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Neosporosis in Animals
Neosporosis in Animals
J. P. Dubey
Animal Parasitic Diseases Laboratory
Beltsville Agricultural Research Center
Agricultural Research Service
U.S. Department of Agriculture
Beltsville, Maryland 20705
A. Hemphill
Institute of Parasitology,
Department of Infectious Diseases and Pathobiology
Vetsuisse Faculty
University of Bern
Länggass-Strasse 122
CH-3012 Bern, Switzerland
R. Calero-Bernal
Animal Parasitic Diseases Laboratory
Beltsville Agricultural Research Center
Agricultural Research Service
U.S. Department of Agriculture
Beltsville, Maryland 20705
G. Schares
Institute of Epidemiology
Friedrich-Loeffler-Institut
Bundesforschungsinstitut für Tiergesundheit
Federal Research Institute for Animal Health
Südufer 10,
17493 Greifswald—Insel Riems, Germany
CRC Press
Taylor & Francis Group
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Boca Raton, FL 33487-2742
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Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for
identification and explanation without intent to infringe.
Names: Dubey, J. P., author. | Hemphill, A., author. | Schares, G., author. |
Calero-Bernal, R. (Rafael), 1983- author.
Title: Neosporosis in animals / J.P. Dubey, A. Hemphill, G. Schares, and R.
Calero-Bernal.
Description: Boca Raton : Taylor & Francis, 2017. | Includes bibliographical
references.
Identifiers: LCCN 2016042634| ISBN 9781498752541 (hardback : alk. paper) |
ISBN 9781498752565 (ebook)
Subjects: LCSH: Veterinary parasitology. | MESH: Parasitic Diseases, Animal
Classification: LCC SF810.A3 D79 2017 | NLM SF 810.A3 | DDC 636.089/696--dc23
LC record available at https://lccn.loc.gov/2016042634
Chapter 1
History of Neospora and Neosporosis................................................................................................ 1
1.1 History......................................................................................................................................1
1.1.1 Discovery of the Organism in Dogs...........................................................................1
1.1.2 In Vitro Cultivation of Viable N. caninum..................................................................2
1.1.3 Linking N. caninum to Abortions in Cattle................................................................2
1.1.4 Retrospective Studies.................................................................................................. 3
1.1.4.1 Dogs in USA�����������������������������������������������������������������������������������������������3
1.1.4.2 Dogs in Norway...........................................................................................3
1.1.5 Cattle and Other Hosts................................................................................................5
1.2 Landmarks in the Biology of Neospora................................................................................... 5
Bibliography....................................................................................................................................... 5
Chapter 2
General Biology.................................................................................................................................. 7
2.1 Introduction..............................................................................................................................7
2.2 Taxonomic Classification......................................................................................................... 7
2.3 Hosts.........................................................................................................................................8
2.3.1 Definitive Hosts..........................................................................................................8
2.3.2 Intermediate Hosts...................................................................................................... 8
2.4 Life Cycle Stages......................................................................................................................8
2.4.1 Tachyzoite������������������������������������������������������������������������������������������������������������������9
2.4.2 Bradyzoites and Tissue Cysts...................................................................................20
2.4.2.1 Conversion of Tachyzoites to Bradyzoites................................................ 23
2.4.3 Oocyst����������������������������������������������������������������������������������������������������������������������26
2.5 Transmission and Epidemiology............................................................................................ 30
2.6 Host–Parasite Relationship.................................................................................................... 32
2.7 Molecular and Cell Biology................................................................................................... 35
2.7.1 Genome and Transcriptome Analysis of N. caninum............................................... 35
2.7.2 Modulation of Host Gene Expression upon Infection..............................................40
2.7.3 Proteomics Approaches............................................................................................ 41
2.7.4 Genetic Manipulation of N. caninum....................................................................... 41
2.7.5 Neospora–Host Cell Interactions and Invasion........................................................ 42
2.7.5.1 Initial Host Cell Contact is Mediated by Parasite Surface
Antigens (SAGs)........................................................................................ 43
2.7.5.2 Secretory Organelle Discharge Governs Host Cell Invasion.................... 50
2.7.5.3 Microneme Proteins.................................................................................. 50
2.7.5.4 ROPs and Rhoptry Neck Proteins (RONs)................................................ 50
2.7.5.5 Dense Granule Proteins............................................................................. 51
2.7.5.6 Intracellular Host Cell Modulation and Parasite–Host Cell Crosstalk..... 52
2.7.5.7 Monoclonal Antibodies............................................................................. 53
2.8 Rodent Models of Neosporosis.............................................................................................. 53
v
vi Contents
2.8.1Mice������������������������������������������������������������������������������������������������������������������������� 53
2.8.1.1 Outbred Mice............................................................................................ 53
2.8.1.2 Inbred Mice���������������������������������������������������������������������������������������������� 57
2.8.1.3 Nude Mice and IFN-γ-KO Mice............................................................... 70
2.8.2 Gerbils���������������������������������������������������������������������������������������������������������������������� 70
2.8.2.1 Common Pet Gerbil (Meriones unguiculatus).......................................... 70
2.8.2.2 Tristram’s Jird (Meriones tristrami).......................................................... 73
2.8.2.3 Wagner Gerbil (Gerbillus dasyurus)......................................................... 73
2.8.3 Multimammate Rat (Mastomys natalensis).............................................................. 73
2.8.4 Sand Rat (Psammomys obesus)................................................................................ 73
2.8.5 Norwegian Rat (Rattus norvegicus).......................................................................... 73
2.8.6 Djungarian Hamster (Phodopus sungorus).............................................................. 74
