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LIBRARY
Michigan State This is to certify that the
University dissertation entitled

 

CONTROLLED INTERVENTIONS USING PYRANTEL TARTRATE
TO PREVENT SARCOCYSTIS NEURONA INFECTION IN
HORSES

presented by

Mary Gordon Rossano

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Large Animal Clinical Sciences

 

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I 0 -' Q - 2003

Date

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CONTROLLED INTERVENTIONS USING PYRANTEL TARTRATE TO
PREVENT SARCOCYSTIS NEURONA INFECTION IN HORSES

By

Mary Gordon Rossano

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Large Animal Clinical Sciences

2003

ABSTRACT

CONTROLLED INTERVENTIONS USING PYRANTEL TARTRATE TO

PREVENT SARCOCYSTIS NEURONA INFECTION IN HORSES
By

Mary Gordon Rossano

This dissertation describes two studies investigating the efficacy of pyrantel tartrate in
preventing infection with the protozoan parasite Sarcocystis neurona in horses.
Sarcocystis neurona is the primary etiologic agent of equine protozoa] myeloencephalitis
(EPM), a neurological disease of horses and ponies of the Americas. A third study
described here details the culture of S. neurona from the blood of an immunocompetent

horse.

In the first study, 24 horses were challenged with sporocysts of S. neurona. Twelve of
the horses received daily doses of pyrantel tartrate, 12 received a look-alike placebo
pellet. Production of serum antibodies against S. neurona was the main outcome of
interest, as this is evidence the parasite was not killed in the gastrointestinal tract by the

drug and that the parasite had penetrated host defenses. Production of antibodies in

cerebrospinal fluid (CSF) was a second outcome of interest. At the end of the study
there was no significant difference between treatment groups with regard to the

proportion of horses testing positive or the days to seroconversion.

In the second study, a field intervention trial was conducted on three Michigan horse
farms to determine whether daily administration of pyrantel tartrate could prevent
infection with S. neurona in horses when the drug was used in an on-farm setting,
according to label instructions for Strongid C® 2x. The outcome of interest was the
production of serum antibodies to S. neurona. Horses were screened negative by
immunoblot for serum antibodies to S. neurona and allocated into two treatment groups:
pyrantel tartrate and placebo. Ten horses were monitored for seroconversion for 6
months. The results of the study were inconclusive, due to the small sample size, and no

treatment effect was detected.

In the third study, six yearling colts given daily doses of S. neurona sporocysts, were
selected for attempted culture of S. neurona from whole blood. Two 10 ml tubes of
blood in EDTA were collected from each horse. Plasma was removed from the tubes and
processed to remove fibrinogen and cultured on equine dermal cells. Thirty-eight days
later the culture from one horse was positive. To our knowledge, this is the first report of

parasitemia in an immunocompetent infected with S. neurona sporocysts.

This dissertation is dedicated to my husband, Greg Wood, for his encouragement and
patience over the 3 ‘/2 years that have passed since I began this degree, especially during
the 6 months we spent in Washington. It is also dedicated to our parents, Helen H.
Rossano and John and Ruth Wood, for their support and understanding. Finally, I
dedicate this dissertation to the future of our son or daughter, not yet born, and all the

promise that future holds.

ACKNOWLEDGMENTS

This project was made possible by the help, support and encouragement of many
people. I am especially grateful to all of those who have provided me with education,
training and guidance.

In particular, I thank my advisor Dr. John B. Kaneene for his optimism, wisdom
and financial support throughout out my graduate education. Through him, I have had
countless opportunities to learn by doing, and I feel the training I have received from him
will serve me well in future endeavors. I especially thank him for helping make it
possible for me to finish my degree in a timely manner, and for his generosity with his
time when I was finishing this dissertation and preparing for my first academic job
interview.

I also express my deepest gratitude to Dr. Linda S. Mansfield for the
extraordinary effort and kindness she has extended while teaching and advising me in
matters pertaining to research and starting a career in academia.

My great appreciation is extended to Dr. Hal Schott, for all the time and effort he
put forth in obtaining the funding for our research, in overseeing the clinical portion of
our research, and for teaching me about equine veterinary procedures. I especially thank
him for is help in getting adjusted to working at Washington State University, and
making important introductions to people there.

I would also like to thank the rest of my guidance committee: Dr. Mat Reeves,
and my external examiner, Dr. Ron Smith for taking the time to participate in my

education and training.

I thank Alice Murphy for her friendship and encouragement, and for contributing
her considerable expertise to all of the studies described here. She has been a great friend
and source of encouragement when the research wasn’t going well, and a wonderful co-
celebrator when it was.

I also that Liz Kruttlin for her assistance with the research, her companionship,
and for her sense of fun.

I thank Dr. Melissa Hines of Washington State University for all her assistance
with the experimental challenge study, and Dr. Deb Sellon for her expertise with the
horse culture experiment. I thank them both for braving snow, rain and whatever else the
weather in Pullman had to offer, to perform neurological examinations on schedule.

F Or the financial support of these studies, I thank and acknowledge Pfizer,
Incorporated, the Population Medicine Center, the MSU College of Veterinary Medicine
and the MSU Graduate School.

I also extend my thanks to the veterinary student who assisted me at Washington
State University, in particular Kurt Johnson, Sonni Trautrnan and Brett Remund. I thank
Jessica Rue for her tireless efforts with the horse adoptions at the end of the study there,
and I thank all the adopters who gave our research horses good homes.

Finally, I thank Ruth Vrable, Nicole Grosjean, Dana Neelis (MSU Diagnostic
Center for Population and Animal Health), RoseAnn Miller (MSU Population Medicine
Center), Fred and Molly Loaiza, Tressa Hochstatter (WSU), John Lagerquist, Bill Foreyt
(WSU Parasitology Laboratory) and Hany Elsheikha (MSU) for the expert technical

assistance they provided.

vi

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. xi
LIST OF FIGURES .......................................................................................................... xii
LIST OF ABBREVIATIONS .......................................................................................... xiv
OVERVIEW ........................................................................................................................ 1
Chapter 1

LITERATURE REVIEW AND STUDY OBJECTIVES

AND HYPOTHESES ......................................................................................................... 2
RATIONALE AND JUSTIFICATION FOR THE STUDIES ............................................ 2
STUDY OBJECTIVES AND HYPOTHESES ................................................................... 4
EQUINE PROTOZOAL MYELOENCEPHALITIS .............................................. 5
CLINICAL SIGNS .................................................................................................. 5
CAUSATIVE AGENT ............................................................................................ 7
EPIDEMIOLOGY ................................................................................................. 1 7
GROSS PATHOLOGY ......................................................................................... 24
HISTOPATHOLOGY ........................................................................................... 24
DIAGNOSIS .......................................................................................................... 25
TREATMENT ....................................................................................................... 3O
PROGNOSIS ......................................................................................................... 32
REFERENCES .................................................................................................................. 33

vii

Chapter 2
DOES DAILY ADMINISTRATION OF PYRANTEL TARTRATE PREVENT

INFECTION WITH S. NE URONA IN HORSES? AN EXPERIMENTAL INFECTION

STUDY. ............................................................................................................................. 40
ABSTRACT ........................................................................................................... 40
INTRODUCTION ................................................................................................. 42
MATERIALS AND METHODS ........................................................................... 44

THE PARASITE ........................................................................................ 44
VERIFICATION OF SPOROCYST VIABILITY .................................... 45
THE HORSES ........................................................................................... 46
SPOROCYST INNOCULATION ............................................................. 47
BIOSECURITY ......................................................................................... 48
PYRANTEL TARTATE AND PLACEBO ............................................... 48
PHYSICAL ASSESSMENT ..................................................................... 49
DIAGNOSTIC TESTS .............................................................................. 49
SPECIMEN COLLECTION ...................................................................... 50
STATISTICAL ANALYSIS OF RESULTS ............................................. 51
RESULTS .............................................................................................................. 52
DISCUSSION ........................................................................................................ 54
CONCLUSIONS .................................................................................................... 58
REFERENCES ...................................................................................................... 63

viii

Chapter 3
DOES DAILY ADMIN STRATION OF PYRANTEL TARTRATE PREVENT S.

