, in addition to inoculating the baseline water with test organisms, Coquitlam particulate material was added to the reactor volumes and left overnight to achieve increments of target turbidity levels. For UV disinfection tests, the direct UV absorbances at 254 nm were measured at each turbidity level to quantify the water correction factors for each exposure time.

Microorganism propagation and assay MS2 was selected because it has been shown to be a conservative surrogate for enteric virus disinfection by UV (Wilson et al., 1992), though it is more sensitive to UV than is adenovirus (Meng and Gerba, 1996). Its UV dose response is well documented (USEPA, 2006). For the present project, MS2 (ATCC No. 15597-B1) was propagated in E. coli Famp, using a liquid culture method. Prior to disinfection challenges, the stock was diluted in sterile PBW to achieve a working stock

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concentration of 1 × 109 PFU/ml. This stock was dispensed to reactor batches in volumes sufficient to achieve 1 × 107 PFU/ml reactor suspensions. MS2 assays Phage concentrations were determined via the double agar layer technique (Adams, 1959). All samples were plated in triplicate. PFU were counted and recorded, and the concentration of MS2 calculated. Disaggregation following UV exposure in elevated NTU In analysing the MS2 surviving UV in elevated turbidity Coquitlam water, dissociation steps were employed to dissociate the phage from the particulate matrix in subsamples of one test set. Cryptosporidium stock Cryptosporidium parvum oocysts (Iowa isolate) were obtained from Waterborne, Inc. (New Orleans, LA). Oocysts were fresh (16 days old post shedding) at the time of testing. Cell culture assay Samples to be analysed for infectious Cryptosporidium oocysts were processed as described by M.S. Johnson (Water Quality Laboratory, Metropolitan Water District of Southern California, 2006, personal communication). UV disinfection results MS2 UV dose response The responses of MS2 seeded into Coquitlam waters at baseline (0.3 NTU) turbidity are presented in Figs 14.2 and 14.3 and indicate that MS2 suspended in MS2 UV dose response curve, Seeded Coquitlam 1 water, baseline conditions (low NTU) 100 UV dose (mJ/cm2)

y = 0.9055x 2 + 17.267x 80 60 40 20 0 0

1

2 3 Log inactivation

4

5

Fig. 14. 2. The UV dose response of MS2 seeded into Coquitlam 1 water at baseline (0.3 NTU) turbidity.

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Coquitlam 1 required 83 mJ/cm2 for 4-log inactivation, while MS2 in the Coquitlam 2 required 92 mJ/cm2 for the same level of disinfection. On introducing particulate matter from the respective samples to achieve elevated turbidity, UV sensitivities at the doses required for 4-log inactivation (83 and 92 mJ/cm2 for Coquitlam 1 and 2, respectively) were found to be as effective as in baseline waters (Figs 14.4 and 14.5). In the latter elevated turbidity test, the sample was disaggregated after UV irradiation to disassociate MS2 from particles. Cryptosporidium disinfection by UV The inactivation of Cryptosporidium oocysts suspended in baseline Coquitlam water is shown in Fig. 14.6. More than 3.6-log inactivation was achieved at 3 mJ/cm2 and more than 5-log inactivation was demonstrated at 4 and 5 mJ/cm2. As 3 mJ/cm2 achieved the GVRD target of 3-log Cryptosporidium inactivation, that UV dose was then applied to seeded reactors with incrementally elevated

MS2 UV dose response curve, Seeded Coquitlam 2 water, baseline conditions (low NTU) 100 UV dose (mJ/cm2)

y = 0.9787x 2 + 19.151x 80 60 40 20 0 0

1

2 3 Log inactivation

4

5

Fig. 14.3. The UV dose response of MS2 seeded into Coquitlam 2 water at baseline (0.3 NTU) turbidity.

UV log inactivation of MS2 vs turbidity Coquitlam sample 1, 83 mJ/cm2 Log inactivation

5.0 4.0 3.0 2.0 1.0 0.0 0

5

10

15 NTU

20

25

(50)

Fig. 14.4. The UV inactivation of MS2 versus turbidity in Coquitlam 1 water.

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UV log inactivation of MS2 vs turbidity Coquitlam sample 2, 92 mJ/cm2 Log inactivation

5.0 4.0 Raw Disaggregated

3.0 2.0 1.0 0.0 0

5

10

15

20

25

(50)

NTU

Fig. 14.5. The UV inactivation of MS2 versus turbidity in Coquitlam 2 water.

UV dose response of Cryptosporidium in baseline Coquitlam water 6

Log inactivation

5 4 3 2 1 0 0

1

2

3

4

5

UV dose (mJ/cm2)

Fig. 14.6. The UV dose response of Cryptosporidium parvum oocysts seeded into Coquitlam baseline water.

turbidity for each water and pellet sample. The results, shown in Table 14.6, indicate that 3-log inactivation of Cryptosporidium by UV was not diminished when oocysts were seeded overnight in Coquitlam particulate matter and subsequently exposed to UV.

Ozone disinfection of MS2 and Giardia Ozone generation and ozone stock production Ozone was produced using a Clearwater Technology CD10/AD corona discharge ozone generator that was modified to accept feed gas from a pressurized source. The feed gas was dry compressed air. The output was delivered to 1 l of 3°C Coquitlam water in an ozone demand-free side arm flask through a glass diffuser

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T.M. Hargy et al. Table 14.6. The UV inactivation of Cryptosporidium with elevated Coquitlam turbidity at 3 mJ/cm2.

Turbidity (NTU) 0 5 10 20 50

Log inactivation in Coquitlam sample 1

Log inactivation in Coquitlam sample 2

>5 >5 >5 >5 >5

>5 >5 >5 >5 >5

inserted through a single-hole stopper. Off-gas was vented from the side arm port and exhausted from the building. Ozone residual measurement All ozone residual analyses were made using a variation of Standard Method 4500-O3 B. To compute the integrated products of disinfectant residual and contact time, ozone residuals recorded over the course of the experiment were multiplied by the contact time interval between each residual measurement. Ozone reactors Batch reactors were prepared by seeding test water in 100 ml ozone demandfree beakers containing stir bars and situated in a water bath (at 3–4°C) over a stir plate. After determining the ozone concentration of the ozone stock, sufficient stock to achieve the target applied ozone or initial ozone residual was pipetted at time zero to the reactors, previously seeded and containing the appropriate volume of test water such that addition of ozone stock would result in a total volume of 50 ml. Ozone reactivity was quenched by adding sodium bisulphite. The effectiveness of the quenching reaction was confirmed by addition of the bisulphite solution to a test reactor containing 3 mg/l ozone and measuring a 0 mg/l ozone residual. The quenching agent was also added to the process control to which no ozone stock had been added. Giardia muris preparation Giardia muris was obtained from the Meyer Laboratory, Oregon Health Sciences, Portland, OR. The purified cysts were stored at 3–5°C in antibiotics for up to 2 weeks post shedding. Sample processing and excystation procedure Samples were received in the laboratory in 40–45 ml volumes and subjected to an excystation procedure as described by Hoff et al. (1985). Cysts were scored as excysted (empty cysts), partially excysted (trophozoite emerging from cyst), and non-excysted (intact cyst containing trophozoite). Cysts which were scored as excysted and partially excysted were considered viable, and those that did not excyst were scored as non-viable.

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Ozone disinfection results MS2 Figure 14.7 shows the inactivation kinetics of MS2 seeded in baseline Coquitlam water and subjected to ozone CT values (concentration of ozone multiplied by contact time with virus) from 0.01 to 0.3 mg min/l. From the resulting curve, the 4-log inactivation benchmark would be achieved by a CT of 0.26 mg min/l. This 0.26 CT level and a slightly higher CT (0.35) were then applied to 50 ml reactor volumes adjusted across the range of turbidity including 0, 2, 5, 10 and 50 NTU with Coquitlam particulate material that had been seeded overnight with MS2 stock (2 × 106 PFU/ml of reactor volume). In all tests, inactivation proceeded to the point where no surviving phages were detected in the highest inoculum volume we tested, demonstrating inactivations of greater than 5.5-log. A lone exception was that a single plaque, indicating one surviving phage, was detected in the lower CT test at 10 NTU, resulting in a reportable log inactivation of 5.5, rather than greater than that level. Ozone disinfection of Giardia Ozone disinfection of Giardia cysts was carried out for both waters under baseline and elevated turbidity conditions. The ozone dose responses of Giardia are shown in Fig. 14.8; a CT of 3.3 mg min/l achieved greater than 3-log inactivation in both waters. That CT level was then targeted to the elevated turbidity reactors. The results, shown in Table 14.7 indicate that for both Coquitlam waters, more than 3-log inactivation was achieved by a CT of approximately 3 (range: 2.7– 3.2) in turbidities of baseline and 5–20 NTU. Evaluation of increasing turbidity effect on ozone/UV synergy The UV transmittance (UVT) of baseline Coquitlam waters (254 nm) and waters adjusted to 5–50 NTU were measured across 1 cm at Clancy Environmental MS2 ozone dose response curve, seeded baseline Coquitlam water

Ozone CT (mg min/L)

0.4 0.3 0.2 0.1 0 0

1

2 3 Log inactivation

4

5

Fig. 14.7. The inactivation kinetics of MS2 seeded into baseline Coquitlam water and subjected to ozone CTs from 0.01 to 0.3 mg min/l.

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T.M. Hargy et al. Log inactivation of Giardiamuris cysts vs ozone CT in baseline Coquitlam waters 1 and 2

Log inactivation

4 3 2 1 0 0

0.5

1

1.5 2 CT (mg min/l)

2.5

3

3.5

Fig. 14.8. The ozone dose responses of Giardia muris cysts seeded into baseline Coquitlam waters 1 and 2. Table 14.7. Ozone disinfection of Giardia muris cysts seeded into Coquitlam water adjusted to a range of turbidities at 3.5°C. Coquitlam sample number

Turbidity (NTU)

CT (mg min/L)

Log inactivation

1 1 1 1 2 2 2 2

Baseline (0.3) 5 10 20 Baseline (0.3) 5 10 20

3.1 2.8 2.7 3.0 2.9 3.2 2.9 3.2

>3.0 >3.0 >3.0 >3.0 >3.0 >3.0 >3.0 >3.0

Consultants (CEC) using direct spectrophotometry (Spectronic Genesys 10UV). Split samples of these waters were ozonated with an applied ozone dose of 2.4 mg/l, and UV absorbances were measured again. Pre- and post-ozone split samples of each of these turbidity levels were shipped chilled overnight to the Department of Civil and Environmental Engineering at Duke University and direct transmittance was again measured using a Cary 300 spectrophotometer. Finally, an integrated sphere device was incorporated to allow measurement of UV absorbance without interference from light scattering by particles. The results of these analyses are shown graphically in Figs 14.9 to 14.12. Figures 14.9 and 14.10 present the pre- and post-ozonated direct UVT data for Coquitlam samples 1 and 2, as measured by CEC and Duke University, and indicate the change in direct UVT as a result of ozonation across the range of turbidities tested. Generally, pre-ozone baseline UVT values were 86%, and decreased with turbidity to as low as 52% at 50 NTU. These figures indicate a significant improvement in UVT post-ozonation in both waters across the turbidity range, with UVT improving in the baseline water by as much as 8 percentage points to 94%.

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Coquitlam 1 direct UV 254% transmittance, pre- and post-ozone, two laboratories 100

CEC DUKE

UV 254%T 1 cm

90 80

Direct %T, Post-ozone

70 Direct %T, Pre-ozone

60 50 0

10

20

30

40

50

Turbidity (NTU)

Fig. 14.9. The pre- and post-ozonated direct UVT data for Coquitlam sample 1, as measured by Clancy Environmental Consultants (CEC) (direct spectrophotometry; Spectronic Genesys 10UV) and Duke University (Cary 300 spectrophotometer). Coquitlam 2 direct UV 254% transmittance, pre- and post-ozone, two laboratories

UV 254%T, 1 cm

100

CEC DUKE

90

Direct %T, Post-ozone 80 70 Direct %T, Pre-ozone 60 50 0

10

20 30 Turbidity (NTU)

40

50

Fig. 14.10. The pre- and post-ozonated direct UVT data for Coquitlam sample 2, as measured by Clancy Environmental Consultants (CEC) (direct spectrophotometry; Spectronic Genesys 10UV) and Duke University (Cary 300 spectrophotometer).

Figures 14.11 and 14.12 compare the integrated sphere (true) pre- and postozone UVT of Coquitlam 1 and 2 waters, respectively. As was noted with direct UVT measurements, ozonation was a beneficial pretreatment for UV. The advantage widens with turbidity in Coquitlam 1, but narrows in Coquitlam 2.

Discussion UV Disinfection of MS2 and Cryptosporidium The sensitivity of MS2 to UV in baseline Coquitlam water was in line with the findings of the numerous studies evaluated in USEPA’s Ultraviolet Disinfection

174

T.M. Hargy et al. Coquitlam 1 integrated sphere (true) UV 254% transmittance, pre- vs post-ozone

UV 254%T, 1 cm

100 90

True %T, Post-ozone

80 70 True %T, Pre-ozone 60 50 0

10

20

30

40

50

Turbidity (NTU)

Fig. 14.11. The integrated sphere (true) pre- and post-ozone UVT of Coquitlam 1 water. Coquitlam 2 integrated sphere (true) UV 254% transmittance, pre- vs post-ozone

UV 254%T, 1 cm

100 True %T, Post-ozone

90 80 True %T, Pre-ozone

70 60 50 0

10

20 30 Turbidity (NTU)

40

50

Fig. 14.12. The integrated sphere (true) pre- and post-ozone UVT of Coquitlam 2 water.

Guidance Manual (UVDGM) (2006). As shown in Table 14.8, all data points generated with baseline Coquitlam water fell well within the recommended limits of the UVDGM. A dose of 83 mJ/cm2 was found to achieve 4-log inactivation of MS2 seeded into the first Coquitlam sample, while 92 mJ/cm2 was required in the second test water. The difference between the curves from the two sample dates is within the range of variability typically seen between collimated beam curves, and is not thought to be indicative of any difference in treatability of the two waters. This interpretation is supported by the results of the test wherein the 4-log inactivation doses (83 and 92 mJ/cm2) were applied to elevated turbidity reactor volumes. In the case of Coquitlam 1, 4-log inactivation was achieved fairly uniformly (range: 3.8–4.1 log) in waters of 0 to 50 NTU. In the second water test, the application of 92 mJ/cm2 uniformly achieved well over 4-log inactivation (range 4.2–4.7).

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Table 14.8. Comparison of MS 2 UV dose response in Coquitlam waters with UVDGM recommendations (USEPA, 2006). UV dose (mJ/cm2) 20 40 60 80 100

Coquitlam 1

Coquitlam 2

UVDGM lower bounds

UVDGM upper bounds

1.2 2.1 3.0 3.7 4.7

1.1 2.0 2.7 3.4 4.3

0.9 1.7 2.4 3.0 3.5

1.5 2.8 4.1 5.2 6.2

This suggests that the 92 mJ/cm2 value determined in the baseline dose response curve for Coquitlam water 2 was high. This is further confirmed by the fact that these higher inactivations achieved in elevated Coquitlam 2 turbidity would be expected by a dose of about 89–100 mJ/cm2 from the first Coquitlam baseline curve. The inactivation data shown in Figs 14.4 and 14.5 indicate that there was no strong negative impact to the inactivation of coliphage MS2 seeded overnight in Coquitlam water at elevated turbidities. That slight shielding did occur is indicated by the results of UV-exposed samples that were put through a dissociation regimen prior to assay. As this process released viable MS2 from the treated matrix, increased counts per ml were realized, decreasing the log inactivation achieved. This exercise, performed on Coquitlam 2 tests from 10 to 50 NTU, demonstrated actual inactivation to be 0.1-log less in all instances. A remaining uncertainty is whether naturally occurring, naturally shielded viral pathogens might pass unaffected through a UV reactor. The low concentrations of indigenous phage and the dominance of mineral, inorganic particles would suggest this is not likely to be a concern for a UV system treating Coquitlam water. Cryptosporidium This study has corroborated the findings of Clancy et al. (2004) and Shin et al. (2001) that approximately 1 mJ/cm2 is necessary for 1-log inactivation of Cryptosporidium. These two references, along with others, form the basis for the USEPA’s inactivation table in UVDGM LT2 (USEPA, 2006), which takes data variability into account and applies safety factors in granting 1-, 2-, 3- and 4-log inactivation credits for validated UV doses of 2.5, 5.8, 12 and 22 mJ/cm2, respectively. Ozone disinfection of MS2 and Giardia The sensitivity to ozone noted here for MS2 masked any impact by Coquitlam particles, as the steepness of the inactivation kinetics exceeded the experimental resolution of CT determination. The application of a CT only 0.1 mg min/l greater than that found to be necessary for 4-log inactivation resulted in at least 5.5-log inactivation across all turbidities.

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The ozone CT table in the Canadian Guidelines for Drinking Water Quality (Health Canada, 2005) requires CTs of 1.9 and 2.9 mg min/l at 5°C and 1°C, respectively, for 3-log ozone inactivation of Giardia cysts. Assuming a linear relationship between temperature and CT, the CT for 3-log inactivation at 3.5°C would be 2.5 mg min/l, which is roughly the effective CT determined here for 3-log inactivation in baseline Coquitlam water. The results of testing in Coquitlam waters 1 and 2 indicate that ozone is effective in Coquitlam waters at 3.5°C and across a range of turbidities. That no apparent impact of turbidity on ozone efficacy could be discerned from the results of this study is supported by the findings of Dow et al. (2006) in their investigation of ozone disinfection of relatively ozone-resistant B. subtilis spores under varying water quality conditions, including turbidity.

Effect of turbidity on ozone and UV UVT measurements indicate that ozonation of Coquitlam water with an applied dose of 2.4 mg/l will be a highly beneficial pretreatment for UV disinfection. Comparisons of direct UVT values measured in pre- and post-ozonated waters across a range of turbidities clearly indicate a strong benefit from ozone, with UVT values increasing by as much as 8 percentage points. True UVT values measured by integrated sphere also noted a significant benefit of ozonation, although this diminished with increasing turbidity in Coquitlam 2 water.

Impact of UVT measurement methods In light of the difference between direct and true UVT values, the results reported above need to be revisited. It was found that 4-log inactivation of MS2 and 3-log inactivation of Cryptosporidium could be achieved in 50 NTU water. The exposure times used were calculated incorporating the direct UV absorbance of the turbidity-adjusted water, a value that would be subject to the light scattering error. Substituting the true UVT for the direct UVT, as measured at the time of the collimated beam tests, the UV doses actually applied can be calculated, and are shown in Table 14.9. For the sake of completeness, this same assessment should be made of the UV dose applied to Cryptosporidium in elevated turbidity. The true applied dose would increase by the same proportion, but as Cryptosporidium is so sensitive to UV, the net effect of using direct UV in determining exposure time necessary for a target dose of 3.0 mJ/cm2 was to increase the actual applied dose to the 50 NTU sample up to 3.2 mJ/cm2.

Summary and Conclusions This project involved the seeding of surrogate or actual pathogenic organisms to low turbidity Coquitlam source water, determining their inactivation kinetics, and

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Table 14.9. UV doses applied to coliphage MS2 corrected for true UV absorbance.

Test water Turbidity (NTU) Baseline 5 10 15 20 50

Coquitlam 1 Targeted: 83 mJ/cm2

Coquitlam 2 Targeted: 92 mJ/cm2

True dose applied (mJ/cm2)

True dose applied (mJ/cm2)

83.0 83.0 83.0 83.0 83.6 85.3

92.0 92.0 92.5 93.3 94.0 98.5

The applied dose values obtained after making the correction for true absorbance (and UVT) are only notably different from the targeted dose for those tested at the highest turbidities.

then applying the disinfectant dose necessary for target inactivation to Coquitlam water that been adjusted with native particulate matter to a range of turbidity levels. In these latter tests, an effort was made to allow the test microorganisms to associate, by passive contact, with Coquitlam particulate matter, although no active flocculation steps were included. In addition to the evaluation of direct impacts to UV and ozone disinfection by turbidity, the ability of ozone to enhance the UV treatability of Coquitlam water was assessed as turbidity was artificially elevated. The results of this project have confirmed that GVRD has selected an effective multi-barrier approach for protecting the health of consumers of unfiltered drinking water. However, caution is recommended if any circumstances arise such that turbidity spikes due to some cause that enriches the organic rather than inorganic fraction of the suspended particles. An algal bloom or terrestrial upset, such as the immediate fallout of a forest fire, might have an effect not tested in this study. The protected nature of the watershed makes a nutrient influx fuelling an algal bloom an unlikely scenario, but a natural or human-induced forest fire is possible. Ozone was shown to be a very effective pretreatment of Coquitlam water for UV, regardless of turbidity. The UV transmittance of Coquitlam water, and thus its UV treatability, was uniformly improved by ozonation.

References Adams, M.H. (1959) Bacteriophages. Interscience Publishers, New York. Bolton, J.R. and Linden, K.G. (2003) Standardization of methods for fluence (UV dose) determination in bench-scale UV experiments. ASCE Journal of Environmental Engineering 129, 209–215. Clancy, J.L., Marshall, M.M., Hargy, T. and Korich, D.G. (2004) Susceptibility of five strains of Cryptosporidium parvum oocysts to UV light. Journal of the American Water Works Association 96, 84–93.

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T.M. Hargy et al. Dow, S.M., Barbeau, B., von Gunten, U., Chandrakanth, M., Amy, G. and Hernandez, M. (2006) The impact of selected water quality parameters on the inactivation of Bacillus subtilis spores by monochloramine and ozone. Water Research 40, 373–382. Health Canada (2005) Canadian Guidelines for Drinking Water Quality. Available at: http://www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/doc_sup-appui/protozoa/ chap_12_e.html. Hoff, J.C., Rice, E.W. and Schaefer, F.W., III (1985) Comparison of animal infectivity and excystation as measures of Giardia muris cyst inactivation by chlorine. Applied and Environmental Microbiology, 50, 1115–1117. Meng, Q.S. and Gerba, C.P. (1996) Comparative inactivation of enteric adenovirus, poliovirus and coliphages by ultraviolet irradiation. Water Research 30, 2665–2668. Shin, G.-A., Linden, K.G., Arrowood, M.J. and Sobsey, M.D. (2001) Low-pressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts. Applied and Environmental Microbiology 67, 3029–3032. USEPA (1992) Consensus Method for Determining Groundwaters under the Direct Influence of Surface Water using Microscopic Particulate Analysis (MPA). EPA 910992029. USEPA Environmental Services Division, Port Orchard, WA. Government Printing Office, Washington, DC. USEPA (2005) Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/ FA. EPA 815-R-05-002. United States Environmental Protection Agency, Office of Water, Washington, DC. USEPA (2006) Ultraviolet Disinfection Guidance Manual for the Final Long Term. 2. Enhanced Surface Water Treatment Rule. Office of Water (4601) EPA 815-R-06-007, United States Environmental Protection Agency, Washington, DC. Wilson, B.R., Roessler, P.F., Van Dellen, E., Abbaszadegan, M. and Gerba, C.P. (1992) Coliphage MS-2 as a UV water disinfection efficacy test surrogate for bacterial and viral pathogens. In: Proceedings of the American Water Works Association Water Quality Technology Conference, Toronto, Canada. American Water Works Association, Denver, CO, USA.

15

Towards Methods for Detecting UV-induced Damage in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

H.V. SMITH1, B.H. AL-ADHAMI1, R.A.B. NICHOLS1, J.R. KUSEL2 AND J. O’GRADY3 1Scottish

Parasite Diagnostic Laboratory, Glasgow, UK; 2Institute of Biomedical and Life Sciences, Glasgow, UK; 3Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, UK

Abstract Water is an important transmission route for cryptosporidiosis, with at least 165 waterborne outbreaks of cryptosporidiosis documented. Cryptosporidium can be controlled in water treatment by physical removal and UV disinfection, and a method that can determine whether individual oocysts in a routine sample exposed to UV irradiation have been disinfected is of benefit as it offers increased confidence to water operators. The major effects of UV radiation on cell membranes are alterations of proteins, particularly protein crosslinking. UV-B radiation progressively inhibits protein synthesis. Specific free radical scavengers protect cells against killing and inhibition of protein synthesis by UV-B. UV light also crosslinks the complementary strands of DNA and causes the formation of single strand breaks and pyrimidine dimers. The major lesions induced are cyclobutyl pyrimidine dimers (CPDs; also known as thymine dimers, TD). UV-induced DNA lesions in living cells and in some microorganisms can be repaired by the enzyme-dependent nucleotide excision repair (NER), also named dark repair, and the light-dependent reaction known as photoreactivation (PHR). Dark repair and PHR enable UV-inactivated microorganisms to recover and may reduce the efficiency of UV inactivation. Cryptosporidium parvum oocysts are inactivated at 3–40 mJ/cm2 using medium- and low-pressure UV light. Cryptosporidium parvum can undertake photoreactivation and dark repair at the genomic level and NER repair genes have been identified in C. parvum and C. hominis. However, UV inactivation of Cryptosporidium oocysts is irreversible, despite the presence of the UV repair genes. © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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H.V. Smith et al. We investigated the following hypotheses for developing a method to demonstrate UV inactivation of Cryptosporidium oocysts: (i) UV disinfection induces the production of reactive oxygen species (ROS); (ii) UV disinfection induces apoptosis; and (iii) UV disinfection causes damage to DNA, which is detectable using fluorogenic DNA reporters. We developed assays for determining UV inactivation of Cryptosporidium oocysts which would remain compatible with, and integrated as much as possible with, existing UK and USA detection methodologies. Our development of suitable methods which can detect UV damage in individual organisms reliably and reproducibly was driven by a search for fluorogenic reporters which could enter and stain UV-killed and damaged Cryptosporidium oocysts. Antioxidants reduce ROS production and the antioxidant glutathione (GSH) plays a significant role in inhibiting the generation of mutagens by ionizing radiation. The presence of GSH, as a putative reporter of UV damage caused by the production of ROS, was investigated in intact, untreated C. parvum oocysts and sporozoites within intact UV-irradiated oocysts (40 mJ/cm2) using the fluorogenic vital dye monochlorobimane (MCB) to detect both GSH levels and activity in oocysts. MCB fluorescence localized GSH in purified, intact, recently excreted and aged C. parvum oocysts, at several nuclear and cytoplasmic sporozoite foci (n = 2–6). We did not demonstrate the function of GSH as an endogenous free radical scavenger in UV-irradiated oocysts, and other free radical scavengers are more active than GSH in UV-treated C. parvum oocysts. MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts. The DNA intercalating dye YO-PRO1 (YP) has been used to determine apoptosis and was used to investigate the role of UV irradiation in inducing programmed cell death/ apoptosis. YP detected DNA damage in UV-treated (40 mJ/cm2) C. parvum oocysts. YP was incorporated into sporozoite DNA of intact, irradiated oocysts (possibly apoptotic) which exhibited no apparent oocyst wall damage. However, control oocysts did not exclude YP entirely. YP is unlikely to provide a reliable estimate of the possible apoptotic changes that can occur in irradiated oocysts. An antibody raised against TDs (α-TD) was used to identify changes induced by UV light in C. parvum sporozoites and oocysts, and its nuclear location was validated by co-localization with DAPI. A freeze-thawing (five cycles) procedure improved α-TD antibody labelling within irradiated C. parvum oocysts. No α-TD localization was seen in non-irradiated oocysts. Both C. parvum and C. hominis oocysts exposed to different doses of UV light (range 10–40 mJ/cm2) demonstrated TD lesions following irradiation. We conclude that an immunofluorescence assay using α-TD antibodies which, for C. parvum, has been validated against a neonatal mouse infectivity assay, is suitable for detecting thymine dimers in air-dried oocysts and air-dried sporozoites of C. parvum and C. hominis oocysts, and that the α-dsDNA antibody is a good candidate for a positive control for the assay.

Introduction Cryptosporidium oocysts are frequent contaminants of water with contributions from infected human and non-human hosts, livestock and agricultural practices, and infected feral and transport hosts (Smith and Rose, 1990, 1998; Smith et al., 1995). Recent genetic analyses have raised doubt about the validity of the current classification of the genus Cryptosporidium and reveal that more than one species of Cryptosporidium can infect susceptible, immunocompetent

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human hosts (Table 15.1). There are 16 ‘valid’ Cryptosporidium species and a further 40+ genotypes, which differ significantly in their molecular signatures but, as yet, have not been ascribed species status (Smith et al., 2007). Seven described Cryptosporidium species (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. suis and C. muris) (Table 15.1) and two undescribed species of Cryptosporidium (cervine and monkey) infect immunocompetent and immunocompromised humans (Xiao et al., 2004; Cacciò et al., 2005), but C. hominis and C. parvum are the most commonly detected (Cacciò et al., 2005). Water is an important transmission route, with at least 165 waterborne outbreaks of cryptosporidiosis documented (Girdwood and Smith, 1999; Fayer et al., 2000; Slifko et al., 2000; Karanis et al., 2007). Cryptosporidium can be controlled in water treatment plants by physical removal and disinfection processes. Physical characteristics, such as size, settling velocities and surface charge affect the behaviour of oocysts in physical treatment processes (Smith et al., 1995). Essentially, oocysts behave as inert, discrete particles in water. Current data indicate that Cryptosporidium is controllable in filtration processes and the performance of individual treatment plants in removing oocysts may be expected to parallel their effectiveness for turbidity control. The major factors that influence a plant’s performance for turbidity control should be expected to affect the efficiency of removal of Cryptosporidium similarly. Cryptosporidium oocysts are insensitive to commonly used disinfectants (chlorine, chlorine dioxide, ozone, etc.), but UV disinfection is effective. Clearly, a method that can determine whether all oocysts in a routine sample exposed to UV irradiation have been disinfected is of benefit, as it offers increased confidence to water operators. Here, we focus on UV disinfection of Cryptosporidium oocysts. Oocysts occur at low densities in water (Smith and Rose, 1990, 1998; Smith et al., 1995) and methods which can detect and determine the UV sensitivity of small numbers of organisms reliably and reproducibly from water concentrates are required. Little can be inferred about the likely impact of oocysts detected in water concentrates on public health without knowing whether they are viable or not. The conventional techniques of animal infectivity and excystation in vitro are not applicable Table 15.1.

Some differences among Cryptosporidium species infecting humans.

Species C. hominis C. parvum C. suis C. felis C. canis C. meleagridis C. muris C. andersoni

Oocyst dimensions (µm)

Site of infection

Major host

4.5 × 5.5 4.5 × 5.5

Small intestine Small intestine

5.05 × 4.41 4.5 × 5.0 4.95 × 4.71 4.5–4.0 × 4.6–5.2 5.5 × 7.4 5.6 × 7.4 (5.0–6.5 × 8.1–6.0)

Small intestine Small intestine Small intestine Intestine Stomach Stomach

Humans Neonatal mammalian livestock, humans Pigs Cats Dogs Turkeys Rodents Cattle

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to the small numbers of organisms found in water concentrates, and much effort has been expended on the development of surrogate techniques that can address, accurately, the viability of individual oocysts. Fluorogenic vital dye methods are based on observing whether specific fluorogenic vital dyes are included into or excluded from Cryptosporidium oocysts as a measure of their viability.

The Usefulness of Existing Fluorogenic Vital Reporters for Determining Disinfection Capability is Compromised A variety of fluorescent dyes have been used to determine Cryptosporidium oocyst viability. In a joint UK/USA initiative to test in vitro (maximized in vitro excystation, fluorogenic vital dyes assay (4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI); DAPI-PI), SYTO9 and SYTO59) and in vivo (neonatal CD-1 mouse infectivity) surrogates (Clancy et al., 2000), high levels of variability were detected with both sets of surrogates for untreated, aged and disinfectant-treated controls. Variability in in vitro surrogates was probably associated with subsampling errors (enumeration of only 100 oocysts from a large oocyst stock), while variability in the in vivo surrogate was attributed to delivery of a standard dose by gavage and the use of outbred animals. In vitro assays overestimated oocyst viability compared with neonatal mouse infectivity, particularly following pulsed UV disinfection. Overall, maximized in vitro excystation and SYTO9 produced closer association with mouse infectivity than DAPI-PI. SYTO59 produced the greatest disparity between in vitro and in vivo assays (Clancy et al., 2000). In the UV treatment studies, SYTO9 and SYTO59 consistently demonstrated higher oocyst viabilities than maximized in vitro excystation and DAPI-PI, even though mouse infectivity failed to demonstrate infectious oocysts. The majority of published data indicate that while fluorogenic vital dyes can be useful predictors of oocyst viability, they also underestimate the degree of oocyst inactivation following chemical disinfection (Bukhari et al., 1999; Clancy et al., 2000; O’Grady and Smith, 2002). Thus, studies evaluating the effectiveness of chemical disinfectants using in vitro viability procedures may have reported higher than necessary disinfection requirements due to the overestimation of viability compared with in vivo procedures.

UV Light Disinfection UV light is classified according to wavelength: UV-A, 315–400 nm; UV-B, 280–315 nm; and UV-C, 100–280 nm; and has been used for drinking water disinfection since the beginning of the 20th century. UV light disinfection does not generate significant disinfection by-products. It does not cause a significant increase in assimilable organic carbon, nor does it convert nitrates to nitrites, nor bromide to bromines or bromates. UV disinfection is relatively insensitive to

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temperature and pH differences. Disadvantages include its unsuitability for water with high levels of suspended solids, turbidity, colour, or soluble organic matter, as they can react with UV radiation, reducing disinfection performance. Turbidity reduces the water penetration of UV radiation and there is no disinfection residual.

Mode of action of UV light disinfection The modes of action of UV light on uni- and multicellular organisms involve oxidative stress resulting from the attack by free radicals of several cellular targets (proteins, DNA and lipids). They are primarily twofold, as follows: 1. The major effects of UV radiation on cell membranes are alterations of proteins, particularly protein crosslinking (detected as high molecular weight protein >200 kDa) (Kochevar, 1990). UV light can also crosslink exposed collagen, making it more resistant to enzymic digestion and less elastic (Lee et al., 2001). UV-B radiation progressively inhibits protein synthesis and kills Staphylococcus aureus. The OH- and 1O2-free radical scavengers protect cells against killing and inhibition of protein synthesis by UV-B, suggesting that such radicals mediate the effects of UV-B on this organism. A similar protective effect using a ferric ion chelator suggests an important role for metallic ions in UV-B lethality (El-Adhami et al., 1994). Additional effects of pulsed UV light damage include increased concentration of eluted proteins, increased cell membrane damage, and structural changes in yeast cells (Takeshita et al., 2003). 2. UV light crosslinks the complementary strands of DNA. It also causes the formation of single strand breaks and pyrimidine dimers (Takeshita et al., 2003). DNA conformation and/or flexibility governs the phenomenon of crosslinking. (GA).(TC) suppresses the crosslink formation in DNA more than any dinucleotide composed of only G and C. (CTAG).(CTAG) promotes crosslinking much more than any other tetranucleotide, including (TATA).(TATA), whereas the closely related (CATG).(CATG) belongs among the tetranucleotides that most suppress the UV-light-induced crosslinks between the complementary strands of DNA (Nejedly et al., 2001a). Nejedly et al. (2001b) also identified that the (ATTTTATA).(TATAAAAT) octamer is a candidate for the hotspot of UV-light-induced crosslinking between the complementary strands of DNA. UV light damages DNA, and the major lesions induced are cyclobutyl pyrimidine dimers (CPDs; also known as thymine dimers, TD) (Mitchell, 1988). The UV-induced DNA base modifications lead to the production of reactive oxygen species (ROS), which can damage cellular elements. The antioxidant glutathione (GSH) plays a significant role in inhibiting the generation of ROS (Fischer-Nielsen et al., 1994). UV-induced DNA lesions in living cells (Roza et al., 1991) and in some microorganisms (Oguma et al., 2001) can be repaired by one or more mechanisms. Such mechanisms include the enzyme-dependent nucleotide excision repair (NER), also named dark repair, and the light-dependent reaction

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known as photoreactivation (PHR). Dark repair and PHR enable UV-inactivated microorganisms to recover and may reduce the efficiency of UV inactivation. Some microorganisms found in water exhibit light and/or dark repair.

UV light and disinfection of Cryptosporidium oocysts The potential of UV irradiation for disinfecting drinking water has been recognized for many years. UV irradiation is effective for killing microorganisms, including bacteria, fungi and oocysts. Various studies have shown that parasitic protozoa are more sensitive to UV irradiation than viruses, but less sensitive than most bacteria (reviewed in Hijnen et al., 2006). Cryptosporidium parvum oocysts can be inactivated using pulsed UV light (Slifko et al., 1999) and low- (Oguma et al., 2001; Shin et al., 2001) and medium-pressure UV (Craik et al., 2001; Rochelle et al., 2004). Pulsed light consists of intense flashes of broad-spectrum white light containing wavelengths from 200 nm (UV) to 1000 nm in the near infrared region (Takeshita et al., 2003). Low-pressure mercury lamps emit peak output at 254 nm, whereas medium-pressure mercury lamps emit radiation at several peaks between 248 and 295 nm. Most studies on C. parvum oocyst inactivation have used low- and medium-pressure lamps (Rochelle et al., 2005). No differences in UV inactivation of C. parvum are apparent using either low- or medium-pressure UV lamps (Craik et al., 2001; Rochelle et al., 2004). Table 15.2 identifies some studies that indicate that UV light is an effective disinfectant for waterborne Cryptosporidium oocysts, particularly when mediumand low-UV-pressure lamps are used. The majority of data on the usefulness of UV disinfection have been accrued using sources which deliver continuous UV irradiation; however, the possibility that pulsed UV light may be more effective for water disinfection has also been put forward (Takeshita et al., 2003).

Assays for Determining UV Inactivation of Cryptosporidium Oocysts A variety of assay methods using a variety of C. parvum oocyst isolates have been used. In one of the earliest published works, C. parvum oocyst inactivation by UV radiation was assessed using maximized in vitro excystation and the fluorogenic vital dyes assay. Oocysts were inactivated (>99%, 2-log10 reduction) using a high dose of UV irradiation (up to 8748 mJ/cm2) (Campbell et al., 1995). Morita et al. (2002) showed that C. parvum oocysts exhibited high resistance to UV irradiation, requiring a dose of 230 mJ/cm2 for a 99% reduction in excystation. Animal infectivity and in vitro cell culture studies indicated that C. parvum oocysts were much more sensitive to UV irradiation than previously identified: the UV dose required for a 99% reduction in mouse infectivity was 1.0 mJ/cm2, whereas a dose of approximately 200 mJ/cm2 was required to achieve 99% reduction by excystation (Morita et al., 2002). Rochelle et al. (2004) showed that 99.9% inactivation (0.001% relative mouse infectivity) was achieved with a UV dose of 7.5 mJ/cm2. Using the CD-1 neonatal mouse infectivity assay, 99.9%

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Table 15.2. UV disinfection of Cryptosporidium oocysts determined by oocyst inactivation. UV pressure

UV dose

Time/mode

Log reduction

Reference

Low

120 mJ/cm2

4 gpm/water device

Drescher et al. (2001)

Not stated

15,000 mW/sec = 15,000 mJ/ cm2

150 min/ bench scale

Medium and low

0.8–119 mJ/cm2

Bench scale

Medium

41 mJ/cm2

Bench scale

Low

20–10 mJ/cm2

Bench scale

5.4 with neonatal mouse infectivity >2 with neonatal mouse infectivity Max. 3.4–4.9 with neonatal mouse infectivity >4 with neonatal mouse infectivity >3 with in vitro infectivity

LorenzoLorenzo et al. (1993) Craik et al. (2001)

Bukhari et al. (1999)

Bukhari and LeChevallier (2003)

inactivation at a UV dosage of 19 mJ/cm2 (Bukhari et al., 1999) and 99.0–99.9% inactivation at 10–25 mJ/cm2 (Craik et al., 2001) could be achieved. Shin et al. (2001) utilized a cell culture infectivity assay combined with epifluorescence detection to determine UV inactivation of C. parvum. They reported 99.9% inactivation at a dosage of 3 mJ/cm2 using a low-pressure UV lamp. Both cell culture and animal infectivity assays gave comparable results with a 99.9% inactivation at a dosage of 3 mJ/cm2. Rochelle et al. (2004) assessed the inactivation of different isolates of C. parvum exposed to low- or medium-pressure UV lamps, using cell culture (monolayers of HCT-8 cells) and a reverse transcriptase polymerase chain reaction (RT-PCR) assay. An average dose of 7.6 mJ/cm2 resulted in 99.9% inactivation of oocysts in five different isolates. Using the same culture-based methods, Johnson et al. (2005) demonstrated that C. hominis display similar levels of infectivity in cell culture and have a similar sensitivity to UV light as C. parvum. Cryptosporidium parvum can undertake photoreactivation and dark repair at the genomic level (Oguma et al., 2001; Morita et al., 2002). In addition, NER repair genes have been identified in C. parvum and C. hominis. However, UV inactivation of Cryptosporidium oocysts is irreversible, despite the presence of the UV repair genes (Rochelle et al., 2004).

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Application to Waterborne Oocysts In order to remain compatible with, and to integrate as much as possible with, existing UK and USA detection methodologies, our development of suitable methods that can detect UV damage in individual organisms reliably and reproducibly was driven by a search for fluorogenic reporters which will enter and stain UV killed and damaged Cryptosporidium oocysts. This was guided by the following hypotheses: 1. UV disinfection induces the production of reactive oxygen species (ROS). 2. UV disinfection induces apoptosis. 3. UV disinfection causes damage to DNA, which is detectable using fluorogenic DNA reporters.

Hypothesis 1: UV induces the production of reactive oxygen species (ROS) Ultraviolet light A (UV-A) radiation induces the production of ROS in cells, which can damage cellular elements. Oxidative modifications to DNA nucleotides (e.g. 8-hydroxyguanine (8-oxoG)) are mutagenic (Cheng et al., 1992). Antioxidants reduce ROS production and GSH plays a significant role in inhibiting the generation of 8-oxoG by ionizing radiation (Fischer-Nielsen et al., 1994). GSH reacts with various ROS and is a cofactor for the H202-removing enzyme, glutathione peroxidase. In cultured mammalian cells, GSH depletion and thermal stress increase endogenous oxidative damage, but the addition of thiols to the medium does not reduce the level of oxidative damage caused by GSH depletion and thermal stress (Will et al., 1999). GSH is a low-molecular-weight tripeptide (gamma-glutamylcysteinylglycine) which is synthesized intracellularly. It plays a critical role in the detoxification of several drugs and xenobiotics (Meister and Anderson, 1983) and in cellular defence against agents that cause oxidative stress (Anderson, 1998). A very well-established technique to measure GSH is to add the cell permeant, fluorogenic vital dye monochlorobimane (MCB) to detect both GSH levels and activity in cells. MCB does not fluoresce, but on reacting with GSH yields GSH-bimane adducts which fluoresce. The enzyme glutathioneS-transferase (GST) exclusively mediates the intracellular conjugation of GSH and MCB. The rate of conjugation between GSH and MCB (which produces the fluorescence signal) is dependent on the abundance of GST (Haugland, 2005). As a putative reporter of UV damage caused by the production of ROS, we investigated the presence of the antioxidant GSH in C. parvum sporozoites in intact, untreated oocysts and in sporozoites within intact UV-irradiated oocysts (40 mJ/cm2). Purified C. parvum oocysts were purchased from Bunch Grass Farm (Idaho, USA; Iowa isolate BGF06-1, 10 days old) and stored between 4°C and 8°C until used. The percentage viability and excystation rate of this isolate using our optimized, fluorogenic vital dyes (DAPI-PI; Campbell et al., 1992) and our maximized in vitro excystation protocols (Robertson et al., 1993) were 95.2 ± 2.3 and 96.0 ± 1.0, respectively.

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UV irradiation was delivered using a high-intensity, low-pressure UV lamp with an output at 254 nm. The procedure was adapted from the method described by Rochelle et al. (2004). Short-wave UV irradiation from a UVGL-58 Mineralight lamp was utilized. The intensity of the UV light, measured using a digital UVX radiometer, was (on average) 350 µW/cm2 at 254 nm. A rig was set up, and 10 cm below the lamp a position was marked where the UV intensity was maximal (350 µW/cm2). The UV dose was then determined from: UV dose (mJ/cm2) = Irradiance (mW/cm2) × Exposure time (s) In all experiments, 1 × 106 oocysts were suspended in 5 ml of Hanks’ balanced salt solution (HBSS). Samples were placed in Petri dishes (diameter 36 mm) which were constantly mixed using a magnetic stirrer during exposure to UV light. To achieve different UV dosages (mJ/cm2), oocysts were exposed to UV light for varying times at a constant distance (10 cm) from the constant intensity UV source. For each experiment, control oocysts were kept under the same conditions without irradiation. Statistical analysis was performed using analysis of variance (ANOVA) with P < 0.05 as the criterion for significance using MINITAB version 11 software. MCB fluorescence localized GSH in intact oocysts and the number of distinctly labelled foci in intact oocysts varied between 2 and 6 both in recently excreted (10 days old) and aged (6 months old) oocysts (Al-Adhami et al., 2006). MCB-labelled excysted sporozoites retained the fluorogenic dye at several intrasporozoite foci. GSH distribution in sporozoites was granular in the apical and posterior (nuclear) regions (Al-Adhami et al., 2006). We used buthionine sulphoximine (BSO), a potent and specific inhibitor of GSH (Meister and Anderson, 1983), to determine whether GSH is synthesized in BSO-treated oocysts, by labelling treated oocysts with MCB. When oocysts were depleted of GSH using BSO for 24 h at room temperature (RT), a significant decrease in fluorescence (~50%) was observed, indicating that MCB binds sporozoite GSH. We enumerated oocysts exposed to 10, 20 or 40 mJ/cm2 doses of UV light for MCB inclusion or exclusion, and quantified their fluorescence (using ANALYSIS software for scientific imaging and calibrated image measurements; Olympus, UK). The percentage inhibition of irradiated versus control oocysts was not significantly different (irradiated group mean ± SD 19.8 ± 10.5, 17.2 ± 12.8, 20.6 ± 8.0 at 10, 20 or 40 mJ/cm2, respectively; control group mean ± SD 15.5 ± 9.6, P > 0.05). No significant differences in fluorescence intensity or distribution of MCB occurred in irradiated or control oocysts (irradiated group mean ± SD mJ/cm2 = 152.3 ± 15.9, 20 mJ/cm2 = 149.6 ± 12.2, 40 mJ/ cm2 = 160.2 ± 18.5; control group mean ± SD 165.1 ± 20.3, P > 0.05). Oocysts subjected to GSH depletion showed significant decrease in GSH-bimane adducts (mean ± SD 62.3 ± 13.4) when compared to the control group (mean ± SD 165.1 ± 20.3, P = 0) as demonstrated by fluorescence quantitization.

Determining oocyst viability using MCB and propidium iodide staining The procedure developed was similar to that used in the fluorogenic vital dyes (DAPI-PI) assay of Campbell et al. (1992). The proportions of MCB+

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PI−, MCB− PI+, MCB− PI− and empty oocysts were quantified by enumerating 100 oocysts in triplicate samples. Oocysts were considered viable if they did not include PI but were stained with MCB (MCB+ PI−). Also, intact oocysts which did not include MCB or PI (MCB− PI−) but contained sporozoites under differential interference contrast (DIC) microscopy were considered viable. MCB incorporation correlated well with DAPI+ PI− oocyst staining. The results of DAPI-PI and MCB-PI staining were not significantly different when labelled oocysts were enumerated (mean ± SD 84.7 ± 4.2 versus 73.3 ± 8.7, P = 0.05, respectively). Results using the maximized in vitro excystation assay were not significantly different from DAPI-PI staining (mean ± SD 89.7 ± 2.1 versus 84.7 ± 4.2, P = 0.1) but were significantly different from MCB-PI staining (mean ± SD 89.7 ± 2.1 versus 73.3 ± 8.7, P = 0.01). These data, based on the MCB labelling of intact C. parvum oocysts, identify the presence of glutathione both in nuclear and cytoplasmic foci of sporozoites, which can be specifically depleted by BSO. Its function as an endogenous free radical scavenger in UV-irradiated oocysts was not demonstrated. Thus it is likely that other free radical scavengers are more active than GSH in UV-treated C. parvum (e.g. cysteine, ascorbic acid). MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts.

Hypothesis 2: UV disinfection induces apoptosis The role of UV irradiation in inducing programmed cell death/apoptosis was investigated using DNA intercalating dyes. Currently, there are no reports available regarding UV-irradiation-induced apoptotic changes in Cryptosporidium oocysts. YO-PRO1 (YP) binds strongly to nucleic acids and has been used to detect apoptosis in mammalian cells without interfering with cell viability (Idziorek et al., 1995). Apoptosis is a highly regulated physiological process that is linked to pathological events such as oxidative stress in which the reactive oxygen species (ROS) play a key role in the initiation of the process (Plantin-Carrenard et al., 2003). The early stages of apoptosis are characterized by chromatin condensation, nuclear fragmentation and mitochondrial clustering, but with no loss of membrane integrity (Cohen, 1993). The plasma membrane becomes slightly permeable during apoptosis, allowing the uptake of YP but not PI, the impermeant dead-cell stain (Gilbert and Knox, 1997). Apoptosis is accompanied by the activation of an endonuclease enzyme that cleaves DNA initially into large fragments (50–300 kb) which yield internucleosomic fragments of 180–200 base pair multimers (Cohen et al., 1994). YP nucleic acid stain forms the basis of an important assay for apoptotic cells and is compatible with both epifluorescence microscopy and flow cytometry. Selective uptake of YP by apoptotic cells of a dexamethasone-treated population of thymocytes, an irradiated peripheral blood mononuclear cell population, and a growth-factor-depleted tumour B cell line was confirmed by cell sorting (Idziorek et al., 1995). Apoptotic cells take up YP, while viable cells exclude it (Haugland, 2005).

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We focused our investigations on the usefulness of YP to detect DNA damage in UV-treated (40 mJ/cm2) C. parvum oocysts. Three nucleic acid stains were used to identify viable (DAPI, blue nuclei; in the absence of PI, red nuclei) oocysts, apoptotic (YP, green nuclei) oocysts and dead (PI, red nuclei) oocysts. UV disinfection procedures were as previously described. Epifluorescence microscopy, flow cytometry and Nomarski differential interference contrast (DIC) microscopy were used to assess outcomes. Oocysts exposed to UV disinfection, those subjected to apoptosis-inducing drug treatment changes, and controls were analysed using this three-colour assay. DAPI (indicator of viability in the absence of PI staining) was taken up by ~95% of the untreated population, while YP stained 35% of the same population. PI was excluded from all DAPI+ YP+ oocysts. PI stained the remaining 5% of the population. This result was consistent with that obtained with the DAPI-PI assay. YP was used in combination with PI to assess the population structure of irradiated, drug-treated and untreated oocysts by epifluorescence microscopy and fluorescence activated cell sorting (FACS). YP was incorporated into sporozoite DNA of intact, irradiated oocysts (possibly apoptotic) which exhibited no oocyst wall damage as determined by DIC microscopy. Apoptosis was induced in oocysts treated with dexamethasone (1 mM) or etoposide (10 µM) prior to labelling with fluorescent probes. Drug treatment induced increased YP signals compared with controls. However, control oocysts did not exclude YP entirely, showing low-level staining. DIC microscopy revealed alterations in the morphology of drug-treated oocysts, compared with irradiated and control (untreated) oocysts. When oocysts were identified as apoptotic (YP+ DAPI+ PI−), no significant differences were detected between irradiated and drug-treated oocysts, compared with controls (Table 15.3). Unlike our epifluorescence microscopy data, FACS revealed that 71.9% of irradiated and 33.7% of drug-treated populations were YP-positive compared with 4.2% of the control population. Despite the increase in YP-positive oocysts by FACS, it is unlikely that YP can provide a reliable estimate of the possible apoptotic changes that can occur in irradiated oocysts or sporozoites, for two main reasons: (i) the staining pattern resulting from the simultaneous use of these Table 15.3. Epifluorescence microscopy and flow cytometric analyses of recently excreted C. parvum oocysts subjected to different treatments, then stained with YP, DAPI and PI for epifluorescence microscopy or YP and PI for flow cytometry. % of labelled oocysts Assay Epifluorescence microscopy (YP+DAPI+PI−) (YP−DAPI+PI−) (YP−DAPI−PI+) Flow cytometry

Irradiated

Etoposide

Dexamethasone

Control

42.2 54.0 3.8 71.9

35.6 54.1 10.3 ND

ND ND ND 33.7

39.3 57.3 3.4 4.2

ND: Not determined. Note: YP stains apoptotic oocysts, PI stains dead oocysts.

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three dyes makes it impossible to distinguish normal from apoptotic oocysts by standard sets of filters used in epifluorescence microscopy; and (ii) differences in the intensity and the rapid quenching of YP make it very difficult to count and photograph YP-labelled oocysts.

Hypothesis 3: UV disinfection causes damage to DNA, which is detectable using fluorogenic DNA reporters The major DNA lesion caused by UV, the cyclobutyl pyrimidine dimer (TD), is responsible for UV-induced cytotoxicity and mutagenicity in living cells and microorganisms (Mitchell, 1988), and the formation of such lesions in genomic DNA inhibits normal replication and transcription of DNA and results in the inactivation of cells. Two known antibody reporters of nuclear UV damage were investigated. Two different antibodies raised against nucleic acids (α-double sDNA (α-dsDNA) and α-TD) were used to identify changes induced by UV light in C. parvum sporozoites and oocysts. Sporozoite nuclei were also stained with DAPI to validate the co-localization of α-dsDNA and α-TD (Al-Adhami et al., 2007). An improved α-TD antibody labelling procedure within irradiated C. parvum oocysts was established following freeze-thawing (five cycles), based on that of Nichols and Smith (2004). No α-TD localization was seen in non-irradiated oocysts. α-dsDNA antibody bound to the nuclei of both irradiated and non-irradiated sporozoites. Both C. parvum and C. hominis oocysts exposed to different doses of UV light (range 4–40 mJ/cm2) were tested using a standardized set of parameters: oocysts dried onto slides prior to freeze-thawing (five cycles), then fixed in methanol and labelled with either α-TD-Ab, a commercially available, fluorescein-labelled, monoclonal antibody reactive with surface exposed epitopes on Cryptosporidium oocysts (FITC-C-mAb) and DAPI stain or α-TD-Ab and DAPI. Our data indicate that UV irradiation at doses ranging from 10 to 40 mJ/cm2 can be detected using α-TD-Ab and DAPI (Al-Adhami et al., 2007). While the combination of α-TD-Ab, FITC-C-mAb and DAPI produced positive outcomes only with high levels of UV irradiation (40 mJ/cm2), if we replaced FITC-C-mAb with Texas Red (TR)-C-mAb and used the combination of α-TD-Ab, TR-C-mAb and DAPI, DNA damage to C. parvum and C. hominis sporozoites within intact oocysts could be detected at a lower limit of 10 mJ/cm2, but not at 4 mJ/cm2. Currently, the combination of α-TD-Ab, TR-C-mAb and DAPI can be used to detect damage in nuclei of oocysts of C. parvum and C. hominis exposed to UV light (range 10–40 mJ/cm2). Validation by comparison with infectivity in neonatal CD-1 mice All procedures and manipulations performed were as described by Korich et al. (2000), and the experimental design is presented in Table 15.4. Mice infected with untreated oocysts (standard curve, Table 15.4) showed varying levels of infection. In the standard curve group, the percentage infection ranged from 5.2% in mice infected with 30 oocysts to 80% in mice infected with 300 oocysts.

Towards Methods for Detecting UV-induced Damage Table 15.4.

191

Experimental design of animal infectivity experiment.

UV dose (mJ/cm2) 10 10 20 20 None (control) None (control) None (control)

No. of animals 20 20 20 20 20 20 20

Dose (No. of oocysts/10 µl HBSS) 300/10 µl (low) 3000/10 µl (high) 300/10 µl (low) 3000/10 µl (high) 30/10 µl (low) 150/10 µl (medium) 300/10 µl (high)

UV-irradiated oocysts of C. parvum at 10 and 20 mJ/cm2 (Table 15.4) failed to cause infection in neonatal CD-1 mice, despite the fact that large doses of oocysts (up to 3000 per os, which is greater than 44 times the ID50 for this strain of mouse; Korich et al., 2000) were given to the animals. We conclude that an immunofluorescence assay using α-TD antibodies, which, for C. parvum, has been validated against a neonatal mouse infectivity assay, is suitable for detecting thymine dimers in air-dried oocysts and air-dried sporozoites of C. parvum and C. hominis oocysts and that the α-dsDNA antibody is a good candidate for a positive control for the assay.

Genotyping Cryptosporidium Oocysts by PCR Following UV Disinfection The species of waterborne Cryptosporidium oocysts recovered from routine Cryptosporidium monitoring of water sources can be determined by molecular methods (see Smith et al., Chapter 17, this volume). One pertinent issue that arises is whether oocysts which have been disinfected by UV light can be amplified by PCR, as UV disinfection damages DNA and the apoptopic cascade activates an endonuclease enzyme that cleaves DNA initially into large fragments (50–300 kb), which yield internucleosomic fragments of 180–200 base pair multimers (Cohen et al., 1994). Statements to the effect that UV light at up to 40 mJ/ cm2 damages DNA to such an extent that the molecular tools used to determine the species of small numbers of oocysts (nested 18S rRNA loci) cannot be used, have been made on numerous occasions. UV irradiation damage and repair is detectable by PCR in mammalian cultured cells and assay sensitivity is dependent on the size of the DNA fragment amplified. Long PCRs (6–24 kb) are required to ensure sensitive and unequivocal detection of DNA-induced lesions. UV irradiation induces DNA helix distortion, caused by the formation of pyrimidine dimmers, and DNA synthesis is impaired both in vivo and in vitro. Wang et al. (2003) demonstrated damage and repair of the p53 gene in human cells by a multiplex long quantitative PCR, designed to co-amply a 7 kb fragment of the gene and a 500 bp fragment control to increase the reliability of the assay. The lesion frequency detected in this gene

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was 0.63 lesions/10 kb/10 J/m2. In most applications, the UV dosage required to induce detectable lesions by PCR inhibition is much higher than the dosage used for Cryptosporidium inactivation; furthermore, the PCR fragment size is much larger than is used in most PCR applications. However, less PCR product was observed after amplifying the 1.7 kb 16S rDNA gene from cyanobacteria after UV-B radiation-induced damage (Kumar et al., 2004). Genomic DNA, extracted from bacteria exposed to 2.5 W/m2 of irradiation (up to 90,000 mw/s/ cm2 = 90,000 mJ/cm2), was tested by random amplification of polymorphic DNA (RAPD) analysis and direct PCR, and a marked decrease in amplification products, which was directly proportional to exposure time, occurred with both assays. Most PCR applications on the Cryptosporidium 18S rRNA gene used for genotyping oocysts from water do not exceed ~1000-bp fragment length for the first PCR amplification and vary from ~400 to 800 bp for the secondary amplification of nested assays. By extrapolation, it is unlikely that UV irradiation of water will have an adverse effect on the PCRs used for detecting oocysts; however, experimental confirmation of this hypothesis must be sought.

Conclusions Water is an important transmission route for cryptosporidiosis, with at least 165 waterborne outbreaks documented. Cryptosporidium can be controlled in water treatment by physical removal, yet Cryptosporidium oocysts are insensitive to commonly used disinfectants, with the exception of UV. Clearly, a method that can determine whether the small numbers of oocysts found in routine samples exposed to UV irradiation have been disinfected is of benefit, as it offers increased confidence of the success of UV disinfection to water operators. Neonatal animal infectivity, in vitro infectivity, excystation in vitro and current fluorogenic vital dye methods cannot be used to address, accurately, the viability of individual UV disinfected oocysts. UV light is classified according to wavelength: UV-A, 315–400 nm; UV-B, 280–315 nm; and UV-C, 100–280 nm; has been used for drinking water disinfection since the beginning of the 20th century, and is relatively insensitive to temperature and pH differences. Turbidity reduces its water penetration radiation and there is no disinfection residual. The modes of action of UV light on uni- and multicellular organisms involve oxidative stress resulting from the attack by free radicals of several cellular targets. The major effects of UV radiation on cell membranes are alterations of proteins, particularly protein crosslinking. UV-B radiation progressively inhibits protein synthesis. Specific free radical scavengers protect cells against killing and inhibition of protein synthesis by UV-B. Additional effects of pulsed UV light damage include increased concentration of eluted proteins, increased cell membrane damage and structural changes in yeast cells. UV light also crosslinks the complementary strands of DNA and causes the formation of single strand breaks and pyrimidine dimers. The major lesions induced are cyclobutyl pyrimidine dimers (CPDs; also known as thymine dimers, TD).

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UV-induced DNA lesions in living cells and in some microorganisms can be repaired by enzyme-dependent nucleotide excision repair (NER), also named dark repair, and the light-dependent reaction known as photoreactivation (PHR). Dark repair and PHR enable UV-inactivated microorganisms to recover and may reduce the efficiency of UV inactivation. C. parvum oocysts are inactivated at 3–40 mJ/cm2 using medium- and low-pressure UV light. C. parvum can undertake photoreactivation and dark repair at the genomic level, and NER repair genes have been identified in C. parvum and C. hominis. However, UV-inactivation of Cryptosporidium oocysts is irreversible, despite the presence of the UV repair genes. We developed assays for determining UV inactivation of Cryptosporidium oocysts which would remain compatible with, and integrated as much as possible with, existing UK and USA detection methodologies. Our development of suitable methods which can detect UV damage in individual organisms reliably and reproducibly was driven by a search for fluorogenic reporters which could enter and stain UV-killed and damaged Cryptosporidium oocysts, and was guided by the following hypotheses: 1. UV disinfection induces the production of reactive oxygen species (ROS). Antioxidants reduce ROS production and the antioxidant glutathione (GSH) plays a significant role in inhibiting the generation of mutagens by ionizing radiation. We investigated the presence of GSH, as a putative reporter of UV damage caused by the production of ROS, in intact, untreated C. parvum oocysts and sporozoites within intact UV-irradiated oocysts (40 mJ/cm2), using MCB to detect both GSH levels and activity in cells. In purified, intact, recently excreted and aged C. parvum oocysts, MCB fluorescence localized GSH in intact oocysts at several nuclear and cytoplasmic sporozoite foci (n = 2–6). The function of GSH as an endogenous free radical scavenger in UV-irradiated oocysts was not demonstrated. Other free radical scavengers are more active than GSH in UV-treated C. parvum oocysts, and MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts. 2. UV disinfection induces apoptosis. The DNA intercalating dye YO-PRO1 (YP) was chosen to investigate the role of UV irradiation in inducing programmed cell death/apoptosis. YP binds strongly to nucleic acids and has been used to detect apoptosis in mammalian cells without interfering with cell viability. We focused our investigations on the usefulness of YP to detect DNA damage in UVtreated (40 mJ/cm2) C. parvum oocysts. YP was incorporated into sporozoite DNA of intact, irradiated oocysts (possibly apoptotic) which exhibited no apparent oocyst wall damage. Dexamethasone- or etoposide-induced apoptosis was localized at higher levels, compared with controls; however, control oocysts did not exclude YP entirely. No significant differences were detected between irradiated and drug-treated oocysts, compared with controls by fluorescence microscopy, but differences were apparent by FACS. Despite the increase in YP-positive oocysts by FACS, YP is unlikely to provide a reliable estimate of the possible apoptotic changes that can occur in irradiated oocysts or sporozoites. 3. UV disinfection causes damage to DNA, which is detectable using fluorogenic DNA reporters. The major DNA lesion caused by UV, the cyclobutyl

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pyrimidine dimer (TD), is responsible for UV-induced cytotoxicity and mutagenicity. It inhibits normal replication and transcription of DNA and results in the inactivation of cells. We used an antibody raised against TDs (α-TD) to identify changes induced by UV light in C. parvum oocysts, and validated its nuclear location by co-localization with the nuclear fluorogen, DAPI. To improve α-TD antibody labelling within irradiated C. parvum oocysts, a freeze-thawing (five cycles) procedure was developed. No α-TD localization was seen in non-irradiated oocysts. Replacing FITC-C-mAb with Texas Red (TR)-C-mAb and using the combination of α-TD-Ab, TR-C-mAb and DAPI, DNA damage to C. parvum and C. hominis sporozoites within intact oocysts could be detected at a lower limit of 10 mJ/cm2, but not at 4 mJ/cm2. Currently, the combination of α-TD-Ab, TR-CmAb and DAPI can be used to detect damage in nuclei of oocysts of C. parvum and C. hominis exposed to UV light (range 10–40 mJ/cm2). We conclude that an immunofluorescence assay using α-TD antibodies, which, for C. parvum, has been validated against a neonatal mouse infectivity assay, is suitable for detecting thymine dimers in air-dried oocysts and air-dried sporozoites of C. parvum and C. hominis oocysts, and that the α-dsDNA antibody is a good candidate for a positive control for the assay.

Acknowledgements This work was funded by the Environmental and Rural Affairs Department, Agricultural and Biological Research Group, Scottish Executive, Scotland, UK. We thank Dr D. Reid, Drinking Water Quality Unit, Scottish Executive, Edinburgh, for managing the project.

References Al-Adhami, B.H., Nichols, R.A.B., Kusel, J.R., O’Grady, J. and Smith, H.V. (2006) Cryptosporidium parvum sporozoites contain glutathione. Parasitology 133, 555–563. Al-Adhami, B.H., Nichols, R.A.B., Kusel, J.R., O’Grady, J. and Smith, H.V. (2007) Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy. Applied and Environmental Microbiology 73, 947–955. Anderson, M.E. (1998) Glutathione: an overview of biosynthesis and modulation. Chemico-Biological Interactions 24, 1–14. Bukhari, Z. and LeChevallier, M. (2003) Assessing UV reactor performance for treatment of finished water. Water Science and Technology 47, 179–184. Bukhari, Z., Hargy, T.M., Bolton, J.R., Dussert, B. and Clancy, J.L. (1999) Mediumpressure UV for oocyst inactivation. Journal of the American Water Works Association 91, 86–94. Cacciò, S.M., Thompson, R.C.A., McLauchlin, J. and Smith, H.V. (2005) Unravelling Cryptosporidium and Giardia epidemiology. Trends in Parasitology 21, 430–437. Campbell, A.T., Robertson, L.J. and Smith, H.V. (1992) Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes. Applied and Experimental Microbiology 58, 3488–3493.

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Campbell, A.T., Robertson, L.J., Snowball, M.R. and Smith, H.V. (1995) Inactivation of oocysts of Cryptosporidium parvum by ultraviolet irradiation. Water Research 29, 2583–2586. Cheng, K.C., Cahill, D.S., Kasai, H., Nishimura, S. and Leob, L.A. (1992) 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G-T and A-T substitution. Journal of Biological Chemistry 267, 166–172. Clancy, J.L., Bukhari, Z., McCuin, R., Clancy, T.P., Marshall, M.M., Korich, D.G., Fricker, C.R., Sykes, N., Smith, H.V., O’Grady, J.E., Rosen, J.P., Sobrinho, J. and Schaefer, F.W., III (2000) Cryptosporidium: Viability and Infectivity Methods. American Water Works Association Research Foundation / American Water Works Association / UK Drinking Water Inspectorate, pp.137 Cohen, G.M., Sun, X.M., Fearnhead, H., MacFarlane, M., Brown, D.G. Snowden, R.T. and Dinsdale, D. (1994) Formation of large weight fragments of DNA is a key committed step of apoptosis in thymocytes. Journal of Immunology 153, 507–516. Cohen, J.J. (1993) Apoptosis. Immunology Today 14, 126–130. Craik, S.A., Weldon, D., Finch, G.R., Bolton, J.R. and Belosevic, M. (2001) Inactivation of Cryptosporidium parvum oocysts using medium- and low-pressure ultraviolet radiation. Water Research 35, 1387–1398. Drescher, A.C., Greene, D.M. and Gadgil, A.J. (2001) Cryptosporidium inactivation by low-pressure UV in a water disinfection device. Journal of Environmental Health 64, 31–35. El-Adhami, W., Daly, S. and Stewart, P.R. (1994) Biochemical studies on the lethal effects of solar and artificial ultraviolet radiation on Staphylococcus aureus. Archives of Microbiology 161, 82–87. Fayer, R., Morgan, U. and Upton, S.J. (2000) Epidemiology of Cryptosporidium: transmission, detection and identification. International Journal for Parasitology 30, 1305–1322. Fischer-Nielsen, A., Jeding, I.B. and Loft, S. (1994) Radiation-induced formation of 8-hydroxy-2-deoxyguanosine and its prevention by scavengers. Carcinogenesis 15, 1609–1612. Gilbert, M. and Knox, S. (1997) Influence of Bcl-2 overexpression on Na+/K+-ATPase pump activity: correlation with radiation-induced programmed cell death. Journal of Cellular Physiology 171, 299–304. Girdwood, R.W.A. and Smith, H.V. (1999) Cryptosporidium. In: Robinson, R., Batt, C. and Patel, P. (eds) Encyclopaedia of Food Microbiology, Vol. 1. Academic Press, London and New York, pp. 487–497. Haugland, R.P. (2005) Probes for cell adhesion, chemotaxis, multidrug resistance and glutathione. In: Spence, M.T.Z. (ed.) The Handbook: A Guide to Fluorescent Probes and Labelling Technologies. Molecular Probes Inc., Oregon, USA, pp. 767–776. Hijnen, W.A.M., Beerendonk, E.F. and Medema, G.J. (2006) Inactivation credit of UV irradiation for viruses, bacteria and protozoan (oo)cysts in water: a review. Water Research 40, 3–22. Idziorek, T., Estaquier, J., De Bels, F. and Ameisen, J.-C. (1995) YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. Journal of Immunological Methods 185, 249–258. Johnson, A.M., Linden, K., Ciociola, K.M., De Leon, R., Widmer, G. and Rochelle, P.A. (2005) UV inactivation of Cryptosporidium hominis as measured in cell culture. Applied and Environmental Microbiology 71, 2800–2802. Karanis, P., Kourenti, C. and Smith, H. (2007) Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. Journal of Water Health 5, 1–38.

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H.V. Smith et al. Kochevar, I.E. (1990) UV-induced protein alterations and lipid oxidation in erythrocyte membranes. Photochemistry and Photobiology 52, 795–800. Korich, D.G., Marshall, M.M., Smith, H.V., O’Grady, J., Bukhari, Z., Fricker, C.R., Rosen, J.P. and Clancy, J.L. (2000) Inter-laboratory comparison of the CD-1 neonatal mouse logistic dose-response model for Cryptosporidium parvum oocysts. Journal of Eukaryotic Microbiology 47, 294–298. Kumar, A., Tyagi, M.B. and Jha, P.N. (2004) Evidences showing ultraviolet-B radiation-induced damage of DNA in cyanobacteria and its detection by PCR assay. Biochemical and Biophysical Research Communications, 318, 1025–1030. Lee, J.E., Park, J.C., Hwang, Y.S., Kim, J.K., Kim, J.G. and Sub, H. (2001) Characterization of UV-irradiated dense/porous collagen membranes: morphology, enzymatic degradation, and mechanical properties. Yonsei Medical Journal 42, 172–179. Lorenzo-Lorenzo, M.J., Ares-Mazas, M.E., Villacorta-Martinez de Maturana, I. and DuranOreiro, D. (1993) Effect of ultraviolet disinfection of drinking water on the viability of Cryptosporidium parvum oocysts. Journal of Parasitology 79, 67–70. Meister, A. and Anderson, M.E. (1983) Glutathione. Annual Review of Biochemistry 52, 711–760. Mitchell, D.L. (1988) The relative cytotoxicity of (6-4) photoproducts and cyclobutane dimers in mammalian cells. Photochemistry and Photobiology 48, 51–57. Morita, S., Namikoshi, A., Hirata, T., Oguma, K., Katayama, H., Ohgaki, S., Motoyama, N. and Fujiwara, M. (2002) Efficacy of UV irradiation in inactivating Cryptosporidium parvum oocysts. Applied and Environmental Microbiology 68, 5387–5393. Nejedly, K., Kittner, R., Pospisilova, S. and Kypr, J. (2001a) Crosslinking of the complementary strands of DNA by UV light: dependence on the oligonucleotide composition of the UV irradiated DNA. Biochimica et Biophysica Acta 1517, 365–75. Nejedly, K., Kittner, R. and Kypr, J. (2001b) Genomic DNA regions whose complementary strands are prone to UV light-induced crosslinking. Archives of Biochemistry and Biophysics 388, 216–224. Nichols, R.A.B. and Smith, H.V. (2004) Optimisation of DNA extraction and molecular detection of Cryptosporidium parvum oocysts in natural mineral water sources. Journal of Food Protection 67, 524–532. O’Grady, J.E. and Smith, H.V. (2002) Methods for determining the viability and infectivity of Cryptosporidium oocysts and Giardia cysts. In: Ziglio, G. and Palumbo, F. (eds) Detection Methods for Algae, Protozoa and Helminths. John Wiley and Sons, Chichester, UK, pp. 193–220. Oguma, K., Katayama, H., Mitani, H., Morita, S., Hirata, T. and Ohgaki, S. (2001) Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair. Applied and Environmental Microbiology 67, 4630–4637. Plantin-Carrenard, E., Bringuier, A., Derappe, C., Pichon, J., Guillot, R., Bernard, M., Foglietti, M.J., Feldmann, G., Aubery, M. and Braut-Boucher, F. (2003) A fluorescence microplate assay using Yopro-1 to measure apoptosis: application to HL60 cells subjected to oxidative stress. Cell Biology and Toxicology 19, 121–133. Robertson, L.J., Campbell, A.T. and Smith, H.V. (1993) In vitro excystation of Cryptosporidium parvum. Parasitology 106, 13–29. Rochelle, P.A., Fallar, D., Marshall, M.M., Montelone, B.A., Upton, S.J. and Woods, K. (2004) Irreversible UV inactivation of Cryptosporidium spp. despite the presence of repair genes. Journal of Eukaryotic Microbiology 51, 553–562. Rochelle, P.A., Upton, S.J., Montelone, B.A. and Woods, K. (2005) The response of Cryptosporidium parvum to UV light. Trends in Parasitology 21, 81–87.

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Roza, L., De Gruijl, F.R., Bergen Henegouwen, J.B.A., Guikers, K., Van Weelden, H., Van Der Schans, G.P. and Baan, R.A. (1991) Detection of photorepair of UV-induced thymine dimers in human epidermis by immunofluorescence microscopy. Journal of Investigative Dermatology 96, 903–907. Shin, G.-A., Linden, K.G., Arrowood, M.J. and Sobsey, M.D. (2001) Low-pressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts. Applied and Environmental Microbiology 67, 3029–3032. Slifko, T.R., Huffman, D.E. and Rose, J.B. (1999) A most-probable-number assay for enumeration of infectious Cryptosporidium parvum oocysts. Applied and Environmental Microbiology 65, 3936–3941. Slifko, T.R., Smith, H.V. and Rose, J.B. (2000) Emerging parasite zoonoses associated with food and water. International Journal for Parasitology 30, 1379–1393. Smith, H.V. and Rose, J.B. (1990) Waterborne cryptosporidiosis. Parasitology Today 6, 8–12. Smith, H.V. and Rose, J.B. (1998).Waterborne cryptosporidiosis: current status. Parasitology Today 14, 14–22. Smith, H.V., Robertson, L.J. and Ongerth, J.E. (1995) Cryptosporidiosis and giardiasis: the impact of waterborne transmission. Aqua – Journal of Water Supply: Research and Technology 44, 258–274. Smith, H.V., Cacciò, S.M., Cook, N., Nichols, R.A.B. and Tait, A. (2007) Cryptosporidium and Giardia as foodborne zoonoses. Veterinary Parasitology 149, 29–40. Takeshita, K., Shibato, J., Sameshima, T., Fukunaga, S., Isobe, S., Arihara, K. and Itoh, M. (2003) Damage of yeast cells induced by pulsed light irradiation. International Journal of Food Microbiology 85, 151–158. Wang, Y.-C., Lee, P.-J., Shih, C.-M., Chen, H.-Y., Lee, C.-C., Chang, Y.-Y., Hsu, Y.-T., Liang, Y.-J., Wang, L.-Y., Han, W.-H. and Wang, I.-C. (2003) Damage formation and repair efficiency in the p53 gene of cell lines and blood lymphocytes assayed by multiplex long quantitative polymerase chain reaction. Analytical Biochemistry 319, 206–215. Will, O., Mahler, H., Arrigo, A. and Epe, B. (1999) Influence of glutathione levels and heat-shock on the steady-state levels of oxidative DNA base modifications in mammalian cells. Carcinogenesis 20, 333–337. Xiao, L., Fayer, R., Ryan, U. and Upton, S.J. (2004) Cryptosporidium taxonomy: recent advances and implications for public health. Clinical Microbiological Reviews 17, 72–97.

16

Effect of Environmental and Conventional Water Treatment Processes on Waterborne Cryptosporidium Oocysts

B. KING, A. KEEGAN, C. SAINT AND P. MONIS Australian Water Quality Centre, Salisbury, Australia

Abstract Cryptosporidium oocysts are prevalent in surface waters as a result of anthroponotic activity and native animal faecal contamination. A sound understanding of the impact of environmental and water treatment processes on the survival of oocysts is required to properly risk-assess the threat posed to public health by the presence of oocysts in water storages. This chapter provides an overview of recently completed work studying the impact of temperature, predation, sunlight and conventional water treatment processes on oocyst survival in water.

Background Cryptosporidium oocysts are frequently found in surface waters (Smith and Rose, 1990; Rose et al., 1997) and are extremely resistant to chlorine and monochloramine at the concentrations used to disinfect water for potable use (Korich et al., 1990; Finch et al., 1993). In addition to being resistant to commonly used disinfectants, it is generally thought that oocysts can persist for several months or more in the aquatic environment (Robertson et al., 1992; Johnson et al., 1997; Medema et al., 1997). These factors, combined with the relatively low infectious dose demonstrated for some isolates of Cryptosporidium parvum (Messner et al., 2001), mean that Cryptosporidium represents a challenge to water utilities responsible for the provision of safe drinking water to the public. Water utilities require accurate data on the fate and transport of oocysts in water and their response to water treatment processes, in order to effectively riskassess and risk-manage the threat posed by the presence of oocysts in source waters. A number of studies have examined oocyst survival, using techniques such as in vitro excystation or vital dye staining (Robertson et al., 1992; Chauret et al., 1998), but such methods are only indicators of viability and are known to overestimate infectivity (Black et al., 1996; Bukhari et al., 2000). As a consequence, 198

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there is a lack of knowledge regarding the inactivation of oocysts in environmental waters, particularly with regard to the effect of biotic and abiotic processes on the retention of oocyst infectivity.

Objectives In order to address knowledge gaps with respect to the survival of oocysts in response to environmental and water treatment conditions, studies were conducted using a cell culture infectivity model (Keegan et al., 2003) to examine the response of oocysts in water to: ● ● ● ● ● ●

Temperature. Microbial activity. Natural light. Chemical flocculation (aluminium sulphate). Dissolved air flotation. Disinfection (chlorination, chloramination).

Approach Details of the methodologies used for the temperature, sunlight and treatment inactivation studies have been described in detail elsewhere (Keegan et al., 2003; King et al., 2005, 2007). Fresh or aged oocysts of Cryptosporidium parvum (Swiss cattle C26) that had been passaged through mice were used for all experiments, and infectivity was assessed using a cell culture infectivity/Taqman PCR assay (CC-PCR) (Keegan et al., 2003). A schematic diagram of the assay is presented in Fig. 16.1. For environmental survival experiments, microcosms containing fresh oocysts and either reagent-grade water, tap water or reservoir water were established in either sterile polycarbonate chambers (for temperature experiments) or methylacrylate chambers (for sunlight inactivation experiments). Oocyst infectivity was measured in triplicate using 10,000 oocysts, and log inactivation was calculated by comparing quantitative PCR results for the treated samples with the non-treatment controls (which represent maximum infectivity). Candidate predators of oocysts were enriched from Hope Valley reservoir water sediment by selecting for organisms with a food preference in the Cryptosporidium oocyst size-range, using cysts of the amoeba Rosculus spp. and baker’s yeast (Saccharomyces cerevisiae). Organisms were collected using micromanipulation and placed in an individual well of a 24-well Cellstar tissue culture plate (Greiner Bio-one, Frickenhausen, Germany) containing 1 ml of autoclaved Hope Valley reservoir water, supplemented with yeast dissolved in Ringer’s solution. Oocysts were pre-stained with a fluorescent antibody (AusFlow Cry 104; BTF, Sydney, Australia) and 4′,6-diamidino-2-phenylindole (DAPI) prior to feeding experiments. Feeding experiments were conducted using 8-well Lab-Tek II Chamber Slides (Nalgene Nunc International, Naperville, Illinois) for amoebae or 1.5 ml microcentrifuge tubes for other organisms in a total volume of 150 µl

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Treat to promote release of sporozoites, apply oocysts to cell monolayer

Incubate 48 hours to allow establishment of infection

Wash monolayer, extract DNA from monolayer, conduct quantitative real-time PCR

Analyse data, compare treatments versus untreated controls

Fig. 16.1. Overview of a Cryptosporidium infectivity assay that combines cell culture and real-time PCR.

of sterile reservoir water containing 5 × 104 oocysts per chamber. Samples were fixed by the addition 150 µl of sodium acetate acetic acid formalin (SAF). Organisms were viewed using an Olympus BX60 system microscope (Olympus, Oakleigh, Australia) and images were captured using an Olympus DP50 digital camera. The parameters used for conventional 2 l jar testing (to assess the effect of coagulation on oocyst infectivity) are listed in Table 16.1. Between 4 × 104 and 1 × 105 oocysts were used per jar, with doses of alum ranging from 0 to 100 mg/l,

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Experimental jar test conditions.

Jar test parameters Oocyst mixing Flash mixing Slow mixing Settling Volume Raw water chemistry pH UV254 Temperature Turbidity Colour

5 min @ 200 rpm 1 min @ 200 rpm 14 min @ 20 rpm 15 min 2 l or 400 ml 8.13–8.40 0.148–0.195/cm 16.8 ± 1.2°C 2.95–3.25 NTU 21–24 HU

which is within the range of concentrations used at conventional water treatment plants in South Australia. For samples further treated by dissolved air flotation (DAF), a 15 min flotation step with 12% recycle at 70 psi was added to the end of the standard jar test procedure. For disinfection, contact times for chlorine and chloramine ranged from 300 to 1200 mg min/l.

Outcomes Temperature inactivation Replicate in vitro experiments were performed in reagent-grade MilliQ water and Hope Valley raw water (with or without sterilization) to assess oocyst removal and inactivation rates at temperatures ranging from 4°C to 25°C. Infectivity for each time point was measured using 10,000 oocysts in triplicate, with inactivation determined by comparison with the level of infectivity observed in the 4°C control at each time point. The inactivation of the 4°C sample over time was calculated using the 4°C sample for t = 0 as the reference for maximum infectivity. The rate of inactivation was related to the temperature, with more rapid inactivation observed at the higher temperatures (Fig. 16.2). Additional experiments were conducted to investigate the mechanism responsible for oocyst inactivation by temperature. Oocysts were stored in reagent-grade water at temperatures between 4°C and 37°C and measurements were made for infectivity and oocyst ATP concentrations to determine whether there was any effect on oocyst energy stores. Changes in ATP concentration closely corresponded with changes in infectivity, with a good correlation observed between these two parameters for all temperatures (Fig. 16.3).

Log inactivation

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0.5 0.0 -0.5 -1.0 -1.5 -2.0 -2.5 -3.0 -3.5 -4.0 -4.5

4 degrees 15 degrees 20 degrees 25 degrees

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0

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4 6 8 Time (weeks)

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0.5 0.0 -0.5 -1.0 -1.5 -2.0 -2.5 -3.0 -3.5 -4.0 -4.5 0

2

4 6 8 Time (weeks)

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0.5 0.0 -0.5 -1.0 -1.5 -2.0 -2.5 -3.0 -3.5 -4.0 -4.5 12

0

2

4 6 8 Time (weeks)

10

12

Fig. 16.2. Inactivation of Cryptosporidium oocysts in water at different temperatures.

Predation Monitoring of oocyst numbers during the initial temperature inactivation study revealed changes in oocyst numbers for some treatments. In the case of the reagent water and autoclaved Hope Valley reservoir water treatments, the oocyst counts remained constant throughout the incubation period for all four temperatures in all replicate experiments. Furthermore, the morphology of the oocysts remained consistent, with a regular shape, good staining with the fluorescent antibody and no clumping observed. However, for one of the replicate experiments conducted on Hope Valley raw reservoir water, a gradual decrease in oocyst numbers was observed for the 15°C treatment and more rapid decreases for both 20°C and 25°C over the 12-week incubation period (Fig. 16.4). Oocysts examined in these samples were often clumped together with variable fluorescent staining and deformations in shape (as many as 18 in an individual clump, data not shown). Oocysts not in clumps exhibited typical staining and morphology, and exhibited infectivity consistent with oocysts in sterile water for the same temperature exposure.

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1E-10

0.5

ATP concentration (M) 1E-9

2E-9 1.8E-9 1.6E-9

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1.4E-9

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1.2E-9

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1E-9

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-1.5 -4

6E-10

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0

20

40

60

80

100

120

140

160

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Time (hours) incubation at 37°C

Fig. 16.3. Comparison of oocyst inactivation measured using cell culture infectivity with ATP concentration, measured during incubation at 37°C. Inset: correlation of oocyst ATP concentration and log inactivation using the combined data from all temperature treatments (15, 20, 25, 30, 37°C) and time points.

Attempts to replicate the oocyst removal in further experiments were variable between replicates, but decreases in oocyst numbers were observed. Reservoir water filtered using an 0.8 micron filter did not exhibit any reduction in oocyst numbers. To further investigate the loss of oocysts, potential predators of oocysts were enriched from the reservoir water and tested to determine whether they could ingest oocysts. A range of ciliates, amoebae and rotifers were identified that could ingest antibody-labelled oocysts, as determined by fluorescent microscopy and DAPI staining, as well as a single platyhelminth and gastrotrich (Table 16.2). In the case of Paramecium, it appeared that oocysts were being digested in food vacuoles (Fig. 16.5). In the case of Blepharisma, it was not possible to view the FITC-labelled oocysts due to autofluorescence, but the oocysts were clearly visible by DAPI staining.

