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TOXOPLASMA GONDII
 TOXOPLASMA
GONDII
The Model
Apicomplexan—Perspectives
and Methods

THIRD EDITION
Edited by

Louis M. Weiss
Department of Medicine, Albert Einstein College of Medicine, New York, NY, United States
Department of Pathology, Albert Einstein College of Medicine, New York, NY, United States

Kami Kim
Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, United States
Global Health Infectious Diseases Research Program, College of Public Health, University of South Florida, Tampa, FL,
United States
Academic Press is an imprint of Elsevier
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This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted
herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding,
changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information,
methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own
safety and the safety of others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury
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methods, products, instructions, or ideas contained in the material herein.

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A catalog record for this book is available from the Library of Congress
ISBN: 978-0-12-815041-2
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visit our website at https://www.elsevier.com/books-and-journals

Cover photograph: Coccidian stage of Toxoplasma gondii in the cat intestine undergoing endopolygeny (asexual division) showing the
formation of multiple daughters within the mother cell cytoplasm, while located within an intestinal epithelial cell. Original
magnification 8000X. Image provided by Dr. David Ferguson, Oxford University.

Publisher: Andre Gerhard Wolff

Acquisitions Editor: Kattie Washington
Editorial Project Manager: Xun Wang
Production Project Manager: Sreejith Viswanathan
Cover Designer: Miles Hitchen
Typeset by MPS Limited, Chennai, India
 Dedication

We would like to thank our families (Lisa, Hannah, Talia, and Oren; Tom, Clayton, and
Vaughan) for their patience, understanding, and tolerance during the completion of this book. In
addition, we want to thank the Toxoplasma research community for their enthusiasm and contribu-
tions to this project. The Toxoplasma research community is legendary for its generosity toward col-
leagues and new investigators. It has been a unique pleasure to be involved with such a
welcoming and intellectually stimulating group of researchers.
There have been many key research groups and individual researchers who have contributed
to the development of the critical knowledge base required for progress on this pathogen. This
book is a testament to these researchers.
We would like to dedicate this book to Elmer Pfefferkorn, PhD, Dartmouth College. Elmer’s
work paved the way for the explosion in molecular biology, cell biology, and genomic research
associated with this organism. Elmer’s intellectual rigor and deep thinking has had a significant
influence on current researchers on Toxoplasma gondii, and we are all indebted to his generosity of
spirit and profound insights into this pathogen.

Louis M. Weiss and Kami Kim

Bronx, NY, 2007
Once again we are deeply grateful for the support of our families, especially Tom and Lisa
who again displayed patience, understanding, and tolerance during the completion of this revised
edition.
The second edition of this book is dedicated to the Toxoplasma scientific community, whose
dedication and productivity have made this updated volume a necessity.

Louis M. Weiss and Kami Kim

Bronx, NY, 2013

Yet again we are profoundly grateful for the patience, understanding, and tolerance of our
families which was critical for the completion of this revised edition.
The third edition of this book is dedicated to the memory of all of our colleagues who have
passed away in the last several years. The work that these various researchers performed laid the
foundation for the incredible explosion of knowledge seen in this updated edition. They will be
deeply missed by the research community.

Louis M. Weiss
Bronx, NY, 2019
Kami Kim
Tampa, FL, 2019
 Contents

List of contributors xiii 3.6 Toxoplasma genotype and biological

characteristics 95
Preface to the third edition xvii 3.7 Toxoplasma gondii genotype and human disease 97
3.8 Conclusion and perspective on Toxoplasma genotype
1. The history and life cycle and human disease 102
of Toxoplasma gondii Acknowledgments 103
References 103
J.P. DUBEY

1.1 Introduction 1 4. Human Toxoplasma infection

1.2 The etiological agent 1 RIMA MCLEOD, WILLIAM COHEN, SAMANTHA DOVGIN,
1.3 Parasite morphology and life cycle 1 LAUREN FINKELSTEIN AND KENNETH M. BOYER
1.4 Transmission 5
1.5 Toxoplasmosis in humans 8 4.1 Clinical manifestations and course 117
1.6 Toxoplasmosis in other animals 10 4.2 Diagnosis of infection with Toxoplasma gondii 143
1.7 Diagnosis 11 4.3 Treatment 159
1.8 Treatment 12 4.4 Prevention 172
1.9 Prevention and control 12 4.5 Other considerations of pathogenesis in human
Acknowledgments 13 infections 174
References 13 4.6 Conclusion, unifying concepts, and toward the
Further reading 19 future 201
Acknowledgments 201
2. The ultrastructure of Toxoplasma gondii References 202
DAVID J.P. FERGUSON AND JEAN-FRANÇOIS DUBREMETZ
Further reading 225

2.1 Invasive stage ultrastructure and genesis 21 5. Ocular disease due to Toxoplasma gondii
2.2 Coccidian development in the definitive host 32
JORGE ENRIQUE GOMEZ-MARIN AND
2.3 Development in the intermediate host 48 ALEJANDRA DE-LA-TORRE
References 58
5.1 Introduction 229
3. Molecular epidemiology and population 5.2 Historical landmarks in ocular toxoplasmosis 229
structure of Toxoplasma gondii 5.3 Epidemiology 231
5.4 Pathophysiology: lessons from animal models and
MARIE-LAURE DARDÉ, AURÉLIEN MERCIER, CHUNLEI SU,
ASIS KHAN AND MICHAEL E. GRIGG clinical studies 234
5.5 Host factors 236
3.1 Introduction 63 5.6 Parasite factors 237
3.2 Genetic markers 64 5.7 Animal models 240
3.3 Evolutionary history 77 5.8 Clinical characteristics 241
3.4 Global diversity and population structure 82 5.9 Diagnostic tests 257
3.5 Outbreak investigations 90 5.10 Differential diagnosis 267

vii
viii CONTENTS

5.11 The treatment and management of ocular
toxoplasmosis 268
5.12 Conclusion 276
Acknowledgments 276
References 276
6. Toxoplasmosis in wild and domestic
animals
DAVID S. LINDSAY AND J.P. DUBEY
6.1 Introduction 293
6.2 Toxoplasmosis in wildlife 293
6.3 Toxoplasmosis in zoos 305
6.4 Toxoplasma gondii and endangered
species 307
6.5 Toxoplasmosis in pets 308
6.6 Domestic farm animals 310
6.7 Fish, reptiles, and amphibians 312
References 312
Further reading 320
7. Toxoplasma animal models and therapeutics
CARSTEN G.K. LÜDER, UTZ REICHARD AND UWE GROß
9. Biochemistry and metabolism of Toxoplasma
gondii: purine and pyrimidine acquisition in
Toxoplasma gondii and other Apicomplexa
BARBARA A. FOX AND DAVID J. BZIK
9.1 Introduction 397
9.2 Purines 399
9.3 Pyrimidines 420
References 436
Further reading 449
10. Metabolic networks and metabolomics
JOACHIM KLOEHN, AARTI KRISHNAN, CHRISTOPHER J.
TONKIN, MALCOLM J. MCCONVILLE AND
DOMINIQUE SOLDATI-FAVRE
10.1 Introduction 451
10.2 Genome-scale metabolic modeling 452
10.3 Central carbon metabolism 456
10.4 Carbohydrate metabolism 468
10.5 Vitamins and cofactor metabolism 475
10.6 Metabolomics approaches 487
10.7 Discussion and outlook 491
References 492

7.1 Introduction 321 11. The apicoplast and mitochondrion of

7.2 Congenital toxoplasmosis 322
7.3 Ocular toxoplasmosis 333
Toxoplasma gondii
7.4 Cerebral toxoplasmosis 342 FRANK SEEBER, JEAN E. FEAGIN, MARILYN PARSONS AND
GIEL G. VAN DOOREN
References 354
11.1 Introduction 499
8. Biochemistry and metabolism of Toxoplasma 11.2 The apicoplast 500
gondii: lipid synthesis and uptake 11.3 The mitochondrion 528
11.4 Conclusion 539
ISABELLE COPPENS AND CYRILLE BOTTÉ
Acknowledgments 539
8.1 Introduction 367 References 539
8.2 Fatty acids 368
8.3 Glycerophospholipids 375 12. Calcium storage and homeostasis in
8.4 Acylglycerols 381 Toxoplasma gondii
8.5 Acylglycerol synthesis and storage in DOUGLAS A. PACE, SILVIA N.J. MORENO AND
Toxoplasma 381 SEBASTIAN LOURIDO
8.6 Sterols and steryl esters 383
8.7 Sphingolipids 386 12.1 Introduction 547
8.8 Isoprenoid derivatives 388 12.2 Fluorescent methods to study calcium in
8.9 Concluding remarks 390 Toxoplasma 548
Acknowledgments 390 12.3 Regulation of [Ca21]i in Toxoplasma gondii 555
References 390 12.4 Transducing Ca21 signals 561
Further reading 395 12.5 Conclusion 568
 CONTENTS ix
Acknowledgments 569 15.5 An integrated model of exocytic trafficking
References 569 through the membrane trafficking system 716
15.6 Dynamics of the endolysosomal system 721
13. Calcium and cyclic nucleotide signaling 15.7 Endocytosis and endocytic trafficking 722
networks in Toxoplasma gondii 15.8 Comparison of Toxoplasma gondii endosomal
trafficking to model systems 724
KEVIN M. BROWN, CHRISTOPHER J. TONKIN,
OLIVER BILLKER AND L. DAVID SIBLEY
15.9 Autophagy 726
15.10 Final remarks 732
13.1 Introduction 577 Acknowledgments 733
13.2 Motility 579 Glossary 733
13.3 Regulated secretion of micronemes 582 References 734
13.4 Release of intracellular calcium as a regulatory
cascade 584 16. The Toxoplasma cytoskeleton: structures,
13.5 Calcium-dependent protein kinases 585 proteins, and processes
13.6 Nucleotide cyclases and cyclic nucleotide
NAOMI MORRISSETTE AND MARC-JAN GUBBELS
phosphodiesterases 590
13.7 Conclusion and future directions 598 16.1 Morphology 743
Acknowledgments 598 16.2 Cytoskeletal elements 750
References 599 16.3 Putting it all together: processes 769
16.4 Summary: a story of adaptation, loss, and novel
14. Toxoplasma secretory proteins and their components 779
roles in parasite cell cycle and infection Acknowledgments 779
MARYSE LEBRUN, VERN B. CARRUTHERS AND References 779
MARIE-FRANCE CESBRON-DELAUW

14.1 Introduction 607

17. Effectors produced by rhoptries and dense
14.2 Motility and invasion 609 granules: an intense conversation between
14.3 Parasitophorous vacuole formation and parasite and host in many languages
maturation 614 JOHN C. BOOTHROYD AND MOHAMED-ALI HAKIMI
14.4 Egress 618
14.5 Micronemes 618 17.1 Background 789
14.6 Rhoptries 642 17.2 Rhoptry effectors—a potent class of host
14.7 Dense granules 668 manipulators 792
14.8 Conclusion 684 17.3 Dense granule effectors—a second wave of
Acknowledgments 684 manipulation 796
References 684 17.4 Conclusion 801
Acknowledgments 802
15. Endomembrane trafficking pathways in References 802
Toxoplasma
SÉBASTIEN BESTEIRO, CHRISTEN M. KLINGER, 18. Bradyzoite and sexual stage development
MARKUS MEISSNER AND VERN B. CARRUTHERS ANTHONY P. SINAI, LAURA J. KNOLL AND LOUIS M. WEISS

15.1 Introduction 705 18.1 Introduction 807

15.2 Sorting signals of secretory proteins 706
15.3 Coding complement of the Toxoplasma gondii
membrane-trafficking system 709
15.4 Organization of the Toxoplasma gondii membrane
trafficking system 713
18.2 Bradyzoite and tissue cyst morphology and
biology 809
18.3 The development of tissue cysts and bradyzoites
in vitro 814
18.4 The cell cycle and bradyzoite development 818
x CONTENTS

18.5 The stress response and signaling pathways for 21. Regulation of gene expression in
bradyzoite formation 820 Toxoplasma gondii
18.6 Heat shock proteins 823
KAMI KIM, VICTORIA JEFFERS AND
18.7 Transcriptional control of bradyzoite genes 826 WILLIAM J. SULLIVAN JR.
18.8 Cyst wall and matrix antigens 829
18.9 Surface antigens 834 21.1 Introduction 941
18.10 Metabolic differences between bradyzoites and 21.2 Transcription in Toxoplasma 942
tachyzoites 836 21.3 Epigenetics in Toxoplasma 954
18.11 Genetic studies on bradyzoite biology 839 21.4 Posttranscriptional mechanisms in
18.12 Sexual stage morphology, biology, and Toxoplasma 966
antigens 841 21.5 Conclusion and future directions 970
18.13 Sexual stage development in cell culture 842 Acknowledgments 971
18.14 Sexual stage development in a mouse model 844 References 971
18.15 Summary 844
Acknowledgments 845 22. Proteomics and posttranslational protein
References 845
modifications in Toxoplasma gondii
Further reading 857
LOUIS M. WEISS, JONATHAN WASTLING,
VICTORIA JEFFERS, WILLIAM J. SULLIVAN JR AND KAMI KIM
19. Development and application of classical
genetics in Toxoplasma gondii 22.1 Introduction to Toxoplasma gondii proteomics 983
MICHAEL S. BEHNKE, JEROEN P.J. SAEIJ AND JON P. BOYLE 22.2 Toxoplasma gondii global proteomics 988
22.3 Toxoplasma gondii subproteomes 990
19.1 Summary 859 22.4 Toxoplasma gondii posttranslational
19.2 Biology of Toxoplasma 860 modifications 993
19.3 Establishment of transmission genetics 863 22.5 Studies on the function of posttranslational
19.4 Development of genetic mapping 867 modifications in Toxoplasma. gondii biology 999
19.5 Mapping phenotypic traits by classical 22.6 Interactions of Toxoplasma gondii posttranslational
genetics 871 modifications 1004
19.6 Future challenges 889 22.7 Conclusion 1009
Acknowledgments 890 Acknowledgments 1010
References 890 References 1010