2.9 General Diagnosis.................................................................................................................. 74
2.9.1 Cytology������������������������������������������������������������������������������������������������������������������� 74
2.9.2 Conventional Histopathology.................................................................................... 74
2.9.3 Transmission Electron Microscopy.......................................................................... 74
2.9.4 Immunohistochemical Staining (IHC)..................................................................... 75
2.9.5 Isolation of Viable N. caninum by Bioassay............................................................. 76
2.9.6 Serological Methods................................................................................................. 77
2.9.6.1 Antibody Detection................................................................................... 77
2.9.6.2 Antigen Detection.....................................................................................90
2.9.7 Detection of DNA (PCR)..........................................................................................90
2.9.7.1 General Aspects of PCR...........................................................................90
2.9.7.2 Target Genes for Diagnostic N. caninum PCRs........................................ 91
2.9.7.3 Quantitative PCR......................................................................................97
2.9.7.4 Typing Different Neospora sp. Strains...................................................... 98
2.10 Treatment............................................................................................................................. 101
2.11 Vaccines............................................................................................................................... 104
2.11.1 Vaccination Studies in Small Laboratory Animals................................................ 104
2.11.1.1 Live Vaccines.......................................................................................... 104
2.11.1.2 Subunit Vaccines..................................................................................... 111
2.12 Prevention, Prophylaxis....................................................................................................... 111
Bibliography................................................................................................................................... 111
Chapter 3
Techniques...................................................................................................................................... 115
3.1 Bioassay of Tissues for Isolation of Neospora..................................................................... 115
3.1.1 Tissues of Acutely Infected Animals...................................................................... 115
3.1.2 Tissues of Chronically Infected Animals............................................................... 115
3.2 Parasite Cultures.................................................................................................................. 116
3.2.1 Obtaining Tachyzoites............................................................................................ 116
3.2.1.1 In Vitro Cultivation................................................................................. 116
3.2.2 Obtaining Tissue Cysts........................................................................................... 119
3.2.3 Obtaining Oocysts.................................................................................................. 119
3.2.4 Cryopreservation..................................................................................................... 120
3.3 Diagnostic Procedures......................................................................................................... 120
3.3.1 Examination of Canine Feces for Oocysts............................................................. 120
3.3.2 Cytology, Histopathologic, and IHC Procedures.................................................... 121
3.3.3 Serologic Procedures.............................................................................................. 124
Contents vii
Chapter 4
Neosporosis in Cattle...................................................................................................................... 133
4.1 Natural Infections................................................................................................................ 133
4.1.1 Serologic Prevalence............................................................................................... 133
4.1.2 Clinical Infections.................................................................................................. 133
4.1.2.1 Abortion and Stillbirth............................................................................ 133
4.1.2.2 Congenitally Infected Calves.................................................................. 175
4.1.2.3 Lesions in Weaned Calves and Adult Cattle........................................... 178
4.1.3 Isolation of Viable N. caninum from Bovine Tissues............................................. 180
4.2 Experimental Infections....................................................................................................... 183
4.2.1 Calves and Adult Cattle.......................................................................................... 183
4.2.2 Cows during Pregnancy.......................................................................................... 191
4.2.2.1 Early Studies������������������������������������������������������������������������������������������ 191
4.2.2.2 Pathogenesis of Neosporosis................................................................... 191
4.3 Diagnosis.............................................................................................................................. 203
4.3.1 General Considerations........................................................................................... 203
4.3.2 Submission of Samples to a Diagnostic Laboratory...............................................204
4.3.3 Routine Histopathological Examination.................................................................204
4.3.4 Immunohistological Examinations.........................................................................205
4.3.5 Demonstration of Viable Neospora........................................................................207
4.3.6 Detection of N. caninum DNA...............................................................................207
4.3.6.1 Clinical Cases..........................................................................................208
4.3.6.2 Asymptomatic Cattle...............................................................................209
4.3.7 Detection of Antibodies..........................................................................................209
4.3.7.1 Antibody Types and Isotypes..................................................................209
4.3.7.2 Ratio of Specific IgG1 and IgG2............................................................. 210
4.3.7.3 Fluctuating Antibody Levels in Relation to Risk of
Abortion and Vertical Transmission....................................................... 210
4.3.7.4 Avidity Maturation.................................................................................. 211
4.3.7.5 Antibodies in Adult Cattle due to Vaccination....................................... 211
4.3.7.6 Antibodies in Fetuses.............................................................................. 211
4.3.7.7 Antibodies in Newborn Calves............................................................... 212