Solar inactivation Outdoor tank experiments and a cell culture infectivity assay were used to measure the effect of solar irradiation on C. parvum oocysts in water. Initial experiments

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70 4°C

60

15°C 50

20°C 25°C

40

4°C autoclaved 30

15°C autoclaved

20

20°C autoclaved 25°C autoclaved

10 0

2

4 6 8 Incubation time (weeks)

10

12

Fig. 16.4. Comparison of oocyst counts in Hope Valley reservoir water (±sterilization) incubated at different temperatures over time.

Table 16.2.

List of phagotrophs capable of ingesting Cryptosporidium oocysts.

Phagotroph group

Taxon

Ciliates

Paramecium Euplotes Blepharisma Oxytricha Holosticha Mayorella Microchlamys Thecamoeba Willaertia Cochliopodium Unidentified rhabdocoel Lepadella Unidentified bdelloid (×3 types) Dicranophorus Chaetonotus

Amoebae

Platyhelminth Rotifers

Gastrotrich

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(B)

(C)

20

Fig. 16.5. Paramecium sp. containing ingested oocysts of Cryptosporidium parvum. (A) Oocysts are clearly visible in the food vacuoles (arrow) when viewed under differential interference contrast (DIC) optics. (B) The sporozoite nuclei within oocysts (arrows) can be seen with DAPI staining. (C) Fluorescent antibody-stained oocysts are visible (small arrow), with oocysts with decreased staining or signs of degradation indicated by a larger arrow. Bar = 20 µm.

Table 16.3. index days. UV indexa 1 3 4 4 7 10 11 12

Inactivation of Cryptosporidium oocysts in tap water on different UV

S90 (kJ/m2)b

T90 (h)c

13,200 8,700 5,300 6,200 11,900 4,400 5,900 76

6.4 3.7 1.8 1.7 2.6 0.9 1.0 0.4

a UV

index = estimated dose at solar noon, 1 = 25 mW/m2 UV between 290 and 400 nm. = the insolation necessary to achieve a 90% reduction in cell culture infectivity. c T90 = the time necessary to achieve a 90% reduction in cell culture infectivity. b S90

used tap water and assessed solar irradiation on days with a different UV index. Days with a UV index of 10 or greater resulted in rapid inactivation (Table 16.3). Dark controls (samples wrapped in aluminium foil), kept under the same conditions as the light-exposed oocysts, exhibited no change in infectivity compared with oocysts stored at 4°C, demonstrating that temperature did not affect oocyst infectivity within the time frame of these experiments.

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Solar inactivation was next assessed for reservoir waters that varied in turbidity, colour and dissolved organic carbon (DOC) (Table 16.4). Waters with high DOC required higher doses of solar irradiation to achieve oocyst inactivation (Table 16.5). Long pass filter experiments were conducted to determine the biologically active components of solar radiation. The component responsible for the majority of inactivation was found to be UV-B, with a small amount of inactivation attributed to UV-A. Visible light had no measurable effect, as determined by the cell culture infectivity assay. The level of UV-induced DNA damage was assessed using an antibody directed against cyclobutane thymine dimers and also by quantitative sequence detection using real-time PCR. The amount of damage detected was considerably less than expected considering the level of inactivation observed when compared to similar levels of oocyst inactivation using UV-C (254 nm), suggesting that other mechanisms in addition to DNA damage are responsible for oocyst inactivation.

Water treatment Conventional water treatment processes remove Cryptosporidium oocysts through coagulation, flocculation, sedimentation and filtration. Little is known regarding the effect of these processes on oocyst infectivity or whether exposure Table 16.4. ments.

Characteristics of reservoir water used for solar inactivation experi-

Water type Bolivar tap Warragamba Prospect Lake Hope Valley Myponga

pH

Dissolved organic carbon (mg/l)

7.80 7.45 7.15 7.78 7.55

2.8 2.6 3.6 8.0 12.3

Turbidity (NTU)

Colour (HU)

0.238 2.94 2.26 7.42 4.12

2 29 47 38 77

Table 16.5. Inactivation of Cryptosporidium oocysts in reservoir waters on different UV index days. Winter: UV index 3 Water type Bolivar tap Warragamba Prospect Hope Valley Myponga

S90 (kJ/m2) 9,300 7,900 13,300 14,751 27,933

T90 (h) 3.2 2.6 4.5 4.88 10.75

Summer: UV index 10 S90(kJ/m2) 4,500 5,800 5,200 13,500 32,599

T90 (h) 0.85 1.1 1.0 2.5 6.5

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to these processes makes oocysts more susceptible to standard disinfection. The effect of the water treatment chemical alum on Cryptosporidium oocyst infectivity was assessed using cell culture infectivity. Coagulation by doses of alum ranging from 40 mg/l to 100 mg/l had no effect on oocyst infectivity (either on oocysts left in suspension or oocysts recovered from flocs). Dissolved air flotation of oocysts following alum coagulation also had no effect on oocyst infectivity. There was no change in the sensitivity of oocysts to chlorine or monochloramine following alum coagulation. The effect of age and temperature on oocyst sensitivity to chlorine was assessed (up to 6 months at 4°C or 15°C). A small increase in oocyst sensitivity to chlorine (Ct = 1200 mg min/l), resulting in 0.5 0 1 log inactivation, was observed for oocysts that had been stored at either 4°C or 15°C for 20 weeks or more (Fig. 16.6).

Summary and Future Directions While environmentally tough and resistant to common disinfection chemicals, Cryptosporidium oocysts are not invincible and are susceptible to environmental stresses. The data produced from this work need to be incorporated into fate and transport models, as current models use inaccurate data for temperature inactivation, and solar inactivation data have either been absent from such models or derived from other organisms. This will not only allow prediction of the location of oocysts in a reservoir but may also allow some prediction of infectivity if environmental conditions (temperature, light, DOC) are known. Importantly we also

1 week 0

week 8

week 12

week 16

week 20

week 24

Log survival

0 -1 -2 -3 -4 -5 Incubation period No treatment 4

JT 4

JT+cl2

No treatment 15

JT 15

JT+cl2 15

Fig. 16.6. Inactivation of oocysts by chlorine as a function of oocyst age and exposure to different temperatures.

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identify the need for further research effort directed towards biotic inactivation and removal. Questions which need to be answered include: ● ● ●

Are excreted oocysts still infectious? How does predation affect oocyst resistance to common disinfectants? Can such oocysts still be efficiently recovered by standard immunomagnetic separation (IMS) concentration?

Such work will provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in water.

Acknowledgements We acknowledge the financial support received from the Co-operative Research Centre for Water Quality and Treatment, Water Services Association Australia, Australian Water Quality Centre, and South Australian Water Corporation. We thank David Daminato, Stella Fanok, Bret Robinson, Daniel Hoefel and Kylie Harvey for their excellent technical expertise and fruitful discussions.

References Black, E.K., Finch, G.R., Taghi-Kilani, R. and Belosevic, M. (1996) Comparison of assays for Cryptosporidium parvum oocysts viability after chemical disinfection. FEMS Microbiology Letters 135, 187–189. Bukhari, Z., Marshall, M.M., Korich, D.G., Fricker, C.R., Smith, H.V., Rosen, J. and Clancy, J.L. (2000) Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts. Applied and Environmental Microbiology 66, 2972–2980. Chauret, C., Nolan, K., Chen, P., Springthorpe, S. and Sattar, S. (1998) Aging of Cryptosporidium parvum oocysts in river water and their susceptibility to disinfection by chlorine and monochloramine. Canadian Journal of Microbiology 44, 1154–1160. Finch, G.R., Black, E.K., Gyürék, L. and Belosevic, M. (1993) Ozone inactivation of Cryptosporidium parvum in demand-free phosphate buffer determined by in vitro excystation and animal infectivity. Applied and Environmental Microbiology 59, 4203–4210. Johnson, D.C., Enriquez, C.E., Pepper, I.L., Gerba, C.P. and Rose, J.B. (1997) Survival of Giardia, Cryptosporidium, poliovirus and Salmonella in marine waters. Water Science and Technology 35, 261–268. Keegan, A.R., Fanok, S., Monis, P.T. and Saint, C.P. (2003) Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection. Applied and Environmental Microbiology 69, 2505–2511. King, B.J., Keegan, A.R., Monis, P.T. and Saint, C.P. (2005) Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infectivity. Applied and Environmental Microbiology 71, 3848–3857. King, B.J., Hoefel, D., Daminato, D.P., Fanok, S. and Monis, P.T. (2007) Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters. Journal of Applied Microbiology 104, 1311–1323.

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Korich, D.G., Mead, J.R., Madore, M.S., Sinclair, N.A. and Sterling, C.R. (1990) Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability. Applied and Environmental Microbiology 56, 1423–1428. Medema, G.J., Bahar, M. and Schets, F.M. (1997) Survival of Cryptosporidium parvum, Escherichia coli, faecal enterococci and Clostridium perfringens in river water: influence of temperature and autochthonous microorganisms. Water Science and Technology 35, 249–252. Messner, M.J., Chappell, C.L. and Okhuysen, P.C. (2001) Risk assessment for Cryptosporidium: a hierarchical Bayesian analysis of human dose response data. Water Research 35, 3934–3940. Robertson, L.J., Campbell, A.T. and Smith, H.V. (1992) Survival of Cryptosporidium parvum oocysts under various environmental pressures. Applied and Environmental Microbiology 58, 3494–3500. Rose, J.B, Lisle, J.T. and LeChevallier, M. (1997) Waterborne cryptosporidiosis: incidence, outbreaks, and treatment strategies. In: Fayer, R. (ed.) Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, Florida, pp. 93–109. Smith, H.V. and Rose, J.B. (1990) Waterborne cryptosporidiosis. Parasitology Today 6, 8–12.

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Methods for Genotyping and Subgenotyping Cryptosporidium spp. Oocysts Isolated During Water and Food Monitoring

H.V. SMITH, R.A.B. NICHOLS, L. CONNELLY AND C.B. SULLIVAN Scottish Parasite Diagnostic Laboratory, Glasgow, UK

Abstract Cryptosporidium oocysts are frequent contaminants of water, with contributions from infected human and non-human hosts, livestock and agricultural practices, and infected feral and transport hosts. Numerous waterborne outbreaks of cryptosporidiosis have been documented and as oocysts occur at low densities in water, methods which can detect and determine the genotype and subgenotype of small numbers of organisms reliably and reproducibly from water and food concentrates are required. Drinking water quality is also an important component of food production. Oocysts can enter the food chain from livestock and agricultural practices and from sewage effluent. Sensitive molecular methods are required for determining Cryptosporidium species, genotypes and subgenotypes in water and in/on foods. Sensitivity of detection can be increased by amplifying loci on multi-copy genes and polymerase chain reaction (PCR) amplification of loci in the 18S rRNA gene is considered to be the most suitable approach, as they can provide information about more species than single-copy loci, and have been more widely accepted worldwide. Representatives of the 16 valid Cryptosporidium species and the 44+ genotypes can be found in environmental, water and food concentrates, everywhere in the world, which raises significant issues regarding approaches to determine their presence, particularly if they are present as mixtures. Primer specificity should be evaluated against organisms that are common contaminants of water and food matrices, particularly those that are closely related to Cryptosporidium phylogenetically. Typing and subtyping systems used for human and non-human samples should also be used for environmental samples, particularly for source and disease tracking and risk assessment, in order to avoid any confusion arising from using different systems for human and non-human hosts and environmental samples for veterinary and public health investigation of disease outbreaks. 210

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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Introduction Cryptosporidium is an apicomplexan, obligate protozoan parasite that causes enteritis in human hosts. First described in the intestines of laboratory mice (Tyzzer, 1912), Cryptosporidium gained importance in the 1970s, when it was found to cause diarrhoea in young calves and humans. Since then it has emerged as a parasite of worldwide importance and has acquired special relevance as an opportunistic pathogen of people with acquired immune deficiency syndrome (AIDS). Recent genetic analyses have raised doubt about the validity of the current classification of the genus Cryptosporidium and reveal that more than one species of Cryptosporidium can infect susceptible human hosts (Table 17.1). There are 16 ‘valid’ Cryptosporidium species and a further 40+ genotypes, which differ significantly in their molecular signatures but, as yet, have not been ascribed species status (Smith et al., 2007). Seven described Cryptosporidium species (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. suis and C. muris) (Table 17.1) and two undescribed species of Cryptosporidium (cervine and monkey) infect immunocompetent and immunocompromised humans (Xiao et al., 2004; Cacciò et al., 2005), but C. hominis and C. parvum are the most commonly detected (Cacciò et al., 2005). Human cryptosporidiosis is characterized by profuse, watery diarrhoea, ’flu-like illness, malaise, abdominal pain, anorexia, nausea, flatulence, malabsorption, vomiting, mild fever and weight loss. In immunocompetent individuals, cryptosporidiosis is self-limiting, but it can become chronic in immunocompromised individuals. There is no recognized effective drug treatment for human cryptosporidiosis. The C. parvum ID50 for seronegative adult human volunteers is isolate-dependent and ranges from 9 to 1042 oocysts (Okhuysen et al., 1999), while the C. hominis ID50 (isolate TU502) is 10–83 oocysts (Chappell et al., 2006).

Table 17.1.

Some differences among Cryptosporidium species infecting humans.

Species

Oocyst dimensions (µm)

Site of infection

Major host

4.5 × 5.5 4.5 × 5.5

Small intestine Small intestine

C. suis

5.05 × 4.41

Small intestine

Humans Neonatal mammalian livestock, humans Pigs

C. felis

4.5 × 5.0

Small intestine

Cats

C. canis

4.95 × 4.71

Small intestine

Dogs

4.5–4.0 × 4.6–5.2

Intestine

Turkeys

5.5 × 7.4

Stomach

Rodents

Stomach

Cattle

C. hominis C. parvum

C. meleagridis C. muris C. andersoni

5.6 × 7.4 (5–6.5 × 8.1–6.0)

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Worldwide, the incidence of human cryptosporidiosis is higher in underdeveloped or developing countries, in children, and in immunocompromised individuals. The incidence of human cryptosporidiosis has been estimated at 3.3 cases per 100,000 individuals in 12 European countries, and in the USA Cryptosporidium infections are estimated to cause disease in 300,000 individuals annually. The incidence of waterborne and foodborne cryptosporidiosis worldwide is difficult, if not impossible, to estimate accurately, since the source of infection for sporadic cases, which constitute the majority of human cryptosporidiosis cases, is very seldom determined. Transmission can occur by direct contact with an infected person or animal, or indirectly via contaminated water or food. Most commonly, zoonotic transmission occurs as a consequence of cryptosporidiosis in neonatal calves and lambs, which can excrete up to 109 oocysts/g faeces (the environmentally resistant and infective form of the parasite). Once in the environment, infectious oocysts can contaminate water sources and food.

Oocyst Contributions to Water and Food Cryptosporidium oocysts are frequent contaminants of water, with contributions from infected human and non-human hosts, livestock and agricultural practices, and infected feral and transport hosts (Smith et al., 1995; Smith and Grimason, 2003). Water is an important transmission route, with at least 165 waterborne outbreaks of cryptosporidiosis documented (Girdwood and Smith, 1999; Fayer et al., 2000; Slifko et al., 2000; Karanis et al., 2007). Oocysts occur at low densities in water (Smith and Rose, 1990, 1998; Smith et al., 1995; Smith and Grimason, 2003), and methods which can detect and determine the genotype and subgenotype of small numbers of organisms reliably and reproducibly from water and food concentrates are required. Oocysts are resistant to numerous disinfectants normally used in water treatment, and water is an important component of food production from crop irrigation, harvesting, sorting, to storage and distribution. In the food industry, potable water, uncontaminated with infectious oocysts, is needed for the preparation of ready-to-eat food and for the dilution of beverages. Oocysts can enter the food chain from agricultural practices such as muck-spreading and slurry spraying of oocyst-contaminated, animal-derived faecal material onto land used for cultivation and from animals pasturing near crops intended for human consumption. The use of contaminated, untreated sewage (and waste stabilization pond) effluents and untreated water for crop irrigation can also contaminate crops. Runoff from, and percolation through, contaminated pasture and soils can contaminate adjacent waterbodies, and oocysts transported into rivers and marine estuaries can lead to the contamination of shellfish, which are frequently eaten raw or lightly cooked. Infectious oocyst contamination of drinking water and foods that are to be eaten raw or with minimum heating is associated with a high risk of transmitting cryptosporidiosis. The number of oocysts which a method can detect should realistically be below its infectious dose. Given that the ID50 for C. parvum and

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C. hominis isolates ranges from 9 to 1042 oocysts, sensitive methods with consistent recovery efficiencies are required for reliable risk assessment. Current Cryptosporidium detection methods include extraction of oocysts from water and food matrices, concentration of oocysts from the eluted material, and detection by epifluorescence and Nomarski differential interference contrast microscopy (Anonymous, 1999a, 1999b, 2005; USEPA, 2001; Smith and Cook, 2008). Experimental recovery efficiencies from water and food matrices range from 1% to 59% (Smith et al., 2001; Nichols and Smith, 2002).

Methods for Water and Foods Standardized methods are available for isolating and enumerating Cryptosporidium oocysts in water, many of whose component parts have been developed in the authors’ laboratory (e.g. Smith et al., 1989, Grimason et al., 1994; Campbell and Smith, 1997). Immunomagnetic separation (IMS) has standardized and increased oocyst recoveries from water concentrates (Smith et al., 2001). The benefits of IMS, in capturing oocysts from crude samples and concentrating and processing them in a buffer free of PCR inhibitors, increases the sensitivity of detection (Smith, 1996), and this approach has been used to genotype oocysts in water and foods (e.g. Xiao et al., 2000, 2001; Nichols et al., 2002, 2003; Jiang et al., 2005). There is no standardized method for isolating and enumerating Cryptosporidium oocysts from foods. Nichols and Smith (2002) reviewed published methods for isolating and enumerating oocysts isolated from various liquid and solid foodstuffs and identified a broad range (1–59%) in the recovery efficiencies of these methods. Some used IMS while others did not. Commercial IMS kits for concentrating oocysts from water concentrates have also been used for concentrating oocysts from food concentrates (Robertson and Gjerde, 2000, 2001; Robertson et al., 2002), but kits optimized for water concentrates are not necessarily optimized for food matrices (Cook et al., 2006a; Smith and Cook, 2008). Cook et al. (2006a) developed and validated (Cook et al., 2006b) a method with the aim of producing standard protocols for detecting Cryptosporidium and Giardia in foods. Component parts were adapted from methods developed for detecting Cryptosporidium and Giardia in water, but specific new sample treatments had to be developed for primary extraction of oocysts and cysts from food samples. Importantly, these treatments were optimized, by varying the physicochemical parameters, to produce sample extracts which were compatible with the IMS and microscopy materials. The optimization was monitored through the recovery efficiencies achieved through parameter variation (Smith and Cook, 2008). The validated method, developed to determine oocyst contamination on lettuce and raspberries, has been used to genotype oocysts on a variety of foods. For C. parvum, the lowest median infectious dose for adult human volunteers is nine oocysts (Okhuysen et al., 1999). To detect this level of contamination, a minimum of one oocyst should be recovered from the extract and end up on the microscope slide where it can be seen. This would necessitate a recovery

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efficiency of at least 11%. The method of Cook et al. (2006a, 2006b) has a higher recovery efficiency, being 59.0 ± 12.0% for lettuce and 41.0 ± 13.0% for raspberries.

What’s Out There? Molecular identification methods can be performed on samples which are suitable for microscopic evaluation, and DNA extraction can be performed either before oocysts are dissociated from magnetizable beads or following their enumeration on microscope slides. One advantage of using microscope slides where oocysts have been detected by microscopy is that both intact and empty oocysts can be observed. Empty oocysts cannot be amplified by molecular methods as they do not contain sporozoite DNA, and this will lead to an underestimation of (i) oocyst contamination and (ii) the efficiency of physical removal processes used for water and food. Since not all Cryptosporidium species that can contaminate waters and foods are infectious to humans, Cryptosporidium species, genotype and subgenotype identification using polymerase chain reaction (PCR)-based methods augments risk assessment. The viability/infectivity of oocysts is also of importance for assessing risk. Currently, there is no accepted method for determining the infectivity/viability of C. parvum oocysts recovered from water or food. Investigation into the extent of the occurrence of different species/genotypes of Cryptosporidium in the environment and in/on foods is only now being addressed. Previously, we showed that oocyst staining with both commercially available fluorescein isothiocyanate (FITC)-labelled monoclonal antibodies that recognize exposed epitopes on oocyst walls and the nuclear fluorogen 4′,6diamidino-2-phenylindole (DAPI), which are used in the standardized methods for water and methods for foods (see below), are amenable to PCR amplification (Nichols et al., 2003), validating the use of these samples. This approach has been adopted in many laboratories investigating environmental contamination by Cryptosporidium. Discordant results between PCR and microscopy were observed during analysis of storm water samples: 10 microscopy-negative samples were PCRpositive (36 of 42 samples were PCR-positive). This occurred within duplicates of the same subsample. Other variables that affected PCR positivity included the volume of template used per reaction and the number of species/genotypes present in the sample (see below). Samples with more than one species/genotype had a PCR positivity rate of 73% compared with 34% for those containing a single species/genotype (Xiao et al., 2006). Methods targeting 18S rRNA gene loci are the most widely used for identifying Cryptosporidium species/genotypes in human and non-human hosts and environmental (water and food) samples, and are advocated here. As there are 20 copies of the Cryptosporidium ribosomal DNA gene present per oocyst (LeBlancq et al., 1997), PCR amplification of multi-copy genes is a practical approach to sensitive molecular detection. Furthermore, as the complete sequences of the SSU rRNA gene from different Cryptosporidium species, originating from a variety

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of hosts, are available from the GenBank database, this gene has been exploited the most for species identification. Much of the earlier data were derived from PCR-restriction fragment length polymorphism (RFLP) analysis, but it has become clear that this approach often has insufficient discrimination for some of the recognized Cryptosporidium species/genotypes found in environmental samples, and that DNA sequencing is the preferred option. Where possible, a multilocus approach to characterizing Cryptosporidium isolates is essential for accuracy, and various 18S and other loci are available for species (and subtype) determination; however, single copy loci may not have sufficient sensitivity for detecting the small numbers (>10) of oocysts frequently found in environmental samples.

Cryptosporidium and Water The analysis of US storm water samples revealed the presence of Cryptosporidium spp. in 27 of 29 samples, mainly wildlife Cryptosporidium genotypes (Xiao et al., 2001). The most common species/genotypes found in surface waters were C. parvum, C. hominis and C. andersoni, with C. andersoni reported to be the most commonly found in wastewater (eight samples). Storm waters are expected to carry greater microbial loads, including larger numbers of Cryptosporidium oocysts (Kistemann et al., 2002). In an analysis of 121 water samples from storm events in three New York area watersheds (Jiang et al., 2005), 107 contained amplifiable Cryptosporidium DNA, and, of the 22 Cryptosporidium species and genotypes identified, only 11 were of known species or genotypes. Reliable detection of oocysts in storm waters using EPA method 1623 (0.5 ml packed pellet volume after filtering 20l samples) depends on the analysis of repeated samples and subsamples, both by microscopy and PCR. Limited subsampling can lead to an underestimation of both the number of oocysts and the number of species/genotypes present (Xiao et al., 2006). An analysis of oocyst-positive water concentrates on microscope slides from four UK water companies/water utilities revealed the presence of C. muris or C. andersoni, C. parvum or C. hominis, C. meleagridis or Cryptosporidium cervine, ferret or mouse genotypes, all of which can be discriminated by further restriction endonuclease digestion (Nichols et al., 2006a). Of a total of 33 slides analysed, 32 produced PCR products, but 10 were in poor condition, making microscopic re-evaluation impossible. All positive slides generated PCR-RFLP patterns consistent with published data. Two slides contained larger (~8 × 6 µm) oocysts, and PCR-RFLP analysis revealed the presence of C. andersoni DNA (Nichols et al., 2006a). In a further study of 1039 oocyst-positive slides, 56.1% of which were from Scottish final water samples and 44% from Scottish raw waters, 601 (57.7%) samples were positive at one or two 18S rRNA gene loci. The most common sample contained one oocyst, which accounted for 45.1% of all samples, and the majority of samples contained between one and five oocysts (81% of the dataset). In 71 of the Scottish final water samples, human-infectious species/genotypes were detected: C. parvum in 67.6%, C. hominis in 18.3%, C. meleagridis in 1.4% and the Cryptosporidium cervine genotype in 30.9% of samples (H.V. Smith et al., unpublished). Other species/genotypes detected

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included C. muris, C. andersoni, C. baileyi, C. bovis, Cryptosporidium muskrat genotypes I and II, Cryptosporidium cervine genotype, Cryptosporidium opossum genotype I, Cryptosporidium deer-like genotype, and six previously undescribed Cryptosporidium genotypes.

Cryptosporidium and Food A preliminary analysis of Cryptosporidium contamination of vegetables in Polish local markets involved analysing 21 samples of local produce (cabbage (various), leek, spinach, lettuce, green onions, broccoli, celery, cauliflower). Samples were purchased from markets in locations based on the number of homesteads in the area which had >30 cows in one herd. Using the method of Cook et al. (2006a), six of the 21 samples were oocyst-positive. By plotting where samples were purchased against the number of homesteads which had >30 cows in one herd, we determined that all oocyst-positive samples were purchased in local markets which had the highest density of homesteads with >30 cows in one herd (range 41–99 homesteads). Oocyst-negative samples were purchased in local markets where the density of homesteads with >30 cows in one herd ranged from 0 to 99 (H.V. Smith and A. Rzezutka, unpublished). The number of oocysts detected ranged from 2 to 35, and two samples contained between 1 and 2 excysted oocysts. Of these six positive samples, three were PCR-positive, and RFLP and sequencing revealed them to contain C. parvum DNA. Cryptosporidium parvum subgenotyping, at the GP60 locus and by multilocus genotyping using mini- and microsatellites (Mallon et al., 2003a, 2003b), is under way. Clearly, these data identify the breadth of species and genotypes detected, and it is likely that representatives of the 16 valid species and the 44+ genotypes can be found in environmental, water and food concentrates worldwide, which raises significant issues regarding approaches to determine their presence. Furthermore, where mixtures of species/genotypes occur, both PCR-RFLP and sequencing options can be compromised. Prior to the routine adoption of molecular methods for investigating Cryptosporidium contamination of the environment, both the variability between methods and the recognized difficulties in amplifying nucleic acids from environmental specimens by PCR must be overcome.

Towards Standardizing Approaches for Determining Cryptosporidium Oocyst Contamination of the Environment, Water and Foods Methods that can genotype small numbers of Cryptosporidium oocysts reliably and reproducibly are required to determine which species/genotypes/ subgenotypes are present, and with what frequency, in water and in/on foods. A prerequisite to identifying sources of human infection and transmission routes is the requirement to define human infective parasites. Environmental matrices contain many inhibitory substances in varying quantities, which will decrease the sensitivity of detection. This demands more effective methods both for

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neutralizing inhibitory effects and for extracting nucleic acids. Much effort has been directed towards the efficient extraction of Cryptosporidium DNA, selection of Cryptosporidium-specific primers and overcoming the effects of inhibitors such as clays, humic and fulvic acids, polysaccharides and other organic compounds, salts and heavy metals, etc. (Sluter et al., 1997; Nichols et al., 2003; Nichols and Smith, 2004; Sunnotel et al., 2006).

DNA extraction DNA extraction is at the centre of efficient PCR amplification and the detection of small numbers of oocysts by molecular methods. A standard, maximized method for DNA extraction from Cryptosporidium oocysts is essential both for detecting small numbers of oocysts and for evaluating the sensitivity of detection by PCR using different primers. Disruption of the robust oocyst wall is a prerequisite for releasing sporozoite nuclei and effective DNA extraction, while the liberation of DNA from bound protein is essential both for efficient primer annealing and for successful PCR amplification. Oocyst wall disruption following freezing by immersion in liquid nitrogen and thawing is the preferred method for the release of sporozoite DNA from small numbers of intact oocysts; however, the optimum temperature reported in the literature for thawing and liberation of oocyst contents varies from 37°C to 100°C (Johnson et al., 1995; Mayer and Palmer, 1996; Rochelle et al., 1997; Sluter et al., 1997; Chung et al., 1998). Furthermore, the number of freeze–thaw cycles and the medium for DNA extraction also vary (Laberge et al., 1996; Leng et al., 1996; Deng et al., 1997; Rochelle et al., 1997; Sluter et al., 1997; Chung et al., 1998; Kaucner and Stinear, 1998). Not only have different C. parvum isolates been used by these researchers, but also different protocols, which underlines the need for maximizing and standardizing oocyst disruption and DNA extraction protocols, especially when dealing with small numbers of oocysts. Nichols and Smith (2004) described a method that maximizes DNA extraction reliably from small numbers of partially purified or purified oocysts. Sporozoite DNA was liberated from C. parvum oocysts by 15 cycles of freezing (liquid nitrogen) and thawing (65°C) in lysis buffer containing sodium dodecyl sulphate (SDS). The inhibitory effects of sodium dodecyl sulphate were abrogated by the addition of Tween 20 to the PCR reaction. Seven different C. parvum oocyst isolates were tested, and the method detected fewer than five oocysts following direct PCR amplification of a segment of the 18S rRNA gene. Older oocysts, which were more refractory to freeze–thawing, were disrupted effectively. These authors recommended 15 cycles of freeze–thawing to maximize oocyst disruption and DNA extraction, particularly when isolate history and oocyst age were unknown.

Sensitivity of 18S rRNA gene loci for genotyping oocysts from water and food In addition to efficient DNA extraction, an optimized protocol for genotyping Cryptosporidium oocysts present on microscope slides must include the use of

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genetic loci that maximize sensitivity. No ‘standard’ genetic locus exists for determining species identity, but RFLP or sequencing of 18S rRNA gene loci provide information about more species than single-copy loci for small numbers of oocysts. For detecting small numbers of oocysts (<100) consistently, a nested PCR is required. The nested assay of Xiao and colleagues (Xiao et al., 1999, 2001) is able to detect most Cryptosporidium species and genotypes by PCRRFLP. Importantly, it has been tested and validated in numerous laboratories. The nested (Nichols–Johnson; Nichols et al., 2003) 18S rRNA assay detects fewer Cryptosporidium species and genotypes by PCR-RFLP, but has also been shown to be very sensitive. Two 18S rRNA PCRs have been tested extensively in the authors’ laboratory (Xiao et al., 1999, 2001; Nichols et al., 2003) and the Nichols–Johnson primers appear to be more sensitive, particularly with small numbers (≤10) of C. parvum, C. hominis, C. felis and C. muris oocysts, probably because of the smaller amplicon size (Nichols et al., 2006a). Although less sensitive, the Xiao and colleagues (Xiao et al., 1999, 2001) assay has the benefit of being able to detect more species than the Nichols–Johnson assay by RFLP, particularly the important human pathogens C. parvum and C. hominis.

Specificity of genetic loci for genotyping oocysts from water and food The need for a judicious assessment of primers as well as knowledge of likely contaminants in a particular matrix is also crucial. Most Cryptosporidium primers have been developed for use with faecal samples, where the range of contaminating species that could co-amplify with the chosen primers is less and better understood than contaminants in water or in/on foods. The specificity of PCR primers, especially those designed for 18S rRNA loci, should be evaluated against organisms that are closely related to Cryptosporidium. Variations in primer or probe binding sites (usually based on sequences from the most, or easiest, studied representative, but which may not be representative of a specific target group) influence the ability of genus-specific primers to amplify all target sequences from all species and genotypes in a genus equally. In our laboratory, we selected the Xiao and colleagues (Xiao et al., 1999, 2001) and the Nichols–Johnson (Nichols et al., 2003, 2006a) primers for the following reasons: (i) the primers are well chosen and have been shown to work in a variety of scenarios; and (ii) Johnson et al. (1995) tested their method for cross-reactions against a total of 23 microorganisms that might be encountered in water concentrates. This reduces the likelihood of potential cross-reactions. The Laxer et al. (1991) Cryptosporidium primers amplify genomic DNA from Eimeria acervulina, whilst the published probe hybridized with E. acervulina amplicons and Giardia intestinalis DNA (Laberge et al., 1996). Obviously, the Laxer et al. (1991) primers cannot be used in matrices where these organisms are likely to be contaminants. Sturbaum et al. (2002) used a nested 18S rRNA PCR (external primers ExCry1 and ExCry2, nested primers NesCry3 and NesCry4)-RFLP and

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sequencing to determine the sensitivity of detection of IMS-PCR in C. parvum oocyst-seeded and unseeded natural waters. Sensitivity was good (≥5 oocysts) but these nested primers also amplified DNA from an unknown (contaminating) dinoflagellate as well as DNA isolated from cultured Gymnodinium fuscum, indicating the requirement for in-depth evaluation of primer sets designed specifically to detect Cryptosporidium in environmental samples. Gymnodinium and Cryptosporidium are members of the protozoan infrakingdom Alveolata. Alveolata is a monophyletic taxon and contains three phyla: Apicomplexa (syn. Sporozoa; apicomplexans), Ciliophora (ciliates) and Dinozoa (dinoflagellates). Alignment of complete 18S rRNA gene sequences of C. parvum and Gymnodinium spp. revealed 86.5% pairwise identity (Sturbaum et al., 2002). Sturbaum et al. (2002) argued that, as C. parvum oocysts, dinoflagellates, and ciliates were common in aquatic habitats and they had a close phylogenetic relationship, it was not entirely surprising to observe that a given 18S rRNA primer set amplified DNA derived from multiple alveolate members which were present together in an environmental sample. Similarly, in a Northern Ireland waterborne cryptosporidiosis outbreak which occurred in the greater Belfast area of Northern Ireland in August 2000 and involved at least 117 cases (Glaberman et al., 2002), C. hominis DNA was detected in 12 of 14 oocyst-positive water concentrates sent to the authors’ laboratory (Nichols et al., 2003; Smith et al., 2004) using the Nichols et al. (2002) nested PCR. Two samples generated amplicons that were larger than the expected 435 bp Cryptosporidium amplicon. Sequencing these PCR products revealed a 96% homology with Gymnodinium simplex and these primers also amplified DNA isolated from cultured Gymnodinium simplex (strain no. CCAP 1117/3). Since we identified these cross-reactions, we have undertaken extensive and in-depth evaluations of primer sets designed specifically to detect Cryptosporidium in environmental samples. Excess template can lead to PCR inhibition. When six different volumes of IMS-concentrated oocyst DNA template (0.5–5 µl) were used for the initial PCR of a nested assay, 4 µl or 5 µl of template produced more negative results compared with 3 µl of the same sample (Xiao et al., 2001). Thus optimization of template is a prerequisite for accurate data. The inclusion of internal positive controls (Stinear et al., 1996; Kaucner and Stinear, 1998; Nichols et al., 2002), to assess inhibitory effects, increases the level of confidence obtained from negative results.

Mixtures of Cryptosporidium species/genotypes Clearly, mixtures of Cryptosporidium species/genotypes can be expected in water and food concentrates, yet PCR products may not reflect the relative proportions of mixtures in the original material, particularly if competition for homologous binding sites occurs. Variation within multiple copies of a target sequence further influences analysis, and heterologous PCR products can present analytical challenges and interpretation bias to the final results (Smith et al., 2006). There has been a wide acceptance of 18S rRNA-based PCR-RFLP

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tools because they are sensitive and can detect and differentiate between a wide range of Cryptosporidium species/genotypes, but their usefulness for detecting mixed infections can be compromised by preferential PCR amplification of the dominant species/genotype in stool specimens (Cama et al., 2006). Increasing the number of replicates performed on a single sample can demonstrate the presence of multiple species/genotypes, where previously only one might have been detected from a single PCR. However, not all replicates will necessarily produce amplicons, probably because of the low number of DNA template copies present and the stochasm associated with pipetting small volumes of template (Ruecker et al., 2005). Strategies for identifying mixed species from a single slide using a nested 18S rRNA PCR-RFLP approach, regardless of species ratio, have included repetitive PCR analysis using either five replicates of the same volume (Ruecker et al., 2005) or five replicates of differing volumes of template (Xiao et al., 2001). In the Ruecker et al. (2005) study, the number of oocysts enumerated on slides was high (17, 24 and 27 oocysts per slide), which may have contributed to the success of this approach. Slides containing low oocyst numbers (1–10) are likely to present a greater challenge to the repetitive PCR-RFLP approaches described (Xiao et al., 2001; Ruecker et al., 2005). In repetitive 18S rRNA PCR-RFLP approaches, the predominant species/ genotype will be amplified preferentially, since the primers are genus-specific, whereas mixtures of species/genotype, present in equal amounts in a sample, are more likely to produce mixed RFLP patterns. We used a repetitive PCR-RFLP approach (five replicates of the same volume of template) of one nested 18S rRNA assay (Xiao et al., 1999, 2001) to determine the composition of species/ genotypes on individual slides, and found that the preferential amplification of a single species/genotype occurred in only 12% of the 25 slides analysed (Nichols et al., 2006b). This is probably due to stochastic sampling of low concentrations of species/genotype-specific DNA (Taberlet et al., 1996). The amplification of all of the mixed species/genotypes in our replicates occurred in 84% of the 25 slides analysed. Interestingly, amplicons were not related directly to the number of oocysts enumerated (range 1–260). We found the multi-PCR-RFLP approach of limited value in separating mixed RFLP patterns into single species/genotypes patterns in our samples. Furthermore, the availability of a restricted amount of DNA hinders repeat analysis. We feel that the use of a two-locus approach is a better option, particularly when large numbers of samples are analysed for epidemiological surveys. When discordant species/genotype RFLP results are obtained from the same slide using two assays based on different loci, all species/ genotypes detected should be recorded as present in the sample analysed. Preferential amplification of the dominant species/genotype is likely to be magnified with environmental samples; therefore, alternative molecular tools are required to determine the frequency of mixed environmental infections and their clinical and epidemiological relevance. Alternative strategies include the use of highly conserved sequences which are identically present throughout the genus Cryptosporidium and/or species-specific tools, and the use of reverse line blotting or microarrays to determine Cryptosporidium species and genotypes in individual environmental samples, as it amplifies DNA from all species within

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a genus in a single reaction. In reverse line blotting, specificity is conferred following hybridization of the amplicon to immobilized species/genotype-specific oligonucleotides (Smith et al., 2006)

Subtyping for disease and source tracking and risk assessment Molecular tools for inter- and intra-species discrimination differ. Genetic markers used to determine Cryptosporidium species/genotypes have insufficient polymorphism to be used to discriminate between isolates (Cacciò et al., 2005; Smith et al., 2006). Investigations into the transmission of genotypes and subtypes, identifying sources of infection and risk factors, require more discriminatory fingerprinting techniques, which can identify individual isolates or clonal lineages (Cacciò et al., 2005; Smith et al., 2006). A fundamental requirement for a typing system for effective tracking of sources of infection, routes of transmission and origins of disease outbreaks is an exquisite discriminatory power. The majority of human infections are caused by either C. hominis or C. parvum. Glycoprotein (GP)60 sequencing, analysis of a double-stranded RNA element and mini- and microsatellite typing have been used to subtype C. parvum and C. hominis, and might offer sufficient subspecies discrimination to address veterinary and public health investigations, either separately or in combination (reviewed in Cacciò et al., 2005; Smith et al., 2006). Recent work has confirmed the usefulness of GP60 sequencing and mini- and microsatellite markers in the study of the population structure of Cryptosporidium, and in understanding the transmission dynamics of infection (Enemark et al., 2002; Xiao et al., 2004; Cacciò et al., 2005; Smith et al., 2006; Grinberg et al., 2007). There is no subgenotyping system available for any of the other human-infectious Cryptosporidium species.

Conclusions Standardized methods are available for isolating and enumerating Cryptosporidium oocysts from water concentrates, which have increased molecular investigations into identifying the species, genotypes and subgenotypes present in water. With the increased interest in testing on foods, and the recent better availability of validated methods, it is expected that standardized methods will soon become available for foods. Methods that can genotype small numbers of Cryptosporidium oocysts reliably and reproducibly are required in order to determine which species/genotypes/subgenotypes are present, and with what frequency, in water and in/on foods. A standard, maximized method for DNA extraction from Cryptosporidium oocysts is essential both for detecting small numbers of oocysts and for evaluating the sensitivity of detection by PCR using different primers. In addition, genetic loci that maximize sensitivity must be used. There is no ‘standard’ genetic locus for determining species identity, but RFLP or sequencing of 18S rRNA gene loci provide information about more species than single-copy loci for small numbers

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of oocysts. The specificity of PCR primers, especially those designed for 18S rRNA loci, should be evaluated against organisms that are common contaminants of water and food matrices, particularly those that are closely related to Cryptosporidium phylogenetically. Mixtures of Cryptosporidium species/genotypes can be expected in water and food concentrates, and the use of genus-specific, 18S rRNA-based PCRRFLP tools can be compromised by preferential PCR amplification of the dominant species/genotype in the sample. Clearly, typing and subtyping systems used for human and non-human samples should also be used for environmental samples, particularly for source and disease tracking and risk assessment, in order to avoid any confusion arising from using different systems for human and nonhuman hosts and environmental samples for veterinary and public health investigation of disease outbreaks (Cacciò et al., 2005; Smith et al., 2006). Species determination should be based on the analysis of at least two loci, since this provides more robust information. Subgenotyping tools are only available for the two species (C. hominis and C. parvum) that cause the majority of human disease. These should offer sufficient subspecies discrimination to address veterinary and public health investigations.

Acknowledgements This work was supported by the Drinking Water Inspectorate, UK, the UK Food Standards Agency, and the Environmental and Rural Affairs Department, Agricultural and Biological Research Group, Scottish Executive, Scotland, UK.

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18

Intervention in Waterborne Disease

G. NICHOLS1, I.R. LAKE2, R.M. CHALMERS3, G. BENTHAM2, F.C.D. HARRISON2, P.R. HUNTER2, S. KOVATS4, C. GRUNDY4, S. ANTHONY5, H. LYONS5, M. AGNEW2 AND C. PROCTOR5 1Health

Protection Agency, London, UK; 2University of East Anglia, Norwich, UK; 3UK Cryptosporidium Reference Unit, Microbiology Swansea, UK; 4London School of Hygiene and Tropical Medicine, UK; 5ADAS Consulting, Wolverhampton, UK

Abstract Waterborne disease resulting from contaminated drinking and recreational waters is preventable. Since 2000 there has been a significant reduction in cryptosporidiosis in the first half of the year in England and Wales, but not in the second. This probably resulted from implementation of new drinking water regulations and substantial investment in water treatment. We estimate an annual reduction in disease of 905 reported cases and approximately 6700 cases in the population as a result of improvements in water treatment, most of which was caused by C. parvum. In order to investigate whether drinking water is still a risk factor for cryptosporidiosis a geographical information system (GIS)-based casecontrol study was conducted, investigating the role of drinking water, as well as wider environmental and socioeconomic factors, upon cryptosporidiosis. Detailed locations of 3368 cases post-2000 were compared to the location of an equal number of controls. Environmental datasets included socioeconomic status, the percentage of the population under 4 years old, water distribution, water treatment, rainfall, animal distribution, urban/ rural distribution, sewage outflows and estimated Cryptosporidium application to land. All cases were genotyped into species, enabling C. hominis and C. parvum to be examined separately. Illness was strongly associated with locations that on average had higher socioeconomic-status individuals, more children aged under 4 years, more livestock and areas with poorer water treatment. Strongly significant risk factors for C. hominis were areas with many higher socioeconomic-status individuals, areas with many children aged less than 4 years and urban areas. Strongly significant risk factors for C. parvum were rural location and a combination of Cryptosporidium in the water catchment and groundwater supply. These results highlight the fact that, in spite of the overall fall in cryptosporidiosis, drinking water is still a risk factor for cryptosporidiosis in England and Wales.

Introduction Cryptosporidium has been reported in England and Wales to national surveillance since 1983 and there have been over 93,000 reported cases. A study of © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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infectious intestinal disease conducted around 1998 suggested that for every case reported to national surveillance there are about 7.4 within the community that go undiagnosed (over 30,000 cases per year) (Adak et al., 2002). A more recent re-examination of the faecal samples from the original study using molecular methods (PCR) found a substantial number of cases that were undiagnosed using conventional microscopic methods (Amar et al., 2007). This implies that the burden of disease may be greater than previously recognized (Nichols et al., 2006). Within England and Wales the distribution of cases of cryptosporidiosis has followed a broadly bi-modal distribution with peaks in the spring and autumn (Fig. 18.1). However, when compared to the seasonality of other enteric pathogens, the distribution is much less even, much less predictable, more driven by outbreaks, and shows substantial differences between C. hominis and C. parvum. Infection is more common in young children than in adults, is more common in young boys than in girls under 15 years of age, and is more common in women of child-bearing age than in men (Nichols et al., 2006). Cryptosporidium spp. and genotypes from 13,112 faecal specimens typed in England and Wales using the Cryptosporidium oocyst wall protein (COWP) gene locus PCR has found approximately equal numbers of C. parvum (5981; 45.6%) and C. hominis (6594; 50.3%), with other species and unidentified isolates making up the remainder (Table 18.1) (Anonymous, 2002; Chalmers et al., 2002; Leoni et al., 2006; Nichols et al., 2006). The routes of transmission are broadly understood for the two main species but not for the minor species and genotypes (Nichols et al., 2006). Waterborne disease has been examined previously using a geographical information system (GIS) (Hughes et al., 2004; Lake et al., 2005; Abubakar et al., 2007; Hunter et al., 2007) and a study was set up to undertake an examination of data from England and Wales.

Cryptosporidium seasonality 1989–2004 Cryptosporidium cases per day of year (7 day rolling mean)

40 2001–2004 1997–2000 1993–1996

35 30

1989–1992 25 20 15 10 5 0 1

Fig. 18.1.

29

57

85

113 141 169 197 225 253 281 309 337 365 Day of year

Cryptosporidium seasonality in England and Wales 1989–2004.

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Table 18.1. Cryptosporidium species/genotypes detected in 13,112 human cases in England and Wales 1989–2005 (based on data from Anonymous, 2002; Chalmers et al., 2002; Leoni et al., 2006; Nichols et al., 2006; Nichols, 2008). Reproduced with kind permission of Springer Science and Business Media. Cryptosporidium species

No of patients

C. hominis C. parvum C. hominis and C. parvum C. meleagridis C. felis C. canis C. suis Cervine genotype Skunk genotype Horse genotype Undetermined genotypes

6,594 (50.29%) 5,981 (45.6%) 65 (0.5%) 99 (0.8%) 22 (0.2%) 2 (0.02%) 1 (0.01%) 6 (0.05%) 1 (0.01%) 1 (0.01%) 337 (2.6%)

Geographical Analysis A study using a GIS was developed to examine the impact of climate, socioeconomic status, employment, urban–rural, healthcare accessibility, animal distribution and water supply variables on the distribution of cryptosporidiosis (Lake et al., 2007b). The study used surveillance data based on all laboratory isolates together with data from the Cryptosporidium Reference Laboratory on the genotyping of isolates that contained postcoded information. The periodic prevalence of cryptosporidiosis in England and Wales, when examined in detail across the country, picked up local outbreaks when the area used was small and showed broad geographical differences that represented more cases in the west of the country than in the east. However, when examined in detail, it became clear that some of the geographical distribution could be accounted for by differences in the completeness of surveillance data. There is an underlying variability in different GPs’ policies for faecal testing and in the laboratories’ policy for testing for Cryptosporidium, together with problems with reporting to surveillance. Moreover, these variables change over time, as do the catchments which laboratories serve. A way around the problem was to look first at Salmonella and Campylobacter data from all the laboratories to determine catchments. GIS was used to draw up catchments (areas that each laboratory served) and to examine periods where the service was not occurring. The catchments were different from the health boundary data and better represented the geographical distribution of cases. Where laboratories served populations in areas close to each other the laboratories were grouped together into larger catchments (this was particularly the case in cities). The distribution maps probably reflected differences in the laboratory protocols more than the disease geography, thus suggesting that direct comparison of prevalence would not be an appropriate approach to take. However, there were data from a number of laboratories that looked robust over a number of

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years when the age distribution of cases was examined (no change in screening policy by age) and the catchment was looked at (no change in Salmonella and Campylobacter data). It was therefore possible to create a predictive model for the occurrence of Cryptosporidium cases for the period. Because climatic variability and community spread from imported travel cases are suggested as the main sources of this interannual variability, predictive models of weekly cryptosporidiosis cases were produced for different periods of the year using weekly incidence data (1989–1999) and national data on temperature, rainfall, river discharges and reported travel cases. Between mid-March and the end of June, cases of cryptosporidiosis were positively associated with river discharges (a surrogate for rainfall) 2 weeks previously. Between July and early September, cryptosporidiosis cases were positively associated with warm dry weather in the previous 2 months. No associations between cryptosporidiosis and weather existed at other times of the year. Travel cases were not significant in any of the models.

Impact of Intervention All cryptosporidiosis cases in England and Wales reported to national surveillance between 1989 and 2005 were analysed, with foreign travel-related cases excluded. The average weekly number of cryptosporidiosis cases pre-regulation (1989–1999) plotted against the same data post-regulation (2000–2005) showed that since the regulations were implemented (2000–2001) there has been less cryptosporidiosis in the first half of the year but more in the second half (Fig. 18.1; Table 18.2). Because the number of cases fluctuated from year to year both before and after the regulations, it is difficult to ascertain whether the changes post-regulation are part of the natural interannual variability or represent real changes in incidence. However, in the first half of the year there was a significant reduction (P < 0.05) in cryptosporidiosis compared with predicted levels for every year since 2000. The largest reduction in cases was in the first half of 2001, coinciding with a foot-and-mouth disease epidemic in the UK. Although the large reduction in cases in 2001 was attributed to this epidemic, our results suggest that cases were already reduced in the first half of 2000; these reductions continued into 2002, and the large reduction in the first half of 2001 was likely to have been due to improvements in drinking water treatment. The pattern of infection is less Table 18.2. Time period 1989–1992 1993–1996 1997–2000 2001–2004 Total

Cryptosporidium in England and Wales by 4-year periods. January to June 11,318 8,667 8,003 4,958 32,946

July to December

Total

12,324 10,517 11,036 11,763 45,640

23,642 19,184 19,039 16,721 78,586

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straightforward in the second half of the year, as the number of cases was significantly (P < 0.05) lower than predicted in 2001, 2002 and 2004, but significantly higher (P < 0.05) in 2000 and 2003. It has been suggested that the excess cases in the second half of 2000 and 2003 may represent travel cases or community spread from these. The average differences between observed and predicted cryptosporidiosis cases (excluding 2001 because of the possible confounding effect of the foot-and-mouth epidemic) indicates that there has been an annual average of 615 fewer reported cases between 2000 and 2005. The majority of the reduction was associated with treatment improvements to a single large supply that was previously unfiltered.

Case-control Study Using postcoded data on Cryptosporidium species detected in England and Wales between 2000 and 2003 from the Cryptosporidium Reference Laboratory, two separate case-control studies were conducted on C. hominis and C. parvum cases using geo-located environmental and socioeconomic factors instead of the questionnaire-derived responses commonly used in outbreak investigations. The postcode residential locations of C. parvum (n = 1623) and C. hominis (n = 1720) cases were compared to the location of an equal number of randomly sampled controls within the same laboratory service area. This allowed difficulties with ascertainment bias between the 135 laboratory service areas to be controlled for. These service areas were consequently large enough (106–1142 km2; average 3770 km2) to contain different drinking water sources and a range of agricultural and socioeconomic environments. Conditional logistic regression multivariable models were used to examine all Cryptosporidium infections, followed by separate models for C. hominis and C. parvum. A forward regression technique added the most significant variable in turn, and standardized forms of the independent variables were entered into the models. Colinearity was avoided by ensuring that the addition of each variable did not lead to significant changes in the coefficients or significance of any other model variable. Explanatory variables were derived for each case and control postcode. The ONS 2001 urban–rural classification groups census areas into one of eight categories ranging from ‘urban areas’ to ‘hamlets and isolated dwellings in sparse surroundings’. A 2.5 km buffer zone was created around each postcode and GIS was used to extract estimates of the total amount of Cryptosporidium in animal manures applied to land which were based on a 1 km2 map of manure applications developed by the Agricultural Land Advisory Service by combining information from the agricultural census, land use data, animal manure management surveys, and estimates of oocyst concentrations in manure. The estimates will consist almost exclusively of C. parvum. The water supplied to households can come from one or more water treatment works that, in turn, derive their source water from one or more abstraction points and are subject to different forms of water treatment. The parameters used to describe the water supply of each case and control postcode included the proportion of water supplied from surface or groundwater sources, different

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treatments (e.g. membrane filtration, simple disinfection) from the England and Wales Drinking Water Inspectorate, and any changes in treatment during the study period. Information was available on public water supplies only. The surface or groundwater abstraction catchments were calculated using GIS, and the density of Cryptosporidium applications to land, sewage discharges and sewage overflows were calculated for each area. Socioeconomic variables were obtained by identifying the 2001 Census Output Area within which each postcode was located and determining the percentage of people in each of the eight socioeconomic status bands, the proportion aged 0–4 years and the percentage employed in agriculture. A measure of healthcare accessibility was derived by calculating the travel time from each postcode to the nearest GP surgery. Areas with higher amounts of Cryptosporidium applied to land in a 2.5 km buffer around each postcode (OR 1.084 P = 0.022), a higher number of people in social classes 1–4 (OR 1.203 P < 0.001) and areas with more individuals aged under 4 years old (OR 1.145 P < 0.001) were significantly more common in all Cryptosporidium cases than in controls (Table 18.3). Drinking water that had superior water treatment (OR 0.770 P < 0.001) and groundwater-sourced drinking water (OR 0.821 P = 0.001) were negatively associated with risk (Table 18.3). Once these two drinking water variables were controlled for the proportion of the water supply which was from non-superiorly treated surface water sources, they were negatively associated with risk (OR 0.869 P = 0.019) (Table 18.3). For infections with C. hominis there were positive associations with living in an urban area (OR 1.261 P < 0.001), an area with a high proportion of individuals from social classes 1–4 (OR 1.297 P < 0.001), and many individuals in the 0–4 age group (OR 1.189 P < 0.001) (Table 18.4). For C. parvum, living in an urban environment was negatively associated with risk (OR 0.852 P < 0.001) (Table 18.5). Living in an area with higher Table 18.3. Multivariate model for all Cryptosporidium cases (Lake et al., 2007b). Reproduced with kind permission of Springer Science and Business Media. Variable

OR

95% CI

P value

Cryptosporidium to land in 2.5 km buffer

1.08

1.012–1.163

0.022

Percentage of population aged 0–4 years Percentage of population in highest socioeconomic groups (1–4)a Percentage of water supply subject to superior treatmentb

1.15

1.090–1.203

<0.001

1.2

1.140–1.270

<0.001

0.77

0.679–0.874

<0.001

Percentage of water supply that is groundwater 0.82 Percentage of water supply from non-superiorly treated surface water 0.87

0.729–0.925

0.001

0.772–0.977

0.019

R2 = 2.23%, estimated from 3368 case and 3368 control postcodes. a Working in managerial and professional occupations, intermediate occupations and small employers and own-account workers. b Defined as water treated with coagulation and flocculation, slow sand filtration, cartridge or membrane filtration.

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Table 18.4. Multivariate model for Cryptosporidium hominis cases (Lake et al., 2007b). Reproduced with kind permission of Springer Science and Business Media. Variable

OR

95% CI

P value

Percentage of population in urban area

1.261

1.154–1.378

<0.001

Percentage of population in highest socioeconomic groups (1–4)a Standardised proportion of population aged 0–4 years

1.297

1.201–1.401

<0.001

1.190

1.112–1.274

<0.001

R2 = 3.57, estimated from 1720 case and 1720 control postcodes.

Table 18.5. Multivariate model for Cryptosporidium parvum cases (Lake et al., 2007b). Reproduced with kind permission of Springer Science and Business Media. Variable

OR

95% CI

P value

Percentage of population in urban area Cryptosporidium to land in 2.5 km buffer

0.852 1.167

0.779–0.932 1.047–1.230

<0.001 0.005

Percentage of population aged 0–4 years

1.094

1.015–1.179

0.018

Percentage of population in highest socioeconomic groups (1–4)a Percentage of water supply subject to superior treatmentb Percentage of water supply that is groundwater

1.109

1.025–1.200

0.010

0.738

0.646–0.842

<0.001

0.679

0.554–0.833

<0.001

Interaction between % groundwater and C. parvum density in catchments Percentage of water supply from non superiorly treated surface water with high C. parvum density in catchment

1.289

1.088–1.527

<0.001

0.846

0.746–0.959

0.009

R2 = 4.24%, estimated from 1623 case and 1623 control postcodes. a Working in managerial and professional occupations, intermediate occupations and small employers and own account workers. b Defined as water treated with coagulation and flocculation, slow sand filtration, cartridge or membrane filtration.

amounts of Cryptosporidium applied to land in a 2.5 km buffer around each postcode (OR 1.167 P < 0.005), having a larger proportion of individuals in the 0–4 age group (OR 1.094 P = 0.018), and having more individuals in the highest socioeconomic status groups (OR 1.109 P = 0.010) were all positively associated with risk (Table 18.5). Drinking water with superior water treatment (OR 0.738 P < 0.001) and groundwater-derived drinking water (OR 0.679 P < 0.001) were negatively associated with risk (Table 18.5). Once these two drinking water variables were controlled for, two further variables became significant. An interaction between the proportion of groundwater supply and the amount of Cryptosporidium applied to land in catchments (OR 1.289 P <0.001) was positively associated with risk (Table 18.5). The proportion of the water supply which was not superiorly treated, from a surface water source with a high amount of Crypto-

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sporidium applied to land in the catchments, was negatively associated with risk (OR 0.846 P = 0.009) (Table 18.5).

Future for Intervention These two studies have demonstrated first that improvements within the water industry that coincide with the changes in regulation have been accompanied by reductions in human cases of cryptosporidiosis. A majority of this reduction was in the north-west of the region and was associated with large-scale investment in drinking water treatment in supplies that had previously received no filtration (Sopwith et al., 2005; Lake et al., 2007a). Regulation appears to have improved process control and many plants now operate with a lower turbidity warning level at which action is taken. At the start of the regulatory period, a number of supplies that failed the risk assessment were taken out of operation. Subsequent examination of problems with risk assessments have hopefully improved these, and water companies have been adopting WHO Water Safety Plans. The regulation has created a dataset of over 400,000 test results that provide an evidence base for understanding the contamination of treated drinking water with Cryptosporidium oocysts. The burden of illness attributed to drinking water prior to these changes was thus in excess of 600 detected cases per year and may be considerably higher than this when the multipliers of undetected cases are applied. However, the GIS case-control studies, which were conducted for the period 2000–2003, indicate that there still appears to be a burden of illness associated with mains drinking water, and that most of this is caused by C. parvum. However, there is a larger burden of illness that is in the second half of the year, where the infections are predominantly C. hominis and the evidence for a source is less clear. There is circumstantial descriptive evidence that swimming pools and travel may be important (Table 18.6). Table 18.6.

Outbreaks of cryptosporidiosis in England and Wales 1987–2005.

Suspected source Public water supply Private water supply Swimming pool Interactive water features Paddling pools Other recreational waters Animal contact Farm Foodborne Person-to-person Unknown Grand total

Outbreaks 55 6 43 3 2 2 16 3 4 10 5 149

Cases 7097 176 799 189 13 27 936 25 140 279 148 9829

(Confirmed) (5821) (30) (490) (70) (6) (12) (294) (19) (81) (116) (141) (7080)

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Swimming Pools There have been 149 outbreaks of cryptosporidiosis between 1983 and 2005, and 56 have been linked to mains water and 46 to swimming pools. Evidence for what management mistakes caused the drinking-water-related outbreaks have been documented (Nichols et al., 2006) and used for improving water treatment. For swimming pools, the problem with cryptosporidiosis results from contamination of pools by bathers and a failure of water treatment to effectively remove oocysts. While pre-swim showering is encouraged, better treatment is also needed. A number of outbreaks in England have been associated with pools where backwashing of filters has been conducted during pool use. During backwashing, the integrity of filters is temporarily disrupted and particulate material, including oocysts, can get into circulation. Backwashing should always be done after the pool has been closed to the public for the day so that any oocysts liberated into pool water through this process will be removed by overnight recirculation. There has been a problem with interactive water features that receive a greater burden of environmental contamination than standard swimming pools, but which often have poorer water treatment arrangements. It is likely that swimming pools, both municipal and those in holiday hotels, play an important role in the autumn increase in cases. There are several problems in intervening to reduce the autumn cases of cryptosporidiosis. There is relatively poor epidemiological evidence for what is causing these cases. If pools are important, the diffuse nature of the swimming pool industry makes intervening and obtaining funding for research difficult.

Conclusions Waterborne disease resulting from contaminated drinking and recreational water is preventable. Since 2000 there has been a significant reduction in cryptosporidiosis in the first half of the year in England and Wales but not in the second half. This probably resulted from the implementation of new drinking water regulations and substantial investment in water treatment. We estimate an annual reduction in disease of 905 reported cases and approximately 6700 cases in the population as a result of improvements in water treatment, most of which was C. parvum. Geographical analysis provides an additional tool for investigating the epidemiology of cryptosporidiosis. In order to conduct this sort of GIS analysis, geocoded or postcoded surveillance data is required. Detailed locations of 3368 cases post-2000 were compared with the location of an equal number of controls. Environmental datasets included socioeconomic status, the percentage of the population aged under 4 years, water distribution, water treatment, rainfall, animal distribution, urban–rural distribution, sewage outflows and estimated Cryptosporidium application to land. All cases were genotyped into species, enabling C. hominis and C. parvum to be examined separately. This approach appears to demonstrate differences in risk by social class and environment for C. parvum and C. hominis, confirming previous evidence that

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the epidemiology of these two species differs. Illness was strongly associated with locations that on average had higher socioeconomic status individuals, more children aged under 4 years, more livestock, and areas with poorer water treatment. Cryptosporidium hominis strongly significant risk factors were areas with many higher socioeconomic status individuals, areas with many individuals aged under 4 years, and urban areas. Cryptosporidium parvum strongly significant risk factors were rural location and a combination of Cryptosporidium in the water catchment and groundwater supply. These results highlight that, in spite of the overall fall in cryptosporidiosis, drinking water is still a risk factor for cryptosporidiosis in England and Wales. The significant reduction in disease associated with improved drinking water treatment that followed regulation suggests that there was a substantial burden of illness attributed to contaminated drinking water and the case-control studies suggest that there remains a burden of infection associated with drinking water. However, swimming pools appear to be an important source of infection, and tackling this may be more difficult.

References Abubakar, I., Myhill, D.J., Hart, A.R., Lake, I.R., Harvey, I., Rhodes, J.M., Robinson, R., Lobo, A.J., Probert, C.S.J. and Hunter, P.R. (2007) A case-control study of drinking water and dairy products in Crohn’s disease: further investigation of the possible role of Mycobacterium avium paratuberculosis. American Journal of Epidemiology 165, 776–783. Adak, G.K., Long, S.M. and O’Brien, S.J. (2002) Trends in indigenous foodborne disease and deaths, England and Wales: 1992 to 2000. Gut 51, 832–841. Amar, C.F., East, C.L., Gray, J., Iturriza-Gomara, M., Maclure, E.A. and McLauchlin, J. (2007) Detection by PCR of eight groups of enteric pathogens in 4,627 faecal samples: re-examination of the English case-control Infectious Intestinal Disease Study (1993–1996). European Journal of Clinical Microbiology and Infectious Diseases 26(5), 311–323. Anonymous (2002) The Development of a National Collection for Oocysts of Cryptosporidium. Final Report to DEFRA: Drinking Water Inspectorate. Foundation for Water Research, Marlow, Bucks, UK. Available at: http://www.fwr.org/. Chalmers, R.M., Elwin, K., Thomas, A.L. and Joynson, D.H. (2002) Infection with unusual types of Cryptosporidium is not restricted to immunocompromised patients. Journal of Infectious Diseases 185, 270–271. Hughes, S., Syed, Q., Woodhouse, S., Lake, I., Osborn, K., Chalmers, R.M. and Hunter, P.R. (2004) Using a Geographical Information System to investigate the relationship between reported cryptosporidiosis and water supply. International Journal of Health Geographics 3, 15. Hunter, P.R., Hadfield, S.J., Wilkinson, D., Lake, I.R., Harrison, F.C.D. and Chalmers, R.M. (2007) Subtypes of Cryptosporidium parvum in humans and disease risk. Emerging Infectious Diseases 13, 82–88. Lake, I.R., Bentham, G., Kovats, R.S. and Nichols, G.L. (2005) Effects of weather and river flow on cryptosporidiosis. Journal of Water and Health 3, 469–474.

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Lake, I.R., Nichols, G., Bentham, G., Harrison, F.C., Hunter, P.R. and Kovats, R.S. (2007a) Cryptosporidiosis decline after regulation, England and Wales, 1989–2005. Emerging Infectious Diseases 13, 623–625. Lake, I.R., Harrison, F.C., Chalmers, R.M., Bentham, G., Nichols, G., Hunter, P.R., Kovats, R.S. and Grundy, C. (2007) Case-control study of environmental and social factors influencing Cryptosporidiosis. European Journal of Epidemiology 22, 805– 811. Leoni, F., Amar, C., Nichols, G., Pedraza-Diaz, S. and McLauchlin, J. (2006) Genetic analysis of Cryptosporidium from 2414 humans with diarrhoea in England between 1985 and 2000. Journal of Medical Microbiology 55, 703–707. Nichols, G., Chalmers, R., Lake, I., Sopwith, W., Regan, M., Hunter, P., Grenfell, P., Harrison, F. and Lane, C. (2006) Cryptosporidiosis: A Report on the Surveillance and Epidemiology of Cryptosporidium Infection in England and Wales. Drinking Water Inspectorate Contract Number DWI 70/2/201. Drinking Water Inspectorate, London. Nichols, G. (2008) Epidemiology. In: Fayer, R. and Xiao, L. (eds) Cryptosporidium and Cryptosporidiosis. CRC Press. Sopwith, W., Osborn, K., Chalmers, R. and Regan, M. (2005) The changing epidemiology of cryptosporidiosis in north west England. Epidemiology and Infection 133, 785–793.

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Occurrence and Control of Naegleria fowleri in Drinking Water Wells

C.P. GERBA1, B.L. BLAIR1, P. SARKAR1, K.R. BRIGHT1, R.C. MACLEAN2 AND F. MARCIANO-CABRAL2 1University

of Arizona, Tucson, AZ, USA; 2Virginia Commonwealth University, Richmond, VA, USA

Abstract Naegleria fowleri is a water-based protozoan found naturally in soil and warm waters. The deaths of two children due to N. fowleri in the Phoenix, Arizona, metropolitan area occurred in 2002, and the drinking water obtained from groundwater was found to be the source of the exposure. A survey was conducted of municipal drinking water wells in central and southern Arizona. N. fowleri was identified in 11 of 143 wells tested. The calculated Ct (chlorine concentration × time) for N. fowleri cysts by free chlorine was 31 for a 99% reduction at room temperature, pH 7.5 and trophozoites 6. Chlorination can be used to control N. fowleri transmission via drinking water with appropriate guidance related to proper dosages and contact times.

Introduction Naegleria fowleri is a water-based parasite found in soil and warm water. Soil is believed to be the primary habitat for N. fowleri (Parija and Jaykeerthee, 1999). It can be found living free in warm bodies of water such as ponds, irrigation ditches, cattle tanks, lakes, coastal waters and hot springs. The organism occurs worldwide (Cabanes, 2001). The majority of identified cases have been reported in countries with tropical and subtropical climates (Parija and Jaykeerthee, 1999). Water sources in which N. fowleri has been detected include domestic water supplies, recreational waters, and thermally polluted runoff from industrial zones. In the USA, N. fowleri has been documented in surface waters in Virginia, Oklahoma, Florida, New Mexico and Arizona (Wellings et al., 1977; Lee et al., 2002; Marciano-Cabral et al., 2003). The deaths of two children due to N. fowleri in the Phoenix metropolitan area in 2002 promoted an interest in understanding its occurrence in well water in Arizona (Marciano-Cabral et al., 2003). No survey of well water had been conducted previously, and it was hoped 238

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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that a broad survey of this type would provide valuable information about the potential occurrence of N. fowleri in water systems using groundwater as a source. The presence of N. fowleri in drinking or bathing water is currently not regulated in the USA. Arizona has the largest number of unchlorinated municipal well-waterdependent systems in the USA (C. Graf, ADEQ, personal communication). Since the children’s deaths in Arizona were linked to unchlorinated well water used for drinking, it is important to develop guidance for the disinfection of N. fowleri to reduce the risk of transmission via drinking water and swimming pools. This was accomplished by developing Ct values/dose requirements for the inactivation of N. fowleri in drinking water. This information will be used to develop a guidance document for Arizona water utilities that use groundwater.

Pathology of Naegleria fowleri Humans and other mammals usually come into contact with N. fowleri via swimming, bathing, or in the case of cattle and domesticated animals, drinking from or swimming in water sources where N. fowleri is present. The organism is inhaled through the nose and then penetrates the nasopharyngeal mucosa and migrates to the olfactory nerves and eventually invades the brain through the cribriform plate (Bottone, 1993). The infection leads to the production of a toxin, which ends up liquefying brain tissue. The immune response leads to swelling and primary amoebic meningoencephalitis (PAM) (Marshall et al., 1997). N. fowleri is the only member of the genus known to be pathogenic to humans, although other amoeboid organisms such as Acanthamoeba spp. are potentially capable of causing similar infections (Cabanes, 2001).

Life Cycle of Naegleria fowleri Naegleria fowleri is an amoeboid flagellate with three morphological stages: trophozoite, flagellate and cyst. The active stage, causing PAM, is the trophozoite, which may be identified by its elongated amoeboid shape and long processes called lobopodia. This stage is motile, survives by engulfing and ingesting bacteria and other detritus in the environment, and can penetrate mucous membranes and digest mammalian tissues (Marshall et al., 1997; Parija and Jaykeerthee, 1999). The flagellate stage is formed when environmental factors such as temperature, food sources, pH, or organic levels are optimal, and is the infective stage in mammals. The flagellate is pear-shaped with multiple flagella and is quite motile, able to migrate up the nasal passages to infect the host. This stage can revert back and forth to the trophozoite stage within a 24 h period (Parija and Jaykeerthee, 1999). Adverse environmental conditions cause N. fowleri to encyst, to form a round, smooth, double-walled cyst approximately 10 µm in diameter (Marshall et al., 1997). Encystation prevents desiccation and enables survival in the soil for

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long periods. When washed into a warm body of water where the temperature is optimal, the organism excysts to become the flagellate stage, and later the feeding, trophozoite stage. While in the trophozoite stage, the organism multiplies by binary fission, known as promotosis (Parija and Jaykeerthee, 1999). Replication of the organism is possible in nasopharyngeal passages, brain tissues, water and soil.

Materials and Methods Hydrogeology and wells sampled This study targeted public drinking water supply wells located in the state of Arizona in the USA. These wells pump groundwater from extensive alluvial aquifers. The temperature of groundwater pumped from the wells varies depending on the depth of the well, the geothermal gradient in the area, the proximity to young buried volcanic rocks, and other factors. Data collected from 500 public water supply wells in the west and east Salt River basins show groundwater temperatures ranging from 13°C to 46°C. All the wells sampled in this study are high-volume public water supply production wells operated by municipal utilities or private water companies. These wells have all been designed and constructed in conformity with all applicable rules, regulations and well construction standards administered by the state of Arizona. Typical well discharges range from hundreds of litres per minute to over 3780 l/min. Although well casing diameters were not specifically recorded in this study, typical diameters for water production wells in these basins ranged from 15 to 61 cm. Well depths vary from several hundred metres to more than 300 m, depending on the depth to groundwater and the production interval selected for the well. The wells are equipped with either an electric submersible pump or a line-shaft turbine pump powered by an electric motor at the surface.

Sample collection Well water samples were collected at or near the wellhead prior to disinfection from September 2004 through to October 2005 from wells in central and southern Arizona. Samples collected in September 2004 were part of an initial survey to identify wells that might be positive for N. fowleri. During a second phase lasting from April 2005 through to October 2005, a subset of wells previously identified as positive or negative for N. fowleri were sampled bi-monthly. Additional selected wells were also sampled for the first time during the second phase. Samples were collected at ‘first flush’ (initial samples) and after each well had been purged three borehole volumes. Grab samples from well water systems were collected using 1 l sterile polyethylene bottles. This sample size was chosen because detection of the organism in this volume was considered to present a potential health risk (Esterman et al., 1984). In phase 1 (September 2004), samples were collected after wells were flushed until the water ran clear from the wellhead.

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A specific purge amount was not defined during this testing period. During phase 2 (April to October 2005), samples were collected as water was turned on from well spigots at or near wellheads initially, and after an approximate three borehole volume had flushed through the system, yielding an ‘initial’ sample and a ‘purged’ sample. Each 1 l sample was held at ambient outside air temperature for transfer to the laboratory.

Sample processing for PCR All samples were manually agitated gently for 2 min, centrifuged, and filtered through 2 µm pore size polyethylene filters (Millipore model L60043; Bedford, MA) to concentrate trophozoites and cysts. Concentrated samples were divided into 1 ml volumes and kept frozen in Page’s saline (Page, 1967) at 80°C for polymerase chain reaction (PCR) assays. Before PCR, samples were concentrated via centrifugation to a volume of 30 µl. Each sample was analysed using PCR a total of six times (triplicate tests for each water sample at the time of collection and triplicate tests following a 2 week incubation period at 37°C).

Polymerase chain reaction protocol Nested PCR was used to determine the presence of N. fowleri in the water concentrates, along with confirmation using genetic DNA sequencing from an independent laboratory. All PCR assays were performed in triplicate using the method of Reveiller et al. (2002) by amplifying a portion of a gene (Mp2C15) producing a protein unique to N. fowleri. Biosafety level II hoods (CBS Scientific, Del Mar, CA) were used to reduce the probability of contamination of oligoprimers (IDT, Coralville, IA) and amplification reagents. Samples were subjected to PCR amplification without prior genomic DNA extraction as per Marciano-Cabral et al. (2003). Cell suspensions were used as the source of genomic DNA rather than purified genomic DNA. The forward primer Mp2C15 (5′-TCTAGAGATCCAACCAATGC-3′) and the reverse primer Mp2c15 (5′-ATTCTATTCACTTCCACAATCC-3′) were used to amplify a 166 bp fragment of the Mp2C15 gene. PCR was performed in a 50 µl volume consisting of Taq DNA polymerase buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2), 0.2 mM concentration of each deoxynucleotide triphosphate (dnTP), 0.6 mM primer and 2.5 units of Amplitaq DNA polymerase (Perkin Elmer, Branchburg, NJ). To increase sensitivity, nested primers, Mp2C15 (5′-GTACATTGTTTTATTAATTTCC-3′) and Mp2C15 rev-in (5′-GGGTCTTTGTGAAAACATCACC-3′) were used to amplify a 110 bp fragment of Mp2C15. Both PCR and nested PCR reactions were performed using the following parameters: 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C for 35 cycles, using a Perkin Elmer PCR Systems 2400 thermal cycler (Perkin Elmer, Norwalk, CT).

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Amplified DNA was demonstrated on 1.5% Gene Pure agarose (Bio Whittaker Molecular Applications, Rockland, ME) via agarose gel electrophoresis (Model FB-SB-2318; Fisher Scientific, Fairlawn, NJ) and stained with ethidium bromide. Positive and negative controls consisted of cell suspensions (10 ng per reaction) of the organisms (N. fowleri ATCC 30894 and N. loveniensis ATCC 30174). Control organisms were grown in culture in the laboratory using the tissue culture method of Marciano-Cabral et al. (2003). Samples were stored and grown in liquid Oxoid modified Nelson’s media (Oxoid Ltd, Sparks, MD). Positive N. fowleri controls were made by the addition of ≤10 N. fowleri cysts to 1 l of sterile de-ionized water and filtered through 2 µm pore size polyethylene filters along with each batch of samples to ensure that filtering protocols were effective. Negative controls using N. loveniensis were made by the addition of 10 µl of cell suspensions from frozen cultures to 40 µl of PCR master mix solution and processed using the aforementioned PCR protocol to ensure that primers were replicating N. fowleri only.

Sequencing analysis Concentrated well water samples that tested positive and those testing negative for N. fowleri were frozen at −80ºC. These were then thawed and vigorously agitated to ensure that pellets were disrupted. Aliquots of 0.5 ml were placed into 1 ml cryovials and refrozen at −80°C for 48 h. Samples were coded for identification to prevent any bias and sent to Dr Francine Marciano-Cabral at the Commonwealth School of Medicine at Virginia Commonwealth University for verification and confirmation by gene cloning and sequencing.

Chlorine Disinfection Experiments Reagents and glassware Glassware was soaked overnight in a solution of at least 100 mg of free chlorine/l to make them chlorine demand-free (CDF). The glassware was then rinsed in CDF water, followed by baking for 2 h at 200°C. Free chlorine stock solution (125 mg/l) was prepared using reagent-grade 5.0% sodium hypochlorite (J.T. Baker Co., Phillipsburg, NJ) diluted in CDF water. The required free chlorine concentration was achieved by diluting the free chlorine stock solution in CDF water.

Culture maintenance A pure culture of N. fowleri (Lee strain, ATCC 30894) was maintained axenically in the laboratory in tissue culture flasks (Corning, Lowell, Massachusetts) in liquid Oxoid modified Nelson’s medium as described by Marciano-Cabral et al. (2003) with incubation at 37°C. Stock cultures were propagated by refreshing the medium every 5 days.

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Experimental procedure Disinfection experiments were conducted with both N. fowleri trophozoites and cysts. Trophozoites were harvested after 5 days of growth, but prior to cyst formation. Cyst suspensions were obtained by adding a 0.5% sodium dodecyl sulphate (SDS) solution to the axenic cultures to induce encystment (Cordingley et al., 1996; Dudley et al., 2005). All trophozoite and cyst suspensions were prepared according to methods previously described by Cassells et al. (1995). Chlorination and Ct were determined by methods previously described by our laboratory (Thurston-Enriquez et al., 2003) in well water samples obtained from municipal wells known to be contaminated with N. fowleri and in CDF phosphate buffer (3.8 mM Na2HPO4, 6.5 mM KH2PO4; pH 7.5). The well water samples were collected from typical production wells located in central Arizona. The concentration of free chlorine was measured at regular intervals during the course of the experiments. These experiments were conducted at the pH and temperature measured at the time of sample collection. Experiments were conducted in sterile chlorine demand-free glassware prepared by the methods mentioned previously. Cyst and trophozoite suspensions were counted using a haemocytometer to obtain appropriate concentrations. Disinfection systems were thoroughly mixed and 1 ml samples were removed at specific time intervals and placed into sterile 1.5 ml polypropylene microcentrifuge tubes (Bristol, CT) containing 10 µl of a neutralizing solution (14.6% sodium thiosulphate, 10% sodium thioglycollate, well water; filtered through a 0.2 µm pore size filter; Acrodisc, Gelman, Ann Arbor, MI). All experiments were conducted in triplicate. Chlorine concentration measurements were taken at the beginning and end of each experiment.

Assay procedure An adaptation of the most probable number (MPN) technique from the Standard Methods for the Analysis of Water and Wastewater (APHA, 1998) was used for the assay of viable N. fowleri immediately after the experiment was concluded (Cassells et al., 1995). Oxoid medium (2 ml) was added to each well of a 24-well tissue culture plate (Nunclon, Roskilde, Denmark). Samples were diluted in Page’s amoeba saline (Page, 1967) and 0.1 ml aliquots were added in triplicate to wells containing medium, and the plates were then incubated at 37°C. An inverted microscope was used to observe the culture plates for growth of N. fowleri for up to 10 days. The concentration of trophozoites/cysts per millilitre was then calculated by counting the number of positive-growth wells for each dilution using the MPN computational program devised by Hurley and Roscoe (1983).

Data Analyses The log10 reduction of the resulting number of trophozoites/cysts after disinfection was calculated as log10Nt/No where Nt and No are the final and

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initial concentrations of amoebae, respectively. The average log10Nt/No values were then incorporated in the EFH model and the kinetic modelling of all experiments was performed using the ‘solver’ function in Microsoft Excel 2000 (Microsoft Corporation) (Gyurek and Finch, 1998; Thurston-Enriquez et al., 2003). Chlorine decay constants, where k′ is the first-order disinfectant decay rate constant (per minute) for each experiment, were obtained in the same manner to regress the first-order kinetic equation using the least squares method: C = Co exp (−k′t)

(19.1)

where C, Co = disinfectant residual (mg/l) at time t and time zero, respectively. The MPN values for each experiment were grouped by the life stage of the organism and the time of inactivation. The values were then fitted into both the ChickWatson (Eqn 19.2) and the efficiency factor Hom (EFH) model (Eqn 19.3): Ln(Nt/No) = −k/k′n(Con − Cn) Ln(Nt/No) = kCon tm (1 − exp (−nk′t/m)(nk′t/m))m

(19.2) (19.3)

where k′ is the disinfectant decay constant, t is the time required to achieve a given level of inactivation, n is the coefficient of dilution k, and m stands for microbial inactivation constants. Ln(Nt/No) is the natural log of survival ratio (number of cysts/trophozoites remaining at time t divided by the number at time zero). The sum of squares of the difference between the observed and predicted Ln(Nt/No) was minimized using the ‘solver’ function of Microsoft Excel 2000 (Microsoft Corporation). The minimized sums of squares of differences were used to determine the values of coefficient of each model. The microbial inactivation curves were also plotted using Microsoft Excel to compare observed and predicted log inactivation values where the observed curve depicts the average log inactivation value of replicate bench-scale experiments versus sampling time.

Results The presence of N. fowleri DNA was confirmed by PCR in 16% of all samples tested and 8% of all wells sampled (Table 19.1). The calculated Ct of N. fowleri trophozoites by free chlorine was 6 for a 99% reduction at pH 7.5. The calculated Ct of the N. fowleri cysts by free chlorine was 31 for a 99% reduction at pH 7.5. All experiments were performed in triplicate and the Ct values were calculated using the efficiency factor Hom model. The Ct values for N. fowleri cysts are comparable to published values for Giardia cysts but lower than those for Cryptosporidium oocysts (Sobsey, 1989; Rose et al., 1997). The results of our research suggest that both the trophozoite and cyst forms of N. fowleri are fairly resistant to free chlorine.

Occurrence and Control of Naegleria fowleri in Drinking Water Wells Table 19.1.

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Occurrence of Naegleria fowleri in well water.

Sample versus wells Total number of samples tested (188) Number of wells tested (143)

Number of samples positive for N. fowleri

Percentage of samples positive for N. fowleri

29

16

11

8

Discussion No previous studies on the occurrence of N. fowleri have been conducted in groundwater, although its presence in surface waters is well documented. In this study, N. fowleri DNA was detected in 8% of the wells tested and 16% of the samples tested. N. fowleri DNA was most often detected after the wells had been purged three borehole volumes, suggesting that the organism was present in the aquifer or was released from the well casing or pump column during this process. N. fowleri naturally feeds on heterotrophic bacteria in water and biofilms and could be growing in the soil or the well casing. It has been reported that the oils used to lubricate well motors may result in the growth of high numbers of heterotrophic bacteria in well water (White and LeChevallier, 1993). These bacteria would serve as an ideal food source for N. fowleri and may explain its colonization of wells. Seasonal changes in temperature, weather, rainfall and soil erosion were not determined as part of this investigation, but may play a role in the organism’s ability to reproduce, to reach well sites, and to survive in wells. The PCR method used for the detection of N. fowleri in water did not determine whether the organisms were alive (infectious). While this method is able to determine differences at the species level by using primers specific for conserved regions of DNA, it is not capable of determining differences in life stage (cyst or trophozoite) in the samples. The trophozoite form is believed to be the infectious form of the organism; however, the presence of cysts in the environment can also be equally harmful because they are able to revert to the trophozoite stage under optimal conditions. The existing data suggest that both trophozoites and cysts are fairly resistant to chlorine (De Jonckheere and van de Voorde, 1976; Chang, 1978; Cursons et al., 1980; Cassells et al., 1995); however, none of these studies established Ct values for practical application because they did not take factors such as the concentration of substrate or disinfectant, the temperature, the pH, or the presence of interfering substances into consideration (Rice and Gomez-Taylor, 1986). Therefore, the disinfection efficacy of our study was modelled using a modification of Hom’s model to account for dynamic disinfectant concentrations (John et al., 2005). Through kinetic modelling, one can determine microbial reductions formed at comparable disinfectant doses. Kinetic modelling also takes into account the non-linearity of disinfection kinetics. Kinetic models have previously been used in the formulation of disinfection design criteria for water treatment

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(Gyurek and Finch, 1998; Thurston-Enriquez et al., 2003). In this study, we used the efficiency factor Hom model, which requires a description of the disinfectant decay kinetics to determine the Ct values for disinfection of N. fowleri. As demonstrated by this study, chlorination can be used as an effective method of control of N. fowleri transmission via drinking water with appropriate guidance related to required dosages and contact times.

Acknowledgements This study was funded by the National Science Foundation Water Quality Center at the University of Arizona and by the University of Arizona’s Water Sustainability Program.