20. Genetic manipulation of 23. ToxoDB: the functional genomic resource

Toxoplasma gondii for Toxoplasma and related organisms
DAMIEN JACOT, SEBASTIAN LOURIDO, MARKUS OMAR S. HARB, JESSICA C. KISSINGER AND DAVID S.
MEISSNER, LILACH SHEINER, DOMINIQUE SOLDATI-FAVRE ROOS
AND BORIS STRIEPEN
23.1 Introduction 1021
20.1 Introduction 897 23.2 Data content 1022
20.2 The mechanics of making transgenic 23.3 Genome in ToxoDB 1023
parasites 898 23.4 Functional data in ToxoDB 1023
20.3 Using transgenic parasites to study the function of 23.5 The ToxoDB home page 1026
parasite genes 907 23.6 The search strategy system 1027
20.4 Perspectives 918 23.7 Genomic colocation 1033
20.5 A selection of detailed protocols for parasite 23.8 The genome browser 1034
culture, genetic manipulation, and phenotypic 23.9 Data analysis and integration into ToxoDB 1037
characterization 918 23.10 Future directions 1039
Acknowledgments 934 Acknowledgments 1039
References 934 References 1040
 CONTENTS xi
24. Cerebral toxoplasmosis 25.8 Additional immune pathways altered by Toxoplasma
ANITA A. KOSHY, TAJIE H. HARRIS AND gondii 1091
MELISSA B. LODOEN 25.9 Conclusion and perspectives 1094
References 1095
24.1 Introduction 1043
24.2 Models for understanding cerebral 26. Adaptive immunity
toxoplasmosis 1043
NICOLAS BLANCHARD, ANNA SALVIONI AND
24.3 Mouse and parasite genotype affect central nervous ELLEN A. ROBEY
system outcomes 1044
24.4 Overview of the central nervous system 1045 26.1 Introduction 1107
24.5 Parasite entry into the central nervous 26.2 How is Toxoplasma gondii “seen” by the adaptive
system 1045 immune system? 1111
24.6 Brain regions and host cells infected in the 26.3 Initiation (priming) of T cell responses by dendritic
brain 1048 cells 1118
24.7 Control of cerebral toxoplasmosis 1050 26.4 Major histocompatibility complex class II
24.8 Physiologic effects of Toxoplasma gondii on the presentation 1118
central nervous system 1060 26.5 Adaptive immune responses in the intestinal
24.9 Conclusion 1062 mucosa and associated lymphoid tissues 1120
Acknowledgement 1062 26.6 Lymphoid system 1123
References 1062 26.7 Adaptive immunity in the brain 1131
26.8 Adaptive immunity in the muscle 1138
25. Innate immunity to Toxoplasma gondii 26.9 Conclusion 1139
DANA G. MORDUE AND CHRISTOPHER A. HUNTER References 1139

25.1 Introduction 1075 Appendix A: The effect of murine gene

25.2 The intimate relationship between Toxoplasma deficiencies on the outcome
gondii and its host cells 1076
of Toxoplasma gondii infection 1147
25.3 Establishment of infection and mucosal
CRAIG W. ROBERTS AND STUART WOODS
immunity 1077
25.4 The role of IL-12-dependent IFN-γ production for
innate resistance 1078 Epilogue 1183
25.5 Antigen processing and presentation 1080
25.6 Molecular basis for innate recognition of Index 1185
Toxoplasma gondii 1081
25.7 IFN-γ-dependent cell autonomous
immunity 1085
 List of contributors
Michael S. Behnke Department of Pathobiological
Sciences, School of Veterinary Medicine,
Louisiana State University, Baton Rouge, LA,
United States
Sébastien Besteiro DIMNP, UMR5235 CNRS,
University of Montpellier, Montpellier, France
Oliver Billker Department of Molecular Biology,
Norrland University Hospital, Umeå, Sweden
Nicolas Blanchard Center for Pathophysiology
Toulouse-Purpan (CPTP), INSERM, CNRS,
University of Toulouse, Toulouse, France
John C. Boothroyd Department of Microbiology
and Immunology, Stanford School of Medicine,
Stanford, CA, United States
Cyrille Botté Apicolipid Team, Institute for
Advanced Biosciences, CNRS UMR5309,
Université Grenoble Alpes, Grenoble, France
Kenneth M. Boyer Rush University Medical
Center, Chicago, IL, United States
Jon P. Boyle Department of Biological Sciences,
University of Pittsburgh, Pittsburgh, PA, United
States
Kevin M. Brown Department of Molecular
Microbiology, Washington University School of
Medicine, St. Louis, MO, United States
David J. Bzik Department of Microbiology and
Immunology, Geisel School of Medicine at
Dartmouth, Lebanon, NH, United States
Vern B. Carruthers Department of Microbiology
and Immunology, University of Michigan
Medical School, Ann Arbor, MI, United States
Marie-France Cesbron-Delauw UMR 5525 CNRS,
University of Grenoble, Grenoble, France
William Cohen The University of Chicago,
Chicago, IL, United States
Isabelle Coppens Department of Molecular
Microbiology and Immunology, Johns Hopkins
xiii
University Bloomberg School of Public Health,
Baltimore, MD, United States
Marie-Laure Dardé INSERM, Univ. Limoges,
CHU Limoges, UMR 1094, Tropical
Neuroepidemiology, Institute of Epidemiology
and Tropical Neurology, Limoges, France; Centre
National de Référence Toxoplasmose/Toxoplasma
Biological Resource Center, CHU Limoges,
Limoges, France
Alejandra de-la-Torre NeURos Group, Escuela de
Medicina y Ciencias de la Salud, Universidad del
Rosario, Bogotá, Colombia
Samantha Dovgin The University of Chicago,
Chicago, IL, United States
J.P. Dubey Animal Parasitic Diseases Laboratory,
United States Department of Agriculture,
Agricultural Research Service, Beltsville
Agricultural Research Center, Beltsville, MD,
United States
Jean-François Dubremetz UMR CNRS 5235,
University of Montpellier, Montpellier, France
Jean E. Feagin Department of Global Health,
University of Washington, Seattle, WA, United
States
David J.P. Ferguson Nuffield Department of
Clinical Laboratory Science, John Radcliffe
Hospital, University of Oxford, Oxford, United
Kingdom
Lauren Finkelstein The University of Chicago,
Chicago, IL, United States
Barbara A. Fox Department of Microbiology and
Immunology, Geisel School of Medicine at
Dartmouth, Lebanon, NH, United States
Jorge Enrique Gomez-Marin GEPAMOL Group,
Centro de Investigaciones Biomédicas,
Universidad del Quindı́o, Armenia, Colombia
xiv LIST OF CONTRIBUTORS
Michael E. Grigg Laboratory of Parasitic Diseases,
Molecular Parasitology Section, National Institute
of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, MD, United States
Uwe Groß Institute for Medical Microbiology,
University Medical Center, University of
Göttingen, Göttingen, Germany
Marc-Jan Gubbels Department of Biology, Boston
College, Chestnut Hill, MA, United States
Mohamed-Ali Hakimi Institute for Advanced
Biosciences (IAB), Team Host-Pathogen
Interactions and Immunity to Infection, INSERM
U1209, University Grenoble Alpes, Grenoble,
France
Omar S. Harb Department of Biology, University
of Pennsylvania, Philadelphia, PA, United States
Tajie H. Harris Department of Neuroscience,
Center for Brain Immunology and Glia,
University of Virginia, Charlottesville, VA,
United States
Christopher A. Hunter Department of
Pathobiology, School of Veterinary Medicine,
University of Pennsylvania, Philadelphia, PA,
United States
Damien Jacot Department of Microbiology and
Molecular Medicine, Faculty of Medicine,
University of Geneva, Geneva, Switzerland
Victoria Jeffers Department of Molecular, Cellular,
and Biomedical Sciences, University of New
Hampshire, Durham, NH, United States
Asis Khan Laboratory of Parasitic Diseases,
Molecular Parasitology Section, National
Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD,
United States
Kami Kim Department of Internal Medicine,
Morsani College of Medicine, University of South
Florida, Tampa, FL, United States; Global Health
Infectious Diseases Research Program, College of
Public Health, University of South Florida,
Tampa, FL, United States
Jessica C. Kissinger Center for Tropical &
Emerging Global Diseases, Department of
Genetics & Institute of Bioinformatics, University
of Georgia, Athens, GA, United States
Christen M. Klinger Division of Infectious
Diseases, Department of Medicine, University of
Alberta, Edmonton, AB, Canada
Joachim Kloehn Department of Microbiology and
Molecular Medicine, Faculty of Medicine,
University of Geneva, Geneva, Switzerland
Laura J. Knoll Department of Medical
Microbiology and Immunology, University of
Wisconsin-Madison, Madison, WI, United States
Anita A. Koshy Department of Neurology,
University of Arizona, Tucson, AZ, United States;
Department of Immunobiology, University of
Arizona, Tucson, AZ, United States; BIO5
Institute, University of Arizona, Tucson, AZ,
United States
Aarti Krishnan Department of Microbiology and
Molecular Medicine, Faculty of Medicine,
University of Geneva, Geneva, Switzerland
Maryse Lebrun UMR 5235 CNRS, University of
Montpellier, Montpellier, France
David S. Lindsay Department of Biomedical
Sciences and Pathobiology, Virginia Maryland
College of Veterinary Medicine, Virginia Tech,
Blacksburg, VA, United States
Melissa B. Lodoen Department of Molecular
Biology and Biochemistry, University of
California, Irvine, CA, United States; Institute for
Immunology, University of California, Irvine,
CA, United States
Sebastian Lourido Whitehead Institute for
Biomedical Research, Cambridge, MA, United
States; Department of Biology, Massachusetts
Institute of Technology, Cambridge, MA, United
States
Carsten G.K. Lüder Institute for Medical
Microbiology, University Medical Center,
University of Göttingen, Göttingen, Germany
Malcolm J. McConville Department of
Biochemistry and Molecular Biology, University
of Melbourne, Parkville, VIC, Australia;
Metabolomics Australia, Bio21 Molecular Science
and Biotechnology Institute, University of
Melbourne, Parkville, VIC, Australia
Rima McLeod The University of Chicago, Chicago,
IL, United States
Markus Meissner Experimental Parasitology,
Department of Veterinary Sciences, Ludwig
Maximilians University, Munich, Germany;
Department of Veterinary Sciences, Experimental
Parasitology, Ludwig-Maximilians-University,
Munich, Germany
 LIST OF CONTRIBUTORS
Aurélien Mercier INSERM, Univ. Limoges, CHU
Limoges, UMR 1094, Tropical
Neuroepidemiology, Institute of Epidemiology
and Tropical Neurology, Limoges, France; Centre
National de Référence Toxoplasmose/Toxoplasma
Biological Resource Center, CHU Limoges,
Limoges, France
Dana G. Mordue Department of Microbiology and
Immunology, New York Medical College,
Valhalla, NY, United States
Silvia N.J. Moreno Department of Cellular Biology
and Center for Tropical and Emerging Global
Diseases, University of Georgia, Athens, GA,
United States
Naomi Morrissette Department of Molecular
Biology and Biochemistry, University of
California, Irvine, Irvine, CA, United States
Douglas A. Pace Department of Biological
Sciences, California State University Long Beach,
Long Beach, CA, United States
Marilyn Parsons Department of Global Health,
University of Washington, Seattle, WA, United
States; Department of Pediatrics, University of
Washington, Seattle, WA, United States; Center
for Global Infectious Disease Research, Seattle
Children’s Research Institute, Seattle, WA,
United States
Utz Reichard Institute for Medical Microbiology,
University Medical Center, University of
Göttingen, Göttingen, Germany; Amedes MVZ
Wagnerstibbe for Medical Microbiology,
Infectious Diseases, Hygiene and Tropical
Medicine, Göttingen, Germany
Craig W. Roberts University of Strathclyde,
Glasgow, United Kingdom
Ellen A. Robey Department of Molecular and Cell
Biology, University of California, Berkeley, CA,
United States
David S. Roos Department of Biology, University
of Pennsylvania, Philadelphia, PA, United States
Jeroen P.J. Saeij Department of Pathology,
Microbiology and Immunology, School of
Veterinary Medicine, University of California,
Davis, CA, United States
Anna Salvioni Center for Pathophysiology
Toulouse-Purpan (CPTP), INSERM, CNRS,
University of Toulouse, Toulouse, France
Frank Seeber FG16: Mycotic and Parasitic Agents
and Mycobacteria, Robert Koch-Institute, Berlin,
Germany
xv
Lilach Sheiner Wellcome Centre for Integrative
Parasitology, Institute of Infection, Immunity &
Inflammation, College of Medical, Veterinary &
Life Sciences, Sir Graeme Davies Building,
University of Glasgow, Glasgow, United
Kingdom
L. David Sibley Department of Molecular
Microbiology, Washington University School of
Medicine, St. Louis, MO, United States
Anthony P. Sinai Department of Microbiology,
Immunology & Molecular Genetics, University of
Kentucky, College of Medicine, Lexington, KY,
United States
Dominique Soldati-Favre Department of
Microbiology and Molecular Medicine, Faculty of
Medicine, University of Geneva, Geneva,
Switzerland
Boris Striepen Department of Pathobiology,
University of Pennsylvania, Philadelphia, PA,
United States
Chunlei Su Department of Microbiology, The
University of Tennessee, Knoxville, TN, United
States
William J. Sullivan, Jr. Departments of
Pharmacology & Toxicology, Microbiology &
Immunology, Indiana University School of
Medicine, Indianapolis, IN, United States;
Department of Microbiology & Immunology,
Indiana University School of Medicine,
Indianapolis, IN, United States
Christopher J. Tonkin Division of Infectious
Disease and Immune Defence, The Walter and
Eliza Hall Institute of Medical Research,
Parkville, VIC, Australia; Department of Medical
Biology, The University of Melbourne, Parkville,
VIC, Australia
Giel G. van Dooren Research School of Biology,
Australian National University, Canberra, ACT,
Australia
Jonathan Wastling Faculty of Natural Sciences,
University of Keele, Keele, Staffordshire, United
Kingdom
Louis M. Weiss Department of Pathology Albert
Einstein College of Medicine, Bronx, NY, United
States; Department of Medicine, Albert Einstein
College of Medicine, Bronx, NY, United States
Stuart Woods University of Strathclyde, Glasgow,
United Kingdom
 Preface to the third edition
Toxoplasma gondii is a ubiquitous, apicom-
plexan parasite of warm-blooded animals and
is one of the most common parasitic infections
of humans. Infection can result in encephalitis
(especially in immune-compromised hosts),
retinochoroiditis, or congenital transmission
with fetopathy if a seronegative pregnant
woman becomes infected. T. gondii has become
a model organism for the study of the
Apicomplexa, as it is the most experimentally
tractable organism in this important group
of intracellular parasites which includes
Plasmodium, Eimeria, Cryptosporidium, Neospora,
and Theileria. T. gondii is readily amenable to
genetic manipulation with refined protocols for
both classic and reverse genetics. It has been
used for testing the biological or biochemical
function of proteins that for one reason or
other cannot be readily expressed in other
organisms. It is easily propagated and quanti-
fied in the laboratory; the mouse animal model
is well established; and reagents for study of
the host response as well as basic biology of
the parasite are widely available. With the
recent development of an in vitro method to
culture the sexual stage of development, as
well as the development of a rodent model that
allows oocyte formation, the entire life cycle
(tachyzoite, bradyzoite, and oocyst) can be
studied in experimental laboratory systems.
Immunity to T. gondii is a complex process
involving innate and adaptive immune
responses. T. gondii has been a useful model
system pathogen for understanding the
immune response to an intracellular pathogen,
xvii
including studies on macrophage function,
cell-mediated immunity, dendritic cells, and
the gut-associated immune response. The ease
with which it can be cultured in vitro, availabil-
ity of reporter parasite lines, and its pathoge-
nicity in mouse models have facilitated these
immunobiology investigations. Because of
these experimental advantages, T. gondii has
emerged as a major model organism for the
study of apicomplexan biology and host-
pathogen interactions.
Since the publication of the second edition
of this book in 2014, the “Omics” revolution
has continued to influence all aspects of the
Toxoplasma field. Research has been accelerated
by the application of CRISPR-Cas9 technology
for the genetic manipulation of T. gondii. This
has facilitated large-scale screens that have
provided, and are providing, critical data on
essential genes as well as genes involved in
survival within the host and those related to
host-pathogen interactions. The application of
BioID and other proteomic technologies is fur-
ther accelerating our understanding of the
composition of various structures within this
pathogen as well as protein complexes that are
essential for its functioning as well as manipu-
lation of its host cell. ToxoDB (https://toxodb.
org/toxo/) continues to be a critical resource
for the analysis and distribution of these vari-
ous “omics” datasets, facilitating new insights
into the pathobiology of this organism. Unique
families of secreted proteins have been identi-
fied as modulators of host signaling and gene
expression, and the mechanisms by which
xviii Preface to the third edition