4.3.7.8 In-House and Commercial Serological Assays....................................... 213
4.3.7.9 Testing of Individual Aborting Dams or Dams at Abortion Risk........... 222
4.3.7.10 Serological Testing on a Herd Level....................................................... 223
4.3.7.11 Avidity Tests to Define the Time Point of Infection...............................224
4.3.7.12 Serological Tests to Estimate the Herd Seroprevalence..........................224
4.4 Epidemiology....................................................................................................................... 225
4.4.1 Transmission and Risk Factors in Cattle................................................................ 225
viii Contents
Chapter 5
Neosporosis in Dogs....................................................................................................................... 261
5.1 Natural Infections................................................................................................................ 261
5.1.1 Sources of Infection and Transmission of N. caninum for Dogs............................ 261
5.1.2 Serologic Prevalence............................................................................................... 261
5.1.2.1 Risk Factors for Infection in Dogs.......................................................... 262
Contents ix
Chapter 6
Neosporosis in Sheep...................................................................................................................... 317
6.1 Natural Infections................................................................................................................ 317
6.1.1 Serologic and DNA Prevalence.............................................................................. 317
6.1.2 Clinical Infections.................................................................................................. 317
6.1.2.1 Histologically Confirmed Abortion and Stillbirth.................................. 317
6.1.2.2 Detection of N. caninum DNA in Aborted Fetuses................................ 323
6.1.2.3 Clinical and Subclinical Neosporosis in Adult Sheep............................ 323
x Contents
Chapter 7
Neosporosis in Goats...................................................................................................................... 329
7.1 Natural Infections................................................................................................................ 329
7.1.1 DNA Serologic Prevalence..................................................................................... 329
7.1.2 Clinical Disease...................................................................................................... 329
7.2 Experimental Infections....................................................................................................... 333
Bibliography................................................................................................................................... 336
Chapter 8
Neosporosis in Water Buffaloes (Bubalus bubalis)........................................................................ 337
8.1 Natural Infections................................................................................................................ 337
8.1.1 Serologic Prevalence............................................................................................... 337
8.1.2 Parasitologic Prevalence......................................................................................... 337
8.1.3 Clinical Disease...................................................................................................... 338
8.2 Experimental Infections....................................................................................................... 339
8.2.1 Infection in Nonpregnant Animals......................................................................... 339
8.2.2 Infection in Pregnant Animals................................................................................ 339
Bibliography................................................................................................................................... 341
Chapter 9
Neosporosis in Pigs......................................................................................................................... 343
9.1 Natural Infections................................................................................................................ 343
9.1.1 DNA Serologic Prevalence..................................................................................... 343
9.1.2 Clinical Infections.................................................................................................. 343
9.2 Experimental Infections....................................................................................................... 343
Bibliography...................................................................................................................................346
Chapter 10
Neosporosis in Camels and South American Camelids................................................................. 347
10.1 Natural Infections................................................................................................................ 347
10.1.1 One Humped Camel (Camelus dromedarius)........................................................ 347
10.1.2 South American Camelids...................................................................................... 347
10.1.2.1 Serologic Prevalence............................................................................... 347
10.1.2.2 Clinical Infections................................................................................... 347
Bibliography................................................................................................................................... 347
Chapter 11
Neosporosis in Felids...................................................................................................................... 351
11.1 Natural Infections................................................................................................................ 351
11.1.1 Serologic Prevalence............................................................................................... 351
11.1.2 Clinical Infections.................................................................................................. 351
11.2 Experimental Infections....................................................................................................... 351
Bibliography................................................................................................................................... 351
Contents xi
Chapter 12
Neosporosis in Avian Species......................................................................................................... 357
12.1 Natural Infections................................................................................................................ 357
12.2 Experimental Infections....................................................................................................... 357
Bibliography................................................................................................................................... 357
Chapter 13
Neosporosis in Humans and Primates............................................................................................ 363
13.1 Neosporosis in Humans....................................................................................................... 363
13.2 Experimental Neosporosis in Rhesus Monkeys................................................................... 363
Bibliography................................................................................................................................... 363
Chapter 14