References APHA (1998) Standard Methods for the Examination of Water and Wastewater, 20th edn. American Public Health Association, Washington, DC. Bottone, E.J. (1993) Free-living amebas of the genera Acanthamoeba and Naegleria: an overview and basic microbiologic correlates. Mt. Sinai Journal of Medicine 60, 260–270. Cabanes, P. (2001) Assessing the risk of primary amoebic meningoencephalitis from swimming in the presence of environmental Naegleria fowleri. Applied and Environmental Microbiology 11, 2927–2931. Cassells, J.M., Yahya, M.T., Gerba, C.P. and Rose, J.B. (1995) Efficacy of a combined system of copper and silver and free chlorine for inactivation of Naegleria fowleri amoebas in water. Water Science and Technology 31, 119–122. Chang, S.L. (1978) Resistance of pathogenic Naegleria to some common physical and chemical agents. Applied and Environmental Microbiology 35, 368–375. Cordingley, J.S., Wills, R.A. and Villemez, C.L. (1996) Osmolarity is an independent trigger of Acanthamoeba castellanii differentiation. Journal of Cellular Biochemistry 61, 167–171. Cursons, R.T.M., Brown, T.J. and Keys, E.A. (1980) Effect of disinfectants on pathogenic free-living amoebae in axenic conditions. Applied and Environmental Microbiology 40, 62–66. De Jonckheere, J. and van de Voorde, H. (1976) Differences in destruction of cysts of pathogenic and nonpathogenic Naegleria and Acanthamoeba by chlorine. Applied and Environmental Microbiology 31, 294–297. Dudley, R., Matin, A., Alsam, S., Sissons, J., Mahsood, A.H. and Khan, N.A. (2005) Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells. Acta Tropica 95, 100–108. Esterman, A., Roder, D.M., Cameron, A.S., Robinson, B.S., Walters, R.P., Lake, J.A. and Christy, P.E. (1984) Determinants of the microbiological characteristics of South Australian swimming pools. Applied and Environmental Microbiology 47, 325–328. Gyurek, L.L. and Finch, G.R. (1998) Modeling water treatment chemical disinfection kinetics. Journal of Environmental Engineering 124, 783–793.

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Hurley, M.A. and Roscoe, M.G. (1983) Automated statistical analysis of microbial enumeration by dilution series. Journal of Applied Bacteriology 55, 159–164. John, D.E., Haas, C.N., Nwachuku, N. and Gerba, C.P. (2005) Chlorine and ozone disinfection of Encephalitozoon intestinalis spores. Water Research 39, 2369–2375. Lee, S.H., Levy, D.A., Craun, G.F., Beach, M.J. and Calderon, R.L. (2002) Surveillance for waterborne-disease outbreaks: United States 1999–2000. MMWR Surveillance Summaries 51, 1–47. Marciano-Cabral, F., MacLean, R., Mensah, A. and LaPat-Polasko, L. (2003) Identification of Naegleria fowleri in domestic water sources by nested PCR. Applied and Environmental Microbiology 69, 5864–5869. Marshall, M.M., Naumovitz, D., Ortega, Y. and Sterling, C.R. (1997) Waterborne protozoan pathogens. Clinical Microbiological Reviews 10, 67–85. Page, F.C. (1967) Taxonomic criteria for limax amoebae, with descriptions of 3 new species of Hartmanella and 3 of Vahlkampfia. Journal of Protozoology 14, 499–521. Parija, S.C. and Jaykeerthee, S.R. (1999) Naegleria fowleri: a free living amoeba of emerging medical importance. Journal of Communicable Diseases 31, 153–159. Reveiller, F.L., Cabanes, P.A. and Marciano-Cabral, F. (2002) Development of a nested PCR assay to detect the pathogenic free-living amoeba Naegleria fowleri. Parasitology Research 88, 443–450. Rice, R.G. and Gomez-Taylor, M. (1986) Occurrence of by-products of strong oxidants reacting with drinking water contaminants: scope of the problem. Environmental Health Perspectives 69, 31–34. Rose, J.B, Lisle, J.T. and LeChevallier, M. (1997) Waterborne cryptosporidiosis: incidence, outbreaks, and treatment strategies. In: Fayer, R. (ed.) Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, FL, pp. 93–109. Sobsey, M.D. (1989) Inactivation of health-related microorganisms in water by disinfection processes. Water Science and Technology 21, 179–195. Thurston-Enriquez, J.A., Haas, C.N., Jacangelo, J. and Gerba, C.P. (2003) Chlorine inactivation of adenovirus type 40 and feline calicivirus. Applied and Environmental Microbiology 69, 3979–3985. Wellings, F.M., Amuso, P.T., Chang, S.L. and Lewis, A.L. (1977) Isolation and identification of pathogenic Naegleria from Florida lakes. Applied and Environmental Microbiology 34, 661–667. White, D.R. and LeChevallier, M.W. (1993) AOC associated with oils from lubricating well pumps. Journal of the American Water Works Association 85, 112–114.

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Environmental Factors Influencing the Survival of Cyclospora cayetanensis

Y.R. ORTEGA University of Georgia, Griffin, GA, USA

Abstract Cyclospora cayetanensis is a parasite responsible for severe diarrhoeal illness; particularly in children, the elderly and immunocompromised patients. In most instances, transmission has been water- and foodborne. The seasonality of Cyclospora in areas of endemicity is well defined, suggesting that it has specific requirements to survive and, under adequate conditions, may be able to start a new endemic cycle. Oocysts are highly resistant to pesticides (Captan 50% W.P., Benomyl 50% W.P., Diazinon 47.5%, Malathion 25% W.P. and Zineb 75% W.P) commonly used on farms and to sanitizers and disinfectants (Timsen, Tsunami, hydrogen peroxide, ammonium hydroxide and gaseous chlorine dioxide) used by the food industry. Although dogs have been epidemiologically associated with cases of cyclosporiasis, in a cohort study we report three dogs excreting oocysts, indicating that it is associated with the coprophagic habits of the dogs and not because of true infection. A survey of ten points of the sewer system serving a community of 450 people taken at different periods of time suggests that sewer testing could well be used as an indicator to determine whether the community has Cyclospora. The sewer information will reflect the Cyclospora activity in the community by assisting in identifying positive communities and to determine seasonality. The specific conditions required for this parasite’s viability in the environment are still not known.

Introduction Cyclospora cayetanensis is a coccidian parasite that causes diarrhoeal illness in humans and has been implicated in various foodborne and waterborne outbreaks. Cyclospora was first described in 1993 (Ortega et al., 1993); however, other reports describing structures morphologically similar were published earlier (Ashford, 1979; Long et al., 1990, 1991; Shlim et al., 1991; Hoge et al., 1993). Cyclosporiasis is characterized by the presentation of watery diarrhoea, anorexia, mild to severe nausea, abdominal cramping and fatigue. 248

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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The fate of Cyclospora oocysts when excreted in the environment is unknown. The life cycle of Cyclospora starts when an individual ingests Cyclospora oocysts from contaminated water or foods. The oocysts are then excysted in the gut of the individual and the sporozoites infect the intestinal epithelial cells of the jejunum and ileum. Asexual multiplication occurs within the parasitic vacuole followed by sexual multiplication. The zygote is then produced and unsporulated or immature oocysts are excreted in the faeces of the infected individuals. It has been reported that Cyclospora can infect the bile ducts and can cause acalculous colecystitis (de Górgolas et al., 2001; Zar et al., 2001). Cyclospora has also been reported in the sputum of infected individuals (Di Gliullo et al., 2000; Hussein et al., 2005). Once in the environment, these oocysts require 7–15 days to fully sporulate and potentially become infectious. In most samples only about 40% of the oocysts will sporulate when induced under laboratory conditions (Ortega et al., 1993). In addition, Cyclospora species present a marked seasonality in the endemic regions of the world and the environmental conditions of each are rather different. The environmental conditions required for these oocysts to sporulate are therefore very specific. This chapter examines the knowledge we currently have with respect to the environmental requirements of Cyclospora as well as the role of animals as reservoirs or vectors and the use of the sewer system to determine the endemicity of Cyclospora in this particular location.

Water- and Foodborne Transmission In 1990, nine housestaff physicians and one member of the administrative staff in a hospital in Chicago reported having watery diarrhoea, abdominal cramping, decreased appetite and low-grade fever, which was found to be caused by Cyclospora. Epidemiological investigation suggested that the probable source of contamination was the tap water from the physicians’ dormitory. Stagnant water from a storage tank could have contaminated the tap water after a pump failure (Huang et al., 1995). In 1994, 12 out of 14 British soldiers and their dependants stationed in Pokhara developed gastrointestinal illness after drinking water that was a combination of municipal and river water. The presence of Cyclospora was laboratory confirmed in six individuals. The residual chlorine levels in the water were between 0.3 and 0.8 ppm. Two litres of the water was concentrated and oocysts were identified (Rabold et al., 1994). Since 1995, foodborne outbreaks have also been reported in the USA and Canada. Sporadic cases of cyclosporiasis have been reported in travellers returning from areas of endemicity. Cases of Cyclospora have been associated with ingestion of imported berries, lettuce and basil (Herwaldt and Ackers, 1997; Caceres et al., 1998; Katz et al., 1999; Herwaldt and Beach, 1999; Doller et al., 2002; Ho et al., 2002; Hoang et al., 2005). In 2004, snow peas were also implicated (Anonymous, 2004). In 1997, more than 1400 individuals acquired Cyclospora and the implicated produce was raspberries produced in Guatemala (Herwaldt and Beach, 1999). This led to the recall of imported berries, a ban on the import of raspberries, and interventions in the farm and produce traceback

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procedure changes. Studies in the Guatemalan raspberry fields determined that irrigation water contained oocysts and could have been the source of contamination of the berries (Bern et al., 1999).

Seasonality, Temperature and Humidity Marked seasonality is one of the characteristics of Cyclospora in areas of endemicity. In Nepal, Cyclospora is found in a tropical region during the rainy season, corresponding to the months of March to July. The rainfall is between 50–350 mm and the ambient temperature is between 15 and 20°C (Hoge et al., 1993). In Guatemala, Cyclospora presents before the rainy season (May to September) with a rainfall between 60 and 275 mm and ambient temperatures of 20–22°C (Bern et al., 2000). In Morelia, Mexico, Cyclospora is found during the rainy season, during the months of July to September, and at an ambient temperature of 23°C (G.E. Orozco-Mosqueda, unpublished). In Lima, Peru, the conditions are rather different from the previous locations. Cyclospora is found during the summer. The endemic region is a desert where rainfall is less than 2 cm/year, and the ambient temperature is 17–22°C (Madico et al., 1997). ●

●

On the farm. Because Cyclospora oocysts were identified in river water used for the application of pesticides in Guatemala, we examined the effects of pesticides on oocyst sporulation. Captan 50% W.P., Benomyl 50% W.P., Diazinon 47.5%, Malathion 25% W.P. and Zineb 75% W.P. at higher and lower than the recommended doses were not effective in inactivating oocyst sporulation. Therefore, if oocysts are present in the water used for pesticide application, these oocysts could survive chemical treatment (Sathyanarayanan and Ortega, 2004). However, it has not been determined how long oocysts could remain viable on the fruits and vegetables and in what temperature and humidity conditions. In food matrices. Cyclospora oocyst sporulation occurs when incubated at 23°C. If stored at 4°C and then brought to 23°C, oocysts will sporulate successfully. If oocysts are incubated at 30°C or 37°C, they will not sporulate. Oocysts are inactivated when incubated at >50°C and <−15°C (Sathyanarayanan and Ortega, 2006).

Reservoirs and the Role of Dogs Unlike other coccidians that infect humans, Cyclospora oocysts require 7–15 days to sporulate. This interval leads to speculation that maybe animals play a role either as reservoirs or vectors. Cyclospora oocysts have been reported in the faeces of chickens, ducks and dogs (Zerpa et al., 1995; Yai et al., 1997; Chu et al., 2004), while other reports of animal surveys in endemic areas did not report the presence of Cyclospora oocysts (Ortega et al., 1997; Eberhard et al., 1999; Carollo et al., 2001). Attempts to infect laboratory animals have also been unsuccessful (Eberhard et al., 2000). As part of a cohort study in an endemic

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area of Lima, Peru, we identified three dogs excreting Cyclospora oocysts in the households where three children were identified with cyclosporiasis. Oocysts were identified only on 1 examination day and the following faecal samples were negative, suggesting that infection had not occurred. Dogs are well known to be coprophagic and this would explain why dogs were passing oocysts for 1 day (Y. Ortega, unpublished).

Sewers as Indicators The epidemiology of Cyclospora has been described in Nepal, Guatemala, Haiti, Peru and Mexico. Whether Cyclospora is endemic in other regions of the world still remains to be determined. In endemic areas, children and travellers are susceptible to acquiring cyclosporiasis, whereas in epidemic situations, individuals of all ages are susceptible to infection. Examination of a large number of clinical samples either in longitudinal or cross-sectional studies needs to be done to determine endemicity. We studied the validity of examining sewer samples as an indicator of the presence of pathogens, in this case Cyclospora. Sewer samples were collected monthly or bimonthly in the endemic area where a Cyclospora human cohort study was in progress. Cyclospora was identified in more sampling points during the months of January to May, which corresponds to the months of the high season for Cyclospora in this community. These results suggest that a community could be monitored to determine whether it is endemic for Cyclospora (Y. Ortega, unpublished).

Sanitizers and Disinfectants Parasites are well recognized for their resistance to environmental conditions and to a variety of sanitizers. Cyclospora is probably more resistant to those described for Cryptosporidium. Oocysts sporulate even after incubation for 15 min using N-alkyl dimethyl benzyl ammonium chloride (400 ppm) and peroxiacetic acidbased (40 µg/ml) sanitizers, hydrogen peroxide 0.5%, and chlorine dioxide gas 4.1 mg/ml (Y. Ortega, unpublished). Cyclospora adheres well to vegetables, and therefore recoveries may not be as efficiently accomplished as with other parasites such as Giardia and Cryptosporidium. For example, when cysts and oocysts are spot inoculated onto basil leaves, about 70–90% of the Giardia cysts can be recovered, 60–80% of Cryptosporidium oocysts, but only 40–60% of Cyclospora oocysts. Whether the cell wall composition may favour attachment needs to be determined. Surveys in endemic areas have demonstrated the presence of Cyclospora oocysts in a variety of vegetables (Ortega et al., 1997; Sherchand et al., 1999; Alakpa et al., 2003). In Peru, the largest amounts of produce contaminated with Cyclospora coincide with the months of high incidence of Cyclospora in the population. Moreover, this coincides with the presence of oocysts in irrigation water and soil samples from the region (Y. Ortega, unpublished).

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Hand washes of food handlers have previously been examined for the presence of foodborne pathogens. Upon examination of vegetable vendor hand washes, Cyclospora oocysts were also identified during the months of the high incidence of Cyclospora in the community (Y. Ortega, unpublished).

Conclusions Cyclospora is highly resistant to environmental conditions. It can resist pesticides, disinfectants and sanitizers, it attaches better to vegetables than other parasites. Whether reservoirs are needed to successfully reach another host still needs to be studied; however, it appears that dogs are not part of this cycle, as demonstrated by experimental attempts to reproduce the infection and by spurious passage of oocysts in dogs from the endemic area. Identification of areas of endemicity is time-consuming and requires significant resources. Examination of sewer samples may prove to be less expensive, simple and effective in identifying endemic communities. Much still remains to be learned about Cyclospora. The long period needed to sporulate in the environment, the specific conditions that favour the survival of these oocysts, and the evidence of marked seasonality all need to be investigated, as well as the host specificity. At present, experimental propagation of Cyclospora is not available, making it challenging to work with, as organisms can only be obtained from naturally infected individuals.

References Alakpa, G.E., Clarke, S.C. and Fagbenro-Beyioku, A.F. (2003) Cyclospora cayetanensis infection: vegetables and water as possible vehicles for its transmission in Lagos, Nigeria. British Journal of Biomedical Sciences 60, 113–114. Anonymous (2004) Outbreak of cyclosporiasis associated with snow peas: Pennsylvania, 2004. Morbidity and Mortality Weekly Report 53, 876–878. Ashford, R.W. (1979) Occurrence of an undescribed coccidian in man in Papua New Guinea. Annals of Tropical Medicine and Parasitology 73, 497–500. Bern, C., Hernandez, B., Lopez, M.B., Arrowood, M.J., de Mejia, M.A., De Merida, A.M., Hightower, A.W., Venczel, L., Herwaldt, B.L. and Klein, R.E. (1999) Epidemiologic studies of Cyclospora cayetanensis in Guatemala. Emerging Infectious Diseases 5, 766–774. Bern, C., Hernandez, B., Lopez, M.B., Arrowood, M.J., De Merida, A.M. and Klein, R.E. (2000) The contrasting epidemiology of Cyclospora and Cryptosporidium among outpatients in Guatemala. American Journal of Tropical Medicine and Hygiene 63, 231–235. Caceres, V.M., Ball, R.T., Somerfeldt, S.A., Mackey, R.L., Nichols, S.E., MacKenzie, W.R. and Herwaldt, B.L. (1998) A foodborne outbreak of cyclosporiasis caused by imported raspberries. Journal of Family Practice 47, 231–234. Carollo, M.C., Amato Neto, V., Braz, L.M. and Kim, D.W. (2001) Detection of Cyclospora sp oocysts in the feces of stray dogs in Greater São Paulo (São Paulo State, Brazil). Revista da Sociedade Brasileira de Medicina Tropical 34, 597–598.

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Chu, D.M., Sherchand, J.B., Cross, J.H. and Orlandi, P.A. (2004) Detection of Cyclospora cayetanensis in animal fecal isolates from Nepal using an FTA filter-base polymerase chain reaction method. American Journal of Tropical Medicine and Hygiene 71, 373–379. de Górgolas, M., Fortés, J. and Fernández Guerrero, M.L. (2001) Cyclospora cayetanensis cholecystitis in a patient with AIDS. Annals of Internal Medicine 134, 166. Di Gliullo, A.B., Cribari, M.S., Bava, A.J., Cicconetti, J.S. and Collazos, R. (2000) Cyclospora cayetanensis in sputum and stool samples. Revista do Instituto de Medicina Tropical de Sao Paulo 42, 115–117. Doller, P.C., Dietrich, K., Filipp, N., Brockmann, S., Dreweck, C., Vonthein, R., WagnerWiening, C. and Wiedenmann, A. (2002) Cyclosporiasis outbreak in Germany associated with the consumption of salad. Emerging Infectious Diseases 8, 992–994. Eberhard, M.L., Nace, E.K. and Freeman, A.R. (1999) Survey for Cyclospora cayetanensis in domestic animals in an endemic area in Haiti. Journal of Parasitology 85, 562–563. Eberhard, M.L., Ortega, Y.R., Hanes, D.E., Nace, E.K., Do, R.Q., Robl, M.G., Won, K.Y., Gavidia, C., Sass, N.L., Mansfield, K., Gozalo, A., Griffiths, J., Gilman, R., Sterling, C.R. and Arrowood, M.J. (2000) Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals. Journal of Parasitology 86, 577–582. Herwaldt, B.L. and Ackers, M.L. (1997) An outbreak in 1996 of cyclosporiasis associated with imported raspberries. Cyclospora Working Group. New England Journal of Medicine 336, 1548–1556. Herwaldt, B.L. and Beach, M.J. (1999) The return of Cyclospora in 1997: another outbreak of cyclosporiasis in North America associated with imported raspberries. Cyclospora Working Group. Annals of Internal Medicine 130, 210–220. Ho, A.Y., Lopez, A.S., Eberhart, M.G., Levenson, R., Finkel, B.S., da Silva, A.J., Roberts, J.M., Orlandi, P.A., Johnson, C.C. and Herwaldt, B.L. (2002) Outbreak of cyclosporiasis associated with imported raspberries, Philadelphia, Pennsylvania, 2000. Emerging Infectious Diseases 8, 783–788. Hoang, L.M., Fyfe, M., Ong, C., Harb, J., Champagne, S., Dixon, B. and Isaac-Renton, J. (2005) Outbreak of cyclosporiasis in British Columbia associated with imported Thai basil. Epidemiology and Infection 133, 23–27. Hoge, C.W., Shlim, D.R., Rajah, R., Triplett, J., Shear, M., Rabold, J.G. and Echeverria, P. (1993) Epidemiology of diarrheal illness associated with coccidian-like organism among travelers and foreign residents in Nepal. Lancet 341, 1175–1179. Huang, P., Weber, J.T., Sosin, D.M., Griffin, P.M., Long, E.G., Murphy, J.J., Kocka, F., Peters, C. and Kallick, C. (1995) The first reported outbreak of diarrheal illness associated with Cyclospora in the United States. Annals of Internal Medicine 123, 409–414. Hussein, E.M., Abdul-Manaem, A.H. and el-Attary, S.L. (2005) Cyclospora cayetanensis oocysts in sputum of a patient with active pulmonary tuberculosis: case report in Ismailia, Egypt. Journal of the Egyptian Society of Parasitology 35, 787–793. Katz, D., Kumar, S., Malecki, J., Lowdermilk, M., Koumans, E.H. and Hopkins, R. (1999) Cyclosporiasis associated with imported raspberries, Florida, 1996. Public Health Report 114, 427–438. Long, E.G., Ebrahimzadeh, A., White, E.H., Swisher, B. and Callaway, C.S. (1990) Alga associated with diarrhea in patients with acquired immunodeficiency syndrome and in travelers. Journal of Clinical Microbiology 28, 1101–1104. Long, E.G., White, E.H., Carmichael, W.W., Quinlisk, P.M., Raja, R., Swisher, B.L., Daugharty, H. and Cohen, M.T. (1991) Morphologic and staining characteristics of a cyanobacterium-like organism associated with diarrhea. Journal of Infectious Diseases 164, 199–202.

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Y.R. Ortega Madico, G., McDonald, J., Gilman, R.H., Cabrera, L. and Sterling, C.R. (1997) Epidemiology and treatment of Cyclospora cayetanensis infection in Peruvian children. Clinical Infectious Diseases 24, 977–981. Ortega, Y.R., Sterling, C.R., Gilman, R.H., Cama, V.A. and Diaz, F. (1993) Cyclospora species: a new protozoan pathogen of humans. New England Journal of Medicine 328, 1308–1312. Ortega, Y.R., Roxas, C.R., Gilman, R.H., Miller, N.J., Cabrera, L., Taquiri, C. and Sterling, C.R. (1997) Isolation of Cryptosporidium parvum and Cyclospora cayetanensis from vegetables collected in markets of an endemic region in Peru. American Journal of Tropical Medicine and Hygiene 57, 683–686. Rabold, J.G., Hoge, C.W., Shlim, D.R., Kefford, C., Rajah, R. and Echeverria, P. (1994) Cyclospora outbreak associated with chlorinated drinking water. Lancet 344, 1360–1361. Sathyanarayanan, L. and Ortega, Y. (2004) Effects of pesticides on sporulation of Cyclospora cayetanensis and viability of Cryptosporidium parvum. Journal of Food Protection 67, 1044–1049. Sathyanarayanan, L. and Ortega, Y. (2006) Effects of temperature and different food matrices on Cyclospora cayetanensis oocyst sporulation. Journal of Parasitology 92, 218–222. Sherchand, J.B., Cross, J.H., Jimba, M., Sherchand, S. and Shrestha, M.P. (1999) Study of Cyclospora cayetanensis in health care facilities, sewage water and green leafy vegetables in Nepal. Southeast Asian Journal of Tropical Medicine and Public Health 30, 58–63. Shlim, D.R., Cohen, M.T., Eaton, M., Rajah, R., Long, E.G. and Ungar, B.L. (1991) An alga-like organism associated with an outbreak of prolonged diarrhea among foreigners in Nepal. American Journal of Tropical Medicine and Hygiene 45, 383–389. Yai, L.E., Bauab, A.R., Hirschfeld, M.P., de Oliveira, M.L. and Damaceno, J.T. (1997) The first two cases of Cyclospora in dogs, São Paulo, Brazil. Revista do Instituto de Medicina Tropical de São Paulo 39, 177–179. Zar, F.A., El Bayoumi, E. and Yungbluth, M.M. (2001) Histologic proof of acalculous cholecystitis due to Cyclospora cayetanensis. Clinical Infectious Diseases 33, E140–E141. Zerpa, R., Uchima, N. and Huicho, L. (1995) Cyclospora cayetanensis associated with watery diarrhea in Peruvian patients. Journal of Tropical Medicine and Hygiene 98, 325–329.

21

Recent Advances in the Developmental Biology and Life Cycle of Cryptosporidium

N.S. HIJJAWI1, A.C. BOXELL2 AND R.C.A. THOMPSON2 1The

Hashemite University, Zarqa, Jordan; 2Murdoch University, WA, Australia

Abstract Cryptosporidium is an apicomplexan parasite that has gained much attention as a clinically important human pathogen since the late 1980s; however, little is known regarding the developmental biology of this parasite. Recent molecular and biological studies provide evidence that Cryptosporidium should be placed in a taxonomic group separate from the coccidia and closer to the gregarines (reviewed in Barta and Thompson, 2006). Furthermore, novel extracellular gregarine-like life cycle stages have been described. In addition to these findings, Hijjawi et al. (2004) reported the cell-free propagation of the life cycle of C. parvum, which also led to the identification of developmental stages similar to those observed in some gregarine species. The completion of the life cycle of Cryptosporidium in the absence of host cells raises many questions about the developmental nature of this parasite and its relationship to lower species of apicomplexans such as gregarines. This chapter covers recent observations on the developmental biology and life cycle of Cryptosporidium in an attempt to highlight more facts on the evolutionary biology of this unique parasite. Similarities between Cryptosporidium and some gregarine species are also included.

Introduction Cryptosporidium has emerged as a well-recognized cause of acute gastrointestinal disease in humans and animals throughout the world and is associated with a substantial degree of morbidity in immunocompromised individuals such as AIDS patients (Hunter and Nichols, 2002). The lack of a well-defined model of Cryptosporidium infection has severely hampered research into the biology and development of this important parasite. Several attempts by scientists to complete the life cycle and maintain propagation of this parasite in different cell lines has failed (see review by Hijjawi, 2003). Continuous culture of the parasite and the production of large numbers of developmental stages in vitro await elucidation on the right combination of growth conditions that stimulate and support the autoinfective cycle. © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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Observations of Cryptosporidium development in in vitro culture and in vivo in mice has led to the description of novel stages in its life cycle which have been shown to be similar to those observed in the gregarine life cycle (Hijjawi et al., 2002; Rosales et al., 2005). Furthermore, a novel finding has shown for the first time that C. parvum can complete its life cycle without the need for host cells (Hijjawi et al., 2004). Molecular phylogenetic studies consistently grouped Cryptosporidium as a separate clade from the other coccidian taxa with which it is recently classified (Carreno et al., 1999; Leander et al., 2003b). Biological and morphological studies have also highlighted similarities between Cryptosporidium and the gregarine group of protozoa (Hijjawi et al., 2002, 2004; Rosales et al., 2005; Barta and Thompson, 2006). Information gained from these studies suggests that Cryptosporidium is most closely related to some of the earliest diverging apicomplexan parasites, the archigregarines.

Cryptosporidium Life Cycle The ability to culture Cryptosporidium and to observe all life cycle stages without the need for host cells (Hijjawi et al., 2002, 2004) makes this technique ideal for following the development of the life cycle without host cell interference. Recent observations in cell-free culture have allowed the clear visualization of processes and life cycle stages that had not previously been described or witnessed. These include: (i) the visualization of aggregates of trophozoites in groups; (ii) two morphologically distinct meronts (I and II); and (iii) the direct observation of the fertilization process and the observation of novel life cycle stages.

Aggregates of Trophozoites Trophozoites have been observed to fuse into aggregates of two or more, and occasionally large aggregates containing 10–20 stages (Fig. 21.1B). Trophozoites within aggregates develop into meronts (meront I) of variable size depending on the number of initially fused trophozoites. This aggregation is a new observation in the life cycle development, and multiple mitotic divisions were considered to occur in single trophozoites following their penetration into host cells (Fig. 21.1C).

Two Morphological Types of Meronts There are two morphological types of meronts (meront I and meront II) (Hijjawi et al., 2002, 2004). Meront I appear as grape-like aggregates. Merozoites released from meront I are actively motile, circular to oval in shape and small in size (1.2 × 1 µm). These merozoites enlarge and clump together to generate type II meronts. The number of merozoites released appears to depend on the initial number of stages that fuse together (either trophozoites or merozoites released from meront I) to form these meronts. Meront II have a rosette-like pattern and

Developmental Biology and Life Cycle of Cryptosporidium

(A)

(B)

257

(C)

Fig. 21.1. (A) Sporozoites transformed to trophozoites upon their release from oocysts. (B) Trophozoites aggregate together in multiple numbers. (A) and (B): Cryptosporidium parvum life cycle in host-cell-free medium. (C) Single trophozoite in HCT-8 cell line 24 h post-inoculation. Scale bar = 5 µm.

(A)

(B)

Fig. 21.2. (A) Sporozoites approaching each other in multiple numbers after 24 h in cell-free culture. (B) Sporozoites fusing end to end in multiple numbers. Ziehl-Nielsen stain. Scale bar = 5 µm.

release type II merozoites which are either broadly spindle-shaped with pointed ends measuring 3.5 × 2 µm in size, or rounded to pleomorphic measuring 1.6 × 1.5 µm in size. In vitro studies have shown that the number of merozoites is variable and therefore is an unreliable means of differentiating between the two types of meronts.

Fertilization The fertilization process in Cryptosporidium had never been directly observed prior to a study by Hijjawi et al. (2004) and this may be attributed to two facts: (i) fertilization is a rapid process; and (ii) the interference of the host cells (in cell culture and in vivo infections) makes it difficult to observe this process occurring

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(A)

(B)

(C)

10 µm

(D)

Fig. 21.3. (A) Gamont-like stage purified from mice 72 h after infection with C. parvum. (B) Two gamont-like stages fused together with two big nuclei confirming their fusion. (C) Gamont-like stage grows bigger with time with numerous nuclei, in 5-dayold cell-free culture. Scale bar = 5 µm. (D) Immunofluorescence image of Cryptosporidium parvum oocysts, purified from murine faecal material, note the pointed gamont-like stage between oocysts (see http://www.hyperionlab.ca/protocols.html).

inside host cells. In cell-free culture, attempts at fertilization were observed many times (Hijjawi et al., 2002, 2004). Microgametes adhering to the surface of macrogamonts were frequently observed and on several occasions a macrogamont with a microgamete inside it was seen. Moreover, what appeared to be developing zygotes resembling unsporulated oocysts were observed, confirming that successful fertilization had occurred in this cell-free culture system (Hijjawi et al., 2002, 2004).

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Novel Life Cycle Stages Two types of novel life cycle stages have been observed in cell-free culture and in vivo. One of these stages can be described as a multinucleated mass which varies in size and has irregular margins. We believe that these may be formed from the aggregation and possible fusion of sporozoites (Fig. 21.2). Sporozoites have been observed to aggregate together in multiple numbers to form a large novel multinucleated mass. The other novel stage was referred as stage 1 in a previous study by Hijjawi et al. (2002). This stage was isolated from the gut contents of mice and frequently observed in cell-free culture (Fig. 21.3). This stage can be distinguished from other stages by having a large posterior nucleus, a rounded circular structure at the anterior end, and granular cytoplasm (Fig. 21.3A). This novel stage appears to enlarge over time and may possibly be regarded as a gamont stage (Fig. 21.3C); however, the origin and fate of this stage is not fully understood. The possible fusion of two gamont stages in a process similar to syzygy in gregarines was also observed (Fig. 21.3B). Interestingly, a similar gamont-like stage was obvious in an image of C. parvum oocysts which were purified from murine faecal material and stained with a commercially available immunofluorescent antibody. Oocysts have an intense apple green fluorescence on the periphery of their oocyst wall and measure 4–6 µm in diameter; however, the gamont-like stage appears elongated, granular, with a characteristic posterior nucleus (Fig. 21.3D).

Phylogeny of Cryptosporidium and its Closest Relatives Aseptate gregarines, particularly those infecting marine worms, are the most important lineage for understanding early apicomplexan evolution and the phylogeny of Cryptosporidium spp. (Leander et al., 2003a). Cryptosporidium was initially considered to be an unusual coccidian (Fayer et al., 1997); however, recent molecular phylogenetic analyses based on the SSU rRNA and several protein coding genes suggest that Cryptosporidium is an early-diverging apicomplexan with affinities to gregarines (Carreno et al., 1999; Leander et al., 2003b; Barta and Thompson, 2006). Cryptosporidium is similar to gregarines in many notable aspects described below and summarized in Table 21.1.

Infection and Location Within the Host Most Cryptosporidium and gregarine species infect epithelial cells of the host gastrointestinal tract and have non-motile zygotes. Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. In contrast, other members of the apicomplexan–protozoan pathogens are usually transmitted by an invertebrate vector or intermediate host and typically invade host cells by using a specialized apical complex.

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Table 21.1. Similarities between Cryptosporidium and some archigregarine species. Properties

Cryptosporidium

Archigregarines/Selenidiidae

Location within the host cell

Occurs in the intestine enterocyte brush border

Occurs in the intestine enterocyte brush border

Extracellular development

Yes

Yes

Presence of parasitiphorous vacuole

Double-membrane

Multi-membranous for Ditrypanocystis species

Presence of apicoplast

Absent

Absent in species studied

Syzygy-like pairing of extracellular stages

Present

Present

Life cycle

Monoxenous

Monoxenous

Life cycle developmental stages

3 schizogonies (merogony, gametogony and sporogony)

3 schizogonies (merogony, gametogony and sporogony)

Oocyst size and layers

2-layered wall

2-layered wall

7.4 × 5.6 µm (C. muris oocysts)

12–18 µm (Selenidium oocysts)

5.0 × 4.5 µm (C. parvum oocysts) No. of sporozoites/ oocyst

Four

Four

Life Cycle Development Cryptosporidium and many gregarines have life cycles with all three schizogonies (merogony, gametogony and sporogony) (Fayer et al., 1997; Carreno et al., 1999; Leander et al., 2003a). All three types of schizogony are present in the eimeriorins and haemosporins, and also in archigregarines and neogregarines; however, merogony is absent in the eugregarines and protococcidia (Levine, 1985). Similarities can be found when comparing life cycle development of Cryptosporidium with two Mattesia species of gregarine (Mattesia dispora and M. geminate) (Fig. 21.4). The behaviour of the sporozoites of M. dispora (Fig. 21.4B, highlighted in the area inside the box) is similar to observations of Cryptosporidium sporozoites in cell-free culture; the formation of circular trophozoites which aggregate together forming type I meronts (Fig. 21.4C, inside the box). Moreover, similarities in the shape of merozoites released from meront I and meront II is also obvious (Fig. 21.4B,C, inside circular area).

Sporozoite

a c1

c

b

Zygote

q

e

d

Trophozoite

b

a

t

c

? Fertilization

p

g

Meront I

s

Gamont-like extracellular stage

o

e1

d

r

Microgamete

Macrogamont

e h

n

g1

Late extrocellular stage

f m

10 µm k

g

l

i

i

k j

l

Microgamont

h

Meront II (early)

Merozoites type II

(A)

(B)

Merozoites type I

Developmental Biology and Life Cycle of Cryptosporidium

Oocyst

Meront II (late)

(C)

Fig. 21.4. (A) Mattesia geminata life cycle (Kleespies et al., 1997). (B) Mattesia dispora life cycle (Levine, 1985). (C) Cryptosporidium parvum life cycle in host-cell-free culture (Hijjawi et al., 2004).

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Oocyst Morphology Like colpodellids and archigregarines (Selenidium), Cryptosporidium have four infective sporozoites within each cyst, whereas all other gregarines have eight or more sporozoites per cyst (Leander et al., 2003b). Cryptosporidium oocysts are small (7.4 × 5.6 µm for C. muris and 5.0 × 4.5 µm for C. parvum) and lack morphological structures such as sporocyst, micropyle and polar granules. Oocysts of Selenidium are similar in shape to those of Cryptosporidium, and possess a two-layered wall 1 µm thick; however, they are larger in size (12–18 µm).

Cross-reactivity of Anti-Cryptosporidium Antibodies Bull et al. (1998) demonstrated that anti-Cryptosporidium monoclonal antibody cross-reacted with oocysts of a monocystid gregarine. It was suggested that the cross-reactivity was due to similar biological properties between the two parasites not shared by other apicomplexan parasites such as the coccidia (Carreno et al., 1999).

Host Cell Interaction There is a unique association of Cryptosporidium with the host cell. The intracellular but extracytoplasmic niche of Cryptosporidium within the host and the attachment of Cryptosporidium within host cells via the feeder organelle are different from all known coccidians. Furthermore, recent molecular and ultrastructural characterization of the marine aseptate gregarines Selenidium and Lecadina (Leander et al., 2003b) provide support for the sister relationship between Cryptosporidium and the gregarines. A recent study by Butaeva et al. (2006) showed similarities in the location in the host cell and the feeding behaviours between Cryptosporidium and the archigregarine Ditrypanocystis (Fig. 21.5). The double-membrane parasitiphorous vacuoles of Cryptosporidium and the multi-membranous vacuoles of Ditrypanocystis appear to be similarly formed, with the involvement of analogous enterocyte outputs, microvilli and cilia (Butaeva et al., 2006). No cases have been reported of a complete enclosing of gregarines within a parasitiphorous envelope made of clustered or fused cilia. Such a type of host parasite interaction is characteristic of the only coccidian genus Cryptosporidium (see Fig. 2 in Barta and Thompson, 2006). Once attached to the top of the enterocyte, zoites of Cryptosporidium stimulate an additional growth of microvilli which eventually fuse around the parasitic cell (Beyer et al., 2000). Both Cryptosporidium (Fig. 21.5A) and Ditrypanocystis sp. (Fig. 21.5B) form a specialized host–parasite interface, reflecting similar modes of adaptations to parasitic survival in similar sites of locations in the enterocyte brush border.

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mvi

mvo mvi cc

om

ci

mv

n

pv om im fo pv smt

aa

f mch

hc (A)

hc

aa hc

hc

hc

(B)

Fig. 21.5. Comparative schematic representation of model of extracytoplasmic intracellular localization in Cryptosporidium and Ditrypanocystis sp. (A) Cryptosporidium in the gut of suckling mouse (from Goebel and Braendler, 1982) possessing a double-membrane parasitophorous vacuole of fused microvilli preserving filaments; feeder organelle is seen in the region of the contact. (B) Ditrypanocystis sp. in the gut of an oligochaete worm possessing a multimembranous parasitophorous vacuole of fused and clusterized cilia lacking microtubules, see membranous structures at the region of contact. aa – attachment area, f – microfilaments of villi, fo – feeder organelle, mv – microvilli, mvi – inner membrane of parasitophorous vacuole, mvo – outer membrane of parasitophorous vacuole, pv – parasitophorous vacuole (Butaeva et al., 2006).

Phylogenetic Similarities Analysis of the complete genome of C. parvum and C. hominis identified neither a plastid genome nor genes with putative plastid-targeting sequences (Abrahamsen et al., 2004; Xu et al., 2004). Furthermore, plastid-like structures have not been revealed through microscopy of Cryptosporidium (Riordan et al., 2003). Most apicomplexans, including Plasmodium, Toxoplasma and Eimeria, possess both plastids and corresponding plastid genomes. Studies on some gregarine species also failed to reveal the presence of an apicoplast (Valigurova and Koudela, 2005; Toso and Omoto, 2007). A recent ultrastructural study of the eugregarine Leidyana failed to find an apicoplast (Valigurova and Koudela, 2005). Moreover, an extensive ultrastructural investigation of the species Gregarina niphandrodes also failed to find evidence of the presence of an apicoplast (Toso and Omoto, 2007). These findings raise the possibility that the plastid was lost in the apicomplexans following the divergence of the gregarines and Cryptosporidium, and further complement the close relationship between the two.

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Concluding Remarks The phylogenetic affinities of Cryptosporidium are intriguing and require reevaluation in the light of developmental, biochemical and genomic data. Cryptosporidium is traditionally considered as one of the coccidian protists (i.e. a taxonomic sister to the intestinal and cyst-forming apicomplexans). In light of these observations of previously undescribed life cycle processes and novel life cycle stages using a cell-free culture system, it is quite evident there is little known about the developmental biology of this parasite. Further research using cell-free culture may unveil the capability of this ambiguous parasite to amplify under different conditions. By combining current and future developmental and phylogenetic studies of this parasite, a clearer understanding of its position within the Apicomplexa and its closeness to the gregarine species can be achieved.

References Abrahamsen, M.S., Templeton, T.J., Enomoto, S., Abrahante, J.E., Zhu, G., Lancto, C.A., Deng, M., Liu, C., Widmer, G., Tzipori, S., Buck, G.A., Xu, P., Bankier, A.T., Dear, P.H., Konfortov, B.A., Spriggs, H.F., Iyer, L., Anantharaman, V., Aravind, L. and Kapur, V. (2004) Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304, 441–445. Barta, J.R. and Thompson, R.C. (2006) What is Cryptosporidium? Reappraising its biology and phylogenetic affinities. Trends in Parasitology 22, 463–468. Beyer, T.V., Svezhova, N.V. Sidorenko, N.V. and Khokhlov, S.E. (2000) Cryptosporidium parvum (Coccidia, Apicomplexa): some new ultrastructural observations on its endogenous development. European Journal of Protistology 36, 151–159. Bull, S., Chalmers, R., Sturdee, A.P., Curray, A. and Kennaugh, J. (1998) Cross-reaction of an anti-Cryptosporidium monoclonal antibody with sporocysts of Monocystis species. Veterinary Parasitology 77, 195–197. Butaeva, F., Paskerova, G. and Entzeroth, R. (2006) Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae): the mode of survival in the gut of Enchytraeus albidus (Annelida, Oligochaeta, Enchytraeidae) is close to that of the coccidian genus Cryptosporidium. Tsitologiya 48, 695–703. Carreno, R.A., Martin, D.S. and Barta, J.R. (1999) Cryptosporidium is more closely related to the gregarines than to coccidia as shown by phylogenetic analysis of apicomplexan parasites inferred using small-subunit ribosomal RNA gene sequences. Parasitology Research 85, 899–904. Fayer, R., Speer, C.A. and Dubey, J.P. (1997) The general biology of Cryptosporidium. In: Fayer, R. (ed.) Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, FL, pp. 1–42. Goebel, E. and Braendler, U. (1982) Ultrastructure of microgametogenesis, microgametes and gametogony of Cryptosporidium spp. in the small intestine of mice. Protistologica 18, 331–344. Hijjawi, N.S. (2003) In vitro cultivation and development of Cryptosporidium in cell culture. In: Thompson, R.C.A., Armson, A. and Morgan-Ryan, U.M. (eds) Cryptosporidium: From Molecules to Disease. Elsevier, Amsterdam, pp. 233–253. Hijjawi, N.S., Meloni, B.P., Ryan, U.M., Olson, M.E. and Thompson, R.C.A. (2002) Successful in vitro cultivation of Cryptosporidium andersoni: evidence for the existence

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of novel extracellular stages in the life cycle and implications for the classification of Cryptosporidium. International Journal for Parasitology 32, 1719–1726. Hijjawi, N.S., Meloni, B.P., Ng’anzo, M., Ryan, U.M., Olson, M.E., Cox, P.T., Monis, P.T. and Thompson, R.C.A. (2004) Complete development of Cryptosporidium parvum in host cell-free culture. International Journal for Parasitology 34, 769–777. Hunter, P.R. and Nichols, G. (2002) Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clinical Microbiology Reviews 15, 145–154. Kleespies, A., Junger, M., Buschinger, A., Nahring, S. and. Schumann, R.D. (1997) Studies on the life history of a neogregarine parasite found in Leptothorax ants from North America. Bio-control Science and Technology 7, 117–129. Leander, B.S., Clopton, R.E. and Keeling, P.J. (2003a) Phylogeny of gregarines (Apicomplexa) as inferred from small-subunit rDNA and B-tubulin. International Journal of Systematic and Evolutionary Microbiology 53, 345–354. Leander, B.S., Harper, J.T. and Keeling, P.J. (2003b) Molecular phylogeny and surface morphology of marine aseptate gregarines (Apicomplexa): Selenidium spp. and Lecudina spp. Journal of Parasitology 89, 1191–1205. Levine, N.D. (1985) Phylum II: Apicomplexa Levine (1970). In: Lee, J.J., Hunter, S.H. and Bovee, E.C. (eds) Illustrated Guide to the Protozoa, Society of Protozoologists, Lawrence, KS, pp. 322–374. Riordan, C., Ault, J., Langreth, S. and Keithly, J. (2003) Cryptosporidium parvum Cpn60 targets a relict organelle. Current Genetics 44, 138–147. Rosales, M.J., Perez-Cordon, G., Sanchez-Moreno, M., Marin-Sanchez, C. and Mascaro, C. (2005) Extracellular like-gregarine stages of Cryptosporidium parvum. Acta Tropica 95, 74–78. Toso, M.A. and Omoto, C.K. (2007) Gregarina niphandrodes may lack both a plastid genome and organelle. Journal of Eukaryotic Microbiology 54, 66–72. Valigurova, A. and Koudela, B. (2005) Fine structure of trophozoites of the gregarine Leidyana ephestiae (Apicomplexa: Eugregarinida) parasitic in Ephestia kuehniella larvae (Lepidoptera). European Journal of Protistology 41, 209–218. Xu, P., Widmer, G., Wang, Y., Ozaki, L.S., Alves, J.M., Serrano, M.G., Puiu, D., Manque, P., Akiyoshi, D., Mackey, A.J., Pearson, W.R., Dear, P.H., Bankier, A.T., Peterson, D.L., Abrahamsen, M.S., Kapur, V., Tzipori, S. and Buck, G.A. (2004) The genome of Cryptosporidium hominis. Nature 431, 1107–1112.

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Basic Biology of Giardia lamblia: Further Studies on Median Body and Funis

M. BENCHIMOL Universidade Santa Úrsula, Rio de Janeiro, Brazil

Abstract Giardia lamblia is a parasitic protozoan that infects thousands of people all over the world, causing a disease known as giardiasis. Giardia trophozoites are tear-shaped cells with two nuclei located in the anterior region of the cell body. Giardia is an amitochondrial flagellate and possesses a complex cytoskeleton based on several microtubular systems. In the interphase, these microtubules include the axonemes of the eight flagella, the median body and the funis, both formed by sets of microtubules, and the ventral adhesive disc built on a helicoidally turned layer of parallel microtubules. Among its components the median body and funis are the least defined microtubular structures. The use of FESEM (field emission scanning electron microscopy) allowed the elucidation of some aspects of the funis, median body and the encystation process. The removal of the plasma membrane and observation by FESEM allowed detailed observations of the median body, resulting in the following findings: (i) it varied in number, shape and position; (ii) it was found in mitotic and interphasic trophozoites; (iii) it was present in about 80% of the cells examined; (iv) it can be connected either to the plasma membrane, to the adhesive disc or to the caudal flagella, and thus it is not completely free in the cells; and (v) it can protrude from the cell surface. Concerning the funis, the following observations were made: (i) it is made of short arrays of microtubules emanating from the axonemes of the caudal flagella, which are anchored to dense rods that run parallel to the posterior-lateral flagella; (ii) after emergence of the posterior-lateral flagella, funis microtubules are anchored to the epiplasm, a fibrous layer that underlies the portion of membrane.

Introduction The diplomonad Giardia lamblia is a parasitic protozoan cell that infects thousands of people all over the world, causing a disease known as giardiasis. The trophozoite form of G. lamblia has a characteristic tear-shaped body, 12–15 µm long and 5–9 µm wide (Figs 22.1–22.3). The ventral disc is composed of a single layer of 250 nm microtubules, forming an asymmetrical spiral, which is very distinct in G. lamblia and G. muris but not as distinct in G. agilis. When observed by routine transmission or scanning electron microscopy (SEM), Giardia 266

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Fig. 22.1. G. lamblia images observed by light microscopy after Panotic staining. Two nuclei (N), the median body (MB) and flagella can be seen, as well as the cell size and shape. Note that in some cells the median body is not clearly visible. Occasionally, condensed chromosomes can be depicted. Bar = 2 µm (M. Benchimol, unpublished).

Fig. 22.2. Scanning electron microscopy of G. lamblia: (a), in trophozoite form (Piva and Benchimol, 2004); (b), as a cyst (M. Benchimol, unpublished ). D denotes disc. Bars = 1 µm.

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BB N

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Fig. 22.3. Schematic diagram showing the arrangements of cell structures in the interphase Giardia. Note the disc (D) situated at the ventral region, the two nuclei (N), the flagella (AF, PLF, VF, CF) and basal body pairs (BB). The median body (MB) is seen transversely to the axonemes, and the funis (F) is composed of microtubules connecting the central axonemes to the postero-lateral flagella axonemes. AF, anterior flagella; PLF, posterior-lateral flagella; VF, ventral flagella; CF, caudal flagella; PV, peripheral vesicles. Bar = 1 µm.

trophozoites showed a half-pear or teardrop-shaped body (Fig. 22.2a). The ventral disc was seen in the anterior region of the cell and was laterally and anteriorly surrounded by the marginal groove and the ventrolateral flange (Figs 22.2–22.4). Giardia presented four pairs of flagella, namely the anterior, posterior-lateral, caudal and ventral (Figs 22.1–22.4). Giardia presents a unique cytoskeleton in which the protein tubulin predominates, especially in the following structures: four pairs of flagella, an adhesive disc (Figs 22.5, 22.6) composed of microtubules and micro-ribbons containing giardins (Fig. 22.7), a median body (Figs 22.8–22.11), and a funis (Figs 22.14, 22.15) made up of sheets of microtubules following the axonemes of the caudal flagella (Figs 22.16, 22.17) (Erlandsen and Feely, 1984; Kulda and Nohýnková, 1995; Upcroft and Upcroft, 1998; Campanati et al., 2002).

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Fig. 22.4. Routine preparation for transmission electron microscopy of G. lamblia trophozoite showing the two nuclei (N), some of the eight flagella (F), flagella axonemes (A), disc (D), profiles of the endoplasmic reticulum (ER) and peripheral vesicles (PV). Bar = 1 µm (M. Benchimol, unpublished).

The origin point of the basal bodies of the eight flagella lies deep in the cell body; this is also the origin of the funis, a microtubular array that follows the axonemes of the caudal flagella and is involved in cell movement.

Fig. 22.5. Routine preparation for transmission electron microscopy of G. lamblia trophozoites showing a bundle of microtubules that constitutes the median body (MB), flagella (F) and peripheral vesicles (PV). Bar = 1 µm (M. Benchimol, unpublished).

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Fig. 22.6. The FESEM (field emission scanning electron microscopy) image of Giardia after detergent extraction. The plasma membrane was partially removed, allowing observation of the cytoskeleton, with the median body (MB) and the funis (Fn) included. The ventral disc (D), the two nuclei (N), MB, anterior (a), caudal (c) and posterior-lateral flagella (P) are seen. Note that every fascicle that constitutes the MB is observed. Bar = 1 µm (from Piva and Benchimol, 2004).

The trophozoite form of this protist lacks the organelles usually found in higher eukaryotes, such as mitochondria and peroxisomes (Gillin et al., 1996), although Tovar et al. (2003) demonstrated that Giardia contains mitochondrial remnant organelles (mitosomes) bounded by double membranes that function in iron–sulphur protein maturation. This finding indicates that Giardia is not primitively amitochondrial and that it has retained a functional organelle derived from the original mitochondrial endosymbiont. Mitosomes are localized in small cellular structures distributed throughout the cytoplasm, including an accumulation of organelles around basal bodies. Structures such as the Golgi complex seem to be absent in trophozoites (Reiner et al., 1990; Luján et al., 1995), although this is controversial (LanfrediRangel et al., 1999). Giardia also presents a system of peripheral vesicles (Figs 22.4, 22.5) which correspond to early and late endosomes and to lysosomes (Lanfredi-Rangel et al., 1998). Giardia is an intriguing parasite, since it has an unusual morphology in having two nuclei (Figs 22.1, 22.3, 22.6).

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Fig. 22.7. Immunofluorescence microscopy localization of β-giardin in Giardia, using the monoclonal antibody 7G9. The disc presents positive labelling as well the median body (arrowheads). (a) DIC visualization; (b) immunofluorescence; (c) overlay. Bar = 2 µm (from Piva and Benchimol, 2004).

Fig. 22.8. Scanning electron microscopy (SEM) of G. lamblia after detergent treatment. (a, b) The median body (arrow) can be seen protruding from the plasma membrane. Two or more fascicles are clearly seen. Bar = 1 µm (from Piva and Benchimol, 2004).

Fig. 22.9. Scanning electron microscopy (SEM) of G. lamblia after detergent treatment.The median body (arrow) is seen in different positions, with different numbers and shapes. (b) The median body occupies the whole cell width. Two or more fascicles are clearly seen. Bar = 1 µm (from Piva and Benchimol, 2004).

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Fig. 22.10. Scanning electron microscopy (SEM) of G. lamblia after detergent treatment. The median body (arrow) can be seen in close proximity to one of the nuclei. Two or more fascicles are clearly seen. N, nucleus. Bar = 1 µm (from Piva and Benchimol, 2004).

Fig. 22.11. Giardia trophozoite observed by FESEM (field emission scanning electron microscopy) after detergent extraction. The plasma membrane was removed, allowing observation of the median body (MB). Note that every fascicle that constitutes the MB is observed. Posterior-lateral axoneme, P; caudal axoneme, C. Bar = 1 µm (from Piva and Benchimol, 2004).

Previous studies have suggested that the peripheral vesicles of Giardia may correspond to early and late endosomes and to lysosomes (Lanfredi-Rangel et al., 1998). Internal vesicles of unknown function have also been described (Benchimol, 2002). There are several questions still open concerning the basic biological properties of Giardia, and one fundamental question is its process of division, which is still under debate. While studies on molecular aspects of gene organization and expression in Giardia have advanced rapidly over recent years, there are few papers dealing with morphological aspects of Giardia. Among these, the behaviour and function of the median body and the funis are still under debate.

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Median Body The most striking feature of Giardia is the presence of a complex and unique cytoskeleton, and among its components the median body (MB) is the least welldefined microtubular structure. In the early literature the MB of G. lamblia was described as being composed of one or two roughly aligned fascicles of microtubules situated transversely to the axonemes (Filice, 1952; Kulda and Nohýnková, 1995), but Piva and Benchimol (2004) discovered that it is not just one or two structures, but that it varies in number, shape and position (see Figs 22.8– 22.12). Thus, nowadays we can state that the MB comprises several small fascicles formed by microtubules, forming larger bundles (Fig. 22.11). The bundle number is variable as well as the number of microtubules found in each fascicle (Figs 22.11, 22.12). The MB can be used as a taxonomic criterion since its morphology varies in different Giardia species such as G. duodenalis, G. muris and G. agilis (Filice, 1952). Our research group used a technique that involved the removal of the plasma membrane and observation of the cytoskeletal structures of G. lamblia by both routine scanning electron microscopy (SEM) and high-resolution field emission SEM (Figs 22.8–22.11). 15 14 13 12 11 Measurement µm

10 9 8 7 6 5 4 3 2 1 0 MB

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Fig. 22.12. Morphometry of median bodies performed in 250 cells stained by Panotic kit. The measurements were made using photographic material or computeracquired images, in order to compare the width and length of the cells and median body (MB). The MB varied in size between 0.2 µm and 1.8 µm (average 0.84 µm) in width and between 0.8 µm and 8.0 µm (average 3.34 µm) in length. About 60% of the cell width is occupied by the MB, whereas only 8.4% of the cell length is occupied by it (from Piva and Benchimol, 2004).

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In the literature, the MB has been described as one or two cytoskeletal structures (Filice, 1952) present in only about 50% of the cells (Bertram et al., 1984). In addition, these authors found isolates in which the MB was absent. In the present study, we took advantage of the Panotic staining technique and showed that at least 80% of the cells displayed MBs (Fig. 22.12). Morphometry performed with Panotic-stained cells showed that of 1820 cells, 1448 exhibited the MBs, which represented almost 80% of the interphasic cell population. Of 1470 mitotic cells, 1257 displayed MBs, which corresponds to 85.5% of MB presence in dividing cells (Piva and Benchimol, 2004). These authors’ results do not agree with the literature and the possible cause may be the methodology used. In fact, some cells presented MBs which were difficult to visualize, since: (i) they can be in a vertical position and could be hidden by the axonemes of the caudal flagella when observed by light microscopy (Fig. 22.1); or (ii) they are so small that are not easily seen (Fig. 22.1). The absence of a MB in some cells led to the assumption that these structures are not permanent but instead represent a temporary storage of tubulin. It is possible that Giardia has different MB subpopulations, or that the size of these structures did not allow their visualization by routine methods. On the other hand, the absence of the MB in 20% of the cells observed in the present work could be due to problems with the Panotic staining method (Fig. 22.1). It is possible that the proportion of cells with a visible MB would be higher if a better staining method could be found. Another possibility is that the lack of a MB could be related to genetic errors in some cells or the existence of a subpopulation which does not present a typical MB. Using high-resolution scanning electron microscopy on detergent-treated cells, the MB was clearly visible (Fig. 22.11). The MB was seen as a structure formed by a variable number of fascicles, which were formed by different numbers of microtubules. The fascicles were gathered together and formed a tight bundle (Fig. 22.11). Our observations by SEM and FESEM demonstrated that there are not one or two MBs, as reported in the literature, but several fascicles; and a variable number between one and six fascicles were found in the present study. It is important to point out that this chapter reports the first observation of MBs using scanning electron microscopy. The SEM used here can achieve a resolution of 3.5 nm at 30 kV, whereas the FESEM shows a resolution of 1.2 nm at 15 kV. In addition, Piva and Benchimol (2004) have presented new observations concerning the MBs such as: (i) they were found both in interphasic (Figs 22.1, 22.8–22.11, 22.13) and mitotic (Fig. 22.13) trophozoites; (ii) they were present in about 80% of the cells (Fig. 22.12); (iii) they could be connected either to the plasma membrane, to the adhesive disc or to the caudal flagella, and thus they are not completely free in the cells, as published previously (Figs 22.8–22.11); (iv) a MB bundle can protrude from the cell surface (Figs 22.8–22.11); and (v) the MB microtubules react with several anti-tubulin and β-giardin antibodies (Fig. 22.7). The microtubules of the MB were found to be dorsally positioned in relation to the funis and were seen as smooth cylinders, rather distinct from those of the funis, which are covered by a rough coat (Figs 22.14, 22.15).

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Fig. 22.13. Localization of tubulin in Giardia by immunofluorescence microscopy after staining with monoclonal no. 357 antibody with the purpose of labelling the median body. Cells are seen from interphase (row a), to different progressive phases of mitosis (b–d) in immunofluorescence, and DIC labelling was observed in the median bodies (arrowheads), axonemes, flagella and disc, in all phases of the cell division. Note the mirror-symmetry of the median bodies in the late mitosis phase (h, i, j, k). Bar = 5 µm (from Piva and Benchimol, 2004).

Friend (1966) observed that the MB measured 2 µm in diameter, and Cheissin (1964) reported that it was 5 µm long. In the work of Piva and Benchimol (2004) the measurements showed that the MB varied between 0.2 µm and 1.8 µm (average 0.84 µm) in thickness and from 0.8 µm to 8.0 µm (average 3.34 µm) in length (Fig. 22.12). This large variation in MB size can be explained by different degrees of polymerization of the microtubules. Also, the strain used in this study (WB) could have been different from those used in previous studies (Piva and Benchimol, 2004). Campanati et al. (2003), using anti-tubulin antibodies with different specificities, suggested that a mixed population of microtubules, both stable and unstable, could form the MBs.

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Fig. 22.14. Giardia trophozoites observed by SEM preparation in a longitudinal view after membrane extraction by detergent. The funis (Fn) is well visualized as microtubules emanating from the axonemes in the direction of posterior-lateral flagella (P). D, disc. Bars, (a) = 1 µm; (b) = 3 µm (M. Benchimol, unpublished).

Funis The funis (Figs 22.14, 22.15) is a poorly studied microtubular structure in Giardia. It was described by Holberton (1973) as short arrays of microtubules emanating from the axonemes of the caudal flagella, one in a dorsal and another in a ventral position, and was given the name ‘funis’ by Kulda and Nohýnková (1978). In the area between the nuclei, the funis consists of well-defined bands of interconnected microtubules. From the point of emergence of the ventral flagella, the individual microtubules detach from each band and fan out laterally in a position dorsal to the posterior-lateral axonemes, down to the tail of the organism (Kulda and Nohýnková, 1978; Campanati et al., 2003). It has been suggested that the funis has a structural function (Kulda and Nohýnková, 1995). Recently, our group has used high-resolution field emission scanning electron microscopy (FESEM) (Benchimol et al., 2004) on material in which the plasma membrane was removed so that the cytoskeleton could be better seen. Images of the funis by FESEM showed that its microtubules do not end in the cytoplasm, but are anchored in the posterior flagella, via the dense rods (Figs 22.14, 22.15). We also showed that after emergence of the posterior-lateral flagella, funis microtubules are anchored to the epiplasm, and attach to filamentous links underlying the membrane where a fibrous layer underlies the portion of membrane where tail contractility occurs. Thus, our group proposed a model for the tail flexion of G. lamblia in which the funis participates. In addition, after the emergence of the posterior-lateral flagella, the funis microtubules irradiate towards the plasma membrane (Figs 22.14, 22.15).

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Fig. 22.15. Scanning electron microscopy (SEM) image of a dorsal view of G. lamblia showing the funis microtubules (Fn), which emanate from each of the axonemes of the caudal flagellum (C), contacting the posterior-lateral flagellum (P) where it attaches to the dense rod. D, disc; A, anterior flagellum. Note that the arrow points to funis microtubules found in distinct levels. Bar = 1 µm (from Benchimol et al., 2004).

How the Plasma Membrane in Giardia was Removed High-resolution field emission scanning electron microscopy (FESEM) Cells were adhered to poly-L-lysine-coated glass coverslips and then treated with a permeabilization buffer solution (PBS) (0.5% Nonidet P-40, 0.1 M Pipes, 1 mM MgSO4, 2 mM glycerol, 2 mM EGTA, 1 mM PMSF (phenylmethylsulphonyl fluoride), and 0.5% Triton X-100) for different periods of time (2–10 min). The cells were washed in PBS and then fixed in 2.5% glutaraldehyde in phosphate buffer, post-fixed for 5 min in 1% OsO4, dehydrated in ethanol, critical-point-dried with CO2, and sputter-coated with carbon. The samples were examined in a Jeol JSM-6340F field emission scanning electron microscope (FESEM) operated at an accelerating voltage of 5 kV, using 5 mm as the working distance and the standard SEI and BEI detectors.

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Ultrastructural examination of detergent-treated Giardia lamblia by SEM allowed the visualization of its microtubular cytoskeleton (Figs 22.6–22.12, 22.15–22.18). The adhesive disc, located in the ventral anterior region of the trophozoite, was mainly made of concentrically arranged microtubules (Figs 22.6, 22.15). The disc microtubules and their associated dorsal ribbons were organized in a spiralized organization, surrounding a central area devoid of microtubules, known as the bare area (Fig. 22.14a).

Fig. 22.16. Conventional scanning electron microscopy (SEM) image of G. lamblia in dorsal view, without membrane extraction (a) and in ventral view, after membrane extraction by detergents (b). Note the caudal bent. D, disc; P, posterior-lateral flagella; C, caudal flagella; A, anterior flagella; V, ventral flagella; Fn, funis. Bars = 1 µm (from Benchimol et al., 2004).

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Fig. 22.17. Field emission scanning electron microscopy (FESEM) image of Giardia after detergent extraction. The plasma membrane was partially removed, allowing observation of the funis (Fn) which is linked to the posterior-lateral flagella (P). The microtubules emanate from each of the axonemes of the caudal flagellum (C), contacting the posterior-lateral flagellum (P) where it attaches to the dense rod (arrow). Posterior-lateral axoneme (P); caudal axoneme (C). Bars: (a) = 1 µm; (b) = 500 nm (from Benchimol et al., 2004).

Fig. 22.18. High-resolution field emission scanning electron microscopy of G. lamblia after immunogold labelling using the monoclonal antibody TAT-1. This is a back-scattered image (BEI). It is possible to see the gold particles labelling the microtubules of the adhesive disc (D), funis (Fn), caudal (C) and posterior-lateral flagella (P). Bar = 400 nm (from Benchimol et al., 2004).

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The thickness of the microtubules observed in our preparations varied as a function of the carbon layer thickness used in the sample preparation and also due to the presence of a coat of fibrous material seen in some microtubular structures. The axonemes of the caudal flagella show emanating microtubules (Figs 22.14, 22.15, 22.17, 22.18), which are part of a structure named the ‘funis’ by Kulda and Nohýnková (1978). At the most anterior region of the caudal flagella, near the basal bodies, the microtubules of the funis are wrapped around the caudal axonemes (Figs 22.17–22.19). Gradually, these microtubules begin to separate from the caudal axonemes and fan out towards the axonemes of the posterior-lateral flagella (Figs 22.15, 22.17–22.19). These microtubules are interconnected by bridges and attached to the fibrous rod (dense rod) present on the axoneme of the posterior-lateral flagella (Fig. 22.14). Thus, the caudal and posterior-lateral axoneme flagella become linked by the funis microtubules (Figs 22.14–22.19). When Giardia was adhered through its ventral region (adhesive disc facing downwards) on poly-L-lysine-coated coverslips, the funis was observed on its dorsal face (Fig. 22.15). This view allowed the observation that the funis microtubules arise from one of the caudal flagella axonemes, running towards the left, contacting the other caudal flagellum axoneme and adhering to the dense rods of the posterior-lateral flagellum axoneme (Figs 22.17–22.19). When Giardia was adhered through its dorsal region (adhesive disc facing upwards) it was possible to observe that the other caudal flagellum axoneme also showed emanating microtubules, which are directed to the opposite posterior-lateral flagellar axoneme (Fig. 22.19). Thus, there are two emanating groups of microtubules from the axonemes of the caudal flagella that migrate in opposite directions, forming a well-structured array (Fig. 22.19). In addition, the funis microtubules were covered by an unidentified material, which gave them a greater thickness. The funis was clearly seen only in cells that had been well extracted by detergent. The microtubules of the funis presented bridges (Figs 22.14–22.18) that were very sensitive to Triton X-100 and were easily lost during cytoskeleton preparation. The caudal flagella axonemes seem to be firmly anchored to the axoneme of the posterior-lateral flagella by means of the funis microtubules. In addition, after the emergence of the posterior-lateral flagella, a few microtubules were seen projecting towards the plasma membrane. They were anchored to a network found underlying the membrane (Figs 22.14–22.17). These structures were preserved during sample preparation, using different times of extraction and detergent concentrations, although the bridges interconnecting the funis microtubules were affected when longer times, high detergent concentrations, or centrifugation were used (Fig. 22.15). In the funis, a linearly aligned material, forming images similar to ‘corn kernels’, covered the posterior-lateral flagella. This coating material was rough and the grains were seen in linear rows (Fig. 22.17). Immunocytochemistry was performed after the detergent extraction and a monoclonal anti-tubulin, TAT-1, was used (Fig. 22.18). The funis microtubules were intensely labelled and were better visualized when back-scattered electrons (BEI) were used, as shown in Fig. 22.18.

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Fig. 22.19. Schematic drawing of a static Giardia lamblia (a–b) and presenting tail movement (c) showing the relationship of the funis and the posterior-lateral and caudal flagella. The caudal flagella pair (C1, C2) presents an array of microtubules that emanate towards the posterior-lateral flagella (P1, P2). Note that in the caudal flagellum (C2) the microtubules emanate towards the dense rods of the posteriorlateral flagellum 1 (P1), whereas in the caudal flagellum 1 (C1) the microtubules are directed towards the posterior flagellum 2 (P2). Note that the funis microtubules are anchored to the dense rods (asterisk) of the posterior-lateral flagella, and are interconnected by filamentous bridges. After the emergence of the posterior-lateral flagella, the funis microtubules are anchored to the epiplasm, a layer of material underlying the plasma membrane. This region presents a network of filamentous structures (F). The pair of caudal flagella is interconnected by thin bridges. During tail flexion (c) the microtubules of the funis found after the posterior-lateral flagella emergence are seen anchored to the material underlying the plasma membrane and presenting different length. They seem to participate in the caudal movement. D, disc; PV, peripheral vesicles. (from Benchimol et al., 2004).

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It is well-known that Giardia exhibits a tail movement (Figs 22.16, 22.19). When cells are well fixed, the caudal region can be seen in a bent position, either by routine SEM (Fig. 22.16a) or after detergent extraction (Figs 22.14a, 22.16b, 22.17, 22.18). Carvalho and Monteiro-Leal (2004) proposed that the funis microtubules participate in Giardia’s tail movement. The authors used videomicroscopy assays of the isolated beating complex together with 3D-reconstruction data, which indicated that the internal portion of the caudal flagella could be the force generator of the movements in this region. Therefore, it was proposed that the funis microtubules play some role in this type of movement (Benchimol et al., 2004; Carvalho and Monteiro-Leal, 2004), as illustrated in a schematic drawing (Fig. 22.19).

Acknowledgements This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa de Núcleos de Excelência (PRONEX), Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES), Fundação Estadual do Norte Fluminense (FENORTE), and Associação Universitária Santa Úrsula (AUSU). The authors thank the Laboratório de Ultraestrutura Celular Hertha Meyer (UFRJ) for the use of the FESEM, and Dr Staffan Svärd, who kindly provided the anti-giardin antibodies.

References Benchimol, M. (2002) Giardia lamblia: a new set of vesicles. Experimental Parasitology 102, 30–37. Benchimol, M., Piva, B., Campanati, L. and de Souza, W. (2004) The funis of Giardia lamblia by high-resolution field emission scanning electron microscopy: new insights. Journal of Structural Biology 96, 291–301. Bertram, M.A., Meyer, E.A., Anderson, D.L. and Jones, C.T. (1984) A morphometric comparison of five axenic Giardia isolates. Journal of Parasitology 70, 530–535. Campanati, L., Holloschi, A., Troster, H., Spring, H., de Souza, W. and Monteiro-Leal, L.H. (2002) Video-microscopy observations of fast dynamic processes in the protozoan Giardia lamblia. Cell Motility and the Cytoskeleton 51, 213–224. Campanati, L., Troester, H., Monteiro-Leal, L.H., Spring, H., Trendelenburg, M.F. and de Souza, W. (2003) Tubulin diversity in trophozoites of Giardia lamblia. Histochemistry and Cell Biology 119, 323–331. Carvalho, K.P. and Monteiro-Leal, L.H. (2004) The caudal complex of Giardia lamblia and its relation to motility. Experimental Parasitology 108, 154–162. Cheissin, E.M. (1964) Ultrastructure of Lamblia duodenalis. I. Body surface, sucking disc and median bodies. Journal of Protozoology 11, 91–98. Erlandsen, S.L. and Feely, D.E. (1984) Trophozoite motility and the mechanism of attachment. In: Erlandsen, S.L. and Meyer, E.A. (eds) Giardia and Giardiasis: Biology, Pathogenesis, and Epidemiology. Plenum Press, New York, pp. 33–64. Filice, F.P. (1952) Studies on the cytology and life history of a Giardia from the laboratory rat. University of California Publications in Zoology 57, 53–146.

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Friend, D.S. (1966) The fine structure of Giardia muris. Journal of Cell Biology 29, 317– 332. Gillin, F.D., Reiner, D.S. and McCaffery, J.M. (1996) Cell biology of the primitive eukaryote Giardia lamblia. Annual Review of Microbiology 50, 679–705. Holberton, D.V. (1973) Fine structure of the ventral disc apparatus and the mechanism of attachment in the flagellate Giardia muris. Journal of Cell Science 13, 11–41. Kulda, J. and Nohýnková, E. (1978) Flagellates of the human intestine and of intestines of other species. In: Kreier, J.P. (ed.) Parasitic Protozoa. Intestinal Flagellates: Histomonads, Trichomonads, Amoeba, Opalinids, and Ciliates, Vol. II. Academic Press, New York, pp. 2–139. Kulda, J. and Nohýnková, E. (1995) Giardia in humans and animals. In: Kreier, J.P. (ed.) Parasitic Protozoa, 2nd edn. Academic Press, San Diego, CA, Vol. 10, pp. 225–422. Lanfredi-Rangel, A., Attias, M., Carvalho, T.M.U., Kattenbach, W.M. and de Souza, W. (1998) The peripheral vesicles of trophozoites of the primitive protozoan Giardia lamblia may correspond to early and late endosomes and to lysosomes. Journal of Structural Biology 123, 225–235. Lanfredi-Rangel, A., Kattenbach, W.M., Diniz, J.A. and de Souza, W. (1999) Trophozoites of Giardia lamblia may have a Golgi-like structure. FEMS Microbiology Letters 181, 245–251. Luján, H.D., Marotta, A., Mowatt, M.R., Sciaky, N., Lippincott-Schwartz, J. and Nash, T.E. (1995) Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia. Journal of Biological Chemistry 270, 4612–4618. Piva, B. and Benchimol, M. (2004) The median body of Giardia lamblia: an ultrastructural study. Biology of the Cell 96, 735–746. Reiner, D.S., McCaffery, M. and Gillin, F.D. (1990) Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia. European Journal of Cell Biology 53, 142–153. Tovar, J., León-Avila, G., Sánchez, L.B., Sutak, R., Tachezy, J., van der Giezen, M., Hernández, M., Müller, M. and Lucocq, J.M. (2003) Mitochondrial remnant organelles of Giardia function in iron–sulphur protein maturation. Nature 426, 172–176. Upcroft, J. and Upcroft, P. (1998) My favourite cell: Giardia. BioEssays 20, 256–263.

23

Giardia intestinalis: a Microaerophilic Parasite with Mitochondrial Ancestry

G. LEÓN-AVILA1, J.M. HERNÁNDEZ2 AND J. TOVAR3 1Escuela

Nacional de Ciencias Biológicas, México DF, Mexico; 2Centro de Investigación y de Estudios Avanzados del IPN, San Pedro Zacatenco, Mexico; 3Royal Holloway University of London, UK

Abstract Highly derived mitochondrion-related organelles originally found in Entamoeba histolytica and known as mitosomes (or cryptons) are heterogeneous in morphology and exist in a range of anaerobic protists including Trachipleistophora hominis and Cryptosporidium parvum. The iron-binding scaffold protein (IscU) and its functional partner IscS participate in the biosynthesis of the iron–sulphur (FeS) cluster. Specific antibodies raised against both proteins detected both IscS and IscU in high-speed pellets obtained from the fractionation of total extracts of Giardia. In an immunoelectron microscopy study using those antibodies labelled, we detected these proteins associated with double-membrane organelles distributed throughout the cytoplasm. Most of the mitochondrial proteins are synthesized as preproteins and are targeted to the organelle by a cleavage of the N-terminal extension composed of positively charged amino acids. The Giardia proteins IscU and ferredoxin have those amino-terminal targeting presequences. Transgenic parasites expressing GFP fusions of the amino terminal presequences of both proteins revealed that such extensions are essential for mitosome targeting. Additionally, the mitosomal targeting presequences were recognized by the mitochondrial and Trichomonas virginalis import machinery. Cpn60 and hsp70 were identified in the mitosomes of transfected trophozoites as well.

Introduction Mitochondria originated approximately 1500 million years ago through the endosymbiosis of an α-proteobacterium by a host cell. Mitochondrial metabolism generates biological energy in eukaryotic cells through pathways such as the Krebs cycle, the electron transport chain, oxidative phosphorylation and the biosynthesis of iron–sulphur clusters (van der Giezen et al., 2004; van der Giezen and Tovar, 2005). 284

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Giardia intestinalis and Entamoeba histolytica had been considered as direct descendants of hypothetical amitochondriate cells (protoeukaryotes) until the finding of genes encoding proteins of mitochondrial origin in their genome, which provided evidence in favour of mitochondrial ancestry for these parasites (Clark and Roger, 1995; Roger et al., 1998). The first evidence against an amitochondriate past for Entamoeba histolytica was the phylogenetic analysis of its small ribosomal subunit that placed it as a late branch in the eukaryotic tree (Sogin et al., 1989; Sogin, 1991). The following significant findings were the identification of genes encoding mitochondrial proteins such as chaperonin 60 (Cpn60) and pyridine-nucleotide transhydrogenase (PNT) (Clark and Roger, 1995). Specific antibodies against EhCpn60 used in cell fractionation experiments allowed the detection of this protein in the microsomal fraction. Using E. histolytica trophozoites transfected with a recombinant vector expressing Cpn60 (tagged with c-myc), it was possible to identify, by fluorescence microscopy, cellular structures of 1–2 µm in diameter denominated mitosomes (Tovar et al., 1999) or cryptons (Mai et al., 1999). The first mitochondrial protein identified in Giardia intestinalis was valyl-t-RNA synthetase, an enzyme encoded by a gene hypothesized to have been transferred to the nucleus by the mitochondrial endosymbiont. The full gene was sequenced and a phylogenetic study was performed using a maximum likelihood analysis over the deduced amino acid sequence. The analysis revealed that Giardia valyl-t-RNA synthetase was situated in the mitochondrial clade (Hashimoto et al., 1998). The following mitochondrial genes identified in Giardia were chaperonin 60 and hp70, which were also placed in the clade of mitochondrial chaperonins by phylogenetic analysis (Roger et al., 1998; Morrison et al., 2001; Arisue et al., 2002). Iron–sulphur clusters act as cofactors of proteins required for electron transfer, enzymatic catalysis, and metabolic regulation, which can take place in the nucleus, cytoplasm or mitochondria of eukaryotic cells (Lill and Muhlenhoff, 2005). Phylogenetic analysis for the Giardia cysteine desulphurase (IscS) sequence placed it in the mitochondrial clade (Tachezy et al., 2001).

From Suggestive Data to Direct Evidence The iron-binding scaffold protein (IscU), the functional partner of IscS, was cloned and characterized. BLAST analysis of IscU using the deduced amino acid sequence revealed a 46.51% overall identity and 63–69% similarity with its bacterial and eukaryotic orthologues. The amino terminal sequence is rich in basic amino acids, and a web tool used to predict mitochondrial targeting sequences predicted the mitochondrial localization of this protein. We raised specific antibodies against both proteins and corroborated the expected molecular weight (which had already been deduced from the open reading frame sequences) by Western blot analysis. In addition, we were able to detect the specific signal in high-speed pellets obtained from the fractionation of total parasite extracts.

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To determine the cellular distribution, or even the possible compartmental localization, of these proteins, we used specific antibodies on fixed trophozoites for confocal scanning fluorescence microscopy. We observed the co-localization of IscS and IscU in small cellular structures randomly distributed over the cytoplasm, and noticed an accumulation of label in the region of the basal bodies at the base of the flagellar axonemes. We estimated that there were 25–100 of these structures per cell. To further characterize these structures, which we called mitosomes (as in Entamoeba histolytica), we performed immunoelectron microscopy and were able to detect the labelling of these proteins over small elongated organelles, which were surrounded by two membranes. The size of the organelles was estimated to be 60 × 140 nm on average and no cristae were observed (Tovar et al., 2003).

Mitosome protein import is in some cases mediated by amino terminal targeting sequences The import of proteins to the mitochondria as well as to the hydrogenosomes (ATP- and hydrogen-generating organelles) (van der Giezen et al., 1998) is carried out through two pathways: one that depends on a presequence consisting of 15–40 positively charged amino acids (Stan et al., 2003), and another that is independent of such sequences. Specifically, the giardial proteins IscU and ferredoxin (Fd) exhibit an amino terminal targeting sequence which was determined by the algorithm MitoProt; however, other proteins also identified in Giardia, such as cpn60, hsp70 and IscS, lack such putative targeting signals (Roger et al., 1998; Morrison et al., 2001; Tachezy et al., 2001; Arisue et al., 2002). To find out whether the Giardia Cpn60 was also localized in mitosomes, homologous antibodies were used. It was observed that Cpn60 co-localized with IscS by confocal microscopy analysis (Regoes et al., 2005). Trophozoites transfected with constructs expressing Cpn60 and mtHsp70 showed co-localization with IscS in mitosomes too. IscU and ferredoxin contain a presequence, which was shown to be necessary for importation into mitosomes. Confocal microscopy analysis of trophozoites transfected with constructs expressing the full open reading frame of IscU or ferredoxin fused to green fluorescent protein (GFP), showed GFP labelling in mitosomes, whereas fusion proteins without the presequence only accumulated GFP label in the cytoplasm (Regoes et al., 2005).

Processing of the Targeting Signal Once the proteins are imported to the mitochondria, the presequence contained in the precursor proteins is cleaved by the mitochondrial processing peptidase (Wiedemann et al., 2004). Western blot analysis of the cell fractionations from trophozoites transfected with IscU and Fd fusion proteins revealed that the mitosome-enriched fraction presented mature peptides of lower molecular weight

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(14.5 kDa for IscU and 27 kDa for Fd) than the immature proteins, as a result of processing. The great majority of the precursor proteins were detected in the cytosolic fraction (Regoes et al., 2005). Dolezal and co-workers (2005) determined the processing of the N-terminal targeting sequence of giardial IscU, using an in vitro translation assay of radiolabelled IscU with either yeast mitochondria or hydrogenosomal lysates. The processing happened in a time-dependent manner and was blocked by EDTA (inhibitor of metalloproteases). The analysis of IscU preprotein incubated with rat MPP (mitochondrial processing peptidase) revealed that it was cleaved between Phe-18 and Leu-19 with arginine at the −2 position. This group also found a putative β-MPP subunit and Pam18 (a TIM complex subunit) in the Giardia genome and reported that both proteins were detected in mitosomes (Fig. 23.1).

Conserved Import Pathways To test whether the ferredoxin presequence was functional in mammalian cells we obtained the 5′-Fd-GFP fusion construct and transfected it into human embryonic kidney cells. The fusion protein was efficiently targeted to the mitochondria by the Giardia ferredoxin targeting sequence, since a co-localization

Hsp70

Pre-sequence IscS

IscU

Outer membrane

Inner membrane

Pam18 Cpn60

β-MPP mt-hsp70

Fig. 23.1. Model of protein import to mitosomes showing the proteins that have been experimentally demonstrated.

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was observed between the GFP and the mitochondrial marker dye MitoTracker Red. Cells transfected with a construct without the mitosomal targeting sequence exhibited the label only in the cytosol. These results proved the functional conservation of targeting sequences between mammalian mitochondria and Giardia mitosomes (Regoes et al., 2005). To analyse whether the IscU and ferredoxin targeting sequence could direct the proteins to hydrogenosomes, Dolezal et al. (2005) expressed the Giardia IscU and ferredoxin open reading frames in Trichomonas vaginalis and analysed their localization. The labelling was detected in hydrogenosomes and co-localized with the malic enzyme, a marker for hydrogenosomes. Deletion experiments, in which the N-terminal targeting sequences of giardial IscU and ferredoxin were removed from the proteins, showed that the transport to the hydrogenosomes was affected and the label was accumulated in the cytoplasm.

Protein Import Mediated by Internal Targeting Sequences It is worth mentioning that a number of luminal mitochondrial proteins are imported into this organelle in the absence of recognizable amino-terminal targeting peptides. Bearing this concept in mind, Giardia IscS fusion with the GFP marker was performed, and we observed the labelling in the mitosomes of transfected trophozoites by confocal microscopy. One possibility is that the IscS sequence contains internal signals for targeting to organelles (Fig. 23.2). This hypothesis is further supported by the findings of Dolezal et al. (2005), who

Fig. 23.2. Confocal microscopy of Giardia trophozoite transfected with the IscSGFP fusion construct.

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expressed the Giardia IscS either as a full sequence or as N-terminal (202 residues) or C-terminal (232 residues) truncated peptides in T. vaginalis. Western blot assays revealed that not only the full sequence but also both truncated fragments were delivered to the hydrogenosomal fraction. Thus they concluded that IcsS contains multiple targeting signals. On the other hand, the deletion of the initial five amino acids from the sequence of Cpn60 did not affect the mitosomal localization; this observation shows that the amino-terminal sequence in Cpn60 is not involved in the targeting to the organelle (Regoes et al., 2005).

Mitosome Division and Inheritance The images of Giardia mitosomes obtained by confocal microscopy display two kinds of mitosomes, which appear morphologically distinct. A rod-shape organelle was always localized between the nuclei and the spherical mitosomes were distributed throughout the cytoplasm (Tovar et al., 2003). According to their cellular distribution they were designated as peripheral mitosomes (PMs) and central mitosomes (CMs) (Regoes et al., 2005). To find out more about the replication and segregation of the mitosomes, microscopy studies were done during the cell cycle. The CM divides during mitosis and is segregated to the daughter cells, whereas the PMs are constant in number during the cell cycle. To determine the possible role of the cytoskeleton in these events, the trophozoites were treated with nocodazole or cytochalasin D. The nocodazole caused loss of the CM but the PMs remained normal in number and position. The cytochalasin D did not affect the CM; however, the PMs apparently increased in number. These findings suggest the participation of the cell cytoskeleton in the positioning and segregation of Giardia mitosomes (Regoes et al., 2005).

Conclusions Although some of the mitosomal components have been identified, much more investigation is still needed in order to find all the constituents, as well as the proteins involved in the biogenesis of these organelles, once thought to be absent in these parasites.

Acknowledgement We thank Dr Ma Isabel Salazar for her critical reading of the manuscript.

References Arisue, N., Sanchez, L.B., Weiss, L.M., Muller, M. and Hashimoto, T. (2002) Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians. Parasitology International 51, 9–16.