NE URONA INFECTION IN HORSES? A CONTROLLED FIELD INTERVENTION

STUDY. ............................................................................................................................. 66
ABSTRACT ........................................................................................................... 66
INTRODUCTION ................................................................................................. 68
MATERIALS AND METHODS ........................................................................... 7O

STUDY DESIGN ....................................................................................... 7O
STUDY POPULATION ............................................................................ 7O
SAMPLE SIZE CALCULATIONS ........................................................... 71
ENROLLMENT OF HORSES ............................................................... 71
ALLOCATION INTO TREATMENT GROUPS ..................................... 72
ANALYSIS OF DATA .............................................................................. 73
STUDY TIMELINE .................................................................................. 73
RESULTS .............................................................................................................. 74
ENROLLMENT OF HORSES .................................................................. 74
STATISTICAL ANALYSIS ..................................................................... 75
DISCUSSION..... ................................................................................................... 75
REFERENCES ...................................................................................................... 78
Chapter 4

PARASITEMIA IN AN IMMUNOCOMPETENT HORSE EXPERIMENTALLY

CHALLENGED WITH SARCOCYSTIS NE URONA SPOROCYSTS .............................. 8O

ABSTRACT ........................................................................................................... 80

INTRODUCTION ................................................................................................. 82
MATERIALS AND METHODS ........................................................................... 83
RESULTS .............................................................................................................. 86
DISCUSSION ........................................................................................................ 86
REFERENCES ...................................................................................................... 91

Chapter 5

SUMMARY AND CONCLUSIONS ................................................................................ 93
SUMMARY ........................................................................................................... 93
CONCLUSIONS .................................................................................................... 93

Appendices ......................................................................................................................... 96
APPENDIX I - PADDOCK LAYOUT ................................................................. 97
APPENDIX 11- PEN CLEANING PROTOCOL ................................................... 99
APPENDIX [11- HORSE SPOROCYST DOSING PROTOCOL ....................... 101
APPENDIX IV - NEUROLOGICAL EVALUATION FORM ........................... 103
APPENDIX V - PROTOCOL FOR CSF COLLECTION .................................. 106
APPENDD( VI - PROTOCOL FOR NECROPSIES .......................................... 107
APPENDD( VII - PRESS RELEASE FOR STUDY .......................................... 112
APPENDIX VIH - INITIAL QUESTIONNAIRES ............................................ 114
APPENDIX IX - FOLLOW-UP QUESTIONNAIRES ....................................... 116

LIST OF TABLES

Table 2.1. Summary of seroconversion for experimental and control horses. NC = no

seroconversion. .......................................................................................... 59

Table 2.1. Summary of red and white blood cell counts from all CSF collections. ...60

Table 2.2. Postmortem examination results for the four horses euthanized at the end

of the study. Only horse #27 exhibited neurological signs. ...................... 61

Table 3.1. Summary of results of antibody tests for horses by the end of the study.
Totals for positive horses are for blood samples drawn on the day they
were dropped from the study, totals for negative horses are for the last

samples drawn. ........................................................................................... 77

Table 4.1. Summary of test results for horses in the study. Treatment groups are (PT)

= pyrantel tartrate and (P) = placebo. ........................................................ 88

xi

Figure 2.1.

Figure 4.1.

Figure 4.2.

LIST OF FIGURES

A map published by the University of Washington depicting the opossum
distribution in the state. Opossums were not reported to be in the

southeast portion of the state, where the study was conducted .................. 62

Picture of ethidium bromide-stained 1.8% agarose gel showing
Sarcocystis neurona-specific PCR products and RFLP analysis of MIHI
and #64. Lane lcontains a 100 bp DNA ladder, lane 2 contains MII-Il,
lane 3 contains MIHl digested with Hinf 1, lane 4 contains MII-Il digested
with Hind HI, lane 5 contains #64, lane 6 contains #64 digested with Hinf
1, lane 7 contains #64 digested with Hind IH, and lane 8 contains the

negative control .................................................................... 89

Sequence alignments for data spanning from 201 bp to 450 bp of the PCR
target from JNB25/JD396. Row 1 contains the sequence for UCDl
(Genbank # AF 093158), row 2 contains the sequence for SN37-R (the
isolate administered to experimentally infected horses), row 3 contains the
sequence from S. neurona cultured from horse #64 and row 4 contains the
sequence of MIHI. The sequences for SN37-R and #64 were identical,

but differed from UCDl and MIHI at the 249 bp position. The rest of the

xii

sequences for SN37-R, #64 and UCDl were identical, and differed from

MIHl at the 267-268 positions and the 419 position ................................. 9O

xiii

OVERVIEW

Chapters 2, 3 and 4 in this thesis are written in a format suitable for publication
independently. Thus, those chapters each have an abstract, introduction, methods and
discussion. Chapter 1, which is a literature review, does not follow this format. A list of
literature cited concludes each chapter.

Chapter 1 is a literature review of equine protozoa] myeloencephalitis (EPM). It
includes the epidemiology, clinical signs, diagnosis and treatment of the disease, as well
as a description of the causative organism, Sarcocystis neurona. Chapter 1 also contains
the rationale, objectives and hypotheses for the studies described here. Chapter 2
describes an experimental challenge study in which horses were dosed with S. neurona in
order to test the efficacy of pyrantel tartrate in preventing infection with S. neurona.
Chapter 3 is a field intervention trial in which pyrantel tartrate was tested to determine
whether it can prevent natural infection with S. neurona in horses in an on-farm setting.
Chapter 4 describes the culture of S. neurona from the blood of an immunocompetent
horse. The dissertation concludes with Chapter 5, which is an overall summary and

synthesis of the findings of this research. Appendices follow Chapter 5.

CHAPTER 1

INTRODUCTION

RATIONALE AND JUSTIFICATION FOR THE STUDY:

Equine protozoa] myeloencephalitis (EPM), a multi-focal neurological disease caused
by the protozoan parasite Sarcocystis neurona, has imposed a significant health threat to
horses, and economic and emotional costs and their owners (MacKay, 1997). The
economic impact of EPM on the horse industry has been estimated between $50 and $100
million annually in the United States (Dubey et al., 2001). The majority of this cost has
been incurred by horse owners pursuing diagnosis and treatment of EPM. Unfortunately,
even when horses respond favorably to treatment, the chance for return to their prior level
of performance appears to be, at best, 50% (MacKay 1997; Dubey 2001). When faced
with a disease with such an uncertain outcome, preventive measures are of great interest
and have a greater potential economic impact, than improved treatments that are
administered once clinical signs have developed.

In areas of the western hemisphere where the definitive host (the Virginia opossum,
Didelphis virginianus) harboring S. neurona is found, detection of serum antibodies to S.
neurona indicates that 30-60% of horses are, or have been, infected with the parasite
(Bentz et a1 1997; Blythe et a] 1997; Saville et a] 1997; Tillotson et a1 1999; Rossano et a]
2001). Consequently, horse owners are interested in any preventive measures that could

possibly decrease the risk of their horses developing EPM.

Despite a concerted research effort at several universities leading to improved
diagnostic tests and treatments for EPM, there has been little progress in the
understanding of its prevention (Dubey 2001; Rossano et a] 2001). In fact, equine
veterinarians have little advice to offer concerned clients besides restricting wildlife
access to feeds and trapping and removing opossums from their farms. Thus, it is not
surprising that some concerned horse owners are using the recently released S. neurona
vaccine (Fort Dodge Animal Health, conditional licensure awarded December, 2000),
despite the fact that the efficacy of the vaccine has not been demonstrated. While the
vaccine offers some hope in the prevention of EPM, identification of other effective
preventive measures against S. neurona infection would be of great interest and benefit to
the equine industry.

In 1999, our research group was asked to submit a research proposal to Pfizer, Inc. to
investigate the validity of anecdotal reports they had received from equine practitioners
that EPM was seen less often in horses that were maintained on their daily deworming
product, Strongid C®. The active ingredient in Strongid C® is pyrantel tartrate. If
pyrantel tartrate proved useful in preventing healthy horses from becoming infected with S.
neurona it would be a great benefit to horses and their owners in the western hemisphere.
Thus, we undertook several studies to determine the efficacy of daily administration of

pyrantel tartrate in preventing infection of horses by S. neurona.

PROBLEM STATEMENT:
Sarcocystis neurona is a ubiquitous parasite in areas of the western hemisphere

where opossums reside, and many horses in those regions are infected with the parasite, a

few of which go on to develop EPM (Dubey et al., 2001a). Preventive options are
limited in the face of high exposure to the parasite, and there is great interest among
horse owners for EPM prevention. Anecdotal reports from field veterinarians suggest
that daily administration of pyrantel tartrate may prevent infection of horses with S.
neurona. Therefore, research to investigate the purported ability of daily pyrantel tartrate

to prevent EPM could address an important concern of the horse industry.

STUDY HYPOTHESIS AND OBJECTIVES:
The hypotheses tested were:

H1: Daily pyrantel tartrate administration prevents infection with S. neurona in
horses.

H2: Daily pyrantel tartrate administration delays infection with S. neurona in
horses.

H3: Daily pyrantel tartrate administration prevents infection with S. neurona in
horses under field conditions.

And, as an additional investigation of how S. neurona infects horses,
H4: S. neurona can be detected in the blood of immunocompetent horses.
Three objectives were conducted to test the stated hypotheses.

1. To determine whether daily administration of pyrantel tartrate decreases the
risk of seroconversion during a period of repeated challenge of seronegative
horses with S. neurona sporocysts.

2. To conduct a controlled field intervention study on equine breeding farms to
determine whether daily administration of pyrantel tartrate decreases the risk
of seroconversion in young horses during the first year of life.

3. To culture live S. neurona from the blood of an immunocompetent horse.

LITERATURE REVIEW

1. EQUINE PROTOZOAL MYELOENCEPHALITIS:

Equine protozoa] myeloencephalitis (EPM) is a progressive, multi-foca]
neurological disease of the horse. It was originally described in the 1960's as focal
myelitis-encephalitis because affected areas of the central nervous system included both
the spinal cord and the brain (Rooney et al., 1969). The disease was renamed in the
1970’s when protozoan parasites were detected in spinal cord lesions of affected horses

(Beech and Dodd, 1974; Cusick et al., 1974; Dubey et al., 1974).

2. CLINICAL SIGNS:

The clinical signs of EPM are highly variable; it can resemble other neurological
diseases, due to the multi-foca] locations of the lesions (MacKay 1997; Dubey et al.,
2001; Furr et al., 2002). Signs that may be apparent include: mild lameness, gait deficits,
head tilt, facial paralysis, limb ataxia, spasticity, weakness, stumbling or falling,
recumbency, atrophy of focal muscle groups (muscles that are enervated by damaged
nerves), abnormal sweating, urinary incontinence and constipation or colic (MacKay
1997; Dubey et al., 2001; Furr et al., 2002). Usually the lameness or paralysis is
asymmetrical; asymmetry of clinical signs is helpful in differentiating EPM from other
neurological diseases (MacKay 1997; Dubey et al., 200]; Furr et al., 2002).