Neosporosis in Cervids and Other Wild Herbivores...................................................................... 365
14.1 Natural Infections in Cervids............................................................................................... 365
14.1.1 White-Tailed Deer (Odocoileus virginianus).......................................................... 365
14.1.1.1 Prevalence���������������������������������������������������������������������������������������������� 365
14.2 Other Cervids....................................................................................................................... 365
14.2.1 Serologic Prevalence............................................................................................... 365
14.2.2 Clinical Infections.................................................................................................. 365
14.3 Other Herbivores.................................................................................................................. 377
14.3.1 Serologic Prevalence............................................................................................... 377
14.3.2 Clinical Neosporosis............................................................................................... 377
Bibliography................................................................................................................................... 377
Chapter 15
Neosporosis in White Rhinoceros (Ceratotherium simum)............................................................ 379
Bibliography................................................................................................................................... 380
Chapter 16
Neosporosis in Wild Canids and Other Carnivores........................................................................ 381
16.1 Wild Canids......................................................................................................................... 381
16.1.1 Australian Dingo (Canis lupus dingo).................................................................... 381
16.1.2 Coyote (C. latrans).................................................................................................. 381
16.1.3 Red Fox (Vulpes vulpes).......................................................................................... 386
16.1.4 Blue Fox (Alopex lagopus)...................................................................................... 386
16.1.5 Gray Wolf (C. lupus)............................................................................................... 386
16.1.6 Other Wild Canids.................................................................................................. 386
16.2 Other Wild Carnivores......................................................................................................... 386
16.2.1 Raccoons (Procyon lotor)....................................................................................... 386
16.2.2 Mustelids���������������������������������������������������������������������������������������������������������������� 391
16.2.3 DNA Detection in Wild Carnivores........................................................................ 391
Bibliography................................................................................................................................... 391
Chapter 17
Neosporosis in Miscellaneous Animals......................................................................................... 393
17.1 Giant Panda (Ailuropoda melanoleuca).............................................................................. 393
xii Contents
Chapter 18
N. hughesi and Neosporosis in Horses and Other Equids.............................................................. 397
18.1 Introduction.......................................................................................................................... 397
18.2 Differences between N. hughesi and N. caninum................................................................ 397
18.2.1 Morphological......................................................................................................... 397
18.2.1.1 Tachyzoites��������������������������������������������������������������������������������������������� 397
18.2.1.2 Tissue Cysts�������������������������������������������������������������������������������������������� 397
18.2.1.3 Antigenic and Molecular Differences..................................................... 397
18.2.1.4 Biological Differences............................................................................. 398
18.3 Natural Infections in Horses................................................................................................ 398
18.3.1 Serologic Prevalence............................................................................................... 398
18.3.2 Transplacental Infection.........................................................................................405
18.3.3 Clinical Infections..................................................................................................405
18.3.3.1 Histologically Confirmed Cases..............................................................405
18.3.3.2 Antemortem Diagnosed Cases................................................................407
18.3.3.3 Abortion�������������������������������������������������������������������������������������������������409
18.3.4 Diagnosis����������������������������������������������������������������������������������������������������������������409
18.3.4.1 Serological Diagnosis..............................................................................409
18.3.4.2 Polymerase Chain Reaction.................................................................... 410
18.4 Experimental Infections....................................................................................................... 410
Bibliography................................................................................................................................... 410
References....................................................................................................................................... 411
Index............................................................................................................................................... 519
Preface
In the 1980s a neuromuscular syndrome of dogs simulating toxoplasmosis was recognized.
In 1988, a new genus, Neospora, and the type species, Neospora caninum, were named, cultivated
in vitro, and differentiated from Toxoplasma gondii. A year later, N. caninum was identified as
an etiological agent for bovine abortions. Considerable progress in understanding the biology of
neosporosis has been made in the last 30 years, resulting in more than 2000 scientific publications.
The economic importance of abortion in cattle, and the availability of knowledge, reagents, and
technology used to study toxoplasmosis have contributed to the rapid progress in understanding
the biology of neosporosis. Whole genome sequencing of N. caninum confirmed close similarities
between N. caninum and T. gondii. However, these 2 protozoans are biologically different: N. cani-
num causes a major disease in cattle, and canids are its definitive hosts, whereas toxoplasmosis is a
major public health problem and felids are its definitive host. Both parasites have a wide host range.
Here we summarize information on the biology of neosporosis, starting with Chapter 1 on the
historical background. Subsequent chapters deal with general aspects of the biology of N. caninum
(Chapter 2), techniques (Chapter 3), and the disease caused by this parasite in cattle (Chapter 4),
dogs (Chapter 5), and all other animals including primates and humans (Chapters 6 through 18).