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G. León-Avila et al. Clark, C.G. and Roger, A.J. (1995) Direct evidence for secondary loss of mitochondria in Entamoeba histolytica. Proceedings of the National Academy of Sciences of the USA 3, 6518–6521. Dolezal, P., Smíd, O., Rada, P., Zubácová, Z., Bursa= , D., Suták, R., Nebesárová, J., Lithgow, T. and Tachezy, J. (2005) Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting. Proceedings of the National Academy of Sciences of the USA 102, 10924–10929. Emelyanov, V.V. (2003) Phylogenetic affinity of a Giardia lamblia cysteine desulfurase conforms to canonical pattern of mitochondrial ancestry. FEMS Microbiology Letters 26, 257–266. Hashimoto, T., Sánchez, L.B., Shirakura, T., Müller, M. and Hasegawa, M. (1998) Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny. Proceedings of the National Academy of Sciences of the USA 95, 6860–6865. Lill, R. and Muhlenhoff, U. (2005) Iron–sulfur protein biogenesis in eukaryotes. Trends in Biochemical Sciences 30, 133–141. Mai, Z., Ghosh, S., Frisardi, M., Rosenthal, B., Rogers, R. and Samuelson, J. (1999) Hsp60 is targeted to a cryptic mitochondrion-derived organelle (“crypton”) in the microaerophilic protozoan parasite Entamoeba histolytica. Molecular and Cellular Biology 19, 2198–2205. Morrison, H.G., Roger, A.J., Nystul, T.G., Gillin, F.D. and Sogin, M.L. (2001) Giardia lamblia expresses a proteobacterial-like DnaK homolog. Molecular Biology and Evolution 2001 18, 530–541. Regoes, A., Zourmpanou, D., Leon-Avila, G., van der Giezen, M., Tovar, J., Hehl, A.B. (2005) Protein import, replication, and inheritance of a vestigial mitochondrion. Journal of Biological Chemistry 26, 30557–30563. Roger, A.J., Svärd, S.G., Tovar, J., Clark, C.G., Smith, M.W., Gillin, F.D. and Sogin, M.L. (1998) A mitochondrial-like chaperonin 60 gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria. Proceedings of the National Academy of Sciences of the USA 95, 229–234. Sogin, M.L. (1991) Early evolution and the origin of eukaryotes. Current Opinion in Genetics and Development 1, 457–463. Sogin, M.L., Gunderson, J.H., Elwood, H.J., Alonso, R.A. and Peattie, D.A. (1989) Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science 243, 75–77. Stan, T., Brix, J., Schneider-Mergener, J., Pfanner, N., Neupert, W. and Rapaport, D. (2003) Mitochondrial protein import: recognition of internal import signals of BCS1 by the TOM complex. Molecular and Cellular Biology 23, 2239–2250. Tachezy, J., Sánchez, L.B. and Müller, M. (2001) Mitochondrial type iron–sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS. Molecular Biology and Evolution 18, 1919–1928. Tovar, J., Fischer, A. and Clark, G. (1999) The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica. Molecular Microbiology 32, 1013–1021. Tovar, J., León-Avila, G., Sánchez, L.B., Sutak, R., Tachezy, J., van der Giezen, M., Hernández, M., Müller, M. and Lucocq, J.M. (2003) Mitochondrial remnant organelles of Giardia function in iron–sulphur protein maturation. Nature 426, 172– 176. van der Giezen, M. and Tovar, J. (2005) Degenerate mitochondria. EMBO Reports 6, 525–530.

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van der Giezen, M., Kiel, J.A., Sjollema, K.A. and Prins, R.A. (1998) The hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis is targeted to mitochondria of the methylotrophic yeast Hansenula polymorpha. Current Genetics 33, 131–135. van der Giezen, M., Cox, S. and Tovar, J. (2004) The iron–sulfur cluster assembly genes iscS and iscU of Entamoeba histolytica were acquired by horizontal gene transfer. BMC Evolutionary Biology 4, 7. Wiedemann, N., Frazier, A.E. and Pfanner, N. (2004) The protein import machinery of mitochondria. Journal of Biological Chemistry 9, 14473–14476.

24

Cytoskeleton-based Lipid Transport in a Parasitic Protozoan, Giardia lamblia

C. CASTILLO, Y. HERNANDEZ, S. ROYCHOWDHURY AND S. DAS University of Texas at El Paso, TX, USA

Abstract The early-divergent protozoan Giardia lamblia, which is a major cause of waterborne enteric disease worldwide, was shown to possess limited lipid synthesis ability and to depend upon preformed lipid molecules for energy production and membrane biosynthesis. Therefore, questions regarding how Giardia imports and utilizes exogenous lipids are important. Using fluorescent lipids and fatty acids as reporter molecules, we show that anti-actin and anti-microtubule agents affect the uptake, intracellular movement and recycling of fluorescent lipids in Giardia, which indicates that lipid transport in this protozoan parasite, as in higher eukaryotes, is dependent upon cytoskeleton filaments. We propose that actin/microtubule-based lipid and vesicular transport, which is operative in Giardia, could be an ancient cellular process that most probably evolved before the emergence of the mitochondrion and other characteristic eukaryotic organelles.

Introduction Giardia lamblia colonizes the luminal surface of the human small intestine and causes gastrointestinal infections (Adam, 2001). While this flagellated and binucleated protozoan parasite is considered an ancient member of the domain eukarya (Sogin et al., 1989), it retains well-organized cytoskeletal structures made from actin and microtubule filaments. The ventral disc, basal bodies, flagella, paraflagellar rods, and median body in Giardia trophozoites are made from microtubules (Crossley et al., 1986; Elmendorf et al., 2003). The basal bodies are the major microtubule organizing centre and functional equivalent of the centrosome of higher eukaryotes (Nohýnková et al., 2000). Each of the eight flagella originates from a basal body near the cellular midline, and the structure of the flagellar axonemes contains the 9 + 2 microtubule arrangements typical of most eukaryotes (Elmendorf et al., 2003). Interestingly, Giardia possesses two copies of β-tubulin and one copy of α-tubulin (Kirk-Mason et al., 1989). Biochemical studies and structural analyses demonstrate that polyglycylation is the major tubulinspecific modification in both α- and β-tubulin. Giardial tubulin contains several of 292

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the posttranslational modifications observed in eukaryotic cells, and polyglutamylation and acetylation of tubulins were also identified (Soltys and Gupta, 1994; Weber et al., 1997). The α-tubulin of G. lamblia is detyrosinated (Soltys and Gupta, 1994) and lacks a detyrosination/tyrosination cycle (Weber et al., 1997). Using various anti-tubulin antibodies, it has been demonstrated that the median body of Giardia, which is considered to be the microtubule organizing centre, is composed of acetylated, mono/polyglycylated, tyrosinated tubulins along with β-giardin and γ-tubulin (see Piva and Benchimol, 2004, for a review). The dynamism of microtubules is involved in various cellular processes, including the maintenance of cell shape and integrity, mitosis, ciliary and flagellar motility, organization of cellular compartments, and membrane/vesicle trafficking. This dynamic behaviour of microtubules is the result of two mechanical processes known as treadmilling and dynamic instability, and both are essential for vesicle transport and targeting and are powered by molecular motor proteins from the kinesin, dynein, and myosin superfamilies (Kamal and Goldstein, 2000). Goode et al. (2000) proposed that short-range vesicle transport takes place on actin filaments by myosin motors, whereas long-range transport takes place along microtubules using kinesin and dynein motors, especially in higher eukaryotes. The literature reveals that Giardia contains a single copy of the actin gene, and its protein sequence reveals an approximately 58% nucleotide identity with other eukaryotic actin sequences (Elmendorf et al., 2003). A large set of kinesin homologues are present in Giardia (Iwabe and Miyata, 2002; Richardson et al., 2006), but so far no putative homologue of myosin has been identified in this binucleated parasite (Elmendorf et al., 2003). Because of its limited lipid synthesis capacity, this parasite depends upon lipids scavenged from the small-intestinal environment (Jarroll et al., 1981; Das et al., 2002). Therefore, it is plausible that, like higher eukaryotic cells, Giardia uses its cytoskeleton network for transporting and trafficking lipid molecules. In a recent study, we demonstrated that Giardia uses clathrin-coated vesicles and actin/ microtubule filaments to internalize and traffic ceramide molecules from the cell exterior (Hernandez et al., 2007). In this chapter, we discuss whether other lipids and the fatty acids of Giardia trophozoites are also dependent upon actin and the microtubule network for internalization, recycling and intracellular targeting.

Uptake and Intracellular Localization of Bodipy and NBD-labelled Membrane Lipids and Fatty Acids Since G. lamblia trophozoites were shown to have limited lipid synthesis ability, it is likely that these intestinal parasites obtain preformed lipid molecules from outside sources and remodel them to generate parasite-specific phospholipids (Stevens et al., 1997; Gibson et al., 1999; Das et al., 2001). Using epifluorescence and confocal microscopy, it was demonstrated that G. lamblia trophozoites are able to internalize fluorescently labelled lipids directly from the culture medium and distribute them into different regions of the cell (Stevens et al., 1997). In the present investigation, we investigated the roles of actin and microtubule filaments in this process. The cellular distribution of fluorescent

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labels was distinctive for each particular lipid analogue studied. For example, fluorescently labelled ceramide (bodipy-Cer) phosphatidylglycerol (NBD-PG) had a preferential localization at perinuclear membranes, whereas phosphatidylcholine (bodipy-PC) was incorporated into plasma and flagellar membranes (Stevens et al., 1997; Gibson et al., 1999; Das et al., 2001). Palmitic acid (bodipyPam) and sphingomyelin (NBD-SM) labelled the nuclear envelopes, various unidentified endomembranes, as well as the plasma membrane. NBD-PE localized at the plasma membrane and in certain cytoplasmic structures adjacent to the plasma membrane (Das et al., 2001). Even though the differences in lipid localization – and thus in composition – between the membranes of the trophozoites are not absolute, some lipids are enriched at particular membranes/organelles compared with others, which indicates that the localization of exogenous lipids in G. lamblia trophozoites is selective. Furthermore, this specific cellular localization is dependent upon the properties of the lipid analogue used, not on the fluorophore (bodipy or NBD) attached to the hydrophobic molecule (Stevens et al., 1997). Given the possibility that lipid molecules could be metabolized during the process of intracellular transport and processing, lipids were extracted from labelled trophozoites 30 and 60 min post-incubation and analysed by thin-layer chromatography (TLC). The results indicate that trophozoites did not modify the fluorescent lipid probes obtained from the culture medium, as seen by their migration as single bands on TLC plates (data not shown). These observations suggest that, unlike other organisms, Giardia is unable to metabolize the majority of the fluorescent lipids used in this investigation during its internalization and intercellular sorting. The cellular distribution of bodipy-Cer, NBD-PG, NBD-SM, and to a lesser extent bodipy-Pam, at nuclear envelopes suggests a role for this organelle in intracellular lipid trafficking. These perinuclear endomembranes have been previously identified by Soltys et al. (1996) as the endoplasmic reticulum of G. lamblia. Using a polyclonal antibody developed against giardial BiP by Gupta and coworkers (Gupta et al., 1994; Soltys et al., 1996) and the fluorescent dicarbocyanine dye DiOC6(3), we confirmed the presence of a perinuclear/reticular network in G. lamblia (not shown). BiP is the hsp70 homologue that resides in the lumen of the ER in higher eukaryotes; it functions as a molecular chaperone in protein folding and translocation across the ER membrane (Gething, 1999). The cyanine dye DiOC6(3) has previously been used to visualize ER and mitochondria in live or fixed mammalian cells (Terasaki et al., 1984; Terasaki and Reese, 1992; Soltys and Gupta, 1994). Since Giardia possesses no mitochondria (Adam, 2001), DiOC6(3) labelling was expected to be primarily in the ER. Immunofluorescence labelling with anti-giardial BiP antibody showed labelling of reticular structures around perinuclear membranes, while DiOC6(3) staining brightly labelled the periphery of the nuclei and other cytoplasmic endomembranes. Our current findings (not shown) support the observations of Soltys et al. (1996) that perinuclear regions of Giardia contain reticular components, as evidenced by staining with DiOC6(3) and antibody labelling. It is possible that the ER in Giardia performs the same functions as the Golgi apparatus, such as protein and lipid sorting, metabolism and remodelling, as in higher eukaryotes.

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The effects of anti-microtubule and anti-actin filament agents on the internalization and distribution of fluorescent lipid analogues To document how anti-microtubule and anti-actin agents affect the internalization and cellular distribution of fluorescent lipid analogues, trophozoites were first exposed to anti-cytoskeletal agents and then labelled with fluorescent lipid probes. The results show that the distribution of bodipy-Cer decreased dramatically in the presence of vinorelbine, while exposure to nocodazole induced the formation of cytoplasmic lipid aggregates. Taxol, which stabilizes microtubules, increased the localization along the plasma membrane. In addition, cytochalasin-D induced the formation of cytoplasmic tubular/vesicular structures. Colchicine and albendazole did not cause significant changes in the lipid localization pattern (not shown). When the mean fluorescence intensity was computed for each treatment and compared with the control (Table 24.1), a decrease in lipid uptake was observed in the presence of colchicine, vinorelbine, albendazole and cytochalasin-D, whereas no significant changes were observed in nocodazole- or taxol-treated cells. Although nocodazole had no significant effects on incorporation, it induced the formation of cytoplasmic lipid aggregates. These observations (Fig. 24.1) indicate that anti-actin as well as anti-microtubule agents affect the uptake and intracellular distribution of bodipy-Cer. The localization pattern of NBD-SM is shown in Fig. 24.1e, f, g and h. A decrease in intracellular labelling was observed in vinorelbine- and nocodazole-treated cells, and no significant changes were observed in trophozoites exposed to

Table 24.1. Internalization and cellular distribution of fluorescent lipids by Giardia trophozoites (measurement of mean fluorescent intensity).

Microtubule (MT) depolymerizing agents

Lipid Bodipy-Cer NBD-SM Bodipy-Pam Bodipy-PG

MT stabilizer

Actin depolymerizer

Colchicine

Vinorelbine

Albendazole

Nocodazole

Taxol

Cytochalsin-D

13.67 ↓ No effect No effect No effect

30.33 ↓ 23.58 ↓ No effect 36 ↓

11.14 ↓ No effect No effect No effect

No effect 19.30 ↓ 11.75 ↓ No effect

No effect No effect 23.34 ↓ 20.24 ↓

11.75 ↓ 14.88 ↓ No effect 18.43 ↑

To measure the amount of fluorescent lipids incorporated by Giardia in the presence or absence of anticytoskeleton agents, trophozoites were treated with anti-microtubule or anti-cytoskeleton drugs for 90 min before labelling with fluorescently labelled lipid molecules for an additional 30 min at 37°C. Confocal images were analysed using the software package Quantity One, version 4.1.1 (Bio-Rad Laboratories, Hercules, CA). The statistical analysis was performed using Graph Pad Prism statistics software. A single-factor analysis of variance (ANOVA) and Dunnett’s multiple comparison tests were used to assess differences between treatment means and control, with P < 0.05 being considered significant. Values are shown as percentage control. Increased (↑) or decreased (↓) intensities are indicated with arrows.

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Fig. 24.1. The role of actin and microtubule filaments in internalization and intracellular targeting of bodipy-ceramide (a, b, c and d), NBD-sphingomyelin (e, f, g and h), bodipy-palmitic acid (i, j, k and l), and NBD-phosphatidylglycerol (m, n, o and p) in the presence of 1 µM vinorelbine (b, f, j and n), 500 nm nocodazole (c, g, k and o) and 80 µM cytochalasin-D (d, h, l and p), compared with controls (a, e, i and m). Trophozoites were treated with anti-microtubule or anti-cytoskeleton drugs for 90 min before labelling with fluorescently labelled lipid molecules for an additional 30 min at 37°C. Photographs were captured with help of an Olympus BX-50 laser scanning confocal microscope (Olympus America, Melville, NY) equipped with a yellow krypton (568 nm) and an argon (488 nm) ion laser. Magnification 600×.

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colchicine, albendazole or taxol. However, in cells treated with anti-actin agent, multiple small, vesicle-like structures scattered throughout the cytosol, and a decrease in the staining of perinuclear membranes were detected. A decrease was also observed for cytochalasin-D-treated trophozoites, but no significant changes were observed for colchicine-, albendazole- or nocodazole-treated cells, as shown in Table 24.1. The previous findings indicate that bodipy-Cer also utilizes the actin filaments and microtubules for its incorporation and intracellular distribution (Hernandez et al., 2007). Summarizing the information presented above, cytochalasin-D significantly reduced the lipid incorporation of both bodipy-Cer and NBD-SM. Moreover, in NBD-SM-labelled cells, cytoskeleton-D induced the formation of small vesiclelike structures scattered throughout the cytosol. These results suggest that both sphingolipids are internalized through actin-dependent mechanisms, and most probably by endocytosis. In addition, vinorelbine, which significantly altered the localization of antitubulin antibodies in trophozoites displaying a collapsed microtubule network (Hernandez et al., 2007), was the most potent inhibitor and drastically reduced the intracellular distribution of these two fluorescent sphingolipids. These observations indicate that bodipy-Cer and NBD-SM require microtubules for intracellular trafficking. However, the high frequency of microtubule polymerization/ depolymerization is not essential for the intracellular transport of these lipids, because taxol had no significant effect on the internalization and cellular distribution of these analogues (Table 24.1). We were also interested in investigating the role of actin and microtubule filaments in the uptake and targeting of bodipy-Pam and NBD-PG, because they are internalized and targeted intracellularly (Fig. 24.1). The fluorescent images show that colchicine, vinorelbine and nocodazole induced the formation of cytoplasmic aggregates of bodipy-Pam (Fig. 24.1i, j, k, l). Interestingly, however, only nocodazole and taxol showed a significant decrease in palmitic acid uptake. The mean fluorescence intensity in colchicine-, vinorelbine- and albendazole-treated cells (Table 24.1) was not significantly affected. The images shown in Fig. 24.1 (m, n, o and p) demonstrate that vinorelbine, as was observed with the other lipid analogues studied, decreased the intracellular incorporation and localization of NBD-PG. It is also interesting that nocodazole-treated cells displayed the formation of large cytoplasmic PG aggregates. Exposure to taxol caused a decrease in lipid incorporation and delivery to perinuclear membranes. This fluorescent lipid vesicle distribution was also observed in cytochalasin-D-treated trophozoites, in which a marked decrease in cytoplasmic fluorescent intensity was also observed.

The Importance of the Microtubule Network and Actin Filaments for the Release and Recycling of Fluorescent Lipid Analogues To document whether microtubules and actin filaments are also involved in the intracellular trafficking and recycling of fluorescent lipid probes in G. lamblia trophozoites, cells were first labelled with lipid analogues, then exposed to anti-cytoskeletal agents. Table 24.2 shows the percentage increase or decrease in

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mean fluorescence intensity for the four lipid analogues studied in the presence of the several anti-cytoskeletal agents used. Treatment of trophozoites with anti-microtubule agents did not affect the intracellular localization of bodipy-Cer in nuclear/ perinuclear regions (Fig. 24.2). However, in the presence of cytochalasin-D the localization of fluorescent ceramide at nuclear envelopes decreased. A reduction in fluorescent staining was observed in nocodazole-treated cells (Fig. 24.2e). These observations suggest that, once bodipy-Cer localizes to perinuclear membranes, its subsequent intracellular trafficking, at least in part, is dependent upon nocodazoleand cytochalasin-D-sensitive microtubule and actin filaments (Table 24.2). Treatment of NBD-SM-labelled trophozoites with vinorelbine and albendazole decreased the intracellular staining; in contrast, however, a brighter cytoplasmic labelling was observed in taxol-treated cells (Fig. 24.3). Exposure of trophozoites to cytochalasin-D reduced the intracellular fluorescence. These changes in fluorescence intensity were also confirmed by computing the mean fluorescence intensity of the images (Fig. 24.3; Table 24.2). For the effects of anti-cytoskeleton agents on recycling of NBD-sphingomyelin, the results suggest that bodipy-Pam localization is not significantly affected in the presence of any anti-cytoskeletal agents, since no noticeable changes in lipid distribution were observed in the presence of anti-microtubule agents (not shown). However, an overall decrease in fluorescence intensity was observed for all the treatments (Table 24.2). These results correlate with the mean fluorescence intensity values obtained for these images and suggest that the involvement of microtubules in intracellular movement of bodipy-Pam in Giardia trophozoites is not yet conclusive. Table 24.2. Intracellular trafficking and recycling of fluorescent lipid analogues by Giardia trophozoites (measurement of mean fluorescent intensity).

Microtubules (MT)-depolymerizing agents

MT stabilizer

Actin depolymerizing agent

Colchicine

Navelbine

Albendazole

Nocodazole

Taxol

Cytochalasin-D

Bodipy-Cer NBD-SM Bodipy-Pam

No effect No effect 35.11 ↓

No effect 14.51 ↓ 9.40 ↓

No effect 8.65 ↓ 23.78 ↓

12.50 ↓ No effect 32.46 ↓

No effect 14.46 ↑ 26.46 ↓

11.25 ↓ 9.94 ↓ 12.95 ↓

NBD-PG

20.01 ↑

12.81 ↓

27.47 ↑

41.23 ↑

No effect

12.99 ↓

Lipid

To measure the amount of fluorescent lipid probes incorporated in the presence or absence of anticytoskeleton agents, trophozoites were first labelled with fluorescent lipid probes for 30 min at 37°C. Samples were then washed twice by centrifugation at 1500 × g for 5 min at 25°C, and were then transferred to flaskette chambers containing anti-actin or anti-microtubule drugs and incubated for 90 min at 37°C. Confocal images were analysed using the software package Quantity One, version 4.1.1 (Bio-Rad Laboratories, Hercules, CA). The statistical analysis was performed using Graph Pad Prism statistics software. A single-factor analysis of variance (ANOVA) and Dunnett’s multiple comparison test were used to assess differences between treatment means and control, with P < 0.05 being considered significant. Values are shown as percentage control. Increased (↑) or decreased (↓) intensities are indicated with arrows.

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Fig. 24.2. Effects of anti-cytoskeleton agents on bodipy-ceramide recycling in Giardia trophozoites. Trophozoites were first labelled with bodipy-ceramide for 30 min at 37°C. Samples were then washed twice by centrifugation at 1500 × g for 5 min at 25°C, and then were transferred to flaskette chambers containing anti-actin or anti-microtubule drugs and incubated for 90 min at 37°C. (a) Control; (b) 250 µM colchicine; (c) 1 µM vinorelbine; (d) 1 µM albendazole; (e) 500 nM nocodazole; (f) 5 nM taxol; and (g) 80 µM cytochalasin-D. Photographs were captured with the help of an Olympus BX-50 laser scanning confocal microscope (Olympus America, Melville, NY) equipped with a yellow krypton (568 nm) and an argon (488 nm) ion laser. Magnification 600×.

Unlike bodipy-Pam, however, the accumulation of NBD-PG in the perinuclear membranes is increased by microtubule depolymerizing or stabilizing agents (Fig. 24.4). In contrast, the depolymerization of actin filaments by cytochalasin-D reduced the localization of fluorescent PG in perinuclear membranes and exhibited increased distribution throughout the cytosol (in a punctate fashion). Table 24.2 shows an increase in NBD-PG in colchicine-, vinorelbine-, albenda-

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Fig. 24.3. Effects of anti-cytoskeleton agents on release of NBD-sphingomyelin from the perinuclear membrane of trophozoites. Trophozoites were first labelled with NBD-SM for 30 min at 37°C. Samples were then washed twice by centrifugation at 1500 × g for 5 min at 25°C, and then were transferred to flaskette chambers containing anti-actin or anti-microtubule drugs and incubated for 90 min at 37°C. (a) Control; (b) 250 µM colchicine; (c) 1 µM vinorelbine; (d) 1 µM albendazole; (e) 500 nM nocodazole; (f) 5 nM taxol; and (g) 80 µM cytochalasin-D. Photographs were captured with the help of an Olympus BX-50 laser scanning confocal microscope (Olympus America, Melville, NY) equipped with a yellow krypton (568 nm) and an argon (488 nm) ion laser. Magnification 600×.

zole- and nocodazole-treated cells, which could be due to the fact that the exit of NBD-PG from perinuclear membranes is microtubule-dependent, since alteration of the microtubule structure by microtubule depolymerizing and stabilizing agents significantly increased the localization of fluorescent PG to nuclear envelopes. However, intracellular targeting of this important phospholipid is not dependent upon actin filaments because cytochalasin-D did not block the release of NBD-PG from the perinuclear membranes.

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Fig. 24.4. Effects of anti-cytoskeleton agents on NBD-phosphatidylglycerol recycling. Trophozoites were first labelled with NBD-PG for 30 min at 37°C. Samples were then washed twice by centrifugation at 1500 × g for 5 min at 25°C, and then were transferred to flaskette chambers containing anti-actin or anti-microtubule drugs and incubated for 90 min at 37°C. (a) Control; (b) 250 µM colchicine; (c) 1 µM vinorelbine; (d) 1 µM albendazole; (e) 500 nM nocodazole; (f) 5 nM taxol; and (g) 80 µM cytochalasin-D. Photographs were captured with help of Olympus BX-50 laser scanning confocal microscope (Olympus America, Inc., Melville, NY) equipped with a yellow krypton (568 nm) and an argon (488 nm) ion laser. Magnification 600×.

Perspective This chapter focuses on understanding the mechanisms of the uptake and trafficking of membrane lipids by the parasitic protozoan Giardia lamblia. Because G. lamblia has limited lipid synthesis ability, it is plausible that the majority of lipids are taken up by this parasite from the human small-intestinal environment,

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where they colonize. It is well established that unicellular and multicellular organisms can internalize lipid molecules from the cell exterior primarily through three major mechanisms: 1. Receptor-mediated endocytosis (Mukherjee et al., 1997). 2. Fluid-phase endocytosis (Nichols and Lippincott-Schwartz, 2001). 3. Trans-membrane transport proteins (flippases) (Dolis et al., 1997) followed by diffusion as monomers, whch may then be facilitated by lipid transfer proteins (van Meer and Op den Kamp, 1982). Using commercially available fluorescent lipid analogues, our laboratory has previously demonstrated (Stevens et al., 1997; Gibson et al., 1999) that G. lamblia trophozoites selectively incorporate and distribute bodipy- and NBD-conjugated lipid/fatty acid molecules into several locations. Important organelles involved in the intracellular lipid distribution were the nuclear envelopes that were identified as the endoplasmic reticulum of the parasite (Soltys and Gupta, 1994). Furthermore, the internalization of fluorescent lipids in a time-dependent manner suggested the presence of well-evolved lipid transport pathways in this early-branching eukaryote. In addition, the presence of actin filaments was observed by immunofluorescence labelling using anti-actin antibody (C4) and was shown to regulate the endo- and exocytosis of FM 4-64 (a fluorescent membrane dye). Like actin filaments, the existence of a well-organized microtubule network was observed using monoclonal antibodies to α- and β-tubulins. This microtubule network was sensitive to various microtubule depolymerizing and stabilizing agents (Hernandez et al., 2007). We have also shown how radioactive and fluorescent ceramide internalization and intracellular targeting was dependent upon the presence of anti-actin and anti-microtubule disrupting/stabilizing agents. In addition, we reported that clathrin- but not caveolae-dependent pathways are involved in internalizing and targeting ceramide in this protozoan parasite (Hernandez et al., 2007). In our current study, we showed for the first time that not only ceramide but also the internalization of fluorescently labelled sphingomyelin (SM), palmitic acid (Pam), and phosphatidylglycerol (PG) are regulated by actin and microtubule filaments to varying degrees. We found that the anti-actin agent cytochalasin-D induced the formation of several tubular/vesicular structures in the cytoplasm of trophozoites labelled with either ceramide or sphingomyelin. Vinorelbine, a microtubule depolymerizing agent, was effective in lowering the intracellular incorporation of ceramide and sphingomyelin significantly, whereas the microtubule stabilizing drug taxol was not effective at all. These observations reflect the possibilities that both ceramide and sphingomyelin are taken up by cells through an endocytic process that depends upon the presence of intact actin and microtubule structures for intracellular targeting. Our recent results on ceramide (Hernandez et al., 2007) confirm these observations and indicate that the dynamic flux of tubulin subunits in microtubule structures is not absolutely essential for transport and targeting of bodipy-Cer and NBD-SM. In higher eukaryotes, the uptake and transport of fatty acids is thought to occur by simple diffusion, but recent results indicate that the presence of several fatty acid-binding proteins facilitates this process. Because palmitic acid is a major fatty acid in Giardia (see Das et al., 2002, for a review), we thought it

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would be interesting to investigate how this 16-carbon, saturated fatty acid is taken up and trafficked by trophozoites. We found that cytochalasin-D and the majority of microtubule-depolymerizing drugs (except nocodazole and taxol) neither altered nor reduced the localization pattern of bodipy-Pam (Table 24.1), suggesting that this fatty acid analogue does not require actin filaments for its internalization. However, at this point the possibility of some involvement of cytoskeletal components cannot be ignored (Table 24.1), because upon internalization, bodipy-Pam undergoes aggregation to form ‘lipid bodies’ that use intact microtubule filaments to be transported and localized in cytoplasm (Fig. 24.1i, j, k, l). Although no metabolic modifications of bodipy-Pam by Giardia were observed by thin-layer chromatography analyses (not shown), previous work from our laboratory has demonstrated that [3H]-palmitate can be esterified into membrane lipids such as phosphatidylglycerol and phosphatidylcholine (Stevens et al., 1997; Gibson et al., 1999), indicating a precise role of exogenous palmitic acid in giardial metabolism. The incorporation and intracellular transport of NBD-PG was investigated in the presence of anti-cytoskeleton agents. Trophozoites exposed to cytochalasin-D displayed an increase in intracellular incorporation (Fig. 24.1p), with a scattered distribution of the lipid throughout the cytoplasm. Treatment with microtubule depolymerizing drugs reduced the perinuclear incorporation of NBD-PG (vinorelbine-treated cells; Fig. 24.1n) or induced the formation of cytoplasmic lipid aggregates (nocodazole-treated cells; Fig. 24.1o). The significant increase in total NBD-PG uptake in the presence of cytochalasin-D indicates that an intact actin filament network is needed for a continuous and regulated endocytic flow. Depolymerization of actin filaments interferes with this process, allowing the uptake of excess NBD-PG and subsequent distribution to the cytoplasm. These results indicate that NBD-PG is taken up by Giardia – most probably through endocytosis – and targeted with the help of microtubule filaments. It will be interesting to investigate whether PG is internalized and trafficked with the help of clathrincoated vesicles such as bodipy-ceramide (Hernandez et al., 2007). In mammalian cells or in the higher eukaryotes, the majority of lipids are synthesized in the endoplasmic reticulum. Our results suggest that fluorescent lipids in Giardia are targeted to perinuclear membranes, which suggests that endoplasmic reticulum/perinuclear regions in this early-diverging eukaryote may contain essential enzymes for lipid metabolism and remodelling (Das et al., 2001). It can be hypothesized that remodelled lipids are targeted through vesicular or non-vesicular pathways to plasma and endomembranes. Therefore, it was our aim to investigate whether the recycling of fluorescent lipids is also dependent upon the cytoskeleton network. Studies in which the recycling of bodipy-Cer and NBD-SM to plasma and endomembranes were investigated (Figs 24.2, 24.3; Table 24.2) have revealed that the exits are not actin- or microtubule-dependent. Similarly, the recycling experiments indicate that the total fluorescence intensity of bodipy-Pam was decreased by the several anti-cytoskeletal agents used in this investigation (Table 24.2), which also supports the idea that the release of palmitic acid is not regulated by actin and microtubule filaments. In contrast to sphingomyelin and palmitic acid, the concentration of NBD-PG in perinuclear regions increased in

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the presence of anti-microtubule agents, which implies that intact microtubule filaments, but not actin filaments, are essential for intracellular trafficking of NBD-PG (Fig. 24.4; Table 24.2). Recently, it has been proposed that short-distance migration of vesicles is actin-dependent, while long-distance vesicular transport is microtubule-mediated (Goode et al., 2000). It is possible that PG in Giardia travels a long distance after its remodelling/modification in perinuclear

Lipid endocytosis

Non-endocytic uptake Bodipy-Cer NBD-SM NBD-PG Bodipy-PM

Bodipy-Cer NBD-SM NBD-PG

Flippase activity

Actin

(4)

MTs

(1)

(2) Lipid transfer

Early endosome

(3) MTs (?)

MTs MTs (?)

Recycling vesicle (?)

Late endosome

MTs

MTs

Perinuclear membrane

Fig. 24.5. The model proposes that bodipy-Cer, NBD-SM and NBD-PG are mainly taken up by actin-dependent endocytosis. Soon after endocytic vesicles pinch off from the plasma membrane containing lipid molecules, they move through microtubules to reach early and late endosomes and finally perinuclear membranes. Transport of sphingolipids and phosphatidylglycerol out of nuclear envelopes through recycling/exocytic pathways is possible and most probably dependent upon intact microtubule filaments (1 and 3). Internalization of sphingolipids, fatty acids and phospholipids may also be possible via non-endocytic pathways through bi-layer flip-flop or other mechanisms. Giardial lipid transport proteins may be involved in translocating lipid molecules to the cytosolic face of perinuclear membranes or vesicles (not shown in the model) that may travel along microtubules to reach perinuclear membranes (2). The vesicular transport of PG and possibly SM out from nuclear envelopes to recycling vesicles may be regulated by intact microtubule filaments. Non-vesicular pathways in Giardia cannot be ignored, especially for palmitate.

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membranes. However, more in-depth experiments are required to validate this point. The current observations encouraged us to propose a working model (Fig. 24.5) for the internalization, intracellular transport, and recycling of fluorescent lipid analogues in G. lamblia trophozoites, as summarized below: ●

●

●

●

Bodipy-Cer, NBD-SM, and NBD-PG are mainly taken up by actin-dependent endocytosis, soon after endocytic vesicles pinch off from the plasma membrane containing lipid molecules, they move through microtubules to reach early and late endosomes and finally perinuclear membranes. Transport of sphingolipids and phosphatidylglycerol out of nuclear envelopes through recycling vesicles to plasma or other endomembranes may be dependent upon intact microtubule filaments. Internalization of phospho- and sphingolipids as well as palmitic acid may occur via non-endocytic pathways through bi-layer flip-flop or other mechanisms located at the plasma membrane. Once lipids have been translocated to the inner leaflet of the plasma membrane, lipid transport proteins may translocate internalized lipid molecules to the cytosolic face of perinuclear membranes or to other vehicles or vesicles that may travel along microtubules to reach perinuclear membranes. The vesicular transport of PG and possibly SM out from nuclear envelopes to recycling vesicles may be regulated by intact microtubule filaments. Non-vesicular pathways for recycling may exist in Giardia, especially for palmitate and sphingolipids. However, it is possible that sphingolipids can be stored in perinuclear membranes and transported to plasma membranes through encystation-specific vesicles for the synthesis of cyst wall biosynthesis during encystation through encystation (Hernandez et al., 2007). There are several questions that remain to be answered regarding the mechanisms of how Giardia incorporates and sorts exogenously acquired lipids to several membrane compartments of the trophozoite. The import of fluorescent membrane markers and lipid analogues can be further documented first by depleting the trophozoites of ATP with chemical agents such as deoxyd-glucose to assess the energy requirements, and second by utilizing proteinmodifying agents like N-ethylmaleimide (NEM), dithiothreitol (DDT), or iodoacetamide to document the participation of membrane proteins, because these chemicals have been shown to inhibit fluorescent lipid uptake by arresting the function of protein translocators (flippases) located at the plasma membrane. Similarly, uptake through endocytic mechanisms can also be studied using several chemical processes. Receptor-mediated endocytosis in fibroblast cells has been observed to be inhibited after intracellular potassium depletion (Larkin et al., 1983).

It is not known whether Giardia has the ability to carry out receptormediated internalization of membrane lipids, but in our recent study we have shown that potassium depletion affects ceramide uptake by this protozoan parasite. Furthermore, the arresting of fluid-phase endocytosis has been documented in cells treated with nystatin (a sterol binding agent that selectively disrupts cholesterol-enriched membrane micro-domains) (Rothberg et al., 1992; Chen

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and Norkin, 1999) or with methyl-β-cyclodextrin (a membrane cholesterol sequestering agent) (Hansen et al., 2000). The proposed experiments should provide further evidence on endocytosis or protein-mediated lipid uptake by Giardia and may provide information regarding the possibility of using lipid transport/trafficking pathways as novel targets for developing new therapy against Giardia and related protozoan parasites.

Conclusions Understanding the process of lipid metabolism and membrane/organelle biosynthesis is important for the study of a protozoan parasite such as Giardia lamblia, which is unable to synthesize its own lipids and cholesterol de novo. The results discussed in this chapter strongly suggest that Giardia trophozoites have the capacity to recruit lipid molecules from their micro-environment and transfer them into appropriate target sites. For this intracellular movement of lipid molecules, Giardia uses actin and microtubule filaments like the higher eukaryotes do. Earlier studies from various laboratories primarily focused on the role of actin and microtubule filaments on Giardia’s attachment to glass or cells. More recent reports reveal that long-term exposure of trophozoites to cytochalasin-D, nocodazole and colchicine induced the fragmentation of the ventral discs of trophozoites and the formation of large vacuoles. The blocking of cytokinesis and membrane blebbing is also possible (Mariante et al., 2005; Correa and Benchimol, 2006). In our current study, we demonstrate that giardial actin and microtubule filaments are also involved in lipid and vesicle trafficking. This could be important from the evolutionary point of view, since Giardia is considered to be an earlydiverging eukaryote. For therapeutic purposes, various derivatives of actin and microtubule inhibitors should be designed and developed to treat giardiasis with better efficiency without harming humans or domestic animals.

Acknowledgements The work in the authors’ laboratory is supported by a grant from the National Institutes of Health (S06 GM 008012-34) to S. Das and an infrastructure development grant from the National Center for Research Resources (RCMI Translational Research Network) (5G112RR08124-09) to the University of Texas at El Paso.

References Adam, R.D. (2001) Biology of Giardia lamblia. Clinical Microbiology Reviews 14, 447–475. Chen, Y. and Norkin, L.C. (1999) Extracellular simian virus 40 transmits a signal that promotes virus enclosure within caveolae. Experimental Cell Research 246, 83–90. Correa, G. and Benchimol, M. (2006) Giardia lamblia behavior under cytochalasins treatment. Parasitology Research 98, 250–256.

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Signalling During Giardia Differentiation

T. LAUWAET AND F.D. GILLIN University of California at San Diego, CA, USA

Abstract Giardia lamblia is a protozoan pathogen of the human small intestine. The life cycle of Giardia and its capacity to differentiate from a motile trophozoite into a dormant, waterresistant cyst, and vice versa, is crucial for its pathogenesis. Although the physiological signals leading to encystation and excystation are known, the intracellular signalling pathways are poorly understood. Several signalling proteins have been reported to be involved in Giardia differentiation, but none have been united in a pathway. In this chapter, we summarize the signalling molecules known to affect Giardia differentiation and discuss their possible cross-talk, based on their localization and expression, and with reference to the current literature on signal transduction in other cell types.

The Importance of Differentiation in the Life Cycle of Giardia Giardia lamblia is an enigmatic pathogen because it causes intestinal disease in humans without invasion, inflammation, conventional toxin or virulence factor. However, the two differentiations that the parasite undergoes in response to external stimuli enable it to persist in the environment and infect a new host. Both giardial life cycle stages are remarkably well adapted to very different hostile environments (Gillin et al., 1996; Svard et al., 2003). The dormant, quadrinucleate, ovoid cyst survives in cold fresh water (Bingham et al., 1979) and ingestion of cysts in faecally contaminated water or food initiates infection (Barnard and Jackson, 1984). When cysts pass through the stomach, exposure to gastric acid triggers excystation, but the trophozoite must not emerge until the cyst enters the small intestine, or it will be killed (Bingham and Meyer, 1979). Thus, excystation is highly regulated. As the parasite emerges, it quickly divides into two binucleate trophozoites that colonize the small intestine. Trophozoites are characterized by a unique microtubule-based cytoskeleton with eight flagella, each nucleated in a basal body, a ventral attachment disc, and a median body © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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(Elmendorf et al., 2003). The intracellular part of each flagellum is accompanied by a dense structure called the paraflagellar dense rod. Trophozoites use their four pairs of flagella to swim in the luminal fluid where they are bathed in changing mixtures of hydrogen ions, bile, proteases and other digestive enzymes, as well as ingested food and digestion products (Davenport, 1977). Trophozoites attach to intestinal epithelial cells using their ventral attachment disc (Friend, 1966; Erlandsen and Feely, 1984; Peattie et al., 1989; Gillin et al., 1996). Relatively few other microbes colonize the small intestine. Trophozoites can persist in this environment, but if they are carried downstream by the flow of intestinal fluid they will die unless they encyst. The mechanisms that enable Giardia to encyst and to secrete the cyst wall are of broad biological interest, as the cyst wall is a model for a simple extracellular matrix (Das et al., 2006; Van Dellen et al., 2006). Excystation, on the other hand, is a model for cellular awakening from dormancy, which is the basis for many diseases. Other intestinal parasites also have an infectious cyst or oocyst form that survives outside the host (Despommier and Karapelou, 1987). Since only the giardial life cycle has been completed in vitro, it is a valuable model for differentiation of other enteric pathogens, such as Entamoeba and Cryptosporidium (Coppi et al., 2002; Van Dellen et al., 2006).

The Biology of Encystation Giardia trophozoites can colonize the human small intestine for long periods of time. However, when they are carried downstream by the flow of intestinal fluid they must encyst in order to survive outside the host. The ‘biological goal’ of encystation is differentiation into a form that can survive in the environment and infect a new host. We induced cultured trophozoites to encyst by exposing them to the luminal intestinal stimuli of increased bile at slightly alkaline pH (Gillin et al., 1989). Because the giardial cyst wall is necessary for survival outside the host and infection, its construction is of primary importance. It is the single structure that allows the parasite to persist in fresh water, resist disinfectants, pass through the new host’s stomach, and infect in the small intestine. This 300 nm thick fibrous structure excludes small molecules such as water, but transmits the physiological stimuli that induce excystation (Davenport, 1977; Bingham et al., 1979; Gillin et al., 1987). Thus, the cyst wall has the dual functions of protecting the parasite, yet allowing the entry of key signals from the host. During cyst formation, the giardial cytoskeleton is greatly remodelled as the flagella and disc are interiorized (McCaffery and Gillin, 1994; Elmendorf et al., 2003; Palm et al., 2005). Moreover, numerous encystation-specific secretory vesicles (ESV) transport cyst wall proteins to the nascent cyst wall (Reiner et al., 1990). Late in encystation, two rounds of DNA synthesis occur to produce a binucleate 16N form (Bernander et al., 2001). We found that the nuclei divide to produce four 4N nuclei late in encystation (McCaffery and Gillin,

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1994). Thus, during encystation, the cytoskeleton is regulated so that cytokinesis and motility cease, while one round of karyokinesis occurs (Bernander et al., 2001).

The Signalling Underlying Encystation Cyclic AMP (cAMP)/protein kinase A (PKA) signalling The cAMP/PKA pathway is activated by the binding of a ligand to a G-proteincoupled receptor, activating adenylyl cyclase and inducing changes in cAMP concentrations (Robinson-White and Stratakis, 2002). PKA is a serine/threonine kinase that functions as an effector downstream of cAMP and which can regulate cell cycle, cell proliferation and differentiation, microtubule dynamics, chromatin (de)condensation, nuclear envelope disassembly and reassembly, regulation of intracellular transport mechanisms and ion fluxes (Taskén and Aandahl, 2004). In ciliated mammalian cells, PKA localizes to the ciliary axoneme and is involved in Encysting

Vegetative

Vegetative

Encysting

bb Calmodulin

PP2A-C

pfr

afr PKAc

PKAr

cf

n vg

ERK1

ERK2

mb

Fig. 25.1. Localization of calmodulin, PP2A-C, PKAc, PKAr, ERK1 and ERK2 in vegetative and encysting trophozoites. Trophozoites are depicted in light grey and localization is shown in dark grey and black. afr, anterior paraflagellar rods; bb, basal bodies; cf, caudal flagella; d, disc; mb, median body; n, nucleus; pfr, posterior-lateral flagellar rods; vg, ventral groove. (Data were obtained from Abel et al., 2001; Ellis et al., 2003; Reiner et al., 2003; Gibson et al., 2006; Lauwaet et al., 2007.)

d

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the regulation of ciliary beating frequency (Salathe, 2007). The PKA holoenzyme is a heterotetramer composed of two catalytic and two regulatory subunits. Specificity and intracellular targeting of the PKA holoenzyme are directed by binding to different A-kinase anchoring proteins (AKAPs). AKAPs are a family of structurally diverse but functionally related proteins (Taskén and Aandahl, 2004). One Giardia PKAc and a PKAr subunit have been characterized (Gibson et al., 2006) and homologues of AKAP-120 and AKAP-550 are present in the Giardia genome (see http://www.giardiadb.org/giardiadb/). In vegetative cells, both PKAc and PKAr localize to the basal bodies and the paraflagellar rods of the anterior and caudal flagella (Abel et al., 2001; Gibson et al., 2006). During encystation, PKA activity was increased and the localization of both PKAc and PKAr gradually changed. Both PKAc and PKAr became more peripheral; however, PKAr staining was reduced more than PKAc staining (Gibson et al., 2006) (Fig. 25.1).

Protein phosphatase 2A signalling Protein phosphatase 2A (PP2A) is a universal serine/threonine phosphatase with a wide variety of substrates, which is involved in many cellular processes including microtubule remodelling, growth control and cellular transformations in a variety of organisms (Janssens and Goris, 2001; Tar et al., 2004; Arroyo and Hahn, 2005). PP2A holoenzyme is a heterotrimer composed of a scaffolding A, one of several regulatory B subunits, and a catalytic C subunit (Lechward et al., 2001; Sontag, 2001). In vegetative Giardia trophozoites, gPP2A-C localizes to the paraflagellar dense rods of the anterior, caudal and posterior-lateral flagella, the disc and the basal bodies (Fig. 25.1). Inhibition of PP2A-C by either okadaic acid or antisense mRNA expression reduced the formation of ESV and cysts. Moreover, gPP2A-C localization changed during encystation and was no longer present in the anterior flagellar rods, while localization to the other structures was constitutive (Lauwaet et al., 2007).

ERK/MAPK signalling The mitogen-activated protein kinase/extracellular-signal receptor kinase (MAPK/ ERK) pathway is a kinase cascade which mediates intracellular signalling in response to a variety of growth factor receptors (GFR) in many cell types (Seger and Krebs, 1995; Bornfeldt and Krebs, 1999). ERK1 (MAPK1) and ERK2 (MAPK2) are activated by dual phosphorylation on Ser and Tyr residues, downstream of GFR-ligand binding and activation of the small GTPase Ras and Raf (MAP3K). Activated ERK1/ERK2 can translocate to the nucleus where they activate specific transcription factors involved in the regulation of cell proliferation and differentiation. In vegetative giardial cells, ERK1 mainly localizes to the basal bodies, the median body, and to a lesser extent to the disc, the ventral groove and the caudal

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flagella. ERK2 localizes to the plasma membrane, nuclei and the anterior and caudal flagella (Ellis et al., 2003). In response to encystation signals, ERK2 localization becomes more cytoplasmic and it no longer localizes to the nuclei and flagella (Fig. 25.1). Changes in ERK1 localization are more subtle and entail loss of median body and caudal flagellar staining and accumulation of staining in the ventral groove. Both ERK1 and ERK2 activities and their phosphorylation are increased early and reduced later in encystation (Ellis et al., 2003). Functional roles for ERK in encystation have not been established.

Protein kinase B (PKB)/Akt signalling Protein kinase B (PKB) is a major serine/threonine kinase that acts downstream of phosphatidyl inositol-3-kinase in response to activation by cytokines, growth factors, neurotransmitters, etc. PKB has key roles in glucose metabolism, cell migration, cell growth, and apoptosis, among others (Brazil and Hemmings, 2001). Disruption of PKB leads to tumour development and aberrant growth and development in mice (Yang et al., 2004; Dummler and Hemmings, 2007). Giardia PKB (gPKB) has protein kinase activity with preferred substrate specificity for histone H1, and PKB activity was not stimulated by cAMP, Ca2+ or Ca2+/calmodulin (Kim et al 2005). The sequence of PKB contains a hydrophobic, putative transmembrane domain, suggesting membrane localization. However, gPKB has not been localized. Although the involvement of gPKB in Giardia encystation has not been investigated so far, its mRNA is increased during encystation (Kim et al., 2005).

Cross-talk between PKA, PKB, PP2A and ERK signalling pathways In human embryonic kidney cells, PKA activates PP2A by phosphorylation of the PP2A regulatory subunit B56delta (Ahn et al., 2007). A homologue of the B56delta subunit is present in the Giardia genome (http://www.giardiadb.org/ giardiadb/). During encystation, PKA enzymatic activity is increased and both PKA and PP2A disappear from the anterior flagellar rods, but remain co-localized to the basal bodies and posterior-lateral flagellar rods (Fig. 25.1). Due to their similar behaviour during encystation, we hypothesize that this serine/threonine phosphatase (PP2A) and kinase (PKA) may function in the same pathway. PP2A can both positively and negatively regulate the ERK signalling pathway by dephosphorylation of Raf1, and MEK (MAPKK), as well as ERK (MAPK) kinases (Ory et al., 2003; Mao et al., 2005; Van Kanegan et al., 2005). In addition to ERK1/2, homologues of Raf, Ras and MAPKK (MEK) can be found in the Giardia genome (http://www.giardiadb.org/giardiadb/). In vegetative as well as in encysting cells, gPP2A-C and ERK1 localize to the basal bodies and the disc. ERK2 on the other hand, only co-localizes with gPP2A-C to the caudal flagellar rods in vegetative cells, but not in encysting cells. Therefore, we hypothesize that PP2A/ERK2 signalling may regulate flagellar dynamics in vegetative cells, while during encystation, PP2A may dephosphorylate and inactivate ERK1 in the

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basal bodies. In adipocytes, PP2A is the main phosphatase that regulates/inactivates PKB (Resjö et al., 2002). PKA has also been reported to interfere with MAPK/ERK signalling, resulting in either growth inhibition or stimulation. The effect of PKA on the MAPK/ERK pathway is dependent on the cell type and the growth factor receptor that was activated. PKA can inhibit the MAPK/ERK pathway in response to growth factor receptor activation by acting downstream of Ras or by phosphorylation of Raf-1. Alternatively, in smooth muscle cells, PKA signalling and inhibition of proliferation can be induced upon activation of the ERK signalling pathway (Bornfeldt and Krebs, 1999). In vegetative Giardia, PKAc and PKAr co-localize with ERK1/2 to the caudal flagella and with ERK2 to the anterior flagellar rods. However, during encystation these signalling proteins are relocated to the periphery of the encysting trophozoite. Since both ERK and PKA activities are increased during encystation, and growth is reduced during encystation, we hypothesize that, as in smooth muscle cells, PKA is activated in response to ERK signalling, leading to inhibition of proliferation.

The Biology of Excystation Although cysts remain dormant in cold water (Bingham et al., 1979), upon ingestion they respond to host signals and awaken. The physiological stimuli of excystation are dramatic: the ambient osmolarity, temperature and H+ concentration increase greatly when cysts enter the host stomach. Although these stimuli trigger excystation, if the cyst wall opens in the stomach, the parasite will be killed. In the small intestine, exposure to slightly alkaline pH and proteases, along with host nutrients, leads to the emergence of the parasite (Gillin et al., 1989). In vitro, we model Giardia’s exposure to the environment by ‘pre-encystation’ in cold distilled water. ‘Stage 1’ models the stomach and ‘Stage 2’ the small intestine (Boucher and Gillin, 1990). During excystation, the motility apparatus rapidly re-assembles for the emerging ‘excyzoite’ to find and attach to an enterocyte in the small intestine. The metabolically active excyzoite undergoes cytokinesis and re-assembles its ventral disc and flagellar apparatus. All structures must be evenly distributed between the daughter cells (Gillin et al., 1996; Adam, 2001; Bernander et al., 2001; Elmendorf et al., 2003; Svard et al., 2003). Each of these key activities requires accurate function and regulation of cytoskeletal elements.

The Signalling Underlying Excystation cAMP/protein kinase A signalling Incubation of cysts with a PKA inhibitor during the early stages of excystation resulted in decreased numbers of excyzoites, suggesting that this pathway is needed for triggering excystation (Abel et al., 2001).

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Protein phosphatase 2A signalling In addition to the intracellular localization, gPP2A-C localizes to the cyst wall in water-treated and excysting cysts (Lauwaet et al., 2007). Surprisingly, gPP2A-C was not found in ESV in encysting cells. This is very unusual, as all previously identified proteins that localize to the cyst wall were found in the ESV. Cyst wall proteins in the cyst wall are phosphorylated and dephosphorylation is needed for excystation (Slavin et al., 2002). This suggests the need for relevant protein kinases in the ESV pathway and for protein phosphatase activity in the cyst wall.

Ca2+/calmodulin signalling Calmodulin is a well-conserved intracellular Ca2+ receptor that regulates diverse intracellular responses to hormones, neurotransmitters and other signals. Differential phosphorylation of calmodulin alters the potency of binding to different effector proteins. Calmodulin-dependent protein kinases (CaMK) are activated by binding to Ca2+/calmodulin and are regulated by phosphorylation (Soderling, 1999). Giardia calmodulin (ORF: 5333) localizes only to the basal bodies, as shown by immunofluorescence microscopy (Reiner et al., 2003). Giardia calmodulin is involved in the late stages of excystation, as specific calmodulin inhibitors (trifluoperazine and chlorpromazine) reduced the number of emerging excyzoites only when they were present in Stage 2 of excystation (Bernal et al., 1998; Reiner et al., 2003).

Cross-talk between PP2A, PKA and calmodulin signalling pathways Ca2+/calmodulin signalling cascades are involved in many cellular processes through cross-talk with other pathways involving PKB, MAPK and cAMP/PKA (Soderling, 1999). Both cAMP/PKA and Ca2+/calmodulin signalling are involved in the regulation of mammalian sperm motility (Nolan et al., 2004; Marin-Briggiler et al., 2005; Schlingmann et al., 2007). Calmodulin and CaMKIIbeta colocalize to the ‘main piece’ of the sperm flagellum. The calmodulin inhibitor W-7 reduced sperm flagellar motility, and its effects were reversed by cAMP stimulation of PKA (Schlingmann et al., 2007). Moreover, male mice lacking the germ-cell-specific PKA-C subunit had sperm cells with reduced motility and ATP content, and were completely infertile (Nolan et al., 2004). Taken together, it is likely that PKA and Ca2+/calmodulin signalling cascades regulate the resumption of flagellar motility during the early and late stages of Giardia excystation, respectively. PP2A can regulate Ca2+/calmodulin signalling both directly by dephosphorylation of calmodulin, and indirectly by dephosphorylation of CaMKI, CaMKII and CaMKIV, as shown in in vitro studies (Millward et al., 1999). The Giardia genome encodes two CaMK genes and one CaMKK (http://www.giardiadb.org/giardiadb/). Moreover, gPP2A-C is, like calmodulin, involved in the later stages of excystation

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(Lauwaet et al., 2007). Thus, gPP2A could regulate Ca2+/calmodulin signalling during the late stages of excystation. Giardia calmodulin is highly divergent and its ‘natural mutations’ might lead to novel interactions.

Conclusions and Future Research The PKA, PKB, PP2A and ERK signalling pathways are universal signalling pathways known to regulate important cellular processes including growth, differentiation and microtubule dynamics in a wide range of eukaryotes. Only parts of the signalling pathways underlying Giardia encystation and excystation have been discovered so far, and it is therefore impossible to assemble a complete map of signalling events. However, based on the current literature on signalling pathways and the localization and the activation patterns during Giardia differentiation, it is highly likely that the signalling proteins, which each independently have been shown to be involved in Giardia differentiation, interact and join forces to induce and coordinate the needed morphological and functional changes in Giardia in response to encystation and excystation signals. Most of the signalling proteins involved in Giardia differentiation localize constitutively to the basal bodies. Therefore, we have undertaken proteomic and transcriptomic analyses of the giardial basal bodies in order to identify additional signalling proteins and potential downstream effector proteins (T. Lauwaet et al., unpublished).

Acknowledgements We wish to acknowledge Dr Barbara Davids for critical reading of the manuscript. This work was supported by grants AI42488, AI51687 and DK31508.

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Preliminary Analysis of the Cryptosporidium muris Genome

G. WIDMER1, E. LONDON1, L. ZHANG1, G. GE4, S. TZIPORI1, J.M. CARLTON3 AND J.C. DA SILVA2 1Tufts

University Cummings School of Veterinary Medicine, New Grafton, MA, USA; 2Institute for Genome Sciences, Baltimore, MD, USA; 3New York University, New York, USA; 4Tufts University School of Engineering, Medford, MA, USA

Abstract In 1999, the National Institute of Health (NIH) recognized the importance of sequencing the genome of Cryptosporidium parvum and Cryptosporidium hominis as a means of addressing the urgent need to better understand these human parasites and identify potential drug targets. Although the genome sequences of these species are providing useful information for understanding their biology and their interaction with host cells, the high degree of similarity between the C. parvum and C. hominis genomes limits the usefulness of comparative methods. C. muris is not an important human pathogen per se, but due to its evolutionary distance to the human parasites, sequencing the C. muris genome will enhance the scientific value of the existing Cryptosporidium genome sequences. We report here on the progress with the C. muris genome project, and a preliminary analysis of the genome of this parasite. Based on a preliminary sequence assembly, the size of the C. muris genome is estimated at 8.7–9.3 Mbp with a 28.6% GC content.

Introduction Together with the recently completed genomic sequences of C. parvum and C. hominis (Abrahamsen et al., 2004; Xu et al., 2004), the genome of C. muris will support comparative genomic studies to improve the annotation of the genome of human infectious Cryptosporidium species, identify regulatory sequence elements in the genome, and identify highly conserved, polymorphic and unique genes between the species. The evolutionary distances between the gastric species C. muris and the intestinal C. parvum/C. hominis species will enable comparative genomic studies which are not feasible with the presently sequenced Cryptosporidium genomes, as C. parvum and C. hominis are 95–98% identical 320

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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at the DNA level. Therefore, comparative analyses will greatly enhance our understanding of the human and animal pathogens C. parvum and C. hominis, and will provide further insight into the biology and evolution of the genus Cryptosporidium, ultimately leading to improvement in strategies for developing therapeutics, vaccines and diagnostics. The genus Cryptosporidium comprises two groups of parasites which have adapted to different environments in the gastrointestinal tract; the small intestine/ colon is where the majority of species multiply, and the stomach is the site of infection by a few species, among them C. muris (Fig. 26.1). As originally suggested by pulsed-field gel karyotype analysis (Widmer et al., 2002), the genome of C. muris has diverged from that of the intestinal species C. parvum and C. hominis, probably as a result of the adaptation to the different host environment. An argument favouring the sequencing of C. muris is the fact that this species readily infects immunodeficient (Nu/Nu) and immunocompetent mice (Uni et al., 1987; Taylor et al., 1999; Miller et al., 2007). Because continuous propagation of Cryptosporidium species in culture is not feasible, DNA for sequencing is obtained from oocysts excreted in the faeces of experimentally infected animals. Oocysts can be purified virtually free of foreign DNA, greatly reducing the level

(a)

(b)

Fig. 26.1. Cryptosporidium muris parasites infecting mouse stomach pits as seen on an original slide prepared by Tyzzer dated 1908 (a), and on a recent electron micrograph (b). Photo credits: Xiaochuan Feng and Christine Pearson, respectively, Tufts Cummings School of Veterinary Medicine.

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of contaminating sequences. Cloned parasite lines are not available for any Cryptosporidium species, but populations of C. parvum have been derived from individual oocysts, which contain four genomes each. Although some genetic heterogeneity within natural and animal propagated laboratory isolates has been observed (Rochelle and Atwill, 2000; Tanriverdi et al., 2003), the lack of clonal populations has not been a problem for sequencing C. parvum and C. hominis, presumably because the continued passage of the parasites amounts to inbreeding, and has led to loss of heterogeneity.

Materials and Methods Parasites Isolate RN66, originally obtained from Waterborne Inc. (New Orleans, LA), was orally inoculated into Nu/Nu homozygous mice (Charles River, Wilmington, MA). Approximately 5000 oocysts were administered to each of 40 mice. Faeces were collected starting approximately on day 30 post-infection until day 120. Faeces were homogenized and the oocysts separated from faecal material by flotation on a saturated NaCl solution. Further purification was achieved by sedimenting the oocysts on a 15–30% Histodenz (Sigma) step gradient as described by Widmer et al. (2004). The oocyst-rich interface was aspirated, diluted with an equal volume of water, and the oocysts pelleted by centrifugation. Exogenous DNA and co-purifying bacteria were removed by suspending the sample for 7–10 min in 10% bleach (0.5% sodium hypochloride) on ice.

Nucleic acid isolation and library construction Faeces obtained from 20 mice over 2–4 nightly collections of 16 h each were used per extraction. To extract oocyst DNA, 5–10 × 107 oocysts were excysted at 37°C, and freeze-thawed three times. The lysate was incubated at 45°C for 1 h in 800 µg/ml proteinase K and 0.2% SDS in a volume of 500 µl. DNA was extracted with phenol/chloroform and precipitated in ethanol. The species was verified by sequencing a portion of the small subunit ribosomal RNA (SSU rRNA) gene and aligning the sequence with BLAST to homologous Cryptosporidium sequences in GenBank.

Preparation of cDNA library To construct a cDNA library, RNA was isolated from oocysts using TRI reagent (Sigma). Briefly, 2 × 107 purified and surface-sterilized oocysts were incubated in water at 37°C to induce excystation. The sample was then subjected to 2–3 cycles of freezing and thawing, and 1 ml of TRI reagent added. The solution was extracted with 200 µl chloroform and the aqueous and organic phases

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separated by centrifugation. The RNA was precipitated from the aqueous phase by adding 0.5 ml isopropanol, and the RNA pelleted by centrifugation. To evaluate the integrity of the RNA, a portion of the sample was fractionated on 1% agarose gel and compared to DNA size and weight standards. RNA was converted into a cDNA library by Express Genomics (Frederick, MD). The library, cloned into NotI-EcoRV restricted plasmid pExpress1, had an estimated titre of 2 × 106 colony forming units/ml and an average insert size of 1.6 kb.

Libraries and sequencing Three libraries were prepared: 2–3 Kb and 6–8 Kb libraries in the vector pHOS2, constructed at TIGR, and a 35–40 Kb fosmid library. Whole genome shotgun (WGS) reads were assembled using Celera Assembler version 3.30, with default parameters, except for minimum genome size, which was set to 4 Mb in order to reduce the number of surrogate and degenerate contigs. Over 97% of the total reads were incorporated in the assembly.

Results and Discussion Preliminary assembly statistics (May 2007) A preliminary assembly was built from approximately 27,000 small-insert library reads, 22,000 fosmid library reads and 1000 medium-insert library reads, for a total of ~50,000 sequence reads. Read length averaged ~851 bp, for a total of 42.3 Mbp and an estimated fivefold genome coverage. The sequence was assembled in 927 contigs, grouped into 595 scaffolds and covering 93% of the genome, estimated at 8.7–9.3 Mbp. This preliminary version of the C. muris WGS sequence has been deposited in GenBank under the accession number AAZY01000000, and can also be accessed from CryptoDB. Recently, the inclusion of sequences from the medium-insert library in the working assembly, corresponding to an additional 0.5X genome coverage, reduced the number of scaffolds by more than half, divided across 669 contigs, and covering an estimated 97% of the genome. Reads corresponding to an additional 1X coverage, from the medium-insert library, and several-fold coverage obtained with 454 pyrosequencing technology, will be used to construct the final assembly.

Comparative analysis of the C. muris genome BLASTN (version 2.2.16; http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare C. muris sequences with C. parvum, and C. parvum with C. hominis. Contigs with BLAST hits with e < 10−4 were identified as homologous sequences. As expected from the Cryptosporidium SSU rRNA phylogeny (Xiao et al., 2004), the

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Alignment length (bp) C. parvum versus C. hominis C. parvum versus C. muris

Fig. 26.2. Distribution of C. parvum versus C. hominis and C. parvum versus C. muris BLAST alignment lengths.

distribution of BLAST length alignments between C. parvum and C. hominis and between C. parvum and C. muris was different (Fig. 26.2). Consistent with a larger divergence time separating the gastric and intestinal Cryptosporidium species than the two intestinal species, alignments longer than 300 bp were more frequent in the C. parvum versus C. hominis alignment, whereas short alignments were more frequent in the C. parvum versus C. muris alignment. An analogous difference in distribution was observed when the percentage similarity was plotted for the two genome comparisons. BLAST hits of 85% similarity or less were more frequent when C. parvum and C. muris were compared, whereas high percentage similarity matches were more frequent in the C. parvum versus C. hominis comparison. A 300 bp fragment of the β-tubulin gene of C. parvum, C. hominis and C. muris, comprising exon 1, an intron, and a portion of exon 2, was aligned to illustrate the extent of coding and non-coding sequence divergence (Fig. 26.3). As expected, numerous mutations were identified in the intron when comparing C. parvum with C. muris. Most striking was the deletion of 18 nucleotides from the C. muris intron, resulting in an intron length of 69 bp, as compared with 85–87 bp in C. parvum and C. hominis. Exonic sequence differences between C. parvum and C. muris also exceeded the number of mutations observed between the intestinal species, although most substitutions were silent. Short regions of sequence conservation at the splice donor and splice acceptor region were noticed, suggesting that these motives are involved in splicing.

10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| ATGAGAGAAATTGTTCATATTCAGGGAGGACAATGTGGGAACCAGATTGGTGCTAAATTCTGGGAAGTCATTTCTGATGAGCACGGGATCGACCCTGTAA ........G...A..........A..T..C........C..T..A........C.....T........T..A...........T.....A..T..G.... 110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| GTTTTGAAAATAATTGAAATAAT--TTGAACTGAATAATTGACTTTTTTTTTTTCTTTTTTTTTCTTTGAAATATGAATTTAGACTGGTACTTATCATGG .....AT.C.-....A.G.....AT...TG-.C....--......GA-------..C...----..G.---....CT-.....T.A..G........... 210 220 230 240 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| AGAATCAGATTTACAGATGGAACGTATTAATGTTTTCTACAATGAAGCTTCGGGTGGAAGATACGTTCCAAGAGCGATTTTGGTAGATCTTGAGCCAGGA G...........G..A.........G.G.....A..T...........AG.A......C.T..T..A..T.....A..A..AA.G...T.A..A..T... C. parvum vs C. hominis

10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| TGAGAGAAATTGTTCATATTCAGGGAGGACAATGTGGGAACCAGATTGGTGCTAAATTCTGGGAAGTCATTTCTGATGAGCACGGGATCGACCCTGTAAG .................................................................................................... 110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| TTTTGAAAATAATTGAAATAATTTGAACTGAATAATTGACTCTTTTTTTTTCTTTTTTTCTCT--GAAATATGAATTTAGACTGGTACTTATCATGGAGA .........................................T.................T...TT...................................

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C. parvum vs C. muris

210 220 230 240 250 260 270 280 290 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|... ATCAGATTTACAGATGGAACGTATTAATGTTTTCTACAATGAAGCTTCTGGTGGAAGATACGTTCCAAGAGCAATTTTGGTAGATCTTGAGCCAGGAA ................................................G.......................G.........................

Fig. 26.3. Alignment of C. parvum, C. hominis and C. muris β-tubulin gene illustrates sequence divergence in coding and non-coding sequences. Boxed sequence indicates intron. Deletions are shown with hyphens, identities with periods. Note short regions of sequence conservation at the splice donor and splice acceptor regions. 325

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To further assess the divergence between Cryptosporidium species, open reading frames (ORFs) showing signs of positive and negative selection in a C. parvum versus C. hominis comparison were used to identify homologues in C. muris, using BLASTP. To interrogate the C. muris sequence we chose all C. parvum sequences with a low frequency of non-synonymous substitutions relative to synonymous substitutions (dN/dS < 1), an indication of negative selection (Yang, 1997). An analogous search was performed with all C. parvum genes showing evidence of positive selection (dN/dS > 1). Whereas C. muris homologues were identified for 42% of the genes from the former group, only 5% of the positively selected C. parvum genes had detectable homologues in the C. muris genome. This comparison indicates that genes under positive selection have evolved to a point where C. parvum and C. muris homologues can no longer be aligned, or may have been lost from the C. muris genome. It is also possible that some of the genes that were not detected are in fact present in the C. muris genome but are not present in their entirety in the current genome assembly and were, therefore, missed in the BLAST searches. A more detailed analysis of these genes will be of particular interest for studying the evolution of Cryptosporidium species and their adaptation to different environments in the gastrointestinal tract.

Conclusions The release of the C. muris genome sequence on the 100th anniversary of the discovery of this species by Edward Tyzzer, the first Cryptosporidium species to be described (Tyzzer, 1907), is a fitting tribute to Tyzzer’s contribution to science in general and the field of parasitology in particular. This milestone, together with the completion of the C. parvum and C. hominis genome sequences 3 years ago, illustrates the dramatic expansion of our knowledge about these parasites. To put these accomplishments in a historical context, the first description of a case of human cryptosporidiosis dates back only 30 years (Nime et al., 1976), and the emergence of these parasites as opportunistic infections in people with AIDS is even more recent (Ma and Soave, 1983). Particularly for parasites which are difficult to manipulate in the laboratory, as is the case for the genus Cryptosporidium, bioinformatics plays an essential role in driving scientific discovery. It is our hope that comparative genome analyses will lead to a better understanding of the metabolism of these parasites, and to the identification of drug targets, as well as to methods for culturing these parasites. The unrestricted availability of these sequences, and the user-friendly interface of sequence databases such as CryptoDB and GenBank, will continue to be essential research tools.

References Abrahamsen, M.S., Templeton, T.J., Enomoto, S., Abrahante, J.E., Zhu, G., Lancto, C.A., Deng, M., Liu, C., Widmer, G., Tzipori, S., Buck, G.A., Xu, P., Bankier, A.T., Dear, P.H., Konfortov, B.A., Spriggs, H.F., Iyer, L., Anantharaman, V., Aravind, L. and Kapur, V. (2004) Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304, 441–445.

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Ma, P. and Soave, R. (1983) Three-step stool examination for cryptosporidiosis in 10 homosexual men with protracted watery diarrhoea. Journal of Infectious Diseases 147, 824–828. Miller, T.A., Ware, M.W., Wymer, L.J. and Schaefer, F.W., III (2007) Chemically and genetically immunocompromised mice are not more susceptible than immunocompetent mice to infection with Cryptosporidium muris. Veterinary Parasitology 143, 99–105. Nime, F.A., Burek, J.D., Page, D.L., Holscher, M.A. and Yardley, J.H. (1976) Acute enterocolitis in a human being infected with the protozoan Cryptosporidium. Gastroenterology 70, 592–598. Rochelle, P.A. and Atwill, E.R. (2000) Intra-isolate heterogeneity and reproducibility of PCR-based genotyping of Cryptosporidium parvum using the beta-tubulin gene. Quantitative Microbiology 2, 87–101. Tanriverdi, S., Arslan, M.O., Akiyoshi, D.E., Tzipori, S. and Widmer, G. (2003) Identification of genotypically mixed Cryptosporidium parvum populations in humans and calves. Molecular and Biochemical Parasitology 130, 13–22. Taylor, M.A., Marshall, R.N., Green, J.A. and Catchpole, J. (1999) The pathogenesis of experimental infections of Cryptosporidium muris (strain RN 66) in outbred nude mice. Veterinary Parasitology 86, 41–48. Tyzzer, E.E. (1907) A sporozoan found in the peptic glands of the common mouse. Proceedings of the Society for Experimental Biology and Medicine 5, 12–13. Uni, S., Iseki, M., Maekawa, T., Moriya, K. and Takada, S. (1987) Ultrastructure of Cryptosporidium muris (strain RN 66) parasitizing the murine stomach. Parasitology Research 74, 123–132. Widmer, G., Lin, L., Kapur, V., Feng, X. and Abrahamsen, M.S. (2002) Genomics and genetics of Cryptosporidium parvum: the key to understanding cryptosporidiosis. Microbes and Infection 4, 1081–1090. Widmer, G., Feng, X. and Tanriverdi, S. (2004) Genotyping of Cryptosporidium parvum with microsatellite markers. Methods in Molecular Biology 268, 177–187. Xiao, L., Fayer, R., Ryan, U. and Upton, S.J. (2004) Cryptosporidium taxonomy: recent advances and implications for public health. Clinical Microbiological Reviews 17, 72–97. Xu, P., Widmer, G., Wang, Y., Ozaki, L.S., Alves, J.M., Serrano, M.G., Puiu, D., Manque, P., Akiyoshi, D., Mackey, A.J., Pearson, W.R., Dear, P.H., Bankier, A.T., Peterson, D.L., Abrahamsen, M.S., Kapur, V., Tzipori, S. and Buck, G.A. (2004) The genome of Cryptosporidium hominis. Nature 431, 1107–1112. Yang, Z. (1997) PAML: a program package for phylogenetic analysis by maximum likelihood. Computer and Applied Biosciences 13, 555–556.

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Proteomic Analyses in Giardia

D. PALM1 AND S.G. SVÄRD2 1Swedish

Institute for Infectious Disease Control, Solna, Sweden; 2Uppsala University, Sweden

Abstract The parasitic protozoan Giardia intestinalis is a worldwide cause of diarrhoea, but the mechanism of disease remains elusive. The parasite colonizes the small intestinal epithelium, known to be a sensor for the presence of enteric pathogens, without invading or causing severe inflammation. Proteomics has been used to study various aspects of Giardia–host interactions. Giardia can differentiate between a cyst and trophozoite stage in response to environmental stimuli. There are relatively large changes in the RNA expression but proteomic analyses showed only small changes in the proteome. This shows that many proteins, used in trophozoites, are stored in the cysts. Secretory antibodies are important for clearance of Giardia. Proteomic analyses have been used to identify the major antigens in Giardia and these proteins can be used in the development of new diagnostic methods or vaccines. Excretory-secretory products of pathogenic microbes often play important roles in host–parasite interactions. Using a proteomics approach, we have identified the major proteins in the culture supernatant after in vitro interaction between Giardia intestinalis and human intestinal epithelial cell lines. The importance of the secreted proteins during host–parasite interactions has been further studied. Giardia genome sequencing and data mining indicate that many basic biological processes are reduced in Giardia compared with the corresponding processes in other eukaryotes. Proteomic analyses of specific cellular structures or molecular complexes will be important in future Giardia research.

Introduction Sequencing of the Giardia genome has revealed approximately 6400 protein encoding genes (McArthur et al., 2000) and this is causing a paradigm shift in Giardia research. It will now be possible to study the function of each gene (functional genomics) encoding proteins in the Giardia genome, instead of a few selected genes. The entire set of proteins encoded by an organism is called the proteome and much of the complexity of different organisms lies at the protein 328

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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level. The analysis of proteomes has been named proteomics and this encompasses measurements of proteins, in a large-scale or global manner, and usually quantitatively. The term proteome was coined 1995 (Wilkins et al., 1996) but many of the tools associated with proteomics were developed much earlier: twodimensional gel electrophoresis (2-DE) in 1975 (O’Farrell, 1975), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization (ESI) MS in the late 1980s (Karas and Hillenkamp, 1988; Fenn et al., 1989). Identification of proteins by sequence database searches of complete genomes with peptide mass fingerprinting was published in 1993 (Yates et al., 1993). Until now, 2-DE combined with MALDI-MS analysis has been the most used method but combinations of liquid chromatography with different MS techniques is becoming more and more common. A search of all publications in PubMed using the keywords Giardia and proteomics in May 2007 generated only one hit (Stefanic et al., 2006). However, several other proteomic-type studies have now been performed and many more are currently being performed. In this chapter we review what has been done so far and also discuss the potential of future proteomic analyses in Giardia.

Identification of Encystation-specific Proteins Giardia has a simple life cycle consisting of a replicative trophozoite stage and an infectious cyst stage. Differentiation from trophozoite to cyst (encystation) has been extensively studied (Luján et al., 1998; Luján and Touz, 2003; Hehl and Marti, 2004) but so far encystation-specific genes have mainly been identified by the biochemical separation of enzymes involved in cyst-wall sugar synthesis (Macechko et al., 1992; Van Keulen et al., 1998; Knodler et al., 1999), using mRNA differential display (Que et al., 1996), screening of cDNA libraries using monoclonal antibodies specific against cyst walls (Luján et al., 1995; Mowatt et al., 1995) and data mining of the Giardia genome using homology searches to the identified cyst-wall proteins (Sun et al., 2003). An alternative approach to identifying genes with encystation-specific expression is protein profiling using 2-DE. Figure 27.1 shows a comparison of the total protein expression pattern in trophozoites and cysts. Computer analysis of the gels in the 4–7 pI range detected around 500 spots per gel and identified 40 trophozoite-specific spots and 18 encystation-specific spots. However, no new encystation-specific proteins could be identified since the highly expressed proteins had already been identified using other approaches, and the low expressed stage-specific proteins were difficult to identify. One interesting observation was that many proteins, highly expressed on the RNA and protein level in trophozoites but downregulated on the RNA level in cysts, still remained among the most abundant proteins in cysts (Fig. 27.1). This shows that many proteins used in trophozoites are stored in cysts; most probably due to the need for a fast differentiation from cyst to trophozoite (excystation) in order to establish an infection in the upper small intestine. Transportation of cyst-wall proteins to the surface requires a specific secretory organelle, the encystation-specific vesicle (ESV) (Gillin et al., 1987; Faubert

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Fig. 27.1. Total protein analysis in the pI range 4–7 using extracts from trophozoites (a) or cysts (b). Differences in protein intensities after silver staining were studied using the PDQuest program and an average of five gels were used in the comparative analysis.

et al., 1991). This secretory organelle has biochemical, but not morphological, similarities to the Golgi apparatus in other eukaryotes (Marti and Hehl, 2003). Regulated expression of the ESV provides the components of the cyst wall. One primary function of the ESV is posttranslational modification of cyst-wall components before transportation and fusion to the outer membrane (Marti and Hehl, 2003). These large rounded compartments are post-ER vesicles sensitive to brefeldin A and associated with peripheral Golgi markers but they have none of the morphological characteristics of Golgi cisternae. We used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles (Stefanic et al., 2006). ESVs were enriched in sucrose gradient fractions and analysed using 2-DE and MALDI-TOF analysis. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on 2-DE gels, i.e. several proteasome subunits and HSP70BiP. Cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to the cytoplasmic side of ESV membranes early during encystation. This suggests that maturation of bulk-exported cyst wall material to early ESVs may require additional recruitment of quality control factors normally associated with ER, in addition to retrograde Golgi to ER transport.

Identification of Giardial Surface Proteins Giardia trophozoites exist in an extremely hostile environment surrounded by proteases, lipases, bile salts, innate defence mediators and components of the adaptive immune system. The outer plasma membrane of the parasite is completely covered by a thick coat of variant-specific surface proteins (VSP) (Nash,

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2002). Genome sequencing showed that there are approximately 225 genes coding for different VSPs in the Giardia WB-C6 genome (McArthur et al., 2000). The cysteine-rich (11–12%), acidic (pI around 4.5) protein products range between 30 and 200 kDa with characteristic structures such as zinc finger motifs and a semi-conserved hydrophobic C-terminus (Nash, 2002). Only one VSP protein is expressed per trophozoite and the cell has the ability to switch surface coats. In vitro trophozoites switch their surface coats every 6–12 generations depending on their initial VSP (Nash, 2002). Antibodies from the host targeting VSP have been shown to be lethal to the parasite in vitro and in vivo, suggesting that the parasite switches surface antigens to avoid immune responses (Stäger et al., 1998). Switching has also been shown to take place as the parasite goes through the life cycle in vitro (Svärd et al., 1998) and in vivo (von Allmen et al., 2004). The VSP proteins were first identified when trophozoites were surfacelabelled with radioactive iodine and analysed on 1-D SDS-PAGE gels (Nash et al., 1983). One possibility is that there are surface-labelled proteins that are covered by the heavily stained VSP proteins in the 1-DE analysis. However, when we performed surface iodination or biotinylation of trophozoites followed by 2-DE analysis, VSP proteins were again the most heavily stained proteins, but we also detected weak reactivity to beta-giardin and alpha- and beta-tubulin (J.E. Palm, unpublished). This could be non-specific labelling, but exposure of the membrane to the degradable environment in the small intestine in vivo can uncover proteins usually hidden by the VSP coat. Surface labelling on parasites not expressing VSP proteins could be a way to identify new giardial surface proteins.

Identification of Adhesive Disc Proteins Attachment of the parasite to the epithelial cells in the lumen is a prerequisite for infection. Binding is mediated by a Giardia-specific organelle, the ventral adhesive disc. The ventral disc is a unique organelle for Giardia not present in the other members of the order Diplomonadida (Elmendorf et al., 2003). The parasite uses the disc as an attachment mechanism to maintain its niche on the intestinal epithelium. The disc is a rigid structure composed of microtubules in the ventral plane with perpendicular micro-ribbons extending into the cytoplasm, creating large laminated structures interconnected by dense networks of crossbridges (Holberton and Ward, 1981; Peattie, 1990). In cross-section the disc is concave with the outer edges forming the lateral crest. Upon attachment, the lateral crest projects downwards and it can produce a visible footprint on the epithelial surface in mice (Khanna et al., 1990). In the centre of the disc is an area called the ‘bare zone’ which is devoid of microtubules (Lanfredi-Rangel et al., 1999). Vesicles are frequently seen in the ‘bare zone’, suggesting that it can be a location for endo- or exocytosis (Lanfredi-Rangel et al., 1999). The intimate contact to epithelial cells makes it tempting to speculate that material can be exchanged between host and parasite at this site. Monoclonal antibodies were raised against water-treated trophozoites and screened for reactivity to the disc. The monoclonal antibodies reacted to the disc

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Trophozoite

8 h Enc

4 h Enc

Cyst

Fig. 27.2. Localization of beta-giardin using the monoclonal antibody 7GE in trophozoites, 4 h encysting, 8 h encysting and water-treated cysts.

structure of trophozoites (Fig. 27.2). 2-DE Western blot analysis and MS analysis of the corresponding spots showed that the monoclonal antibodies recognized beta-giardin, a previously described disc protein (Aggarwal et al., 1989). We used one of the monoclonal antibodies in immunofluorescence microscopy to investigate what consequences differentiation has on the disc structure. Microscopic analysis showed that the adhesive disc is disassembled early in encystation and that the building blocks of the disc are stored in fragments (Fig. 27.2). The disassembly of the disc coincides with the observation that the cells lose their ability to attach early in encystation. It is possible that the parasite uses the disassembly of the disc to escape a stressful environment. The cyst form of the parasite carries four fragments of disc components (Fig. 27.2) ready to be recycled again when excysted. All major proteins in a classical cytoskeleton fractionation according to Holberton (Holberton and Ward, 1981) of Giardia trophozoites were identified (Palm et al., 2005). The fraction contained earlier described disc-proteins such as alpha- and beta-tubulin and beta- and delta-giardin (Palm et al., 2005). SALP-1, a beta-giardin homologue found in the fraction, was AU-1 tagged and localized to the parasite adhesive disc (Palm et al., 2005). The protein concentrations of three disc proteins over the life cycle were quantified from silver-stained 2-DE gels using the PDQuest program. Compared with a protein extract from exponentially growing trophozoites, the disc proteins are slightly upregulated in the cyst stage. This indicates that the disc is already duplicated when the parasite goes into encystation. Giardia differentiates from the G2 stage in the cell cycle and the majority of the trophozoites in an exponentially growing culture are in the G2 stage (Bernander et al., 2001). This suggests that a doubling of the disc proteins is not to be expected when comparing the intensities of a protein between the cyst stage and an exponentially growing trophozoite population. An increase by a factor of two should only be expected if the majority

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Table 27.1. Cytoskeleton gene transcript abundance during differentiation as determined by serial analysis of gene expression (SAGE). Numbers represent percentages of all SAGE tags sampled, a function of the percentage abundance of the original transcripts in the sampled cells. Time points used: trophozoite, 4 and 12 h encysting cells; cyst, 30 and 60 min excysting cells.

Trophozoite

4 h enc.

12 h enc.

Cyst

30 min exc.

60 min exc.

37,927

36,975

37,748

36,361

38,576

38,598

Alpha 1-giardin

1.012

0.808

0.792

0.138

0.293

0.321

Beta-giardin

0.226

0.082

0.107

0.024

0.043

0.027

Total sampled tags Gene

Gamma-giardin

0.752

0.480

0.392

0.129

0.295

0.254

Delta-giardin

0.099

0.069

0.047

0.000

0.012

0.004

SALP-1

0.127

0.074

0.050

0.021

0.027

0.035

Alpha-2 tubulin

1.154

0.541

0.487

1.903

1.299

1.020

Beta-tubulin

0.266

0.157

0.095

0.495

0.334

0.275

of the cells in the exponentially growing culture were in the G1 phase. We used serial analysis of gene expression (SAGE) to quantify the RNA expression levels from the disc-related genes (Table 27.1). These results did not correlate with the protein quantification data. Expression was downregulated 6–10 times in the cyst stage, compared with trophozoites, for beta-giardin and SALP-1 (Palm et al., 2005). Late in excystation, the RNA levels are upregulated again, but by this time the cell already has a functional disc. The upregulation of disc proteins late in excystation is probably to produce cytoskeletal material for the second cytokinesis of the excyzoite. This indicates that there is a change in protein stability during differentiation. The turnover of important proteins is altered so that key proteins are stored within the cyst. Excystation is a rapid event and the adhesive disc is needed immediately upon exit inside the new host. There is not enough time for transcription, translation and assembly of a new adhesive disc, so proteins are stored as fragments and recycled. These results are in agreement with our earlier results that showed storage of trophozoite proteins in cysts even if the mRNAs were downregulated. It also shows that proteomic analyses are crucial during studies of differentiation of Giardia since the RNA levels often do not accurately reflect the protein levels.

Identification of Immunodominant Giardia Proteins Giardia colonization of the small intestine can result in symptoms that include nausea, epigastric pain and diarrhoea. Although the inflammatory response is low, the human immune system initiates the production of antibodies against the parasite. The humoral response to Giardia infection is known to be important in humans (Perlmutter et al., 1985), as well as in mice (Langford et al., 2002;

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Fig. 27.3. 2-DE Western blot analysis of immunoreactive proteins in the pI range 5–8. (A) Western blot probed with serum from a patient with acute giardiasis. (B) G. lamblia WB clone C6 trophozoite extract separated in the same pI range and stained with silver.