The rate at which EPM progresses differs by individual case. It can appear as a
subtle lameness or gait abnormality in its early stages (Mayhew and Greiner, 1986;

MacKay et al., 1992). Later, the horse may become ataxic and weak (Rooney et al.,

1969; Mayhew et al., 1978; Mayhew and Greiner, 1986; Madigan and Higgins, 1987;
MacKay et al., 1992). Ultimately, the horse can become recumbent or be found sitting in
a dog-like position (Rooney et al., 1969; Mayhew et al., 197 8; Mayhew and Greiner,
1986; Madigan and Higgins, 1987; MacKay et al., 1992). In some cases the disease is
slow and chronic, and the lesions may remain quiescent for months or years without
treatment (Mayhew et al., 1978; Mayhew and Greiner, 1986; Madigan and Higgins,
1987; MacKay et al., 1992). In other instances the rate of progression is very rapid, with
the lesions fuhninating in hours or days (Mayhew et al., 1978; Mayhew and Greiner,
1986; Madigan and Higgins, 1987).

Other diseases that cause peripheral or central nerve damage can present signs
that are similar to those for EPM. In making a diagnosis of EPM, a clinician must be
differentiate EPM from: West Nile virus encephalitis, compressive myelopathy
(wobblers), equine herpes myelitis, multi-focal bacterial infections, rabies, tetanus, poly
neuritis equi, neoplasia, eastern and western equine encephalitis, equine degenerative
myeloencephalopathy, leukoencephalomalacia (moldy corn poisoning), helrninth parasite
migration and head or spinal trauma (Rooney et al., 1969; Mayhew et al., 1978; Mayhew
and Greiner, 1986; Madigan and Higgins, 1987; MacKay et al., 1992; Furr et al., 2002.).
Of course, it is possible that a horse may have more than one neurological disease at once
(e. g. EPM and wobblers); thus, identification of one problem may not necessarily rule out

all others.

3. CAUSATIVE AGENT:

In early investigations, after protozoa were identified in neural tissue of affected
horses, Toxoplasma gondii was considered to be a possible causative organism of the
disease (Beech and Dodd, 1974; Cusick et al., 1974; Dubey et al., 1974). In Brazil, T.
gondii infection has been associated with equine neurological disease, but it was likely
that it was mistakenly associated with cases of EPM (Mayhew et al., 1978; Mayhew and
Greiner, 1986; Simpson and Mayhew, 1980; Masri et a1, 1992). By 1980, there was
strong evidence that the causative agent of EPM was a Sarcocystis spp, not Toxoplasma
gondii (Simpson and Mayhew, 1980). In 1991, the parasite was cultured from central
nervous system tissue from a naturally infected horse, described and named Sarcocystis
neurona (Dubey et al., 1991). Sarcocystis neurona was later confirmed as the primary
etiologic agent of EPM (Bowman et al., 1992; Fenger et al., 1994; Dubey and Lindsay
1998; Dubey et al., 1998). Despite the predominance of S. neurona-related EPM cases,
four cases of EPM have been attributed to Neospora spp. infection (Marsh et al., 1996;
Daft et al., 1997; Harnir et al., 1998; Cheadle et al., 1999). In one of the cases, the
parasite was cultured from neural tissue and was determined to be distinct from Neospora
caninum (Marsh et al., 1998b). This new species of Neospora was described,
characterized, and named N. hughesi (Marsh et al., 1998). Although this was a
noteworthy discovery, additional cases of Neospora-induced myeloencephalitis must be
reported before Neospora spp. can be considered a significant cause of EPM.

Sarcocystis species have an obligatory, heteroxenous life cycle (Levine 1985;
Urquhart et al., 1996). That is, in order to reproduce, the parasite must go through

replication and development within two different hosts: an intermediate host and a

definitive host. In the intermediate host, the terminal asexual stage is formed, and in the
definitive host, sexual reproduction occurs (Levine 1985; Urquhart et al., 1996). The life
cycle of Sarcocystis species is circular, and this description will start with the definitive
host.

Cysts (sarcocysts) in muscle tissue of the intermediate host are consumed by the
definitive host, usually a carnivore (Levine 1985). The cyst wall is digested, releasing
bradyzoites that actively penetrate the host’s intestinal epithelium, where they undergo
gametogeny and develop into the sexual stages, microgarnetes and macro gametes (Levine
1985). The macrogarnete is fertilized by the microgarnete, and the resulting zygote
matures to become an oocyst that contains two sporocysts (Levine 1985). The oocyst is
released from its host epithelial cell, in most cases the oocyst wall is ruptured as it travels
down the intestine, freeing the sporocysts which are then excreted in the feces (Urquhart
et al., 1996). At this stage, each sporocyst contains four sporulated sporozoites capable
of infecting an appropriate intermediate host or an aberrant host (Levine 1985).

The intermediate host, usually a prey species, becomes infected when it ingests fecal
material, or food or water contaminated with feces, which contain sporocysts from the
definitive host (Levine 1985). Upon ingestion, the sporocysts release their sporozoites in
the intermediate host’s gastrointestinal tract, which then migrate to endothelial cells of
the host’s internal organs (Urquhart et al., 1996). The tissues affected depend on the
particular Sarcocystis species and intermediate host infected (Levine 1985; Urquhart et
al., 1996). In most cases, the parasite undergoes two rounds of asexual schizogony and
forms tachyzoites, and eventually, motile merozoites (Urquhart et al., 1996). Merozoites

are subsequently released from schizonts, then travel in circulating lymphocytes to

muscle tissue (Urquhart et al., 1996). There they undergo fixed rounds of merogony
(asexual reproduction) and develop into the terminal asexual stage, bradyzoites, in
intramuscular cysts (Urquhart et al., 1996). The sarcocysts are tightly packed with
bradyzoites, which are infective to the appropriate definitive host (Levine 1985). The
cycle begins anew when the intermediate host is eaten as prey or carrion, and the
bradyzoites emerge in the carnivorous host’s intestine. (Levine 1985; Urquhart et al.,
1996)

Sarcocystr's species are usually very specific to their hosts (Levine 1985; Urquhart et
al., 1996). Although aberrant infections are possible, usually Sarcocystis spp. cannot
form terminal sexual or asexual stages in an aberrant host (Levine 1985). One notable
exception to this tendency is Sarcocystis neurona, which is thus far found to be specific
to its definitive host, the opossum (Didelphis virginiana)(Dubey, et al., 2001a), but has
been shown to utilize a variety of unrelated species (the nine-banded armadillo (Dasypus
novemcinctus) (Cheadle et al., 2001a), the domestic cat (F elis domesticus) (Dubey et
al.,2000), the striped skunk (Mephitis mephitis) (Cheadle et al., 2001b), the raccoon
(Procyon lotor) (Dubey et al., 20010) and the sea otter (Enhydra lutris) (Dubey et al.,
2001b) as its intermediate hosts. Another exception to the tendency of Sarcocystis spp.
specificity to their intermediate host is Sarcocystisfalcatula, which is specific to its
definitive host, also the opossum (Didelphis virginiana), but has been shown to utilize a

variety of bird species as its intermediate host (Box and Smith, 1982).

Control of Sarcocystis spp. can be problematic. Tissue cysts are enclosed in a
thick wall comprised of 2 membranes (Levine, 198 5). Bradyzoites within the cysts can

survive incomplete cooking, but are killed by freezing (Levine, 1985). Sarcocystis spp.

sporocysts are incased in a relatively impervious wall and are extremely well adapted to
survive in the environment (Levine, 1985). Sarcocystis gigantea sporocysts have been
shown to have remarkable resistance to freezing, desiccation, and various chemical
agents, including 2.5% hydrochloric acid, 10% formalin, and 0.5% ammonia solution
(McKenna and Charleston, 1992). Sarcocystis neurona sporocysts can survive a
temperature of 50°C for 1 hour (Dubey et al., 2002). Treatment with bleach (10, 20, and
100%), 2% chlorhexidine, 1% betadine, 5% o-benzyl-p—chlorophenol, 12.56% phenol,
6% benzyl ammonium chloride, and 10% formalin does not kill them (Dubey et al.,
2002). Exposure to undiluted ammonium hydroxide (29.5% ammonia) for 1 hour kills
sporocysts, as does heating them to 55°C for 15 min and 60°C or more for 1 minute
(Dubey et al., 2002). Unfortunately, none of the lethal treatments is practical for
disinfecting barns or feedstuffs.

The horse is a natural intermediate host to three Sarcocystis species. S. bertrami,
S. equicanis and S. fayeri form cysts in the muscle tissue of equids and complete their life
cycles in canid definitive hosts (Levine, 1986). The distribution of S. bertrami and S.
equicanis is speculated to be worldwide; S. fayerr' has only been reported in North
America (Levine, 1985). It is speculated that the three species may be synonomous
(Levine, 1985). These species of Sarcocystis are not normally pathogenic to either their
definitive or intermediate hosts, and in Britain, where the practice of feeding hunt horses
to hounds exists, the infection rate in horses has been estimated to be between 60 and 70
percent (Edwards, 1984).