T. gondii alters its host cell and the exquisite
dance of host-pathogen interplay is gradually
being teased out. Finally, sophisticated imag-
ing techniques are yielding critical insights into
the neurobiology and immunology of T. gondii;
providing new insights.
The first edition of this book was an out-
growth of discussions held at the Seventh
International Congress on Toxoplasmosis in
Tarrytown, New York in 2003 and the publica-
tion of papers and review articles from this
congress in March 2004 in the International
Journal for Parasitology [volume 34, number 3,
pages 249 432]. It was evident at this congress
and confirmed at the Eighth International
Congress in 2005 (Corsica, France) that the
field of study of this pathogen had matured
considerably since the First International
Congress occurred in 1990 at Dartmouth
University (Hanover, NH). This was paralleled
by the progressive increase in attendance at
this International Congress, which grew from
an initial group of 26 investigators (Fig. P1) to
meetings with over 200 participants (see
Epilogue Figure E1 from the 15th International
Toxoplasmosis Congress), which reflects the
increasing number of laboratories working on
this organism.
The third edition of “Toxoplasma gondii: the
model apicomplexan” was assembled to
address the numerous advances in the
Toxoplasma field in the past 5 years and pro-
vide a single volume which would continue to
represent the breadth of research efforts and
data on this important pathogenic protist. We
are fortunate that so many members of the
Toxoplasma research community have gener-
ously donated their time and expertise to
update this book. New authors were added to
this edition to revise chapters and provide
fresh perspectives on various topics. To this
end, the second edition contains other perspec-
tives on several of these topics and provides
complementary data to what is found in this

FIGURE P1 Participants in the First International Congress on Toxoplasmosis held 1990 at the Minary Conference
Center of Dartmouth College, Squam Lake, Holderness New Hampshire, United States.
Bottom: Lloyd Kasper, Rima McLeod, Jean Francois Dubremetz, John Boothroyd, Abott Laboratories (unknown).
Middle: Joseph Schwartzman, Elmer Pfefferkorn, Takuro Endo, Louis M. Weiss, Francoise Darcy, Phillippe Thuiliez, Marie
France Cesbron, Judy Smith, Alan Johnson, Yasu Suzuki, Takashi Asia, J.P. Dubey.
Top: Greg Felice, James L. Fishback, David Sibley, Jack Remington, Alan Sher, Jack Frenkel, David Roos, Keith Joiner,
Benjamin Luft, Bill Current.
 Preface to the third edition
new edition. Authors of the third edition were
encouraged to review older literature compre-
hensively so that their chapters could serve as
free-standing reference articles.
In our own laboratory groups, this book has
become our standard educational resource. We
hope that this new edition will continue to pro-
vide important insights into this pathogen, a
useful synthesis and summary of the current
literature, and suggest new avenues of investi-
gations to current and future investigators in
this field.
xix
We look forward to the ongoing tsunami of
research that will enable the field to more thor-
oughly understand this important parasite and
its relationship with its animal and human
hosts giving us further insights into how T.
gondii has evolved into such a successful intra-
cellular parasite.
Louis M. Weiss
Bronx, NY, United States, 2019
Kami Kim
Tampa, FL, United States, 2019
 C H A P T E R

1
The history and life cycle of
Toxoplasma gondii
J.P. Dubey
Animal Parasitic Diseases Laboratory, United States Department of Agriculture, Agricultural Research
Service, Beltsville Agricultural Research Center, Beltsville, MD, United States

1.1 Introduction based on the morphology (mod. L. toxo 5 arc or

Infections by the protozoan parasite
Toxoplasma gondii are widely prevalent in
humans and other animals on all continents.
There are many thousands of references to this
parasite in the literature, and it is not possible
to give equal treatment to all authors and dis-
coveries (Dubey, 2008). The objective of this
chapter is, rather, to provide a history of the
milestones in our acquisition of knowledge of
the biology of this parasite.
1.2 The etiological agent
bow, plasma 5 life) and the host (Nicolle and
Manceaux, 1909). Thus its complete designation
is T. gondii (Nicolle and Manceaux, 1908, 1909).
In retrospect the correct name for the parasite
should have been T. gundii, as Nicolle and
Manceaux (1908) had incorrectly identified the
host as Ctenodactylus gondi. Splendore (1908, see
also English translation Splendore, 2009) discov-
ered the same parasite in a rabbit in Brazil, also
erroneously identifying it as Leishmania, but he
did not name it. It is a remarkable coincidence
that this disease was first recognized in labora-
tory animals and was first thought to be
Leishmania by both groups of investigators.

Nicolle and Manceaux (1908) found a proto-

zoan in tissues of a hamster-like rodent, the
gundi, Ctenodactylus gundi, which was being
1.3 Parasite morphology and life cycle
used for leishmaniasis research in the laboratory
of Charles Nicolle at the Pasteur Institute in
1.3.1 Tachyzoites
Tunis. They initially believed the parasite to be The tachyzoite (Frenkel, 1973) is lunate
Leishmania but soon realized that they had dis- (Figs. 1.1 and 1.2A) and is the stage that
covered a new organism and named it T. gondii Nicolle and Manceaux (1909) found in the

Toxoplasma Gondii
DOI: https://doi.org/10.1016/B978-0-12-815041-2.00001-3 1 © 2020 Elsevier Ltd. All rights reserved.
2 1. The history and life cycle of Toxoplasma gondii
FIGURE 1.1 Life cycle of Toxoplasma gondii.
gundi. This stage has also been called tropho-
zoite, the proliferative form, the feeding form,
and endozoite. It can infect virtually any cell in
the body. It divides by a specialized process
called endodyogeny, first described by
Goldman et al. (1958). Gustafson et al. (1954)
first studied the ultrastructure of the tachy-
zoite. Sheffield and Melton (1968) provided a
complete description of endodyogeny when
they fully described its ultrastructure.
1.3.2 Bradyzoite and tissue cysts
The term “bradyzoite” (Gr. brady 5 slow)
was proposed by Frenkel (1973) to describe the
stage encysted in tissues. Bradyzoites are also
called cystozoites. Dubey and Beattie (1988)
proposed that cysts should be called tissue
Toxoplasma Gondii
cysts (Figs. 1.1, 1.2B, and 1.2C) to avoid confu-
sion with oocysts. It is difficult to determine
from the early literature who first identified
the encysted stage of the parasite (Lainson,
1958). Levaditi et al. (1928) apparently were the
first to report that T. gondii may persist in tis-
sues for many months as “cysts”; however,
considerable confusion between the term
“pseudocysts” (group of tachyzoites) and tis-
sue cysts existed for many years. Frenkel and
Friedlander (1951) and Frenkel (1956) charac-
terized cysts cytologically as containing organ-
isms with a subterminal nucleus and periodic
acid Schiff-positive granules (Fig. 1.2C) sur-
rounded by an argyrophilic cyst wall
(Fig. 1.2B). Wanko et al. (1962) first described
the ultrastructure of the T. gondii cyst and its
contents. Jacobs et al. (1960a) first provided a
biological characterization of cysts when they
 1.3 Parasite morphology and life cycle 3

FIGURE 1.2 Life cycle stages of Toxoplasma gondii. (A) Tachyzoites (arrowhead) in smear. Giemsa stain. Note
nucleus dividing into two nuclei (arrow). (B) A small tissue cyst in smear stained with Giemsa and a silver stain. Note the
silver-positive tissue cyst wall (arrowhead) enclosing bradyzoites that have a terminal nucleus (arrow). (C) Tissue cyst in
section, PAS. Note PAS-positive bradyzoites (arrow) enclosed in a thin PAS-negative cyst wall. (D) Unsporulated oocysts in
cat feces. Unstained. PAS, Periodic acid Schiff.

found that the cyst wall was destroyed by pep-
sin or trypsin, but the cystic organisms were
resistant to digestion by gastric juices (pepsin-
HCl), whereas tachyzoites were destroyed
immediately. Thus tissue cysts were shown to
be important in the life cycle of T. gondii
because carnivorous hosts can become infected
by ingesting infected meat. Jacobs et al. (1960b)
used the pepsin digestion procedure to isolate
viable T. gondii from tissues of chronically
infected animals. When T. gondii oocysts were
discovered in cat feces in 1970, oocyst excretion
was added to the biological description of the
cyst (Dubey and Frenkel, 1976).
Dubey and Frenkel (1976) performed the
first in depth study of the development of
tissue cysts and bradyzoites and described
their ontogeny and morphology. They found
that tissue cysts formed in mice as early as 3
days after their inoculation with tachyzoites.
Cats excrete oocysts (Fig. 1.2D) with a short
prepatent period (3 10 days) after ingesting
tissue cysts or bradyzoites, whereas after they
ingested tachyzoites or oocysts, the prepatent
period was longer ($18 days), irrespective of
the number of organisms in the inocula (Dubey
and Frenkel, 1976; Dubey, 1996, 2001, 2006).
Prepatent periods of 11 17 days are thought to
result from the ingestion of transitional stages
between tachyzoite and bradyzoite (Dubey,
2002, 2005).
Wanko et al. (1962) and Ferguson and
Hutchison (1987) reported on the ultrastructural
of the development of T. gondii tissue cysts. The
biology of bradyzoites including morphology,
development in cell culture in vivo, conversion
of tachyzoites to bradyzoites, and vice versa, tis-
sue cyst rupture, and distribution of tissue cysts
in various hosts and tissues was reviewed criti-
cally by Dubey et al. (1998).