Davids et al., 2006). Experimental infections have shown that antibodies can mediate protection from secondary infection or reduce symptoms (Belosevic and Faubert, 1983; Lewis et al., 1987; Davids et al., 2006). When we started our studies, knowledge about Giardia antigens targeted by the human immune system was very limited and most of the studies had been conducted on one-dimensional gels without any identification of proteins in the reactive bands (Janoff et al., 1989; Soliman et al., 1998; Hasan et al., 2002). The best-characterized Giardia antigens were members of the VSP family (Nash, 2002). These proteins cover the complete surface of the parasite, but their high variability decreases their role in protective immunity. In order to identify immunoreactive Giardia proteins, we used sera collected from patients infected with Giardia during a waterborne outbreak in a non-endemic country (Sweden), and this gave us an opportunity to explore the non-variable antigens during acute giardiasis (Palm et al., 2003). Serum samples from 328 individuals exposed to the parasite were collected within 3 weeks of infection, negative control sera from 428 healthy individuals were also collected. Antibody titres were measured by indirect immunofluorescence, and infected patients showed substantially higher anti-Giardia titres than uninfected controls. The 93 highest titre sera were screened for anti-Giardia activity by 1-DE Western blot, using Giardia strain WB C6 trophozoites as the antigen. Sera reacted with proteins with a molecular size between 20 and 180 kDa (Palm et al., 2003). Sera showing similar reaction patterns on 1-DE Western blots were grouped. Representative sera from each group were analysed by 2-DE Western blotting, and reactive spots were mapped on silver-stained 2-DE gels (Fig. 27.3). Immunoreactive proteins were excised from Coomassie-stained 2-DE gels and In-Gel digested with trypsin. Proteins were identified by MALDI and/or tandem mass spectrometry. As some of the excised spots contained isoforms of the same protein, 16 individual proteins were identified from the 24 spots. Immunoreactive proteins are summarized in Table 27.2.

Spot number

MW (kDa)

pI

Identity

Function

1

20

5.2

GTA-1

Unknown – signal transduction?

2 and 6

55 and 35

5.3 and 5.7

β-tubulin

Cytoskeleton-associated

3 and 9

36 and 39

5.5 and 6.1

α-2-tubulin

Cytoskeleton-associated

4 and 5

29 and 27

5.5 and 5.6

β-giardin

Disc protein

7

27

5.8

SALP-1

Unknown

8

50

5.9

Enolase

Metabolic enzyme – secreted

10, 22 and 23

33.5, 44 and 46

6.5, 7.7 and 8.0

OCT

Metabolic enzyme – secreted

11, 14 and 16

25, 68 and 46

6.7, 7.0 and 7.2

ADI

Metabolic enzyme – secreted

12 and 15

33 and 33

6.9 and 7.1

α-2-giardin

Cytoskeleton-associated

13

34

7.0

α-7.1-giardin

Cytoskeleton-associated

17

32

7.2

α-1-giardin

Cytoskeleton-associated

18

27

7.3

GTA-2

Unknown

19

38

7.3

UPL-1

Unknown – U/T phosphorylase?

20

37

7.4

FBA

Glycolytic enzyme

21

33

7.6

α-7.3-giardin

Cytoskeleton-associated

24

25

8.0

TSA-417

VSP protein

Proteomic Analyses in Giardia

Table 27.2. Identified immunoreactive proteins. SALP, SF-assemblin like protein; OCT, ornithine carbamoyl transferase; ADI, arginine deiminase; UPL, uridine/thymidine phosphorylase-like; FBA, fructose-1,6-biphosphate aldolase; U/T, uridine/thymidine; VSP, variant-specific surface protein.

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Arginine deiminase (ADI), ornithine carbamoyltransferase (OCT), alpha-1, alpha-2 and 7.3 giardin were expressed as recombinant proteins in E. coli. Sera from patients with acute giardiasis also recognized the recombinant forms of the proteins (Palm et al., 2003). Cytoskeleton-associated components are over-represented in the list of immunoreactive proteins. This can partly be explained because they are highly expressed proteins, but also because most of them are located closely to the membrane of the parasite and therefore are partly exposed to the immune system. The alpha-giardin gene family (Weiland et al., 2005) was represented by four proteins. Alpha-1 and alpha-2 giardin have been characterized as the proteins responsible for the trypsin-activated Giardia lectin activity (Taglin) (Weiland et al., 2003) described by Ward et al. (1987). These proteins are also associated with the surface of the parasite, but their function in the cell or role in infection has not yet been completely elucidated (Weiland et al., 2003). Members of the giardin gene families possibly build up the reactive band around 30 kDa observed in earlier studies using 1-DE Western blotting (Janoff et al., 1989). One of the identified proteins was a semi-conserved part of the major VSP expressed in G. lamblia strain WB C6. Patients were most probably not infected by a strain expressing this VSP but cross-reactivity from other VSPs can also explain this reactivity. The identified VSP fragment has been shown to contain an immunodominant, conserved sequence (Müller et al., 1996). We conducted a similar analysis using sIgA antibodies in breast milk from mothers living in an endemic area – Leon, Nicaragua (Tellez et al., 2005). Interestingly, the presence of anti-Giardia antibodies in mothers’ milk could be connected to protection against symptomatic giardiasis (Tellez et al., 2003). The dominant antibody reactivity observed in the milks was to full-length VSP proteins, and even recombinant VSPs produced in bacteria were recognized. The milk donors had most probably been infected multiple times by several strains of Giardia during their lifetime, which could explain the broad recognition of VSP proteins. It is possible that the protective effect of the antibodies in breastfed children is related to the broad reactivity of VSP antibodies. However, the milk antibodies also targeted several of the immunoreactive proteins (ADI, OCT, alpha-giardin and enolase) found in the earlier study of serum from acute giardiasis patients, and they are also candidates as targets for protective antibodies. Mice incapable of secreting antibodies into the intestinal lumen (pIgR knockout mice) were infected with G. lamblia strain GS/M (Davids et al., 2006). These mice generate high serum titres of Giardia-specific sIgA antibodies. Passive immunization studies showed that systemic and oral transfer of these sera could induce protection in mice from infection with G. lamblia strain GS/M. Analysis of the specificity of sIgA antibodies in serum from the pIgR mice showed similar results as in the earlier two studies. Thus, using 2-DE Western blotting with anti-Giardia antibodies from three different sources (human serum, human breast milk and mice serum) and MS analysis to identify immunoreactive protein spots has generated a very complete picture of the conserved immunoreactive Giardia proteins. These antigens can be used in the development of vaccines and diagnostic tools. Further studies using parasites from different assemblages can potentially identify assembly-specific

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antigens that can complement the conserved antigens to achieve specificity in diagnosis and/or vaccination.

Identification of Secreted Proteins Several studies have searched for secreted proteins correlated to disease and colonization by G. lamblia. The VSPs were originally identified as excretorysecretory proteins because, VSPs, the major surface iodinated trophozoite proteins, were released into the media, apparently spontaneously (Nash et al., 1983; Nash and Keister, 1985). However, surface iodination would not efficiently detect secretion of internal proteins. A 58 kDa non-VSP protein, also reported to localize to the surface of G. lamblia (Kaur et al., 2001; Shant et al., 2002, 2005), was found in the culture supernatant of trophozoites incubated in serum-free medium for 6 h without interaction with host cells (Kaur et al., 2001; Shant et al., 2002, 2005). This is an interesting protein since it induces fluid accumulation in vivo and IEC cytotoxicity in vitro (Shant et al., 2002). Several proteins with masses ranging from 15 to 225 kDa and cysteine protease activities have also been reported in the supernatant of Giardia cultures, (Jimenez et al., 2004, 2000). One interesting recent observation was the identification of cysteine–proteinase activities specifically secreted into the growth medium after host–parasite interaction in vitro (Rodriguez-Fuentes et al., 2006). However, none of these proteins have been identified. We investigated whether exposure of G. lamblia to human intestinal epithelial cells (IEC) might lead to the release of specific proteins into the medium. An in vitro interaction model was set up using G. lamblia strain WB C6 and the human intestinal cell lines Caco-2, HT-29 and T-84. In the secretion experiment, G. lamblia interacted with intestinal epithelial cells in vitro for 3 h in serum-free medium 199 supplemented with ascorbic acid and cysteine. After interaction, cells were separated from the medium by centrifugation and filtering. Released proteins were precipitated, separated by 2-DE gel electrophoresis and analysed by mass spectrometry. The silver-stained 2-DE gel seen in Fig. 27.4 shows the protein found in the culture medium after these interactions. Proteins were identified by mass spectrometry and are summarized in Table 27.3. Peptide mass fingerprints were compared to theoretical digests of proteins from all species at NCBI (National Center for Biotechnology Information). Tandem mass spectrometry analysis was performed for final identification of spot number 4 (see Fig 27.4), which revealed Giardia enolase. Encircled spots (see Fig. 27.4) were identified as medium components (mainly bovine serum albumin) and were also visible in the control experiment with Giardia and epithelial cells alone. One human protein was also identified, alpha-enolase (syn. enolase-1). In addition, the heat shock 70 kDa protein 8 (HSPA8) was identified, but it is too highly conserved to distinguish between human and bovine origin. HSPA8 was only detected in the medium of co-incubated cells, indicating that it was specifically released by the human cells during the interaction. Recombinant forms of the Giardia proteins were produced in E. coli and used to raise antibodies in mice. Each serum reacted with one band of the

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MW (kDa) 97 3

5

66 2 1 45

4

31

21 3

4

5

6

7

8

9

pI

Fig. 27.4. Silver-stained 2-DE gel in the pI range 3–10 of proteins found in the culture medium after co-incubation with human intestinal cells. Encircled spots were identified as medium components (mainly bovine serum albumin) and were also visible in the control experiment with Giardia and epithelial cells alone.

Table 27.3.

Identified medium proteins after Giardia–intestinal cell interaction.

Spot

MW (kDa)

pI

1

37

6.8

Giardia OCT

2

52

7.1

Human enolase

3

66

6.2

Giardia ADI

4

48

5.6

5

69

4.1

Amino acid sequence

LGPQEYMIAPTK

Identity

Giardia enolase Human HSP70

expected molecular weight in Western blots (data not shown). Immunofluorescence microscopy showed that ADI and enolase exhibited a punctate pattern within the cytoplasm, while OCT localized prominently at the plasma membrane (data not shown). The localization was similar during co-incubation with intestinal cells, except for a higher concentration of protein at the interaction points between the parasite adhesive disc and epithelial cells. ADI and OCT are members of the arginine dihydrolase pathway found in many bacteria including Streptococci and Enterococci (Knodler et al., 1998). These enzymes metabolize arginine, producing ornithine. A possible function for the secreted proteins could be the extracellular transformation of arginine to ornithine. This inhibits uptake and decreases the concentration of available arginine.

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Enolase is a metabolic enzyme that has been found on the surface or secreted from bacteria. In its secreted form, enolase has been shown to suppress the adaptive immune response (Veiga-Malta et al., 2004) and bind to mucin, the major component in mucus (Ge et al., 2004). The vast majority of reported organisms with these proteins on the surface or secreted are living on or in mucosal surfaces. The bacteria given as examples also belong to the normal flora in the upper part of the small intestine. Potentially, these enzymes can be involved in a protective mechanism shared by the microbes living in this environment.

Future Directions The ongoing rapid evolution in separation science, mass spectrometry and bioinformatics will continue to stimulate the investigation of the giardial proteome and will lead to new insights in the near future. LC-LC-MS-MS (tandem mass spectrometry)-based techniques such as multidimensional protein identification technology (MudPIT) have advantages over gel-based techniques in speed, sensitivity, reproducibility and applicability to different samples and conditions. Currently, several MudPIT-based studies are being performed in Giardia (see Lauwaet and Gillin, Chapter 25, this volume) using fractions of different cellular compartments, and the number of similar studies is likely to increase. Posttranslational modifications play a crucial role in cell signalling and protein function. More than 200 protein modifications have been described (Mann and Jensen, 2003) and important modifications include phosphorylation, acetylation, glycosylation, ubiquitination and nitration. The analysis of posttranslational modifications is still an analytical challenge due to the fragility of chemical bonds, signal suppression of negatively charged (e.g. phosphorylated peptides) molecules in the commonly used positive detection mode, and difficulty in obtaining full sequence coverage. Phosphorylation pattern changes in Giardia are especially interesting, since genome sequencing revealed a large number of Giardia-specific kinases and phosphatases (McArthur et al., 2000), and changes in the phosphorylation pattern have been detected during differentiation (Slavin et al., 2002; Lauwaet et al., 2007). In a recent study, Lalle et al. were able to show phosphorylation and glycylation at specific positions of the 14-3-3 protein (Lalle et al., 2006). VSP proteins have been shown to be palmitoylated (Touz et al., 2005) and glycosylated (Hiltpold et al., 2000). Future studies will most probably reveal other protein modifications in Giardia with important features, especially during different stages of differentiation. The major finding in the analysis of the Giardia genome is the minimalized composition of molecular complexes in Giardia (McArthur et al., 2000). Molecular complexes or machines have been studied in other organisms using tandemly tagged bait proteins expressed in vivo followed by purification and MS analysis of the purified complexes (Rigaut et al., 1999). This kind of study has not yet been performed in Giardia, but we have recently developed a vector system with three different affinity tags for the purification of complexes. This system, in combination with gel-based analysis – MALDI-TOF and/or MudPIT, will be very useful in future studies of giardial molecular machines.

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We can conclude that we have only seen the start of proteomic analyses in Giardia. Classical genetic methods will be difficult in this polyploid organism (Bernander et al., 2001) and functional genomic approaches followed by specific biochemical experiments will be very important in future Giardia research projects.

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Jimenez, J.C., Fontaine, J., Grzych, J.M., Dei-Cas, E. and Capron, M. (2004) Systemic and mucosal responses to oral administration of excretory and secretory antigens from Giardia intestinalis. Clinical and Diagnostic Laboratory Immunology 11, 152–160. Karas, M. and Hillenkamp, F. (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Analytical Chemistry 60, 2299–2301. Kaur, H., Ghosh, S., Samra, H., Vinayak, V.K. and Ganguly, N.K. (2001) Identification and characterization of an excretory-secretory product from Giardia lamblia. Parasitology 123, 347–356. Khanna, R., Joshi, K., Kum, K., Malik, A.K. and Vinayak, V.K. (1990) An ultrastructural analysis of changes in surface architecture of intestinal mucosa following Giardia lamblia infection in mice. Gastroenterology Japan 25, 649–658. Knodler, L.A., Sekyere, E.O., Stewart, T.S., Schofield, P.J. and Edwards, M.R. (1998) Cloning and expression of a prokaryotic enzyme, arginine deiminase, from a primitive eukaryote Giardia intestinalis. Journal of Biological Chemistry 273, 4470–4477. Knodler, L.A., Svärd, S.G., Silberman, J.D., Davids, B.J. and Gillin, F.D. (1999) Developmental gene regulation in Giardia lamblia: first evidence for an encystation-specific promoter and differential 5′ mRNA processing. Molecular Microbiology 34, 327–340. Lalle, M., Salzano, A.M., Crescenzi, M. and Pozio, E. (2006) The Giardia duodenalis 14-3-3 protein is post-translationally modified by phosphorylation and polyglycylation of the C-terminal tail. Journal of Biological Chemistry 281, 5137–5148. Lanfredi-Rangel, A., Diniz, J.A., Jr and de Souza, W. (1999) Presence of a protrusion on the ventral disk of adhered trophozoites of Giardia lamblia. Parasitology Research 85, 951–955. Langford, T.D., Housley, M.P., Boes, M., Chen, J., Kagnoff, M.F., Gillin, F.D. and Eckmann, L. (2002) Central importance of immunoglobulin A in host defense against Giardia spp. Infection and Immunity 70, 11–18. Lauwaet, T., Davids, B.J., Torres-Escobar, A., Birkeland, S.R., Cipriano, M.J., Preheim, S.P., Palm, D., Svärd, S.G., McArthur, A.G. and Gillin, F.D. (2007) Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation. Molecular and Biochemical Parasitology 152, 80–89. Lewis, P.D., Jr, Belosevic, M., Faubert, G.M., Curthoys, L. and MacLean, J.D. (1987) Cortisone-induced recrudescence of Giardia lamblia infections in gerbils. American Journal of Tropical and Medical Hygiene 36, 33–40. Luján, H.D. and Touz, M.C. (2003) Protein trafficking in Giardia lamblia. Cellular Microbiology 5, 427–434. Luján, H.D., Mowatt, M.R., Conrad, J.T., Bowers, B. and Nash T.E. (1995) Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats: implications for secretory granule formation and protein assembly into the cyst wall. Journal of Biological Chemistry 270, 29307–29313. Luján, H.D., Mowatt, M.R. and Nash, T.E. (1998) The molecular mechanisms of Giardia encystation. Parasitology Today, 14, 446–450. Macechko, P.T., Steimle, P.A., Lindmark, D.G., Erlandsen, S.L. and Jarroll, E.L. (1992) Galactosamine-synthesizing enzymes are induced when Giardia encyst. Molecular and Biochemical Parasitology 56, 301–309. Mann, M. and Jensen, O.N. (2003) Proteomic analysis of post-translational modifications. Nature Biotechnology 21, 255–261. Marti, M. and Hehl, A.B. (2003) Encystation-specific vesicles in Giardia: a primordial Golgi or just another secretory compartment? Trends in Parasitology 19, 440–446. McArthur, A.G., Morrison, H.G., Nixon, J.E., Passamaneck, N.Q., Kim, U., Hinkle, G., Crocker, M.K., Holder, M.E., Farr, R., Reich, C.I., Olsen, G.E., Aley, S.B., Adam, R.D.,

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Proteomic and Genomic Approaches to Understanding the ‘Power Plant’ of Cryptosporidium

L. PUTIGNANI1, S.J. SANDERSON2, C. RUSSO1, J. KISSINGER3, D. MENICHELLA1 AND J.M. WASTLING2 1Bambino

Gesù Hospital, Rome, Italy; 2University of Liverpool, UK; of Georgia, Athens, GA, USA

3University

Abstract Cryptosporidium spp. are important parasites of humans and animals for which current therapies are extremely limited. In order to target the biology of this unusual organism, a fuller understanding of its biochemistry, including the role of its subcellular organelles, is required. Genomics has enabled us to produce predictive biochemical maps of Cryptosporidium spp. and recently proteomic confirmation of many of these pathways has been achieved with the first full-scale proteomic analysis of Cryptosporidium parvum. These proteomic data, which cover over one-third of the entire predicted proteome, have now been made publicly available via CryptoDB (http://cryptodb.org/cryptodb/). Proteomic studies confirm that the parasite appears to retain a remnant organelle that fulfils some, but not all, of the roles of a fully functioning mitochondrion. Initial attempts to describe the sub-proteome of this organelle have met with limited success, and further work is required to improve the enrichment of the mitochondria-related organelle prior to proteomic analysis.

Introduction Cryptosporidium spp. are widespread enteric pathogens of humans and animals and the causative agent of cryptosporidiosis, a disease which results in sickness and severe diarrhoea, especially in the young. In humans, infection can be lifethreatening in the very young, elderly and in immunosuppressed individuals, particularly those with HIV infection (Chen et al., 2002). Contamination of drinking water supplies by Cryptosporidium can result in major waterborne outbreaks of cryptosporidiosis (McKenzie et al., 1994; Solo-Gabriele and Neumeister, 1996); in addition, Cryptosporidium is now considered as an increasingly important foodborne pathogen (Cacciò and Pozio, 2001). 344

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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Fig. 28.1. Life cycle of Cryptosporidium parvum. Following ingestion, the oocyst excystates (a), releasing sporozoites which invade enterocytes (b, c). The excysted parasites undergo asexual (merogony) (d–g) and sexual multiplication (gametogony) producing undifferentiated gamonts (h), microgamonts (i), macrogamonts (l) and microgametes (m). Upon fertilization of the macrogamonts by microgametes, a zygote (n) develops, which sporulates, producing thinwalled oocysts (o), involved in autoinfection, and thick-walled oocysts (p), excreted from the host. Adapted from http://dpd.cdc.gov/dpdx/HTML/Cryptosporidiosis.htm, Center for Disease Control and Prevention (CDC).

Cryptosporidium spp. are protozoan parasites classified as members of the phylum Apicomplexa, class Sporozoea, subclass Coccidia, order Eucoccidida, and family Eimeriidae (Levine, 1988). The parasite possesses an apical complex and shares a life history similar to other Coccidia, comprising merogony, gametogony and sporogony, the formation of macro- and microgamonts which develop independently to form a non-motile zygote, and the development of oocysts (O’Donoghue 1995) (Fig. 28.1). However, Cryptosporidium also exhibits several important peculiarities that distinguish it from other Coccidia. These include: 1. The location of Cryptosporidium within the host cell where the endogenous developmental stages are confined to the apical surfaces of epithelial cells with a unique intracellular, but extra-cytoplasmic, position. 2. The attachment of the parasite to the host cell, where a multi-membranous attachment (the ‘feeder organelle’) is formed at the base of the parasitophorous vacuole to facilitate the uptake of nutrients from the host cell. 3. The presence of two morpho-functional types of oocysts, thick-walled and thin-walled (both lacking sporocyst, micropyle and polar granules; Barta and

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Thompson, 2006), with the latter responsible for the initiation of an autoinfective cycle in the host (Fig. 28.1). 4. Insensitivity of Cryptosporidium parasites to many common anticoccidial agents (O’Donoghue, 1995). These unique biological and morphological features have been further investigated via phylogenetic studies, which increasingly suggest that Cryptosporidium spp. should be grouped as a separate clade from other Coccidia (Morrison and Ellis, 1997). A study by Carreno et al. (1999) based on the analysis of the smallsubunit ribosomal RNA gene (SSU rRNA), indicated that Cryptosporidium spp. are distinct from the Coccidia in a sister group of the monophyletic clade of the gregarines, suggesting an early-divergent apicomplexan lineage. This phylogenetic analysis may be further substantiated by recent morphological observations including both in vitro and in vivo observations of apparent extracellular stages in the life cycle of Cryptosporidium, with some similarity to stages found in gregarines (Hijjawi et al., 2002, 2004; Rosales et al., 2005). A syzygy-like pairing of extracellular stages has been observed in Cryptosporidium, reinforcing the possible relationship with the gregarines, which are also characterized by a syzygy stage in their life cycle (Barta and Thompson, 2006). Molecular and morphological studies have suggested the absence of the plastid-like organelle (apicoplast) in Cryptosporidium parvum (Zhu et al., 2000), which is common in other Apicomplexa such as Plasmodium spp. and Toxoplasma gondii. When present, the apicoplast is the elective site of parasitespecific biochemical pathways such as type II fatty acid synthesis (FAS) (Goodman and McFadden, 2007). Again, the absence of this organelle might support the idea of an independent evolutionary history for Cryptosporidium. Thus it would appear that the ancestor to extant Cryptosporidium species lost its morphologically identifiable plastid during evolution, but the parasite has retained functional genes associated with the apicoplast in its nuclear genome (Huang et al., 2004). It is not certain whether any gregarines retain a morphologically distinct apicoplast, but a recent ultrastructural study of the eugregarine Leidyana failed to find one (Valigurova and Koudela, 2005) and the same evidence was reported for Gregarina niphandrodes (Toso and Omoto, 2007). Cavalier-Smith and Chao (2004) suggested that multiple plastid losses and replacements could have occurred in the alveolates and related eukaryotes, but that the ancestor to all alveolates was derived from a plastid-bearing eukaryote. Although there is no evidence for a plastid, both C. parvum and C. hominis possess an atypical mitochondrion that has now been extensively characterized by molecular and ultrastructural studies (Riordan et al., 1999; Putignani et al., 2004; Keithly et al., 2005; Putignani, 2005). Intriguingly, the mitochondrion closely resembles the structure and morphology reported for the mitochondria of G. niphandrodes (Toso and Omoto, 2007) and Blastocystis hominis (Nasirudeen and Tan, 2004), rather than mitochondria from other Coccidia. While the structure and substructure of this organelle has been described (Keithly et al., 2005), the expressed protein repertoire and corresponding metabolic functions of the Cryptosporidium mitochondrion are yet to be confirmed. Moreover, a full understanding of Cryptosporidium metabolism has not been attained and many

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biochemical pathways remain to be elucidated, especially those related to aerobic and/or anaerobic metabolism (Henriquez et al., 2005). The sequencing of the genome of Cryptosporidium spp. (Abrahamsen et al., 2004; Xu et al., 2004; G. Widmer, Mexico, 2007, personal communication), has substantially increased our understanding of the biology of the parasite and enabled the inference of various hypothetical metabolic pathways (Abrahamsen et al., 2004; Putignani et al., 2004; Xu et al., 2004; Putignani, 2005). The recent application of proteomics to the study of Cryptosporidium has the potential to provide protein expression evidence to translate hypothetical models into substantiated proteome maps and thus help provide a clearer picture of the hitherto controversial metabolism of the parasite. This effort has been considerably supported by the introduction of advanced bioinformatics resources for the handling of genomic and, more recently, proteomic data (ApiDB, http://eupathdb.org/ eupathdb/, see Aurrecoechea et al., 2007; CryptoDB, http://cryptodb.org/cryptodb/, see Heiges et al., 2006). Expression analysis of Cryptosporidium genes, including proteomic studies, are hampered by the difficulty of propagating the parasite in vitro due to the lack of a cell culture system capable of producing sufficient quantities of each life stage (Girouard et al., 2006). Culture systems described so far (Hijjawi et al., 2002, 2004) produce yields that are generally too low for many proteomic applications. The best source of material is from neonatal calves that shed the oocysts in their faeces in high numbers (i.e. 107 oocysts per gram of faeces, see Upton, 1997), although the analysis is therefore limited to that of the oocyst and the sporozoite. Furthermore, faecal contaminants of oocysts must be carefully minimized as they may contribute to a misleading protein profile and inaccurate proteomic analysis. Optimized oocyst faecal purification procedures are therefore required which combine high recovery and purity, such as a sucrose and Percoll (SP) combined method (Truong and Ferrari, 2006). Despite these limitations, C. parvum sporozoite and oocyst/sporozoite proteome analyses have recently been performed (Snelling et al., 2007; Sanderson et al., 2008; see also http://cryptodb.org/cryptodb/). This chapter discusses recent advances in the proteomics of Cryptosporidium and focuses on the implications of these data for understanding the role of the modified mitochondria and mitochondrial-related pathways in the cellular and evolutionary biology of this parasite. It is hoped that analysis of the Cryptosporidium proteome will confirm predictions of the biochemistry of this parasite and provide new insights into its unique biology.

Global Protein Expression Profiling High-throughput global protein expression analyses using gel- or non-gelbased protein separation technologies, coupled with mass spectrometry and bioinformatic interpretation, have enabled the study of several parasite proteomes and sub-proteomes including the apicomplexan parasites Plasmodium falciparum (Florens et al., 2002; Lasonder et al., 2002), T. gondii (Cohen et al., 2002) and Eimeria tenella sporozoites (de Venevelles et al., 2004).

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Fig. 28.2. Operational workflows of global and differential proteomics analyses of the Cryptosporidium parvum sporozoite stage. Technological platforms and experimental strategies exploited to generate current global and differential expression profiling (Snelling et al., 2007; Sanderson et al., 2008) are represented here. For details see text. The C. parvum sporozoite representation is adapted from http://www.saxonet.de/coccidia/et-spz.htm; the oocyst image was retrieved from http://www.dep.state.pa.us/dep/deputate/watermgt/wsm/wsm_dwm/FPPE/MPA.htm

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Until recently, virtually no proteome data were available for Cryptosporidium. A report describing 217 non-redundant proteins from the sporozoite stage of C. parvum, equivalent to 6% coverage of the entire predicted proteome, has been published (Snelling et al., 2007), and a more comprehensive proteome analysis of a sporozoite/oocyst mixture which expands the expressed dataset to more than 1200 non-redundant proteins (30% coverage) (Sanderson et al., 2008). Sanderson et al. employed a multidirectional approach in order to maximize the coverage of the sporozoite proteome based on the combination of three independent platforms: (i) a mono-dimensional 1-DE LC-MS/MS; (ii) a bi-dimensional 2-DE LC-MS/MS; and (iii) a gel-free multidimensional protein identification technology (MudPIT), where peptides digested with trypsin are separated by multidimensional liquid chromatography followed by MS/MS (Fig. 28.2). This strategy balances the strengths and weaknesses of each approach, with the further advantage that overlapping datasets provide greater confidence in protein identifications. Protein identifications were based upon the probabilistic matching of in silico-generated tryptic peptides and the in vitro-derived tryptic peptides, using searching algorithms such as MASCOT, SEQUEST and ProID. Protein databases used included NCBI and the CryptoDB bioinformatics resources, which integrate whole genome sequence and annotation with expressed sequence tag and genome survey sequence data and provide supplemental bioinformatics analyses and data-mining tools (Heiges et al., 2006). Additional verification and characterization was obtained by using protein BLASTP algorithms at NCBI (www.ncbi.nlm.nih.gov/BLAST/) and ApiDB, a bioinformatics resource combining genomic and expression data for the phylum Apicomplexa. In the study of Snelling et al. (2007), differential protein expression in excysted and non-excysted oocyst fractions of C. parvum, were also analysed. Stable isotope labelling using iTRAQ to quantify protein expression levels in both excysted and non-excysted sporozoite soluble fractions was employed. Tryptic peptides were analysed using nano-flow liquid chromatography – linked on-line to an electrospray tandem mass spectrometer (LC-MS/MS) (Fig. 28.2). To increase the identification rate, insoluble fractions were analysed by performing shotgun peptide sequencing (Wolters et al., 2001). These two studies represent the first global proteomic analysis of Cryptosporidium species. The number of proteins that are expected to be expressed from the sporozoite/oocyst stages of the parasite is not known and it is therefore difficult to assess the coverage of the present proteomic data. The current estimate of 30% of the predicted proteome (Sanderson et al., 2008) is based on an estimated 3900 genes predicted by gene models, and assumes that all proteins will be expressed at this stage of the life cycle (which is unlikely to be the case). Until more expression data become available, all that can be said is that the present coverage of the proteome is not less than 30% of all expected genes, but could be considerably higher, given the currently undetermined relationship between gene models and actual gene expression.

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Accessibility of Cryptosporidium spp. Genomics and Proteomics Data The database, CryptoDB, is a community bioinformatics resource for Cryptosporidium spp., including C. parvum, C. hominis and Cryptosporidium muris. CryptoDB integrates whole genome sequence and annotation with expressed sequence tags and provides supplemental bioinformatics analyses and data-mining tools. A simple yet comprehensive web interface is available for mining and visualizing the data. ApiDB integrates the existing CryptoDB, ToxoDB (http://toxodb.org/toxo/) and PlasmoDB (http://plasmodb.org/plasmo/) component resources; database links allow integration of genomic survey with other data sources, such as transcriptomic and proteomic annotations (Heiges et al., 2006). To enable the proteomic data generated for Cryptosporidium spp. to act as a community proteomics resource, Sanderson et al. (2008) have developed a publicly accessible database in which proteomics data can be viewed in the context of the genome scaffold of Cryptosporidium by mapping the peptide coordinate locations for each identified protein to their corresponding locations on the genomic contigs in CryptoDB. By using this interface, users may search for annotated gene models or ORFs that have evidence of expression based on peptides identified by MS/MS analysis. A graphical representation of the peptides in the context of the protein and genomic sequences is shown in addition to the extent of peptide coverage. Importantly, the results can be combined with other searches supported by CryptoDB, including searches for genes with EST expression evidence, or GO categories, Pfam domains, etc. The proteomics interface in CryptoDB has been designed to enable additional proteomics data from the research community to be deposited, with the aim of being able to ensure that a full proteomics picture for all life stages of the parasite can be built with time. In the majority of cases, the proteomic data from Snelling et al. (2007) and Sanderson et al. (2008) confirmed predicted genes, providing conclusive evidence for the existence of these hypothetical genes and of their expression at the protein level in the sporozoite stage of C. parvum. The MS data of Sanderson et al. (2008) were searched against both hypothetical annotated genes as well as against all possible open reading frames (ORFs) greater than 50 amino acids in the C. parvum genome, thus protein identification was not dependant on the present annotation of the C. parvum and C. hominis genome (Abrahamsen et al., 2004; Xu et al., 2004) and the analysis retained the flexibility to match to alternative annotations.

Global Protein Analysis Provides Insights into the Metabolism of Cryptosporidium The recently completed global analysis of the expressed proteome of excysted C. parvum sporozoites of Sanderson et al. (2008) provided more than 4800 protein

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Biogenesis of cellular components Cell cycle and DNA processing Cell fate Cell rescue, defence, and virulence Cellular communication, signal transduction Cellular transport Energy Interaction with the environment Metabolism Protein folding, modification, destination Binding function or cofactor dependence Regulation of metabolism and protein function Transcription Protein synthesis Unclassified

Fig. 28.3. Functional categorization of the Cryptosporidium parvum sporozoite global proteome. This is a graphical representation of the cell function distribution for the overall 1237 proteins identified by global expression profiling (Sanderson et al., 2008). Proteins were assigned to MIPS (Munich Information Center for Protein Sequences, http://mips.gsf.de/projects/funcat) categories based on the GO annotation of the CryptoDB database (http://cryptodb.org/cryptodb/) and additional annotations. Proteins with unknown function are listed as unclassified. Categories shown in bold are potentially correlated to mitochondrial pathways.

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identifications representing 1237 non-redundant proteins. The 1-DE and 2D-gelbased proteomics workflows identified 115 and 642 non-redundant C. parvum sporozoite expressed genes, respectively. Excysted sporozoites were also processed by MudPIT analysis, which resulted in 1154 non-redundant proteins. By combining both gel-based and gel-free technologies, a total of 1237 unique proteins were detected. The complete dataset was ascribed to functional categories by assigning GO classifications listed on CryptoDB to specific MIPS categories (Munich Information Centre for Protein Sequences, see http://mips.gsf.de/projects/funcat). However, for a large proportion of the proteins (39%) it was not possible to assign a putative function, and these proteins are listed as unclassified (Fig. 28.3). In the study of Snelling et al. (2007), 217 non-redundant proteins expressed in the excysted and non-excysted sporozoites of C. parvum were identified. Of these, 26 proteins exhibited increased expression associated with the excystation process, comprising many ribosomal (40S and 60S) and heat shock chaperonin (Hsp70 and Hsp90) proteins. This is perhaps expected for a parasite preparing for differentiation to further asexual and sexual stages. Chaperonins function in the folding of newly synthesized proteins and are likely to be crucial immediately before and/or during the sporozoite invasion process. Also differentially expressed were the glycolytic enzymes, lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, suggesting a highly activated glycolytic cycle for energy generation. Proteins involved in modification (thioredoxin peroxidase-like protein; protein-disulphide isomerase and glutathione S-transferase EF-1γ) were also modulated, suggesting a repertoire of posttranslational protein modification events during excystation and invasion stages of C. parvum. Significantly, no alterations in the expression levels of proteins involved in other energy pathways were detected, including potential mitochondrial components.

Genomic and Proteomic Evidence for the Function of the Remnant Mitochondria In eukaryotes many specialized functions are compartmentalized within organelles, and protein localization to membrane-enclosed organelles is a central feature of cellular organization. Recently, much attention has been paid to these organelles in protozoan parasites, chiefly because of their unique properties and therefore potential to act as novel therapeutic targets. Additionally, the mitochondria of protozoa have been of particular interest because of their potential to help inform the evolutionary origin of an organism. Although C. parvum and C. hominis spp. do not possess a mitochondrial genome (Abrahamsen et al., 2004; Xu et al., 2004; Putignani, 2005), as already discussed, a remnant organelle is present and has been described in C. parvum sporozoites (Riordan et al., 1999; Putignani et al., 2004; Putignani, 2005; Keithly et al., 2005), in C. hominis (Putignani et al., 2004), and also in the merozoite stage of C. parvum (Beyer et al., 2000). A hypothetical mitochondrial proteome was provided by Putignani et al., (2004) based on c-DNA cloning, public sequence annotation (http:// cryptogenome.umn.edu/; http://www.hominis.mic.vcu.edu/g_overview.html), and the existing literature (Strong and Nelson, 2000; Abrahamsen et al., 2004;

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Xu et al., 2004). Important pathways associated with this organelle were inferred, including: (i) protein import and folding; (ii) iron–sulphur cluster biosynthesis and assembly; (iii) transport facilitation system; (iv) electron transport and ATP synthesis; (v) pathways linking with the cytosol; and (vi) oxygen radical scavenging. Evidence for the existence of mitochondrial import and folding pathway components was corroborated by combining immunoelectron microscopy (IMEM) and immunofluorescence (IMF) Hsp60 localization data (Putignani et al., 2004; Šlapeta and Keithly, 2004), Hsp60 cloning from sporozoite cDNA, and phylogenetic analysis (Riordan et al., 1999; Putignani et al., 2004). In addition, the cloning of the Hsp70 (dnaK-like) partial coding sequence from a C. parvum expression library (Putignani et al., 2004) and the Hsp60 and Hsp70 phylogenetic analysis, showing monophylesis with the Apicomplexa clade, (Putignani et al., 2004; Šlapeta and Keithly, 2004) support the existence of a dynamic system of inner and outer mitochondrial membranes, previously characterized by Riordan et al. (1999). Consistent with this, a set of sequences coding for inner and outer transport system components (TIM, ABC, ATM carriers and ADP shuttles), a pyridine nucleotide proton pump transhydrogenase (PNT) usually involved in inner membrane proton gradient regulation and electron transport, a peptidase for imported molecule processing (MPP), and a NifS-like protein involved in iron transport and in the respiratory complex Fe-S protein assembly (LaGier et al., 2003) were described (Abrahamsen et al., 2004; Xu et al., 2004; Putignani et al., 2004) and are available for data mining (http://cryptodb.org/cryptodb/). The presence of PNT may suggest a functionally active membrane system associated with electron gradient and membrane potential difference. Its role could be related to ATP generation and pump activity of the ATP-synthesis, despite the fact that apparently only the ATP-synthase β-subunit of the multicomplex ATP–synthase complex V has been identified in the C. parvum and C. hominis genomes (Abrahamsen et al., 2004; Putignani et al., 2004; Xu et al., 2004). Cryptosporidium parvum is the only microorganism described to date that has a highly reduced mitochondrial organelle retaining a modified terminal cytochrome oxidase, the so-called alternative oxidase (AOX) (Roberts et al., 2004). Usually this plant enzyme lowers the production of mitochondrial reactive oxygen species (ROS) under stress conditions, activating an alternative respiratory pathway (Maxwell et al., 1999). An intriguing question is how the electrons enter the respiratory chain of the C. parvum mitochondrion based on the expression of the alternative NADH-dehydrogenase and on the lack of the succinate– dehydrogenase complex II. However, alternative routes have been described in other organisms. For example, certain bivalves, polychaete worms and crustaceans use sulphide oxidase (Tielens et al., 2002), and some trypanosomatids (including Trypanosoma brucei and Trypanosoma vivax) use glycerol-3-phosphate dehydrogenase for this purpose (Chaudhuri et al., 1998). Intriguingly, phylogenetic analysis showed the clustering of the C. parvum sequence with that of T. brucei (Putignani et al., 2004; Putignani, 2005) with two main tree clades originated by plants (Viridiplantae, Streptophyta) and fungi (Basidiomycota and Ascomycota) with the protozoa (C. parvum, Dyctiostelium discoideum and T. brucei) being a subgroup of the latter clade, confirming previous data on the Trypanosoma AOX (Chaudhuri et al., 1998).

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GLYCOLYSIS

PEPC

PNO Hsp70cytosolic

Oxaloacetate Oxoglut/malate translocator

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e transport

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Hsp60, matrix chaperonin

Glycerol 3-P shuttle TR-II

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MD

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CYTOSOL ADP shuttle

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G Ubi ase l thy

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E Ubi ase l thy

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Intermembrane space O-

Me

2

Fe-S protein, Fe traffic

FeSOD

H2

OUTER MEMBRANE GLUTATHIONE PEROXIDASE

THIOREDOXIN REDUCTASE

Fig. 28.4. Proteome chart of the main mitochondrion pathways of Cryptosporidium parvum inferred from genomics and proteomics data. Protein import and folding: pyridine nucleotide transhydrogenase (PNT) proton pump, Hsp70-MI (dnaK), mitochondrial Hsp60, mitochondrial processing peptidase (MPP). Iron–sulphur cluster biosynthesis and assembly: NifS. Transport facilitation: TIM (inner transport system) components, ABC, ATM transporters, phosphate carrier, ADP shuttle system (AK2), mitochondrial carriers (MCP1, MCP2). ROS scavenging system: superoxide dismutase (FeSOD), glutathione peroxidase, thioredoxin reductase and thioredoxin II (TR-II). Electron transport and ATP synthesis: alternative respiratory oxidase (AOX), alternative NADH-dehydrogenase, thioredoxin II, ATP synthase, F1 β-subunit. Links with the cytosol:malate dehydrogenase (MDH), enzyme phosphoenolpyruvate-carboxylase (PEPC), glyceraldehyde-3-P dehydrogenase (GPDHC), pyruvate:ferredoxin oxidoreductase (PNO). Stippled-shaded components represent expressed proteins (CryptoDB database, see http://cryptodb.org/cryptodb/). The picture was modified from Putignani et al. (2004).

The proteomic analysis described in this chapter confirms the expression of the following mitochondrial proteins in C. parvum sporozoites (Sanderson et al., 2008): Hsp60 and Hsp70 dnaK-like heat-shock proteins; carrier mitochondrial protein transporters; the alternative oxidase; the alternative mitochondrial NADH-dehydrogenase enzyme; the glutaredoxin-like (thioredoxin 2-like) protein; and the mitochondrial metabolism-related pyruvate:ferredoxin oxidoreductase. The current proteomic picture appears to be consistent with the presence of a partially functional relic mitochondrion, retaining only major functions such as an active import and folding pathway, iron–sulphur protein activity and electron transport (Fig. 28.4).

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Organellar Sub-proteomics in Cryptosporidium spp. Recently, sub-proteomics approaches have been applied to study organelle composition and define specific protein catalogues, starting with organelle-enriched fractions in protozoan parasites. However, due to the high sensitivity of mass spectrometry and the difficulties inherent in purifying organelles to homogeneity, it is often challenging to distinguish bona fide organellar proteins from those that are contaminating. To date, subcellular proteomes have mostly centred upon the invasion organelles of Apicomplexa, including analysis of the rhoptries and secretory proteome of T. gondii (Bradley et al., 2005; Zhou et al., 2005), the micronemes and rhoptries of E. tenella (Bromley et al., 2003), and the rhoptries of Plasmodium spp. (Sam-Yellowe et al., 2004). An equivalent study of the apical organelles of Cryptosporidium ssp. is yet to be produced. Preliminary work on lysates derived from intact organelles, resolved by density gradient centrifugation, has been designed to address the mitochondrial subproteomics of C. parvum (L. Putignani, Mexico, 2007, personal communication). Establishing the profile of functional mitochondrial proteins would reveal fundamental biochemical pathways central to Cryptosporidium spp. metabolism (Henriquez et al., 2005; Putignani, 2005). As a model of fractionation techniques and gradient concentration, the mitochondrion of the protist B. hominis (Nasirudeen and Tan, 2004) was exploited to enrich the mitochondrial phase of C. parvum, on the basis of similar size, density and morphology of the organelle. Preliminary protein expression profiling of a sucrose-gradient-enriched mitochondrial fraction of C. parvum has been undertaken adopting both gel-based separation using 1-DE followed by analysis by LC-MS/MS (Fig. 28.2) and direct LC-MS/MS analysis of the enriched sample following solution-phase tryptic digestion (L. Putignani, Mexico, 2007, personal communication). However, technical issues relating to low mitochondrial protein abundance and difficulties in enriching and isolating mitochondrial fractions (Putignani et al., 2004; Keithly et al., 2005) have so far prevented a complete sub-proteomic analysis of the C. parvum mitochondrion. A major issue affecting the enrichment procedure was contamination by oocyst wall proteins and additional subcellular organelles. The key challenges for an effective proteomic analysis of the mitochondrion-related organelle itself remain in the optimization of procedures for organellar isolation and enrichment.

Concluding Remarks Genomics has allowed us to produce predictive biochemical maps of Cryptosporidium spp. parasites. Proteomic confirmation of these pathways is now needed in order to describe fully the proposed biochemistry of this organism, and recently considerable steps have been made with the first full-scale proteomic analyses of Cryptosporidium spp. However, further work is required, especially with proteomic studies of subcellular organelles such as the mitochondrial-related organelle of Cryptosporidium spp. Such studies remain a challenging task as the intimate structure of the organelle, whose functionality is often embedded within

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membrane structures, presents particular difficulties with respect to organelle isolation and protein fractionation. Furthermore, the low abundance of proteins associated with the organelles further hinders the identification of the real mitochondrial sub-proteome. If we are to gain further insights into the complete metabolism of Cryptosporidium spp., we need to persist with the development of further and more precise sub-proteome studies.

Acknowledgements The work was supported by a BBSRC Project Grant (BBS/B/03807) awarded to J.M. Wastling; D. Menichella Ricerca Corrente Grant (200502P001617); Wastling-Putignani STSM-COST-ACTION 857. We thank DiaSorin SpA Molecular Diagnostics. This work is dedicated to Giuliano.

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Toso, M.A. and Omoto, C.K. (2007) Gregarina niphandrodes may lack both a plastid genome and organelle. Journal of Eukaryotic Microbiology 54, 66–72. Truong, Q. and Ferrari, B.C. (2006) Quantitative and qualitative comparisons of Cryptosporidium faecal purification procedures for the isolation of oocysts suitable for proteomic analysis. International Journal for Parasitology 36, 811–819. Upton, S.J. (1997) In vitro cultivation. In: Fayer, R. (ed.) Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, Florida, pp. 181–207. Valigurova, A. and Koudela, B. (2005) Fine structure of trophozoites of the gregarine Leidyana ephestiae (Apicomplexa: Eugregarinida) parasitic in Ephestia kuehniella larvae (Lepidoptera). European Journal of Protistology 41, 209–218. Wolters, D.A., Washburn, M.P. and Yates, J.R., III (2001) An automated multidimensional protein identification technology for shotgun proteomics. Analytical Chemistry 73, 5683–5690. Xu, P., Widmer, G., Wang, Y., Ozaki, L.S., Alves, J.M., Serrano, M.G., Puiu, D., Manque, P., Akiyoshi, D., Mackey, A.J., Pearson, W.R., Dear, P.H., Bankier, A.T., Peterson, D.L., Abrahamsen, M.S., Kapur, V., Tzipori, S. and Buck, G.A. (2004) The genome of Cryptosporidium hominis. Nature 431, 1107–1112. Zhou, X.W., Kafsack, B.F., Cole, R.N., Beckett, P., Shen, R.F. and Carruthers, V.B. (2005) The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins. Journal of Biological Chemistry 280, 34233–34244. Zhu, G., Marchewka, M.J. and Keithly, J.S. (2000) Cryptosporidium parvum appears to lack a plastid genome. Microbiology 146, 315–321.

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Energy Metabolism and Carbon Flow in Cryptosporidium parvum

G. ZHU Texas A&M University, College Station, TX, USA

Abstract Cryptosporidium lacks a Krebs cycle in the remnant mitochondria, thus probably solely relies on glycolysis to generate energy. The glycolytic pathway in this parasite consists of enzymes with a complex evolutionary history and diverse phylogenetic affinities. A number of enzymes are closely related to those of plants (e.g. PGluM, PGI, aldolase and GDH) or bacteria (e.g. PGM, MDH, LDH, ME, ADH1 and ADH-E). Some enzymes are characteristic to the microanaerobic lifestyle, such as PPi-PFK, which uses pyrophosphate rather than ATP. Glycolysis also provides precursors for at least two synthetic pathways: acetylCoA can be converted to malonyl-CoA for synthesizing fatty acid(s) and polyketide(s); and glycerol-3P may be used for synthesizing phospholipids. On the other hand, glycolysis may also produce three organic end-products (i.e. lactate, ethanol and acetate), which is important for maintaining the carbon flow and recycling NAD(P)H. The glycolytic pathway may produce 3–5 net ATP molecules, which is much fewer than those generated by complete aerobic metabolism (up to 36 ATP). Some glycolytic enzymes are expressed at relatively consistent levels during the parasite’s life cycle (e.g. GAPDH and GPI), while others may be differentially expressed. For example, the expression of one of the two PPiPFK orthologues is increased during the life cycle, while that of the other is decreased. The PGluM and LDH genes are expressed in free sporozoites at much higher levels than at the intracellular stages, while ADH1 and ADH-E are expressed at much higher levels in the late developmental stages. The preliminary biochemical features have been characterized for some enzymes using recombinant proteins, including LDH and MDH. Inhibitors targeting this pathway are able to inhibit the parasite’s growth, indicating that glycolytic and fermentative enzymes could be explored as drug targets against cryptosporidiosis.

Introduction Despite the fact that Cryptosporidium has been recognized for 100 years, most of our knowledge about the biochemical features of this genus of apicomplexans has only been acquired since the late 1990s, although there were a few earlier biochemical works focusing on the primary characterization of some common enzymes, such as those involved in glycolysis in the oocysts (Denton et al., 1996; 360

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Entrala and Mascaro, 1997). This type of work was severely limited by the difficulty in obtaining sufficient pure parasite material for biochemical analysis, and by uncertainty about the biochemical nature of the parasite. Therefore, subsequent effort was focused on molecular cloning using various approaches including informed guesswork about conserved regions for designing degenerate probes or PCR primers (e.g. Vasquez et al., 1996; Cacciò et al., 1997; Zhu and Keithly, 1997; Perkins et al., 1999). However, this approach also has its limitations. First, it could be difficult or impossible to clone genes that were highly divergent from their orthologues, such as some of the polyamine metabolic enzymes (Keithly et al., 1997; Yarlett et al., 2007). Second, due to the lack of pre-knowledge on the metabolic pathways, one could only guess the metabolic enzymes in the parasite based on what was known about other related species, thus, in many cases, wasting effort and resources on searching for genes that were actually absent in the parasite. A good example is the hunt for the apicoplast genomes and those encoding plastid-specific pathways that are actually absent in C. parvum (Zhu et al., 2000a). Finally, it was extremely difficult to clone species-unique genes that were absent from related species but interesting to the unique biology and to the development of therapeutics, such as type I fatty acid and polyketide synthase that was ‘accidentally’ identified in an effort to clone other genes (Zhu et al., 2000b; Zhu et al., 2002). More recently, the completion of the C. parvum genome sequencing project (Abrahamsen et al., 2004), followed by the near complete genome sequence data for C. hominis (Xu et al., 2004), has turned the molecular and biochemical work on this unique apicomplexan genus inside out. Today, we are able to study the metabolic enzymes at molecular, biochemical and pathway levels. We have also learned from the genomes that Cryptosporidium not only lacks almost all de novo biosynthetic capacities, but also possesses some unique pathways absent in many other apicomplexans (Abrahamsen et al., 2004). In this chapter, we discuss some current knowledge and progress on the glycolytic pathway and carbon-flow in Cryptosporidium, which includes both published data and unpublished observations.

General Features of Energy Metabolism in Cryptosporidium There were very limited early biochemical data on the Cryptosporidium metabolism. It was first observed that C. parvum oocysts were insensitive to respiratory inhibitors (Brown et al., 1996), followed by the detection of the activities of a complete set of glycolytic enzymes (Entrala and Mascaro, 1997). Together with the lack of a recognizable mitochondrial organelle at an ultrastructural level, these early observations suggested that C. parvum probably relies on glycolysis as its sole energy source. The most recent genome sequence analysis has confirmed this notion (Abrahamsen et al., 2004). Genomic analysis has clearly shown that C. parvum indeed lacks a cytochrome-based respiratory chain, but possesses all the glycolytic enzymes to convert polysaccharides (amylopectin/ amylose) or glucose to pyruvate and acetyl-CoA (Abrahamsen et al., 2004) (Fig. 29.1).

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Sugars

Maltose GGH

Intestinal lumen or host cell

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Fructose

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GK

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Glycerate-2P

Glycerol Enolase Complex Lipids: phospholipids Glycolipids, GPI, etc.

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PDC Aldehyde ADH1

adhE Aldehyde

Oxaloacetate MDH

ME

Pyruvate PNO

Fatty acid/ polyketide biosynthesis

PEPCL

Phosphoenolpyruvate

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Ethanol

Fig. 29.1. Glycolytic and fermentative pathways in Cryptosporidium based on current genomic and biochemical data. Solid arrow lines indicate one-step reactions, while dashed lines indicate multiple steps of reactions or directions. End-products are boxed. Plant- and bacterial-type enzymes are highlighted in dark grey and black boxes. Abbreviations: ACC, acetyl-CoA carboxylase; AceCL, acetate-CoA ligase (also termed acetyl-CoA synthetase); ADH, alcohol dehydrogenase; adhE: type E alcohol dehydrogenase; GAPDH, glyceraldehyde phosphate dehydrogenase; GDH, glycerol phosphate dehydrogenase; GGH, glucoside glucohydrolase; GK, glycerol kinase (probably absent); HK, hexokinase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; PDC, pyruvate decarboxylase; PEPCL, phosphoenolpyruvate carboxylase; PFK, phosphofructokinase; PGI, phosphoglucose isomerase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PGluM, phosphoglucose mutase; PK, pyruvate kinase; PMI, mannose-6-phosphate isomerase; PMM, phosphomannomutase; PNO, pyruvate:NADP+ oxidoreductase; TIM, triosephosphate isomerase.

There are at least five transporters responsible for the uptake of sugars from the host intestinal lumen and/or host cells, and a hexokinase (HK) to activate various hexoses. The parasite utilizes two homologues of pyrophosphatedependent phosphofructokinase (PPi-PFK), rather than ATP-dependent PFK (ATP-PFK) to economize the use of ATP, for which the activity was previously detected in oocysts (Denton et al., 1996). It also possesses a unique bifunctional pyruvate:NADPH oxidoreductase (PNO) that is otherwise only observed in

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another distant protist, Eugnela gracilis, to convert pyruvate to acetyl-CoA (Rotte et al., 2001). There are three potential organic end-products: 1. Lactate produced by a bacterial-type lactate dehydrogenase (LDH) from pyruvate. 2. Acetic acid produced by an AMP-dependent acetate-CoA ligase (AceCL, also termed as acetyl-CoA synthetase). 3. Ethanol produced from acetyl-CoA by a bifunctional type E alcohol dehydrogenase (adhE) or from pyruvate by pyruvate decarboxylase (PDC) and a monofunctional ADH1 (see Fig. 29.1; end-products are boxed). Glycolysis is a low-efficiency process to produce energy. In comparison with the complete aerobic oxidation of one hexose that produces up to 36 ATP molecules, the anaerobic metabolism can only produce 3–5 ATP molecules for C. parvum, depending on whether it starts from sugars or amylopectin and whether it ends with acetate or other end-products or malonyl-CoA (Fig. 29.1; middle inset). While discussing energy metabolism in C. parvum, one needs to remember that this parasite apparently lacks the β-oxidation pathway and is unable to utilize fatty acids as its energy or carbon source (Zhu, 2004).

Other Synthetic Pathways Connected to the Glycolytic Pathway As well as producing organic end-products, acetyl-CoA may be converted to malonyl-CoA by a cytosolic acetyl-CoA carboxylase (ACC) for synthesizing fatty acids and polyketides, which appears to be the only major synthetic pathway connected to the end of glycolysis (Fig. 29.1). Cryptosporidium does not have a plastid-type ACC, which is consistent with the lack of an apicoplast organelle (Zhu et al., 2000a). It also lacks type II enzymes for synthesizing fatty acids de novo, but possesses a multifunctional type I fatty acid synthase (FAS) and a polyketide synthase (PKS) that prefer long-chain fatty acids as their substrates, suggesting that the malonyl-CoA would be used for fatty acid and polyketide elongation, rather than de novo synthesis (Zhu et al., 2000b, 2002, 2004; Fritzler and Zhu, 2007). Additionally, fructoase-6P and glycerol-3P produced in the early stage of glycolysis may be utilized for synthesizing N-glycans and complex lipids before ATP is produced (Fig. 29.1). It has been observed that the Cryptosporidium genome appears to not encode a glycerol kinase (GK) for generating an ATP, although its weak activity was previously reported (Entrala and Mascaro, 1997). Therefore, glycerol is unlikely to be an end-product and cannot be used as an energy source. The above observations, together with the fact that Cryptosporidium lacks de novo synthetic capacity for amino acids and nucleotides, indicate that the carbon flow in Cryptosporidium starting from the uptake of sugars is only channelled through glycolysis to produce three potential end-products and to synthesize fatty acids, polyketides and complex carbohydrates (e.g. N-glycans), glycolipids or glycoproteins. Because the syntheses of fatty acids, polyketides and complex molecules are not unlimited, the production of the three end-products

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appears to play a more important role in maintaining the necessary carbon flow for the highly variable energy needs and the essential recycling of NAD(P)H during the parasite development.

Glycogenesis in Cryptosporidium Cryptosporidium is capable of synthesizing amylopectin and trehalose. Amylopectin and amylose are common energy storage forms among apicomplexans. This parasite may simply synthesize polysaccharides using glucose or other sugars as a precursor. Although PPi-PFK is able to catalyse reversible reactions and can be used in glycogenesis, there is no evidence to show that this parasite could produce or take up either pyruvate or acetyl-CoA (or other glycolytic mid-products) from other sources to serve as precursors for glycogenesis. The ‘reversed’ glycolytic pathway for glycogenesis does not appears to be energy-efficient, due to the fact that this parasite relies mostly (if not solely) on glycolysis for producing ATP. However, this notion requires further experimental validation. The ability to synthesize trehalose is less common among apicomplexans, as up until now, this pathway has only been found in Cryptosporidium and Theileria (Abrahamsen et al., 2004; Gardner et al., 2005). It is proposed that trehalose might play a similar role as mannitol in Eimeria coccidia (i.e. in desiccation and stress tolerance), due to the lack of the mannitol cycle pathway in this parasite (Abrahamsen et al., 2004; Thompson et al., 2005).

Bacterial and Plant-type Cryptosporidium Glycolytic Enzymes Among the glycolytic enzymes, malate and lactate dehydrogenases (MDH and LDH) were the first to be cloned and sequenced. Phylogenetic analysis surprisingly indicated that all apicomplexan MDH/LDH were probably originated from a single α-proteobacterial MDH (Zhu and Keithly, 2002). The separation of apicomplexan LDH from MDH had occurred before species expansion within the phylum, whereas CpLDH further differs from other apicomplexan LDHs by evolving from CpMDH via a separate gene duplication event (Madern et al., 2004). With the availability of the entire genome, further analysis indicates the presence of bacterial- and plant-type glycolytic enzymes in C. parvum (Abrahamsen et al., 2004). As shown in Fig. 29.1, there are at least seven bacterial-type enzymes and five plant-type enzymes in the glycolytic and fermentative pathways, indicating a complex evolutionary history of the energy metabolism in this parasite (Abrahamsen et al., 2004; Huang et al., 2004). Several enzymes involved in amylopectin synthesis are also of bacterial affinity, such as glucan branching enzyme and amylase (Huang et al., 2004). Both types of enzymes were probably acquired by various lateral gene transfer (LGT) events, in which the plant-type genes might possibly be associated with the ancient acquisition of the apicoplast via a secondary endosymbiosis (Huang et al., 2004), although the genus Cryptosporidium appears to have lost this plastid organelle and its organellar genome (Zhu et al., 2000a).

Energy Metabolism and Carbon Flow in Cryptosporidium parvum M

1

2

3

4

5

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M bp

GAPDH– 18S–

PGIuM– 18S–

– 500 – 200

– 500 – 200

PPi-PFK1–

– 500

18S– – 200

PPi-PFK2– 18S–

– 500 – 200

Fig. 29.2. Gene expression of selected glycolytic enzymes during various parasite life cycle stages as determined by semi-quantitative RT-PCR using total RNA as templates. The levels of 18S rRNA were used as an internal control in each reaction. Lane 1 = oocysts, lane 2 = sporozoites, lane 3 = intracellular parasite at 24 h postinfection (PI), lane 4 = 48 h PI, lane 5 = 72 h PI, lane 6 = negative control containing mixed RNA and all other reagents except for the reverse transcriptase. Results are representative from at least three duplicates.

Using semi-quantitative RT-PCR, our unpublished observations have indicated that some glycolytic enzymes are expressed at relatively consistent levels in different C. parvum life cycle stages (e.g. GAPDH), while others are differentially expressed (e.g. PGluM) (Fig. 29.2). PGluM is expressed at much higher levels in oocysts and sporozoites, indicating that amylopectin is an essential energy source for the Cryptosporidium extracellular life cycle stages. The two PPi-PFK genes in C. parvum are also differentially expressed – one gene is highly expressed in extracellular stages, but at lower levels in the intracellular development; whereas the other gene is expressed in the opposite manner (Fig. 29.2), indicating the differential roles played by these two PFK homologues during different life cycle stages. Differential expression of other genes, such as the two ADH genes, have also been observed in our laboratory, and the data will be presented in forthcoming publications.

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Drug Development against Cryptosporidium Glycolytic Enzymes Because Cryptosporidium mainly (if not solely) relies on glycolysis for its energy, and many enzymes within this pathway are highly divergent from the orthologues in animals, targeting this pathway may be a good choice for drug development against cryptosporidiosis in humans and animals (Abrahamsen et al., 2004; Thompson et al., 2005). For example, we have previously shown that pyrozole, an inhibitor of alcohol dehydrogenase (ADH), could inhibit the growth of C. parvum in vitro, which provides a proof of concept in exploring this and other glycolytic and fermentative enzymes as valid drug targets (Cai et al., 2005). As mentioned earlier, there are a number of bacterial- and plant-type enzymes within the pathway to serve as a first set of molecular targets for functional and therapeutic explorations. On the other hand, little research has been done on the biochemical characterizations of these enzymes using purified fusion proteins. Among these, the enzyme kinetics and activities for MDH and LDH have been reported (Madern et al., 2004), and the crystals have been generated for the fusion proteins of GAPDH, PK and LDH (Senkovich et al., 2005). PNO has been expressed as a fusion protein and its localization in cytosol and the crystalloid body has been observed, but its biochemical features remain to be further characterized (Ctrnacta et al., 2006). Our laboratory has several ongoing projects to explore the biochemical and therapeutic features of glycolytic and fermentative enzymes in C. parvum, such as LDH, MDH, adhE, ADH1 and PPiPFK, and will report detailed results in future publications.

Conclusions In summary, Cryptosporidium differs from most apicomplexans in its energy metabolism by lacking a Krebs cycle and relying mainly on glycolysis and fermentation to produce ATP. Many glycolytic enzymes are either bacterial- or plant-type, indicating a complex evolutionary history of the pathway. Some enzymes are expressed at relatively consistent levels, while others are differentially expressed during the complex parasite life cycle. The essentialness of glycolysis in all organisms, the molecular divergence of this pathway in this parasite from humans and animals, and published and unpublished observations, support the notion that the core energy metabolism may be explored as a rational molecular target for drug development against cryptosporidiosis, for which no completely effective drugs are yet available.

Acknowledgements The studies mentioned in this chapter were supported in part by the National Institute of Allergy and Infectious Diseases (NIAID) and National Institutes of Health (NIH), USA (grants R01 AI44594 and R21 AI055278 to G. Zhu) and through a developmental project funded by the West Regional Center for Excel-

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lence in Biodefense and Emerging Infectious Diseases (WRCE, NIH Center grant U54 AI057156 to D.H. Walker).

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The Surface Protein Repertoires of Cryptosporidium spp. and Other Apicomplexans

T.J. TEMPLETON Weill Medical College of Cornell University, New York, USA

Abstract Whole-genome and high-coverage nucleotide sequence information is now available for three species of Cryptosporidium: C. parvum, C. hominis and C. muris. In this chapter I introduce the repertoires of Cryptosporidium surface and secreted proteins as revealed by complete annotations and whole-genome comparisons. These descriptions are also extended to comparisons with the apicomplexans, Plasmodium, Theileria and Toxoplasma, all of which have available complete nucleotide sequence information. Cryptosporidium possesses a large repertoire of multi-domain surface proteins that align it with the Coccidia, to the exclusion of Haemosporidia (Plasmodium spp.) and Piroplasmida (Theileria). The proposed phylogenetic affinity of Cryptosporidium with Gregarina indicates that these two early-diverging apicomplexans might uniquely share surface protein repertoires; however, analyses of sequence information from a GSS project for the gregarine, Ascogregarina taiwanensis, did not identify proteins that are conserved with Cryptosporidium to the exclusion of Toxoplasma. Cryptosporidium possesses numerous lineage-specific proteins, many of which are expanded within gene families and within loci of paralogous genes. These rapidly evolving surface proteins may have conferred adaptations to the specialized parasitic niche within the intestinal epithelium, and perhaps mediate direct physical interactions with the host. The lineage-specific proteins are conserved, perhaps without exception, in C. hominis, but are highly divergent and apparently not universally conserved as orthologues in the newly available genome nucleotide sequence database for C. muris.

A Brief Introduction to Apicomplexan Surface Proteins The availability of whole-genome or high-coverage nucleotide sequences for Cryptosporidium parvum, C. hominis and, most recently, C. muris, has opened up a landscape of comparative studies both within the Cryptosporidium genus and across the apicomplexan clade. These studies will aid in the forming of hypotheses in the description of metabolic pathways, the identification of drug © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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targets and drug discovery, genotyping, and the topic of this chapter, the characterization of parasite surface antigens. C. parvum possesses a repertoire of over 300 predicted surface or secreted proteins, the breadth of which outstrips the ability of researchers to characterize life-cycle stages of expression, cellular localizations, and predicted functions. For this reason it is necessary to prioritize surface proteins for study, and one goal of this chapter is to provide information from intra-genus and apicomplexan-specific comparative studies. This study will use whole-genome or high-coverage nucleotide sequence databases for the above Cryptosporidium species, plus those of Plasmodium falciparum, Theileria parvum and T. annulata; and the unreleased databases for Toxoplasma gondii, Eimeria tenella and Babesia bovis. This study will not consider integral membrane proteins such as transporters or ion channels, nor will it describe proteins with predicted enzymatic activities such as proteases. Surface or secreted proteins are herein defined as predicted proteins that possess a signal peptide sequence but lack ER retention signals or intracellular trafficking motifs, and do not have similarity to known intracellular proteins via BLAST screenings of GenBank. These proteins may additionally possess one or multiple transmembrane (TM) regions or a GPI anchor signal sequence; mucin-like stretches of threonine or serine residues; a cysteine-rich character that is typical of extracellular (EC) globular domains; or be comprised of multi-domain architectures, including those with complexities that are typically associated with extracellular proteins of animals or plants. Many EC globular domains and proteins are of lineage-specific or apicomplexan origin, whereas others are of foreign origin, such as the domains thrombospondin type 1, EGF, Notch, Apple, CCP (Sushi), vWA, SCP and Kringle, to name a few. Many of the foreign EC domains probably had an origin within the metazoa or bacteria and their presence in the Apicomplexa arose via lateral transfer rather than vertical inheritance (Striepen et al., 2004; Templeton et al., 2004a; Templeton, 2007). The presence of these EC domains within complex multi-domain architectures is a feature typical of the Apicomplexa and is not generally found in other protozoans that have been analysed. A whole-genome comparison of predicted orthologues that are shared between Cryptosporidium and Plasmodium revealed that surface proteins are the most rapidly evolving class of cellular proteins, in marked contrast to the repertoires of proteins that constitute more conserved cellular machineries such as transcription, translation, DNA replication and repair, and intracellular protein trafficking and degradation (Templeton et al., 2004a). Thus the ‘invention’ and expansion of lineage-specific EC proteins probably played an important role in the adaptation of apicomplexan parasites to highly evolved parasitic niches. For example, P. falciparum possesses a large, lineage-specific family of over 60 var genes that encode the erythrocyte surface protein, termed PfEMP1, which mediates sequestration of infected erythrocytes via adherence to the endothelium of post-capillary venules (Deitsch and Hviid, 2004; reviewed in Kraemer and Smith, 2007). Similarly, the majority of predicted EC proteins in Cryptosporidium do not have recognizable orthologues outside of this genus. As described below, many of these proteins are expanded within gene families, sometimes within loci of amplified genes in which neighbouring paralogues are highly divergent from each other.

Surface Protein Repertoires of Cryptosporidium spp.

371

(A) cgd6_2090 Chro.60244 C_muris

VCPPGYTMEAGVAQGTRRSLGTASNHPHHSSGHHHALGHHHHHHAVTQEVSIVRTTVCS 1495 VCPPGYTMEAGVAQGTRRSLGTASNHPHHSSGHHHALG-HHHHHAVTQEVSIVRTTVCS 1492 VCPPGYTMESGVAAGNPKNIAVSS-----HSGHHHSLG--HHQRSVMAPTNIVRTTVCI 1477 *********:*** *. :.:..:* *****:** **:::* ..*******

(B) cgd7_3860 Chro.70431 C_muris_4328 cgd7_3870

QLRYHLDDVNEQWSFIASEARKPGIFMAPTMPTDSSLLGDVWRKLYVDQHIATSSVNEV QLRYHLDDVNEQWSFIASEARKPGIFMAPTMPTDSSLLGDVWRKLYVDQHIATSSVNEV QQVYRLDNVNEQWAFIAKEAKKPGVFYAPTLPSDSSLLGDVWRNIYMKQQLVTSTEKTV SFYKSINDREKEWHAVVNEAR------ISPLVSSKDLTEEIVPYTFIDERIKR--ESNL . ::: :::* :..**: ..: :...* :: ::.::: . :

cgd7_3860 Chro.70431 C_muris_4328 cgd7_3870

DAKISILTDMCSKAIRNLMQKKN--YYGEQLYKMVYDGEN--SIEVFCHDVSRR DAKISILTDMCSKAIRNLMQKKN--YYGEQLYKMVYDGEN--SIEVFCHDVSRR ESKITVLTEMCIQAINDLKRKKN--YYGDPLYKMTYDSQD--SIEIFCSDIAGR EKKESLLSDVCFKTINTLRKERSQGSSDIPLYNIEIGSENEVSIAKFCKGLFER : * ::*:::* ::*. * :::. . **:: ..:: ** ** .: *

1059 1059 1067 568

Fig. 30.1. Examples of conservation of EC proteins in Cryptosporidium spp. Panel (A) amino acid divergence is typically found in repeat regions, such as within a histidine-rich region within COWP1. This region also exemplifies the greater divergence that is observed within a predicted COWP1 orthologue in C. muris. Shown is an amino acid alignment of C. parvum COWP1 (cgd6_2090) with the C. hominis orthologue (Chro.60244) and the predicted C. muris orthologue. Residues that are shared are indicated by grey shading and residues that are shared by the three orthologues are indicated by an asterisk in the consensus line below the alignment. The open star above the sequence indicates a site of sequence divergence between C. parvum and C. hominis. Panel (B) conservation of amino acids within a region of a CpLSP protein that is has orthologues in C. parvum (cgd7_2090), C. hominis (Chro.60244), and C. muris (an ORF on contig4328). Also shown is conservation within a paralogue (cgd7_3870) that is adjacent to cgd7_2090 within a locus of CpLSP proteins. Residues that are shared with cgd7_2090 are indicated by grey shading. Residues that are shared by the three orthologues are indicated by an asterisk in the consensus line below the alignment.

Comparisons of C. parvum with C. hominis and C. muris Annotation of the open reading frames (ORFs) encoded within the complete nucleotide sequence of C. parvum identified over 300 predicted EC proteins (Abrahamsen et al., 2004). Two hundred of these proteins were compared with C. hominis via BLAST analyses, in an attempt to understand the range and character of sequence divergence within the repertoire of EC proteins that are shared between these two species. The average sequence identity of the 200 proteins was 95% (the median was 96%), and the range was 70–100%. Sequence divergences were largely confined to conservative amino acid changes, such as valine to alanine, or lysine to arginine, as well as variations in low-complexity regions including repeat regions such as the abundant stretches of threonine, serine, or proline-rich regions. For example, the oocyst wall protein, COWP1, is identical at 1603 out of 1623 residues, and 9 of the 20 changes are within

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low-complexity histidine-rich spacers that are a feature of COWP proteins (Templeton et al., 2004b). The amino acid alignment in Fig. 30.1A shows an example of divergence within a histidine-rich spacer of COWP1, as well as the greater divergence for the predicted orthologue in C. muris. Via TBLASTN analyses, approximately 5% of the C. parvum EC proteins did not identify orthologues in the C. hominis proteins that were submitted to GenBank, although there is no indication that the observed vacancies are due to species-specific differences rather than the incompleteness of the C. hominis genome nucleotide sequence (as discussed further below). Thus it remains unknown whether EC proteins underpin differences in virulence or host specificities of C. parvum versus C. hominis. Cryptosporidium muris was initially identified as a parasite of rodents and is now known to infect a range of mammals, including animals of veterinary interest and, importantly, immunocompromised individuals such as AIDS patients (reviewed in Xiao et al., 2004). Based upon the tissue tropism of C. muris, which targets epithelial microvilli within the harsh environment of the stomach, it might be anticipated that this parasite is highly divergent with respect to its repertoire of EC proteins. To test this hypothesis, comparisons of C. muris and C. parvum were conducted via local TBLASTN using the complete set of 927 contigs, containing ~8.31 Mb of nucleotide sequence, that were submitted to GenBank by the C. muris genome sequencing project (see Acknowledgements). The genome sequence is high-coverage, as revealed by an analysis of a 2945-aa-long concatamer of C. parvum ribosomal proteins (Templeton et al., 2004a) that indicated that 95% of these sequences have orthologues that are identifiable in the C. muris. Similarly, 97% of C. hominis ribosomal protein subunit orthologues are identifiable. Therefore, observed absences of EC proteins in C. muris are probably due only in part to fragmentation of the C. muris genome sequence database. Of 244 C. parvum EC proteins that were screened against C. muris, only 152 (62%) were identified as having predicted orthologues. Thus it is likely that the species divergence of C. parvum and C. muris has led to numerous lineagespecific ‘inventions’ and expansions of EC proteins that are not shared between these divergent species. Annotation of the C. muris surface protein repertoire is likely to similarly reveal numerous proteins that are found in C. muris, but not in C. parvum. The high divergence of C. parvum and C. muris is evident in the fact that the average amino acid identity was only 41%, versus 95% between the EC proteins of C. parvum and C. hominis. The glycoprotein, GP60 (cgd6_1080 in C. parvum), is an exceptional example and is perhaps the most highly divergent EC protein between C. parvum and C. hominis, sharing only about 70% sequence identity, and is conserved and also highly divergent in C. muris, with only 20% identity. This gene is frequently used in genotyping analyses of Cryptosporidium isolates (Chalmers et al., 2005; Abe et al., 2006; Gatei et al., 2006) although, due to the extremely low-complexity character of the protein, it is best used in the context of genotyping using large panels of genes. Many of the COWP proteins appear to have orthologues in C. muris, and their amino acid conservation ranges from approximately 40% (COWP2 and COWP3) up to 63% in the case of COWP1 and 78% for COWP6.

Surface Protein Repertoires of Cryptosporidium spp.

373

Lineage-specific Expansions of EC Domains and Proteins The completion of genome nucleotide sequences for several apicomplexans has revealed the presence of lineage-specific amplifications of gene families. Examples include the families of over 60 var and 130 rifin genes that were identified in Plasmodium falciparum (Gardner et al., 2002), and the prediction of over 100 GPI-linked SAG glycoproteins in Toxoplasma (Manger et al., 1998; Lekutis et al., 2001; Jung et al., 2004). Lineage-specific amplification of EC proteins was also identified in C. parvum, most notably within numerous sub-telomeric and internal clusters of predicted EC proteins (Abrahamsen et al., 2004). Comparison of C. parvum with C. muris reveals that some gene clusters are shared in synteny, whereas others are absent in C. muris (again, with the caveat that the C. muris genome sequence database is incomplete). Thus, it is likely that the absence of orthologues is either due to lineage-specific gene loss in C. muris, or lineagespecific gene ‘invention’ and amplification in C. parvum. Of the amplified families of C. parvum EC proteins (see the Supplementary Table in Abrahamsen et al., 2004) C. muris possesses WYLE, GGC, HCD, and members of the CpLSP (Cryptosporidium Large Secreted Protein) family, in many cases within syntenic loci of genes, but appears to lack genes within the families FGLN, SKSR and MEDLE. The CpLSP gene family is remarkable because of its genus-specific provenance and expansion to 14 family members in C. parvum. Seven of the genes are localized within a highly compact locus (described in Figure 1 of Abrahamsen et al., 2004) that arose via gene duplication. The expansion was accompanied by profound divergence in which adjacent genes encode proteins having amino acid conservation of less than 30% and similarities spanning less than 10% of the protein lengths (examples shown in the top panel of Fig. 30.2 and alignment in Fig. 30.1B). Extrapolating from this high degree of divergence, it is likely that paralogous genes have diverged, perhaps via functional or immunological pressures, beyond the ability of BLAST analyses to detect similarities. In this manner, proteins would appear to be ‘invented’ de novo in a lineage-specific fashion, with no clues to their ancestral origin. The CpLSP locus is conserved with identical synteny of gene locations and orientations in both C. hominis and C. muris (Fig. 30.2, middle and bottom panels, respectively). Whereas the C. parvum and C. hominis orthologues within this locus share amino acid identities ranging from 96% to 98%, the conservation of residues between C. parvum and C. muris range from less than 30% to a maximum of 46%. An example of amino acid conservations within the LSP proteins is shown for a short region within the C. parvum CpLSP gene, cgd7_3860, which is absolutely conserved in C. hominis (Chro.70431) and highly conserved in C. muris (Fig. 30.1B). Thus this locus of expanded EC protein-encoding genes mirrors the general theme that C. parvum and C. hominis have far greater sequence similarities within the EC protein repertoire in comparison with C. muris. Selective pressures also result in the expansion of domains within genes, such as architectures containing tandem arrays of EGF (e.g. five tandem domains in cgd2_1590), Sushi (e.g. cgd7_4560), or TSP1 (e.g. cgd5_3429 and cgd5_3420) domains. A dramatic example of lineage-specific domain ‘invention’ coupled

374

850k

860k

870k

(28%)

880k (22%)

cgd7_3800

cgd7_3810

Chro.70424 (97%)

Chro.70425 (97%)

cgd7_3820

cgd7_3830 cgd7_3840

cgd7_3860

cgd7_3870

C. parvum

C. hominis

Chro.70429 Chro.70431 Chro.70426 (97%) (97%) (98%) Chro.70428 (98%)

Chro.70432 (96%)

contig4328 (38%)

C. muris

contig3519 +contig4327 (<30%)

contig4327 (37%)

contig4327 + contig4328 (42%)

contig4328 (43%)

contig4328 (46%)

contig4328 (37%)

T.J. Templeton

Fig. 30.2. Synteny and amino acid divergences of orthologues within a locus of Cryptosporidium-specific large secreted proteins (CLSP; see Fig. 30.1B of Abrahamsen et al., 2004) that are conserved in C. parvum (top panel), C. hominis (middle panel) and C. muris (bottom panel). The numbers in parentheses for the C. hominis and C. muris panels represent the percentage amino acid identities shared with C. parvum; for example, the C. hominis gene Chro.70424 shares 97% identical residues with the C. parvum orthologue, cgd7_3800. In noted contrast, the C. muris predicted orthologue shares less than 30% amino acid identity with the C. parvum gene, cgd7_3800. The numbers in parentheses above the C. parvum panel demonstrate that adjacent genes within the locus share limited sequence similarity; for example, cgd7_3800 shares only 28% identity with cgd7_3810, and shares identity within a region that is less than 10% of the protein length. No gene identifiers are given for predicted C. muris genes; instead the last four digits of the contig number are shown, and some genes are indicated as spanning two contigs (e.g. the C. muris orthologue of cgd7_3800 spans contig3519 and contig4327). The scale at the top refers to the position of the C. parvum orthologues on chromosome 7 near the right-hand telomere.

Surface Protein Repertoires of Cryptosporidium spp.

375

Apple

TSP1 TSP1 TSP1 TSP1 TSP1

Cys

Cys

Thr

TM

TSP1

Rep

GP900 (cgd7_4020)

TRAP-C1 (cgd1_3500)

TM

Apple

TM

Cryptosporidium TRAP superfamily members

EGF

TSP1 TSP1 TSP1

CpTSP7 (cgd5_4470)

TSP1 TSP1 TSP1 TSP1 TSP1 TSP1

TSP1

TSP1 TSP1 TSP1

TM

EGF EGF EGF EGF EGF EGF EGF EGF EGF

EGF EGF EGF EGF EGF

TM

EGF EGF EGF EGF EGF EGF EGF EGF EGF EGF

vWA vWA

Repeats

TM

76.m01679

AAK19757 (signal peptide?)

TM

Repeats

TSP1 TSP1 TSP1 TSP1

EGF EGF EGF

TM

55.m04840 (signal peptide?)

49.m03396

(31)

TM

25.m01843 (one or more Apple domains?)

TgMIC2 (AAB63303)

TSP1 TSP1 EGF

TM

vWA

TM

Toxoplasma TRAP superfamily members

AAD28185.1

57.m01872 (contains 32 EGF domains)

TRAP (PF13_0201)

TSP1

vWA

TRAP4 (PFF0800w)

TM

TSP1

vWA

TSP1

PTRAP (PFL_0870w)

MTRAP (PF10_0281)

vWA vWA vWA vWA vWA vWA

TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1

TM

Rep

TM

TSP1

TM

vWA

TM

Plasmodium TRAP superfamily members

CTRP (PFC0640w)

Fig. 30.3. Domain organizations of the predicted TRAP superfamily proteins identified in annotations and molecular and cellular studies of Cryptosporidium (top panel), Toxoplasma (middle panel), and Plasmodium (bottom panel). TRAP superfamily proteins are defined as having a signal peptide (open box); extracellular adhesive domains that include TSP1, vWA, and Apple; a transmembrane region that may have a rhomboid protease cleavage site near the external face of the TM region; and a short acidic cytoplasmic domain possessing a conserved C-terminal proximal tryptophan residue. The panel of proteins included here is guided by liberal criteria in an attempt to be as inclusive as possible in the identification of candidate TRAP superfamily proteins. For example, in Toxoplasma only MIC2 has been confirmed to be a member of the TRAP family, and the Cryptosporidium mucin, GP900, does not have typical extracellular adhesive domains and has an atypical cytoplasmic domain.

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with domain expansion is the Cryptosporidium 11,700-aa-long protein, cgd3_720, which is composed of 17 repeats of an approximately 600-residuelong all-β-strand globular domain that contains 12 cysteine residues. This protein is specific to Cryptosporidium, and is also found in C. muris (30% sequence identity) and is highly conserved in C. hominis (98% sequence identity).

Lateral Transfer of EC Domains and Accretion into Multi-domain Proteins As described above, apicomplexans contrast with other protozoans that have been analysed in that they possess a wide variety of EC proteins that are composed of multiple domains whose architectural complexities are typically observed only in animals and plants. Many of the component EC domains probably arose via lateral transfer, such as the SCP, MAM and Hedgehog-type HINT domains (Templeton et al., 2004a). Within the Apicomplexa the phylogenetic distributions vary for different EC domains, as well as for the multi-domain architectures of EC proteins (Tomley and Soldati, 2001; Templeton et al., 2004a; Templeton, 2007). For example, the MAM and Notch domains are found only in coccidians and are absent in Plasmodium and Theileria, both genera for which complete genome sequence information is available. For some proteins it is impossible to assign orthologues across genus boundaries, such as members within the functionally homologous TRAP superfamily of adhesive proteins that are involved in apicomplexan gliding motility and invasion (Sibley et al., 1998; Spano et al., 1998; Menard, 2001; Kappe et al., 2004; Sibley, 2004), including multiple predicted members in Cryptosporidium (Fig. 30.3; described in Spano et al., 1998; Deng et al., 2002). The Cryptosporidium Hedgehog-type HINT predicted secreted protein (in C. parvum, cgd7_5290) is a remarkable example of lineage-specific lateral gene transfer. This protein is not found in any protozoan other than Cryptosporidium sp., and BLAST analyses of GenBank using the protein as a query identifies only metazoan Hedgehog-like proteins. In metazoans, the Hedgehog proteins are secreted and function in diverse roles in embryonic and adult tissues, such as morphogens in embryonic pattern formation. The Cryptosporidium version possesses the C-terminal HINT intein domain that mediates auto-proteolytic cleavage, but is divergent within the N-terminal region that functions as a secreted protein that confers signalling. The Cryptosporidium HINT protein is conserved in C. muris (37% amino acid identity, versus 97% amino acid conservation between C. parvum and C. hominis), and therefore its role is probably orthologous in the parasitic strategies of these divergent species.

What is a Coccidian? The phylogenetic position of Cryptosporidium with respect to the Coccidia remains the subject of debate, although well-supported phylogenies are showing

Surface Protein Repertoires of Cryptosporidium spp.

377

Tox1

NOTCH

NOTCH

Tox1

NOTCH

TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 Tox1

NOTCH

Tox1

NOTCH

Surface protein domain architectures shared with Toxoplasma Tox1 Tox1

Tox1 Tox1 Tox1 Tox1 SUSHI SUSHI SUSHI SUSHI

TM

ARC ARC

SUSHI SUSHI SUSHI SUSHI SUSHI SUSHI

NOTCH

TRAP-C2 (cgd5_3420) SUSHI SUSHI

Cu-amine oxidase

MAM

cgd3_3430 KAZ KAZ KAZ

PENTRAXIN

KAZ

cgd4_2420

CLOSTRIPAIN

EGF

EGF

TM

NOTCH

NOTCH

NOTCH

NOTCH

NOTCH

NOTCH

TM

ANK ANK ANK ANK ANK

SUSHI

KAZ KAZ KAZ KAZ KAZ KAZ KAZ KAZ KAZ

cgd4_3550

cgd1_1520

NOTCH

EGF

TM TM TM TM TM TM TM TM TM TM

cgd7_4560

cgd6_670

cgd8_2800

RICIN

Glycotransferase-2

Cys Cys Cys Cys Cys Cys Cys

cgd5_690

Oocyst wall protein (COWP; e.g. cgd6_2090)

Tox1

Tox1 Tox1

NOTCH

Tox1

NOTCH

TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 TSP1 Tox1

NOTCH

NOTCH

Tox1

NOTCH

Surface protein domain architectures shared with Ascogregarina and Toxoplasma Tox1 Tox1 Tox1 Tox1 SUSHI SUSHI SUSHI SUSHI

SUSHI SUSHI

TM

ARC ARC

SUSHI SUSHI SUSHI SUSHI SUSHI SUSHI

NOTCH

TRAP-C2 (cgd5_3420)

cgd3_3430 Cys Cys Cys Cys Cys Cys Cys

RICIN

Glycotransferase-2

Cu-amine oxidase

MAM

cgd7_4560

Oocyst wall protein (COWP; e.g. cgd6_2090)

cgd5_690

Apicomplexan surface protein domain architectures DISCOIDIN

NEC

LCCL

LEVANASE

LEVANASE

//

ApiECA

RICIN

ApiECA

//

CCp1 and CCp2 (cgd7_300; cgd7_1730)

//

LH2

LCCL

SR

SR

LCCL

PENTRAXIN

LCCL

LCCL

CCp3 (cgd2_790)

Fig. 30.4. Domain organization of a representative set of surface proteins that are conserved in Toxoplasma and Cryptosporidium, to the exclusion of Plasmodium and Theileria (top panel); present in Toxoplasma, Cryptosporidium and Ascogregarina, to the exclusion of Plasmodium and Theileria (middle panel); versus widespread distribution in the apicomplexans (i.e. conserved as predicted orthologues in all of the above apicomplexans; bottom panel). All proteins have a signal peptide, indicated by an open box at the amino terminus. Domains present within the protein architectures include: Tox1 (ShKT); Notch (NL); TSP1; Sushi (CCP); ARC; MAM; Cu-amine oxidase (Cu_amine_oxidase); Glycotransferase-1 (glycos_transf_1); Ricin; Cys; Discoidin (F5_F8_type_C); NEC; LCCL; Levanase; ApiECA; LH2 (PLAT_SR); SR; and Pentraxin (PTX). Many of these domains are described further at one of the following domain browsers: http://smart.embl-heidelberg.de/browse.shtml and http://pfam. janelia.org/browse. Not described in these browsers is the Cys domain found in the highly cysteine-rich coccidian oocyst wall proteins, and the ARC domain, which is a small domain with characteristically spaced cysteine residues that is fused to a papain-like protease domain in the secreted protein AF1946 from the archaean, Archaeoglobus fulgidus.