Solving the life cycle of S. neurona was a slow process that involved researchers

at a number of different centers. S. neurona was first shown to utilize the opossum as its

10

definitive host in 1995 (F enger et al., 1995) but it was not until the early 21St century that
the intermediate hosts were identified. In 1995, a phylogenetic study suggested that S.
neurona was synonymous with S. falcatula (Dame et al., 1995), which had been reported
to use the opossum as its definitive host, and various species of birds as intermediate
hosts (Box and Smith, 1982). Later investigations revealed the two species of
Sarcocystis did not infect the same intermediate host species. S. neurona was shown to
not be infective to budgerigars (Melopsittacus undulates), while S. falcatula proved to
be highly pathogenic to buderigars (Marsh et al., 1997b). S. neurona produced
neurological disease in immunocompromised nude and gamma-interferon knockout mice
(Marsh et al., 1997a; Dubey et al., 1998a; Dubey et al., 1998b), but S. falcatula did not
(Dubey et al., 1998a; Dubey et al., 1998b). In an infection challenge of S. falcatula in
horses, no clinical disease was produced (Cutler et al., 1999). A series of investigations
revealed that the opossum serves as the definitive host for at least 3 distinct species of
Sarcocystis: S. falcatula, S. neurona, and S. speeri (Dubey et al., 1998a; Dubey et al.,
1998b; Dubey and Lindsay, 1999).

Horses are aberrant hosts to S. neurona, in which the parasite cannot undergo its
final stage of schizogony (Dubey et al., 2001a). The schizonts formed during the course
of EPM contain merozoites (Dubey et al., 2001a). The terminal asexual form, the
sarcocyst, has not been found in infected horses (Dubey et al., 2001a). A carnivore
cannot become infected by eating S. neurona merozoites, thus the horse is a dead-end
host (MacKay et al., 1992). Recently, however, there was a report of a young colt with
neurological disease in which both mature schizonts in the brain and spinal cord and

mature sarcocysts in the tongue were found (Mullany et al., 2003). Electron microscopy

11

revealed that the sarcocysts had characteristics that were consistent with published
characteristics of S. neurona. Testing for genetic markers showed that the sarcocysts
produced Sarcocystis-specific PCR products and these products exhibited banding
patterns characteristic of S. neurona. Further studies will be required to determine
whether horses are true intermediate hosts to S. neurona or if this colt was abnormally
susceptible due to his young age, or some form of immunosuppression.

Since 1997, a number of experimental infection studies have been performed in
which specific pathogen free horses were given doses of S. neurona sporocysts in an
attempt to reproduce clinical EPM. (F enger et a]. 1997, Saville et a]. 2001, Cutler et a].
2001, Sofaly et al., 2003). In the first study (F enger et al., 1997), the investigators were
operating under the assumption that S. neurona was synonymous with S. falcatula and
thus produced sporocysts by feeding tissue from birds infected with sporocysts harvested
from feral opossums to specific pathogen free opossums. These sporocysts, most likely
S. falcatula, were pooled with sporocysts from feral opossums and administered to foals
in doses ranging from 1 x 10° to 2 x 107, with some foals receiving repeat doses, and one
foal receiving daily doses of dexamethasone for 2 weeks to induce immunosuppression.
Despite the fact that sporocysts were counted, it was not possible to ascertain the dose of
S. neurona given to the foals, due to the likely presence of S. falcatula sporocysts in the
inoculum. Foals first produced serum antibodies to S. neurona from 19-42 days and CSF
antibodies 28 days after the first inoculation, with 1 seropositive foal failing to produce
CSF antibodies. Neurological signs were reported in four of five foals and at post
mortem examination microscopic inflammatory lesions suggestive of EPM were found in

neural tissue from 3 of the foals with neurological signs, but not the fourth. That foal had

12

 

spinal cord lesions that were more suggestive of spinal cord compression. Two
uninfected control foals remained seronegative and neurologically normal throughout the
study. No S. neurona was identified in or cultured from any neural tissue specimen
tested in this study (F enger et al., 1997).

Another published equine infection study studied the progression of
irnmunoconversion in horses with and without dexamethasone-induced
irnmunosuppression (Cutler et al., 2001). When this study was conducted, molecular
markers identified by Tanhauser et a]. (1999) could be used to identify pure infections of
S. neurona in feral opossums, enabling investigators to administer known doses of
sporocysts to experimentally infected horses. Yearling specific pathogen free horses
were divided into two treatment groups, in which four horses received daily
dexamethasone and 4 horses did not. Both groups were challenged with with 7 daily
consecutive doses of 5 x 105 sporocysts; two additional horses were maintained as
unchallenged controls. The horses were isolated in stalls for 11 days following dosing,
then transferred to a group enclosure. Horses were observed for neurological signs and
serum and CSF were tested for antibodies to S. neurona. In addition, attempts were made
to identify or isolate S. neurona from whole blood, neural tissue, heart, lung, liver, kidney
spleen, thyroid, tongue, esophagus, stomach, small and large intestine, mesenteric lymph
nodes, and skeletal muscle. Horses produced serum antibodies 17-32 days and CSF
antibodies 17-61 days after the first inoculation. Horses receiving dexamethasone tended
to irnmunoconvert sooner than those that did not, but this difference was not statistically
significant. One control horse seroconverted 75 days after the experimental horses were

challenged. Mild neurological signs were observed in horses in both treatment groups,

13

but the mean scores for each treatment group never exceeded 1.5 (on a scale of 0-4) for
either group, and the horses not receiving dexamethasone appeared to be improving
without treatment as time progressed. Microscopic inflammatory lesions were found in
some challenged horses, but were not considered characteristic of active EPM. No
attempt to culture or detect S. neurona postmortem was successful. The authors
suggested that despite the fact that the horses were challenged with extremely high doses
of sporocysts, they experienced transient CNS infections that were progressing toward
clinical recovery without antiprotozoal treatment. (Cutler et al., 2001).

Two studies conducted in Ohio attempted to induce EPM in horses
experimentally infected with S. neurona after being subjected to the stress of long-
distance transport (Saville et al., 2001; Sofaly et al., 2003). In the first study (Saville et
a1. 2001), sporocysts used in the experimental challenges were obtained from 24 wild-
caught opossums and pooled into one inoculum. Because the opossums likely had mixed
Sarcocystis infections, the S. neurona content was estimated using a bioassay with
gamma-interferon knockout mice. The lethal mouse dose was determined from the
proportion of mice in each group that developed neurological disease, and it was
estimated that the horses were administered at least 80,000 times the lethal mouse dose.
Twelve horses were divided into four groups of three, with one group receiving
sporocysts immediately following a 55-hour trailer ride, one group receiving sporocysts
after having 14 days to acclimate following a 55-hour trailer ride, one group receiving
sporocysts and daily dexamethasone for three days after having 14 days to acclimate
following a 55-hour trailer ride and a control group received no sporocysts after a 55-

hour trailer ride. Horses were evaluated for neurological signs by blinded equine

14

clinicians who were not aware of dose treatment assignments. Horses in the transport-
stressed group seroconverted 2 weeks post-inoculation, horses in the group that had 14
days to acclimate seroconverted 2-4 weeks post-inoculation, horses in the dexamethasone
group seroconverted 4 weeks post-inoculation, and 2 of the 3 controls seroconverted, 6
and 9 weeks afier entering the study. All horses that received sporocysts tested positive
for CSF antibodies at the end of the study, approximately 44 days post-inoculation and
one of the two seropositive controls tested positive for CSF antibodies after 61 days in
the study. The transport-stressed horses developed mild to moderate neurological signs
10-11 days after dosing that improved at least one grade by the final examination. The
acclimated group developed mild neurological signs 9-16 days post-inoculation, and the
signs did not change thereafter. The dexamethasone-treated horses developed mild
neurological signs 9-16 days after dosing, and one horse from the group improved one
neurological grade by the final examination. One control horse developed mild
neurological signs 30 days prior to seroconversion. No attempts to recover S. neurona
postmortem in neurological and other tissues were successfir], although some
microscopic inflammatory lesions were found in spinal cord and brainstem specimens.

In the second Ohio study (Sofaly et al., 2002), 24 seronegative weanling horses
were subjected to the stress of transport from Saskatchewan, Canada to Columbus, Ohio
and immediately dosed with sporocysts upon arrival at the research center. The strain of
S. neurona used in the study was SN 37-R, derived from raccoon tissue fed to laboratory-
reared opossums. Horses were divided into six groups of four horses, with one group
serving as uninfected controls and the remaining five groups receiving 100, 1,000, 10,000

100,000 or 1,000,000 sporocysts in a single dose. No control horses seroconverted or

15

tested positive for CSF antibodies to S. neurona. Two of four horses that received 100
sporocysts produced serum antibodies 4-5 weeks post-inoculation. Horses that received
1,000 or 10,000 sporocysts seroconverted 3-4 weeks after dosing. One horse that
received 10,000 sporocysts did not seroconvert. Horses that were given 100,000
sporocysts produced serum antibodies 3 weeks post inoculation, and horses receiving
1,000,000 sporocysts seroconverted 2 weeks post-inoculation. All seropositive horses
had produced CSF antibodies to S. neurona when they were tested at days 28-35, except
for two horses that received the 1,000 sporocyst dose. Neurological examinations by
blinded evaluators revealed that horses in all groups developed mild to moderate
neurological signs, but no dose-response trend of severity or time to onset (8-29 days)
was detected. Additionally, one control developed neurological disease consistent with
equine degenerative myelopathy (EDM) and one horse receiving 100,000 sporocysts did
not develop neurological signs. Neurological signs were seen in 5 horses that tested
negative for CSF antibodies to S. neurona. No attempt to recover or identify S. neurona
postmortem was successful, although some horses in the study had microscopic
inflammatory lesions suggestive of protozoan infection.