Toxoplasma Gondii
4 1. The history and life cycle of Toxoplasma gondii

1.3.3 Enteroepithelial asexual and sexual
stages
Asexual and sexual stages (Figs. 1.3 and
1.4) were reported in the intestine of cats in
1970 (Frenkel, 1970). Dubey and Frenkel
(1972) described the asexual and sexual devel-
opment of T. gondii in enterocytes of the cat
and designated the asexual enteroepithelial
stages as Types A through E (Figs. 1.3 and 1.4)
rather than as generations conventionally
known as schizonts in other coccidian para-
sites. These stages were distinguished mor-
phologically from tachyzoites (Fig. 1.3D) and
bradyzoites, which also occur in cat intestine.
The challenge was to distinguish different
stages in the cat intestine because there was
profuse multiplication of T. gondii 3 days post-
infection (Fig. 1.4A). The entire cycle was com-
pleted in 66 hours after feeding tissue cysts to
cats (Dubey and Frenkel, 1972). There are
reports on the ultrastructure of schizonts
(Sheffield, 1970; Piekarski et al., 1971;
Ferguson et al., 1974), gamonts (Ferguson
et al., 1974, 1975; Speer and Dubey, 2005),
oocysts, and sporozoites (Christie et al., 1978;
Ferguson et al., 1979a,b; Speer et al., 1998;
Freppel et al., 2019). In 2005 Speer and Dubey
described the ultrastructure of asexual
enteroepithelial Types B through E and distin-
guished their merozoites.

FIGURE 1.3 Asexual and sexual stages of Toxoplasma gondii in sections of small intestine of cats fed tissue cysts.
H&E stain. (A) Type C (arrow) schizont with a residual body and a Type B schizont with a hypertrophied host cell nucleus
(arrowhead). 52 h p.i. (B) Heavily infected small intestine with schizonts in and gamonts in the epithelium. Five days p.i.
(C) Type D and E schizonts (a and d), a mature female gamont (e), young female gamont (b), and two male gamonts (c) in
the epithelium. (D) Tachyzoites in the lamina propria (arrows). Types B and D schizonts are below the enterocyte nucleus
and often cause hypertrophy of the parasitized cell whereas Types D and E schizonts are always above the enterocyte
nucleus and do not cause hypertrophy of the host cell even in hyperparastized cases. Tachyzoites are found in the lamina
propria of the cat intestine.