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that it diverges at the base of the apicomplexan clade and perhaps has an affinity with a genus of intestinal parasites of invertebrates, the apicomplexan Gregarina (Carreno et al., 1999; Zhu et al., 2000; Barta and Thompson, 2006). Despite this apparent early divergence, both Cryptosporidium and the gregarines share numerous features with the coccidians, such as infection of intestinal epithelial cells, and transmission via an excreted, environmentally resistant cyst stage. In contrast, Haemosporida and Piroplasmida lack an excreted cyst stage and share a life cycle that alternates between a warm-blooded host and transmission via an insect definitive host. With regard to EC proteins, Cryptosporidium and Toxoplasma share many orthologous multi-domain architectures to the exclusion of Plasmodium, Theileria and Babesia (Fig. 30.4, top panel). That said, Toxoplasma and Plasmodium also share EC protein domains to the exclusion of Cryptosporidium, such as the vWA domain that is found in members of the TRAP superfamily, and the ‘6-cys’ domain that is the building block of both the Toxoplasma SAG surface proteins and Plasmodium surface coat proteins such as the gamete surface antigens P48/45 and P230 (Gerloff et al., 2005). Cryptosporidium also lacks examples of the macrophage attack complex perforin (MACPF)-like secreted proteins that are thought to be involved in membrane lysis during tissue traversal and which are found in Plasmodium (Kaiser et al., 2004; Ishino et al., 2005; Ecker et al., 2007), Theileria and Toxoplasma. In general, it is difficult to draw phylogenetic conclusions on the absence of select EC domains and proteins in Cryptosporidium, because gene loss has played such an important role in the evolution of this parasite. To better understand the relationship of Cryptosporidium with the gregarines, the conservation of select EC domains and proteins was assessed within a recent GSS project for the gregarine, Ascogregarina taiwanensis, a parasite of the mosquito, Aedes albopictus (in collaboration with Guan Zhu, Shin Enomoto and Mitch Abrahamsen). Similar to Cryptosporidium, it is possible to purify gregarine parasite oocyst stages on sucrose buoyancy gradients, and to then treat them with bleach to remove contaminating bacteria and host tissues. A highquality genomic DNA library was constructed from this DNA source, via the phi29 polymerase whole-genome amplification (WGA) methodology, to yield an unlimited amount of material for sequencing. This GSS project was performed in order to determine the feasibility of a genome sequence project, and over 20,000 sequence reads have been performed and assembled into contigs. This nucleotide sequence database was screened for EC domains and proteins via TBLASTN. The analyses were qualitative, due to the fragmented state of the database, and negative data on the absence of genes or domains are not informative or conclusive. No Cryptosporidium EC proteins were found that were conserved in Ascogregarina to the exclusion of Toxoplasma, whereas several proteins that are conserved in coccidians were also found in this gregarine (Fig. 30.4, middle panel). Again, this supports the hypothesis that although Cryptosporidium and Ascogregarina may have diverged at the base of the apicomplexan clade they share EC proteins that group them within a ‘coccidian’ natural history. Survey of Ascogregarina also revealed two examples of multi-domain EC proteins that are thought to be conserved throughout the apicomplexan clade; namely, members of the LCCL domain-containing family, which include

Surface Protein Repertoires of Cryptosporidium spp.

379

in Cryptosporidium cgd7_300, cgd7_1730 and cgd2_790 (Fig. 30.4, bottom panel). Thus the LCCL domain proteins are perhaps the most ‘royal’ of EC proteins, in that their complex multi-domain architectures arose prior to the split of the apicomplexans and their roles are probably conserved in fundamental apicomplexan biology.

Conclusions Our understanding of the natural history of Cryptosporidium began a century ago with microscope-based studies of morphology (Tyzzer, 1907), which only within the past few decades has crystallized – using molecular, cellular and epidemiological tools – into descriptions of roughly 20 species (Fayer, 2004; Xiao et al., 2004; reviewed in Barta and Thompson, 2006). Whole-genome comparison of Plasmodium and Cryptosporidium indicated that EC proteins are the most highly divergent class of parasite proteins (Templeton et al., 2004a), and it is therefore anticipated that the invention and amplification of EC proteins also played an important role in the species-specific distinctions within Cryptosporidium. Indeed, a comparison of C. parvum and C. muris indicates that perhaps over 30% of the EC proteins are not conserved as orthologues between these two species. In contrast, C. parvum and C. hominis appear to have 100% orthology, as well as substantial amino acid similarity within orthologues. The availability of a GSS nucleotide sequence database for the gregarine, Ascogregarina, as well as the whole-genome nucleotide sequence for the coccidian, Toxoplasma, allows comparisons of the EC repertoire for these parasites. Such an analysis identified numerous multi-domain EC proteins that are shared as orthologues between these three parasites to the exclusion of Plasmodium and Theileria. Thus, with respect to EC proteins, there is substantial EC protein repertoire information that links the early-diverging apicomplexans, Cryptosporidium and Gregarina, within the coccidian clade.

Acknowledgements The GSS project for Ascogregarina taiwanensis was conducted by Guan Zhu (Texas A&M University), Shin Enomoto (University of Minnesota) and Mitch Abrahamsen (University of Minnesota). The C. muris project is a superb resource and was conducted by Giovanni Widmer (Tufts University) and Joana Silva (J. Craig Venter Institute).

References Abe, N., Matsubayashi, M., Kimata, I. and Iseki, M. (2006) Subgenotype analysis of Cryptosporidium parvum isolates from humans and animals in Japan using the 60-kDa glycoprotein gene sequences. Parasitology Research 99, 303–305.

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T.J. Templeton Abrahamsen, M.S., Templeton, T.J., Enomoto, S., Abrahante, J.E., Zhu, G., Lancto, C.A., Deng, M., Liu, C., Widmer, G., Tzipori, S., Buck, G.A., Xu, P., Bankier, A.T., Dear, P.H., Konfortov, B.A., Spriggs, H.F., Iyer, L., Anantharaman, V., Aravind, L. and Kapur, V. (2004) Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304, 441–445. Barta, J.R. and Thompson, R.C. (2006) What is Cryptosporidium? Reappraising its biology and phylogenetic affinities. Trends in Parasitology 22, 463–468. Carreno, R.A., Martin, D.S. and Barta, J.R. (1999) Cryptosporidium is more closely related to the gregarines than to coccidia as shown by phylogenetic analysis of apicomplexan parasites inferred using small-subunit ribosomal RNA gene sequences. Parasitology Research 85, 899–904. Chalmers, R.M., Ferguson, C., Cacciò, S., Gasser, R.B., Abs El-Osta, Y.G., Heijnen, L., Xiao, L., Elwin, K., Hadfield, S., Sinclair, M. and Stevens, M. (2005) Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species. International Journal for Parasitology 35, 397–410. Deitsch, K.W. and Hviid, L. (2004) Variant surface antigens, virulence genes and the pathogenesis of malaria. Trends in Parasitology 20, 562–566. Deng, M., Templeton, T.J., London, N.R., Bauer, C., Schroeder, A.A. and Abrahamsen, M.S. (2002) Cryptosporidium parvum genes containing thrombospondin type 1 domains. Infection and Immunity 70, 6987–6995. Ecker, A., Pinto, S.B., Baker, K.W., Kafatos, F.C. and Sinden, R.E. (2007) Plasmodium berghei: Plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi. Experimental Parasitology 116, 504–508. Fayer, R. (2004) Cryptosporidium: a water-borne zoonotic parasite. Veterinary Parasitology 126, 37–56. Gardner, M.J., et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511. Gatei, W., Hart, C.A., Gilman, R.H., Das, P., Cama, V. and Xiao, L. (2006) Development of a multilocus sequence typing tool for Cryptosporidium hominis. Journal of Eukaryotic Microbiology 53 (Suppl. 1), S43–S48. Gerloff, D.L., Creasey, A., Maslau, S. and Carter, R. (2005) Structural models for the protein family characterized by gamete surface protein Pfs230 of Plasmodium falciparum. Proceedings of the National Academy of Sciences of the USA 102, 13598–13603. Ishino, T., Chinzei, Y. and Yuda, M. (2005) A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection. Cellular Microbiology 7, 199–208. Jung, C., Lee, C.Y. and Grigg, M.E. (2004) The SRS superfamily of Toxoplasma surface proteins. International Journal for Parasitology 34, 285–296. Kaiser, K., Camargo, N., Coppens, I., Morrisey, J.M., Vaidya, A.B. and Kappe, S.H. (2004) A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites. Molecular and Biochemical Parasitology 133, 15–26. Kappe, S.H., Buscaglia, C.A., Begman, L.W., Coppens, I. and Nussenzweig, V. (2004) Apicomplexan gliding motility and host cell invasion: overhauling the motor model. Trends in Parasitology 20, 13–16. Kraemer, S.M. and Smith, J.D. (2007) A family affair: var genes, PfEMP1 binding, and malaria disease. Current Opinion in Microbiology 9, 374–380. Lekutis, C., Ferguson, D.J., Grigg, M.E., Camps, M. and Boothroyd, J.C. (2001) Surface antigens of Toxoplasma gondii: variations on a theme. International Journal for Parasitology 31, 1285–1292.

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Manger, I.D., Hehl, A.B. and Boothroyd, J.C. (1998) The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1. Infection and Immunity 66, 2237–2244. Menard, R. (2001) Gliding motility and cell invasion by Apicomplexa: insights from the Plasmodium sporozoite. Cellular Microbiology 3, 63–73. Sibley, L.D. (2004) Intracellular parasite invasion strategies. Science 304, 248–253. Sibley, L.D., Håkansson, S. and Carruthers, V.B. (1998) Gliding motility: an efficient mechanism for cell penetration. Current Biology 8, R12–R14. Spano, F., Putignani, L., Naitza, S., Puri, C., Wright, S. and Crisanti, A. (1998) Molecular cloning and expression analysis of a Cryptosporidium parvum gene encoding a new member of the thrombospondin family. Molecular and Biochemical Parasitology 92, 147–162. Striepen, B., Pruijssers, A.J., Huang, J., Li, C., Gubbels, M.J., Umejiego, N.N., Hedstrom, L. and Kissinger, J.C. (2004) Gene transfer in the evolution of parasite nucleotide biosynthesis. Proceedings of the National Academy of Sciences of the USA 101, 3154–3159. Templeton, T.J. (2007) Whole-genome natural histories of apicomplexan surface proteins. Trends in Parasitology 23, 205–212. Templeton, T.J., Iyer, L.M., Anantharaman, V., Enomoto, S., Abrahante, J.E., Subramanian, G.M., Hoffman, S.L., Abrahamsen, M.S. and Aravind, L. (2004a) Comparative analysis of apicomplexa and genomic diversity in eukaryotes. Genome Research 14, 1686–1695. Templeton, T.J., Lancto, C.A., Vigdorovich, V., Liu, C., London, N.R., Hadsall, K.Z. and Abrahamsen, M.S. (2004b) The Cryptosporidium oocyst wall protein is a member of a multigene family and has a homolog in Toxoplasma. Infection and Immunity 72, 980–987. Tomley, F.M. and Soldati, D.S. (2001) Mix and match modules: structure and function of microneme proteins in apicomplexan parasites. Trends in Parasitology 17, 81–88. Tyzzer, E.E. (1907) A sporozoan found in the peptic glands of the common mouse. Proceedings of the Society for Experimental Biology and Medicine 5, 12–13. Xiao, L. Fayer, R., Ryan, U. and Upton, S.J. (2004) Cryptosporidium taxonomy: recent advances and implications for public health. Clinical Microbiological Reviews 17, 72–97. Zhu, G., Keithly, J.S. and Philippe, H. (2000) What is the phylogenetic position of Cryptosporidium? International Journal of Systematic and Evolutionary Microbiology 50, 1673–1681.

31

Giardan: Structure, Synthesis, Regulation and Inhibition

K. S¸ENER1, H. VAN KEULEN2 AND E.L. JARROLL1 1Northeastern

University, Boston, MA, USA; 2Cleveland State University, Cleveland, OH, USA

Abstract During encystment, Giardia trophozoites become encased in a filamentous extracellular matrix of their own making that consists of novel cyst wall proteins (Cwp) 1, 2 and 3, and a novel 2-acetamido-2-deoxy-d-galactan we are naming giardan. Giardan is synthesized from glucose via sugar phosphate intermediates to UDP-GalNAc by inducible, cytosolic enzymes. The UDP-GalNAc is fixed into giardan apparently by an inducible, particleassociated transferase. Regulation of this synthesis appears to centre around pyrophosphorylase, epimerase and cyst wall synthase (Cws) activities. Pyrophosphorylase seems to be involved in making sufficient UDP-N-acetylglucosamine (GlcNAc) to drive the epimerase kinetics toward UDP-GalNAc synthesis, while the Cws removes intracellular UDP-GalNAc, extruding it as giardan and thus preventing an increased intracellular concentration of UDP-GalNAc that could drive the reaction toward GlcNAc synthesis. Cyst wall proteins have been localized to encystment-specific vesicles (ESVs), but whether or not this is true for giardan is unknown. Also unknown is whether or not the cyst wall proteins and giardan are covalently linked. It remains unknown how or whether Giardia degrades giardan during excystation.

Introduction Many protozoa share the ability to differentiate from vegetative forms to cyst forms (encystment), triggered by factor(s) unfavourable to the vegetative form’s survival; one of these factors is nutrient deprivation. Cyst formation entails the secretion of an extracellular matrix by the vegetative form. Giardia encysts in the lower small intestine, with trophozoites rounding up, elaborating a cyst wall and forming intact cysts by the time they reach the colon (Meyer and Jarroll, 1980). Gillin et al. (1987) and Schupp et al. (1988) achieved in vitro encystment when axenically grown trophozoites were subjected to a growth medium with a slightly alkaline pH and the addition of bovine bile. Originally, bile (or its derivatives) was believed to induce encystment directly, but Luján et al. (1996a, 1997) demonstrated that trophozoites probably encyst because bile sequesters 382

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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Fig. 31.1. (A) Scanning electron micrograph (SEM) of Giardia cyst minus the trophozoite material inside. Note the outer wall filament flexibility. (B) SEM of Giardia cyst wall filaments. (C) Transmission electron micrograph (TEM) of an intact Giardia cyst. (D) TEM of Giardia cyst minus trophozoite material. Trophozoite material was removed as described by Gerwig et al. (2002) leaving only cyst wall filaments (black arrows). Micrographs courtesy of Stanley Erlandsen.

the cholesterol required by Giardia, a lipid auxotroph (Jarroll et al., 1981). In vitro, cholesterol is supplied to Giardia by the serum used in the culture medium. Giardia encystment undoubtedly requires a series of signalling events that trigger the differentiation of a binucleate trophozoite into a tetranucleate cyst, which result from karyokinesis but not cytokinesis, in trophozoites. Encysting trophozoites encase themselves in a cyst wall composed of an inner membranous and an outer filamentous portion (Feely et al., 1990). The filamentous

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outer portion has a thickness of 0.3–0.5 µm, is composed of filaments measuring 7–20 nm in diameter (Fig. 31.1A) and is arranged in a tightly packed meshwork (Fig. 31.1B) (Erlandsen et al.., 1989, 1990, 1996). Figure 31.1C shows transmission electron micrographs that depict a section through an intact Giardia cyst and one (Fig. 31.1D) that has been treated with sodium dodecylsulphate and various glycosidases and proteinases (Gerwig et al., 2002) to remove the internal components of the cyst, leaving only the purified cyst wall filaments. Approximately 15-nm-diameter blebs appear on the encysting trophozoite’s cell membrane about 10 h after encystment is induced. Formed cysts begin to appear about 14 h after induction and increase by 16 h. Carbohydrate-specific tags indicated that the polysaccharide exposure on the trophozoite surface precedes fibril patch assembly, and that the pattern of filament assembly during Giardia encystment resembles that for microfibrils of the β(1,3) glucan in Candida albicans (Argüello-García et al., 2002). The appearance of encystment-specific vesicles (ESV; cytoplasmic vesicles specific to encysting trophozoites) is the earliest detectable microscopic change during encystment (Reiner et al., 1989). Cyst wall antigens, localized to these vesicles, suggest that the ESVs function is in the export of at least some cyst wall constituents (Reiner et al., 1990). Regulated transport and cyst wall antigen secretion in ESVs occur during encystment (Reiner et al., 1989). Encystment antigens localize to Golgi membrane stacks in cisternae and within ESVs. These are not seen in non-encysting trophozoites or in cysts after completion of encystment. Lanfredi-Rangel et al. (2003) observed that the endoplasmic reticulum cisternae dilate to form clefts that enlarge into ESVs. Stefanic et al. (2006) reported that ESVs lack the morphological characteristics of Golgi cisternae or their sorting functions. Giardia cyst walls contain novel cyst wall proteins (Cwps) (Luján et al., 1996b, 1997; Reiner et al., 2001; Sun et al., 2003). Each is encoded by a single-copy gene, the transcripts for which increase substantially during encystment. These proteins contain leucine-rich repeats (LRRs) as well as a cysteine-rich conserved region. Cwps localize to ESVs as well as to the mature cyst wall (Luján et al., 1995a, 1995b, 1997; Luján and Touz, 2003; Sun et al., 2003). Hehl et al. (2000) demonstrated that during encystment Cwp 1 formed dense granule-like vesicles and showed subsequent incorporation into the cyst wall, and that the N-terminal domain of Cwp 1 is required for targeting it to secretory compartments. Sun et al. (2003) showed that the LRRs and the N-terminal region were needed for targeting Cwp 3 into secretory compartments and that the C-terminus was necessary for its incorporation into the cyst wall. ESV formation appears to be due to Cwp2 aggregating Cwp1 and Cwp3 via its conserved LRR (Gottig et al., 2006).

Structure Originally, the Giardia cyst wall polysaccharide was identified as chitin, a β(1,4) N-acetylglucosamine polymer (2-acetamido-2-deoxy-d-glucan) (IUPAC-IUB JCBN, 1982; Ward et al., 1985; Gillin et al., 1987). Later studies showed that the cyst wall was not made of chitin but of a β(1,3) N-acetyl-d-galactosamine polymer

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Fig. 31.2. Model polysaccharide chains constructed from disaccharide and linkage information randomly selected from appropriate conformational regions. All polysaccharides contain 30 monosaccharide residues. The polymer constructed from region Ia forms a right-handed (clockwise) helix, whereas the polymer built from region Ib has a left-handed helical conformation. The polysaccharide assembled from regions Ia + Ib (4:1) shows a random coil conformation. From Gerwig et al. (2002) by permission of Oxford University Press.

(2-acetamido-2-deoxy-d-galactan) we are naming giardan, and that the chitin synthase activity is a β(1,3) UDP-GalNAc transferase we termed cyst wall synthase (Cws) (Jarroll et al., 1989; Gerwig et al., 2002; Karr and Jarroll, 2004). Analysis of purified Giardia cyst wall filaments (Fig. 31.1D) revealed that ~63% of its cyst wall filament is composed of giardan (Fig. 31.2) (Gerwig et al., 2002). A covalent linkage between giardan and some Cwps may play a role in its insolubility, although this is unproven (Gerwig et al., 2002).

Synthesis Extracellular formation of giardan requires intracellular synthesis of UDP-GalNAc (Fig. 31.3). Macechko et al. (1992) showed that glucose is incorporated into

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UDP-GalNAc by inducible, cytosolic enzymes: glucosamine 6-P deaminase (Gpi, EC 5.3.1.10), glucosamine 6-P N-acetylase (Gna, EC 2.3.1.4), phospho N-acetylglucosamine mutase (Pgm, EC 2.7.5.2), uridine diphospho (UDP) N-acetylglucosamine pyrophosphorylase (Uap, EC 2.7.7.23) and UDP-N-acetylglucosamine 4′-epimerase (Uae, EC 5.1.3.7).

Glucosamine 6-P deaminase (Gpi) Giardia’s Gpi was purified from encysting trophozoites and is labile to freezing and thawing but stable for up to 2 months when stored in 50% glycerol at −20°C (Steimle et al., 1997).The molecular mass was 29 kDa with a pH optimum of 8.9. Van Keulen et al. (1998) identified two genes, gpi1 (798 bp) and gpi2 (789 bp), encoding glucosamine 6-phosphate deaminases in Giardia, but only one, gpi1, was expressed. The transcript for gpi1 appeared no earlier than 6 h after cells were induced with bile. Using a different nomenclature for the same genes, Knodler et al. (1999) showed that the Gln6PI genes have distinct patterns of expression: Gln6PI-A (789 bp) has a short 5′ untranslated region, and was expressed at a low level during vegetative growth and encystation. The other gene, Gln6PI-B (798 bp), has two transcripts: one of which was expressed

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constitutively, with the other being upregulated during encystment. gpi is under transcriptional control (Lopez et al., 2003), and in mature cysts Gpi is removed by a ubiquitin-mediated pathway (Lopez et al., 2002).

Glucosamine 6-P N-acetylase (Gna) The gene for Gna has been cloned and sequenced (Lopez et al., 2003) from Giardia; its coding region is 615 bp and is transcriptionally regulated (Lopez et al., 2003). Gna has a predicted pH optimum of 6.45 and molecular mass of 22.8 kDa. The functional Gna has not yet been characterized kinetically.

Phospho N-acetylglucosamine mutase (Pgm) Giardia’s pgm gene has an open reading frame (ORF) of 1513 bp and exhibits a predicted molecular mass of 56.4. Pgm was partially purified and characterized by Lindmark and Schmidt (1992) with a pH optimum of 8.0 and a temperature optimum of 37°C. The native Pgm increases 12-fold during encystment and requires Mg2+, glucose 1, 6-diP and diethyldithiocarbamate (hydroxyquinoline may substitute). The recombinant Pgm is active but has not been characterized kinetically. Lopez et al. (2003) showed that the regulation of the mutase is also at the transcriptional level.

Uridine diphospho (UDP) N-acetylglucosamine pyrophosphorylase (Uap) Evidence exists that there are two Uap activities in encysting Giardia. Lopez et al. (2003), Mok et al. (2005), and Mok and Edwards (2005) reported and characterized the inducible pyrophosphate (iUap). Lopez et al. (2003) showed that iUap (1308 bp; a molecular mass of 48.2) regulation is transcriptional. Figure 31.4 shows a phylogenetic tree in which Giardia’s iUap clusters with those of other eukaryotes, unlike the Giardia epimerase (see below), and appears as the most primitive of the eukaryotic UDP-N-acetylglucosamine pyrophosphorylases sequenced to date. A structural model for the iUap has been proposed (Fig. 31.5) using the human pyrophosphorylase Ax1 (Peneff et al., 2001) as a template and based on a structural protein modelling program developed by Abyzov et al. (2005). Verify3D analysis scores revealed that this model was an acceptable fit (all residues have scores greater than zero) except in the region that spans the residues from 294 to 314. In this region, unlike the template, the iUap model has an extra loop of amino acids 292–299 that seems to overlap with the active site. In addition to the iUap (riUap is the recombinant iUap), Bulik et al. (2000) reported a ~66 kDa Uap which is constitutive (cUap) and stimulated by GlcN6-P anabolically toward UDP-GalNAc synthesis. To date, we have been unable to clone the gene encoding cUap. We believe that it may not be a typical Uap but

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Fig. 31.4. Phylogenetic tree of UDP-GlcNAc pyrophosphorylases constructed using the r group method with arithmetic mean (UPGMA). Bar shows the change in protein sequences.

rather another enzyme capable of behaving as a Uap. One candidate for such an enzyme is one of the UDP-sugar pyrophosphorylases seen in some plants (Litterer et al., 2006a, 2006b). While these have not been reported in Giardia previously, we are exploring this possibility.

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Fig. 31.5. Structural model of Giardia’s induced UDP-GlcNAc pyrophosphorylase. An extra loop (black arrows) does not exist in the human template and appears to overlap with the active site of the iUap.

In an attempt to sort out whether or not there are two Uap activities, total Uap activity was measured in whole lysates of encysting and non-encysting cells under various pH conditions. In non-encysting trophozoites (Fig. 31.6A), the pH optimum of total Uap activity was at nearly 8.0, which is close to the pH optimum of the cUap activity, with lower activity observed around pH 6.1, the pH optimum for riUap. With 24 h encysting cells (Fig. 31.6B), the activity at pH 6.1 increased slightly, while the activity at pH 8.0 remained high. By 48 h (Fig. 31.6C), the activity at pH 6.1 increased to the level of that at pH 8.0, while activity around pH 8.0 remained relatively constant. Interestingly, when we stimulated the total Uap activity with GlcN 6-P, the allosteric effector of cUap, the only increase in relative activity was observed at the initiation of encystment (0 h encysting cells) at pH 8.0 (Fig. 31.7) indicating that it had an effect on the cUap but not the iUAp. These data suggest that there are two Uap activities, but efforts are still under way to identify the gene responsible for cUap activity.

UDP-N-acetylglucosamine 4′-epimerase (Uae) The gene for Uae exists as a single copy in Giardia regulated at the level of transcription (Lopez et al., 2003). Expressed Uae exhibits a molecular mass of ~42 kDa, an open reading frame of 1155 bp, and a pH optimum between 7.6

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and 8.6 (Lopez et al., 2007). Conversion of UDP-GalNAc to UDP-GlcNAc is favoured in in vitro assays; an excess of UDP-GlcNAc is required to drive the reaction towards the synthesis of UDP-GalNAc (Lopez et al., 2007). Uae has been detected in a number of organisms and is capable of catalysing distinct and reversible reactions: UDP-GlcNAc to UDP-GalNAc and UDP-Glc to UDP-Gal (EC 5.1.3.2) depending upon the organism from which it originates. In some organisms it can catalyse the conversion of UDP-Glc/GlcNAc to UDP-Gal/ GalNAc, while in others it can only convert UDP-Gal to UDP-Glc or UDP-GalNAc to UDP-GlcNAc (Ishiyama et al., 2004). Macechko et al. (1992) detected UDP-GlcNAc/GalNAc Uae activity in crude Giardia lysates but could not detect UDP-Gal to Glc activity. Giardia Uae catalyses the reversible epimerization of UDP-GlcNAc to UDP-GalNAc and so phylogenetically aligns with the Group 3 prokaryotes, rather than the eukaryotes (Ishiyama et al., 2004).

Cyst wall synthase (Cws) The UDP-GalNAc synthesized in the cytoplasm must be incorporated into the giardan portion of the cyst wall. This is accomplished apparently by an inducible, particle-associated Cws, the activity of which increases ~1245-fold during the first 24–36 h of encystment (Karr and Jarroll, 2004). The particles with which it associates are different from the lysosome-like organelles (Fig. 31.8). In fact, the vesicles are possibly ESV, but this remains to be demonstrated. Cws has been partially purified about 156-fold. It exhibits a pH optimum of 7.5, a temperature optimum between 30 and 37°C, a requirement for divalent cations with Ca2+ and Mg2+ significantly preferred over Co2+, Mn2+ and Zn2+. The addition

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of 1 mM EDTA inhibits Cws by 99% over control activity and DTT is not required for its activity. Cws exhibits specificity for UDP-GalNAc and not other UDPsugars, with the apparent Km for UDP-GalNAc being 48 × 10−6 M.

Regulation S ¸ ener et al. (2004) demonstrated changes in the amount of sugar phosphate intermediates generated by these enzymes during encystment. The largest absolute increase (~5-fold) was in the level of GlcN-6-P. However, the greatest relative increase in concentration of amino sugars in the pathway (~9-fold) is in UDP-GlcNAc, which is produced by Uap activity. The relatively large increase in UDP-GlcNAc could be due to the presence of two Uap activities in Giardia during encystment. Lopez et al. (2007) showed that Giardia’s Uae has a preference (at least in vitro) for the catabolic reactions, with a larger Vmax and smaller Km for UDPGalNAc than for UDP-GlcNAc. In that case, the productive synthesis of UDPGalNAc would be difficult, if not impossible. They speculated that two conditions are needed to drive the reaction toward UDP-GalNAc synthesis during encystment: (i) the intracellular levels of UDP-GlcNAc brought about by the Uap activity(ies) must be high; and/or (ii) the UDP-GalNAc produced must be removed by Cws. Together these suggest that the epimerase is a regulatory step in giardan biosynthesis. The measured sugar phosphate intermediates (S ¸ ener et al., 2004) and the observed Km values of riUap and rUae prove this observation. Assuming a cell volume of 80 fl, which is the mean cell volume of a red blood cell, the most accurately measured cell, the concentration of GlcNAc 1-P at 0 h (51 amoles/ cell) is 6.4 × 10−4 M. This value becomes (216 amoles/cell) at 24 h: 2.7 × 10−3 M. The Km measured for riUAP in the anabolic direction is 9.7 × 10−5 M (K. S ¸ ener et al., unpublished). Thus the substrate concentration is 6.5 × Km and 27 × Km, respectively, indicating that the enzyme operates at Vmax in 24-h-induced cells. The situation is different for the epimerase reaction. At 0 h the substrate concentration (UDP-GlcNAc, 7 amoles/cell) is 8.75 × 10−5 M and is 8.12 × 10−4 M (65 amoles/cell) at 24 h. With a Km of 1.22 × 10−3 M for the epimerase (Lopez et al., 2007), this means that the substrate concentration is well below the Km values (0.088 × Km and 8.12 × Km at 0 and 24 h, respectively). After 24 h in encystment the enzyme is not operating even at ½ Vmax. This suggests that removal of the end product by Cws is absolutely required to drive giardan synthesis. If the Cws is not present, the entire pathway will halt and intermediates are no longer shunted from the glycolytic pathway.

Inhibition The drug of choice for treating giardiasis remains metronidazole (MTZ) (Medical Letter, 2004). MTZ is reduced by the low redox potential of pyruvate ferredoxin oxidoreductase present in Giardia and other anaerobes, and the reduction

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products are apparently toxic to the trophozoites. MTZ does not inhibit oxygen uptake by encysting Giardia after about 12 h or in completely formed cysts; whereas oxygen uptake by Giardia trophozoites is significantly inhibited by MTZ (Paget et al., 1989, 1993, 1998). Paget et al. (2004) reported that menadionegenerated radicals kill Giardia intestinalis trophozoites and cysts. Jarroll and S ¸ ener (2003) speculated that since the trigger for encystment is usually depletion of a vital nutrient, and assuming that encystment is irreversible at some point, then inhibiting encystment, especially late in the process, could cause the encysting trophozoites to die rather than just stop encysting. While this concept has yet to be proven experimentally, it does seem likely that there exist targets in the pathways for potential drug design. Two likely targets are Uae and Cws, since both are at the end of the encystment synthetic pathway when the cell is most probably committed. In addition, Giardia’s Uae differs significantly from the human Uae, and Cws is most probably not found in humans or any other mammal. Inhibition of these key components of filament synthesis could, at the very least, render encysting cells incapable of surviving osmotic pressures inside or outside the host. Unfortunately, few inhibitors of these enzymes are known. Steimle et al. (1997) demonstrated that 2-amino-2-deoxyglucitol-6-phosphate, a GlcN-6-P analogue, inhibited the activity of glucosamine 6-phosphate deaminase, while E.L. Jarroll (unpublished) observed that this same analogue at 1 mM reduced encystment in vitro from ~70% to ~2–3%. Inhibition by this analogue in vitro did not appear to cause trophozoite death during the 4-day period for which it was observed. Cws requires a divalent cation, and EDTA, a chelating agent, inhibits Cws activity in vitro at a contration of 1 mM or higher (Karr and Jarroll, 2004). Studies are currently under way to determine whether echinocandin, a β(1,3) glucan synthase inhibitor for fungi, will inhibit Cws.

References Abyzov, A., Errami, M., Leslin, C.M. and Ilyin, V.A. (2005) Friend, an integrated analytical front-end application for bioinformatics. Bioinformatics 21, 3677–3678. Argüello-García, R., Argüello-López, C., González-Robles, A., Castillo-Figueroa, A.M. and Ortega-Pierres, M.G. (2002) Sequential exposure and assembly of cyst wall filaments on the surface of encysting Giardia duodenalis. Parasitology 125, 209–219. Bulik, D., Lindmark, D. and Jarroll, E. (1998) Purification and characterization of UDPN-acetylglucosamine pyrophosphorylase from encysting Giardia. Molecular and Biochemical Parasitology 95, 135–139. Bulik, D., van Ophem, P., Manning, J., Shen, Z., Newburg, D. and Jarroll, E. (2000) UDPN-acetylglucosamine pyrophosphorylase: a key enzyme in encysting Giardia is allosterically regulated. Journal of Biological Chemistry 275, 14722–14728. Erlandsen, S., Bemrick, W. and Pawley, J. (1989) High resolution electron microscopic evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis. Journal of Parasitology 75, 787–797. Erlandsen, S., Bemrick, W., Schupp, D., Shields, J., Jarroll, E., Sauch, J. and Pawley, J. (1990) High-resolution immunogold localization of Giardia cyst wall antigens using

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field emission SEM with secondary and backscatter electron imaging. Journal of Histochemistry and Cytochemistry 38, 625–632. Erlandsen, S., Macechko, P.T., van Keulen, H. and Jarroll, E. (1996) Formation of the Giardia cyst wall: studies on extracellular assembly using immunogold labeling and high resolution field emission SEM. Journal of Eukaryotic Microbiology 43, 416– 420. Feely, D., Holbertson, D. and Erlandsen, S. (1990) The biology of Giardia. In: Meyer, E.A. (ed.) Giardiasis. Elsevier, New York, pp. 11–50. Gerwig, G.J., van Kuik, J.A., Leeflang, B.R., Kamerling, J.P., Vliegenthart, J.F.G., Karr, C.D. and Jarroll, E.L. (2002) The Giardia intestinalis filamentous cyst wall contains a novel β(1-3)-N-acetyl-d-galactosamine polymer: a structural and conformational study. Glycobiology 12, 499–505. Gillin, F.D., Reiner, D.S., Gault, M.J., Douglas, H., Das, S., Wunderlich, A. and Sauch, J.F. (1987) Encystation and expression of cyst antigens by Giardia lamblia in vitro. Science 235, 1040–1043. Gottig, N., Elías, E., Quiroga, R., Nores, M., Solari, A., Touz, M. and Luján, H. (2006) Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia. Journal of Biological Chemistry 281, 18156–18166. Hehl, A.B., Marti, M. and Köhler, P. (2000) Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia. Molecular Biology of the Cell 11, 1789–1800. Ishiyama, N., Creuzenet, C., Lam, J. and Berghuis, A. (2004) Crystal structure of WbpP, a genuine UDP-N-acetylglucosamine 4-epimerase from Pseudomonas aeruginosa. Journal of Biological Chemistry 279, 22635–22642. IUPAC-IUB JCBN (Joint Commission on Biochemical Nomenclature) (1982) Polysaccharide nomenclature. European Journal of Biochemistry 126, 439–441. Jarroll, E. and S ¸ ener, K. (2003) Potential drug targets in cyst-wall biosynthesis by intestinal protozoa. Drug Resistance Updates 6, 239–246. Jarroll, E.L., Muller, P.J., Meyer, E.A. and Morse, S.A. (1981) Lipid and carbohydrate metabolism of Giardia lamblia. Molecular and Biochemical Parasitology 2, 187– 196. Jarroll, E.L., Manning, P., Lindmark, D., Coggins, J. and Erlandsen, S. (1989) Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine. Molecular and Biochemical Parasitology 32, 121–132. Karr, C. and Jarroll, E. (2004) Cyst wall synthase: N-acetylgalactosaminyl-transferase activity is induced to form the novel GalNAc polysaccharide in the Giardia cyst wall. Microbiology 150, 1237–1243. Knodler, L., Svard, S., Silberman, J., Davids, B. and Gillin, F. (1999) Developmental gene regulation in Giardia lamblia: first evidence for an encystation-specific promoter and differential 5′ mRNA processing. Molecular Microbiology 34, 327–340. Lanfredi-Rangel, A., Attias, M., Reiner, D.S., Gillin, F.D. and de Souza, W. (2003) Fine structure of the biogenesis of Giardia lamblia encystation secretory vesicles. Journal of Structural Biology 143, 153–163. Lindmark, D. and Schmidt, K. (1992) Phosphoacetylglucosamine mutase of Giardia duodenalis. Society of Protozoology Annual Meeting, University of British Columbia, Vancouver, Canada (abstract). Litterer, L.A., Plaisance, K.L., Schnurr, J.A., Storey, K.K., Jung, H.-J.G., Gronwald, J.W. and Somers, D.A. (2006a) Biosynthesis of UDP-glucuronic acid in developing soybean embryos: possible role of UDP-sugar pyrophosphorylase. Physiologia Plantarum 128, 200–211.

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K. S¸ener et al. Litterer, L.A., Schnurr, J.A., Plaisance, K.L., Storey, K.K., Gronwald, J.W. and Somers, D.A. (2006b) Characterization and expression of Arabidopsis UDP-sugar pyrophosphorylase. Plant Physiology and Biochemistry 44, 171–180. Lopez, A., Hossain, M. and van Keulen, H. (2002) Giardia intestinalis glucosamine 6-phosphate isomerase: the key enzyme to encystment appears to be controlled by ubiquitin attachment. Journal of Eukaryotic Microbiology 49, 134–136. Lopez, A., ¸Sener, K., Jarroll, E. and van Keulen, H. (2003) Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis. Molecular and Biochemical Parasitology 128, 51–57. Lopez, A., ¸Sener, K., Trosien, J., Jarroll, E. and van Keulen, H. (2007) UDP-N-acetylglucosamine 4′-epimerase from the intestinal protozoan Giardia intestinalis lacks UDPglucose 4′-epimerase activity. Journal of Eukaryotic Microbiology 54, 154–160. Luján, H.D. and Touz, M.C. (2003) Protein trafficking in Giardia lamblia. Cellular Microbiology 5, 427–434. Luján, H.D., Marotta, A., Mowatt, M.R., Sciaky, N., Lippincott-Schwartz, J. and Nash, T.E. (1995a) Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia. Journal of Biological Chemistry 270, 4612–4618. Luján, H.D., Mowatt, M.R., Conrad, J.T., Bowers, B. and Nash T.E. (1995b) Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats: implications for secretory granule formation and protein assembly into the cyst wall. Journal of Biological Chemistry 270, 29307–29313. Luján, H.D., Mowatt, M.R., Byrd, L.G. and Nash, T.E. (1996a) Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia. Proceedings of the National Academy of Sciences USA 93, 7628–7633. Luján, H.D., Mowatt, M.R. and Nash, T.E. (1996b) Lipid requirements and lipid uptake by Giardia lamblia trophozoites in culture. Journal of Eukaryotic Microbiology 43, 237–242. Luján, H.D., Mowatt, M.R. and Nash, T.E. (1997) Mechanisms of Giardia lamblia differentiation into cysts. Microbiology and Molecular Biology Reviews 61, 294–304. Macechko, P.T., Steimle, P.A., Lindmark, D.G., Erlandsen, S.L. and Jarroll, E.L. (1992) Galactosamine-synthesizing enzymes are induced when Giardia encyst. Molecular and Biochemical Parasitology 56, 301–309. Medical Letter (2004) Tinidazole (Tindamax): A new antiprotozoal drug. Medical Letter on Drugs and Therapeutics 46, e1–e12. Meyer, E. and Jarroll, E. (1980) Giardiasis. American Journal of Epidemiology 11, 1–12. Mok, M. and Edwards, M. (2005) Kinetic and physical characterization of the inducible UDP-N-acetylglucosamine pyrophosphorylase from Giardia intestinalis. Journal of Biological Chemistry 280, 39363–39372. Mok, M., Tay, E., Sekyere, E., Glenn, W., Bagnara, A. and Edwards, M. (2005) Giardia intestinalis: molecular characterization of UDP-N-acetylglucosamine pyrophosphorylase. Gene 357, 73–82. Paget, T., Jarroll, E.L., Manning, P., Lindmark, D.G. and Lloyd, D. (1989) Respiration of the cyst and trophozoite forms of Giardia muris. Journal of General Microbiology 135, 145–154. Paget, T., Manning, P. and Jarroll, E. (1993) Oxygen uptake in cysts and trophozoites of Giardia lamblia. Journal of Eukaryotic Microbiology 40, 246–250. Paget, T., Macechko, P. and Jarroll, E. (1998) Giardia intestinalis: metabolic changes during cytodifferentiation. Journal of Parasitology 84, 222–226. Paget, T., Maroulis, S., Mitchell, A., Edwards, M., Jarroll, E. and Lloyd, D. (2004) Menadione-generated radicals kill Giardia intestinalis trophozoites and cysts. Microbiology 150, 1231–1236.

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Peneff, C., Ferrari, P., Charrier, V., Taburet, Y., Monnier, C., Zamboni, V., Winter, J., Harnois, M., Fassy, F. and Bourne, Y. (2001) Crystal structure of two human pyrophosphorylase isoforms in complexes with UDPGlc (Gal) NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture. EMBO Journal 20, 6191–6202. Reiner, D., Douglas, H. and Gillin, F. (1989) Identification and localization of cyst-specific antigens of Giardia lamblia. Infection and Immunity 57, 963–968. Reiner, D., McCaffery, M. and Gillin, F.D. (1990) Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia. European Journal of Cell Biology 53, 142–153. Reiner, D., McCaffery, J. and Gillin, F. (2001) Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway. Cellular Microbiology 3, 459–472. Schupp, D., Erlandsen, S., Januschka, M., Sherlock, L., Meyer, E., Bemrick, W. and Stibbs, H. (1988) Production of viable Giardia cysts in vitro: determination by fluorogenic dye staining, excystation, and animal infectivity in the mouse and Mongolian gerbil. Gastroenterology 95, 1–10. S ¸ ener, K., Shen, Z., Newburg, D. and Jarroll, E. (2004) Amino sugar phosphate levels change during formation of the Giardia cyst wall. Microbiology 150, 1225–1230. Stefanic, S., Palm, D., Svard, S.G. and Hehl, A.B. (2006) Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia. Journal of Biological Chemistry 281, 7595–7604. Steimle, P., Lindmark, D. and Jarroll, E. (1997) Purification and characterization of glucosamine 6-phosphate isomerase from encysting Giardia. Molecular and Biochemical Parasitology 84, 149–153. Sun, C.-H., McCaffery, J., Reiner, D. and Gillin, F. (2003) Mining the Giardia lamblia genome for new cyst wall proteins. Journal of Biological Chemistry 278, 21701– 21708. van Keulen, H., Steimle, P.A., Bulik, D.A., Borowiak, R.K. and Jarroll, E.L. (1998) Cloning of two putative Giardia lamblia glucosamine 6-phosphate isomerase genes only one of which is transcriptionally activated during encystment. Journal of Eukaryotic Microbiology 45, 637–642. Ward, H., Alroy, J., Lev, B., Keusch, G. and Pereira, M. (1985) Identification of chitin as a structural component of Giardia cysts. Infection and Immunity 49, 629–634.

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Protein Kinase C in Giardia duodenalis: a Family Affair

M.L. BAZÁN-TEJEDA1, R. ARGÜELLO-GARCÍA1, R.M. BERMÚDEZ-CRUZ1, M. ROBLES-FLORES2 AND G. ORTEGA-PIERRES1 1CINVESTAV-IPN,

Mexico City, México; 2UNAM, Mexico City, México

Abstract The induction of Giardia duodenalis encystment entails an exquisite interplay among transducer elements, which proceeds in a highly ordered manner in response to external stimuli and leads to changes in gene expression. However, little is known about how this parasite transduces the signals of its external environment during encystment. We have identified in Giardia duodenalis protein kinase C-like isoforms (β, δ, ε, θ and ζ) using specific polyclonal antibodies raised against mammalian PKC isoforms, and these proteins showed a differential expression pattern during encystment. Moreover, this kinase family has been shown to have a role during encystment because PKC inhibitors blocked the differentiation process. In particular, the PKCβ-like molecule (gPKCβ) with an atypical high molecular weight (150 kDa), showed a gradual increase in expression during encystment of the parasite and also redistributed from cytoplasm to plasma membrane soon after encystment induction, suggesting that it may play an important role during the differentiation process. Additional biochemical characterization of gPKCβ showed that this isoform displayed in vitro kinase activity dependent on cofactors required by conventional PKCs, i.e. phospholipid, diacylglycerol and calcium. In the Giardia genome database, an ORF that encodes for a homologue of PKCβ catalytic domain was found and cloned. The gPKC features, together with its divergent nature, indicate that this enzyme may constitute a promising target for drug design to block the encystment, and therefore the transmission, of this parasite.

Introduction The protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis) is the aetiological agent of giardiosis, a common enteric infection of human and other vertebrates. This disease is endemic worldwide and several hundred million cases are reported every year (Lane and Lloyd, 2002). Due to its high prevalence and pathogenicity, this parasite represents an important medical and veterinary problem. 398

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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Giardia has a direct life cycle that alternates between two stages: trophozoites and cysts, which are remarkably well adapted to survive in different and hostile environments (Adam, 2001). To achieve this, two different differentiation processes occur within the host, namely encystment and excystment. Infection begins with ingestion of a cyst from contaminated water or food, or by personto-person transmission. Excystation in the host’s duodenum results in the release of an excyzoite that develops into two trophozoites which rapidly colonize the proximal small intestine, causing acute or chronic diarrhoea, dehydration, abdominal discomfort and weight loss (Farthing, 1994, 1997). Encystment occurs when trophozoites are carried down to the jejunum and ileum where they are exposed to changes in nutritional micro-environment that induce their differentiation into infective cysts. The cysts are then excreted in the faeces and are able to infect a new host (Gillin et al., 1987). Encystment is therefore a process that enables the continuity of giardial infections. It has been reproduced in vitro with stimuli that mimic gastrointestinal conditions using several methods such as gas infusion, cholesterol deprivation and through the addition of high bile concentrations to cultures (Gillin et al., 1987; Sterling et al., 1988; Luján et al., 1996). Encystment involves a series of inductive events that begin with the transduction of external stimuli and end with the extracellular assembly of the cyst wall (Argüello-García et al., 2002). However, the signalling pathways regulating this process are not yet well defined. The sequencing of the Giardia genome has provided a survey for several kinds of transduction molecules including potential serine/threonine protein kinases (McArthur et al., 2000), but only a few signalling elements have been characterized so far. Protein kinases are key signalling mediators that connect environmental cues with the corresponding intracellular processes. In Giardia duodenalis, different protein kinases have been identified, cloned and shown to be associated with giardial cellular processes. Protein kinase A (gPKA) is involved in trophozoite motility during vegetative growth, in the activation of excystment (Abel et al., 2001), and it also probably regulates encystment because the regulatory subunit shows changes in protein expression during the differentiation process (Gibson et al., 2006). Protein homologues of ERK1 and ERK2 have been identified in trophozoites and encysting cells, and it has been suggested that these kinases may be involved in the induction of cyst morphogenesis (Ellis et al., 2003). In addition, a lipid signalling pathway may be associated with encystment activation, since several components of the phosphoinositide signalling pathway have been identified and characterized. These are GiPI3K1 and GiPI3K2 (PI3K homologues) and also gPKB. GiPI3K1 and GiPI3K2 are expressed in trophozoites and encysting cells, suggesting a possible role in proliferation and encystment regulation (Cox et al., 2006), while gPKB was detected upregulated during encystment (Kim et al., 2005). More recently, several protein kinases related to the protein kinase C (PKC) family were identified in Giardia duodenalis and were proposed to participate during encystment induction (Bazán-Tejeda et al., 2007). Giardial PKC isoforms may exhibit interesting evolutionary features that could provide an opportunity to design new strategies for encystment inhibition.

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Fig. 32.1. Structural features of the PKC family. Members of the PKC family present a regulatory and a kinase domain at the amino-and carboxy-terminus, respectively. C1 and C2 domains (membrane-targeting modules) bind diacylglycerol or phorbol esters (black boxes) and anionic lipids in conventional PKCs, and Ca2+ (grey box), respectively. Domains such as C2 (V0) (dotted box), PH, which binds phosphoinositides (chequered box), and pseudosubstrate (white boxes) are indicated. The kinase domain (hatched boxes) comprises the ATP-binding lobe (C3) and substrate-binding lobe (C4). Subclasses and members of each isozyme subclass are listed on the left.

PKC Family from Giardia Protein kinase C (PKC), a phospholipid-dependent serine/threonine kinase, has a crucial role in signal transduction for a variety of cellular responses, including cell proliferation and differentiation (Nishizuka, 1986). This multigene kinase family is an evolutionarily conserved group of enzymes that have been found in diverse eukaryotic organisms from yeasts to humans (Kuo, 1994). Members of this family are single polypeptide chains containing two functional domains: N-terminal regulatory and C-terminal kinase domains (Dekker and Parker, 1994). PKC isoforms have been subdivided into classes based on their structural similarities and cofactor requirements. In mammals, twelve isoforms are categorized into three major subfamilies: conventional PKCs (cPKCs), which are activated by Ca2+, phosphatidylserine and diacylglycerol; novel PKCs (nPKCs), which are Ca2+-independent and are regulated by diacylglycerol (DAG) and phosphatidylserine (PS); and atypical PKCs (aPKCs), which are Ca2+-independent and do not require DAG for activation, although PS regulates their activity (Nishizuka, 1986). Thus, the catalytic domain is highly conserved, while the regulatory domain shows polymorphism in size and structure. The catalytic domain comprises two functional regions: the ATP binding module (C3), and the substrate

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Encystment (% of control)

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Fig. 32.2. Effect of PKC inhibitors on encystment of G. duodenalis. Trophozoites were induced to encyst for 17.5 h in the presence or absence of the PKC inhibitors BIM-I at 7.5 µM (white bars), chelerythrine chloride at 7.5 µM (grey bars) or calphostin C 5 µM (black bars). Encystment ability was compared to controls without the compounds in the presence of vehicle (DMF) which corresponds to 100% of encystment ability. Data are presented as mean values ± SEM from three independent experiments. From Bazán-Tejeda et al. (2007) with kind permission from Springer and Business Media.

binding module (C4). The PKC N-terminal domain presents one or two types of membrane-targeting modules to provide sensitive, specific and reversible regulation of protein function. These two modules are the C1 domain, which binds diacylglycerol; and the C2 domain, which binds acidic lipids and Ca2+ (Fig. 32.1) (Newton, 1995; Dieterich et al., 1996; Newton and Johnson, 1998). In Giardia duodenalis, members of the PKC family were identified using polyclonal antibodies raised against mammalian-specific PKC isoforms (BazánTejeda et al., 2007). Five PKCs were detected: three novel (δ, ε and θ), one atypical (ζ), and one conventional PKC. Selective PKC inhibitors blocked the encystment process in a dose-dependent manner, suggesting that PKC isozymes may play important roles during the encystment differentiation process (Fig. 32.2). This pharmacological approach also showed that PKC inhibitors specifically reduced the trophozoites’ ability to encyst without affecting their capacity to grow (data not shown). The PKC inhibitors used were bisindolylmaleimide I (BIM I), chelerythrine chloride and calphostin C, which bind to different PKC regions; the first and second compounds recognize the kinase domain while the third binds to the regulatory domain (Kobavashi et al., 1989; Hayashi, 1992). These results also confirmed that the PKC family is present in Giardia duodenalis and that these proteins bear homologue regions for these inhibitors. It is worth mentioning that all giardial PKC isoforms displayed changes in their protein expression pattern in encysting trophozoites. β, δ and ε isoforms increased their expression during encystment, while the expression of PKCs θ and ζ was reduced (Fig. 32.3). The giardial novel and atypical PKCs had a similar molecular weight (75–85 kDa) to those of their mammalian counterparts (Mellor and Parker, 1998). However, gPKCβ had a molecular weight of 150 kDa, which is higher than mammalian PKCβ-type, although similar to PKC1 from S. cerevisiae (Watanabe et al., 1994; Antonsson et al., 1994).

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Fig. 32.3. Detection of PKC isoforms in G. duodenalis. Protein extracts from trophozoites (lanes T) or encysting trophozoites 17.5 h after encystment induction (lanes E) were separated by SDS/PAGE and analysed by Western blot using polyclonal antibodies directed against the indicated mammalian PKC isoforms (α, β, δ, ε, θ, ζ and λ). Positive controls corresponding to extracts from mouse brain are included in lanes C+. Results are representative of three independent experiments. The size of protein standards in kDa are indicated to the left of the Figure. From Bazán-Tejeda et al. (2007) with kind permission from Springer and Business Media.

The three classes of PKC are conserved in a variety of organisms from metazoan species to yeasts. For example, in Drosophila, there are at least two cPKCs (PKC53E and eyePKC), two nPKCs (PKCdelta and PKC98E) and one aPKC (DaPKC), and in C. elegans, there are at least one cPKC (PKC-2), two nPKCs (PKC-1 and TPA-1) and one aPKC (PKC-3) (Ohno and Nishizuka, 2002; Tabuse, 2002). In contrast, there is only a single PKC gene in some fungal genomes, including S. cerevisiae, Trichoderma resei and Candida albicans (Levin et al., 1990; Morawetz et al., 1996; Paravicini et al., 1996), and this isoform is homologous to cPKCs and PRKs. However, in S. pombe there are two isoforms: SpPck1p and SpPck2p (Perez and Cologne, 2002). These pieces of evidence indicate the evolutionary conservation of PKC isoforms from yeast to mammals, and that this kinase family is involved in specific cellular mechanisms such as cell differentiation, i.e. in protozoa encystment. In agreement with this, it has been reported that selective PKC inhibitors reduced encystment of a closely related protozoan, Entamoeba invadens (Makioka et al., 2000, 2003).

PKCb-like Molecular Characterization The expression of gPKCβ was monitored by Western blot analysis at several time periods (0.33, 1, 2, 3, 5, 7.5 and 17.5 h) after encystment induction, and CWP1was analysed in parallel as an inducible and upregulated marker protein. Interestingly, gPKCβ expression that was barely detected in trophozoites increased 20 min following encystment induction, and thereafter it continuously increased until 17.5 h post-induction. Additionally, by indirect immunofluorescence assays, a redistribution of gPKCβ from cytoplasm to the plasma membrane of trophozoites was observed at 10 and 20 min post-encystment induction. Since membrane translocation is commonly used as an index of PKC activation (Shirai and Saito,

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Mr (kDa) 37 25 37

Specific phosphorylation (% of control)

25 120 100 80 60 40 20 0

Fig. 32.4. In vitro phosphorylation of histone H1 by gPKCβ. Kinase activity was measured with purified gPKCβ in the presence or absence of cofactors, using histone H1-IIIS as substrate. Upper panel, histone H1 (doublet) detected by Coomassie blue staining. Middle panel, autoradiograph of phosphorylated histone. Results are representative of three independent experiments. Lower panel, autoradiograph and Coomassie blue staining of gels were quantified with an image densitometer, and the specific phosphorylation was determined as the ratio of 32 P-labelled protein to dyed protein. Results are representative of three independent experiments. From Bazán-Tejeda et al. (2007) with kind permission from Springer and Business Media.

2002), these results indicate that gPKCβ activation may play an important role during G. duodenalis cyst formation. As expected, this isoform presented biochemical properties of cPKC when its phosphorylation abilities were assayed in vitro. Using the typical PKC substrate histone H1-IIIS, it was demonstrated that gPKCβ activity was modulated by cofactors required by conventional PKCs (Fig. 32.4), since kinase activity was diminished in the absence of any cPKC cofactor (Ca+2 or phospholipids) and strongly reduced in their absence. gPKCβ kinase activity was also blocked by a general selective PKC inhibitor (BIM-I at 1 µM) and by the PKCβ selective inhibitor hispidin at 4 µM (Gonindard et al., 1997). Taken together, these data indicate that native gPKCβ from G. duodenalis behaved catalytically as a cPKC isoform. In the Giardia genome database, a homologue candidate of PKCβ type that corresponds to open reading frame (ORF) 86444 at contig 3550 was identified. This ORF encodes a protein similar to the carboxy-terminal end of the PKCβ catalytic domain. The amino-acidic sequence of ORF 86444 was aligned with other PKCβ sequences from mammals and other PKCβ-like kinases as PKC2B

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from C. elegans and PKC1 from S. cerevisiae. The alignment analysis revealed that the ORF 86444 exhibits a significant homology with the catalytic domain of other β-type PKCs, for example gPKCβ presents 37% identity and 60% homology with the catalytic domain of human PKCβ (Bazán-Tejeda et al., 2007) as well as all 12 characteristic subdomains of serine/threonine kinases (Hanks and Hunter, 1995). Additionally, the expressed recombinant protein was recognized by the mammalian anti-PKCβ II antibody. Therefore, ORF 86444 encodes the catalytic domain of gPKCβ.

PKC as a Potential Pharmacological Target Human protein kinases C have been considered as a promising target for the treatment of human diseases such as cancer and diabetes. Thus the regulatory and catalytic domains of PKC offer potential targets for designing specific inhibitors. In general, protein kinase inhibitors fall into four main groups: (i) substratespecific; (ii) ATP-competitive; (iii) activation; and (iv) irreversible inhibitors. A suitable protein kinase inhibitor prevents activation rather than competing with the ATP cofactor or the substrate domains (Naula et al., 2005). In this context, progress has been made on the design of ATP-competitive inhibitors of PKC. Although the ATP site is well conserved, a number of more diverse sequence pockets surrounding it have been extensively used in the design of selective inhibitors. This has provided the basis for the development of a bisindolylmaleimide group of PKC inhibitors. These inhibitors were derived from the earlier PKC inhibitor staurosporine by the removal of the 12a–12b bond of staurosporine aglycone and by the introduction of a second carbonyl group into the lactam ring. These inhibitors exhibit a high degree of selectivity for PKC over other closely related protein kinases (Davis et al., 1992). Recently, new protein kinase C beta inhibitors have been developed and used in clinical trials. Two examples of these are the bisindolylmaleimides enzastaurin (LY317615.HCl) and ruboxistaurin (LY333531). Enzastaurin was developed as an ATP competitive, selective inhibitor of PKCβ, and in clinical trials it exhibited a direct effect on human tumoural cells by inducing apoptosis and suppressing proliferation of a wide array of cultured human cancer cells (Graff et al., 2005). A similar compound, ruboxistaurin, is now under investigation in clinical trials for diabetic complications. These studies have demonstrated a delay in the progression (and even a reversal) of diabetic retinopathy, nephropathy and neuropathy (Shen, 2003; Joy et al., 2005; Sledge and Gokmen-Polar, 2006). In lower eukaryotes, it has been reported that cercosporamide is a specific CaPKC1p inhibitor. This molecule affects the human opportunistic pathogen Candida albicans and, interestingly, it does not affect mammalian PKC isoforms or other mammalian kinases (Sussman et al., 2004). Recently, the availability of genome databases for protozoan parasites has allowed the prediction of their kinome for inhibitor design in order to target protein kinases of parasitic protists (Doerig et al., 2005; Parsons et al., 2005). The rationale for targeting the protein kinases of protozoan parasites is based on

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the vast phylogenetic distance between unicellular parasites and their vertebrate hosts, a feature reflected by divergence in the properties of their protein kinases, which may be exploited for specific inhibition of parasite enzymes (Doerig et al., 2005). Nevertheless, there is still controversy regarding the phylogenetic position of some protozoa. Giardia duodenalis may present a divergent kinome since this protozoan parasite represents one of the earliest divergent lineages in the evolutionary history of eukaryotic organisms, and it is a member of the excavate subgroup, a deeper eukaryotic branch (Baldauf, 2003). Moreover, its phylogenetic distance is reflected by important divergences in comparison with mammalian kinases. These divergences may exist at different levels: in the properties of protein kinases, in the genes encoding these kinases, and in the organization of signalling pathways. For instance, the genes that encode for GiPI3K1 and GiPI3K2 exhibit long insertions in their kinase domains; thus these regions may also be considered as possible drug targets (Cox et al., 2006). gPKCβ was the only conventional PKC isoform found in Giardia duodenalis; this enzyme possesses important characteristics that may contribute towards its consideration as a novel kind of kinase, as well as leading to further study of this kinase in more detail as an attractive target for pharmacological design. This is due to its special characteristics, such as its higher molecular weight than mammalian PKCβ-type molecules, and consequently the probable divergence from its counterparts in the host organism. Another important aspect to be considered is the involvement of gPKCβ in encystment: in this respect, gPKCβ may be a good target for blocking the transmission of Giardia duodenalis.

Acknowledgements This work was supported in part by CONACyT Grant No. M-49724 (México).

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Secretory Granule Biogenesis and the Organization of Membrane Compartments via SNARE Proteins in Giardia lamblia

E.V. ELÍAS, N. GOTTIG, R. QUIROGA AND H.D. LUJÁN Mercedes and Martin Ferreyra Institute for Medical Research, Catholic University of Cordoba, Argentina

Abstract Giardia lamblia trophozoites lack organelles typical of higher eukaryotes such as mitochondria, peroxisomes and compartments involved in intracellular protein trafficking and secretion, the Golgi apparatus and secretory granules. Nevertheless, the minimal machinery for protein transport and sorting is present in this parasite. When Giardia undergoes encystation, the biogenesis of secretory organelles necessary to transport cyst wall constituents to the cell surface takes place. Although both constitutive and regulated pathways for protein secretion exist in Giardia, little is known about the molecules and mechanisms specifically involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) of the vesicle-associated membrane protein (VAMP) and syntaxin proteins play an essential role in these processes through the formation of complexes between proteins present on donor and target membranes. Recent studies in both vegetative and encysting trophozoites have provided interesting information regarding the secretory pathway of this important pathogen.

Introduction Giardia lamblia, a parasitic protozoan of humans and other vertebrates, is a major source of waterborne disease worldwide. Clinical signs of giardiasis vary from asymptomatic infection to acute or chronic disease associated with diarrhoea and malabsorption. Giardia is also of biological interest because it derives from one of the earliest branches of the eukaryotic line of descent (Adam, 2001). Giardia undergoes important biological changes to survive in hostile environments, alternating between the motile trophozoite and the environmentally resistant cyst (Adam, 2001). Trophozoites inhabit the upper small © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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intestine and are responsible for the symptoms of the disease, whereas cysts develop in the lower intestine and are excreted with the faeces. This allows Giardia survival outside the intestine and transmission between susceptible hosts (Adam, 2001). The encystation process includes cyst wall component synthesis and secretory organelle biogenesis. Encystation-specific secretory vesicles (ESVs) (Luján et al., 1995a), which are absent in non-encysting trophozoites, are necessary to transport cyst wall secretion components, leading to assembly of the extracellular cyst wall (Adam, 2001). We previously characterized two Giardia cyst wall proteins (CWPs): CWP1 and CWP2 (Mowatt et al., 1995). Recent Giardia genome database mining identified a new cyst wall protein, CWP3 (Sun et al., 2003). CWP1, CWP2 and CWP3 expression increases after trophozoites are exposed to the encystation stimulus (Luján et al., 1995b). CWP1, CWP2, and CWP3 are acidic proteins. A hydrophobic N-terminal signal peptide targets them to the secretory pathway (Mowatt et al., 1995). The central region of CWP1 and CWP2 consists of a tandem of five leucine-rich repeats, whereas CWP3 has four complete and one incomplete leucine-rich repeat (Sun et al., 2003). Leucine-rich repeat motifs in both prokaryotic and eukaryotic proteins have diverse functions and cellular localizations, but are always implicated in protein/protein interactions (Kobe and Kajava, 2001). The C-terminus of CWPs has a cysteine-rich domain involved in the formation of disulphide-bonded oligomers (Luján et al., 1995b). Although CWPs are closely related to each other, CWP2 is distinguished from CWP1 and CWP3 by the presence of a basic 121-residue C-terminal extension. In CWP2, this C-terminal region is present within ESVs, but is proteolytically cleaved before cyst wall assembly. After synthesis in the endoplasmic reticulum (ER), CWPs are shuttled to the cell exterior within ESVs. Their presence is the earliest morphological change observed during Giardia encystation (Faubert et al., 1991). In cells from higher eukaryotes, regulated secretory proteins concentrate into a dense core that buds off, forming an immature secretory granule in the last portion of the Golgi apparatus (Bauerfeind and Huttner, 1993). Evidence suggests that Giardia may possess organelle(s) in which typical Golgi functions take place, even though they do not have a Golgi-like appearance (Adam, 2001). Constitutive and regulated mechanisms for protein transport exist in Giardia, suggesting Golgi functions, because the sorting and selection processes generally occur in the trans-Golgi network (TGN) (Rothman and Orci, 1990). Detailed structural analyses of encysting cells (Lanfredi-Rangel et al., 2003) and the presence of BiP, in these organelles (Luján et al., 1996) suggest that ESVs arise from modified ER cisternae. Whether these specific secretory granules form from an uncharacterized trans-Golgi or through condensation within the ER is unclear (Luján and Touz, 2003). Previously, Mowatt et al. (1995) and others (McCaffery and Gillin, 1994) observed that CWPs aggregate within membrane-bound clefts. These aggregates appear to grow by the direct addition of newly synthesized CWPs, forming large ESVs, suggesting that CWP accumulation is an important factor for granule formation. Our results suggest that secretory granule formation is a direct consequence of CWP synthesis (Luján and Touz, 2003) and requires complex interactions between granule components (aggregation/condensation)

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and granule membrane receptors (sorting), involving both passive and active mechanisms. Since the late 1990s, many proteins involved in the Giardia secretory pathways have been identified: ER-chaperones such as BiP/GRP78 (Luján et al., 1996), protein disulphide isomerase (PDI) (Knodler et al., 1999), encystationspecific cysteine protease (ESCP) (Touz et al., 2002a), and granule specific protein (GSP) (Touz et al., 2002b), among others (Hehl and Marti, 2004). Recently, by taking advantage of the completion of the Giardia genome project (McArthur et al., 2000) and with the use of specific antibodies, a number of molecules involved in protein trafficking, such as βCOP, clathrin, Rabs and SNARES proteins, were identified but not functionally analysed (Marti et al., 2003a). Even though other proteins involved in vesicular transport, such as ARF (Luján et al., 1995b) and the adaptor protein complex 1 (AP1) (Touz et al., 2004), have been more deeply characterized in Giardia, little is known about the process of vesicle transport and membrane fusion in this parasite. Intracellular membrane fusion is a complex, multistage process essential for cell growth, proliferation and differentiation. Both the homotypic and heterotypic fusion of intracellular membranes along the secretory and endocytic pathways are mediated by a family of proteins called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) (Rothman, 1994). These proteins have been found to mediate vesicle fusion in essentially all organisms, from yeasts to humans (Ferro-Novick and Jahn, 1994). SNAREs share α-helical coiled-coil domains, called SNARE motifs, that probably evolved from a common ancestor and are composed of a repeated hydrophobic heptad register (Weimbs et al., 1998) interrupted at a cross-section (so-called ‘zero’) layer by a conserved arginine or glutamine polar residue (Sutton et al., 1998). On the basis of this observation, SNAREs are often referred to as R- or Q-SNAREs for arginine or glutamine, respectively (Fasshauer et al., 1998). Alternatively, they are known as v- or t-SNAREs, depending on their localization to either the transport vesicle (v) or the target (t) membrane (Ungar and Hughson, 2003). Functional analysis of these proteins in higher eukaryotes has led to the ‘SNARE hypothesis’, which states that interactions between R-SNAREs and Q-SNAREs mediate vesicle fusion with target membranes (Sollner et al., 1993). Specific R-SNAREs will only bind to a limited number of Q-SNAREs (Pevsner et al., 1994). Such restricted interaction of SNARE proteins is thought to form the basis for specificity of vesicle targeting. SNARE-mediated fusion typically results from the formation of a ternary complex that comprises a supercoil of four α-helical coils, consisting of one R-SNARE and three Q-SNARE motifs (Sutton et al., 1998). In this context, Q-SNAREs can be subdivided into Qa- (syntaxins), Qb- (25-kDa synaptosomalassociated protein (SNAP-25) N-terminal SNARE motif), or Qc- (SNAP-25 C-terminal SNARE motif) SNAREs (Bock et al., 2001). The R-SNAREs can be also subdivided into short VAMPs (vesicle-associated membrane proteins) or ‘brevins’ (e.g. synaptobrevin) and long VAMPs or ‘longins’ (Filippini et al., 2001). Since Giardia is an early-diverged eukaryote (Adam, 2001), the study of the secretory organelle biogenesis and the organization of membrane compartments and membrane fusion processes in this organism may allow a better understanding of the evolution of transport pathways from primitive to higher eukaryotes.

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Alternatively, due to its parasitic lifestyle, Giardia is an interesting system to analyse how microorganisms could have lost complex organelles.

Molecular Basis for Secretory Granule Biogenesis in Giardia G. lamblia possesses an interesting secretory system lacking an identifiable Golgi complex, and secretory granules are present only during trophozoite differentiation into cysts (Hehl and Marti, 2004). Because higher eukaryotic cells contain secretory granules during their entire lifetime, the ability to regulate secretory granule formation in Giardia by changing the culture medium composition makes this parasite an exceptional model system (Tooze, 1991). We investigated the functional role of CWPs in Giardia ESV biogenesis (Gottig et al., 2006). Epitope-tagged CWPs were expressed individually or in combination in nonencysting trophozoites, a situation that is comparable with the knock-out methodology. In this life cycle stage, CWPs are not expressed. When we expressed CWP1, CWP3, and CWP2 lacking its basic extension in non-encysting cells, they entered the secretory pathway without forming granules and were secreted into the culture medium. Conversely, transgenic expression of CWP2 and CWP1 plus the CWP2 basic extension in non-encysting trophozoites induced the biogenesis of large secretory granules that shared characteristics with the typical ESVs that normally transport cyst wall materials during Giardia encystation. These granules never discharged their contents to the cell exterior, and no cyst wall was formed in transgenic non-encysting trophozoites. These data agree with previous reports showing that additional proteins are required for secretion and cyst wall assembly. These results also demonstrate that the CWP2 basic extension is necessary for ESV biogenesis during the regulated pathway occurring during Giardia encystation. Additional results show that expression of the CWP2 basic extension alone (TCWP2) or the chimeric protein VSPH7(−TM+TCWP2)-HA did not induce ESV biogenesis and that these constructs localized predominantly to the ER in non-encysting cells. Because the basic extension alone is not sufficient to induce ESV biogenesis, other CWP2 domains are probably necessary for this process. The leucine-rich repeats and the cysteine-rich region of CWP2 are potential candidates for this activity because they are known to be involved in protein/protein interactions (Kobe and Kajava, 2001). In contrast, TCWP2-HA and VSPH7(−TM+TCWP2)-HA were not sorted to the ESVs and did not form aggregates under the same conditions, suggesting that the aggregation of CWPs is also necessary for ESV biogenesis. Thus, we propose that interactions between CWP1 and CWP3 with CWP2 are also needed for ESV biogenesis. In addition, co-expression of CWP1-HA and CWP2-HA in non-encysting trophozoites changed CWP1-HA localization from the ER to ESV-like vesicles, probably as a result of their interaction with CWP2. Interestingly, ESVs induced by expression of CWP2-HA or CWP1(+TCWP2)-HA co-localized with endogenous CWPs. Our results implicate CWP2 as an aggregation nucleation point for other CWPs destined for the ESVs. If this is true, interaction between the basic extension and an anionic receptor could tether CWP2 to segregation compartment membranes. Subsequent CWP interactions with CWP2 could lead to complex formation and secretory granule budding. Thus, this compartment could effectively act as a

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trans-Golgi, excluding proteins from ESVs not destined for these granules. It is well known that many proteins contain basic amino acid clusters that can bind to acidic phospholipids, which are preferentially located on the luminal cell membrane surface or on acidic proteins (Buser et al., 1995). Supporting this idea is the finding that a receptor in luminal membranes is indispensable for secretory granule formation. We observed in encysting trophozoites expressing TCWP2-HA or VSPH7(−TM+TCWP2)-HA that the ESV number was reduced. Possibly in these cases, the CWP2 basic extension competes with endogenous CWP2 for an anionic receptor. Localization of TCWP2-HA and VSPH7(−TM+TCWP2)-HA moved from the ER to a more limited clamp-shaped pattern adjacent to the ER/nuclear envelope compartment, in a pattern similar to the Golgi specific marker 12-(Nmethyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-ceramide (Luján et al., 1995a). In these experiments, the presence of the basic extension inhibited extracellular secretion of chimeras containing the CWP2 basic extension. Retention of these expressed proteins in that specific compartment led us to speculate that the intracellular receptor for the basic extension is localized in a region near the nuclei in encysting trophozoites. This specialized compartment is derived from the ER because blockage of the ER exit at 15°C inhibits formation of ESVs both in encysting trophozoites (Marti et al., 2003b) and in transgenic non-encysting trophozoites expressing CWPs containing the CWP2 basic domain. Moreover, co-expressed CWP1 and CWP2 lacking its basic extension co-localized to the ER. However, biogenesis of ESVs was not induced, suggesting that interaction among CWPs is not sufficient to trigger ESV formation. The intrinsic characteristics of the CWPs, in addition to the requirement of CWP2 basic extension interaction with a receptor, are important for ESV biogenesis. On the basis of our results, we propose that membrane selection is the initial step in secretory granule biogenesis (Gottig et al., 2006). Because the synthesis of one receptor for each protein destined for granules or a unique receptor for all is highly unlikely, our results support the possibility that the elusive sorting receptor necessary for sorting proteins to secretory granules in eukaryotes may well be lipid molecules (Thiele et al., 1997). In Giardia, the concentration of CWP1, CWP2 and CWP3 in heteroaggregates previously sorted by the CWP2 basic extension drives secretory granule biogenesis. The dense granules formed by CWP aggregation might then mature by further addition of CWPs and other granule components (Luján et al., 1997). Because CWP exit from the ER is necessary for ESV biogenesis, the presence of BiP at later steps of the regulated secretory pathway is an indication that CWP sorting to the ESVs occurs directly from an ER-derived compartment that could be a specialized ER organelle. This view is supported by data from detailed electron analysis of encysting cells with ESVs arising from modified ER cisternae (Knodler et al., 1999).

SNARE Proteins that Mediate Membrane Fusion Events in Giardia Selective protein transport between organelles is essential for the organization of membrane compartments. This process is largely mediated by the budding of transport vesicles from a donor compartment followed by vectorial trafficking to

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and fusion with an acceptor compartment. The SNARE proteins are the core fusion machinery for intracellular vesicle trafficking, and specific SNARE pairing is required for appropriate membrane fusion (Chen, and Scheller, 2001). Although this molecular machinery is evolutionarily conserved (Ferro-Novick and Jahn, 1994) in Giardia and other protozoa, the knowledge of molecules involved in the process of membrane docking and fusion during protein trafficking is limited (Dacks and Doolittle, 2004). By searching the Giardia genome database (McArthur et al., 2000), we identified 17 putative Giardia SNAREs involved in intracellular membrane trafficking in this parasite (Elias et al., 2008). The highly specific nature of transport from the ER to Golgi, Golgi to lysosome/vacuole, Golgi to plasma membrane, etc., appears to require the use of distinct combinations of mutually recognizable SNAREs in order to allow targeted membrane fusion. In this sense, the participation of SNAREs in intracellular trafficking in Giardia seems to contain common features that are essential for parasite growth. The finding of a complete set of syntaxin and VAMP proteins and the characterization of their subcellular localization prompted us to build a model of how the SNAREmediated intracellular trafficking is accomplished in this early-branching eukaryote, taking into special consideration the fact that changes in expression and/or subcellular redistribution of Giardia SNAREs might indicate additional requirements for an existing function of a particular SNARE or the acquisition of new functional roles during trophozoite differentiation into a cyst. Our results indicate that each Giardia SNARE localizes to a particular subcellular compartment and that some of them are involved in the regulated secretory pathway occurring during cyst wall formation. By antisense knock-down expression, we also found that many SNARES are essential for parasite viability, demonstrating the importance of these SNARE proteins in constituting the minimal machinery required for vesicle fusion. Since Giardia is an early-branching protist, these results indicate that many membrane trafficking pathways have been conserved throughout evolution. In addition, our results suggest that Giardia has the minimal machinery for vesicle docking and fusion, composed of 17 SNARE proteins. These results clearly imply that an increase in organelle complexity from ancestral cells to multicellular organisms, which have a larger number of SNAREs proteins (Bock et al., 2001), might have occurred during evolution. But it is also possible that a secondary loss of SNAREs from Giardia could have been the result of the loss of ‘unnecessary’ organelles due to its parasitic lifestyle (Elias et al., 2008).

Conclusions Since Giardia is one of the earliest-branching eukaryotes, knowledge of the secretory organelle biogenesis that occurs during its differentiation into cysts offers novel insights into the molecular machinery required for regulated protein transport in higher organisms. Further studies regarding the functional role of SNARE proteins in Giardia are important, considering that vesicle trafficking in this human pathogen is essential in maintaining infection and disease. Additionally,

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some gSNAREs proteins could be considered specific drug targets, since outside the SNARE motif they are very divergent compared with those of the human host.

References Adam, R.D. (2001) Biology of Giardia lamblia. Clinical Microbiology Reviews 14, 447–475. Bauerfeind, R. and Huttner, W.B. (1993) Biogenesis of constitutive secretory vesicles, secretory granules and synaptic vesicles. Current Opinion in Cell Biology 5, 628–635. Bock, J.B., Matern, H.T., Peden, A.A. and Scheller, R.H. (2001) A genomic perspective on membrane compartment organization. Nature 409, 839–841. Buser, C.A., Kim, J., McLaughlin, S. and Peitzsch, R.M. (1995) Does the binding of clusters of basic residues to acidic lipids induce domain formation in membranes? Molecular Membrane Biology 12, 69–75. Chen, Y.A. and Scheller, R.H. (2001) SNARE-mediated membrane fusion. Nature Reviews: Molecular Cell Biology 2, 98–106. Dacks, J.B. and Doolittle, W.F. (2004) Molecular and phylogenetic characterization of syntaxin genes from parasitic protozoa. Molecular and Biochemical Parasitology 136, 123–136. Elias, E.V., Quiroga, R., Gottig, N., Nakanishi, H., Nash, T.E., Neiman, A. and Lujan, H.D. (2008) Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia. Available at http://www.ncbi. nim.nih.gov/pubmed/18930915 Fasshauer, D., Sutton, R.B., Brunger, A.T. and Jahn, R. (1998) Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proceedings of the National Academy of Sciences of the USA 95, 15781–15786. Faubert, G., Reiner, D.S. and Gillin, F.D. (1991) Giardia lamblia: regulation of secretory vesicle formation and loss of ability to reattach during encystation in vitro. Experimental Parasitolology 72, 345–354. Ferro-Novick, S. and Jahn, R. (1994) Vesicle fusion from yeast to man. Nature 370, 191–193. Filippini, F., Rossi, V., Galli, T., Budillon, A., D’Urso, M. and D’Esposito, M. (2001) Longins: a new evolutionary conserved VAMP family sharing a novel SNARE domain. Trends in Biochemical Sciences 26, 407–409. Gottig, N., Elias, E.V., Quiroga, R., Nores, M.J., Solari, A.J., Touz, M.C., and Lujan, H.D. (2006) Active and passive mecahnisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia. Journal of Biological Chemistry 281, 18156–18166. Hehl, A.B. and Marti, M. (2004) Secretory protein trafficking in Giardia intestinalis. Molecular Microbiology 53, 19–28. Knodler, L.A., Noiva, R., Mehta, K., McCaffery, J.M., Aley, S.B., Svard, S.G., Nystul, T.G., Reiner, D.S., Silberman, J.D. and Gillin, F.D. (1999) Novel protein–disulfide isomerases from the early-diverging protist Giardia lamblia. Journal of Biological Chemistry 274, 29805–129811. Kobe, B. and Kajava, A.V. (2001) The leucine-rich repeat as a protein recognition motif. Current Opinion in Structural Biology 11, 725–732.

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Molecular Mechanisms of Cryptosporidium-induced Host Actin Cytoskeleton Dynamics

S.P. O’HARA1, X.-M. CHEN2 AND N.F. LARUSSO1 1Mayo

Clinic College of Medicine, Rochester, MN, USA; 2Creighton University School of Medicine, Omaha, NE, USA

Abstract Early ultrastructural observations of Cryptosporidium parvum-infected cells consistently described the accumulation of microfilaments beneath the dense band, suggesting the initiation of actin reorganization within the host cell. Immunolocalization and forced expression of β-actin green fluorescent protein confirmed the rearrangement of host actin filaments, and recruitment of actin-binding proteins to infection sites. Subsequent studies have demonstrated the importance of host signalling cascades leading to actin reorganization during early invasion events. Therefore, this chapter focuses on actin reorganization during sporozoite internalization and development, concentrating primarily on the molecular mechanisms of actin reorganization.

Introduction The luminal surface of epithelial cells is often the point of entry for intracellular pathogens. Intracellular microbes utilize a variety of mechanisms that exploit host cell signalling cascades to gain entry into non-phagocytic cells, most of which result in the modulation of the host actin cytoskeleton. To initiate host cell signalling cascades some microbial pathogens express surface proteins that can interact with host cell surface receptors often involved in cell–matrix or cell–cell adherence. Activation of these receptors initiates signalling cascades culminating in protein phosphorylation cascades, recruitment of effectors and the actin polymerization-dependent formation of a vacuole that engulfs the pathogen. Other microbes have subverted the necessity to interact with host cell receptors through the injection of cytoskeletally active effectors. The pathogenic bacteria, Salmonella enterica and Shigella spp. induce localized cytoskeleton rearrangements through the insertion of actin effectors, by means of a dedicated secretory system, directly into the host cytoplasm. The result is an intricate interplay between pathogen and host cell factors resulting in transient, actin-rich membrane ruffles that engulf the bacteria (reviewed in Cossart and Sansonetti, 2004). 418

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Previous studies of the apicomplexan parasite, Toxoplasma gondii, demonstrated the active invasion of host cells, independent of host cell actin dynamics or tyrosine kinase activity in the host cell (Morisaki et al., 1995). Subsequent studies demonstrated that the Toxoplasma actomyosin cytoskeleton, the force behind apicomplexan gliding cell motility, provided the motive force behind host cell penetration and intracellular localization (Dobrowolski and Sibley, 1996). Investigations of Cryptosporidium sporozoite entry into host cells by Wetzel et al. (2005) have demonstrated that the initial entry into the host cell depends on the conserved penetrative force of the parasite actomyosin cytoskeleton. However, modulation of the host actin cytoskeleton plays an essential role in the early events associated with the formation of a productive infection site, as inhibition of host actin cytoskeleton dynamics diminishes the infectivity as assessed by immunofluorescence. Based on the observations of Wetzel et al. (2005), it seems likely that the observed host actin remodelling and resultant membrane alterations are secondary to the initial, parasite-driven internalization process. Host actin remodelling most probably contributes to modifications of the parasitophorous vacuole membrane (PVM), promoting the formation of a productive infection site (Forney et al., 1999; Elliott et al., 2001; Chen et al., 2003, 2004a, 2004b). Ultrastructurally, internalization of sporozoites and merozoites is similar, and it is assumed that both distinct developmental stages utilize the same invasion machinery and drive actin reorganization through similar mechanisms. Less is known about host cell actin reorganization during the sexual stages of parasite development. This chapter focuses on signalling cascades, initiated by the interaction of Cryptosporidium sporozoites with the apical domain of epithelial cells, which culminate in the reorganization of the actin cytoskeleton and the establishment of infection.

Ultrastructural Observations Reveal Host-membrane Modifications during Cryptosporidium Infection Following excystation from oocysts, the sporozoites immediately exhibit gliding cell motility, a conserved method of motility throughout the apicomplexans (Arrowood et al., 1991; Gut and Nelson, 1994; Forney et al., 1998). Through sequential zoite motility, attachment to the surface of an epithelial cell, and internalization, the zoite is ultimately encapsulated in host-derived membrane. The host-derived bi-membrane structure encapsulating the parasite is composed of the outer and inner PVM, between which is a thin layer of cytoplasm (Current and Reese, 1986; Marcial and Madara, 1986; Lumb et al., 1988; Tzipori and Griffiths, 1998). During the initial aspects of host–sporozoite interactions, a tunnel connection is formed between the apical region of the parasite and the surface of the host cell, suggesting the transfer of parasite molecules to the level of the host cell (Huang et al., 2004). As the invasion process ensues, a unique structure is formed at the base of the host–parasite interface, containing electrondense material (dense band) with an adjacent filamentous network (Marcial and Madara, 1986), later determined to be polymerized actin (Bonnin et al., 1999; Elliott and Clark, 2000). Therefore, the obligate intracellular parasite proceeds

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through developmental stages on the surface of epithelial cells in a unique niche that is intramembranous yet extracytoplasmic.