The most consistent findings of the experimental infection studies published to
date (Fenger et a]. 1997, Saville et a1. 2001, Cutler et a1. 2001, Sofaly et al., 2003) are that
seroconversion appears to occur two to four weeks afier horses receive a single dose of S.
neurona sporocysts, neurological signs, when seen, are mild to moderate and do not
appear to follow a dose-response relationship, and the parasite has yet to be identified or
recovered in experimentally infected horses postmortem. All the studies suffered from

low sample sizes. In the first study (Fenger et al., 1997), no two horses got the same dose

16

of sporocysts, although due to the fact that the inoculum was of mixed Sarcocystis
species, this may not have made a difference in the interpretation of the results. In the
other three studies (Saville et a]. 2001, Cutler et a]. 2001, Sofaly et al., 2003), there were
no more than 4 horses per treatment group. This did not allow sufficient statistical power
to detect differences between treatment groups with respect to days to seroconversion,
onset of neurological signs or mean severity of neurological signs. Thus, while the data
suggests that dexamethasone does not cause irnmunosuppression that increases
susceptibility to EPM, there has not been a sufficiently powerful study to date that can
demonstrate this with 95% confidence (Saville et a]. 2001, Cutler et a]. 2001). Likewise,
in the study that evaluated sporocyst dose and neurological disease in transport-stressed
horses, in which there were only four horses per group, it was not possible to determine
whether sporocyst dose or CSF antibody status were associated with the severity of
neurological signs (Sofaly et al., 2002). It is possible that horses that develop EPM from
natural infections have some immunological characteristic that makes them different
fi'om the general horse population. That is, they may be more susceptible to infection or
less able to clear the infection from the CNS than the experimental horses studied to date.

At this time, a reliable equine disease model for EPM has yet to be established.

4. EPIDEMIOLOGY:

EPM is the most commonly diagnosed infectious equine neurological disease in
the US (Dubey et al., 2001a). It has been reported in horses and ponies, but not in
donkeys or mules (MacKay et a] 1992; Dubey et al., 20013). EPM usually occurs

sporadically, and has not been shown to be transmissible fi'om horse to horse (Dubey et

17

al., 2001a). Clusters of cases are unusual (Madigan and Higgins, 1987; MacKay et al.,
1992). Rarely does one farm report more than one case, but some farms have had cases
annually (Madigan and Higgins, 1987; MacKay etal., 1992), and there are two reports in
the literature of clusters of cases on farms (Granstrom et al., 1992; Fenger et al., 1997b)

EPM is considered a New World disease. It has been diagnosed in horses in
Canada, the United States, Panama and Brazil (F ayer et al., 1990; Granstrom et al., 1992;
Masri et al., 1992). Cases reported in other parts of the world have been horses that
originated from the United States (F ayer etal., 1990; MacKay et al., 1992; Katayama et
al., 2003) or had traveled there prior to being diagnosed (Goehring and Sloet van
Oldruitenborgh-Oosterbaan, 2001). EPM has been diagnosed in horses ranging in age
from 2 months to 24 years (F ayer et al., 1990; MacKay et al., 1992). The possibility of
transplacental transmission is suggested, given the fact that a 2-month-old foal was
diagnosed by histopathology with EPM.

In a retrospective study of 364 histologically confirmed EPM cases from the US
and Canada (F ayer et al., 1990), clinical cases were found to range in age from two
months to greater than 19 years, but more than 60% of the cases were 4 years old or
younger. No association was identified between the occurrence of EPM and the horses’
geographic location or gender. There was also no apparent seasonal pattern to EPM. The
breeds most commonly affected were Thoroughbred, Standardbred, and Quarter Horse,
respectively. Other breeds in the study were: Appaloosa, Arabian, Morgan, Paint, Belgian
and Crossbred.

In a smaller 1990 study of 82 EPM cases confirmed by histopathology (Boy et al.,

1990), Standardbreds were overrepresented compared to the normal hospital population

18

at the University of Pennsylvania. In that study, 87.5% of the EPM cases were aged 6 or
younger. Also, male horses appeared to be at greater risk of developing EPM. The
authors acknowledged that there were associations within the data that may have
confounded the results; young racehorses were most likely to be male at that hospital, and
the mean age of the hospital population for the study period was not available for
comparison.

In a case-control study of living horses in Ohio, putatively diagnosed with EPM,
several factors were associated with the development of clinical disease (Saville et al.,
2000). In comparison to controls, cases were at greater odds of being presented for
diagnosis in spring, summer or fall, and cases were at greater odds of residing on
operations where a previous case of EPM had occurred (Saville et al., 2000). Cases were
at greater odds of being involved in racing or showing than controls, cases were at greater
odds of having had a previous health event (such as lameness, injury or parturition) than
controls, and cases were at greater odds of residing on operations where opossums were
known to be present (Saville et al., 2000). In comparison to controls, cases were at lower
odds of being 1 year of age or younger, being fi'om an operation with a creek or river on
the premises, or residing on an operation where all feeds and forages were secure from
wildlife (Saville et al., 2000).

The incidence of clinical EPM cases appears to be low, despite the fact that
antibody seroprevalence studies suggest many horses have been infected with S. neurona
at some time in their lives. The incidence of EPM has been estimated to be 0.014% for
the United States (N .A.H.M.S., 2001). In a Michigan seroprevalence study, 10 of 1121

(0.9%) horses had been diagnosed with clinical EPM within one year of participating in

19

the study, although none was considered an active case at the time the questionnaire was
administered (Rossano et al., 2001). Thus, EPM can be characterized as a disease of high
antibody seroprevalence and low clinical disease incidence.

S. neurona antibody seroprevalence studies have been performed on populations
of presumably neurologically normal horses in Oregon (Blythe et a], 1997), Ohio (Saville
etal., 1997), Michigan (Rossano et al., 2001), northern Colorado (Tillotson et al., 1999),
and Chester County, Pennsylvania (Bentz et al., 1997). Serum antibody prevalence in
Chester County, Pennsylvania was approximately 45.3% (Bentz et al., 1997). In Oregon,
seroprevalence ranged fi'om 22% to 65% from east to west, and overall prevalence was
approximately 45% (Blythe et a], 1997). Seroprevalence was associated with geographic
region; horses tested in the temperate, coastal regions were more likely to be infected
than horses from the colder, arid eastern regions (Blythe et a], 1997). This difference was
attributed to opossum prevalence and climate, since opossums were more abundant in the
temperate climates (Blythe et a], 1997). In Michigan, seroprevalence followed a north-
south gradient that also reflected the opossum distribution in the state (Rossano et al.,
2001). There, 47% of the horses in the northernmost region were seropositive, 56% of the
horses in the middle region were seropositive, and 62% of the horses in the southernmost
region were seropositive (Rossano et al., 2001). Overall average statewide seroprevalence
in Ohio was approximately 53.6%, and seroprevalence was negatively associated with
days below freezing (Saville et al., 1997). This protective effect was hypothesized to be
due to decreased sporocyst survival in cold conditions, as well as poor sarcocyst (tissue
cyst) survival, causing a reduction of infections in the definitive host population. In

northern Colorado, the antibody prevalence was 33.6%. There, a seasonal trend was

20

reported (Tillotson et al., 1999). Seroprevalence was higher among horses tested in
spring, summer and fall than horses tested in winter. Two breed groups, warmbloods,
and a category comprised of ponies and non-horse equids, had higher antibody
prevalence than hot-blooded breeds and stock breeds. It was acknowledged by the
authors, however, that the breed category variables might have indirectly accounted for
other factors associated with the management of different types of equids. In that study,
it was also speculated that there was an association between low opossum density and
low antibody prevalence (Tillotson et al., 1999) In all of the cited studies, age was
positively associated with seroprevalence (Bentz et al., 1997; Blythe et a], 1997; Saville
et al., 1997; Tillotson et al., 1999; Rossano et al., 2001). No association has been
identified between the risk of infection and horse gender (Bentz et al., 1997; Blythe et a],
1997; Saville et al., 1997; Tillotson et al., 1999; Rossano et al., 2001), or indoor versus
outdoor housing Blythe et al., 1997; Rossano et al., 2001). In the Michigan study, which
examined the most management factors of the studies to date, a mild association was
found between seropositivity and access to pasture (Rossano et al., 2001). Feeding sweet
feed was associated with decreased seropositivity, but this finding was questioned by the
authors due to confounding with age and geographic region (Rossano et al., 2001). No
association was found between farm size, animal gender, hay type or access to natural
surface water and seropositivity (Rossano et al., 2001). Another analysis of the same
study population did not reveal any herd-level management practices (applied to all
horses on a given farm) and the proportion of animals seropositive (Rossano et al., 2002).
The prevalence studies published to date have been useful in quantifying S.

neurona infection in normal horses, but they have suffered from notable limitations and