Abortion is a worldwide problem in the livestock industry accounting for annual economic
losses of billions of dollars, and N. caninum is a major cause of this. Neosporosis causes abortion
in both dairy and beef cattle. Abortions not only occur in cattle that have been exposed recently but
also in chronically infected cattle, which poses a major challenge for vaccine development. There is
no effective vaccine or therapy to eliminate N. caninum in cattle, but progress is being made.
In this book, we provide an up-to-date account of structure, biology, clinical disease, diagnosis,
epidemiology, treatment, attempts at immunoprophylaxis, and control in all hosts. There are 175
illustrations on the life cycle, structure of parasitic stages, and of lesions. More than 2100 references
are cited.
It is hoped that this book will be useful to biologists, veterinarians, and researchers.
We would like to acknowledge those who made this book possible; we feel we cannot possibly
list all. Camila K. Cerqueira-Cézar was a big help in compiling bibliography, and coordinating
efforts among the 4 of us; we are truly grateful to her for this. Many scientists contributed illus-
trations, unpublished information, and help with this book; chief among them being S. Almería,
I. Bjerkås, J. F. Edwards, A. L. Hattel, D. S. Lindsay, M. M. McAllister, L. M. Ortega-Mora, C.
A. Speer, and W. Wouda. Many others contributed to the making of this book including D. Alves,
M. Anderson, B. C. Barr, W. Basso, C. Björkman, D. Buxton, J. L. Carpenter, O. Cabezón, P. A.
Conrad, F. J. Conraths, B. Daft, T. Dijkstra, J. T. Ellis, E. A. Innes, L. C. Gasbarre, C. Genchi, S. M.
Gennari, B. Gottstein, C. E. Green, M. E. Grigg, W. J. Hartley, D. K. Howe, M. C. Jenkins, A. Khan,
O. C. H. Kwok, M. R. Lappin, A. E. Marsh, D. P. Moore, J. A. Morales, D. O’Toole, K. Peperkamp,
M. P. Reichel, J. R. Šlapeta, C. Sreekumar, J. P. Thilsted, M. J. Topper, W. Tuo, A. J. Trees, A. Uggla,
C. Venturini, and I. Villena.
J. P. Dubey
A. Hemphill
R. Calero-Bernal
G. Schares
xiii
Authors
J. P. Dubey, MVSc, PhD, was born in India. He earned his veterinary degree in 1960, and master
in veterinary parasitology in 1963, from India. He earned his PhD in medical microbiology in 1966
from the University of Sheffield, England. Dr. Dubey received postdoctoral training from 1968 to
1973 with Dr. J. K. Frenkel, Department of Pathology and Oncology, University of Kansas Medical
Center, Kansas City. From 1973 to 1978, he was an associate professor of veterinary parasitology,
Department of Pathobiology, Ohio State University, Columbus and a professor of veterinary parasi-
tology, Department of Veterinary Science, Montana State University, Bozeman, from 1978 to 1982.
He is currently a senior scientist, Animal Parasitic Diseases Laboratory, Beltsville Agricultural
Research Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville,
Maryland.
Dr. Dubey has spent more than 50 years researching protozoa, including Toxoplasma, Neospora,
Sarcocystis, and related cyst-forming coccidian parasites of humans and animals. He has published
more than 1400 research papers in international journals, more than 250 of which are on neosporo-
sis. In 1985, he was chosen to be the first recipient of the “Distinguished Veterinary Parasitologist
Award” by the American Association of Veterinary Parasitologists. Dr. Dubey is recipient of the
1995 WAAVP Pfizer Award for outstanding contributions to research in veterinary parasitology. He
also received the 2005 Eminent Parasitologists Award by the American Society of Parasitologists.
The Thomas/Institute for Scientific Information identified him as one of the world’s most cited
authors in plant and animal sciences for the last decade. In 2003, he was selected for the newly
created Senior Science and Technology Service (SSTS), and is one of the few scientists and execu-
tives within the USDA’s Agricultural Research Service; selection for this position is by invitation
only, on approval by the Secretary of Agriculture. In 2010, Dr. Dubey was elected to the U.S.
National Academy of Sciences, Washington, DC, and inducted in the USDA-ARS Hall of Fame.
He has made seminal contributions to the biology of neosporosis, including naming of the parasite,
Neospora caninum.
Andrew Hemphill, PhD, was born in Lucerne, Switzerland. He grew up in central Switzerland,
studied microbiology with an emphasis on cell and molecular biology at the University of Bern,
and completed his PhD on cytoskeletal elements of African trypanosomes (Trypanosoma brucei) in
1991, at the University of Bern. He did postdoctoral training at the University College London and at
the London School of Hygiene and Tropical Medicine and continued research on trypanosomiasis.