Toxoplasma Gondii
 1.4 Transmission
FIGURE 1.4 Smears of intestinal epithelium of a cat 7
days after feeding tissue cysts (Giemsa stain). (A) Note
different sizes of merozoites (a c), schizont with three
nuclei (d), schizont with six or more nuclei and merozoites
are budding from the surface (e), and a multinucleated
schizont (f). (B) Four biflagellated microgametes (arrows)
and merozoites (arrowhead) for size comparison.
1.4 Transmission
1.4.1 Congenital
The mechanism of transmission of T. gondii
remained a mystery until its life cycle was dis-
covered in 1970. Soon after the initial discovery
of the organism, it was found that the C. gundi
were not infected in the wild and had acquired
T. gondii infection in the laboratory. Initially,
transmission by arthropods was suspected, but
this was never proven (Frenkel, 1970, 1973).
Congenital T. gondii infection in a human child
was initially described by Wolf et al. (1939a,b)
and later found to occur in many species of
Toxoplasma Gondii
5
animals, particularly sheep, goats, and rodents.
Congenital infections can be repeated in some
strains of mice (Beverley, 1959), with infected
mice producing congenitally infected offspring
for at least 10 generations. Beverley discontin-
ued his experiments because of high mortality
in some lines of congenitally infected mice and
because the progeny from the last generation
of infected mice was seronegative and pre-
sumed not to be infected with T. gondii. Jacobs
(1964) repeated these experiments and found
that congenitally infected mice may be infected
but not develop antibodies because of immune
tolerance. Dubey et al. (1995a) isolated viable
T. gondii from seronegative naturally infected
mice. These findings are of epidemiological
significance.
1.4.2 Carnivorism
Congenital transmission occurs too rarely to
explain widespread infection in man and ani-
mals worldwide. Weinman and Chandler
(1954) suggested that transmission might occur
through the ingestion of undercooked meat.
Jacobs et al. (1960a) provided evidence to sup-
port this idea by demonstrating the resistance
to proteolytic enzymes of T. gondii derived
from cysts. They found that the cyst wall was
immediately dissolved by such enzymes but
the released bradyzoites survived long enough
to infect the host. This hypothesis of transmis-
sion through the ingestion of infected meat
was experimentally tested by Desmonts et al.
(1965) in an experiment with children in a
Paris sanitorium. They compared the acquisi-
tion rates of T. gondii infection in children
before and after admission to the sanitorium.
The 10% yearly acquisition rate of T. gondii
antibody rose to 50% after adding two portions
of barely cooked beef or horse meat to the daily
diet and to a 100% yearly rate after the addi-
tion of barely cooked lamb chops. Since the
prevalence of T. gondii is much higher in sheep
6 1. The history and life cycle of Toxoplasma gondii
than in horses or cattle, this illustrated the
importance of carnivorism in transmission of
T. gondii. Epidemiological evidence indicates it
is common in humans in some localities where
raw meat is routinely eaten. In a survey in
Paris, Desmonts et al. (1965) found more than
80% of the adult population sampled had anti-
bodies to T. gondii. Kean et al. (1969) described
toxoplasmosis in a group of medical students
who had eaten undercooked hamburgers.
1.4.3 Fecal oral
While congenital transmission and carnivor-
ism can explain some of the transmission of
T. gondii, it does not explain the widespread
infection in vegetarians and herbivores. A
study in Bombay, India found the prevalence
of T. gondii in strict vegetarians to be similar to
that in nonvegetarians (Rawal, 1959).
Hutchison (1965), a biologist at Strathclyde
University in Glasgow, first discovered T. gon-
dii infectivity associated with cat feces. In a
preliminary experiment, Hutchison (1965) fed
T. gondii cysts to a cat infected with the nema-
tode Toxocara cati and collected feces contain-
ing nematode ova. Feces floated in 33% zinc
sulfate solution and stored in tap water for 12
months induced toxoplasmosis in mice. This
discovery was a breakthrough because, until
then, both known forms of T. gondii (i.e., tachy-
zoites and bradyzoites) were killed by water.
Microscopic examination of feces revealed only
T. cati eggs and Isospora oocysts. In Hutchison’s
report, T. gondii infectivity was not attributed
to oocysts or T. cati eggs. He repeated the
experiment with two T. cati infected and two
T. cati free cats. T. gondii was transmitted only
in association with T. cati infection. On this
basis, Hutchison (1967) hypothesized that T.
gondii was transmitted through nematode ova.
He suspected transmission of T. gondii through
the eggs of the nematode Toxocara similar to
the transmission of the fragile flagellate
Toxoplasma Gondii
Histomonas through Heterakis eggs. Hutchison
initially wanted to test the nematode theory
using Toxocara canis and T. gondii transmission
in the dog but decided on the cat and T. cati
model because there was no place to house
dogs (J.P. Dubey, 1965, personal communica-
tion). Transmission of T. gondii by T. canis eggs
made more sense because of the known zoo-
notic potential of T. canis; T. cati was not, at
that time, known to infect humans, but T. canis
was. Discovery of the life cycle of T. gondii
would have been delayed if Hutchinson had
worked with dogs instead of cats.
Hutchison’s (1965) report stimulated other
investigators to examine fecal transmission of
T. gondii through T. cati eggs (Dubey, 1966,
1968; Jacobs, 1967; Hutchison et al., 1968;
Frenkel et al., 1969; Sheffield and Melton,
1969). The nematode egg theory of transmis-
sion was discarded after Toxoplasma infectivity
was dissociated from T. cati eggs (Frenkel
et al., 1969), and Toxoplasma infectivity was
found in feces of worm-free cats fed T. gondii
(Frenkel et al., 1969; Sheffield and Melton,
1969). Finally, in 1970, knowledge of the T. gon-
dii life cycle was completed by discovery of the
sexual phase of the parasite in the small intes-
tine of the cat (Frenkel et al., 1970). T. gondii
oocysts, the product of schizogony and game-
togony, were found in cat feces and character-
ized morphologically and biologically (Dubey
et al., 1970a,b).
Several group of workers independently
and about the same time found T. gondii
oocysts in cat feces (Hutchison et al., 1969,
1970, 1971; Frenkel et al., 1970; Dubey et al.,
1970a,b; Sheffield and Melton, 1970;
Overdulve, 1970; Weiland and Kühn, 1970;
Witte and Piekarski, 1970). The discovery of
T. gondii oocyst in cat feces and its implications
has been reviewed by Frenkel (1970, 1973) and
Garnham (1971).
In retrospect the discovery and characteriza-
tion of the T. gondii oocyst in cat feces was
delayed because (1) T. gondii oocysts were
 1.4 Transmission
morphologically identical to oocysts of the
previously described coccidian parasite of cats
and dogs (Dubey et al., 1970a) and (2) until
1970, coccidian oocysts were sporulated in
2.5% potassium dichromate. Chromation of the
oocysts wall interfered with excystation of the
sporozoites when oocysts were fed to mice and
thus the mouse infectivity titer of the oocysts
was lower than expected from number of
oocysts administered (Dubey et al., 1970a).
These findings led to the use of 2% sulfuric
acid as the best medium for sporulation and
storage of T. gondii oocysts. Unlike dichromate,
which was difficult to wash off the oocysts, sul-
furic acid could be easily neutralized, and the
oocysts could be injected without washing into
mice (Dubey et al., 1972). Unlike other cocci-
dians, T. gondii oocysts were found to excyst
efficiently when inoculated parenterally into
mice and thus alleviated the need for oral inoc-
ulation for bioassay of oocysts (Dubey and
Frenkel, 1973).
Ben Rachid (1970) fed T. gondii oocysts to
gundis which died 6 7 days later from toxo-
plasmosis. This knowledge about the life cycle
of T. gondii probably explains how gundis
became infected in the laboratory of Nicolle. At
least one cat was present in the Pasteur
Laboratory in Tunis (Dubey, 1977).
Of the many species of animals experimen-
tally infected with T. gondii, only felids excrete
T. gondii oocysts (Miller et al., 1972; Jewell
et al., 1972; Janitschke and Werner, 1972;
Polomoshnov, 1979; Dubey, 2010). Oocysts
excreted into the environment have caused
several outbreaks of disease in humans
(Teutsch et al., 1979; Benenson et al., 1982;
Bowie et al., 1997; de Moura et al., 2006). T.
gondii oocysts found in the feces of naturally
infected cougars (Aramini et al., 1998) were
epidemiologically linked to the largest
recorded waterborne outbreak of toxoplasmo-
sis in humans (Bowie et al., 1997).
Seroepidemiological studies on isolated islands
in the Pacific (Wallace, 1969, for full story see
Toxoplasma Gondii
7
Dubey, 2016), Australia (Munday, 1972), and
the United States (Dubey et al., 1997) have
shown an absence of Toxoplasma on islands
without cats, confirming the important role of
the cat in the natural transmission of T. gondii.
Vaccination of cats with a live mutant strain of
T. gondii on eight pig farms in the United
States reduced the transmission of T. gondii
infection in mice and pigs (Mateus-Pinilla
et al., 1999), thus supporting the role of the cat
in natural transmission of T. gondii.
Historically, before the discovery of the cocci-
dian cycle of T. gondii, coccidian parasites were
considered host and site specific and to be trans-
mitted by the fecal oral route. After the discov-
ery of the sexual cycle of T. gondii, several other
genera (e.g., Sarcocystis and Besnoitia) were
found to be coccidian. Although T. gondii has a
wide host range, it has retained the definitive-
host specificity restricted to felids. Dr. J.K.
Frenkel deserves the credit for initiating testing
of many species of animals, including wild
felids, for oocyst excretion under difficult (it was
not easy handling bob cats and ocelots in cages)
housing conditions (Dubey, 2009). Only the
felids were found to excrete T. gondii oocysts
(Frenkel et al., 1970; Miller et al., 1972).
Although T. gondii can be transmitted in several
ways, it has adapted to be transmitted most effi-
ciently by carnivorism in the cat and by the
fecal oral (oocysts) route in other hosts. Pigs
and mice (and presumably humans) can be
infected by ingesting even one oocyst (Dubey
et al., 1996), whereas 100 oocysts may not infect
cats (Dubey, 1996). Cats can excrete millions of
oocysts after ingesting only one bradyzoite,
while ingestion of 100 bradyzoites may not
infect mice orally (Dubey, 2001, 2006). This infor-
mation has proved very useful in conducting
epidemiological studies and for the detection by
feeding to cats of low numbers of T. gondii in
large samples of meat (Dubey et al., 2005).
Understanding the biology of T. gondii
oocyst excretion by cats (both wild and domes-
tic is essential for reducing prevalence of
8 1. The history and life cycle of Toxoplasma gondii
T. gondii in humans and animals. Although
cats are thought to excrete oocysts only once in
their life after primary infection, there are no
guaranties that they will not do so the second
time. Immunity can wane with time (Dubey,
1995); cats have excreted millions of oocysts
the second time after challenge with unrelated
organisms (Dubey, 1976) or challenge with het-
erologous T. gondii strains (Zulpo et al., 2018).
Therefore pregnant women should avoid con-
tact with cat feces, irrespective of the serologi-
cal status of the cat.
After the discovery of the life cycle of
T. gondii in the cat, it became clear why
Australasian marsupials and New World mon-
keys are highly susceptible to clinical toxoplas-
mosis. The former evolved apparently in the
absence of the cat (there were few or no cats in
Australia and New Zealand before settlement
by Europeans) and the latter lives on tree tops,
not exposed to cat feces. In contrast, marsu-
pials in America and Old World monkeys are
resistant to clinical toxoplasmosis (Dubey and
Beattie, 1988).
1.5 Toxoplasmosis in humans
1.5.1 Congenital toxoplasmosis
Three pathologists, Wolf, Cowen, and Paige
from New York, United States, first conclu-
sively identified T. gondii in an infant girl who
was delivered full term by cesarean section on
May 23, 1938, at Babies Hospital, New York
(Wolf et al., 1939a,b). The girl developed con-
vulsive seizures at 3 days of age, and lesions
were noted in the maculae of both eyes
through an ophthalmoscope. She died when a
month old and an autopsy was performed. At
postmortem, brain, spinal cord, and right eye
were removed for examination. Free and intra-
cellular T. gondii were found in lesions of
encephalomyelitis and retinitis of the girl.
Portions of cerebral cortex and spinal cord
Toxoplasma Gondii
were homogenized in saline and inoculated
intracerebrally into rabbits and mice. These
animals developed encephalitis, T. gondii was
demonstrated in their neural lesions, and
T. gondii from these animals was successfully
passaged into other mice.
Wolf, Cowen, and Paige reviewed in detail
their own cases and those reported by others,
particularly Janků (1923), and Torres (1927)
of T. gondii like encephalomyelitis and chor-
ioretinitis in infants (Wolf and Cowen, 1937,
1938; Wolf et al., 1939a,b, 1940; Cowen et al.,
1942; Paige et al., 1942). Janků (1923), an oph-
thalmologist from Czechoslovakia, was cred-
ited earlier with finding a T. gondii like
parasite in a human eye. The following
description of the case of Janků is taken from
the English translation published by Wolf
and Cowen (1937): “The patient was born
with left microphthalus and became blind at
the age of 3 months, and had hydrocephalus.
The child died when 11 months old. The eyes
and brain were removed at autopsy. Grossly,
the child had internal hydrocephalus but the
brain was not available for histopathological
examination. Chorioretinitis was present in
both eyes and cysts-like structures (termed
sporocysts by Janků) were seen in the right
eye.” Janků (1923) thought that this parasite
was Encephalitozoon (a microsporidia). The
material from this case is thought to be
destroyed in World War bombing, and so
confirmation of these findings is not possible.
Torres (1927) found protozoa in lesions of
encephalitis in a 2-day-old girl in Rio de
Janeiro, Brazil. Numerous organisms were
seen, but these were thought to be a new
species of Encephalitozoon. This patient
also had myocarditis and myositis. In the
Netherlands, de Lange (1929) found protozoa
in sections of the brain of a 4-month-old
child that was born with hydrocephalus.
These sections were reexamined by Wolf and
Cowen, and a full account was reviewed by
Sabin (1942).
 1.5 Toxoplasmosis in humans
Sabin (1942) summarized all that was
known of congenital toxoplasmosis in 1942
and proposed typical clinical signs of
congenital toxoplasmosis: hydrocephalus or
microcephalus, intracerebral calcification, and
chorioretinitis. These signs helped in the clini-
cal recognition of congenital toxoplasmosis.
Frenkel and Friedlander (1951) published a
detailed account of five fatal cases of toxoplas-
mosis in infants that were born with hydro-
cephalus; T. gondii was isolated from two of
these infants. They described the pathogenesis
of internal hydrocephalus as a blockage of the
aqueduct of Sylveus due to ventriculitis result-
ing from a T. gondii antigen antibody reaction.
This lesion is unique to human congenital toxo-
plasmosis and has never been verified in other
animals (J.P. Dubey, unpublished). This report
was the first in depth description of lesions of
congenital toxoplasmosis not only in the
central nervous system but also other organs.
Hogan (1951) also provided the first detailed
clinical description of ocular toxoplasmosis.
1.5.2 Acquired toxoplasmosis
1.5.2.1 Children
Sabin (1941) reported toxoplasmosis in a
6-year-old boy from Cincinnati, Ohio. An
asymptomatic child with initials of RH was hit
with a baseball bat on October 22, 1937. He
developed a headache 2 days later and convul-
sions the day after. He was admitted to the
hospital on the 7th day but without obvious
clinical signs. Except for lymphadenopathy
and enlarged spleen, nothing abnormal was
found. He then developed neurological signs
and died on the 30th day of illness. The brain
and spinal cord were removed for histopatho-
logical examination and bioassay. Because of
the suspicion of polio virus infection a homog-
enate of cerebral cortex was inoculated into
mice. T. gondii was isolated from the inoculated
mice, and this isolate was given the initials of
Toxoplasma Gondii
9
the child and became the famous RH strain.
Only small lesions of nonsuppurative encepha-
litis were found microscopically in the brain of
this child. Neither gross lesions and nor any
viral or bacterial infections were found. This
child most likely had acquired T. gondii infec-
tion recently, and the blow to the head was
coincidental and unrelated to the onset of
symptoms. It is noteworthy that some mice
infected with the original RH strain did not die
until day 21 postinoculation. By the third-
passage mice died in 3 5 days after inocula-
tion. The RH strain of T. gondii has since 1938
been passaged in mice in many laboratories.
After this prolonged passage, its pathogenicity
for mice has been stabilized (Dubey, 1977), and
it has lost the capacity to produce oocysts in
cats (Frenkel et al., 1976). Additional details of
history of toxoplasmosis in humans were given
by Dubey (2008) and Weiss and Dubey (2009).
1.5.2.2 Toxoplasmosis in adults
Pinkerton and Weinman (1940) identified
T. gondii in the heart, spleen, and other tissues
of a 22-year-old patient who died in 1937 in
Lima, Peru. The patient exhibited fever and con-
comitant Bartonella sp. infection. Pinkerton and
Henderson (1941) isolated T. gondii from blood
and tissues of two (50 and 43 years old) indivi-
duals who died in St. Louis, Missouri. Recorded
symptoms included rash, fever, and malaise.
These were the first reports of acute toxoplas-
mosis in adults without neurological signs.
1.5.2.2.1 Lymphadenopathy
Siim (1956) drew attention to the fact that
lymphadenopathy is a frequent sign of acquired
toxoplasmosis in adults, and these findings
were confirmed by Beverley and Beattie (1958)
who reported on the cases of 30 patients. A full
appreciation of the clinical symptoms of
acquired toxoplasmosis was achieved, when
outbreaks of acute toxoplasmosis were reported
in adults in the United States (Teutsch et al.,
1979) and in Canada (Bowie et al., 1997).
10 1. The history and life cycle of Toxoplasma gondii
1.5.2.2.2 Ocular disease
Before 1950 virtually all cases of ocular toxo-
plasmosis were considered to result from con-
genital transmission (Perkins, 1961). Wilder
(1952) identified T. gondii in eyes that had been
enucleated. The significance of this finding lies
in the way this discovery was made. These eyes
were suspected of being syphilitic, tuberculous,
or of having tumors. Wilder was a technician in
the registry of Ophthalmic Pathology at Armed
Forces Institute of Pathology, Washington, DC,
and she routinely microscopically examined the
sections that she prepared. She put enormous
effort into identify microbes in these “tubercu-
lous” eyes but never identified bacteria or spiro-
chetes by special staining. Then she found T.
gondii in the retinas of these eyes. She subse-
quently collaborated with Jacobs and Cook and
found most of these patients with histologically
confirmed T. gondii infection had low levels of
dye test antibodies (a titer of 1:16), and in one
patient antibodies were demonstrable only in
undiluted serum (Jacobs et al., 1954a). Jacobs
et al. (1954b) made the first isolation of T. gondii
from an eye of a 30-year-old male hospitalized
at the Walter Reed Army Hospital, Washington,
DC. The eye had been enucleated because of
pain associated with elevated intraocular
pressure. A group of ophthalmologists from
southern Brazil initially discovered ocular toxo-
plasmosis in siblings. Among patients with
postnatally acquired toxoplamosis who did not
have retinochoidal scars before, 8.3% developed
retinal lesions during a 7-year follow-up
(Silveira et al., 1988, 2001). Ocular toxoplasmosis
was diagnosed in 20 of 95 patients with acute
toxoplasmosis associated with the Canadian
waterborne outbreak of toxoplasmosis in 1995
(Burnett et al., 1998; also see Holland, 2003).
1.5.2.2.3 Acquired immunodeficiency syndrome
epidemic
Before the epidemic of the acquired immuno-
deficiency syndrome (AIDS) in adults in the
Toxoplasma Gondii
1980s, neurological toxoplasmosis in adults was
rarely reported and essentially limited to
patients treated for tumors or those given trans-
plants. Luft et al. (1983) reported acute
toxoplasmosis-induced encephalitis that was
fatal if not treated. In almost all cases, clinical
disease occurred as result of reactivation of
chronic infection initiated by the depression of
intracellular immunity due to HIV infection.
Initially, many of these cases of toxoplasmosis
in AIDS patients were thought to be lymphoma.
1.6 Toxoplasmosis in other animals
Mello (1910) in Turin, Italy, first reported
fatal toxoplasmosis in a domestic animal (a
4-month-old dog) that died of acute visceral
toxoplasmosis. Over the next 30 years canine
toxoplasmosis was reported in Cuba, France,
Germany, India, Iraq, Tunisia, USSR, and the
United States (Dubey and Beattie, 1988).
Campbell et al. (1955) found that most cases of
clinical toxoplasmosis were in dogs infected
with the canine distemper virus (CDV) infec-
tion. Even vaccination with live-attenuated
CDV, vaccine can trigger clinical toxoplasmosis
in dogs (Dubey et al., 2003a,b). The incidence
of clinical toxoplasmosis in dogs has decreased
dramatically after vaccination with CDV vac-
cine became a routine practice.
Strangely enough the first case of toxoplas-
mosis was not reported in a cat until 1942
when Olafson and Monlux found it in a cat
from Middletown, New York, United States. In
the 1950s and 1960s Galuzo and Zasukhin pub-
lished in Russian their own studies and those
of other researchers on many species of ani-
mals from the former USSR. This information
was made available to scientists in other coun-
tries, when their book was translated into
English by Plous Jr. and edited by Fitzgerald
(1970). Jı́ra and Kozojed (1970, 1983) published
the most comprehensive bibliography of toxo-
plasmosis, listing more than 12,000 references
 1.7 Diagnosis
and categorizing them by hosts and topics.
This work proved useful for literature searches
before electronic databases.
Toxoplasmosis in sheep deserves special
attention because of its economic impact.
William Hartley, a well-known veterinary
pathologist from New Zealand, and his associ-
ates J.L. Jebson and D. McFarlane, discovered
T. gondii like organisms in the placentas and
fetuses of several unexplained abortions in
ewes in New Zealand. They called it New
Zealand Type II abortion. Hartley and
Marshall (1957) finally isolated T. gondii from
aborted fetuses. Hartley (1961) and Jacobs and
Hartley (1964) experimentally induced toxo-
plasmic abortion in ewes. The identification of
T. gondii abortion in ewes was a landmark dis-
covery in veterinary medicine. Prior to that
protozoa were not recognized as cause of epi-
demic abortion in livestock. Subsequently,
Beverley and Watson (1961) recognized epi-
demics of ovine abortion in the United
Kingdom. Dubey and Towle (1986) and Dubey
and Beattie (1988) summarized all that was
known about toxoplasmosis in sheep and its
impact on agriculture. Millions of lambs are
still lost throughout the worldwide due to this
infectious disease.
Sanger and Cole (1955) were first to isolate
T. gondii from a food animal. Dubey and
Beattie (1988) and Dubey (2010) reviewed the
worldwide literature on toxoplasmosis in
humans and other animals. The discovery and
naming of two new organisms, Neospora cani-
num (Dubey et al., 1988) and Sarcocystis neurona
(Dubey et al., 1991), that were previously
thought to be T. gondii, resulted in new infor-
mation on the host distribution of T. gondii. We
now know that cattle and horses are resistant
to clinical T. gondii; there have been no con-
firmed cases of clinical toxoplasmosis in either
cattle or horses (Dubey, 2010). N. caninum was
found to be a common cause of abortion in
cattle worldwide (Dubey, 2003; Dubey et al.,
2017), and S. neurona was found to be a
Toxoplasma Gondii
11
common cause of fatal encephalomyelitis in
horses in the Americas (Dubey et al., 2001).
S. neurona was also found to cause systemic
disease entities involving eyes, lungs, muscles,
and neural tissues in pets and wildlife (Dubey
et al., 2015).
The finding of T. gondii in marine mammals
deserves special mention. Before the discovery
of T. gondii oocyst, no one would have sus-
pected that the marine environment would be
contaminated with T. gondii and that fish-
eating marine mammals would be found
infected with T. gondii (Dubey et al., 2003a,b;
Conrad et al., 2005; Thomas et al., 2007; Miller
et al., 2008; Dubey, 2010). Thomas and Cole
(1996) and Cole et al. (2000) isolated viable
T. gondii from sea otters in the United States.
Several reports have now appeared that con-
firm that T. gondii can occur in many species of
marine mammals (Dubey, 2010).
1.7 Diagnosis
The current management, diagnosis, and
clinical manifestations of T. gondii infection in
humans are discussed in Chapter 4, Human
Toxoplasma infection.
1.7.1 Sabin Feldman dye test
Development of a novel serologic test, the
dye test, by Sabin and Feldman (1948), was
perhaps the greatest advancement in the field
of toxoplasmosis. The dye test is highly sensi-
tive and specific with no evidence for false
results in humans. Even titers as low as 1:2 are
meaningful for the diagnosis of ocular disease.
The ability to identify T. gondii infections based
on a simple serological test opened the door
for extensive epidemiological studies on the
incidence of infection. It became clear that
T. gondii infections are widely prevalent
in humans in many countries. It also
12 1. The history and life cycle of Toxoplasma gondii
demonstrated that the so-called tetrad of
clinical signs considered indicative of clinical
congenital toxoplasmosis occurred in other
diseases and assisted in the differential diagno-
sis (Sabin and Feldman, 1949; Feldman and
Miller, 1956).
1.7.2 Detection of IgM antibodies
Remington et al. (1968) first proposed the
usefulness of the detection of IgM antibodies in
cord blood or infant serum for the diagnosis of
congenital toxoplasmosis since IgM antibodies
do not cross the placenta, whereas IgG antibo-
dies do cross the placenta. Remington (1969)
modified the indirect fluorescent antibody test
and the ELISA (Naot and Remington, 1980) to
detect IgM in cord blood. Desmonts et al.
(1981) developed a modification of IgM-ELISA,
combining it with the agglutination test (IgM-
ISAGA) to eliminate the necessity for an
enzyme conjugate. Although IgM tests are not
perfect, they have proved useful for screening
programs (Remington et al., 2001).
1.7.3 Direct agglutination test
The development of a simple direct aggluti-
nation test has aided tremendously in the sero-
logical diagnosis of toxoplasmosis in humans
and other animals. In this test, no special
equipment or conjugates are needed. This test
was initially developed by Fulton (1965) and
improved by Desmonts and Remington (1980)
and Dubey and Desmonts (1987) who called it
the modified agglutination test (MAT). The
MAT has been used extensively for the diagno-
sis of toxoplasmosis in animals (Dubey, 2010).
The sensitivity and specificity of MAT has
been validated by comparing serologic data
and isolation of the parasite from naturally and
experimentally infected pigs (Dubey, 1997;
Dubey et al., 1995a,b) and naturally infected
Toxoplasma Gondii
chickens (Dubey et al., 2016). Even low MAT
titers (,1:10) were found specific.
1.7.4 Detection of Toxoplasma gondii
DNA
Burg et al. (1989) first reported detection of
T. gondii DNA from a single tachyzoite using
the B1 gene in a polymerase chain reaction
(PCR). Several subsequent PCR tests have been
developed using different gene targets.
Overall, this technique has proven very useful
in the diagnosis of clinical toxoplasmosis
(Remington et al., 2011).
1.8 Treatment
Sabin and Warren (1942) reported the effec-
tiveness of sulfonamides against murine toxo-
plasmosis, and Eyles and Coleman (1953)
discovered the synergistic effect of combined
therapy with sulfonamides and pyrimeth-
amine; the latter is the standard therapy for
toxoplasmosis in humans (Remington et al.,
2001). Garin and Eyles (1958) found spiramycin
to have antitoxoplasmic activity of in mice.
Since spiramycin is nontoxic and does not
cross placenta, it has been used prophylacti-
cally in women during pregnancy to reduce
transmission of the parasite from mother to
fetus (Desmonts and Couvreur, 1974b).
1.9 Prevention and control
1.9.1 Serologic screening during
pregnancy
Georges Desmonts initiated studies in
Paris, France in the 1960s looking at serocon-
version in women during pregnancy and the
transmission of T. gondii to the fetus. Blood
was obtained at the first visit, at 7 months,
 References
and at the time of parturition. Desmonts initi-
ated prophylactic treatment of women who
seroconverted during pregnancy. Results of
the 15-year study demonstrated that (1) infec-
tion acquired during the first two trimesters
was most damaging to the fetus, (2) not all
women that acquired infection transmitted it
to the fetus, (3) women seropositive prior to
pregnancy did not transmit infection to the
fetus, and (4) treatment with spiramycin
reduced congenital transmission, but not clini-
cal disease in infants (Desmonts and
Couvreur, 1974a,b). At about the same time,
Thalhammer (1973, 1978) initiated a similar
screening program for pregnant women in
Austria. In addition to scientific knowledge,
these screening programs have helped to dis-
seminate information for the prevention of
toxoplasmosis.
A neonatal serological screening and early
treatment for congenital T. gondii infection was
initiated in Massachusetts, United States in
1980s (Guerina et al., 1994). The efficacy of
treatment of T. gondii infection in the fetus and
newborn is not fully delineated, and many
issues related to the cost and benefit of screen-
ing and treatment in pregnancy and in new-
borns remain to be examined.
1.9.2 Hygiene measures
After the discovery of the life cycle of T. gon-
dii in 1970, it became possible to advise preg-
nant women and other susceptible populations
on avoiding contact with oocysts (Frenkel and
Dubey, 1972). Studies were conducted to con-
struct thermal curves showing temperatures
required to kill T. gondii in infected meat by
freezing (Kotula et al., 1991), cooking (Dubey
et al., 1990), and by gamma irradiation (Dubey
et al., 1986). These data are now used by regu-
latory agencies to advise consumers about the
safety of meat. Freezing of meat overnight in a
household freezer before human or animal
Toxoplasma Gondii
13
consumption remains the easiest and most eco-
nomical method of reducing transmission of
T. gondii through meat.
1.9.3 Animal production practices
Extensive epidemiological studies on pig
farms in the United States in 1990s concluded
that keeping cats out of the pig barns and rais-
ing pigs indoors can reduce T. gondii infection
in pigs (Dubey et al., 1995a,b; Weigel et al.,
1995). Because of changes in pig, husbandry
prevalence of viable T. gondii in pigs is reduced
to ,1% (Dubey et al., 2005). Because ingestion
of infected pork is considered the main meat
source of T. gondii for humans (at least in the
United States), the prevalence of T. gondii in
humans in the United States has decreased in
the past decade (Jones et al., 2007).
1.9.4 Vaccination
Vaccination of sheep with a live cyst-less
strain of T. gondii reduces neonatal mortality in
lambs, and this vaccine is available commer-
cially (Wilkins and O’Connell, 1983; Buxton
and Innes, 1995). To date, there is no vaccine
suitable for human use.
Acknowledgments
I would like to dedicate this paper to the late Profs. J.K.A.
Beverley and C.P. Beattie who were my PhD advisors
(1964 67) and to the late Prof. J.K. Frenkel who was my
postdoctoral studies supervisor (1973 78).
References
Aramini, J.J., Stephen, C., Dubey, J.P., 1998. Toxoplasma
gondii in Vancouver Island cougars (Felis concolor van-
couverensis): serology and oocyst shedding. J. Parasitol.
84, 438 440.
Benenson, M.W., Takafuji, E.T., Lemon, S.M., Greenup,
R.L., Sulzer, A.J., 1982. Oocyst-transmitted toxoplasmo-
sis associated with ingestion of contaminated water.
N. Engl. J. Med. 307, 666 669.
Another random document with
no related content on Scribd:
[Inhoud]
 RATTEN, KOGELFLESCHJES EN LADDERS!