21

biases. For example, in Ohio (Saville et al., 1997) and northern Colorado (Tillotson et
al., 1999), the sera used to determine antibody prevalence were obtained from equine
infectious anemia (EIA) testing laboratories. In those studies, sample sizes were robust
(1,056 equids in Ohio (Saville et al., 1997), 608 in northern Colorado (Tillotson et al.,
1999), and to reduce bias, the researchers made random selections from the serum
samples. However, the authors acknowledged that horses tested for EIA are apt to travel,
and EIA samples were not necessarily representative of the actual horse populations of
those geographic regions (Saville et al., 1997; Tillotson etal., 1999). Another limitation
of using EIA samples is that only general descriptive data could be obtained about the
horses tested. Although information regarding the location, age, breed and gender of the
horse was recorded at testing (Saville et al., 1997; Tillotson et al., 1999), it was not
possible to know how long the horse had been at the location where it was tested, and no
horse management information was available for analysis. In Oregon, 334 horses were
selected by a modified cluster-sampling technique (Blythe et al., 1997). The clusters
were organized according to four distinct geographic regions, and veterinarians were
recruited to systematically select neurologically normal horses from their practices for
serum testing. No more than two horses from a given farm could be included in the
study. Although this sampling technique may have resulted in a more representative
sample of a region’s horses than BIA testing samples, it was not a random selection
process. In the Oregon study, information regarding individual horses and their housing
was collected (Blythe et al., 1997). While this provided more detailed information,
compared to EIA testing records, no information regarding feeds or watering systems was

collected. Finally, the study of seroprevalence in Chester County, Pennsylvania was

22

performed on 117 serum samples collected from thoroughbred breeding farms (Bentz et
al., 1997). Due to the small sample size this study was lacking in statistical power, and
due to the small geographic area tested and the exclusion of all breeds but thoroughbred,
the results have limited extema] validity beyond the study population. The Michigan
study benefited from large sample size (98 farms, 1121 equids) and a stratified-random
sample of farms on which all horses were tested, but the study was limited by the fact that
the information on the opossum distribution in the state was 16 years old when the study
was conducted (Rossano et al., 2001).

It is assumed that horses are infected with S. neurona by consuming hay, grain,
pasture grass or water that has been contaminated with feces from the opossum (Dubey et
al., 2001a). In other Sarcocystis species, the severity of infection has been related to the
number of sporocysts fed (Urquhart et al., 1996), however the dose response is not
known for S. neurona in equids. Since 1997, a number of experimental infection studies
have been performed in which specific pathogen fiee horses were given doses of S.
neurona sporocysts in an attempt to reproduce clinical EPM or document the life cycle of
S. neurona (Fenger et a]. 1997; Saville et al. 2001; Cutler et a]. 2001; Cheadle et al.
2001a; Cheadle et al. 2001b). In the first study sporocyst doses were calculated, but were
most likely sporocysts from opossums with mixed Sarcocystis spp. Infections (F enger et
al. 1997). In that study, although clinical neurological disease was produced by feeding
large doses of sporocysts to foals, and inflammatory lesions consistent with EPM were
found in three of the five experimental animals, at the end of the experiment no parasites
were found by histopathology, and none could be cultured from the foals’ neural tissue

(F enger et a]. 1997). A recent study that utilized transport stress as a means of producing

23

immunosuppression in weanling horses reported that mild to moderate clinical signs were
produced in horses who received sporocyst doses ranging from 100 to 1,000,000, with the

most consistent signs seen in the 1,000,000-sporocyst group (Sofaly et al., 2002).

5. PATHOLOGY:
a.) GROSS PATHOLOGY:

S. neurona causes non-suppurative inflammation of the CNS, resulting in gross
lesions in both gray and white matter and necrosis of affected neural tissue (Rooney et
al., 1969; Mayhew et al., 1978; Fayer et al., 1990). The lesions are random in occurrence
and vary in size from microscopic to several centimeters in diameter (Rooney et al.,
1969; Mayhew et al., 1978; F ayer et al., 1990). Lesions are most common in the spinal
cord or brain stem (Rooney et al., 1969; Mayhew et al., 1978; Fayer et al., 1990). When
they are macroscopic, the lesion will appear discolored (pink, brown or yellow), the
tissue may be sofi and swollen, and it may have (multi-) focal hemorrhages (Rooney et
al., 1969; Mayhew et al., 1978; Fayer et al., 1990). Treatment with anti-protozoa] drugs is
reported to decrease the likelihood that gross lesions will be detected upon postmortem

examination (Dubey et al., 1991; Boy et al., 1990).

b.) HISTOPATHOLOGY:

The mechanism by which S. neurona gains access to the central nervous system
has not been described, but the parasite has been identified in mononuclear cells, and
occasionally giant cells, neutrophils and eosinophils (Rooney et al., 1969; Simpson and

Mayhew, 1980; Dubey and Miller, 1986, Bowman et al., 1992). Perivascular cuffing

24

with inflammatory cells is evident in the affected areas and in the meninges that cover the
lesions (Rooney et al., 1969; Mayhew et al., 197 8). Demyelination of axons by
macrophages has been reported, and degeneration of neurons and axons is commonly

seen (Rooney et al., 1969; Bowman et al., 1992).

6. DIAGNOSIS:

A definitive diagnosis of EPM requires finding the parasite in neural tissue
sections at postmortem examination (Mayhew et al., 1978; Hamir et al., 1993). Because
the lesions can be microscopic in size, histopathology can be unrewarding (Hamir et al.,
1993). In horses, the length of the spinal cord and size of the brain makes it difficult to
adequately sample for lesions, even when clinical signs are used to localize the most
likely area(s). Animals that have been treated with anti-protozoa] drugs are even more
difficult to diagnose by this method (Boy et al., 1990; Hamir et al., 1993).
Immunohistochemical staining using polyclonal anti-S. neurona or anti-S. cruzi
antibodies has been shown to increase success in locating the parasite, even in horses
treated with sulfonarnides (Hamir, et al., 1993). It is also possible to culture the parasite
fi'om neural tissue collected from horses suspected of having EPM (Davis et al., 1991;
Bowman et al., 1992; Mansfield et al., 2001), but this process is elaborate and not
normally done as part of a routine necropsy.

Antemortem diagnosis of EPM has evolved since the disease was first described.
Because a variety of neurological signs may be present, clinical signs alone are not
sufficient for accurate diagnosis of EPM (Mayhew and Greiner, 1986; Madigan and

Higgins, 1987; Mackay et al., 1992; Furr et al., 2002).

25

The first diagnostic test available to veterinarians was an indirect fluorescent
antibody (FIAX) titer performed on serum. The test measured reactivity to Sarcocystis
cruzi bradyzoites from bovine cardiac tissue cysts, and was used to indicate the presence
of Sarcocystis spp. infections, but was not specific to S. neurona (MacKay et al., 1997).
In 1993, a western blot test that could detect antibodies to S. neurona in serum or
cerebrospinal fluid (CSF) was developed (Granstrom et al., 1993). The western blot test
used reactivity to proteins of 22, 13 and 10.5 kDa as the criteria for a positive test. These
proteins were asserted to be specific to S. neurona, however the sensitivity and specificity
of the test were not estimated. A subsequent western blot test that employed bovine-
derived anti-S. cruzr’ antibodies to block cross-reacting proteins on blots was estimated to
have sensitivity and specificity of _<_ 100% and 98%, respectively, for detecting antibodies
to S. neurona in equine serum when simultaneous reactivity to the 30- and 16-kDa
proteins was used as the positive criterion (Rossano et al., 2000).

A polymerase chain reaction (PCR) test has been offered since 1995 (F enger et
al., 1994). The advantage of the PCR test is that it can directly detect parasite DNA,
rather than antibodies, in CSF (F enger et al., 1994). The PCR test targeted the 188 small
subunit ribosmal RNA (SSURNA) gene of S. neurona and related species, and
production of a 484 base-pair product was the criteria for a positive test (Fenger et al.,
1994; Fenger et al., 1995). Later investigations have shown that this portion of the target
gene was not unique to S. neurona, and that the PCR test could not distinguish S. neurona
from S. falcatula or S. speeri, but that S. neurona could be distinguished from the other
species by using different primers and subjecting the 334-bp and 1,085-bp amplicons to

restriction fragment length polymorphism enzyme digestion (Tanhauser et al., 1999).

26

Although it is a valuable research tool, in clinical use on CSF samples the PCR test
appears to suffer from low sensitivity, making negative results unhelpful in a diagnosis
(Dubey et al., 2001a). A positive result is strong evidence of active infection of the CNS
with S. neurona, however (Dubey et al., 2001a). The apparently low sensitivity may be

. due to poor DNA extraction techniques in the laboratory, degradation of samples during
shipping, or low numbers of S. neurona merozoites present in the small quantities of CSF
that are usually submitted for testing. In one study, it was recommended that test
sensitivity could be enhanced if veterinarians submitted CSF samples of greater than 5
m1, rather than the customary 1.5 ml (Marsh et al., 1996b). In 2001, a direct
agglutination test using S. neurona merozoites was developed that was estimated to have
100% sensitivity and 90% specificity in experimentally infected mice (Lindsay and
Dubey, 2001). Although the test has proved useful for wildlife antibody seroprevalence
studies, it is not commercially available for use by equine practitioners as a diagnostic
test.