In 1994, Dr. Hemphill was appointed as a researcher at the Institute of Parasitology of the University
of Bern. From 2002 to 2007, he was the president of the Swiss Society of Tropical Medicine and
Parasitology. He became an associate professor in 2008 and is teaching parasitology at the Vetsuisse
Faculty, University of Bern. Dr. Hemphill has been an external advisor for Swissmedic since 2013.
He has been acting as peer reviewer and editorial board member of several journals dealing with
infectious diseases, guest-edited several special issues, and is currently an editor of Parasitology,
an international peer-reviewed journal in the field. His main research interests relate to the develop-
ment of options for the prevention and treatment of protozoan and helminth infections, especially
Neospora caninum and Echinococcus multilocularis, the causative agent of alveolar echinococ-
cosis in humans. Echinococcosis is an important public health problem and its treatment options
are limited. He established a parasite culture and efficacy assessment system for drug screening
activities. He pioneered studies on cell biology of N. caninum, including the mechanism of para-
site interaction with host cells, identification, and characterization of parasite proteins for vaccine
development, studies on drug targets in both parasite and host cells, and immunoprophylaxis. He is
an author and coauthor of more than 200 peer-reviewed research publications, and contributed to
several reviews and book chapters.
xv
xvi Authors
Rafael Calero-Bernal, DVM, MSc, PhD, was born in Badajoz, Spain. He earned his degree in
veterinary medicine at the University of Extremadura, Spain in 2006. One year later he attended
the Official Master in Meat Science and Technology at the same institution. In 2011, Dr. Calero-
Bernal earned PhD in European framework in Veterinary Medicine. From 2008 to 2015 he was a
professor of the Animal Health Department at the University of Extremadura, developing teach-
ing periods at the Faculty of Veterinary Medicine of the University of Lisbon (Portugal). He has
been a researcher in the Spanish National Microbiology Centre and the Tropical Medicine National
Centre, both belonging to the Instituto de Salud Carlos III (Spain). Dr. Calero-Bernal has devel-
oped several research protocols at Istituto Superiore di Sanità (Italy), Instituto Nacional de Saúde
(Portugal), Fundação Oswaldo Cruz (Brazil), and Centro de Referencia para el Control de Endemias
(Equatorial Guinea). Currently he is a postdoctoral researcher in the Agricultural Research Service,
United States Department of Agriculture. He has authored more than 50 articles and 4 books related
to veterinary sciences, the latest being Sarcocystosis of Animals and Humans, CRC Press. His
research interests are wildlife parasites and zoonoses, especially meat-borne pathogens and tissue
cysts forming coccidia.
Gereon Schares, DVM, was born in Bitburg, Germany. He is a veterinary parasitologist with pri-
mary interests in the diagnosis and epidemiology of various parasitic diseases of animals, including
those with zoonotic importance such as toxoplasmosis. He earned a degree in veterinary medicine
in 1987 at Justus-Liebig-University, Giessen, Germany and a doctorate in veterinary medicine at the
Institute of Parasitology, Justus-Liebig-University in 1992. As a postdoctoral trainee at the Institute
of Parasitology and Tropical Veterinary Medicine, Free University, Berlin, Germany, Dr. Schares
worked on the diagnosis and typing of African trypanosomes. In 1995, he joined the Institute
of Epidemiological Diagnostics and the Institute of Epidemiology, Federal Research Centre for
Virus Diseases of Animals, Wusterhausen, Germany as an independent scientist. There he initi-
ated research on tissue cyst forming coccidia, including Toxoplasma, Neospora, Sarcocystis, and
Besnoitia. He is a senior researcher at the Institute of Epidemiology, Friedrich-Loeffler-Institute,
Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany, since 2013, and
the head of the National Reference Laboratories for Toxoplasmosis and Dourine in Germany. He has
authored and coauthored more than 150 peer-reviewed research publications. He has made seminal
contributions to the diagnosis of neosporosis and besnoitiosis.
Abbreviations Commonly Used
GENERAL
Amylopectin granules am
Conoid co
Golgi body go
Ground substance layer gs
Inner membrane im
xvii
xviii Abbreviations Commonly Used
Micronemes mn
Micropore mp
Microtubules mt
Mitochondrion mc
Nucleus nu
Outer membrane om
Plasmalemma membrane pm
Rhoptries rh
Subpellicular microtubules st
Chapter 1
1.1 HISTORY
Three Norwegian veterinarians, Inge Bjerkås (an anatomic pathologist), Svein Fredrik Mohn
(serologist and diagnostician), and John Presthus (a neurologist, now deceased) reported in a
short communication the finding of an unidentified protozoan in a litter of 6 congenitally infected
pups born to a Boxer dog in Norway. 205 The pups appeared to be healthy until 2 months old.