Cryptosporidium induces the Localized Accumulation of Actin and Actin-binding Proteins to Invasion Sites Early ultrastructural observations consistently described a localized aggregation of microfilaments beneath the dense band, suggesting the initiation of actin reorganization within the host cell. Several groups localized actin to infection sites by immunolocalization or phalloidin staining of infected neonate mouse ileal tissues (Bonnin et al., 1999) or in cell culture (Forney et al., 1999; Chen and LaRusso, 2000; Elliott and Clark, 2000). Bonnin et al. (1999), using confocal microscopy and phalloidin staining, localized a thin, weakly fluorescent layer of polymerized actin surrounding each internalized parasite suggestive of localization to the PVM. Based on the early observations of actin recruitment to infection sites, and the suggestion that the PVM is derived from microvilli, several studies focused on the recruitment of actin binding and remodelling proteins to infection sites. Actin itself was consistently observed at infection sites, but early reports from several laboratories produced conflicting results with respect to the distinct pattern of actinbinding proteins accumulating at invasion sites. Forney et al. (1999), using immunofluorescent techniques, suggested immediate tyrosine phosphorylation upon inoculation of C. parvum sporozoites to bovine fallopian tube epithelial cells, with subsequent accumulation of polymerized actin and the actin-bundling, microvilli-associated protein, villin, to invasion sites. In contrast, Elliott et al. (2001) described the inability to localize early phosphorylation events or villin to sites of infection in the ileocaecal cell line HCT-8. Additionally, ezrin, a protein that links transmembrane molecules, especially adhesion receptors and ion channel proteins, to actin-cytoskeleton, was immunolocalized to the PVM by both immunogold electron microscopy and immunofluorescence (Bonnin et al., 1999). On the other hand, Elliott et al. (2001) described the exclusion of ezrin from regions subjacent to developing parasites. The actin-bundling protein α-actinin, which localizes to active actin-dependent membrane protrusions, was localized to invasion sites during the early invasion process, but immunoreactivity was not evident during later stages of development (i.e. meront stage). The varied results obtained through immunolocalization studies may reflect the difficulties of localizing, spatially and temporally, specific proteins required for the formation of the transient machinery necessary for actin polymerization during infection. The host-cell origin of the accumulated actin to the cytoplasm subjacent to the infection site was confirmed by the transfection of host cells with a plasmid containing β-actin green fluorescent protein (GFP) (Elliott et al., 2001). Here the researchers used both phalloidin staining and the forced expression of β-actinGFP to confirm the aggregation of actin filaments to the region of parasite internalization. The studies documented the aggregation of actin to a sharply circumscribed plaque of actin directly beneath the internalized parasite, and suggested that host cell microvilli elongation was not the source of the increased actin fluorescence observed by Forney et al. (1999).

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Cryptosporidium-induced Signalling Cascades Trigger Host Cell Actin Reorganization Immunolocalization studies confirmed the aggregation of actin filaments, and recruitment of actin-binding proteins to infection sites, yet the signalling cascades promoting this rearrangement remained elusive. Infectivity studies, using a series of pharmacological inhibitors of signalling pathways, demonstrated that the protein kinase inhibitors genistein and staurosporine significantly inhibited invasion in a concentration-dependent manner. Furthermore, pretreatment of host cells with the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin, at nanomolar concentrations, also dramatically reduced parasite numbers, while pretreatment with the G-protein uncoupling agent, suramin, had no effect on the number of parasites detected by microscopy (Forney et al., 1999). The accumulation of proteins directly involved in dendritic nucleation of actin filaments, often observed in regions of active membrane protrusion events, and in the actin comet tails of motile, intracellular pathogenic bacteria and viruses, including the neural WiskottAldrich syndrome protein (N-WASp), have been detected by immunofluorescence at sites of parasite internalization. Upon activation, N-WASp binds to and activates the Arp2/3 complex of proteins, which in turn binds to the side of an existing actin filament and promotes the formation of a branching network of actin filaments. Indeed, Arp3 of the Arp 2/3 complex was detected, by immunofluorescence, at sites of internalization, implying parasite-induced actin polymerization at these sites (Elliott et al., 2001). The first mechanistic approaches to determine the molecular pathways of C. parvum-induced host cell actin polymerization demonstrated a requirement for the actin branching and nucleation machinery of the Arp2/3 complex of proteins. In addition to the accumulation of actin and associated structural components to infection sites, Elliott et al. (2001), provided clear evidence of the importance of host actin polymerization during the establishment of an infection site by overexpressing the C-terminal fragment of the protein Scar1 (Scar-WA) in the human ileocaecal cell line (HCT-8). The C-terminal portion of this protein contains the WH2/acidic (WA) domain: the acidic domain binds the Arp2/3 complex and the WH2 domain binds globular actin. Overexpression of this fragment activates Arp2/3 throughout the cytoplasm, making it less available for processes at the cell periphery. Infections in cells expressing this fragment of Scar, as detected by immunofluorescence microscopy, were significantly reduced. In addition, forced expression of a dominant negative form of N-WASp, in which the acidic domain required for Arp2/3 binding is deleted, revealed the accumulation of this protein to sites of parasite–host cell interaction; however, since it lacked the Arp2/3 binding domain, actin polymerization at these sites and the number of parasites detected was again dramatically reduced (Elliott et al., 2001). Subsequent work by our group, using a model of biliary cryptosporidiosis, confirmed the importance of N-WASp activation during the development of an infection site and further demonstrated that functional Cdc42 was necessary for N-WASp activation and accumulation at infection sites (Chen et al., 2004a, 2004b). Several Rho GTPases were analysed for accumulation at infection sites

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and activation. It was determined, by both confocal immunofluorescence and immunogold electron microscopy, that both Cdc42 and RhoA accumulated at sites of parasite–host cell interaction, yet we could demonstrate, by GST pulldown assays, the activation of only Cdc42. The functional significance of Cdc42 activation was demonstrated by the dramatic decrease in the number of infection sites detected by immunofluorescence in cells expressing either a GTPasedefective dominant negative mutant of Cdc42 (Cdc42-17N) (30% of control) or an shRNA vector targeting Cdc42 (20% of control), which effectively suppressed Cdc42 protein levels. Conversely, transfection of a constitutively active Cdc42 (Cdc42-61L) significantly increased the number of parasites detected by immunofluorescence following a 1 h infection. Attachment of the parasite to the host cell was not inhibited. Importantly, when observed by scanning electron microscopy (SEM), parasites attached to cells expressing either the dominant negative form of Cdc42 or the shRNA targeting Cdc42 revealed limited instances of host membrane remodelling at the region of parasite–host interaction. Additionally, when these attached parasites were analysed by transmission electron microscopy (TEM), the actin plaque and dense band typically observed in the host cytoplasm did not form. It was therefore proposed that parasite interaction with the apical surface of host cells initiates a signalling cascade leading to Cdc42 recruitment and activation. The activated, GTP-bound Cdc42 then binds the GTP-binding domain (GBD) of N-WASp and alleviates N-WASp autoinhibition. The release of intramolecular inhibition exposes the WA-domain of N-WASp, which interacts with the Arp2/3 complex of proteins to promote actin nucleation and polymerization. The upstream events leading to Cdc42 activation were determined by using both pharmacological inhibitors of host cell PI3K or by cellular expression of a mutant p85 regulatory subunit of class IA PI3K, and assessing activation of Cdc42 following C. parvum invasion (<1 h) (Chen et al., 2004a, 2004b). It was determined that both the pharmacological inhibitors and mutant PI3K decreased the aggregation of Cdc42 to infection sites and inhibited the activation of this Rho GTPase. Given that the function of Cdc42 requires guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of GDP from the GDPbound inactive form and promote the formation of the GTP-bound active form, we looked for the known Cdc42-associated GEF, frabin. Frabin not only accumulated at infection sites, but the aggregation was dependent on functional PI3K, and frabin activity was necessary for activation of Cdc42 and subsequent polymerization of actin at infection sites, as determined by infection of cells expressing the functionally deficient mutants of frabin. Therefore, a molecular pathway of actin reorganization has emerged, involving a phosphorylation cascade initiated by parasite interaction with the apical domain of epithelial cells, which involves PI3K activation, and the Rho-GTPase, Cdc42, which, interestingly, has been implicated in membrane protrusion events associated with microvilli and filopodial elongations, and also with lamellipodial extensions. In addition to the PI3k/Cdc42 signalling cascade, an alternative (or complementary) signalling cascade is initiated at the host–parasite interface. c-Src, a kinase associated with the cytoplasmic face of the plasma membrane, accumulates at sites of Cryptosporidium internalization. c-Src exists in an autoinhibited

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state in which the kinase domain is masked until an inhibitory tyrosine near the C-terminus is phosphorylated. During Cryptosporidium infection, c-Src, and the downstream effector cortactin, are phosphorylated as determined by immunoprecipitation analyses (Chen et al., 2003). Cortactin is a filamentous actinbinding protein known to be a central regulator in cortical actin remodelling, and can enhance N-WASp-stimulated actin branching and polymerization. The functional importance of c-Src and cortactin during Cryptosporidium infection was further supported by the observation that transfection and forced expression of the kinase inactive c-Src (c-Src K297M) diminished parasite internalization, cortactin phosphorylation, and the accumulation of actin to infection sites. Furthermore, forced expression of a cortactin dominant negative mutant diminished C. parvum internalization. Therefore, two separate, but not necessarily independent, kinase cascades are activated during Cryptosporidium invasion. The potential contribution of the two pathways, and the localized events leading to their activation, is unknown; however, as with other mechanisms of membrane alterations, the pathways may converge to synergize in actin branching or filament stabilization (Fig. 34.1).

Potential Role of Actin Reorganization during Cryptosporidium Invasion Forney et al. (1999) and Chen and LaRusso (2000) first suggested an active role for actin polymerization-dependent membrane protrusion in the establishment of infection sites on the surface of epithelial cells. Elliott and Clark (2000) described the formation of an actin plaque, subjacent to the developing parasite within the host cytoplasm, but suggest that the accumulation of actin plays a structural role in maintaining the parasite in its intramembranous, yet extracytoplasmic, niche. Previous studies looking at the apicomplexan parasite Toxoplasma gondii demonstrated the active invasion of host cells, independent of cytoskeletal rearrangement or tyrosine kinase activity in the host cell (Morisaki et al., 1995). Subsequent studies demonstrated that the Toxoplasma actomyosin cytoskeleton, the force behind apicomplexan gliding cell motility, provided the motive force behind host cell penetration and intracellular localization (Dobrowolski and Sibley, 1996) and a similar mode of entry has been attributed to Plasmodium spp. (Field et al., 1993). Cryptosporidium also exhibits actindependent gliding cell motility (Gut and Nelson, 1994; Forney et al., 1998). Based on the observation that pretreatment of sporozoites with cytochalasin D, which effectively abrogated C. parvum motility yet did not significantly affect parasite numbers after a 24 h infection, and the unique niche occupied by C. parvum, Forney et al. (1998) proposed that C. parvum utilized a unique mechanism of invasion, independent of parasite actin-based motility and membrane penetration. However, more recent investigations by Wetzel et al. (2005), which used time-lapse video microscopy to visualize the early events of parasite–host interactions, demonstrated that the initial entry into the host cells depends on the conserved penetrative force of the parasite actomyosin cytoskeleton. When an

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Cryptosporidium Membrane alteration

Actin plaque

Transport proteins

Actin branching and nucleation ?

C-Src

PI3k Cdc42-GDP Frabin (GEF)

Cortactin

Arp2/3

Cdc42-GTP

Myosin II dependent transporter aggregation?

N-WASP

Fig. 34.1. Model of Cryptosporidium-induced actin reorganization and membrane alteration. A phosphorylation cascade is initiated within the host cytosol upon initial interaction with Cryptosporidium sporozoites, culminating in actin nucleation and polymerization via the Arp2/3 complex of proteins and cortactin. Transporters and channels aggregate in the immediate vicinity of parasite internalization, a process that may depend on the contractile activity of myosin II.

assessment of the initial invasion was performed in the presence of cytochalasin D, using a cytochalasin D-resistant cell line, a complete block of rapid internalization was observed, demonstrating the requirement of parasite actin polymerization. Based on the observations by Wetzel et al. (2005) it seems likely that the protein phosphorylation cascades initiated by interaction between Cryptosporidium and the host plasma membrane resulting in host actin remodelling and resultant membrane alterations are secondary events that contribute to the establishment of a productive infection site. The demonstration of the requirement of a dynamic network of actin filaments at sites of Cryptosporidium internalization strongly suggests the requirement of host actin polymerization for some aspect of the early infection process. Actin depolymerizing factors, including ADF/cofilin, have not been investigated. Given the involvement of both actin polymerizing and depolymerizing factors in the model of actin treadmilling and membrane protrusion, these factors are

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worthy of investigation. Our observations have led us to conclude that actin reorganization is intimately involved in parasite retention at the plasma membrane, and post-invasion actin-dependent processes are required for successful parasite development. We have extended our initial studies on the signalling cascades responsible for rearrangement of the host actin cytoskeleton to factors that may influence the efficiency of membrane extension and parasite development. Using localized membrane extensions as models, we have assessed the recruitment of myosins and various transporters which may serve to expedite membrane extension. The machinery involved in localized membrane protrusions, which involves localized actin polymerization, is often exploited by intracellular pathogens. The overall rate of membrane protrusion depends on both the actin polymerization rate and the increase in localized cell volume. Interestingly, we determined that the Na+/glucose cotransporter, SGLT1, and aquaporin 1, AQP1, a channel protein selective for the movement of water and other small non-ionic molecules (Agre et al., 2002), accumulate at C. parvum invasion sites and participate in efficient membrane protrusion events induced by the parasite (Chen et al., 2005). Concordantly, the region of attachment displays localized glucose-driven water influx that is inhibited by either suppression of AQP1 by means of AQP1-small interfering RNA or the inhibition of SGLT1 by a specific pharmacological inhibitor, phlorizin. By inhibiting either of these proteins, we were able to diminish the efficiency of membrane protrusions as determined by both SEM and TEM. Thus, an important role for AQPs and solute transporters in the membrane remodelling associated with the development of the intramembranous/extracytoplasmic niche within host cells by C. parvum seems plausible, if not likely. Interestingly, pretreatment of host cells with the myosin light chain kinase inhibitor, ML-7, as well as the myosin II ATPase inhibitor, 2,3-butanedione monoxime (2,3-BDM), both exhibited inhibitory effects on infectivity, while parasite attachment was unaffected (Forney et al., 1999). Our recent, preliminary studies support the observations of the inhibitory effects of myosin inhibitors on invasion. We add that pretreatment of host cells with the myosin II-specific inhibitor, blebbistatin, and inhibition of myosin light chain kinase, significantly decreases parasite infectivity and transporter accumulation to infection sites as observed with immunofluorescence, suggesting a potential role for host cell actomyosin function during the formation of the unique niche occupied by C. parvum (unpublished data). It seems likely, therefore, that C. parvum-induced actin reorganization, and the recruitment of actin-binding proteins, including myosins, may serve several functions during the development of C. parvum.

Conclusions Cryptosporidium exhibits a complex strategy to invade and establish productive infection sites in epithelial cells, involving complementary parasite and host cell processes. While the work regarding host cell actin remodelling has greatly enhanced our understanding of the molecular pathways involved in the parasiteinduced actin reorganization, the specific function of host cell actin remodelling is still equivocal. Indeed, the potential contribution of the host cytoskeleton in other

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aspects of parasite development is not well studied. In particular, trafficking events required for the efficient transport of channels and transporters to sites of parasite internalization, and the retention of these to sites of infection, may be critical for parasite development. Our observations have led us to propose that host cell actin polymerization contributes to productive C. parvum infection sites by generating membrane protrusion events, which may assist in the retention of the parasite at the apical surface within the unique extracytoplasmic niche. Furthermore, the aggregation of transporters and channels to a localized region suggests that the activation of signalling cascades by Cryptosporidium may not only assist in the initial internalization events, but may promote the acquisition of necessary factors for parasite development. With our current understanding of the molecular pathways initiating actin remodelling upon C. parvum interactions with host cells, the next logical step is to determine the upstream events resulting in PI3K and c-Src activation and the specific role of actin remodelling in parasite development, a process that may have implications beyond host–pathogen interactions.

Acknowledgements The work reported in this chapter was supported by National Institutes of Health, grants DK57993 and DK24031, and by the Mayo Foundation to N.F.L.

References Agre, P, King, L.S., Yasui, M., Guggino, W.B., Ottersen, O.P., Fujiyoshi, Y., Engel, A. and Nielsen, S. (2002) Aquaporin water channels: from atomic structure to clinical medicine. Journal of Physiology 542, 3–16. Arrowood, M.J., Sterling, C.R. and Healey, M.C. (1991) Immunofluorescent microscopical visualization of trails left by gliding Cryptosporidium parvum sporozoites. Journal of Parasitology 77, 315–317. Bonnin, A., Lapillonne, A., Petrella, T., Lopez, J., Chaponnier, C., Gabbiani, G., Robine, S. and Dubremetz, J.F. (1999) Immunodetection of the microvillous cytoskeleton molecules villin and ezrin in the parasitophorous vacuole wall of Cryptosporidium parvum (Protozoa, Apicomplexa). European Journal of Cell Biology 78, 794–801. Chen, X.-M. and LaRusso, N.F. (2000) Mechanisms of attachment and internalization of Cryptosporidium parvum to biliary and intestinal epithelial cells. Gastroenterology 118, 368–379. Chen, X.-M., Huang, B.Q., Splinter, P.L., Cao, H., Zhu, G., McNiven, M.A. and LaRusso, N.F. (2003) Cryptosporidium parvum invasion of biliary epithelia requires host cell tyrosine phosphorylation of cortactin via c-Src. Gastroenterology 125, 216–228. Chen, X.-M., Huang, B.Q., Splinter, P.L., Orth, J.D., Billadeau, D.D., McNiven, M.A. and LaRusso, N.F. (2004a) Cdc42 and the actin-related protein/neural Wiskott-Aldrich syndrome protein network mediate cellular invasion by Cryptosporidium parvum. Infection and Immunity 72, 3011–3021. Chen, X.-M., Splinter, P.L., Tietz, P.S., Huang, B.Q., Billadeau, D.D. and LaRusso, N.F. (2004b) Phosphotidylinositol 3-kinase and frabin mediate Cryptosporidium parvum cellular invasion via activation of Cdc42. Journal of Biological Chemistry 279, 31671–31678.

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Chen, X.-M., O’Hara, S.P., Huang, B.Q., Splinter, P.L., Nelson, J.B. and LaRusso, N.F. (2005) Localized glucose and water influx facilitates Cryptosporidium parvum cellular invasion by means of modulation of host-cell membrane protrusion. Proceedings of the National Academy of Sciences USA 102, 6338–6343. Cossart, P. and Sansonetti, P.J. (2004) Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 304, 242–248. Current, W.L. and Reese, N.C. (1986) A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice. Journal of Protozoology 33, 98–108. Dobrowolski, J.M. and Sibley, L.D. (1996) Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite Cell 84, 933–939. Elliott, D.A. and Clark, D.P. (2000) Cryptosporidium parvum induces host cell actin accumulation at the host–parasite interface. Infection and Immunity 68, 2315–2322. Elliott, D.A., Coleman, D.J., Lane, M.A., May, R.C., Machesky, L.M. and Clark, D.P. (2001) Cryptosporidium parvum infection requires host cell actin polymerization. Infection and Immunity 69, 5940–5942. Field, S.J., Pinder, J.C., Clough, B., Dluzewski, A.R., Wilson, R.J. and Gratzer, W.B. (1993) Actin in the merozoite of the malaria parasite, Plasmodium falciparum. Cell Motility and the Cytoskeleton 25, 43–48. Forney, J.R., Vaughan, D.K., Yang, S. and Healey, M.C. (1998) Actin-dependent motility in Cryptosporidium parvum sporozoites. Journal of Parasitology 84, 908–913. Forney, J.R., DeWald, D.B., Yang, S.G., Speer, C.A. and Healey, M.C. (1999) A role for host phosphoinositide 3-kinase and cytoskeletal remodeling during Cryptosporidium parvum infection. Infection and Immunity 67, 844–852. Gut, J. and Nelson, R.G. (1994) Cryptosporidium parvum sporozoites deposit trails of 11A5 antigen during gliding locomotion and shed 11A5 antigen during invasion of MDCK cells in vitro. Journal of Eukaryotic Microbiology 41, 42S–43S. Huang, B.Q., Chen, X.-M. and LaRusso, N.F. (2004) Mechanisms of attachment and internalization of Cryptosporidium parvum by biliary epithelia: a morphological study. Journal of Parasitology 90, 212–221. Lumb, R. Smith, K., O’Donoghue, P.J. and Lanser, J.A. (1988) Ultrastructure of the attachment of Cryptosporidium sporozoites to tissue-culture cells. Parasitology Research 74, 531–536. Marcial, M.A. and Madara, J.L. (1986) Cryptosporidium: cellular localization, structural analysis of absorptive cell–parasite membrane–membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells. Gastroenterology 90, 583–594. Morisaki, J.H., Heuser, J.E. and Sibley, L.D. (1995) Invasion of toxoplasma-gondii occurs by active penetration of the host-cell. Journal of Cell Science 108, 2457–2464. Tzipori, S. and Griffiths, J.K. (1998) Natural history and biology of Cryptosporidium parvum. Advances in Parasitology: Opportunistic Protozoa in Humans 40, 5–36. Wetzel, D.W., Schmidt, J., Kuhlenschmidt, M.S., Dubey, J.P. and Sibley, L.D. (2005) Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites. Infection and Immunity 73, 53–79.

35

Pathogenic Mechanisms in Giardiasis and Cryptosporidiosis

A.G. BURET Department of Biological Sciences, University of Calgary, Canada

Abstract This chapter elaborates on pathogenic processes responsible for the production of symptoms during giardiasis and cryptosporidiosis. To date, research findings indicate that both infections share a number of these processes. Infection appears to cause diarrhoea via a combination of intestinal malabsorption and hypersecretion. Malabsorption and maldigestion mainly result from a loss of total epithelial brush border surface area, and diffuse microvillous shortening is mediated by activated host T lymphocytes. This activation is secondary to Giardia- and Cryptosporidium-induced disruption of epithelial tight junctions, which in turn increases intestinal permeability. Both parasites may breach the epithelial barrier by inducing enterocyte apoptosis. These effects may facilitate the development of other enteric disorders in infected patients, including inflammatory bowel disease, irritable bowel syndrome and allergies, via mechanisms that remain obscure. The regulatory processes of epithelial apoptosis are also discussed, including the role of parasite products as well as host factors. In this context, recent observations indicate that host epithelial nitric oxide responses, as well as a newly discovered glucose-mediated cytoprotective mechanism, may represent effective modulators of the epithelial apoptosis induced by these parasites. This review of the various pathogenic mechanisms of giardiasis and cryptosporidiosis sheds light on potential therapeutic targets that may help control the disease associated with these infections as well as a variety of other enteric disorders.

Introduction Giardia and Cryptosporidium are ubiquitous intestinal protozoan parasites. Infection with these entero-pathogens may cause diarrhoea, dehydration, abdominal discomfort and weight loss. Symptoms can be present in the absence of any significant morphological injury to the intestinal mucosa, and infections may remain asymptomatic or become chronic for reasons that remain obscure. Despite the worldwide prevalence of these diseases, the pathophysiological mechanisms underlying intestinal disturbances in giardiasis and cryptosporidiosis remain incompletely understood. Giardia causes disease without penetrating the epithelium. In contrast, Cryptosporidium goes through a life cycle that 428

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includes an intra-enterocytic stage. Research data available to date indicate that giardiasis and cryptosporidiosis share similar pathophysiological features, and that at least part of these may also resemble those of other enteric disorders, including bacterial enteritis, chronic food anaphylaxis, Crohn’s disease and coeliac disease (Rubin et al., 1966; Dvorak, 1988; Buret et al., 1990, 1998; Curtis et al., 1990; Gunasekaran and Hassall, 1992; Savioli et al., 2006). A better understanding of these pathophysiological processes may therefore help identify new therapeutic targets for a variety of gastrointestinal diseases. This chapter presents an overview of our current knowledge of the mechanisms of intestinal abnormalities and disease production during Giardia or Cryptosporidium infections.

Diarrhoea is Due to Epithelial Malabsorption and Hypersecretion In an attempt to understand the pathophysiological basis of diarrhoeal symptoms in giardiasis or cryptosporidiosis, a number of studies have investigated the effects of Giardia or Cryptosporidium on intestinal epithelial transport. Previous studies using models in vivo and in vitro have established that Giardia causes malabsorption of glucose, sodium and water, and reduced disaccharidase activity, due to a loss of epithelial absorptive surface area (Belosevic et al., 1989; Buret et al., 1992, 2002a; Cevallos et al., 1995; Müller and von Allmen, 2005; Gascon, 2006). Observations from humans chronically infected with this parasite recently confirmed these findings (Troeger et al., 2007). Other reports had previously suggested that this parasite may alter chloride secretory responses in human colonic cells in vitro, as well as in murine models (Gorowara et al., 1992; Resta-Lenert et al., 2000) . It has now been demonstrated that Giardia-induced hypersecretion of chloride may also occur in the intestine of human patients (Troeger et al., 2007). Similarly, malabsorption of electrolytes and nutrients, combined with hypersecretion of chloride and water, are the driving force of diarrhoeal disease in cryptosporidiosis (Argenzio et al., 1990; Goodgame et al.,1995; Huang and White, 2006). Together, these observations support the view that a combination of malabsorption and secretion of electrolytes are responsible for fluid accumulation in the intestinal lumen during infection with Giardia or with Cryptosporidium. Both parasites have a predilection for the small intestine, where most of their pathophysiological effects take place. These effects contribute to the production of watery diarrhoea seen in symptomatic patients.

Epithelial Malabsorption Results from Injury to the Brush Border Microvilli The overall malabsorptive effects of infection, as well as small intestinal disaccharidase impairments, suggest the occurrence of a diffuse mucosal injury. Consistent with this observation, epithelial injury during giardiasis or cryptosporidiosis is evidenced by diffuse shortening and/or loss of brush border microvilli (Argenzio et al., 1990; Buret et al., 1992; Goodgame et al., 1995; Müller and von Allmen, 2005; Huang and White, 2006). These microscopic alterations, even in the absence of

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villous atrophy, explain the overall malabsorption and maldigestion seen during infection. This pathophysiological process is also observed in a variety of other enteric disorders, including bacterial enteritis, chronic food anaphylaxis, Crohn’s disease and coeliac disease (Rubin et al., 1966; Dvorak, 1988; Buret et al., 1990, 1998; Curtis et al., 1990; Gunasekaran and Hassall, 1992; Savioli et al., 2006). Therefore, rather than representing an insult from direct exposure to parasitic products, brush border injury probably results from a common host-mediated event.

Brush Border Abnormalities are Mediated by Host Lymphocytes Host T lymphocytes play a central role in the host–parasite relationship during giardiasis and cryptosporidiosis. Epithelial brush border injury and disaccharidase deficiencies in giardiasis appear to be mediated by CD8+ T cells, while CD4+ T cell activation contributes to parasite clearance (Faubert, 2000; Eckmann, 2003; Scott et al., 2004) (Fig. 35.1). Consistent with these observations, microvillus brush border abnormalities and parasite clearance do not occur in hosts devoid of functional T lymphocytes (Faubert, 2000; Scott et al., 2000; Eckmann, 2003). The finding that athymic mice infected with Giardia do not exhibit microvillous injury and dysfunction despite the presence of live parasites again refutes the hypothesis that intestinal malfunction solely results from trophozoite attachment or parasite virulence factors. A recent study confirmed that increased numbers of intraepithelial lymphocytes are associated with the sodium/glucose malabsorption detected in Giardia-infected patients (Troeger et al., 2007). Similarly, increased numbers and activation of the CD8+ TCRgamma-delta T cell population have been reported in the intra-epithelial compartment during intestinal infestation with Cryptosporidium (Chai et al., 1999; Guk et al., 2003). Therefore, findings to date imply that during infection, activated T lymphocytes cause microvillous injury, which in turn causes the disaccharidase deficiencies and epithelial malabsorption contributing to diarrhoea. Consistent with these observations, increased cytolytic activity of intraepithelial CD8+ T cells has been implicated in the epithelial tissue damage observed during Crohn’s disease (Mueller et al., 1998; Nüssler et al., 2000). Findings from these studies have prompted investigations into the role of increased epithelial permeability as an initiating event during this immunopathogenic process.

Stimulated Epithelial Apoptosis Facilitates Translocation of Luminal Antigens Observations from models in vitro and in vivo have established that Giardia and Cryptosporidium parasites increase intestinal permeability (Dagci et al., 2002; Scott et al., 2002; Huang and White, 2006) (Fig. 35.2). Infection with Giardia muris in mice has been associated with elevated macromolecular uptake in the jejunum during the period of peak trophozoite colonization, but not during the parasite clearance phase (Hardin et al., 1997) (Fig. 35.2). Using impedance spectroscopy, recent observations from human patients with chronic giardiasis

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Fig. 35.1. Transmission electron micrographs (TEM) obtained at the same magnification, and illustrating the jejunal microvillous brush border of control mice (A), and of naïve non-infected mice that received (intravenously) mesenteric lymph node lymphocytes from Giardia muris-infected animals (B, C, D). Transfer of whole mesenteric lymph node lymphocyte populations (B) and of CD8+-enriched T cells (D), but not of CD4+ T cells (C) from infected mice significantly shortens brush border microvilli, consistent with a central role of CD8+ T cells in the loss of brush border surface area seen during the infection (modified from Scott et al., 2004).

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Fig. 35.3. Exposure of epithelial monolayers to Cryptosporidium andersoni significantly increases cell apoptosis in association with its disruptive effects on tight junctional zonula occludens-1. Values are expressed as (A) percentage (versus controls) of epithelial cells showing nuclear chromatin condensation after Hoechst staining, or (B) percentage (versus controls) of epithelial cells showing ZO-1 abnormalities after staining with a polyclonal rabbit anti-ZO-1 antibody (modified from Buret et al., 2003).

confirmed the loss of epithelial barrier function in the infected intestine (Troeger et al., 2007). Cryptosporidium is known to disrupt epithelial tight junctions, via mechanisms similar to those described for Giardia and other enteric microbes (Zhang et al., 2000; Buret et al., 2003; Huang and White, 2006). Again, increased intestinal permeability caused by Cryptosporidium can be very significant, although temporary (Zhang et al., 2000). Infection-associated loss of epithelial barrier function allows luminal antigens to activate host immune-dependent pathological pathways. Therefore, such events are of great clinical significance. Not surprisingly, intense research efforts are trying to identify the molecular events regulating epithelial tight junctional function in gastrointestinal health and in disease (Laukoetter et al., 2006; Shen et al., 2006). Giardia is known to disrupt epithelial F-actin and zonula occludens-1, as well as alpha-actinin and claudin proteins, all critical components of the sealing properties of tight junctions (Teoh et al., 2000; Buret et al., 2002b; Scott et al., 2002; Troeger et al., 2007) (Fig. 35.2). These structural alterations, as well as the resulting increase in transepithelial permeability, appear to be modulated at least in part by myosin light chain kinase and pro-apoptotic caspase-3 (Chin et al., 2002; Scott et al., 2002; Panaro et al., 2007) (Fig. 35.2). Epithelial barrier dysfunction in patients with chronic giardiasis is also associated with increased rates of enterocyte apoptosis (Troeger et al., 2007). Consistent with these observations, recent findings from microarray analyses on the effects of G. duodenalis on human CaCo2 cells found that the parasite–host interactions lead to a significant upregulation of genes implicated in the apoptotic cascade and the formation of reactive oxygen species (Roxström-Lindquist et al., 2005). Similarly, infection with Cryptosporidium significantly increases levels of epithelial apoptosis, and this phenomenon has also been implicated in disruptions of tight junctional integrity and disease pathogenesis (Chen et al., 1999; McCole et al., 2000; Buret et al., 2003) (Fig. 35.3).

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Together, these findings indicate that, in giardiasis and in cryptosporidiosis, heightened epithelial apoptosis is associated with a loss of intestinal barrier function, which in turn facilitates the activation of intra- or sub-epithelial immune cells, including those responsible for intestinal abnormalities.

Parasitic Products Cause Epithelial Apoptosis Strain-dependent activation of enterocyte apoptosis, as well as loss of epithelial barrier function induced by Giardia, may occur in the absence of any other cell type, and small intestinal permeability returns to baseline upon parasite clearance in murine giardiasis (Cevallos et al., 1995; Teoh et al., 2000; Chin et al., 2002; Scott et al., 2002; Panaro et al., 2007). Giardia virulence products that may instigate these cascades of events remain obscure. In addition to expressing surface glycoproteins able to induce fluid accumulation in the intestine, Giardia is known to contain and/or release a variety of potentially ‘toxic’ substances, such as proteinases and lectins that may be responsible for direct epithelial injury (Chen et al., 1995; Kaur et al., 2001; Sousa et al., 2001; Jimenez et al., 2004). Recent findings suggest that a 58 kDa Giardia ‘enterotoxin’ may induce chloride secretion in a model of murine giardiasis (Kaur et al., 2001; Shant et al., 2004). Whether or not such a product may be implicated in the secretory response seen in human patients (Troeger et al., 2007) needs to be clarified. Also, proteinases have long been recognized as important virulence factors in a variety of microbial pathogens, including Giardia (Hare et al., 1989; Parenti, 1989). The publication of the complete genome sequence of Cryptosporidium parvum also revealed several lineage-specific expansions for proteases (Abrahamsen et al., 2004). While some of these are known to facilitate biological processes such as Cryptosporidium oocyst excystation (Forney et al., 1996a), others have been shown to be of critical importance in host cell invasion by this parasite (Forney et al., 1996b). The specific contributions of parasitic proteases to pathogenesis remain incompletely understood. Proteinase-activated receptors (PAR) are members of a unique class of G-protein-coupled signalling receptors that can modulate enterocyte apoptosis and increase intestinal epithelial permeability in a caspase3-dependent fashion (Chin et al., 2003). Much remains to be learned of the ability of Giardia proteinases to activate host PARs in the gastrointestinal tract, and whether or not this mechanism may directly contribute to pathogenesis. Full characterization of the Giardia genome should facilitate the identification of putative Giardia ‘enterotoxin(s)’ (Morrison et al., 2008). Such advances may help identify novel pharmacological targets for the treatment of giardiasis and cryptosporidiosis.

Other Host–parasite Interactions Related to Epithelial Apoptosis The biological basis of Giardia- or Cryptosporidium-induced epithelial apoptosis remains incompletely understood. Some evidence points to a role for strainselective parasite products, at least in giardiasis (Chin et al., 2002). However,

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Fig. 35.4. Effects of Giardia duodenalis on cell death and epithelial sugar uptake in human colonic CaCo2 cells transfected with the sodium-glucose co-transporter-1 (SGLT-1). (A) in low-glucose media (5 mM), Giardia significantly increases apoptosis (i.e. levels of DNA fragmentation) compared with control. This increase is abolished in presence of high-glucose media (25 mM). This glucose-mediated cytoprotection of human intestinal epithelial cells against Giardia-induced apoptosis is abolished by phloridzin (phlor), which competitively binds to SGLT-1, hence confirming specificity of this phenomenon to the SGLT-1 transporter. n = 3/group. *P < 0.05 compared to controls, P < 0.05 compared to data from ‘high glucose, Giardia alone’. (B) confocal XZ-plane serial imaging illustrating the increased expression of SGLT-1 on the apical surface of epithelia cells exposed to Giardia, compared to control. Observations from these micrographs are consistent with a direct stimulation of SGLT-1 apical translocation upon exposure to the pro-apoptotic Giardia stimulus. Cell silhouettes are identified by actin staining using phalloidin-conjugated fluorescence. Representative images obtained from three individual experiments at the same magnification.

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recent findings have also established an important role for host factors in the regulation of microbially induced epithelial apoptosis. For example, Giardia can prevent the formation of epithelial nitric oxide, a compound known to inhibit giardial growth, by consuming local arginine, which effectively removes the substrate needed by enterocytes to produce nitric oxide (NO) (Eckmann et al., 2000). This mechanism may contribute to Giardia-induced enterocyte apoptosis, since arginine starvation in these cells is known to cause programmed cell death (Potoka et al., 2003). Other findings also described a novel biological process in which sodium-coupled-glucose transporter-1 (SGLT-1) activation may rescue enterocytes from Giardia-induced epithelial cell apoptosis by enhancing glucose uptake (Fig. 35.4). This host defence mechanism is characterized by increased apical expression and Vmax of the SGLT-1 transporter, and requires an intact microtubular network (Yu et al., 2008). As this effect also seems to occur upon exposure to bacterial LPS (Yu et al., 2005, 2006), more research needs to determine whether it may represent a generic feature of the intestinal response against parasitic and bacterial infection. Interactions between Cryptosporidium and host NO responses, as well as effects of SGLT-1 activation on the epithelial apoptosis induced by this parasite, have yet to be explored. Also, more research is needed to determine whether individual variations in these processes may, at least in part, explain the broad spectrum of symptoms associated with these infections. A better understanding of these cytoprotective mechanisms may help to establish a rational basis for the development of therapeutic interventions in various intestinal disorders where excessive epithelial apoptosis is central to pathogenesis.

Giardiasis and Cryptosporidiosis Facilitate Other Enteric Disorders An ever increasing number of reports support the hypothesis that host–microbial interactions underlie the pathogenesis of a variety of intestinal disorders, including inflammatory bowel disease (IBD). Indeed, infectious agents, including Salmonella or Campylobacter jejuni, may initiate and/or exacerbate IBD via mechanisms that remain unclear (Rodriguez et al., 2006). Consistent with a pathogenic consequence from this association, others have demonstrated that acute microbial infections may be directly responsible for relapse in IBD (Weber et al., 1992). As this aspect of IBD seems to be conserved across the global distribution of the disease, the possible pathogenic role for ubiquitous microbial products in IBD, including Giardia and Cryptosporidium, needs to be investigated. Similarly, toxigenicity in vitro of enteropathogens responsible for bacterial dysentery has been associated with post-infectious irritable bowel syndrome (Thornley et al., 2001; Marshall et al., 2006). A recent report has now demonstrated that Giardia may also elicit symptoms similar to IBS (D’Anchino et al., 2002). In view of the worldwide prevalence of IBS, mechanisms whereby enteric infections may contribute to this disease have become a topic of intense investigation. Finally, giardiasis and cryptosporidiosis have been associated with an increased incidence of allergic disease via mechanisms that remain incompletely understood (Clyne and Eliopoulos, 1989; Di Prisco et al., 1993). Giardia is known to cause an increase in intestinal permeability and macromolecular

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uptake, which in turn induce hyperplasia of mucosal and connective tissue mast cells, the effector cells involved in anaphylaxis (Hardin et al., 1997). The mechanisms of how infection may exacerbate a clinically silent IBD and or initiate disease, including IBS and allergy, warrant further investigation. That microbially induced loss of intestinal barrier function represents a central feature in these disorders may well become a promising working hypothesis for such research.

Summary In summary, the mechanisms by which Giardia or Cryptosporidium cause diarrhoea are similar and include a combination of factors, including parasiteinduced chloride hypersecretion, epithelial apoptosis, and loss of barrier function leading to lymphocyte-mediated reduction of absorptive surface area, which is ultimately responsible for malabsorption of nutrients, sodium and water. Data available to date indicate that some of these processes are strain-dependent. The pathogenic role of parasite products such as surface glycoproteins, lectins, proteinases or other types of ‘enterotoxins’ still requires further clarification. Host enterocytes have evolved elaborate innate responses, which may also modulate these pathophysiological pathways. These include the production of antimicrobial products such as nitric oxide, as well as a recently discovered anti-apoptotic cell rescue mechanism involving the activation of SGLT-1-mediated glucose absorption. More research is needed to assess whether individual variations of these innate responses in the infected host, beyond the more traditional genetic factors responsible for immunodeficiencies, may explain the wide clinical presentation of giardiasis and cryptosporidiosis.

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Interferon-gamma (IFN-g) in Immunological Control of Cryptosporidial Infection

N. CHOUDHRY, M. BAJAJ-ELLIOTT AND V. MCDONALD University of London, UK

Abstract Immunological control of cryptosporidial infection is dependent on a cell-mediated immune response involving CD4+ T cells. These cells produce interferon-gamma (IFN-γ), and a deficiency in the activity of this cytokine is associated with an inability to clear the infection. Enterocyte cell lines treated with IFN-γ have been shown to have a degree of resistance to Cryptosporidium parvum infection. However, new studies suggest that the parasite may be able to counteract IFN-γ-mediated activation of enterocytes. This effect may be an important means by which C. parvum subvert immunological mechanisms generated to kill the parasite.

Introduction Once a microbial pathogen finds its developmental niche in a host, its primary goal is to survive long enough to reproduce and transmit progeny to a new host. The efficiency of the host immune response is potentially a major obstacle to achieving this aim. Under laboratory conditions the pattern of Cryptosporidium reproduction in otherwise healthy susceptible humans or animals is typically acute, presumably reflecting the ability of these hosts to establish effective immunological protection. The immune responses that develop to counteract cryptosporidial infection have become reasonably well characterized for one species in particular, C. parvum, although some important details remain to be established. Comparing observations with different parasite species suggests that the host responses leading to suppression of infection are broadly similar. Whether cross-immunity between species exists, however, is unclear. A cell-mediated type of acquired immune response involving CD4+ T lymphocytes appears to be necessary for the elimination of cryptosporidia. The cytokine interferon-gamma (IFN-γ) that is produced by these cells plays an 442

© CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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important part in driving the development of cell-mediated immune activity, including the effector arm of this immune response that activates antimicrobial killing mechanisms. Most investigations of the role of IFN-γ in the control of Cryptosporidium infection implicate its involvement in establishing immunity and, in addition, the cytokine has been shown to directly inhibit parasite replication in host cells Despite the capacity of the host to generate strong protective immune responses, the potentially pathogenic intestinal species of Cryptosporidium, as well as non-pathogenic gastric species, are clearly able to propagate. It is possible that, like other protozoan parasites, cryptosporidia have evolved ways of counteracting key protective host immunological responses in order to maximize parasite reproduction. Immuno-evasion by Cryptosporidium is a topic that has not been thoroughly researched. New evidence, however, suggests that C. parvum may improve its chances of survival by interfering with IFN-γ-mediated activation of enterocytes.

The Role of Lymphocytes and Cytokines in Immune Responses to Infections The adaptive immune response elicited by T (thymus-derived) and B (bursa or bone marrow-derived) lymphocytes that require recognition of specific antigens is essential for the elimination of virulent infectious microorganisms. T cells may be subdivided into two major subsets identified by the expression of either CD4 or CD8 molecules on the surface. CD4+ T cells usually initiate the adaptive immune response and influence the form it takes. CD8+ T cells can be important in the control of intracellular infections, as they recognize and kill infected cells. B cells differentiate into plasma cells that produce antibodies and the class of antibody expressed is determined in part by signals from CD4+ T cells (Kalia et al., 2006). Cytokines are small proteins that are important in the ontogeny of lymphocytes and other cells of the immune system. They also play a pivotal role in the establishment and regulation of different types of immune responses. They are produced not only by immune cells such as lymphocytes or macrophages but also by other cell types including fibroblasts and epithelial cells. There are many different cytokines and their action is dependent on ligation of receptors on the surface of cells, that are usually specific for individual cytokines. Following infection by viral, bacterial or protozoan pathogens, immunological control is often established under the direction of CD4+ T helper (referred to as Th) cells. These T cells may differentiate in a polarized fashion to develop either a cell-mediated (Th1) or humoral/allergic (Th2) type of response. In general, Th1 responses are normally required for the elimination of pathogens that invade cells, including intracellular protozoan parasites, whereas Th2 responses control extracellular infection (Romagnani, 2006). The Th1 or Th2 response is characterized by the spectrum of cytokines produced by the CD4+ T cells. CD4+ T cells are activated by processed microbial antigens presented to them on MHC class II molecules by dendritic cells or macrophages, often referred to as antigen-presenting cells (APCs). Other signals are required for activation, including cytokines from the

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APCs. The types of cytokines produced by APCs may be influenced by conserved signature molecules expressed by the infecting microorganism. In the case of an intracellular infection, dendritic cells and macrophages produce interleukin (IL)-12, which plays an important part in the establishment of the Th1 response (Seder and Ahmed, 2003). Cytotoxic CD8+ T cells often also play an important part in a Th1 response to infection and CD4+ T cells may help in the activation of these cells. The cytokine expressed by T cells that embodies Th1 development is IFN-γ (Romagnani, 2006), and its production is highly upregulated by IL-12. The functions of IFN-γ include promoting the differentiation of T cells to the Th1 pathway, inhibiting the establishment of the Th2 pathway that may prevent elimination of an intracellular pathogen, and stimulating production of other proinflammatory cytokines such as TNF-α and IL-1 by monocytes and other cells (Romagnani, 2006). In addition, IFN-γ, acting alone or in concert with other cytokines such as TNF-α, activates antimicrobial killing mechanisms, the nature of which may depend on the cell type. In macrophages, for example, a major mechanism for parasite killing is the production of highly toxic nitric oxide through the enzyme inducible nitric oxide synthase (iNOS) (Oswald et al., 1994). Alternatively, microbes may be killed indirectly by deprivation of molecules essential for microbial metabolism such as Fe ions or the essential amino acid l-tryptophan (Rottenberg et al., 2002). IFN-γ kills Toxoplasma gondii in fibroblasts by inducing the enzyme indoleamine 2,3-dioxygenase, which catabolizes intracellular tryptophan required by the parasite for development (Pfefferkorn et al., 1986). In addition to T cells, the natural killer (NK) cell is another important source of IFN-γ. NK cells form part of the T-cell-independent or innate immune system that responds more rapidly to infection than T-cell-driven immunity. NK cells have been shown to be important in early control of viral as well as intracellular bacterial and protozoan infections through IFN-γ production and, in certain cases, cytolytic activity against infected cells (Lodoen and Lanier, 2006).

Adaptive (T- and B-cell-dependent) Immune Responses to Cryptosporidial Infection Much of the body of information on immune responses to cryptosporidia has been obtained from experimental investigations with murine infection models. Findings from large animal (mainly bovine) and human studies are usually compatible with those involving mice. Microbial pathogens reproducing in the gastrointestinal tract inhabit the largest organ of the immune system. The lamina propria is rich in both T and B cells and other immune cells, while the epithelium is occupied by lymphocytes, particularly T cells. T cells are essential for establishing sterile immunity to C. parvum or C. muris infection as athymic nu/nu mice that lack these cells were unable to clear infections (Heine et al., 1984; McDonald et al., 1992). Similarly, SCID mice that have no T or B cells failed to control parasite development, but injection of these animals with functional T cells allowed them to develop control of infection (Mead et al., 1991; Chen et al., 1993a). CD4+ T cells appear to be more

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important than CD8+ T cells in host resistance. Mutant mice lacking CD4+ T cells were unable to eliminate C. parvum infection, whereas other mutant animals deficient in CD8+ T cells were as capable of controlling infection as wild-type control mice (Aguirre et al., 1994). The same conclusions were obtained after measuring infections in wild-type mice depleted of CD4+ or CD8+ cells using specific antibodies: i.e. a deficiency in CD4+ T cells prolonged infection while loss of CD8+ T cells had no major effect on the course of infection (Ungar et al., 1991). Specific immunity directed at C. muris was manifest in SCID mice after the transfer of lymphoid cells from previously infected wild-type mice; depletion of CD4+ T cells from the donor cell population abrogated this protection but depletion of CD8+ T cells reduced the protective effect only moderately (McDonald et al., 1994). However, a cytokine-dependent resistance to C. parvum infection that was detected very early during infection of mice appeared to be associated with CD8+ T cells (Leav et al., 2005). In bovine calves, the establishment of immunity to C. parvum infection was associated with increased traffic of both CD4+ and CD8+ T cells into the intestinal lamina propria (Abrahamsen et al., 1997), implying that both T cell subpopulations might have a protective role. The susceptibility to cryptosporidial infection in AIDS patients increased with declining numbers of CD4+ cells (Blanshard et al., 1992; Flanigan et al., 1992), while recovery of this cell population in the intestine after antiretroviral therapy coincided with clearance of cryptosporidial parasites (Schmidt et al., 2001). T cells express one of two types of T cell antigen receptor (TCR): peptide antigens are normally recognized by T cells with TCRαβ while either peptides or other types of molecules may be recognized by cells with TCRγδ. The latter cells are common within epithelium and may be important for immunoregulation in the skin and mucosal surfaces (Hayday and Tigelaar, 2003). However, transgenic mice that fail to express the TCRαβ receptor were observed to be highly susceptible to C. parvum infection, whereas mice deficient in TCRγδ could readily recover. This indicates that TCRαβ is more important for T cell recognition of cryptosporidial antigens (Waters and Harp, 1996). Different host species, including humans and cattle, develop Cryptosporidium-specific antibodies in serum and in mucosal secretions following cryptosporidial infection (Ungar et al., 1986; Peeters et al., 1992).The titres rise to a peak during the recovery phase of infection, then decline, suggesting that antibodies might be important in the clearance of parasites. However, the patterns of infection in mice depleted of B cells following administration of anti-µ chain antibodies, or in transgenic mice that lack B cells, were no different from those in control mice (Takhi-Kilani et al., 1990; Chen et al., 2003). These results indicated that B cells and antibodies were not essential in the development of host resistance to Cryptosporidium.

IFN-g and Other Cytokines in Immunity There is strong evidence that IFN-γ activity is essential for the control and elimination of cryptosporidial infection. Treatment of mice infected with C. parvum or

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C. muris with anti-IFN-γ-neutralizing antibodies caused a significant increase in levels of parasite reproduction (Ungar et al., 1991; McDonald et al., 1992). Transgenic animals that lack the IFN-γ gene derived from two mouse strains, C57BL/6 and BALB/c, had increased susceptibility to C. parvum infection (Theodos, 1998; Mead and You, 1998). Mice of the former strain died within a few days as a result of the infection but those of the latter strain recovered, hence indicating that the extent of dependence on IFN-γ for resistance may depend on the genetic make-up of the host. As noted previously, recovery from cryptosporidial infection occurs only in the presence of CD4+ T cells and the development of immunity to C. parvum in mice has also been associated with the production of IFN-γ by these cells (Harp et al., 1994). In immunocompetent humans, recovery from cryptosporidial infection was uniformly associated with production of IFN-γ by peripheral blood T cells when cultured with parasite antigen, whereas T cells from HIV-infected patients, who were more susceptible to infection, were less likely to produce the cytokine (Gomez Morales et al., 1999). IFN-γ produced by NK cells may also play a major role in the T-cell-independent innate immune response to C. parvum infection. SCID mice, which have normal innate immune functions including NK cell activity, develop chronic C. parvum infection. They eventually succumb to infection, but for a period display a capacity to prevent overwhelming parasite reproduction. The protective response of these animals is at least partly dependent on IFN-γ, since treatment with anti-IFN-γ-neutralizing antibodies resulted in an exacerbation of infection (Chen et al., 1993b; McDonald and Bancroft, 1994). In addition, infection spread across the epithelium more rapidly in SCID mice that lacked the IFN-γ gene compared with control SCID mice (Hayward et al., 2000). With in vitro experiments it was shown that spleen cells from SCID mice produced IFN-γ when exposed to sporozoite antigen but the cytokine was not produced if NK cells had been depleted (McDonald et al., 2000). This suggests that NK cells are the most likely source of IFN-γ in this T-cell-independent mechanism of immunity, although it has yet to be demonstrated that intestinal NK cells are the main source of the cytokine during infection. Investigations have indicated that IL-12 expression is necessary for the early control of C. parvum infection in both adaptive and innate immunity. Adult immunocompetent mice are normally refractory to infection with this parasite but transgenic mice lacking the IL-12 gene developed a heavy acute infection (Ehigiator et al., 2005). However, the related cytokine IL-23, which promotes a type of inflammatory response associated, for example, with certain autoimmune diseases, appeared to be less essential for the control of infection. Both neonatal wild-type and SCID mice treated with recombinant IL-12 prior to inoculation of oocysts were highly resistant to infection, while treatment with antiIL-12 neutralizing antibodies enhanced the infection level (Urban et al., 1996). The protective effect of IL-12 correlated with an increase in expression of IFN-γ. IL-12, therefore, promotes both the innate immune response (observations with SCID mice) and the adaptive immune response to infection. The involvement of both IL-12 and IFN-γ in the development of early resistance to C. parvum infection implies that a Th1-cell-mediated immune response is mainly responsible for host control of infection.

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Effect of IFN-γ on Parasite Development in Enterocytes An important function of IFN-γ in the effector stage of cell-mediated immunity against infection is to upregulate antimicrobial killing mechanisms by infected cells or other cells in the environment. As C. parvum readily infects human and animal enterocyte cell lines for at least a few days, it has been possible to study the effects of IFN-γ on parasite replication. It was shown that treatment of human HT-29 and Caco-2 cells with IFN-γ for 24 h before infection could significantly inhibit C. parvum reproduction in a dose-dependent manner (Pollok et al., 2001). Inhibition required the presence of the IFN-γ receptor on the host cell, as the cytokine did not influence parasite development in a cell line which did not express the receptor. Two antimicrobial mechanisms were identified. First, cytokine treatment made cells more resistant to invasion by sporozoites. Second, there was evidence of cellular deprivation of Fe required for parasite development, since the addition of exogenous Fe reversed the inhibitory effect of IFN-γ. Two other important mechanisms of killing that can be induced by the cytokine appeared not to be involved. The enzyme iNOS, which produces nitric oxide from arginine, is induced in macrophages, but the use of enzyme inhibitors suggested that nitric oxide was not important in IFN-γ-mediated inhibition of C. parvum development in enterocytes. Another enzyme upregulated by IFN-γ, indoleamine 2,3-dioxygenase (IDO), catabolizes the essential amino acid tryptophan required for microbial metabolism (Pfefferkorn et al., 1986). A decrease in IFN-γ killing activity in the presence of excess tryptophan is indicative that IDO is important in parasite killing. In the case of C. parvum infection, increasing the concentration of tryptophan had no effect on IFN-γ-mediated killing, suggesting that IDO was not involved (Pollok et al., 2001). However, we observed that the IDO mechanism of killing could operate in the same types of cell, as it was required for the inactivation of another parasite that infects enterocytes, the microsporidian Encephalitozoon intestinalis, an important opportunistic pathogen in AIDS-related diarrhoea (Choudhry et al., 2008). This suggested the possibility that C. parvum might be able to directly inhibit the upregulation of IDO in enterocytes by IFN-γ. Further investigation showed that C. parvum development in enterocytes reversed the induction of IDO mRNA by IFN-γ in enterocytes and that IDO enzymic activity was also significantly reduced following infection (Choudhry et al., 2008). Another molecule that is expressed by enterocytes and is upregulated by IFN-γ is MHC class II. Activation of CD4+ T cells requires interaction of the TCR with peptide antigen fragments presented by MHC class II molecules on APCs such as dendritic cells. The physiological significance of MHC class II expression by enterocytes is not entirely clear, but in vitro studies suggest that under inflammatory conditions, such as Crohn’s disease, enterocytes can activate CD4+ T cells in a MHC class II-dependent manner (Dotan et al., 2007). In studies with C. parvum, IFN-γ-mediated expression of MHC class II by enterocytes was abrogated by infection (N. Choudhry et al., unpublished). These findings, therefore, suggest that although IFN-γ activity is required for the control of C. parvum reproduction, the parasite may have evolved a

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mechanism that comprehensively inhibits IFN-γ activation of enterocytes. This may be an important strategy to facilitate the survival of the parasite in an immunologically hostile environment. However, cytokines produced by other cells and whose expression may be induced by IFN-γ, including TNF-α or IL-1, may also inhibit parasite development in enterocytes (Pollok et al., 2001). The effect that C. parvum has on the action of these cytokines on enterocytes remains to be determined.

References Abrahamsen, M.S., Lancto, C.A., Walcheck, B., Layton, W. and Jutila, M.A. (1997) Localization of alpha/beta and gamma/delta T lymphocytes in Cryptosporidium parvum-infected tissues in naive and immune calves. Infection and Immunity 65, 2428–2433. Aguirre, S.A., Mason, P.H. and Perryman, L.E. (1994) Susceptibility of major histocompatibility (MHC) class I- and class II-deficient mice to Cryptosporidium parvum infection. Infection and Immunity 62, 697–699. Blanshard, C., Jackson, A.M., Shanson, D.C., Francis, N. and Gazzard B.G. (1992) Cryptosporidiosis in HIV-seropositive patients. Quarterly Journal of Medicine 85, 813–823. Chen, W., Harp, J.A. and Harmsen, A.G. (1993a) Requirements for CD4+ cells and gamma interferon in resolution of established Cryptosporidium parvum infection in mice. Infection and Immunity 61, 3928–3932. Chen, W., Harp, J.A., Harmsen, A.G. and Havell, E.A. (1993b) Gamma interferon functions in resistance to Cryptosporidium parvum infection in severe combined immunodeficient mice. Infection and Immunity 61, 3548–3551. Chen, W., Harp, J.A. and Harmsen, A.G. (2003) Cryptosporidium parvum infection in gene-targeted B cell-deficient mice. Journal of Parasitology 89, 391–393. Choudhry, N., Korbel, D.S., Zaalouk, T.K., Blanshard, C., Bajaj-Elliott, M. and McDonald, V. (2008) Interferon-γ-mediated activation of enterocytes in immunological control of Encephalitozoon intestinalis infection. Parasite Immunology 30, in press. Dotan, I., Allez, M., Nakazawa, A., Brimnes, J., Schulder-Katz, M. and Mayer, L.F. (2007) Intestinal epithelial cells from inflammatory bowel disease patients preferentially stimulate CD4+ T cells to proliferate and secrete interferon-gamma. American Journal of Physiology: Gastrointestinal and Liver Physiology 292, G1630–G1640. Ehigiator, H.N., Romagnoli, P., Borgelt, K., Fernandez, M., McNair, N., Secor, W.E. and Mead, J.R. (2005) Mucosal cytokine and antigen-specific responses to Cryptosporidium parvum in IL-12p40 KO mice. Parasite Immunology 27, 17–28. Flanigan, T., Whalen, C., Turner, J., Soave, R., Toerner, J., Havlir, D. and Kotler, D. (1992) Cryptosporidium infection and CD4 counts. Annals of Internal Medicine 116, 840–842. Gomez Morales, M.A., La Rosa, G., Ludovisi, A., Onori, A.M. and Pozio, E. (1999). Cytokine profile induced by Cryptosporidium antigen in peripheral blood mononuclear cells from immunocompetent and immunosuppressed persons with cryptosporidiosis. Journal of Infectious Diseases 179, 967–973. Harp, J.A., Whitmire, W.M. and Sacco, R. (1994) In vitro proliferation and production of gamma interferon by murine CD4+ cells in response to Cryptosporidium parvum antigen. Journal of Parasitology 80, 67–72. Hayday, A. and Tigelaar, R. (2003) Immunoregulation in the tissues by gamma delta T cells. Nature Reviews in Immunology 3, 233–242.

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Hayward, A.R., Chmura, K. and Cosyns, M. (2000) Interferon-gamma is required for innate immunity to Cryptosporidium parvum in mice. Journal of Infectious Diseases 182, 1001–1004. Heine, J., Moon, H.W. and Woodmansee, D.B. (1984) Persistent Cryptosporidium infection in congenitally athymic (nude) mice. Infection and Immunity 43, 856–859. Kalia, V., Sarkar, S., Gourley, T.S., Rouse, B.T. and Ahmed, R. (2006) Differentiation of memory B and T cells. Current Opinion in Immunology 18, 255–264. Leav, B.A., Yoshida, M., Rogers, K., Cohen, S., Godiwala, N., Blumberg, R.S. and Ward, H. (2005) An early intestinal mucosal source of gamma interferon is associated with resistance to and control of Cryptosporidium parvum infection in mice. Infection and Immunity 73, 8425–8428. Lodoen, M.B. and Lanier, L.L. (2006) Natural killer cells as an initial defense against pathogens. Current Opinion in Immunology 18, 391–398. McDonald, V. and Bancroft, G.J. (1994) Mechanisms of innate and acquired resistance to Cryptosporidium parvum infection in SCID mice. Parasite Immunology 16, 315–320. McDonald, V., Deer, R., Uni, S., Iseki, M. and Bancroft, G.J. (1992) Immune responses to Cryptosporidium muris and Cryptosporidium parvum in adult immunocompetent or immunocompromised (nude and SCID) mice. Infection and Immunity 60, 3325–3331. McDonald, V., Robinson, H.A., Kelly, J.P. and Bancroft, G.J. (1994) Cryptosporidium muris in adult mice, adoptive transfer of immunity and protective roles of CD4 versus CD8 cells. Infection and Immunity 62, 2289–2294. McDonald, V., Smith, R., Robinson, H. and Bancroft, G. (2000) Host immune responses against Cryptosporidium. Contributions in Microbiology 6, 75–91. Mead, J.R. and You, X. (1998) Susceptibility to Cryptosporidium parvum infection in two strains of gamma interferon knockout mice. Journal of Parasitology 84, 1045–1048. Mead, J.R., Arrowood, M.J., Healey, M.C. and Sidwell, R.W. (1991) Cryptosporidial infections in SCID mice reconstituted with human or murine lymphocytes. Journal of Protozoology 38, 59S–61S. Oswald, I.P., Wynn, T.A., Sher, A. and James, S.L. (1994) NO as an effector molecule of parasite killing: modulation of its synthesis by cytokines. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology 108, 11–18. Peeters, J.E., Villacorta, I., Vanopdenbosch, E., Vandergheynst, D., Naciri, M., Ares-Maras, E. and Yvorre, P. (1992) Cryptosporidium parvum in calves: kinetics and immunoblot analysis of specific serum and local antibody responses (immunoglobulin A [IgA], IgG, and IgM) after natural and experimental infections. Infection and Immunity 60, 2309–2316. Pfefferkorn, E.R., Eckel, M. and Rebhun, S. (1986) Interferon-gamma suppresses the growth of Toxoplasma gondii in human fibroblasts through starvation for tryptophan. Molecular and Biochemical Parasitology 20, 215–224. Pollok, R.C., Farthing, M.J., Bajaj-Elliott, M., Sanderson, I.R. and McDonald, V. (2001) Interferon gamma induces enterocyte resistance against infection by the intracellular pathogen Cryptosporidium parvum. Gastroenterology 120, 99–107. Romagnani, S. (2006) Regulation of the T cell response. Clinical Experimental Allergy 36, 1357–1366. Rottenberg, M.E., Gigliotti-Rothfuchs, A. and Wigzell, H. (2002) The role of IFN-γ in the outcome of chlamidial infection. Current Opinion in Immunology 14, 444–451. Schmidt, W., Wahnschaffe, U., Schafer, M., Zippel, T., Arvand, M., Meyerhans, A., Riecken, E.O. and Ullrich, R. (2001) Rapid increase of mucosal CD4 T cells followed

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Immune Response to Giardia Infection: Lessons from Animal Models

S.M. SINGER AND J. KAMDA Georgetown University, Washington DC, USA

Abstract Infection with Giardia intestinalis is one of the most common parasitic infections among humans. In many parts of the world ~20% of the population is infected with Giardia at any given time, and most children show evidence of having been infected by the age of 3 years. The majority of these cases are controlled within 2–3 weeks by host immune responses. However, chronic infections can develop in otherwise healthy patients. Variability also exists in the clinical presentation of Giardia infection. Some individuals experience severe cramps, nausea and malabsorptive diarrhoea while infected, while others have no obvious symptoms. Nutrient malabsorption and changes in enterocyte activity may still occur in the absence of overt symptoms. Understanding the roles of host immune responses and parasite genetics in this variability in duration and presentation of Giardia infection are thus major challenges to our ability to deal with this disease. This chapter focuses on the role of immune responses in contributing to both parasite elimination and disease pathogenesis in this infection.

Health Burden and Management Giardia is one of the most prevalent causes of diarrhoeal disease in humans. The World Health Organization (WHO) estimates that of 4 billion diarrhoea cases worldwide yearly (Farthing, 2000), 1 billion are associated with Giardia, although a cause–effect relationship has not been established. It is the most commonly identified intestinal parasite globally and is particularly prevalent in children in developing countries (Bryan et al., 1994). The developed world is not spared either, including the USA (Kappus et al., 1994), where the Center for Disease Control (CDC) estimates 2.5 million cases of giardiasis yearly (Furness et al., 2000). The prevalence of parasites in stool specimens submitted for ova and parasite examination is 2–5% in industrialized countries, and up to 40% in some developing countries (Ortega and Adam, 1997). The parasite is transmitted by © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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cysts that are extremely resistant to the environment and to normal chemical treatment of drinking water, As few as ten cysts are able to initiate infections and the parasite exhibits a wide host range. Together these account for the easy transmissibility and thus the worldwide distribution of Giardia. Most Giardia infections result in no symptoms to the host. One study determined that 60–80% of infected children in daycare centres and their household contacts had asymptomatic giardiasis (Keystone et al., 1978). Indeed, the parasite was only added by WHO to its list of pathogens in 1981, three centuries after its discovery (Faubert, 2000). The acute phase of symptomatic disease usually resolves spontaneously within 2–3 weeks, suggesting an effective immune response against Giardia infection. However, a small percentage of patients progress to chronic giardiasis, even with a healthy immune system (Faubert, 2000). Apart from diarrhoea, symptomatic giardiasis presents with headaches, low-grade fever, abdominal cramps, nausea and vomiting, flatulence, pale and fatty and foul-smelling stools, and a malabsorption syndrome, severe forms of which result in weight loss and interference with normal mental and physical development in children. Malabsorption due to giardiasis has been linked to intestinal villus and microvillus shortening, reduction in intestinal disaccharidases especially lactase, and physical interference with absorption in cases of high parasite burden (Buret, 2007). Human mortality from giardiasis is rare but has been described, particularly in children (USEPA, 1999). Animals, both wild and domestic, are especially vulnerable to the disease, given the easy contact with contaminated food and water. Mortality is much higher in animals. The highest incidence of Giardia was found in dogs. Other animals with very high incidences include cattle, horse, pigs, sheep and beavers (USEPA, 1999).

Lessons from Animal Models Our knowledge of the role of host immunity in human Giardia infection is limited and a great deal has been learned by using animal models of Giardia infection. These models include infections in adult mice with the rodent-specific species G. muris (Roberts-Thomson et al., 1976), as well as with the human G. intestinalis isolate GS(M) (Byrd et al., 1994). GS(M) has been used to successfully infect humans, mice and gerbils in experimental contexts (Aggarwal and Nash, 1987; Nash et al., 1987; Byrd et al., 1994). Other models include infection of neonatal mice (Hill et al., 1983) and infection of gerbils (Belosevic et al., 1983). While neonatal mice are susceptible to infection with most isolates of G. intestinalis, they lack mature immune responses, thus reducing their usefulness. Gerbils are also susceptible to infection with most human isolates, but this model suffers from a lack of available reagents compared with the use of mice. The GS(M) model has been criticized for failing to induce overt disease in mice and for not being the natural host for this parasite. As mentioned above, however, G. intestinalis commonly does not induce overt disease in humans and, as described in other chapters in this volume, G. intestinalis clearly does have a zoonotic lifestyle. None the less, it is important always to remember that none of the results from these models should be extrapolated directly to human infection

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without attempting to confirm them. Rather, these models all provide ways to generate hypotheses that can then be addressed in patient populations.

Humoral Immunity Infection with Giardia in humans and adult animals leads to a robust production of antibodies, particularly IgA, which is secreted into the intestinal lumen. Antibodies have some effect on parasite viability in vitro (Nash et al., 1990; Heyworth, 1992), but the ability of the parasite to undergo antigenic variation may reduce the effectiveness of antibodies in vivo, especially at the early stages of infection (Nash, 1997). While chronic infections do occur in otherwise healthy patients, they are particularly common among individuals with primary immunodeficiencies such as common variable immunodeficiency (CVID) and X-linked agammaglobulinelia (XLA) (Washington et al., 1996). Both of these syndromes are marked by defects in the production of antibodies, indicating a role for antibody in the control of chronic infection. The primary defect in XLA is mutation in the gene Bruton’s tyrosine kinase (btk). Btk is expressed in B cells, where it contributes to signalling through the B cell antigen receptor complex. Btk is expressed in other types of immune cells, such as mast cells, where it also contributes to signalling and activation (Hata et al., 1998; Horwood et al., 2006). Some cases of CVID have been linked to mutations in the gene TAC1, which is involved in signalling B cells in response to the TNF superfamily members BAFF and APRIL (Castigli et al., 2005). Again, this pathway is not restricted to B cells, and other immune responses may also be affected in this syndrome. Indeed, defects in dendritic cell and T cell function are quite common in CVID patients (Di Renzo et al., 2004; Cunningham-Rundles and Radigan, 2005). Thus, these observations cannot definitively show a role for antibodies in the control of giardiasis. Evidence from mouse models has helped to clarify the role of antibodies in fighting Giardia. Initial studies were performed using G. muris infections in adult mice. To test the role of antibodies, mice carrying a mutation in btk, the XID mouse, or wild-type mice treated from birth with anti-IgM antisera to deplete B cells were used (Snider et al., 1985, 1988). Both resulted in chronic G. muris infections, although the levels of anti-parasite IgA were actually greater in the XID mice than in wild-type controls (Snider et al., 1988). More recently, Stäger et al. (1998) showed that G. intestinalis GS(M) infected neonatal mice which nursed from infected dams, presumably receiving IgA passively, exhibited rapid selection for antigenic variants, whereas antigenic variation was not observed in neonates nursing from naïve dams. Thus, while antibody appeared to exert selective pressure on parasites, transfer of antibody alone was unable to eliminate the infection. In contrast, infection of B-cell-deficient adult µMT mice with G. intestinalis GS(M) showed that parasites were eliminated from mice with kinetics similar to that observed in wild-type controls (Singer and Nash, 2000). µMT mice lack B cells due to deletion of the µ exons of the Ig heavy chain locus, but have been reported to express IgA in some circumstances (Macpherson et al., 2001). However, we were unable to detect anti-parasite IgA in infected µMT

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mice using indirect immunofluorescence, whereas these antibodies were readily detected in wild-type controls (P. Zhou and S.M. Singer, unpublished). We also examined IgA responses in IL-6-deficient mice infected with GS(M), since these mice fail to control infections rapidly (Bienz et al., 2003; Zhou et al., 2003). By day 10 post-infection these mice began to produce anti-parasite IgA that reacted with a subset of parasites present during in vitro cultures. High parasite numbers nevertheless persisted within the small intestine until day 60 post-infection (Zhou et al., 2003). Interestingly, at this time, the IgA present in intestinal fluid now reacted with all the parasites present in the same in vitro cultures, indicating recognition of new epitopes on the parasite. Again, this is consistent with the notion that antigenic variation precludes antibodies from effectively eliminating G. intestinalis infections until the antibody response is able to recognize either epitopes expressed by all parasites in the population or all of the different variant-specific surface proteins (VSPs) encoded in the parasite’s genome. In contrast, infection with G. muris of µMT mice or mice lacking IgA due to deletion of the α exon found that parasites persisted in both strains at higher levels for longer than in wild-type mice (Langford et al., 2002). While a VSP-like gene has been identified in G. muris (Ropolo et al., 2005), it is unclear whether these parasites undergo antigenic variation similar to what has been described in G. intestinalis. Moreover, Eckmann and colleagues recently showed that mice lacking the polyIg receptor, the molecule responsible for transcytosis of IgM and IgA across the epithelial barrier and into the intestinal lumen, exhibited enhanced sensitivity to G. muris infections, but less of an effect when infected with GS(M) (Davids et al., 2006). Together, these data suggest that GS(M) is sensitive to killing by mechanisms present in mice to which G. muris is resistant. Thus, antibodies are both necessary and sufficient for control of G. muris infections. In contrast, antibodies are not necessary for the control of the early phase of G. intestinalis infections, although they may play an important role in combating chronic infection.

Cellular Immunity Very little is known about the role of cellular responses in the control of Giardia infections in humans. Peripheral blood leucocytes (PBL) from uninfected individuals have been shown to proliferate and make interferon-γ in response to parasite extracts (Ebert, 1999). While anecdotal evidence suggests that Giardia is common in HIV-infected populations, most studies now indicate that low CD4 counts in these patients do not make them more susceptible to giardiasis. However, these studies involved very small numbers of patients and IFN-γ responses occurred in PBL from naïve individuals. Athymic nude mice develop chronic infections with both G. muris and G. intestinalis, as do mice treated with antiCD4 antibodies (Roberts-Thomson and Mitchell, 1978; Heyworth et al., 1987; Singer and Nash, 2000). Using the GS(M) model, we also found that while TCRβ gene deletion produced the same phenotype as nude mice, deletion of the TCRδ locus had no effect on parasite elimination (Singer and Nash, 2000). Consistent with the role for CD4+ T cells, the deletion of β2-microglobulin also had no effect

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on infection (Singer and Nash, 2000). These studies indicate that MHC class II restricted, CD4+, αβ-TCR-expressing T cells are important, but do not indicate what their role is. Indeed, it has been suggested that it may be as simple as providing help for B cells so that they can produce IgA (Heyworth et al., 1987). However, anti-CD4 depletion in B-cell-deficient mice prolonged GS(M) infection, indicating a clear role for T cells outside of antibody production in this system (Singer and Nash, 2000). Direct measurement of T cell responses in mice has been limited. Restimulation of mesenteric lymph node (MLN) cells from G. muris-infected mice produced little in the way of cytokines (Venkatesan et al., 1996). Stimulation with the mitogen ConA revealed production of both IFN-γ and IL-5 by MLN cells from C57BL/10 mice, while BALB/c mice produced only IL-5. Furthermore, Anti-IFN-γ treatment exacerbated G. muris infections in C57BL/10 but not BALB/c mice, indicating a potential protective role for this cytokine (Venkatesan et al., 1996). In contrast, IFN-γ-deficient mice on a C57BL/6 background were able to eliminate GS(M) infections as well as wild-type mice (Singer and Nash, 2000). Mice deficient in IL-4, IL-4 receptor and the signal transducer and activator of transcription (STAT) 6 molecule also exhibited rapid elimination of GS(M) (Singer and Nash, 2000). As mentioned previously, mice deficient in the cytokine IL-6 have a significant defect in eliminating G. intestinalis infections, although they produce normal anti-parasite IgA responses. It remains unclear, however, which cells produce the relevant IL-6 and what the exact defect is in these mice. One study suggests that mast cells may contribute to IL-6 production during infection (Li et al., 2004), and another showed that intestinal epithelial cells expressed IL-6 during infection (von Allmen et al., 2006). Neither study showed that depletion of this IL-6 specifically led to the observed defect in parasite elimination. A recent report from our laboratory showed that mice deficient in TNFα are also more susceptible to G. intestinalis infections than their wild-type counterparts (Zhou et al., 2007). The initial parasite burdens in TNFα-deficient mice or wild-type mice treated with anti-TNFα were ~10 times greater than those in control wild-type mice, similar to what has been seen in IL-6-deficient mice. However, in TNF-deficient mice, infections were eliminated by day 28, whereas infections in IL-6-deficient mice did not resolve until day 60. Thus, the defect in the TNFα-deficient mice appears to be less severe than that in the IL-6-deficient mice. Moreover, while IL-6-deficient mice have reduced TNFα mRNA levels in the small intestine following infection compared with wild-type mice, TNFαdeficient mice had no decrease in IL-6 mRNA levels. This suggests that the defect in the IL-6-deficient mice occurs upstream of the TNFα response, and is consistent with the longer duration of infection in the IL-6-deficient animals. Little is known about T cell and cytokine responses during human giardiasis. Using peripheral blood and intestinal lymphocytes from naïve individuals, one study demonstrated that CD4+ T cells proliferated and secreted IFN-γ in response to Giardia, suggesting the presence of a T cell mitogen (Ebert, 1999). Peripheral blood leucocytes from infected individuals were also shown to proliferate to Giardia antigens and to secrete IL-2 (Gottstein et al., 1991). Recently, sera from infected individuals were analysed and elevated levels of IL-2 were found, while little IL-4 or IL-10 was detected (Bayraktar et al., 2005a). In a separate study,

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these authors found elevated TNFα in uncomplicated paediatric giardiasis cases (Bayraktar et al., 2005b). In giardiasis with associated urticaria, levels of several cytokines, including IL-6, TNFα, IL-1β and IL-8, as well as IgE, C-reactive protein (CRP) and nitrite were even more elevated in patient sera (Bayraktar et al., 2005a). Elevated levels of IL-6 in sera from giardiasis patients, with and without urticaria, were also found in a separate study (Mahmoud et al., 2004). These data suggest that animal models showing important roles for mast cells, IL-6 and TNFα in protection against this infection are accurate reflections of human infections. They also argue against highly polarized Th1 or Th2 responses in human disease, but instead a response with some type I inflammatory characteristics as well as hallmarks of type 2 immunity.

Mast Cells As noted above, an interesting and common complication of giardiasis is presentation with urticaria (McKnight and Tietze, 1992). Food allergies and irritable bowel syndrome have also been associated with clinical giardiasis in some cases (Di Prisco et al., 1993, 1998; D’Anchino et al., 2002). A number of studies have demonstrated the presence of elevated anti-parasite and/or total IgE levels in these patients. In mice and gerbils, an intestinal mastocytosis has been demonstrated following infections with G. muris and G. intestinalis (Erlich et al., 1983; Leitch et al., 1993; Hardin et al., 1997; Venkatesan et al., 1997; Li et al., 2004). Elevated IgE responses have not yet been examined in these models, however. Moreover, in animals lacking intestinal mast cells, either due to genetic defects in the c-kit locus or treatment with blocking antibodies for the c-kit receptor, Giardia infections do not resolve (Erlich et al., 1983; Li et al., 2004). Several possibilities exist as to how mast cells might participate in parasite elimination, including production of cytokines such as IL-6 or TNFα, release of pre-formed mediators such as histamine or 5-hydroxytryptamine that can affect intestinal motility, and production of lipid mediators such as prostaglandins that can also increase motility responses. Unfortunately, no data directly link mast cells to any of these mechanisms during Giardia elimination. We have recently found that alterations in contractile responses of mouse intestinal smooth muscle are induced during G. intestinalis infection (Li et al., 2007). Moreover, pharmacological blockade of mast cell degranulation and histamine H-1 receptors with ketotifen, or pretreatment with compound 48/80 to deplete granule contents, reduced the spontaneous contractile force of these muscles and also the response to the peptide hormone cholecystokinin (CCK) (Li et al., 2007). These results suggest that mast cells function to increase the motility response during Giardia infection, and also suggest that CCK is a mediator of mast cell activation in this system. We and others have also shown that increased intestinal transit participates in parasite elimination during mouse infections (Andersen et al., 2006; Li et al., 2006), suggesting that the increased contractile response to CCK mediated by mast cells is important for parasite elimination. Interestingly, increased transit was not observed after infection of SCID mice, indicating that adaptive immune responses are required for generating changes in intestinal motility. While CCK and mast

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cells contribute to increased contractile forces, nitric oxide production by the neuronal isoform of nitric oxide synthase (NOS1) is known to function as an inhibitor of muscle responses in the intestinal tract. Mice lacking the NOS1 gene or treated with inhibitors of NOS1 have elevated and prolonged infections with both G. intestinalis and G. muris. The simplest explanation for this is that increased propulsion through the intestine aids in parasite elimination and that propulsive force is generated by carefully orchestrated contraction and relaxation of intestinal muscles. Increased contractions of intestinal muscles and increased propulsive forces are protective, but also contribute to the overt symptoms of disease. Comparisons between symptomatic and asymptomatic patients will be required to better understand the role of these responses in humans.

Epithelial Cell Responses The intestinal epithelium itself is also a major immune organ in giardiasis. It is the first tissue contacted by the parasite, and thus could play an important role in detection of the infection and in its eventual eradication. In addition, changes in the epithelium probably contribute significantly to the pathology associated with this infection. The initial interaction between Giardia and the host occurs when parasites attach to the epithelial cells of the intestinal tract. Epithelial cells are known to play a significant role in the innate response to infections by producing chemokines and cytokines, and by restricting access of pathogens to the body (Rumbo et al., 2004; Fasano and Shea-Donohue, 2005). Early studies of Giardia interaction with the human colon carcinoma cell line Caco2 found that Giardia did not induce expression of IL-8, MCP-1, TNFα or GM-CSF, whereas several other intestinal pathogens did (Jung et al., 1995). However, a more recent study using microarrays to analyse this response found that Caco2 cells responded to Giardia by secreting the chemokines CCL2, CCL20 and CXCL1-3 (RoxströmLindquist et al., 2005). We have verified induction of CCL20 from a murine small intestinal cell line by Giardia (E. Li and S.M. Singer, unpublished) and experiments to determine the role of CCL20 during in vivo infections remain to be performed. Giardia has also been shown to effect the integrity of the intestinal epithelium. Increased macromolecular uptake was first observed in a gerbil model of giardiasis, although no mechanistic analyses were performed in this study (Hardin et al., 1997). In a more recent set of experiments, certain strains of Giardia were shown to reduce the barrier function of epithelial monolayers by inducing apoptosis (Chin et al., 2002). This same research group also showed reduced epithelial barrier function in mice infected with G. muris (Scott et al., 2002). Interestingly, this epithelial effect in vivo was not observed in nude mice, although adoptive transfer of CD8+ T cells from infected wild-type mice could induce these changes in uninfected nude recipients (Scott et al., 2004). We have also recently reported that intestinal permeability increases in wild-type mice infected with G. intestinalis (Zhou et al., 2007). While TNFα expression can lead to increased permeability in vivo and in vitro in several systems, this cytokine was not required in this infection. A role for CD8+ cells was not examined, however.

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Thus, it remains to be determined whether direct effects of the parasite on the epithelium or immune-mediated changes are more important for changes in epithelial barrier function during this infection.

Conclusions Studies of Giardia infections in animal models are providing major new insights into immunological mechanisms of parasite control as well as disease pathology. Importantly, several recent studies have helped demonstrate parallels between the human disease and these model infections. For example, epithelial barrier defects similar to those observed in animal models were recently reported in human giardiasis patients (Hardin et al., 1997; Scott et al., 2002; Troeger et al., 2007; Zhou et al., 2007). Future studies in animals must aim to further demonstrate exactly which aspects of the human disease are being represented. Major differences in the importance of antibodies and IgA for the elimination of G. muris and G. intestinalis exist in mice; IgA being more important in G. muris than G. intestinalis. It is therefore likely that reciprocal differences may exist, with cellular mechanisms playing a larger role in eliminating the human pathogen than its murine counterpart. Finally, studies need to determine which immunological pathways participate in parasite elimination, which in immune pathology, and which in both. This knowledge is essential for improved vaccine development as well as possible immuno-based therapeutics for reducing symptoms in chronically infected patients.

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Drug Treatment and Novel Drug Targets Against Giardia and Cryptosporidium

J.-F. ROSSIGNOL Stanford University School of Medicine, Palo Alto, CA, USA

Abstract Against Cryptosporidium parvum only nitazoxanide and paramomycin show some activity in immunocompromised patients such as those with AIDS. In immunocompetent children and adults, nitazoxanide receives regulatory approval for this indication in the USA. New targets include the epidermal growth factor (EGF) receptor, pp60v-src and pp110gag-fes, and new isoflavone derivatives have shown in vitro and in vivo activities against C. parvum. The nitroimidazoles such as metronidazole and tinidazole are effective treatments against giardiasis and could be given in a single dose. Albendazole and nitazoxanide are two effective antigiardial drugs but require multiple doses. Compounds active against protein disulphide isomerases, more specifically PDI2 and PDI4, as recently reported, could also represent promising targets for new drugs effective against Cryptospororidium spp. and Giardia intestinalis, respectively. There is little doubt that more antiprotozoal drugs are needed.

Introduction The outbreak of cryptosporidiosis that occurred in Milwaukee in 1993 was a serious wake-up call for the medical community. It was not only the first significant emergence of Cryptosporidium spp. as a major human pathogen but also a huge waterborne outbreak affecting thousands of people from a major city in North America. In the past, most of the protozoan outbreaks have been somewhat limited in the number of people infected and were more frequently caused by Giardia duodenalis. Since then, outbreaks of giardiasis and cryptosporidiosis have occurred regularly throughout the world. At that time, there was no treatment for cryptosporidiosis and some metronidazole resistance reported for G. duodenalis. Consequently, new drugs against these two parasites were needed urgently. Giardiasis is particularly prevalent in developing countries. Currently it is treated with nitroimidazole compounds such as metronidazole, tinidazole and © CAB International 2009. Giardia and Cryptosporidium: From Molecules to Disease (eds G. Ortega-Pierres et al.)

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secnidazole. As a second line, benzimidazole carbamates, such as albendazole or mebendazole, have proven to be among the most effective in their class, but are somewhat less effective than the nitroimidazoles. Conversely, furazolidone is one of the least effective anti-giardial drugs. Paramomycin, a non-absorbable aminoglycoside, is somewhat less effective, but can be used during pregnancy. The recently approved drug, nitazoxanide, is effective against both cryptosporidiosis and giardiasis. Results from well-designed controlled clinical studies suggest that it could be an alternative treatment for drug-resistant giardiasis and an option for individuals with metronidazole or albendazole intolerance. Recently, formononectin, a halogenated isoflavone, was reported to possess anti-giardial activity in vitro. The emergence of cryptosporidiosis triggered the screening of many compounds for potential anti-cryptosporidial activity, but the majority were ineffective. Among the most commonly used treatments against cryptosporidiosis are paramomycin and azithromycin, which are partially effective. The effectiveness of nitazoxanide has been demonstrated in vitro, and in vivo using several animal models and finally in clinical trials. It significantly shortened the duration of diarrhoea and decreased mortality in adults and in malnourished children. Nitazoxanide is not effective in the absence of an appropriate immune response. Consequently, in patients with acquired immunodeficiency syndrome (AIDS), treatment with highly active antiviral therapy (HAART) is recommended. Recent investigations have focused on the potential for molecular-based immunotherapy against this parasite. Probiotic bacteria have also been tested, but were unable to eradicate the parasite. The most recent advance in cryptosporidiosis treatment is the identification of a new synthetic isoflavone derivative (RM-6427), which demonstrated excellent activity against C. parvum in vitro and in vivo in the gerbil model of infection. Since the advent of HAART, opportunistic infections in patients with AIDS have decreased markedly in developed countries. However, opportunistic infections remain the most important cause of death in HIV-infected people. While antiretroviral therapy should increase patients’ CD4+ count above the risk threshold, and improve patients’ tolerance and response to treatment, antimicrobial therapy targeting opportunistic organisms remains essential to prevent ongoing morbidity. Thus, combination therapy, restoring immunity in AIDS patients along with antimicrobial treatment of Cryptosporidium infection, is necessary.