At present, the western blot test is the most commonly used EPM test today
(Dubey et al., 2001 a). A positive serological test result indicates past or current infection
with S. neurona, but does not confirm that the parasite has reached the central nervous
system. Given the high estimated rate of serum antibody prevalence to S. neurona in
clinically normal horses (>50% in some areas), testing of CSF is usually preferred
(Fenger, 1997; MacKay, 1997; Furr et a]. 2002). Clinical signs of multifocal
neurological disease, accompanied by a positive CSF test allow for a reasonably reliable
approach toward diagnosis, but do not constitute a definitive diagnosis of EPM (F enger,

1997; MacKay, 1997; Furr et al. 2002). This is partly because IgG antibodies are only

27

indirect evidence of the presence of the parasite, and can remain after an infection has
cleared. Also, false positive western blot results on CSF samples can be produced by the
leakage of seropositive blood into the CSF, either at the time of collection or due to a
disease process that causes leakage of serum proteins across the blood-brain barrier, such
as EHV-l encephalitis (Miller et al., 1999; Furr et. a12002). In a recent analysis of
paired serum and CSF western blot test results from horses being tested for diagnostic
(not research) purposes, 29% of the horses that had serum antibodies tested negative for
CSF antibodies (Rossano et al., 2003). In that study, the degree of blood contamination
in the CSF samples was not known. Recent evidence suggests that horses with very high
serum antibody titers to S. neurona could produce false-positive CSF tests, due to a
minute amount of blood contamination (8 RBC/ul of CSF) (Miller et al., 1999) or by
passive movement of serum antibodies across an intact blood-brain barrier (F urr, 2002).
Fortunately, because S. neurona spends most of its time inside cells, not in the
bloodstream (Dubey et al., 2001a), serum titers tend to be low, and high titers are not
common (Duarte et al., 2003). It has been recommended that samples of CSF should
have 5 SORBC/ul (Furr et al., 2002) to 5 100 RBC/u] (Daft et al., 2002) to be considered
suitable for western blot testing.

A negative western blot test result of a CSF sample is generally accepted to be
useful for ruling out EPM, due to the high sensitivity of the test (Furr et al., 20013). One
study based on histologically confirmed EPM cases supports this assumption, reporting
the sensitivity of the western blot test on CSF to be 87% for horses with and 88% for
horses without neurological abnormalities (Daft et al., 2002). In the same study, the

specificity of the western blot was estimated to be 44% for horses with and 60% for

28

horses without neurological abnormalities. One weakness of the study, however, is that
its definition of a “gold standard” negative was a horse in whom inflammatory lesions or
parasite were not found in numerous CNS sampling points. Since it was not feasible to
sample the entire brain and spinal cord of a horse, and histopathology is a technique of
low sensitivity (Hamir et al., 1993), it is likely that there were horses in the study that
were misclassified as gold standard negatives. This undoubtedly biased estimates toward
lower specificity. Another study based on 48 samples from the same group of
histologically confirmed cases found that a S. neurona indirect fluorescent antibody
(IFA) test performed on serum was more accurate overall under receiver-operating
characteristic analysis than the western blot on serum or CSF when a titer of 1:80 was
classified as a positive result (Duarte et al., 2003). Again, the same weaknesses hold true
in terms of the potential for rnisclassification of gold standard negatives, and now that a
S. neurona vaccine has come to market, the value of serum IFA titers for diagnosis of
EPM is questionable at this time.

Today, equine clinicians are encouraged to perform a thorough physical
examination and order ancillary tests (such as radiographs and diagnostic tests for EPM
and other diseases) to rule out musculoskeletal lameness and other neurological diseases
before arriving at a diagnosis of EPM (Furr et al., 2002). This diagnosis should ideally
be supported by clinical signs that are consistent with EPM and a positive western blot

test result on uncontaminated CSF from the horse (Furr et al., 2002).

7. TREATMENT:

29

Drugs that interfere with folic acid synthesis in protozoa, by inhibiting
dihydrofolate reductase/thymidylate synthase and dihydropteroate synthase, were the first
to be employed in the treatment of EPM, and thus EPM was considered to be a treatable
disease (Mayhew et al., 1978; Mayhew and Greiner, 1986; Madigan and Higgins, 1987;
F enger, 1997; Mackay, 1997). Pyrimethamine and sulfa combinations were the standard
therapy and it was recommended that treatment be given for 3 to 5 months (Fenger, 1997;
Mackay, 1997). Relapses have been known to occur after this therapy, and treatment of
these cases is usually less successful than it was during the treatment of the initial case
(F enger, 1997; Mackay, 1997). It has been estimated (but no published study supports
this estimate) that approximately 70% of horses treated by this therapy respond favorably
(Dubey et al., 2001a).

Prolonged antifolate therapy can result in undesirable side effects, including
reduced spermatogenesis in stallions, possible harm to the fetus in pregnant mares, and
bone marrow suppression in adult horses (MacKay, 1997). Blood counts may be used to
monitor for evidence of toxicity due to the folic acid inhibitors (Dubey et al., 2001a).
When leukopenia is detected in its early stages, lowering the dosages of the antifolates
can prevent further progression (Mayhew and Greiner, 1986). Although oral
supplementation of folic acid in horses treated for EPM has been recommended (F enger
et al., 1997), one study suggests that it may cause congentia] defects in foals born to dams
treated with antifolates during pregnancy (Toribio et al., 1998). F clinic acid
supplementation can reverse the folic acid inhibiting effect of the medication, but it is too

costly for most equine applications (Mackay, 1997). Folinic acid can be provided to

30

horses at much lower cost in a diet of fresh green forage (Mackay, 1997; Toribio et al.,
1998).

Since the late 1990’s coccidiocidal drugs used in poultry species have been tested
in clinical trials in horses. The first, diclazuril, had shown efficacy in treating acute T.
gondii infections in mice (Lindsay et al., 1995). For reasons not made public, the drug
has not yet been approved for use in horses. The second coccidiocide to enter clinical
trials was ponazuril, which can be given to horses as a 15% oral paste by their owners
(Furr et al., 2001). At present, it is the only drug labeled for the treatment of EPM.
Ponazuril is given daily for 28 days, less time than the sulfa/pyrmethamine therapy
requires (Furr et al., 2001). When the response to therapy in 10] horses was evaluated,
62% of the horses treated met the criteria for a successful treatment after 3 months of
observation (Furr et al., 2001). (Horses who converted to a negative CSF antibody
western blot result or who demonstrated an improvement of at least 1 neurological grade
on a 0-5 scale were classified as treatment successes).

The final compound tested for EPM therapy is nitazoxanide (NT Z), a broad-
spectrum anthehnintic used in human medicine, which is formulated as an oral paste for
horses (Dubey et al., 2001a). The treatment course for NTZ was also 28 days. An early
report based on 7 horses showed the efficacy of NTZ to be comparable in efficacy to the
sulfa/pyrimethamine therapy (Vatistas et al., 1999). A subsequent study involving 70
horses reported that 63% of horses on the treatment improved one neurological grade or
more or becoming negative for CSF antibodies on the western blot test, but the drug had
a narrow margin of safety (McClure and DePalma, 1999). To date, NTZ has not been

approved for use in horses.

31

Ira-.29“

In acute cases of EPM, anti-inflammatory drugs, such as phenylbutazone,
dimethylsulfoxide and glucocorticosteroids may be used in conjunction with anti-
protozoal drugs (MacKay, 1997). Corticosteroids used alone have been shown to hasten
the progression of EPM in naturally infected horses (Mayhew et al., 1978; Mayhew and
Greiner, 1986; Madigan and Higgins, 1987), but did not appear to affect neurological
signs in experimentally infected horses (Cutler et al., 200]). In situations where EPM is
thought to be related to a poor immune response in the horse, use of immunostimulants,
such as levamisole, alpha-interferon, mycobacterial wall extract or killed
Propionibacterium acne, have been recommended (MacKay, 1997). Vitamin E
supplementation may help minimize damage from inflammation and aid healing of the
central nervous system, although a standard equine diet of flesh or dried forage usually

contains excess levels of vitamin E (MacKay et al., 1997).

8. PROGNOSIS:

In cases where EPM is treated with anti-protozoa] drugs and the horse survives,
no definitive post-mortem diagnosis is possible. Therefore, cure rates cannot accuately
be determined. Horses that respond to treatment usually stabilize within a week
(MacKay et al., 1992). Improvement may begin at that point and can continue for
months or years following cessation of drug therapy (MacKay et al., 1992). Recovery is
often incomplete, but if treatment begins soon after the onset of signs, the likelihood of a
full recovery is greater (MacKay et al., 1992). Brain disease seems more likely to
improve than spinal cord disease and muscle atrophy is most often permanent (MacKay

et al., 1992).

32

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39

CHAPTER 2

DOES DAILY ADMINISTRATION OF PYRANTEL TARTRATE PREVENT
INFECTION WITH S. NE URONA IN HORSES? AN EXPERIMENTAL

CHALLENGE STUDY.

2.0 ABSTRACT:

Equine protozoal myeloencephalitis (EPM) is caused by the protozoan parasite
Sarcocystis neurona. In vitro studies have shown that pyrantel tartrate is lethal to S.
neurona merozoites. The purpose of this study was to determine whether daily
administration of pyrantel tartrate prevents S. neurona infection in horses, as determined
by the presence of antibodies in the serum. Twenty-four mixed breed weanling horses,
confirmed free of infection by absence of semm antibodies to S. neurona, were group-
housed at the Washington State University (W SU) Animal Resource Unit in outdoor pens
and fed locally produced feeds. Horses were randomized into two groups: group A
received pyrantel tartrate (Strongid C 2x) at the label dose and group B received a
placebo pellet. Both groups were orally inoculated with 100 sporocysts per day for 28
days, 500 sporocysts per day for 28 days and 1,000 sporocysts per day for 56 days.
Pyrantel tartrate or placebo was administered to the horses immediately following dosing
with sporocysts. Blood was collected weekly and spinal fluid was collected monthly.
Eleven adult horses from WSU that also tested negative for serum antibodies to S.
neurona and were monitored as untreated, uninfected controls. Control horses were bled

weekly on the same schedule as the experimental horses. All serum and CSF samples

40

were tested for antibodies to S. neurona. After 134 of sporocyst dosing, 10/ 12 horses in
each treatment group were seropositive for antibodies to S. neurona. 4/12 horses in
group A and 3/12 horses in group B had antibodies to S. neurona in CSF. No statistically
significant difference was found between groups A and B for serum or spinal fluid tests
(Fisher’s exact test). Ten of 11 control horses remained seronegative. We conclude that
daily administration of pyrantel tartrate does not prevent infection with S. neurona in

horses when given according to label dose.