Het is voor negenen in den morgen. Een gedeelte van de bende staat voor de
school te knikkeren.

Een schel gefluit kondigt de nabijheid van Ambro aan en deze komt in vollen
draf aangerend, z’n knikkerzak boven z’n hoofd zwaaiend, als een cavalerist,
die op den vijand aanrukt.

Hijgend nadert hij het troepje.

„Potje schoffelen, Ambro,” vraagt Karel, „of pompen?”

Maar Ambro luistert niet naar hem. Hij is vervuld van het groote nieuws, dat
hij ze moet vertellen.

„Jongens, ik heb een brief, van wie denk je?”

„Van Margootje,” veronderstelt Paul.

„Bê-je,” zegt Ambro diep beleedigd.

„Daar zou ik zoo’n drukte van maken? Neen, ’t is geen zij, maar een hij.”

„Ik weet ’t,” roept Piet. „Van Dokter Reens!”

„Wat zou die mij te schrijven hebben!”

De jongens branden van nieuwsgierigheid.

„Nog eenmaal raden,” roept Ambro en hij zet een gewichtig gezicht.

„Nou,” zegt Chris. „Dan is er nog maar een die ’t kan zijn … de directeur van
den Dierentuin.”

„Mis vader! Hij is van Wim Bolk.”

„Wim Bolk!” roepen ze in koor.

„Dat is waar, die leeft ook nog,” zegt Puckie droog. [142]

Wim Bolk was met de noorderzon vertrokken, want z’n vertrek viel juist in de
groote vacantie, toen het meerendeel der jongens uit de stad was.

Zijn vader, ambtenaar der posterijen, was plotseling overgeplaatst naar

Haarlem en na het afscheid op school, vóór de groote vacantie, had geen van
de jongens Wim teruggezien.

Den eersten tijd had hij nog wel met hen gecorrespondeerd, ofschoon die
correspondentie uitsluitend bestond uit een prentkaart, met zijn naam er
onder. Maar vrij spoedig hielden ook deze levensteekenen op en dachten de
jongens niet meer aan elkaar.

„Zeg, jongens, hij komt weer hier wonen,” zei Ambro. „Z’n vader is weer
overgeplaatst.”

„Waar was ie ook weer?”

„In Haarlem. Hij vraagt me of jullie ’t allemaal goed maken en of het Hol van
Kaan nog bestaat. Met September gaat hij naar de H.B.S.”

„We hebben veel keet met hem gehad,” zegt Chris. „’t Was een eerste-klas-
keet-schopper.”

„Zou-die veranderd zijn?”

„An z’n brief zou je ’t niet zeggen,” zegt Ambro die den brief voor den dag
haalt.

De jongens steken de hoofden bijeen en lezen:

Hooggeachte bende van het Hol van Kaan,

Gij zult waarschijnlijk Uw oud mede-lid, die thans de pen heeft opgenomen om zijne
vroegere bondgenooten te verwittigen van zijn … barst!… ik kan er niet uit komen.
[143]

Zeg, kerels, ik kom fijn weer in Rotterdam wonen, en dan zullen we de peentjes
opscheppen.

In Haarlem is ’t niks gedaan, hoor! Een saaie bedoening!

 Op school trof ik allemaal brave Hendrikken, die niks durven. En om alleen de beest
uit te hangen … daar had ik geen lol in.

Dan wij, hè! Weet jullie nog, die keet aan de haven, toen Ambro aan de kraan hing.

Toen ik ’t die brave Haarlemmer halletjes vertelde, wilden ze ’t niet eens gelooven.

Een was wel eens in Rotterdam geweest en had, bij z’n terugkeer, z’n voeten
geveegd, vóór hij in Haarlem aankwam.

Nou, ik heb wat vaak naar de Rotterdamsche modder verlangd! Nog twee weekie’s,
dan ben ik er weer!

Leeft Alebes nog? Maken jullie het allemaal goed? En hoe staat ’t met het Hol van
Kaan? Met September ga ik naar de H.B.S. Jullie ook zeker? Ik hoop maar, dat we
allemaal op dezelfde komen. Een reuzen-keet, zeg!