Five of these pups had neurological signs for several months. All 6 pups were examined at nec-
ropsy and were diagnosed with encephalitis and myositis with protozoa in lesions. There were
numerous tachyzoites and a few tissue cysts in the brain. Ultrastructurally, tachyzoites were like
Toxoplasma gondii but with more rhoptries (up to 11) than seen in T. gondii. Tissue cysts were
not examined ultrastructurally. Antibodies to T. gondii were not found in the sera of 5 dogs by
the dye test but the dilution of the serum tested was not stated. Several attempts to culture the
parasite failed.
The report of Bjerkås et al.205 remained vague until one of us, JPD, requested a tissue slide of
the brain of the affected dog for inclusion in the book on Toxoplasmosis of Animals and Man that
was being prepared. 527,563 Because of no reply from Dr. Bjerkås, JPD examined tissue sections
and case histories from all dogs and cats that had died of toxoplasmosis-like illness from 1952 to
1987 and were archived at the Angell Memorial Animal Hospital (AMAH), Boston, Massachusetts,
USA. The AMAH is the largest hospital for dogs and cats in USA and meticulously keeps records
of pathology cases. Dr. James Carpenter, one of the pathologists at AMAH, and JPD examined
thousands of slides from dogs, and also cats, and reached a conclusion that the syndrome recog-
nized by Bjerkås et al. was not toxoplasmosis. In addition to neuromuscular clinical signs, the dogs
suffered severe disease involving the heart, lungs, liver, and the skin. The parasite in dogs did not
react in immunohistochemical tests to T. gondii antibodies; sera were not available for antibody
determination. The presence of thick walled (up to 4 μm thick) tissue cysts was considered an iden-
tifying feature and inspired naming the organism as a new genus and species Neospora caninum
in collaboration with Drs. Carpenter, Speer, Uggla, and Topper. 529 The focus of this paper “Newly
recognized fatal protozoan disease of dogs” was to draw attention to the new clinical syndrome529;
and the name N. caninum helped to achieve that. Thanks are due to Dr. A. J. Koltveit, then the
editor of the Journal of the American Veterinary Medical Association, for making an exception to
publish our article with new taxa and numerous histopathology illustrations in a clinical journal.
1
2 Neosporosis in Animals
He also advised JPD to separately publish findings of neosporosis and toxoplasmosis in dogs and
cats. Clinically, neosporosis was found to be a primary disease of dogs versus canine toxoplasmosis
often associated with tumors or canine distemper virus infection. 536 Additionally, neosporosis was
not found to be a disease of cats. 553
The name N. caninum aroused considerable scientific controversy because the description was
based on parasites in tissue sections, and many scientists felt that it should have been a species of
Toxoplasma. In collaboration with many scientists around the world the parasite was redescribed
and specimens were deposited in museums. 589
Luck, opportunity, perseverance, and confidence in one’s own findings are an integral part of
discovery; all of these were a factor in the discovery of N. caninum. JPD contacted practicing
veterinarians and pathologists in USA for help to send tissues from paralyzed dogs for isolation
of N. caninum. Such an opportunity arose when Dr. E. J. Stanley, a veterinarian in Pennsylvania,
telephoned JPD that one of his clients had a litter of dogs with hind limb paralysis. He also indicated
that the previous litter from the same bitch had died of toxoplasmosis-like illness.404 JPD became
very interested in this case because there were no previous confirmed cases of congenital toxoplas-
mosis in dogs in sequential pregnancies. JPD contacted Dr. Arthur Hattel, a veterinary pathologist
at the Animal Diagnostic Laboratory, University Park, Pennsylvania who made arrangements for
the donation of the affected litter, necropsy, and sending of fresh, unfixed tissues to JPD. By luck,
Dr. David Lindsay (DL) who was a postdoctoral scientist in JPD’s laboratory had expertise in cell
culture. DL succeeded in growing the parasite on first attempt. For several months DL and JPD
could not decide if the parasite was N. caninum or T. gondii, until bioassay results in mice became
available. Twenty-five outbred Swiss Webster mice had been inoculated with homogenized tissues
of the 4 affected dogs. The mice remained seronegative for T. gondii. Three thick-walled tissue
cysts528 were found in unstained brain smears of the brains of 25 mice; entire brains of all 25 mice
had been examined microscopically. This is an example of perseverance; it was subsequently dis-
covered that outbred mice are not susceptible to N. caninum unless immunosuppressed. In vitro cul-
tivation of N. caninum made it possible to develop diagnostic tests to fulfill Koch’s postulates, and
induce clinical disease in several hosts within 3 years of the discovery of the parasite (Table 1.1).
None of this would have been possible without the flexibility in the Agricultural Research Service,
U.S. Department of Agriculture system to pursue new areas of research and the support of JPD’s
supervisors who allowed him to perform this boot-legged research while performing his assigned
research on toxoplasmosis.