Cryptosporidiosis Initially, C. parvum was believed to be an occupational hazard affecting only those people exposed to infected animals, but it is now known to cause illness or disease both in immunocompetent and immunocompromised people. After its description in 1976, human cryptosporidiosis became increasingly important in the 1980s and 1990s, not only because of the emergence of waterborne outbreaks of disease worldwide but also because it was associated with the AIDS epidemic as a complication in severely immunocompromised patients. Cryptosporidiosis is one of the most common, and certainly the most devastating,

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gastrointestinal infections in people with AIDS. While the small intestine is the site most commonly affected, symptomatic Cryptosporidium infections have also been found in other organs including the gall bladder, the pancreas, the lungs and the conjunctiva. Compared with digestive disorders, respiratory disease linked to Cryptosporidium has not been very well documented in patients with HIV/AIDS. In 1996, Roussel et al. studied the effect of C. parvum infection in immunosuppressed rats, and found that, in addition to the intestine, the biliary tract was a major site for C. parvum infection and was potentially a protected reservoir which could sustain chronic infection. There is strong evidence for an increased risk of infection in patients with compromised immunity. In patients with AIDS, cryptosporidiosis is self-limited in individuals with CD4+ cells count higher than 180 cells/mm3, chronic in patients with CD4+ cell depletion to less than 100 cells/mm3, and fulminant in some of those with a count below 50 cells/ mm3. X-linked immunodeficiency with hyperimmunoglobulin M, caused by a defect in CD40 ligand (also termed CD154), is associated with increased frequency and severity of Cryptosporidium infection. Patients with biliary tract involvement and profound immunodeficiency may present with acalculous cholecystitis, sclerosis cholangitis or pancreatitis (Vakil et al., 1996; Hashmey et al., 1997; Teare et al., 1997). This condition is usually associated with markedly shortened survival. Cryptosporidiosis continues to be a serious problem in immunocompromised patients and on a worldwide scale in malnourished infants and children. The propensity of the parasite to survive and be transmitted through source waters makes it an important public health threat. Some drugs have proven toxic at the doses required to reduce parasite multiplication, others have shown some efficacy only in animal models, and most others have shown no efficacy at all. After many years of research on strategies and drugs for the treatment and control of cryptosporidiosis, nitazoxanide is the only drug that has been approved in the USA for the treatment of cryptosporidiosis in adults and children.

Drug Treatment for Cryptosporidiosis In 1993, Lemeteil et al. tested more than 30 drug candidates for curative or preventive activity in an immunocompromised rat model that mimics severe human cryptosporidiosis. No significant anticryptosporidial activity was observed when using sulphadoxine–pyrimethamine, quinacrine, trimethoprim–sulphamethoxazole, bleomycin, elliptinium, daunorubicin, pentamidine, alpha-difluoromethylornithine, diclazuril or N-methylglucamine. Vitamin A appeared to reduce oocyst shedding. Active agents included sinefungine (2–10 mg/kg/24 h), lasalocid A (2–10 mg/kg/24 h), metronidazole (25–50 mg/kg/24 h) and sulphadimethoxine (10–100 mg/kg/24 h). Sinefungine (10 mg/kg/24 h) and lasalocid A (10 mg/kg/24 h) displayed the highest anticryptosporidial activity. Currently, the most used drugs for cryptosporidiosis are paramomycin, azithromycin, nitazoxanide and, lately, antiretroviral therapy.

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Paramomycin Paramomycin is an aminoglycoside antibiotic poorly absorbed from gut epithelium, but that can apparently be absorbed in small quantities across the limiting apical membrane surrounding the extracytoplasmic parasite. Its mechanism of action is targeting the bacterial ribosome, where it binds to the A-site and disrupts protein synthesis. In vitro studies showed that paramomycin has some activity against C. parvum at concentrations achievable in the intestinal lumen. Based on experimental data and clinical experience, paramomycin has a modest activity against C. parvum (Griffiths et al., 1998). Four small and two large open uncontrolled studies have evaluated the efficacy of this drug prospectively. In most studies there was a good clinical and parasitological response. However, after paramomycin cessation, many patients relapsed (Ciezy et al., 1991; Armitage et al., 1992; Fichtenbaum et al., 1993; Wallace et al., 1993; Bissuel et al., 1994), moreover a meta-analysis of data from several studies performed from 1990 to 1996 in immunocompromised patients concluded that it was only partially effective. Well-controlled clinical studies are limited. Hewitt et al. (2000) conducted a prospective, randomized, double-blind, placebo-controlled study to evaluate the efficacy of paramomycin in the treatment of symptomatic cryptosporidial enteritis in AIDS patients. Thirty-five adults with CD4+ cell counts of <150 cells/mm3 were included in the study. The clinical course of cryptosporidiosis was variable, and paramomycin was not shown to be more effective than placebo. White et al. (1994) performed a double-blind clinical study in ten patients with AIDS and cryptosporidiosis. Oocyst excretion decreased significantly, along with a good clinical response, and they suggested that patients should receive maintenance therapy to prevent relapse. Combination studies reported good results with the association of paramomycin and azithromycin in immunocompromised individuals; however, its effectiveness has not been definitively established. Adverse events are limited to the gastrointestinal tract. Lesions in the gut, including those due to cryptosporidiosis, can increase systemic absorption of paramomycin and could result in renal toxicity and ototoxicity. In addition, paramomycin has a narrow therapeutic index and toxicity associated with long-term therapy is not unusual and could include renal failure, hypocalcaemia, myasthenia gravis, and conditions that depress neuromuscular transmission.

Azithromycin In animal models, azithromycin, an azalide antibiotic, is the most active among the macrolides. Azithromycin acts by binding the 50S ribosomal subunit of susceptible microorganisms, thus interfering with microbial protein synthesis. Giacometti et al. (1999) described a case of disseminated cryptosporidiosis in a patient with AIDS. In order to monitor the in vitro susceptibility of the parasite, specimens from various sites were collected periodically. These C. parvum clinical isolates were subjected to in vitro susceptibility to paramomycin at 1 mg/kg concentration, azithromycin at a concentration of 8 mg/l, and nitazoxanide at 10 mg/l. At these concentrations azithromycin, paramomycin and nitazoxanide

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showed 26.5%, 63.4% and 67.2% reduction of the parasite’s growth, respectively. Subsequent isolates of C. parvum showed similar susceptibilities. The weakest activity occurred with azithromycin. Nitazoxanide at a concentration of 10 mg/l showed higher activity than paramomycin at its higher concentration. Kadappu et al. (2002) performed a randomized, controlled trial to study the efficacy of short-term use of azithromycin in the management of cryptosporidiosis in 13 AIDS patients. There was a good clinical improvement but parasitological benefit was doubtful. Dionisio et al. (1998) reported, after a study in 13 patients, that ‘long term, low dose azithromycin is well tolerated and may induce stable remission of chronic cryptosporidiosis in patients with AIDS. It may lead to probable eradication of the infection in some patients, even those with severe immunodeficiency’. Most studies of azithromycin for cryptosporidiosis in AIDS have reported good results when combined with other antimicrobials such as paramomycin. A small open-level study of a combination of paramomycin and azithromycin for 4 weeks followed by paramomycin alone for 8 weeks in 11 AIDS patients with CD4+ cell counts of <100 cells/mm3 showed a significant reduction in symptoms and oocyst excretion. Azithromycin may increase the toxicity of theophylline, warfarin and digoxin; these effects could be reduced by a co-administration of aluminium and/or magnesium antacids; nephrotoxicity and neurotoxicity may occur when co-administered with cyclosporin. Diarrhoea is a common adverse effect, and pseudo-membranous colitis has also been reported (Zithromax prescribing information, Pfizer). Reactions can occur with intravenous administration, and bacterial or fungal overgrowth may result with prolonged antibiotic use. Also, it may increase hepatic enzymes and cholestatic jaundice.

Antiretroviral therapy The use of highly active antiretroviral therapy (HAART) in people with AIDS has dramatically reduced the prevalence of cryptosporidiosis and the length and severity of its clinical course. This effect was attributed to the recovery of host immunity, as demonstrated in other cases of cryptosporidiosis associated with other immunodeficiencies such as primary immunodeficiencies, organ transplantation, cancer, diabetes and malnutrition for which antiretroviral therapy is not indicated. A prime example is the extremely high prevalence of cryptosporidiosis in the setting of X-linked immunodeficiency with hyper-immunoglobulin M, a rare form of primary immunodeficiency disease, whereby T lymphocytes cannot induce B cells to undergo immunoglobulin class-switching from immunoglobulin M (IgM) to IgG, IgA and IgE. In this specific case, the key mediator of the immune response to Cryptosporidium is the production of interferon-gamma (IFN-γ). Some studies using protease inhibitors such as ritonavir, saquinavir and indinavir claim a drastic reduction of C. parvum infection both in vivo and in vitro (Hommer et al., 2003; Mele et al., 2003). Whether or not aspartyl proteases could have some important functions is not known, as there are no reports of its presence in C. parvum. Despite the reduction of opportunistic infection in patients with AIDS under HAART, opportunistic infections continue to be

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the most important cause of death in HIV-infected individuals. While HAART should increase patients’ CD4+ cells above risk thresholds, concomitant targeting of the opportunistic infection remains important in order to prevent ongoing morbidity.

Roxithromycin Roxithromycin is another macrolide that is being studied as a possible treatment option for cryptosporidiosis; however, clinical data are extremely limited. Good results were reported from four individuals in Argentina. Diarrhoea stopped after 2–10 days of treatment in all four individuals, but diarrhoea recurred in one person after treatment had been stopped. Stools were negative for Cryptosporidium oocysts after treatment in three of the individuals, and oocyst numbers were reduced in the fourth individual. No side-effects were reported. The efficacy of roxithromycin was also investigated in 26 patients with AIDS, 22 of whom completed the study; 15 patients (68.2%) were cured, 6 (27.3%) improved, and treatment failed in 1 patient (Uip et al., 1998). No conclusions can be drawn from these results, and additional controlled clinical trials are necessary.

Antibody therapy The close relationship between Cryptosporidium infection and the host immune response led to investigations into antibody therapy. Some investigators hypothesized that immunoglobulins in bovine colostrum immunized against particular pathogens may help protect against some specific pathogens such as Cryptosporidium spp. Unfortunately, bovine colostrum supplements vary widely in terms of their specific constituents. Results obtained from bovine colostrum antibody therapy are mostly contradictory. Riggs et al. (2002) suggested that targeting the apical complex and surface antigens of Cryptosporidium zoites (CSL, GP25-200 and P23) could passively immunize against cryptosporidiosis. Monoclonal antibodies (mAbs) were evaluated for therapeutic efficacy against persistent infection in adult IFN-γ-depleted SCID mice. The results indicated that anti-CSL mAb 3E2 had highly significant efficacy in reducing, but not in eliminating, persistent C. parvum infection.

Probiotics Experimental study of the effects of probiotics on Cryptosporidium infection in neonatal rats showed a trend to a more rapid clearance of parasites in rats treated with probiotics. No significant effect of probiotic administration was observed in terms of weight gain, parasite burden, mucosal damage, or kinetics of mucosal cytokines during the course of infection. Overall, the results showed that the daily administration of L. casei-containing mixtures was unable to eradicate the parasite in the neonatal rat model (Guitard et al., 2006).

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Nitazoxanide Nitazoxanide is a thiazolide anti-infective that is effective against anaerobic protozoa and bacteria, and interferes with the pyruvate:ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reaction, which is essential for anaerobic energy metabolism. Nitazoxanide and its two metabolites, tizoxanide and tizoxanide glucuronide, inhibited the growth of C. parvum sporozoites and oocysts at concentrations lower than 10 µg/ml (Theodos et al., 1998; Gargala et al., 2000). In vivo it was subsequently found to be effective against C. parvum in suckling mice, nude mice, gerbils, rats and piglets (Blagburn et al., 1998; Theodos et al., 1998; Li et al., 2003; Baishanbo et al., 2005). The efficacy of nitazoxanide and its two metabolites was tested against three stages of the life cycle of C. parvum (asexual and sexual stages and oocysts) in HCT-8 enterocytic cells (Gargala et al., 2000). Nitazoxanide, tizoxanide, and tizoxanide glucoronide were inhibitory for up to 46 h when added after sporozoite invasion (MIC50 1.2, 22.6 and 2.2 mg/l, respectively). Tizoxanide had only limited activity, but nitazoxanide and tizoxanide glucuronide strongly inhibited the asexual and sexual stages, respectively. The inhibitory concentrations on complete parasite development using this methodology were consistent with results obtained by others (Theodos et al., 1998; Giacometti et al., 1999, 2000). In experimentally infected suckling mice, SCID mice, rats and piglets, nitazoxanide showed various degrees of efficacy when compared to paramomycin. The in vivo efficacy of nitazoxanide and paramomycin was tested in biliary tract cryptosporidiosis in immunosuppressed gerbils (Baishanbo et al., 2005). In nitazoxanide-treated and paramomycin-treated groups, as compared with untreated animals, oocyst shedding was partially suppressed in a similar manner (P > 0.05). Parasites were present in histological sections of the ileal mucosa of untreated animals compared with 3/14 and 6/15 in the nitazoxanide-treated (2/14, P < 0.01) and the paramomycin-treated groups, respectively. No histological alteration of the biliary mucosa was observed in either treated or untreated infected gerbils. The efficacy of nitazoxanide in treating cryptosporidiosis in immunocompetent patients was well established by three double-blind placebo-controlled clinical studies carried out in more than 140 immunocompetent adults and 150 immunocompetent children from Egypt and Zambia (Rossignol et al., 2001a, 2006; Amadi et al., 2002). Clinical and parasitological cures were recorded following a 3-day course of treatment using 500 mg of nitazoxanide twice a day in adults and adolescents (>12 years of age), 200 mg/10 ml (4–11 years of age) or 100 mg/5 ml (12–36 months of age) twice a day for 3 days using an oral suspension containing 20 mg of nitazoxanide/ml of suspension. In addition, there was a statistically significant reduction in the mortality of malnourished infants. Two double-blind placebo-controlled studies of nitazoxanide in the treatment of cryptosporidial diarrhoea in adult AIDS were carried out on 66 Mexican and 50 Thai patients. The drug was given as 1000 mg (two tablets) twice a day for 2 weeks in patients with CD-4+ cell counts of >50 cells/mm3, and for 8 weeks in patients with CD-4+ counts of <50 cells/mm3. The results reached statistical significance (P < 0.05) in both clinical resolution of diarrhoea and suppression of oocyst shedding. The onset of HAART made this type of trial design both

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difficult to carry out and unethical. The programme was therefore discontinued (Rossignol et al., 1998; Rossignol, 2006). In total, 365 patients were enrolled at 165 study centres throughout the USA. The duration of treatment ranged from 1 to 1528 days (median 62 days). Among the 357 patients included in the ‘intent to treat’ analysis, 209 (59%) achieved a sustained clinical response while on treatment. Clinical responses were closely associated with Cryptosporidiumnegative stools (P < 0.0001). No safety issues were identified at doses up to 3000 mg/day or for long duration of treatment. (Rossignol, 2006) Nitazoxanide and tizoxanide account for activity in the intestine, while in other locations tizoxanide glucoronide is the most important agent (i.e. bile). High concentrations of tizoxanide glucuronide are excreted in bile. This latter metabolite has been assayed at concentrations up to 204 µg/ml in human bile after a single oral administration of 1000 mg of nitazoxanide, and is believed to be responsible for the activity of the compound in Cryptosporidium-induced cholangitis in immunocompromised patients with disseminated cryptosporidiosis. In a recent review that assessed the efficacy of interventions for the treatment and prevention of cryptosporidiosis in immunocompromised patients by electronically searching Medline, Embase and other electronic databases up until August 2005, nitazoxanide was found to reduce parasite load and possibly be useful in immunocompetent individuals (Abubakar et al., 2007). De la Tribonnière et al. (1999) reported a case of systemic cryptosporidiosis that resolved with oral nitazoxanide and paramomycin inhalation. A 31-year-old man with AIDS presented with cough, fever, chronic diarrhoea and cholestasis, without pancreatic abnormalities. Sclerosis cholangitis was diagnosed, and Cryptosporidium spp. oocysts were detected in stools and bronchoalveolar lavage. CD4+ lymphocyte count was 38 cells/mm3. The patient was treated with paramomycin inhalation for 15 days, and 1 month later bronchoalveolar lavage was negative for Cryptosporidium spp. oocysts. In addition, he received 500 mg of nitazoxanide twice daily for 12 weeks and a marked reduction in cholestasis was observed without adverse effects. Triple antiretroviral therapy was added and the CD4+ cell count increased to 70 cells/mm3. One year later, the CD4+ count had fallen to 12 cells/mm3 and the cholangitis reappeared. Cryptosporidium spp. oocysts were isolated from the stools, and following further nitazoxanide therapy, the cholestasis decreased. Faraci et al. (2007) described a case of Cryptosporidium infection occurring in a child after allogeneic stem cell transplantation (SCT) for acute non-lymphoblastic leukaemia. This patient presented with an intestinal, biliary and pancreatic Cryptosporidium disease associated with an intestinal acute graft versus host disease (aGvHD). The increase in CD3+/CD4+ cells secondary to the reduction of steroid therapy associated with the improvement of aGvHD and the use of anti-parasitic treatments (especially nitazoxanide) improved the infection-related symptoms and led to the complete clearance of Cryptosporidium.

Isoflavone derivatives In 1987, Akiyama et al. described the tyrosine-specific, protein kinase activity of the epidermal growth factor (EGF) receptor, pp60v-src and pp110gag-fes, which

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Table 38.1. Inhibitory activities of dihydroxyisoflavone and trihydroxy-deoxybenzoin derivatives on the in vitro development of Neospora caninum, Sarcocystis neurona and Cryptosporidium parvum. Results fora:

Neospora caninum

Agent RM-6403 RM-6411 RM-6424 RM-6425 RM-6426 RM-6427 RM-6428 RM-6430 RM-6431 RM-6433 RM-6434 RM-6435 RM-6436 RM-6439 RM-6440 RM-6441 RM-6442 RM-6443 RM-6446 RM-6448

Sarcocystis neurona

Cryptosporidium parvum

IC50 (µg/ml)

MI (%)

IC50 (µg/ml)

MI (%)

IC50 (µg/ml)

2.1 3.0 1.9 2.0 2.5 2.1 1.8 1.9 2.9 0.7 0.9 0.6 1.6 3.1 0.7 2.5 0.7 3.0 0.7 2.1

97 ± 3.0 97.5 ± 2.10 96 ± 2.5 98 ± 3.8 98 ± 3.3 98.5 ± 2.10 100 100 100 100 100 100 100 98.4 ± 2.30 73 ± 6.3 89.9 ± 5.20 83.2 ± 5.30 100 96.8 ± 2.40 98.4 ± 2.10

2.65 4.25 2.7 2.6 2.8 1.9 2.0 0.8 1.3 2.2 2.0 0.7 0.9 2.9 2.75 2.1 1.5 2.4 0.55 2.95

95 ± 1.6 59 ± 4.8 93 ± 2.3 95 ± 1.8 86 ± 2.8 99.7 ± 0.4 95.3 ± 0.3 96.9 ± 4.3 100 100 90.8 ± 13.0 99.6 ± 0.6 100 100 100 100 73.3 ± 3.6 100 93.9 ± 2.1 97.2 ± 1.8

1.25 2.3 2.4 0.9 3.75 0.75 0.9 3.9 6.9 7.75 7.75 4.75 1.8 5.75 0.8 3.4 2.4 0.85 3.0 2.85

MI (%) 98 99.5 98.8 99.4 96.5 97 99 68.1 52 66.6 69 63.9 96.9 77.6 98.1 97.1 100 90.1 91.2 83.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.4 0.3 1.7 0.8 2.2 2.6 1.4 3.9 9.5 7.8 10.2 6.7 1.6 2.6 2.6 3.5 0 6.2 3.3 4.5

aMI,

maximum inhibition of Neospora caninum or Sarcocystis neurona merozoite or Cryptosporidium parvum form development. MI results are expressed as means ± standard deviations.

was inhibited in vitro by the isoflavone, genistein. Inhibition was competitive with respect to ATP and non-competitive to a phosphate acceptor, histone H2B. By contrast, genistein scarcely inhibited the enzyme activities of serine- and threonine-specific protein kinases such as cAMP-dependent protein kinase, phosphorylase kinase, and the Ca2+/phospholipid-dependent enzyme, protein kinase C. When the effect of genistein on the phosphorylation of the EGF receptor was examined in cultured A431 cells, EGF-stimulated serine, threonine, and tyrosine phosphorylation was decreased. Phospho-amino acid analysis of total cell proteins revealed that genistein inhibited the EGF-stimulated increase in phosphotyrosine levels in A431 cells. Stachulski et al. (2006) identified two isoflavone derivatives among several derivatives synthesized. Excellent in vitro activity against C. parvum was observed

472

Table 38.2. In vivo efficacies of RM-6427, RM-6428, nitazoxanide, and paramomycin against Cryptosporidium parvum in immunosuppressed Mongolian gerbils. No. of animals with no oocyst shedding (day post-infection)

% Reduction in mean oocyst sheddinga (day post-infection)

No. of animals (day 12 postinfection) Illeum

Biliary Tract

Agent

No.

Dose

None

10

None

2

4

RM-6427

10

200 mg/kg/day/8 days

6 (8)b

90.5 (8)e

5f

5f

RM-6427

10

400 mg/kg/day/8 days

6 (8)b

92.0 (8)e

7i

10k

Nitazoxanide

14

200 mg/kg/day/12 days

2 (8)c

55.0 (8)b

11i

9l

100 mg/kg/day/12 days

(8)c

17.6

(8)b

9f

9f

1

0

45.5

(4)d

11g

11k

Paromomycin None RM-6428

14 9 15

0

2

None 400 mg/kg/day/12 days

0 2

(12)d

53.6 (8)g 67.0 (12)h oocyst count in treated animals) / (mean oocyst count in untreated animals)) x 100. bP < 0.001 c P = 0.0001 d P < 0.05 < 0.0001 f P < 0.05 g P < 0.01 h P < 0.02 i P = 0.067 j P = 0.011 k P < 0.002 i P < 0.005.

a((Mean

J.-F. Rossignol

eP

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for both compounds in cell culture, and in vivo in experimental infections. In a novel approach for identifying anticoccidial agents, Gargala et al. (2005) studied the activities of EGF against Sarcocystis neurona, Neospora caninum and Cryptosporidium parvum grown in BM and HCT-8 cell cultures. Fifty-two EGFR PTK inhibitor isoflavone analogues, including dihydroxyisoflavone and trihydroxydeoxybenzoin derivatives, were investigated. As shown in Table 38.1, dihydroxyisoflavone and trihydroxy-deoxybenzoin derivatives induced a maximum development inhibition (MI) of more than 95% for at least one parasite. It was found that 17 inhibited N. caninum, with IC50s ranging from 0.6 to 3.1 µg/ml; 13 inhibited S. neurona, with IC50s ranging from 0.7 to 2.95 µg/ml; and 11 inhibited C. parvum, with IC50s ranging from 0.75 to 7.75 µg/ml. For five agents (RM-6403, RM-6425, RM-6427, RM-6428 and RM-6436), the MI was ≥95% for all three parasites. The activity of two of these isoflavones was confirmed in vivo using a C. parvum-infected gerbil model. RM-6427 and RM-6428 were quite effective, as indicated in Table 38.2. Both spiramycin and clarithromycin failed to manage disease in immunocompromised individuals and failed to prevent the development of cryptosporidial enteritis. Octreotide acetate, atovaquone, letrazuril and lasolacid have been investigated for use in immunosuppressed individuals, but have poor efficacy and gave inconsistent results (Zardi et al., 2005).

Giardiasis Giardia duodenalis is both the most common human intestinal parasite in the USA and the most common cause of chronic diarrhoea in travellers. Research into its epidemiology, pathogenesis and treatment has intensified since G. duodenalis waterborne outbreaks were reported in Europe and the USA during the 1960s and 1970s (Moore et al., 1969; Craun, 1986; Farthing, 1992). Currently, G. duodenalis is responsible for the largest number of waterborne outbreaks of diarrhoea in the USA (Craft et al., 1981; Kramer et al., 1996). Giardia infects approximately 2% of adults and 6–8% of children in developed countries, worldwide. It infects more than 40 animal species and is regarded as zoonotic by the World Health Organization (WHO), although animal reservoirs of human outbreaks have not yet been clearly identified. Giardia cysts can survive in water for several months in cold climates, and although many outbreaks have been linked to contaminated drinking water (Marshall et al., 1997), the faecal–oral route is the major route of infection, particularly in countries where source waters are warm or where there is little drainage and no reticulated water supply (Ortner et al., 1997). This evidence is supported by the fact that giardiasis is prevalent in childcare centres and in nursing homes (Wittner and Tanowitz, 1992; Cheney and Wong, 1993; Nash and Weller, 1998). WHO, in a press release in 1998, reported that 3000 million people live in unsewered communities in developing countries, where the rate of giardiasis approaches 30%, suggesting that there are close to 1000 million cases of giardiasis at any one time, contributing to 2.5 million deaths which occur annually from diarrhoeal disease. Giardia duodenalis infection causes a decrease in

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small-intestinal brush border surface area, microvillus and villus atrophy, enterocyte immaturity, disaccharidase and luminal enzyme deficiencies, and malabsorption of electrolytes: a multifactorial pathogenesis which is not well understood. Roberts-Thomson et al. (1982) described granulomatous hepatitis and cholangitis associated with chronic diarrhoea attributed to giardiasis (Roberts-Thomson et al., 1982; Adam, 1991). Persistent infection and diarrhoea may occur in immunocompromised individuals (such as those with immunoglobulin abnormalities and AIDS).

Drug Treatment for Giardiasis Despite the fact that giardiasis is an important human disease, there have been relatively few agents used for its therapy. It is difficult to evaluate the clinical efficacy of anti-giardial agents because they vary in methodology, populations studied, clinical conditions, measured outcomes and follow-up.

Quinacrine Quinacrine was the first used anti-giardial drug in the mid-1930s. Initially introduced as an anti-malarial (Hartman and Kyser, 1942), it was used as an antigiardial drug until it was replaced by metronidazole. Its production in the USA was discontinued in 1992 as the market for the treatment of giardiasis was limited. The mode of action of quinacrine is not completely understood. The drug intercalates readily with G. duodenalis DNA, and it is this interaction which is thought to inhibit nucleic acid synthesis.

Nitroimidazoles The main agents used against giardiasis are metronidazole, tinidazole, ornidazole and secnidazole, which have been used for the treatment of giardiasis since the mid-1950s. Following its discovery in the mid-1950s, metronidazole was used to treat Trichomonas vaginalis and Entamoeba histolytica, and in 1962 Darbon et al. found it useful for treating giardiasis. Since then, clinicians have used metronidazole and other nitroimidazoles as the mainstay of therapy for giardiasis. Despite its acceptance and widespread use for giardiasis, it has never been approved by the USA Food and Drug Administration (FDA) for this application. Of the nitroimidazoles, metronidazole is the most commonly used drug worldwide. The drug is activated by reduction of its nitro group. Reduced metronidazole serves as a terminal electron acceptor which binds to DNA macromolecules, damaging the DNA helical structure. However, metronidazole does not affect cyst viability (Paget et al., 1989). Metronidazole is completely absorbed after oral administration and penetrates body tissues and secretions such as saliva, breast milk, semen and vaginal secretions. The drug is metabolized mainly in the liver and is excreted in urine (Lau et al., 1992). Its median reported

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efficacy is 92% (Levi et al., 1977; Cimerman et al., 1989; Gorbea et al., 1989; Kalyci et al., 1995). Metronidazole is prescribed as a 250 mg/dose 2–3 times daily for 7–10 days or as a 3-day course, daily single-dose therapy of 2.0 or 2.4 g/dose, or a single dose that increases compliance but reduces efficacy and carries strong adverse effects. Metronidazole is not available in a standard liquid form. A suspension can be prepared by thoroughly crushing tablets using a drop of glycerin as a lubricant, and suspending the mixture in Cherry Syrup NF (Lerman and Walker, 1982). Adverse effects have been the major limitation for metronidazole use, and the most common include headache, vertigo, nausea, and an unpleasant metallic taste, which can result in poor compliance. In addition, pancreatitis, central nervous system toxicity at high doses (Kusumi et al., 1980; Roe, 1985), and transient, reversible neutropenia have all been attributed to metronidazole (Lau et al., 1992). The inhibition of aldehyde dehydrogenase by metronidazole can cause severe vomiting, flushing, headache and gastrointestinal pain following alcohol ingestion (Gardner and Hill, 2001). Tinidazole, another nitroimidazole derivative, is given as a 2 g single dose for adults or equivalent for children. Tinidazole has a clinical efficacy of 80–100% with a median efficacy of 92% (Bakshi et al., 1978; Jokipii and Jokipii, 1979, 1982). The recommended dosage for paediatric populations is usually 50 mg/kg as a single dose, and the drug is available as a liquid suspension. Adverse effects reported for tinidazole are not as common as for metronidazole but do include unpleasant taste, vertigo and gastrointestinal upset (Jokipii and Jokipii 1979, 1982) Ornidazole is another nitroimidazole with few clinical trials, but studies indicate an efficacy similar to tinidazole (92–100%) when given over several days or as a single dose (Sabchareon et al., 1980; Suntornpoch and Chavalittamrong, 1981; Bassily et al., 1987; Kuzmicki and Jeske, 1994). Single-dose ornidazole (40 mg/kg) has also been given to children, with obvious benefits regarding compliance and cost (Oren et al., 1991). Secnidazole is also member of the nitroimidazole family similar to tinidazole and ornidazole, usually given in a single dose. Clinical studies demonstrate efficacy rates of over 85% as a single dose in adults. Secnidazole is rapidly and completely absorbed, has a half-life of 17–29 h, and is metabolized via oxidation in the liver (Gillis and Wiseman, 1996). Common side-effects are gastrointestinal disturbances such as nausea, anorexia and abdominal pain. At the molecular level, resistance to metronidazole is associated with changes in DNA. DNA probes that hybridize with specific chromosomes and repetitive sequences indicate that rearrangements both at the chromosome and repetitive DNA level occurred concurrently with the development of metronidazole resistance (Upcroft et al., 1990).

Benzimidazoles The suggested mechanism of action of benzimidazoles is an interaction with the colchicine site of tubulin in microtubules, resulting in the disruption of their assembly and disassembly. Mebendazole was the first benzimidazole used to treat

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giardiasis, and was ineffective, killing only 37% of parasites. In 1982, albendazole was introduced as anthelmintic. The first published reports of the use of albendazole for giardiasis came from China in 1986 (Zhang et al., 1986; Zhong et al., 1986); other benzimidazoles were also tested prior to 1990 with mixed success. The first large-scale human study was in Bangladesh, where the average efficacy of albendazole was 62–95% compared with 97% for metronidazole. Albendazole resistance in giardiasis is correlated with cytoskeletal changes but not with a mutation at amino acid 200 in beta-tubulin (Upcroft et al., 1996).

Paramomycin Paramomycin inhibits protein synthesis in G. duodenalis by interacting with the 50S and 30S ribosomal subunits, resulting in misreading of mRNA codons. It is poorly absorbed, with about 60–70% efficacy against G. duodenalis. Paramomycin has been considered a possible drug for Giardia infections in pregnant patients as it is excreted in faeces without being metabolized (Tomoko and Michaell, 2003).

Nitazoxanide The activities of nitazoxanide and its metabolite tizoxanide were compared to metronidazole in vitro in microplates against six axenic isolates of G. duodenalis. Tizoxanide was eight times more active than metronidazole against metronidazolesusceptible isolates and twice as active against a resistant isolate. The susceptible

Table 38.3. IC50 values of nitazoxanide, tizoxanide and tizoxanide glucuronide compared with metronidazole for Giardia duodenalis. Strain JKH-1 EBE VNB1 VNB5 EBC VNB2 Mean Mean (S) t-test versus S MTZ ratio (S) MTZ ratio (R) MTZ ratio (total) R/S

MTZ (µM) ± S.D. NTZ (µM) ± S.D. 15.42 ± 4.3 5.95 ± 5.8 5.72 ± 3.4 5.20 ± 3.5 4.55 ± 2.2 3.21 ± 1.2 6.68 ± 4.4 4.93 ± 1.1

3.1

7.81 ± 1.7 2.25 ± 2.5 1.17 ± 1.1 1.69 ± 0.9 1.07 ± 0.7 1.20 ± 0.9 2.53 ± 2.6 1.48 ± 0.49 P = 0.0009 3.3 2.0 2.6 5.3

TIZ (µM) ± S.D.

TIZG (µM) ± S.D.

8.18 ± 1.4 0.641 ± 0.5 0.603 ± 0.2 0.641 ± 0.4 0.716 ± 0.3 0.528 ± 0.4 1.89 ± 3.1 0.63 ± 0.07 P = 0.0009 7.8 1.9 3.5 13.0

14.00 ± 1.3 4.28 ± 1.2 33.2 ± 0.9 14.66 ± 5.4 11.87 ± 0.6 15.22 ± 1.9 15.54 ± 9.5 15.85 ± 10.6 NS 0.3 1.1 0.4 0.9

Abbreviations: MTZ, metronidazole; NTZ, nitazoxanide; TIZ, tizoxanide; TIZG, tizoxanide glucuronide.

Drug Treatment Against Giardia and Cryptosporidium

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strains were isolated from diagnostic faecal samples from cases of giardiasis returning from the Indian subcontinent, which were subsequently successfully treated with metronidazole. All of these strains were determined to be susceptible to metronidazole in vitro. A further isolate of G. duodenalis (JKH-1) was isolated from a chronic human infection refractory to metronidazole (Adagu et al., 2002) (Table 38.3). Three well-controlled clinical trials were conducted in adults and children using a placebo or metronidazole as controls. Clinical and parasitological cures were recorded following a 3-day course of treatment using 500 mg of nitazoxanide twice a day in adults and adolescents (>12 years of age), 200 mg/10 ml (4–11 years of age) or 100 mg/5 ml (12–36 months of age) twice a day for 3 days using an oral suspension containing 20 mg of nitazoxanide per millilitre of suspension. Nitazoxanide was as effective as a 5-day course of metronidazole, with clinical cure observed in 85% of patients and with parasite eradication observed in 71–80% of patients (Ortiz et al., 2001; Rossignol et al., 2001b). Giardia resistance to metronidazole is negatively correlated with the intracellular concentration of pyruvate:ferredoxin oxidoreductase, leading to a concomitant decrease in the uptake of free metronidazole into the cell, while resistance to furazolidone appears to be due to an increase in thiol cycling enzymes. Currently, the nitroimidazoles are the drugs of choice for giardiasis. However, problems of cross-resistance and treatment failure occurring in absence or resistance are additional difficulties, which have important implications for the treatment of individual cases. In such cases, nitazoxanide, which has recognized activity against metronidazole-resistant and metronidazole-sensitive strains, represents the best option for treating refractory giardiasis.

Conclusions: Drug Targets and Future Development Nitazoxanide alone, or perhaps combined with paramomycin, can be used for treating cryptosporidiosis. The nitroimidazoles, such as metronidazole and tinidazole, are effective treatments for giardiasis and can be administered as a single dose. Albendazole and nitazoxanide are also effective anti-giardial drugs, but require multiple doses. There is little doubt that more anti-protozoal drugs are needed. Compounds targeting the epidermal growth factor (EGF) receptor, pp60v-src and pp110gag-fes, could be effective against Cryptosporidium spp., while compounds active against protein disulphide isomerases, specifically PDI2 and PDI4 as recently reported (Müller et al., 2007), could also represent promising targets for new drugs effective against Cryptosporidium spp. and G. duodenalis, respectively.

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Index

Page numbers in bold refer to illustrations and tables Acetate, octreotide 473 Acetic acid 363 Acetyl-CoA conversion 363 Acquired immune deficiency syndrome (AIDS) 133, 211, 464–465, 467, 469 Actin anti-actin filament agents 295–301 binding proteins 420, 421, 423, 425 cytoskeleton 418–426 dependent, endocytosis 304 depolymerization 295, 298, 299 host cell 418–426 lipid molecules movement 306 reorganization 418–426 role 293, 296, 302 Actomyosin 419, 423 Adenosine triphosphate (ATP) concentrations 201, 203 production 362, 363, 364, 366 synthesis 353 Age-related infection cattle 68–71, 110, 112, 125 children 51, 228, 233 Albendazole bodipy-ceramide recycling effect 299 effectiveness 464, 476, 477 fluorescence intensity 295, 296

NBD-phosphatidylglycerol recycling effect 301 NBD-sphingomyelin increase 300 Algorithms 148–152, 154 Allelic sequence heterozygosity 86–87 Allergies 436–437, 456 Alum 207 Alveolata 219, 346 Amino acids alignment 371, 372 changes 371 conservation 373, 376 Cryptosporidium synthesis capacity lack 363 divergences 374 identity 372, 373 tryptophan 447 Amino terminal targeting sequences 286 Amplicons 219, 220 Amplification 101, 102, 220–221 Amylopectin 364, 365 Analyte 153, 154 Animals companion 29, 109 Cryptosporidium spp. zoonotic genotypes 124 domestic 4–5 health 94–104

483

484

Index Animals continued human-to-animal infection 111–112 immune response models 452–453, 454, 455 infections, diagnostic methods guidance 149 infectivity experiment 191 reservoirs, zoonotic 65 see also Cattle; Pigs; Sheep; Wildlife Anthropogenesis 101–102, 142 Anti-cytoskeleton agents 299, 300, 301 Anti-microtubule agents 295–297 Antibiotics 466–467 see also Azithromycin; Paramomycin Antibodies 2-DE Western blotting use 336–337 anti-tubulin 293 cross-reactivity 262 giardiasis control 453–454 host development 445 humoral immunity role 453–454 immunoglobulin A 453–454, 458 infection level enhancement 446 labelling 180, 190 milk 336 production 333–334 proteins detection 285–286 reactivity, adhesive disc proteins 331–332 staining 194, 199, 214, 294 therapy 468 thymine dimers detection 191 UV-induced damage detection 206 Anticoccidial agents insensitivity 346 see also Disinfection, resistance Antigens detection 151–152 encystment 384 Giardia 334 host immune-dependent pathological pathways activation 433 luminal, translocation 430–434 peptide 445 vaccines and diagnostic tools development 336–337 variation 453 see also Serotypes; Variant-specific surface proteins (VSP) Antioxidants 180, 183, 186, 193 Antiretrovirals 464, 465, 467–468

Apicomplexans 259, 263, 355, 369–379 Apicoplast 263, 346, 364 Apoptosis 188–191, 193, 430–436 Aquaporin 425 Archigregarines, Cryptosporidium similarities 260 Archives 103 Arginine 338–339 Arp2/3 complex proteins 421 Ascogregarina 377, 378, 379 Assemblages Giardia 2, 3, 6–7, 81–82, 84, 108 naming 28 swapping 87 see also Species Atovaquone 473 Attachment 1, 309, 345, 411–415 see also Discs Axonemes, caudal 279, 280, 281, 292 Azithromycin 464, 465, 466–467

Babesia bovis 378 Bacteria 160–161, 163, 165, 245, 338 Barcode approach 36–45 Basil, Cyclospora infection 249 Beavers 5, 84–85, 95, 96, 97, 101 Benzimidazoles 464, 475–476 see also Albendazole; Mebendazole Berries, Cyclospora infection 249–250 b-giardin 268, 271, 332 b-tubulin gene 325 b2-microglobulin 454–455 Binomen 26 Biogenesis 409–415 Biology cells 409 Cryptosporidium 255 division, process 272 excystation 314 Giardia lamblia 266–282 typing methods 139–140, 159 Birds 59, 73–74 Blebbistatin 425 Blepharisma 203, 205 Boron-dipyrromethene (bodipy) 293–294 see also Ceramide (bodipy-cer); Dyes Bovinae 12–20, 45, 47 see also Cattle; Ungulates

Index

485 Calmodulin 311, 315–316 Cancer 404, 470 Candida albicans 404 Carbamates benzimidazole 464 see also Albendazole; Mebendazole Carbon-flow 360, 361, 363, 364 Catchments, determination 229 Cattle age-related infection 68–71, 110, 112, 125 cryptosporidiosis 12–20, 68–71 giardiasis 5, 85, 95, 111–112 human health risk 124 infection prevalence 110–111 infection susceptibility 112, 125 mouse genotype susceptibility 45–46 oocysts shedding 125–127 zoonotic transmission 85, 97, 124 Cdc42 protein 421, 422–423 cDNA library construction 322–323 Cells biology 409 death 188, 435 see also Apoptosis entry, host 423 epithelial 443, 457–458 immune activity 443 intestinal epithelial 337 mast 456–457 motility 419, 423 natural killer 444, 446 responses 457–458 structure, Giardia 268 Ceramide (bodipy-Cer) distribution 295 internalizing and targeting 302 intracellular trafficking role 297, 298, 305 lipid trafficking role 294 recycling 299, 303–304 uptake 305 see also Boron-dipyrromethene (bodipy); Dyes Cercomonas intestinalis 147 Cercosporamide 404 Cervids, Norwegian 98 Cetaceans 99 Chaperonins 352 Chemokines 457 Children cryptosporidiosis

age-related 51, 228, 233 dehydration 132 developing counties 55, 57, 212 growth, shortfall 60, 134, 135 mental development 134 multiple episodes 59–60 nutritional effects 60 gardiasis childcare centres, prevalence 473 clinical impact 4 prevalence 2, 451 transmission 3–4 malnourished 465 Chloride 429, 434 Chlorination 158, 207, 242–243, 244, 245 Cholangitis 470, 474 Cholecystokinin (CCK) 456–457 Cholesterol 383 Citations, molecular edidemiology 139 Clarithromycin 473 Clones 88–89, 353, 361 see also Reproduction Coccidian 346, 376–379 Colchicine bodipy-Pam cytoplasmic aggregates formation 297 ceramide recycling role 299 lipid uptake decrease 295 phosphatidylglycerol recycling 301 sphingomyelin recycling 300 tubulin site 475 ventral discs fragmentation 306 Coliphages 160, 161, 163, 165, 177 see also MS2 phage Collagen, crosslinking 183 Common variable immunodeficiency (CIVD) 453 Complexes, molecular 339 Consensus 46–47 Contamination determination methods 216–221 drinking water 83, 235, 236, 344 environmental 83 food 83, 212–213 human source 96, 99, 101–102, 115 manure use 109 recreational water 85, 109, 234, 235, 236 vegetables 216, 252 see also Water, contamination

486

Index Control methods 158–177, 181, 238–246 see also Disinfection; Drugs; Intervention; Treatment; Ultraviolet (UV) disinfection Copro-antigen tests 152 Coquitlam watersheds 158–177 Cortactin 423 Cost, diagnostic tests 153 Coyotes 71, 95, 101 Crosslinking 183, 192 CryptoDB database 350 Cryptons see Mitosomes Cryptosporidiosis causes 344 clinical signs 13 distribution 55, 56, 57, 228 drug treatment 464–473 genotyping 141–142 intervention impact 230–231 outbreaks 234 risk, drinking water 236 seasonality 228, 230 treatment, see also Drugs Cryptosporidium C. andersoni 17 C. felis 29, 33 C. hominis 57, 179, 233, 371–372 C. muris 320–326, 371–372, 373 C. parvum comparisons other Cryptosporidium species 371–372, 373 differential proteomics analyses 348, 351 energy metabolism and energy flow 260–266 immune responses 442 infectious dose 213–214 transport 126 UV-induced damage detection 179–194 Cryptosporidium Large Secreted Protein (CpLSP) gene family 373 Cryptosporidium parvum virus (CPV) 16 Cryptosporidium Reference Unit, UK 149, 154 Culture maintenance 242–243 Cyclobutyl pyrimidine dimers (CPDs) 179, 180, 183, 190, 192, 193–194 Cyclospora cayetanensis 248–252 Cyst, formation see Encystation

Cyst wall proteins (CWPs) aggregation 410, 412 differentiation processes 399 encystation-specific vesicles biogenesis role 412-413 environmentally resistant 409 functional role 412 giardin link 385 novel 384 organelle biogenesis into 310, 414–415 synthesis 410–411 transporation 329–330 Cyst wall synthase (CWS) 385, 391–393, 394, 410 Cysteine 410, 412 Cysteine desulphurase (IscS) 285, 286 Cytochalasin-D ceramide recycling 299 fluorescent intensity decrease 298 lipid incorporation reduction 297 lipid uptake decrease 295 motility abrogation 423 phosphatidylglycerol recycling 301 phosphatidylglycerol uptake increase 303 rapid internalization block 424 Sphingomyelin (NBD-SM) release 300 tubular/vesicular structures formation induction 302 ventral discs fragmentation 306 Cytokines deficiency 455 immunity role 443–444, 445–446, 457 Interferon-gamma 442–448 interleukin-23 gene 446 parasite elimination role 456 production 457 protective role 455 tumour necrosis factor 455 Cytoskeleton actomyosin 419, 423 anti-cytoskeleton agents 299, 300, 301 dynamics, Cryptosporidium induced 418–426 effectors injection 418 gene transcript abundance 333 giardial 268, 273, 310

Index

487 lipid transport 292–306 modulation, actin 418 network 292–306 Cytosol 353

Dairies 110, 128 Databases 36, 40, 350, 370 Definitions, need, Giardia; Cryptosporidium 135–136 Dehydrogenase 352, 353, 364, 366, 475 see also Enzymes, glycolytic Detection methods DNA extraction 217, 221–222 fluorescent staining 151 guidance 151 kits 151–152, 213 molecular 17–18, 81–90, 217 protein kinase C isoforms 402 protocols 213 sensitivity determination 219 UV-induced damage 179–194 see also Labelling; Microscopy; Staining Detergent 270, 271, 272, 279 see also Disinfection Diagnosis 147–155 Diamidino-2-phenylindole (DAPI) 189, 194, 199 Diarrhoea AIDS-related 447, 469 cause mechanisms 437 cryptosporidiosis 13, 14, 58, 60, 131, 134 Cyclospora cayetanensis infection 248 daycare centre outbreak 57 definition need 136 drugs 132, 473–474 drugs adverse effect 467 environmental factors 473 giardiasis 5, 7, 451, 473, 474 hypersecretion 429 immunocompromised people 133 malabsorption 429 mortality 473 resolution 469 roxithromycin treatment effect 468 severity variation 15 see also Symptoms

Differentiation gene transport abundance 333 Giardia secretory organelle biogenesis 414 IFN-y role 444 importance, Giardia life cycle 309–310 polyphyletic 54 processes 399 protein expression 349 signalling 309–316 trophozoites into cysts 329, 383, 412 see also Encystation; Encystment; Excystation; Life cycle Dihydroxyisoflavone 471, 473 Dimers 180, 183, 190, 191, 192, 193–194 Disaggregation 167 Discriminatory power 140–141 Discs adhesive 268, 269, 270, 278, 279, 280, 331 attachment 1, 309 protein 332–333 ventral 1, 266, 267, 268, 309, 331 Disease birds, Cryptosporidium manifestation 73–74 foodborne 85, 249–250, 344 intestinal, cause 309 reporting 155 risk assessment 221 sources tracking 221 waterborne 227–236 see also Infection; Symptoms Disinfection capability determination 182–184 chlorine 158, 242–243 coliphages 163, 165–166 Cryptosporidium 163, 165, 173 degradation 165 disinfectant decay 244 guidance 239 kinetics 245–246 MS2 phage 163, 165, 169–170, 171–173, 173–174 Naegleria fowleri 243 ozone 158, 169–171, 171–173, 175–176, 177 resistance 198, 212, 245, 251–252 turbidity levels 165–166, 169

488

Index Disinfection continued water 158–177, 181, 192–194 see also Chlorination; Detergent; Ultraviolet (UV) disinfection Dissolved organic carbon (DOC) 206 Ditrypanocystis sp. 262, 263 DNA (deoxyribonucleic acid) crosslinking 183, 192 damage assessment 206 evidence 41 extraction 217, 221–222 lesions 193–194 reporters 186, 190 sequencing 34, 52, 215 UV disinfection damage 183–184, 190, 191, 193–194 Dogs 2, 7, 29, 250–251, 252 Domains 373–376, 378–379 Double-stranded RNA (dsRNA) virus 16, 89 Drinking Water Management Plan (DWMP) 158 Drugs adverse effects 475 anti-diarrhoeal 132, 468, 473–474 antiprotozoal 463–477 see also Nitazoxanide cryptosporidiosis treatment 464–473 Cryptosporidium glycolytic enzymes, against 366 giardiasis treatment 4, 393–394, 473–477 microtubule-depolymerizing 303 see also Nocodazole; Taxol resistance 463, 464, 475, 476, 477 targets 369–370, 394, 404 see also Treatment Dyes apoptosis investigation 188–190 boron-dipyrromethene 293–294 fluorescent 182, 293–294 fluorogenic 186, 187–188, 193 Mito Tracker Red 288 YO-YPRO1180, 188, 189, 193 see also Ceramide (bodipy-Cer); Markers; Palmetic acid (bodipypam); Stain

Electrons 353, 419 Emergence, Giardia 97, 100

Encephalitozoan intestinalis 447 Encystation 239–240, 310–314, 410 see also Encystment; Vesicles, encystation-specific Encystment 382–394, 399, 401 see also Encystation Endemicity 85, 87, 251 Endocytosis 302, 304, 305, 306 Endoplasmic reticulum (ER) 294, 410, 411, 413 Energy 360, 361, 363, 366 see also Metabolism Enolase 338, 339 Entamoeba histolytica 152, 285, 474 Enterocytes 433, 434, 437, 447–448 Enterotoxins 434, 437 Enumeration 160–161, 213–214 Environment 3, 4, 216–221 Enzastaurin 404 Enzyme immunoassay assay (EIA) 151–152 Enzymes 230, 234, 366, 386–391, 392, 447 see also Dehydrogenase; Enolase; Hexokinase; Proteases; Protein kinase; Proteins Epidemiology cryptosporidiosis 51–61, 154 giardiasis 83–85 molecular 6–7, 51–61, 65–75, 138–144 outbreak investigations 143 tools 54 Epidermal growth factor (EGF) receptor 470–471, 473, 477 Epimerase reaction 393 Epithelium 429–436, 443, 457–458 Escherichia coli 139, 140, 159, 336 Ethanol 363 Evaluation 152–154 Evolution see Phylogenetics Exchanges, genetic enhancement 89 Excystation biology 314 cellular awakening from dormancy model 310 procedure 170 proteins 333, 352 reduction, UV irradiation 184 regulation 309 signalling 314–316

Index

489 Excystment 399 Excyzoites 314 Ezrin 420

Fatty acids 293–297, 302–303, 305, 346, 363 Fecundity 16 Feeding 262, 345 Ferredoxin 286, 287–288, 469, 477 Fertilization 257–258 Fish, Cryptosporidium 41, 74 Flagella caudal 279, 280, 281, 282, 292 giardial 267, 268, 269, 292, 309–310 posterior-lateral 276, 279, 281 see also Microtubules Flagellates, intestinal 81 Fluorescence actin 420 dyes 182, 293–294 green fluorescent protein 286 intensity 295, 296, 297, 298 labelling 286 lipids analogues 295–301, 298, 303 microscopy, epifluorescence 189 stains 151, 199 Food contamination 114–115, 213–214, 216–221 infection, transmission 85, 107–116, 249–250, 344 livestock, infection transmission role 107–116 monitoring 210 iciest contribution 212–213 Formononectin 464 Foxes 101 Frabin 422 Fructaose-6P 363 Funis 266, 272, 276–277, 279, 280, 281 Furazolidone 464, 477

Gastroenteritis 132–133, 134, 148, 249, 255, 292 Genes differential expression 365 divergence 373 expression, analysis 347

identification use 18 mapping 82 marker 18, 19, 36, 40, 52 mitochondrial 285 protein encoding 328 sequencing 40, 53, 285, 320–326 tubulin 292–293, 302 see also Genetics Genetic loci 82–83, 218 Genetics 89–90, 138–144 see also Genes Genistein 421, 471 Genomics 320–326, 344–356, 361 Genotypes avian 73–74 bovine 45, 47 cattle 17, 68–71 fish 74 humans 45, 47, 54–56, 215 livestock 111–113 mouse 45–46, 47, 48, 54 pig 66–68 reptiles 74–75 wild animal 71 zoonotic 111–113, 123–128 Genotyping Code of nomenclature exclusion 28 cryptosporidiosis 141–143 Cryptosporidium 60, 65, 210–222 distribution 229 Giardia 8, 82–83, 143–144 identification 214 infection sources indicator 143 methods 21–26, 139–140, 154, 210–222 microbial 143 molecular 82–83, 86–89, 133–135 multilocus 53–54 new species naming 46–47, 48 polymerase chain reaction 191–192 sample 154 scheme standards 140 tools 52 typability 140 variables 141 see also Markers; Subtyping Giardan 382–394 Giardia G. canis 28, 29 G. duodenalis (syn. G. intestinalis; G. lamblia) 83, 84–85, 107, 435

490

Index Giardia continued G. enterica 148 G. intestinalis 148, 284–289, 452, 453 G. lamblia 266–279, 292 G. muris 170, 172 Giardia specific virus (GLV) 89 Giardiasis cause 266, 398 drug treatment 4, 393–394, 473–477 effect 432 foodborne transmission 85 immune response 451–458 inhibition 393 prevalence 451 symptoms 3, 409, 452 Giardins 268, 271, 332 Glucosamine 386–387, 389–390, 393, 394 Glucose 385–386, 436, 437 glutathione 180, 183, 186, 187, 193 Glycerol 363 Glycogenesis 364 Glycolipids 262, 363 Glycolysis 360, 361, 363, 366 Glycoproteins 19–20, 52, 221, 363, 372, 434 Glycylation 292, 339 Golgi function 410 Golgi markers 330 Gorillas 5 Greater Vancouver Regional District (GVRD) 158, 177 Gregarines 259, 262, 346, 378 Guanine nucleotide exchange factors (GEFs) 422 Guidance Cryptosporidium symptomatic patients 149 disinfection 173–174, 239 Gymnodinium 219

Haemosporidia 378 Hapantotype 35 Harmonization of Laboratory Diagnostic Methods of the National Surveillance Group on Diseases and Infections of Animals 149 Health animal 94–104

burden and management 451–452 human 94, 124 Heat shock protein (Hsp60) localization data 353 Hedgehog-type HINT predicted secreted protein 376 Hegner, R.W. 28 Height-for-age (HAZ) Z scores 60 Hepatitis, granulomatous 474 Heterotrophic plate count (HPC) 159, 160–161, 163, 165 Hexokinase (HK) 362 see also Glucose Hexoses 362 Highly active antiretroviral therapy (HAART) 133, 464, 467, 469–470 Histone, H1 phosphorylation 403 Holotype 35 Hom’s model 245, 246 Horses 112 Host-parasite relationship 262, 419, 434–436 Hosts actin cytoskeleton dynamics 418–426 adapted species 124 cell entry 423 cell interaction 262–263 common names use 41 giardial 3, 4–5, 99, 100, 101 human 181, 211, 229 immunocompromised 55, 132–133, 181, 211, 212, 255, 372 infection location within 259 infection susceptibility markers 138 ranges 2, 37–39, 108–109 specificity 33–34, 46, 72, 82, 94–95, 96, 108 sporozoite interactions 419 switching 101 variety 65 Human Cryptosporidium spp. differences 181 food contamination source 115 genotypes 47, 54–56, 58–60, 215, 229 giardiasis 2–4, 84 health 94, 124 molecular epidemiology, infections 51–61

Index

491 mouse genotype susceptibility 45–46 susceptibility 211 to-animal transmission 111–112 to-human transmission 47, 113 transmission cycle inclusion 96 water contamination 96, 99 zoonotic strains in wildlife implications 101–102 Human immunodeficiency virus (HIV) antiretroviral treatment 464, 468 asymptomatic cryptosporiodosis 134 Cryptosporidium 55, 56 cryptosporiodosis attack rate 133 Giardia susceptibility lack 454 Humidity 250 Hybridization 88

Identification infection sources 216–217, 221 markers 138, 139 methods 17–18, 31–48, 214–215, 219–220 proteins 328–340, 349, 350 species 31, 214 strains 103 transmission routes 216–217 see also Detection IFN-y 445–448, 454, 455 Immune response 133, 442, 443–448, 451–458, 464 Immunity cell-mediated 14–15, 443 cellular 454–456 compromised 133, 465 cytokines role 443–444, 445–446, 457 humoral 453–454 immuno-invasion,443 lymphocytes role 443–444, 454–455 role, Cryptosporidium clinical presentation 135 status 14–15 suppression 132–133, 344, 465 see also Hosts, immunocompromised; Lymphocytes Immunoassay 150, 151–152 Immunochomatography 151, 152 Immunodeficiency 132–133, 321, 453, 464, 465, 474

see also Acquired immune deficiency syndrome (AIDS); Hosts, immunocompromised; Human immunodeficiency virus (HIV) Immunodominance 336 Immunoflorescence microscopy (IFM) 151, 153, 154 Immunoglobulin 453–454, 456, 458 Immunogold labelling 279 Immunomagnetic separation 154, 160, 213 Inactivation chlorine 207 kinetics 171 MS2 174–175, 176 Naegleria fowleri 239 solar 199, 203–206 table 175 temperature effect 199, 201–202, 204 see also Disinfection; Ultraviolet (UV disinfection) Incubation period 207 Index, discriminatory power 140 Indinavir 467 Indoleamine 2, 3-dioxygenase (IDO) 447 Inducuble pyrophosphate (iUap) 387 Infection asymptomatic 5, 134–135 establishment 419, 425 immunological control 442 impact 148 method, cyst ingestion 249, 259, 309, 399 mixed 59–60, 86 molecular epidemiology 51–61, 138–144 oocysts role 33 pathogenic mechanisms 428–437 poverty link 3 prevalence 18–19, 66, 110–111, 114–115 see also, Transmission; Disease severity 15 sources anthropogenic 101–102, 115, 142, 234 foodborne 115–116, 234 identification 216–217, 221 water 234

492

Index Infection continued sources continued zoonotic 97, 101, 111–112, 127, 142, 234 see also Animals; Food; Water susceptibility 14–15, 45–46, 112, 125, 138, 180–181, 211 symptomatic 4 time to 126 waterborne 85, 107, 181, 186, 227–236, 249–250 see also Cryptosporidiosis; Giardiasis; Reservoirs, zoonotic Infectious dose, Cryptosporidium parvum 213–214 Infectivity 199, 200, 207, 425 Inflammatory bowel disease (IBD) 135, 436 Ingestion 204, 205, 249, 259, 309, 399 Inheritance 289 Inhibitors infectivity 425 protein kinase 401, 403, 404–405, 421, 425 pyrozole 366 respiratory 361 Injury, mucosal 429 iNOS, enzymes 447 Insensitivity anticoccidial agents 346, 361 see also, disinfection, resistance Interferon-gamma (IFN-y) 442–448, 454, 455 Interleukin (IL) 446, 455 International Code of Zoological Nomenclature 26, 27–28, 35 International Commission on Zoological Nomenclature (ICZN) 26, 32 International Giardia and Cryptosporidium Conference 135 Intervention 133, 227–236 see also Treatment Intestine barrier function 434 disease, cause 309 disorders 429, 436–437 see also Diarrhoea epithelial cells 337 flagellates 81 parasites 4 permeability 430, 434

small, mesenteric lymph node 455 Introgression 88 Invasion mechanisms 420, 423 see also Discs Inventory 102–103 Investigations of Specimens other than Blood for Parasites 149 Ions, metallic 183 Iron-binding scaffold protein (IscU) 285, 286, 287, 288–289 Iron-sulphur 284, 285, 353 Irritable bowel syndrome (IBS) 135, 456 Isoflavones 464, 470–473 Isolates 15, 65–75 Isolation methods 213–214, 322 Isomerases, protein disulphide 477

Kinase see Protein kinase Kinetics 171, 366 Kits 152, 213 Krebs cycle 360, 366

Labelling 180, 279, 286, 288, 293–297 Laboratory Detection of Parasites 149 Lactate 363, 364, 366 Lactoferrin 134 Lasalocid 465, 473 Lateral gene transfer (LGT) 364 Lesions 190, 191 Letrazuril 473 Lettuce, Cyclospora infection 249 Leucine 410, 412 Leucocytes, peripheral blood 454 Leukemia 470 Libraries 322, 323 Life cycle Cryptosporidium 148, 255–264, 345, 428–429 Cyclospora 249 gamont stage 258, 259 Giardia 309–310, 329, 399 Mattesia spp. 260, 261 Naegleria fowleri 239–240 polymorphisms 142, 143 stages detection 149, 151 see also Differentiation; Merozoites; Oocysts; Trophozoites Lipid/fatty acid molecules incorporation and distribution 302

Index

493 Lipids fluorescent analogues 295–301, 298, 303 glycolipids 262, 363 membrane 293–297 protein-mediated uptake 306 signalling pathway 399 sphingolipids 305 transport 292–306 Livestock 2, 96, 107–116 see also Cattle Localization b-giardin 271, 332 extracytoplasmic intracellular 263 protein kinase 311 protein phosphatase 2A-C 311 proteins 352, 353 tubulin 275 Location, Cryptosporidium 345 Loci, genetic 217–219 Lymphocytes brush border abnormalities mediation 430 CD4+ T cells activation 447 adaptive immune response initiation 443 anti-CD4 depletion 455 antiretroviral therapy effect 464, 468 cellular immunity role 454–455 count levels 465, 466, 470 cryptosporidia elimination 442 immunity establishment 445 CD8+T cells 443, 445, 457–458 immune response role 442, 443–448, 454–455 parasites response 14 Lysates 355

Machines, molecular 339 Macrophages 443, 444 Major histocompatibility complex (MHC) expression 447, 455 Malabsorption 134, 135, 429–430, 452 Malate 364, 366 Malnourishment 465 Malonyl-CoA 363 Mammals 46, 82, 95, 97, 99–100

Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 149 Manure 109, 112, 124, 125, 127, 231 Maps, biochemical, Cryptosporidium 355–356 Marine ecosystems 99–100 Markers 60-kDA glycoprotein precursor (GP 60) 40 Cryptosporidium detection 36, 41, 134, 135, 142 faecal lactoferrin 134 genetic 221 Golgi 330 identifying 138, 139 import 305 microsatellite 142 Mito Tracker Red 288 results unreliability 87 satellite 221 virulence 139 see also Actin; Dyes; SSU rRNA (small subunit ribosomol RNA) gene; Stains Marsupials 71 Mastocytosis 456 Mattesia spp. life cycle 260, 261 Meat 115 see also Livestock Mebendazole 464, 475–476 Mechanism, molecular 418–426 Median body 266, 268, 269, 271–272, 273–275, 309–310 Meiosis 88 see also Replication; Reproduction Membranes alteration 424 compartments organization 409–415 extensions 425 fusion, SNARE-mediated 411–412, 413–414 host, modification 419–420 intracellular, fusion 411 lipid 293–297 mitochondrial 353 parasitophorous vacule membrane 419 plasma membrane removal 277–282 protrusion 425, 426 selection 413 vesicles-associated 414

494

Index Membranes continued see also Organelles, membraneenclosed Merogony 260 Meronts 256–257, 260, 261 Merozoites 256–257, 260, 261, 419 Mesenteric lymph node (MLN) 455 Metabolism 346–347, 350–352, 360, 361–363, 366 Metabolites 469, 470 see also Cytochalasin-D Metronidazole (MTZ) 393–394, 463–464, 474–475, 476–477 Mice 46, 47–48, 54, 72, 455 Microbiology, typing methods 139–140, 159 Micronemes 355 Micronutrients deficiencies 135 Microorganisms 159–160, 166–167 Microscopic particulate analysis (MPA) 159, 160–173 Microscopy confocal 288 diagnosis benchmark 147–155 epifluorescence 189 field emission scanning electron microscopy 266, 274, 277–282 immunoflorescent 179–194 oocysts detection 17–18, 214 Microtubules antimicrotubules agents 295 bundles 269 cytoskeleton 270, 271, 272, 273, 277, 278, 279 depolymerization 295, 298, 299, 303 dynamism 293 filaments role 292, 293, 296, 297, 302, 306 funis 266, 272, 276–277, 280, 281 network 297–301, 302 thickness 280 see also Flagella; Median body Microvilli 262, 429–430 Milk, antibodies 336 Mitochondria 284–285, 346–347, 352–354 Mitosomes 270, 284, 286, 287, 289 Mitrogen-activated protein (MAPK) signalling 312–313

Mixtures, Cryptosporidium species/ genotypes 219–221, 222 Modifications, posttranslational 339 Molecules, deprivation 444 Molluscs 115 Monochlorobimane (MCB) dye 186, 187–188, 193 Morbidity 16–17 Morphology, oocysts 262 Mortality 16–17, 452, 473 Most probable number (MPN) technique 243 Motility 419, 423, 456 Mouse genotype 45–46 MS2 coliphage assays 167 disinfection dose response 163 inactivation 171, 174–175, 176 ozone disinfection 169–170, 171–173 UV disinfection 166–169, 173–175, 177 Multilocus sequence typing (MLST) 54 Multilocus typing (MLT) 53–54 Multiplication 249 see also Biogenesis; Reproduction; Sex Muskoxen (Ovibos moschatus) 5–6, 98–99 Mysosins 425

N-acetylglucosamine pyrophosphorylase (Uap), activities 389, 393 N-glycans synthesis 363 N-WASp activation 421–422 NADPH oxidoreductase 362–363, 364 Naegleria fowleri 238–246, 245 Names Cryptosporidium spp. 25–30, 35–36 see also Nomenclature; Species; Taxonomy Natural killer cells (NK) 444, 446 Neglectd Disease Initiative 2–3, 102, 135, 148 Neospora caninum 471 Neotype 27–28 Nitazoxanide anti-giardial action 476–477 concentration 466–467 cryptosporidiosis treatment 132–133, 464–465

Index

495 efficacy 472 metabolites 469, 470, 476 uses 469–470 Nitric oxide 436, 444, 447, 457 Nitroimidazoles 463–464, 474–475, 477 Nocodazole 295, 297, 298, 299, 300, 301 Nomenclature 25–30, 37–39, 82–83 see also Taxonomy Nuclei 267, 268, 270 Nucleic acid isolation 322 Nucleotides 36–45, 179, 183, 185, 193, 363 see also DNA Nursing homes 473 Nutrients, deficiencies 135, 305, 394 Nystatin 305

plastid-like 346–347 proteins 355 remnant 270, 352 secretory 410, 411–412, 414–415 structure 355–356 sub-proteomics 355 see also Discs; Flagella; Mitochondria; Mitosomes; Vesicles, encystationspecific Ornidazole 474, 475 Ornithine 338 Orthologues 370, 372, 374, 376, 379 see also Phylogenetics Oxidase 353 Oxidative stress 183, 188 Oxidoreductase 362-363, 364, 469, 477 Ozone 158, 159, 169–173, 175–176, 177

Occurrence, Cryptosporidium species/ genotypes 214 Oocysts behaviour 181 detection methods 215, 217 faecal purification 347 inactivation, solar 203, 205–206 infection role 33 ingestion 205 morpho-functional 345–346 morphology 262 number needed to cause infection 135 production rates 15, 16, 17 resistant 114 shedding 60, 125–127, 134–135, 465, 469 size range 65 survival 125–127, 198 temperature effect 204 typing methods 210–222 UV disinfection 186–191 viability determination 182, 187–188 water treatment effect 198–208 Open reading frame (ORF) 403–404 Organelles apicoplast 263, 346, 364 biogenesis 410, 411–412, 414–415 composition 355 feeder 345 membrane-enclosed 352, 353–354 mitochondrial 270, 353

Palmetic acid (bodipy-Pam) 294, 295, 297, 302–303, 305 Paramecium sp. 203, 205 Paramomycin cryptosporidiosis treatment 465, 466–467 efficacy 132, 464, 469, 472 giardiasis treatment 476, 477 inhalation 470 Parasitology classical 103 enteric 4 host-parasite relationship 262, 419, 434–436 replication 447 web 102 Parasitophorous vacule membrane (PVM) 419, 420 Particulate analysis data, microscopic 162 Pathogenesis 1–2, 15, 16, 60, 428 Pathogens, access restriction 457 Pathology, Naegleria fowleri 239 Pathways b-oxidation 363 biochemical 347 conserved import 287–288 fatty acid synthesis 363 fermentative 362 glycolytic 361, 362 lipids synthesis 363 membrane-enclosed organelle 353–354

496

Index Pathways continued metabolic 369 mitochondrian 354 N-glycans synthesis 363 oocysts shedding, watersheds fault-tree model 125 oxygen radical scavenging 353 protein import 287–288 proteome chart 354 source to water reservoir transport pathway 126 synthetic 363–364, 386 see also Signalling Peptides 349, 350 Permeability, epithelial 430, 434 Pesticides 250 Phage, typing, Staphyloccoccus aereus 140 Phagotrophs 204 Phenotypes, Giardia 6–7, 8 Phlorizin 425 Phosphatases 339 Phosphate, sugar 393 Phosphatidylcholine (bodipy-PC) 294 Phosphatidylglycerol labelling 294, 297, 299 recycling 301, 303–304 regulation 302 transport 305 Phospho N-acetylglucosamine mutase (Pgm) 386, 387 Phosphoinositide, signalling pathway 399 Phosphorylation 339, 424 Photoreactivation 179, 184, 185, 193 Phylogenetics Cryptosporidium 53, 259, 263, 264 divergences 405 Giardia 1, 84 pyrophosphorylases 388 Pigs 66–68, 111, 112 Pinnipeds 99 Piroplasmida 378 Plasma, membrane removal 277–282 Plasmodium spp. genes 373 genome comparison with Cryptosporidium spp. 376, 379 host cell entry mode 423 orthologues 370 proteins, TRAP superfamily 375 surface coat proteins 378

Plastids 346–347 Polyglycylation 292 Polyketide synthase (PKS) 363 Polymerase chain reaction (PCR) analysis amplification 214, 217 detection method 154 genotyping 191–192 inhibition 219 Naegleria fowleri detection 245 PCR-restricted fragment length polymorphism analysis 215, 216, 218 primers 52, 221, 222 protocol 19, 241–242 species identification role 18 Polymorphisms 142, 143 see also Life cycle Polysaccharides 364, 384, 385 Pools swimming 234, 235 see also Water, recreational Potassium depletion 305 see also Nutrients, deficiencies Poverty link, Giardia infection 3 Power plant, Cryptosporidium spp. 344 Predation 199, 203, 204 Primates, non-human 5, 100 Primers 59, 218–219, 222 Priority principle of names 27 Probiotics 464, 468 Procedures 243 Proteases 434, 467–468 Protein disulphide isomerases 477 Protein kinase b-like molecular characterization 400, 402–404 c-SRC 422–423, 426 cascades 423 Giardia duodenalis 398, 400–405 Giardia-specific 339 glycerol 363 hexokinase 362 inhibitors 401, 403, 404–405, 421, 425 isoforms detection 402 localization 311 molecular weight 401 pharmacological target potential 404–405 Pp60v-src 470, 477 Pp110gag-fes 470–471, 477

Index

497 pyrophosphate-dependent phosphofructokinase 362, 364, 365 signalling 311–312, 313–316, 399 tyrosine-specific 470–471 Protein phosphatase 311, 312, 313–314, 315–316 Proteinases 434, 437 Proteins conservation 377 encystation-specific 329–330, 410 expansions 372 expression 347–352, 354, 401 extracellular 370, 371, 372, 373–376, 378, 379 fatty acid-binding 302 fusion 286, 366 gene encoding 373 import 286, 287–288, 288–289, 353 inhibitors 425 iron-binding scaffold 284, 285 LCCL domain-containing family 378–379 lineage-specific 370, 372, 373–376 lipid transport 305 multi-domain 376 orthologues 376 precursor 287 secretory 337–339, 370, 373, 411 signalling 309–316 surface 330–331, 334, 336, 337, 369–379, 418 synthesis inhibition 183, 476 trafficking 411, 414 translocators 305 transport role 302, 329–330, 410, 413–414 TRAP superfamily 375, 376 ultraviolet (UV) alteration 183, 192 see also Actin; Cytokines; Enzymes; Kinase; Proteomics Proteomics 8, 328–340, 344–356 see also Proteins Proton pump transhydronase (PNT) 353 Pyrophosphate 360, 362, 364, 365 Pyrophosphorylases 388, 389, 390, 391, 393 Pyrozole 366 Pyruvate 362, 363

Quality test 152–154 Quenda (Isoodon obesulus) 6, 101 Quinacrine 474

Reactive oxygen species (ROS) 183, 186–188, 193, 353 Reagents 242 Receptors 411–415, 434, 445, 470–471, 477 Recombination 7 Regulations 230, 234 Repair 179, 183–184, 185, 191, 193 Replication 447 see also Reproduction; Sex Reporters 186, 190 Reproducibility 140, 141 Reproduction 7–8, 88–89, 260, 447 see also Sex Reptiles 74–75 Reservoirs water, characteristics 206 zoonotic cattle 13, 85, 111–113 dogs 250–251 farm animals 65 livestock 113–114 sheep 113 wildlife 65, 101–102, 124 Resistance chlorine 245 disinfection 184, 198, 212 drugs 463, 464, 475, 476, 477 environmental conditions, Cyclospora 252 host, Cryptosporidium 445 metronidazole 463 oocysts 114 sanitizers 251–252 Restricted fragment length polymorphism (RFLP) analysis 54, 215, 216, 218, 221 Reticulum, endoplasmic 294 Reversibility 46–47 Rho factor 421–422 Rhoptries 355 Ribonucleic acid (RNA) 333 Risk assessment 221, 232, 234 cryptosporidiosis 123–128, 132, 135, 142

498

Index Risk continued factors 4, 235–236 groups 135 human health 101–102, 124 identification 221 Ritonavir 467 Rodents 71–72 Rods (fibrous) 280, 310 Roxithromycin 468 Ruboxistaurin 404 Ruminants, asymptomatic Giardia infection 5

Salmonella, serotyping 140 Samples, testing 151, 152, 154 Sampling data, Coquitlam 161 Sanitizers 251–252 see also Disinfection Saquinavir 467 Sarcocystis neurona merozoite 471 Scanning electron microscopy (SEM) analysis 273, 276, 277, 278 Scar1 (Scar-WA), proteins 421 Schizogony 260 see also Reproduction Seasonality 95, 228, 230, 249, 250, 251 Secnidazole 464, 474, 475 Secretion chloride 429, 434 electrolytes 429 granule biogenesis 409–415 hypersecretion, epithelial 429 organelles 410, 411–412, 414–415 pathway 411 proteins 337–339, 370, 376, 411 rhoptries 355 system 418 vesicles 310, 329–330, 384, 391, 410, 412–413 Selection 15–16 Selenidiidae 260 Sensitive testing 154 Sensitivity, analytical 151 Sequences 18S rRNA gene loci amino terminal targeting 286, 289 analysis GP60 19–20, 52 barcode approach 36–45 Cryptosporidium genome 214–215, 347

Cryptosporidium muris 16, 40–41, 44, 320, 323, 326 Cryptosporidium spp. comparisons 371 Giardia spp. 8, 328–340 incomplete 41, 45 internal targeting 288–289 Naegleria fowleri 242, 286, 288–289 oocysts genotyping 217, 218, 219, 221 reference numbers 41 Giardia duodenalis 83 reference sequences, Cryptosporidium spp. 42–43 SSU rRNA (small subunit ribosomol RNA) gene 214–215 see also Genotyping Serotypes 140 Serum 334, 336 Sewers 251, 473 Sex 7–8, 88, 249 see also Reproduction Sheep 66, 72, 111, 113 Side-effects 475 Signalling cell, posttranslational modifications role 339 Cryptosporidium-induced cascades 421–423 differentiation trigger 383 extracellular signal-regulated kinase 311, 312–314, 399 host 418, 419, 422–423, 425, 426 lipids 399 organelle targeting 288–289 phosphoinositide 399 protein 309–316, 399, 422–423, 426 targeting, processing 286–287 transduction, protein kinase C role 400 Sinefungine 465 Sodium-coupled-glucose transporter-1 (SGLT-1) 436, 437 Solar inactivation 203–206 inactivation, see also Ultraviolet (UV) disinfection Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 411–415

Index

499 Species aggregates 26 behaviour differences in humans 60 classification 34–35 definition 34–35 detection, human-infectious 215 determination 222 Giardia 2, 3, 108 identification 31, 214 infecting cattle 17–18, 18–19, 68–71 infecting humans 54–56, 58–60, 108–109, 229 infecting sheep 66 livestock infection 111–113 names 25–30, 36, 37–39 prevalence 18–19 see also Assemblages; Nomenclature; Taxonomy Specificity 153, 154 Sphingolipids recycling 305 Sphingomyelin (NBD-SM) 294, 295–297, 300, 302, 303–304, 305 Spill-back 101 Spiramycin 473 Splitters 28–29, 35 Spores, total aerobic 159, 160, 161, 163, 165 Sporozoites aggregation 256, 259 differential proteomics analyses 348 differentiation 257 DNA 217 functional categorization 351 fusion 259 host cell entry 419 infective 262 internalization 419 motility 419 proteome 349 SSU rRNA (small subunit ribosomol RNA) gene analysis 60-kDA glycoprotein precursor (GP 60) 40, 52 complete sequence 214–215 Cryptosporidium detection 18, 19, 36, 40, 41, 52 misidentification problem 54 satellite, complete sequence 214–215 Staining iodide 180, 187–188, 189 iodine 152

techniques 148, 151, 199, 214 see also Dyes; Labelling; Stains Staining Procedures 149 Stains antibodies 214 diamidino-2-phenylindole (DAPI) 189, 194, 199, 214 fluorescent antibodies 199 nucleic acid 189 YO-YPRO1 nucleic acid 188 see also Dyes; Staining Standards 140, 153, 216–217, 221–222 Staurosporine 421 Stomach flu 98 Strains identification 103, 142 Subgenera 26 Subgenotyping 210, 222 Subspecies 26, 142–143 Subtyping Cryptosporidium 19–20, 57, 60, 221 Giardia duodenalis 83 tools 52–53, 59 see also Genotyping Sugars 362–363, 393, 435 Sunlight inactivation 199 see also Ultraviolet (UV) disinfection Suramin 421 Surveillance schemes 148, 155 Survey 102–103 Survival Cyclospora spp. 248–252 encystation role 310 Giardia 410 Naegleria fowleri 239–240 oocysts 125–127, 198 see also Encystation Swine see Pigs Symptoms Cryptosporidium infection 131–136, 211, 344, 428 Cyclospora cayetanensis infection 248 Giardia infection 4, 7, 333, 399, 409, 428, 452 lack 5, 134–135 see also Diarrhoea Syntaxin 414 Synteny 373, 374 Synthesis adenosine triphosphate 353

500

Index Synthesis continued cyst wall proteins 410–411 fatty acid 363 giardan 385–393 inhibition 183, 476 lack, Cryptosporidium 363 N-glycans 363 trehalose 364 Systematics 34–36

T cell antigen receptor (TCR) 445, 447 T cells see Lymphocytes Tail, movement, Giardia 279, 280, 281, 282, 292 Taxol 295, 297, 299, 300, 301 Taxonomy 2, 28–29, 31, 32–33, 108–109 see also Names; Nomenclature; Species Taxonomy of the genus Cryptosporidium website 45 Temperature effect 199, 201–202, 204, 240, 250 Templates, mixed 86 Terrestrial-marine cycle, Giardiasis 98 Tests and testing specialist 154 choice 152–154 collimated beam 166 compliance, Cryptosporidium spp. 149 costs 153 diagnosis 154 evaluation 152–154 performance monitoring 152 quality assurance 152–154 reference 154 samples 151, 152, 154 sensitive 154 Th cells see Lymphocytes Theileria spp. 376, 378, 379 Thymine dimers (TD) 179, 180, 183, 191, 192, 193–194 Tinidazole 463, 474, 475, 477 Tizoxanide 469, 470, 476 Tizoxanide glucuronide 469, 470, 476 Tools 18S rRna-based PCR-RFLP 219–220 alternative molecular 220–222

genotyping 52 molecular epidemiological 54 proteomic 329 subgenotyping 222 subtyping 59 Total aerobic spores (TAS) 159, 160, 161, 163, 165 Toxoplasma spp. 375, 377, 378, 419, 423, 444 Transmission complexicity 57 cyst ingestion 204, 205, 249, 259, 309, 399 cysts ingestion 451–452 developing countries 51–61 direct and indirect 84 faecal-oral 107, 109 faeces excretion 410 food 85, 107–116, 249–250, 344 human 3–4, 34, 47, 59, 96, 111–112, 113 investigations 221 livestock 2, 96, 107–116 paths identification 216–217 pathways 138 see also Infection, sources; Reservoirs, zoonotic; Water Transport channels 426 facilitation system 353 lipids 292–306 pathways 126, 413–414 proteins role 302, 329–330, 410, 413–414 sugars uptake 362–363 system components 353 vesicles 293, 304, 305, 411 Transporters 425, 426, 436, 437 Travel, infection source 234, 249, 473 Treatment cryptosporidiosis 464–473 detergent 271, 272 diarrhoea 468 drinking water improvement 230, 231–232, 234 efficacies 472 giardiasis 393–394, 473–477 highly active antiretroviral 133 targets 463–477 water 158, 179, 198, 206–207 see also Disinfection; Drugs

Index

501 Trehalose, synthesis 364 Trichomonas vaginalis 474 Trihydroxy-deoxybenzoin 471, 473 Triple Faeces Test 152 Trophozoites aggregates 256 body shape 266, 267 cytochalasin-D exposed 303 detergent extraction effect 270, 272 differentiation into cysts 412 emergence 309 encystation 310–311 encysting 383–384 Golgi complex lack 220 IscS transfected 288 organism infectious form 245 survival 409–441, 3990 visibility 148, 149, 151 see also Life cycle Trypsin-activated Giardia lectin (Taglin) activity 336 Tryptophan 447 Tubulin 275, 292–293, 302, 324, 325, 475 Tumour necrosis factor (TNF) 455, 456, 457 Turbidity adjustment 166 causes 159 control 181 disinfection 169 disinfection degradation 165 effect 167–168 evaluation 171–173 increase 161–162, 171–173 MS2 effect 169 ozone effect 171–173, 176 UV effect 166, 167–168, 176, 177, 183, 192 warning level 234 Type definition 27 Typing see Genotyping Tyrosine 470–471 Tyzzer, Ernest T. Cryptosporidium genus description 31, 131, 326 host-specificity 33 life cycle stages 33, 148 mouse genotype 32, 33, 46, 47, 321

UDP-GlcNAc pyrophosphorylases 388, 389, 390, 391, 393 UDP-N-acetylglucosamine 4’-epimerase (Uae) 386, 389–391, 393, 394 UDP-N-acetylglucosamine (GlNAc) 385–386, 393 Ultraviolet Disinfection Guidance Manual 173–174 Ultraviolet (UV) disinfection DNA damage 183–184, 190, 191, 193–194 doses 169 downstream of ozone treatment 158–159 excystation reduction 184 inactivation 170, 180, 184–185, 193, 205 induced damage detection 179–194, 206 MS2 inactivation versus turbidity 168 ozone synergy, turbidity increase effect 171–173 proteins alteration 183, 192 resistance 184 transmittance 171–172, 176 doses 166, 177 MS2 coliphage 166–169, 173–175, 177 transmittance data 173, 174 Ungulates 97–99 see also Bovinae; Cattle; Pigs Universality 46–47 Unsewered communities 473 Uptake Uridine diphospho (UDP) N-acetylglucosamine pyrophosphorylase (Uap) 386, 387–389 Urticaria 456 UV see Ultraviolet (UV)

Validation 139 Van Leeuwenhoek, Antony 147 Variant-specific surface proteins (VSP) 330–331, 336, 337, 454 Vectors 94–104 see also Reservoirs, zoonotic Vegetables contamination 216, 251, 252

502

Index Vesicles docking 414 encystation-specific 310, 329–330, 384, 391, 410, 412–413 fusion 414 migration 304 peripheral 269, 270, 272 secretion 310, 329–330, 384, 391, 410, 412–413 trafficking 414 transport 293, 304, 305, 411 Vesicles-associated membrane protein (VAMP) 414 Vinorelbine bodipy-Pam uptake and targeting 297 ceramide effect 299, 302 lipid fluorescent analogues release and recycling 298 lipid uptake decrease 295 NBD-phosphatidylglycerol recycling 301 sphingomyelin effect 300, 302 Virulence 7, 60, 138, 139 Viruses 16, 89–90 see also Human immunodeficiency virus (HIV) Vitamin A 465

Water contamination Cryptosporidium spp. typing methods 213–214, 215–221 Cyclospora cayetanensis 249 dairy farms role 128 determination 215–221 dissolved organic carbon (DOC) 206 drinking 83, 158, 235, 236, 238, 344 human source 96, 99, 101–102, 115 livestock role 107, 112, 113, 114 manure use 109 oocyst contribution 128, 212–213

drinking water improvement 230, 231–232, 234 monitoring 210 ozone treatment 159 pesticides application, oocysts survival 250 quality measurement methods 159 recreational 85, 109, 234, 235, 236 treatment processes 198–208 wells 240–241 zoonotic genotypes risk 123–128 see also Disinfection; Turbidity Waterborne disease 85, 107, 181, 186, 227–236, 249–250 Web, parasite 102 Websites Cryptosporidium 350, 352, 353 diagnostic test algorithms guidance 149 genomics 352 proteomics 347, 349, 352 Taxonomy of the genus Cryptosporidium 45 Wells 240–241 WHO Water Safety Plans 234 Wildlife 71 genotypes 71, 124, 215 Giardia impacts 100–101 humans, zoonotic strains implications 101–102 infection by humans 5–6 oocysts shedding 125 transmission role 65, 94–104, 109, 124 Wortmannin 421

X-linked agammaglobulinelia (XLA) 453

YO-YPRO1 (YP) 180, 188, 189

Ziehl-Neelsen test 153 Zoite see Sporozoite