Komen jullie me allemaal van den trein halen, dan schrijf ik nog hoe laat ik kom.

Adi, ik smeer ’m. Tot kijks,

Wim.

„Nee, hoor, hij is nog niks veranderd,” zegt Chris blij.

„Ik had ’m ook geen gezicht meer aangekeken,” zegt Puckie. [144]

Ze vonden het allen eenparig fijn, dat ie terugkwam en Ambro vond, dat zijn
terugkeer feestelijk gevierd moest worden.

Daar ging de schoolbel.

„Fijn, vanmiddag vrij, kunnen we verder vergaderen in het Hol,” zegt Ambro.

In korten tijd ligt het schoolplein, eerst zoo vol lawaai van joelende jongens,
nu stil en verlaten daar en wordt de schooldeur hard en onverbiddelijk achter
ze gesloten.

Ze zijn weer voor drie lange uren opgeborgen, de woelige rakkers.

De elementen waren hen echter dien middag niet gunstig gezind.

Toen de school uitging plasregende het en de jongens, hierop onvoorbereid,
rennen in woeste vaart naar huis.

Op school hebben ze afgesproken samen te komen in het oude apen-

gebouw, dat bij slecht weêr als vergaderzaal dienst deed.

Om één uur zijn ze allen present.

„Wat een snert-weêr,” zegt Ambro.

„Maar, dat is niets, dan maar wat turnen.” En meteen trekt hij zich op aan een
horizontale ijzeren stang en maakt den buik-zwaai.

In minder dan geen tijd bengelen nu alle jongens aan de zijstangen naast de
leege hokken.

Dan komt Chris met een plan voor den dag.

„Jongens, we gaan ratten vangen.” [145]

„En zeker weer d’r uit getrapt worden,” roept Puckie.

„Neen,” zegt Ambro. „In dien kletsregen is ’t niks gedaan, dan zijn ze d’r niet
eens.”

„Wel neen, niet buiten,” schreeuwt Chris terug. „Hier zitten d’r bij de vleet.
Maar we moeten voorzichtig te werk gaan,… ik weet precies door welk gaatje
ze ’m piepen. Daar ginds,” wijst hij. „Tegen ’t schot bij den chimpansee.”

„Hoe wil je ze dan pakken? Zeker met je handen.”

„Dat ’s te gevaarlijk,” zegt Paul. „Een rat in angst vliegt je naar de keel.”

„O, daar heb je juffrouw Piepzak! Hoor je ’t jongens, Paul knijpt ’m alweer,”
plaagt Puckie.

„Neen,” zegt Chris. „Onder de cape krijgen we ze te pakken,” en hij wijst op

het eerste hok van den ingang af.

„Als we ons nou koest houden, komen ze daar maïs vreten, want je begrijpt
wel, dat ze, als we blijven schreeuwen en lawaai schoppen, kalm in hun
holletje blijven.”

„Ik begrijp er niks van,” zegt Ambro. „Wat mot je nou eigenlijk met die cape?”

„Laat me dan ook uitspreken. Achter deze deuren stellen we ons op, dan zal
je zien, dat de ratten spoedig komen, want toen we daarnet binnenkwamen
zag ik ze „weg-peesen”. Dan gaan ze, zòò, langs den heelen muur en aan het
einde moeten ze een gangetje oversteken voor ze bij hun hol zijn. [146]

„Wij loopen met een cape daar ook naar toe, zoo hard we kunnen, hè! dan
gooien we de cape bliksemsnel op een rat en gaan er op dansen. Ben
reuzen-bak, jongens! ik heb ’t al meer gedaan.”

Nu deze ingewikkelde geschiedenis duidelijker vormen voor ze aanneemt,

vinden de jongens het plan kapitaal en ze besluiten dan maar direct te
beginnen.

„Als de oppasser ’t maar niet merkt,” waagt Paul het te zeggen.

„Och, Lutjebroek, als je bang bent, hoepel dan maar op. Trouwens, we doen
er een goed en nuttig werk meê.”

„Ja,” merkt Paul schamper op. „Een maand den tuin uit als je een dooje rat op
het kantoor brengt.”

„Verdelgt de ratten en ge zult spinazie eten,” zegt Ambro zalvend. „Vooruit

knullen, beginnen!”

„Hier moeten we staan,” en Chris neemt het opper-commando op zich.

„En één ding, jongens, kop dicht! Wie kabaal maakt, krijgt een peut in z’n
ribben.”

Onbewegelijk en dood-stil staan ze achter de deuren. Chris houdt een oog in

’t zeil.

„Er kaikt mit één oogie!” imiteert Ambro een clown waarvan hij pas genoten
heeft, als hij Chris zoo naar één punt ziet staren.
Maar vijf stompen doen hem er aan herinneren, dat het wachtwoord „kop
dicht” is.

„Nog één keer,” dreigt veldheer Chris. „En dan smeer je ’m maar, jij bederft
altijd alles met je flauwe kul.” [147]

Wel vijf minuten lang heerscht er de diepste stilte en met wederzijdsche

bewondering voor deze algeheele rust, kijken de jongens elkaar in spanning
aan.

Hun geduldig wachten wordt beloond. Een stevige rat schiet met een telkens
afgebroken vaart rrrtsss langs den muur.

Zóó is bij op zijn hoede voor dreigend gevaar, dat hij zich telkens
onbewegelijk houdt, en, geen onraad bespeurend, weer snel vooruit schiet.

De jongens behoeven zich nu geen geweld meer aan te doen om stil te zijn.
Ademloos bespieden ze, met groote tintelende oogen, iedere beweging van
hun prooi.

De rat loopt z’n noodlot tegemoet, hij is den bak met maïs genaderd.

Als een pijl uit den boog, schiet Chris met zijn cape vooruit, de jongens volgen
hem. En de rat vliegt in volle vaart terug langs den muur, maar Chris is hem
vóór, en juist, als het dier het gangetje oversteekt werpt hij handig de cape
over hem heen en begint luid schreeuwend, een woesten oorlogsdans boven
op hem.

De jongens volgen direct zijn voorbeeld en als een troepje Indianen om een
gevangen blanken broeder, zoo voeren ze hun krijgsdans uit.

De rat, die niet weet, hoe hij ’t heeft, wijst door sprongen de plaats aan waar
hij zich onder de cape bevindt en iedere sprong, wordt beantwoord met een
trap naar zijn kant.

„Hou op, jongens, da’s wreed,” roept Paul met [148]ongewone stemverheffing.
„As-t-ie dood moet, best, maar dan ineens.”

Ze zien hem daar ineens, bleek en klein tegen den muur gedrukt staan.
„Bah! hij is bang,” zegt smalend Chris.

„Dat ben ik niet,” zegt Paul heftig.

„Maar ik vind jullie laf. Je hebt ’m nou toch gepakt, laat ’m nou weer los.”

Deze woorden van Paul hebben de jongens toch even tot kalmte gebracht, en
de rat heeft deze tijdelijke rust benut om onder de cape weg te schieten en
vliegensvlug in zijn hol te verdwijnen.

„Hoe was ie?” vraagt Chris zegevierend.

„Nog een keertje,” roept Karel.

„Dàt kê-je denken,” antwoordt Chris. „Nou duurt ’t uren voor-ie weer komt.”
„Paul kijkt er beteuterd van,” zegt Ambro. „Jij [149]moet maar rokken gaan
dragen,” smaalt Piet.

Paul, nu gerustgesteld over het lot van de rat, trekt zich van al die gezegden
bitter weinig aan.

„Zou ’t nog regenen?” vraagt Ambro.

Chris, die inziet, dat er niet veel meer in het oude apenhuis te doen valt, zegt:
„We zullen zien,” en met loopt hij naar den ingang en constateert het heugelijk
feit, dat de zon is doorgebroken.

„Pracht-weêr, jongens! Kom mee, we gaan er uit!”

„Naar ’t Hol,” roept Karel.

„Wel nee! daar is ’t nog kletsnat. Goed om de pip te krijgen.”

„Wat moeten we dan doen?”

Geen van hen weet iets te bedenken en de bende slentert doelloos voort in
de richting van de groote sociëteit.

„Willen we honderd meter hardloopen,” stelt Piet voor.

„Eerst me vriendin bezoeken,” zegt Ambro. „Ik weet geen raad van den dorst.”

De vriendin van Ambro is een oude juffrouw, meer bekend onder den naam
van „het watervrouwtje”. Ze verkoopt glazen water voor 1 cent per stuk en is
tevens opper-„privaat”-meesteres.

Ambro stond bij haar in een goed blaadje, omdat hij dikwijls voor haar op de
torenklok ging kijken hoe laat het was. Als belooning mocht hij dan zooveel
water drinken als hij wilde.

Maar nu, zonder haar opdracht af te wachten, bracht hij haar de tijding, dat
het zeven minuten [150]over drieën was, waarop ze hem vriendelijk bedankte
en vroeg of hij niet wat wilde drinken.
„Zes glazen drink ik vandaag,” was het antwoord van Ambro. „En als ik ze niet
alleen op kan, zullen me vrienden wel helpen.”

En zich tot dezen richtend, riep hij: „Jongens, ik geef een rondje water.”

Zes groote glazen werden naar binnen geklokt. De jongens namen afscheid
van het oudje en vervolgden hun weg.

Toen ze de sociëteit genaderd waren, riep Puckie: „Kijk es, het kelderluik staat
open, laten we stiekum naar binnen kruipen en lol maken.”

Dáár waren ze voor te vinden.

Een ontelbaar aantal stoeltjes stond in lange rijen tegen den achtermuur van
de sociëteit, klaar, om vervoerd te worden naar het groote terrein bij de
muziektent.

„Zou er niemand zijn,” fluistert Ambro en hij luistert aandachtig aan het
kelderluik.

„Ik hoor niks. Nou stil, jongens, ik zal wel voor gaan.” En hij daalt
behoedzaam het trapje af, dat naar den kelder voert. De rest van de bende
volgt. En nu staan ze in den half-duisteren kelder.

„Krimmenikkie, wat is ’t hier donker,” roept Paul.

„En vol ratten,” plaagt Ambro. „Volgen jullie me maar, straks zitten we in stik-
duisteren nacht.”

Blijkbaar is er niemand in den kelder en vol moed trekken ze nu voorwaarts.

De kelder loopt door onder de geheele sociëteit en heeft dus een verbazende
omvang. [151]

De jongens staan nu voor een vierkant gat in den muur, ongeveer een meter
boven den beganen grond.

Het schaarsche licht, dat door een raampje valt, maakt, dat ze nog net
kunnen zien, waar ze zich bevinden.
„Hier moeten we inkruipen,” zegt Ambro. „Maar pas op, dat je je kop niet
stoot, ’t is hier bar laag.”

Achtereenvolgens kruipen ze nu door het gat.

„Een mooie boel hier,” gromt Chris. „Waar kruip ik eigenlijk over heen?
Kolengruis of zand?”

„Dat zal je wel zien als je weer buiten bent,” zegt Ambro en hij waarschuwt
voor een gaspijp, die dwars over hun weg ligt.

„Zoo zullen we er nog een heeleboel tegen komen,” zegt hij.

„Waar ga je eigenlijk naar toe,” roept Karel, die evenmin als Chris erg veel
pleizier schijnt te hebben in dit duistere avontuur.

„Nog veertig meter ongeveer moeten we vooruit kruipen, dan komen we uit de
benauwenis en vinden als belooning voor ons ploeteren een heelen stapel
kogelfleschjes.”

„Hij zeit wat!” roept Chris ongeloovig.

„Op me woord van eer,” zegt Ambro.

Dit „prikkelende” vooruitzicht doet de jongens verder zonder morren hun weg
vervolgen over stof en kolengruis, gaspijpen en glasscherven en dat alles,
kruipend op hun buik in volslagen duisternis.

„We zijn er haast,” zegt Ambro. [152]

„Ik proef ’t al,” roept Puckie.

„Ik zoek frambozen uit,” zegt Piet. „Als je tenminste zien kan wat je te pakken
hebt.”

„Ik gember,” zegt Paul verrukt.

Ze zijn nu in de nabijheid gekomen van het kelderraampje, aan de voorzijde

van de sociëteit en tot aller verademing wijkt de lage zoldering boven hun
hoofden en staan ze wederom in een half-verlichte ruimte.
„Kijk jongens, daar staan ze,” roept Ambro triomfantelijk.

De jongens rennen er op af.

„Ver …, da’s een leege,” bromt Piet.

„Die ik heb ook,” roept Puckie.

„Ze zijn allemaal leeg,” schreeuwt Karel.

En Ambro, die dit reeds lang wist, maar zonder dit aanlokkelijk vooruitzicht
geen kans zag de bende mee te krijgen op dezen luguberen tocht door de
catacomben, zegt met het onnoozelste gezicht van de wereld:

„Heb ik dan gezegd, dat ze vol waren?”

„Flauwe bak,” bromt Piet.

„Hebben we daarvoor zoo gezwoegd? Als je nog eens wat weet,” zegt Chris,
z’n handen van kolengruis reinigend.