Although there were isolated reports of protozoa-associated encephalitis in calves (Table 1.1),
protozoa were not known to be a major bovine abortifacient. In 1987, Dr. John Thilsted, a veterinar-
ian pathologist from New Mexico, USA contacted JPD concerning abortions in a 240 dairy cow herd
where 29 (12%) cows had aborted; and 4 to 8 cows aborted in mid gestation during 5 months. Tests
for bacterial, fungal, and viral causes were negative. Histologically, focal necrotic encephalitis and
nonsuppurative myocarditis were the main lesions in 7 of 9 fetuses examined histologically. When
an immunohistochemical test became available1157 the slides were stained with N. caninum antibod-
ies. Neospora parasites were found in the brain of 2 fetuses and in the kidney of 1 fetus.1954 This
was the first report of an epidemic type of abortion in cattle associated with protozoa. Subsequently,
Barr et al.,130,131 and Anderson et al.61,62 documented that N. caninum is a major cause of abortion in
cattle, accounting for 18% of all bovine abortions in California, so much so that some dairies went
out of business because of these abortions (Table 1.2).
HISTORY OF NEOSPORA AND NEOSPOROSIS 3
After the discovery of N. caninum in 1988, one of the questions asked was whether neosporosis
is a new disease. In late 1970s while a faculty member at the Ohio State University, JPD recalled
reading Richard Piper’s PhD thesis (1960, The Ohio State University, http://library.ohio-state.edu/
record=b2781251∼S7) where Piper mentioned finding small thick-walled tissue cysts in the retina of
dogs. In an outbreak starting in 1957, 4 litters of German Shorthaired Pointers from 1 owner devel-
oped clinical illness, initially diagnosed as toxoplasmosis; 29 of the 39 dogs had pelvic limb paraly-
sis. Six pups from 2 litters were necropsied and their tissues had been studied by several pathologists
at the Ohio State University, including Koestner and Cole1084 and Piper et al.1620 However, Piper
did not mention these thick-walled tissue cysts in a formal publication of his findings in a refer-
eed journal.1620 In collaboration with Drs. Piper and Koestner, JPD reevaluated the case histories
and original histological sections of these 6 dogs. Paraffin sections were stained with N. caninum
antibodies. The presence of N. caninum, and not T. gondii, was confirmed in all 6 dogs, including
thick-walled tissue cysts in the retina. 541 This finding in 1957 is the earliest record of neosporosis in
any host, worldwide.
Bjerkås published details of the cases he reported initially in 1984207–209 allowing the follow-
ing conclusions to be drawn. (i) The 6 dogs were born to a single Boxer bitch from 3 successive
4 Neosporosis in Animals
litters, starting in 1982. (ii) The dogs developed ataxia starting at 2–5.5 months of age. (iii) Lesions
were confined to the central nervous system (CNS) and muscles, and protozoa were found in the
histological sections of tissues of all 6 dogs, including the thick-walled tissue cysts demonstrated
by electron microscopy. (iv) There was another Saluki dog in Norway with similar disease that had
died in 1967. (v) Comparison of the parasite in dogs in Norway and USA revealed that both organ-
isms were identical. JPD invited Dr. Bjerkås to collaborate with scientists at USDA; his studies in
the laboratory of Dr. Mark Jenkins resulted in the first characterization of antigens of N. caninum
using the culture derived tachyzoites.211
If this case205 had not been reported it is likely that the recognition of neosporosis and N. cani-
num would have been delayed several years.205
Encephalitis in sheep and cattle associated with undiagnosed protozoa had been recorded as
early as 1974 by Hartley and Bridge854 (Table 1.1). In 1976, Bill Hartley (now deceased) from New
Zealand sent paraffin blocks of his sporozoan-associated encephalitis cases in sheep and cattle
for further diagnosis to JPD. Some of these turned out to be Sarcocystis.622 One case in sheep and
one in cattle were confirmed neosporosis (Table 1.1). After the discovery of neosporosis in cattle
in 1989, JPD contacted veterinary pathology laboratories in several countries seeking tissues from
aborted bovine fetuses for retrospective studies; none was available earlier than 1980. The reason
given was that brains of aborted fetuses were not examined routinely. Thus, 1974 remains as the
earliest case reported.
BIBLIOGRAPHY
61, 62, 130, 131, 136, 164, 205, 207, 208, 209, 211, 213, 373, 404, 445, 527, 528, 529, 530, 532, 533, 535,
538, 541, 542, 545, 552, 553, 563, 564, 568, 589, 615, 622, 781, 854, 1067, 1084, 1113, 1154, 1157,
1158, 1159, 1160, 1161, 1162, 1180, 1284, 1309, 1495, 1553, 1555, 1560, 1620, 1673, 1698, 1849, 1954.
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