„Nou, wees maar koest,” kalmeert Ambro. „Jullie krijgen ieder van mij een
mooi glazen knikkertje,” en meteen ketst hij met kracht een kogelfleschje
tegen den muur.

„Hier heb je er al een,” zegt hij, het glazen kogeltje aan Paul gevend. [153]

„Da’s een idee,” zegt Chris getroost

De jongens, na allen Ambro’s voorbeeld volgend, pakken ieder een projectiel

en de onderaardsche gewelven daveren van uiteenspattende granaten in den
vorm van kogelfleschjes.

In minder dan geen tijd, heeft ieder een stuk of tien fleschjes gebroken en met
rijken buit belaân, wordt langs denzelfden moeilijken weg de terugtocht
aanvaard.

„Ik zal blij zijn als we weer buiten staan. Au!” schreeuwt Puckie, die vergeten
is, dat de zoldering maar een halve meter hoog is.

„Puckie denkt, dat z’n kop een kogelfleschje is,” lacht Ambro.
„Zouden ze niets gehoord hebben,” vraagt Paul bevreesd.

„Vast en zeker,” stelt Ambro hem gerust. „We vinden nu de deur gesloten en
een man tot de tanden toe gewapend er voor. Maar dat is niets, ik weet een
geheimen uitgang.”

Eindelijk zijn ze weer bij het vierkante gat gekomen, waardoor heen ze hun
tocht begonnen zijn.

„Zie je wel,” zegt Ambro verschrikt. „Het kelderluik is dicht! En de geheime

gang is versperd.”

„Wat moeten we nou beginnen?” zegt Paul en ook de andere jongens zien de
toekomst donker in.

In werkelijkheid, wilde Ambro z’n makkers slechts schrik aanjagen, want, het
kelderluik stond nog open en de geheime gang bestond slechts in zijn
verbeelding.

„Hij liegt,” roept Chris, die direct na Ambro [154]het gat bereikte. „We kunnen er
uit, hoor!”

„Hè, gelukkig maar,” zucht Paul opgelucht „Ik was al bang den tuin uitgetrapt
te worden.”

Behouden staat het zestal weer buiten en als ze elkaar bekijken, schateren ze
het uit. Ze zijn veranderd in negers, hun kleeren, bedekt met een dikke laag
stof en spinrag, de gezichten zwart van kolengruis.

„Ja, jullie hebben lol,” zegt Paul beteuterd. „Maar ik heb pas een nieuw pakkie
an, daar zit weer wat op.”

„Kolengruis zit er op,” plaagt Ambro. „En dat zal er wel af gaan ook,” en hij
begint Paul zóó hardhandig af te kloppen, dat deze het uitschreeuwt van pijn.

„Tjonge, jonge, wat stuift het? Weet je wat we doen, jongens? we gaan straks
naar het weiland achter het spoor en brengen onze kleertjes weer netjes in
orde, en …”
„Da’s van later zorg,” valt Piet Ambro in de rede. „Ik zie een hooge ladder
staan.”

„Waar?” vraagt Ambro haastig. En de angst voor hun besmeurde kleêren

vermindert sterk bij het gezicht van dit verleidelijk voorwerp.

„Daar moeten we op,” zegt Ambro, „niks an te doen!”

„Ik heb er voorloopig genoeg van,” zegt Paul knorrig.

„Des te beter, dan ga jij op den uitkijk staan en waarschuwt als er iemand
aankomt,” zegt Piet.

„Hij gaat heelemaal tot het dak, zie je wel. Ik [155]zal wel weer voorgaan.” En
Ambro begint de ladder te beklimmen.

„Goed uitkijken, Paul,” roept hij toen hij midden op de ladder is gekomen.

Piet wil hem onmiddellijk volgen, maar de ladder begint bij deze beweging zoo
onregelmatig te zwiepen, dat Ambro, een beetje benauwd, naar beneden kijkt
en tot Piet schreeuwt:

„Even wachten tot ik boven ben, stommert!”

En Piet houdt zich nu stil en hoeft niet lang te wachten, want Ambro is het
bovenste terras van de sociëteit genaderd.

„Hè, wat een fijn uitzicht!” zegt hij uitblazend. „Piet, nou kan je komme.”

En achtereenvolgens klimt de bende, behalve Paul, naar boven.

„Wat nou?” vraagt Chris. „Daar staan we! Vriezen we dood, dan vriezen we
dood!”

„O, we kunnen nog hooger,” is Ambro’s antwoord. „En dan zullen we wel
verder zien.”

„Jij commandeert ons maar den heelen middag,” zegt Chris. „En wij doen
maar als makke schapen je zin.”

„Stil maar, ik heb alweer een plan. We gaan rangeeren.”

„Rangeeren?” vraagt Puckie, die zich al de gekste sprongen van Ambro
voorstelt, nu ze daar zoo hoog staan.

Ambro heeft nu een tweede ladder ontdekt, veel kleiner dan die ze
beklommen hebben en die naar het dak voert. [156]

Blijkbaar zijn er op het oogenblik geen werklieden aanwezig en gretig maakt

de bende van deze gelegenheid gebruik en als ze eindelijk boven staan, zegt
Ambro tevreden: „Ziezoo, jongens, hooger kunnen we niet, en nou gaat ’t spul
beginnen.”

Hij haalt een groote fluit voor den dag en wijst naar een paar
goederenwagens op het spoorterrein achter den Dierentuin, die gerangeerd
moeten worden.

De fluit van den rangeerder had hem de booze gedachte ingegeven, ook zijn
fluit te gebruiken en daarmee den boel in de war te sturen.

De jongens zitten nu naast elkaar langs de daklijst. Plotseling laat Ambro een
langgerekt gefluit hooren en zóó zuiver was de nabootsing van het rangeer-
signaal, dat de machinist in den waan werd gebracht en de locomotief tot
staan bracht, met het gevolg, dat onmiddellijk een tegen-signaal volgde van
den rangeerder.

En toen Ambro voor de tweede maal deze grap uithaalde, kreeg de man de
bende op het dak in de gaten en begon luid vloekend met zijn vuist te dreigen,
terwijl hij ze den raad gaf gauw met dat spelletje op te houden.

De waarschuwing van den man werd ontvangen met een waar hoongelach
van de brutale bengels, maar plotseling werden ze opgeschrikt door een
alarmkreet van Paul, die hen toeriep dat de werklieden in aantocht waren.

„Hij houdt ons voor ’t lappie,” zegt Ambro. [157]„Hij is natuurlijk bang, dat we
weer den tuin uitgetrapt worden, blijf maar gerust zitten, jongens, en die kerel
aan den overkant kan toch niet bij ons komen, laat die maar vloeken.”

En ook op de tweede waarschuwing van Paul werd geen acht geslagen,

zoodat de werklui, die intusschen vlak bij waren gekomen, de jongens op het
dak ontdekten.
„Kijk daar een stelletje spreeuwen,” lachte een goedmoedige, oude metselaar.
„Ik zal ze daar es effe nemen!”

Paul staat duizend doodsangsten uit, doch durft niets te zeggen.

„Help es, Jan,” zegt de metselaar tot z’n zoon, een stevige, jonge borst. „We
zullen es effe dat laddertje weghalen,” en luid voegde hij er aan toe: „Dan
hebbe de heere daarbofe gratis losjies!”

„Zie je wel,” zegt Chris boven nijdig tot Ambro. „Paul hield ons niet voor de
mal, da’s niks voor hem.”

„Hij heeft weer es wat gedaan,” vult Puckie aan en de heele bende is ineens
tegen Ambro.

„Leggen jullie nou niet an me kop te kletsen,” zegt deze. „Maar laten we liever
een middel bedenken, hoe we zonder die ladder naar beneden kunnen
komen.”

„Dat wordt nek breken,” zegt Karel. „Ik heb geen zin in een doodval.”

„Al gevonden,” juicht Ambro. „Ze zullen een reuzen-strop hebben! Vlug
jongens, volg me!” en hij daalt de kleine ladder af, naar ’t eerste terras.

Ambro’s beslist optreden en z’n résolute manier [158]van spreken heeft de

gewenschte uitwerking op de jongens en ze gehoorzamen hun hoofdman
weer blindelings.

Ze staan nu op het terras, waarop de deuren uitkomen van het Indisch

Museum.

Ambro had reeds ontdekt, dat één ervan open stond en fluisterend tot de
jongens zegt hij: „Dàt deurtje is onze redding, maar we moeten voorzichtig
zijn en goed kijken of die vent van het Museum ons niet in de gaten heeft.”

„Die maft het grootste deel van den dag,” zegt Piet. „Dus we hebben kans.”

De rolgordijnen voor de deuren zijn neergelaten, om de zon buiten te houden,

dus voor ontdekking behoeven de jongens voorloopig niet bang te zijn.
Op handen en voeten sloop Ambro naar de openstaande deur en keek
behoedzaam naar binnen.

„Inderdaad, de mummie maft,” zegt hij blij-verrast. „Nou zachtjes, jongens. Als
we hem eenmaal voorbij zijn, is het zaakie gezond.”

De bewaker van het museum, evenals de edele Alebes, een Oud-Gediende,

sliep als een Turk, hetgeen niet te verwonderen was, want het was een
warme dag en van bezoek was in dezen tijd van het jaar geen sprake.

Geruischloos sluipt nu het vijftal de deur binnen en of ze op vilten toffeltjes

loopen, inplaats van op stevige moddertrappers, zóó zacht passeeren ze den
slapenden waakhond.

Nu is de uitgang van het museum gauw bereikt en als ze eenmaal de trap

genaderd zijn, [159]holt de bende juichend naar beneden, terwijl het laatste
gedeelte afgegleden wordt langs de breede trapleuning.

„Wat een strop,” lacht Ambro. „Dat hadden ze niet gedacht, de kevers! Ik ga
ze vertellen, dat er vijf jongens in nood zitten, want mij hadden ze toch niet in
de gaten.”

„Ja, ja, een reuzen-bak,” roepen de jongens.

En met z’n zoetsappigste snuitje gaat hij naar de werklieden, terwijl de

overige jongens zich verscholen houden.

„Mijnheer,” spreekt hij den ouden metselaar aan. „Aan den anderen kant van
de sociëteit staan vijf jongens te schreeuwen, ze kunnen d’r niet af. Boven op
het dak, mijnheer, heelemaal boven.”

„Ja, ja, ik weet ’t,” zegt de oude man. „Laat ze maar schreeuwen, me hebbe
den tijd. Asse-me naar huis gaan, magge ze d’r af, en dat duurt nog een paar
uurtjes.”

„Hè toe, laat u ze d’r af, ’t huilen staat ze nader dan ’t lachen.”

„Zeur niet, jong, anders kâ-je een pak op je broek krijgen.”

Ambro geeft het op en slentert langzaam weg. En als hij op een behoorlijken
afstand van den metselaar is gekomen, wenkt hij de jongens.

En als ze weer allemaal bij elkaar zijn, maakt hij van z’n hand een toeter en
schreeuwt tot den metselaar:

„Meheer! hier zijn ze al! De spreeuwen kenne vliege, leg ze zout op hun
staart!” [160]

En in woeste vaart rennen ze weg, bang, achtervolgd te worden door de

werklui.

„Naar ’t Hol, naar ’t Hol!” gilt Ambro.

En als ze daar, hijgend en blazend aangekomen zijn, vinden ze Paul, die

rustig zit te lezen en laconiek zegt: „Ik dacht wel dat jullie ontsnappen zouden,
Ambro was er toch bij.”

Dit volkomen vertrouwen streelde den hoofdman niet weinig.

[Inhoud]
 AMBRO EN PAUL OP REIS.

De jongens hebben Paasch-vacantie. De meeste van hen zijn uit de stad.

Ambro en Paul zijn de eenigen die in stad bleven en ze hebben zich

voorgenomen veel samen uit te gaan.

Allerlei tochten zijn al op touw gezet om toch zooveel mogelijk te kunnen

genieten van de vacantie.

Op een dezer dagen komt Paul al ’s morgens vroeg naar Ambro toe om hem
te vragen of hij lust heeft hem te vergezellen naar Delft, waar hij een pakje
moet brengen naar zijn grooten broer, die daar in garnizoen ligt.

„’t Is wèl een heel eind, heen en terug,” zegt Ambro, die niet veel voelt voor
zoo’n reuzenloop.

Maar Paul stelt hem spoedig gerust.

„Bê-je mal! niet loopen! we gaan heen en terug met het bootje.”

„’t Zal niet gaan, man, ik heb nog maar twee kwartjes en die ga ik niet in één
dag opmaken.” [161]

„Dat hoeft ook niet; moeder fuift ons. Ik vond ’t saai om alleen te gaan en
vroeg of ik jou mee mocht nemen. Dat vond moeder best en ze gaf me
reisgeld voor ons allebei.”

„Dan is ie fijn en nu ben ik je man,” zei Ambro. „Ik zal ’t even aan moeder gaan
zeggen.”

In een wip is ie terug en zegt: „Wanneer gaan we?”

„Over een uur gaat het bootje.”

„Kom mee, Paul, dan tracteer ik op appelen, dan hebben we wat te bikken
onderweg.”