41

2.1 INTRODUCTION:

Equine protozoal myeloencephalitis (EPM) is a multi-focal neurological disease
affecting horses and other equids of the Americas. Clinical signs of EPM may include
weakness, ataxia, loss of proprioception, muscle wasting, behavioral changes,
recurnbancy and/or death (Dubey et al., 2001 a; MacKay, 1997).

The most important etiologic agent of EPM is the protozoan parasite, Sarcocystis
neurona (Dubey et al. 1991). S. neurona requires a definitive and intermediate host to
complete its life cycle. Intermediate hosts of S. neurona identified to date include the
domestic cat (Dubey et al., 2000), the nine-banded armadillo (Cheadle et al., 2001a), the
striped skunk (Cheadle et al., 2001b), the raccoon (Dubey et al., 2001c) and the sea otter
(Dubey et al., 2001b). Opossums serve as the only known definitive host of S. neurona
(Dubey et al., 2001a). Horses and ponies are infected when they ingest sporocysts of S.
neurona that are present in contaminated feed or water (Dubey et al., 2001a). To date,
mature sarcocysts of S. neurona have not been detected in equids, therefore equids are
considered to be dead end hosts (Dubey et al., 2001a). Recently, however, there was a
report of a young colt with neurological disease in which both mature schizonts in the
brain and spinal cord and mature sarcocysts in the tongue were found (Mullany et al.,
2003). Electron microscopy revealed that the sarcocysts had characteristics that were
consistent with published characteristics of S. neurona. Testing for genetic markers
showed that the sarcocysts produced Sarcocystis-specific PCR products and these
products exhibited banding patterns characteristic of S. neurona. Further studies will be

required to determine whether horses are true intermediate hosts to S .neurona or if this

42

colt was abnormally susceptible due to his young age, or some form of
immunosuppression.

S. neurona can infect the central nervous system (CNS) of equids, which may
lead to the development of neurological disease. The US. Department of Agriculture
estimated the incidence of EPM in 1998 to range from 0.06% in the southern US. to 0.43
in the central US, with a national average of 0.014% (N .A.H.M.S.). Cross-sectional
studies conducted in several geographic areas of the United States revealed that in
regions of western Oregon and southern Michigan, seroprevalence was as high as 60-
65% (Blythe, et al., 1997; Rossano et al., 2001) and as low as 22-34% in regions of
eastern Oregon and northern Colorado (Blythe et al., 1997; Tillstone et al., 1999). Taken
together, the disease incidence and antibody seroprevalence data suggest that many
horses are infected with S. neurona at some time in their lives, but very few of them
develop clinical disease. Despite the low incidence of EPM, there is great interest among
members of the horse industry to find effective preventive strategies. This interest to
develop effective strategies can be attributed to the potential loss of use of the horse and
the high cost of treating the disease. In 2001, a whole-cell killed vaccine of S. neurona
merozoites was granted a conditional license as an aid for the prevention of EPM (Fort
Dodge Animal Health) but the efficacy of the vaccine in horses has yet to be established.
An important drawback of the vaccine is that vaccinated animals will test positive for
serum antibodies, thus confounding the diagnosis of neurological disease in vaccinated
horses.

The drug pyrantel tartrate has shown promise as a preventive agent in in vitro

studies against the merozoite and sporozoite stages of S. neurona (Kruttlin et al., 2001;

43

Kruttlin et al., 2003). In those studies, the drug was lethal to the parasite when exposed
to concentrations higher than 0.005M. In contrast, a study using gamma-interferon
knockout mice, the drug was not effective in preventing infection with S. neurona at a
dose of 4-5 mg pyrantel tartrate per mouse, however the drug may have been inactivated
by being kept in a water solution for long periods of time (Lindsay and Dubey, 2001).
Because of the encouraging results of Kruttlin et al. (2001 and 2003) it was decided to
evaluate the efficacy of this drug in preventing infection in horses. Therefore, the
purpose of this study was to test whether daily pyrantel tartrate (Strongid C 2x) could
prevent infection by S. neurona in horses. The specific hypothesis tested in this study
was that daily administration of pyrantel tartrate prevents infection with S. neurona in

horses when given at the label dose.

2.2 MATERIALS AND METHODS:

2.2.1 The parasite:

S. neurona sporocysts were produced using a specific-pathogen-free laboratory-
reared opossum. The opossum was fed muscle tissue from a raccoon that had been
experimentally infected with S. neurona strain SN-3 7R (Sofaly et al., 2002), which was
kindly provided by J .P. Dubey. At day 14 post-infection, the opossum was euthanized
using C02 asphyxiation and the small intestine was removed, flushed with saline
solution, and stripped of its mucosa. Sporocysts were harvested fi'om the resulting tissue
slurry according to previously described methods (Murphy and Mansfield, 1999) and

stored in Hank’s balanced salt solution with amikacin sulfate (100 ug/ml), penicillin G

(100U/m1) and amphotericin B (1.25 jig/ml) in a refi'igerator for 1 year prior to the start
of the study. Sporocysts were confirmed to be S. neurona by PCR with restriction
fiagment length polymorphism (RF LP) digestion (Tanhauser et al., 1999). Before being
administered to animals, the sporocysts were further purified of cell debris and bacteria
using a previously described potassium bromide gradient separation technique (Elsheikha

et al., 2003.).

2.2.2 Verification of sporocyst viability:

Two months before the study, 3 female gamma-interferon knockout mice
(Jackson Laboratories, Strain Name: C.129S7(B6)-Ifirg""’ T3) were inoculated with
subsamples of SN-37R sporocysts. Two mice received 1,000 sporocysts and 1 mouse
received 1,500 sporocysts. The doses were gavaged to the stomach in sterile water
solution using a 5 French rubber catheter. The mice were provided free choice food and
water and were observed daily for general health and development of neurological signs.
When neurological disease was observed, the mice were euthanized using C02
asphyxiation and homogenized samples of brain tissue were placed on equine dermal cell
culture and observed weekly for growth of S. neurona. Merozoites harvested from the
cell cultures were confirmed to be S. neurona using PCR with RFLP (Tanhauser et al.
1999). Mice were also submitted for histopathologic examination to confirm protozoal
encephalitis.

Thirty-five days after initiation of the study, viability of the sporocysts used in the
inoculum given to horses was confirmed again in 3 female 'y-IFN knockout mice (Jackson

Laboratories, Strain Name: C.129S7(B6)-Ifng""’ T‘). On this occasion, 2 mice received

45

100 sporocysts and 1 mouse received 1,000 sporocysts in sterile water using a 5 French
rubber catheter. Mice were euthanized as mentioned above when neurological signs were
apparent, and brain tissue was harvested and placed on equine dermal cell culture and
observed for parasite growth. Once again, mice were submitted for histopathologic

examination and confirmed to have protozoal encephalitis.

2.2.3 The horses:

Twenty-four mixed breed weanlings, approximately 5 months old at the start of
the study, were obtained from an equine ranch in North Dakota. The horses were
delivered to the Animal Research Unit at Washington State University (W SU), in
Pullman, Washington and allowed to acclimate in isolation on pasture for 2 weeks.
Throughout the horses were fed alfalfa and grass hays grown in eastern and central
Washington, an arid region where opossums are not reported to live (Figure 2.1.). Some
locally produced sweet feed was provided to the horses as a training motivator. The
horses were subsequently group-housed in adjacent outdoor paddocks with sparse
vegetation, according to their treatment group, with access to shelter and free choice hay
and water. A woven wire fence with an electrified top wire separated the two pens,
limiting, but not excluding, horse-to-horse contact. (A diagram of the paddock layout is
in Appendix 1.) Eleven adult horses, maintained at WSU for teaching and research, were
also studied as untreated, uninfected controls to insure that horses maintained on the same
feeds as the experimental horses did not become infected with S. neurona. Five of the
controls were housed in a paddock directly adjacent to the experimental horses, 6 were

housed at another horse facility on the same campus, approximately 2 miles away.

46

Prior to arrival at WSU, the 24 experimental horses were dewormed with
moxidectin and vaccinated against eastern and western encephalitis, tetanus, influenza
and equine herpes virus I. They were dewormed again with iverrnectin at the start of the
study and booster vaccines were administered approximately 1 month after the first
vaccine (10 days prior to the start of sporocyst dosing). West Nile Virus vaccines were

given at days 10 and 35 after the start of sporocyst dosing.

2.2.4 Sporocyst inoculation:

Sporocysts were counted for the stock solution using a hemacytometer. In order
to obtain the most accurate estimate possible, this was accomplished by keeping the
solution constantly moving with stirring and by determining the mean of 32 counts.

The sporocyst inoculum for the horses was prepared daily, immediately prior to
dosing. Sporocysts from the stock solution were suspended at the desired dose in a 70%
corn syrup and 30% water mixture with stirring. A 2 m1 dose was delivered to the horses
using an oral syringe.

The goal of the dosing scheme was to mimic a low-dose, chronic infection
situation and use the minimum number of sporocysts needed to achieve seroconversion.
The minimum number of SN-37R sporocysts used to produce seroconversion in
experimentally infected horses subjected to transport stress was reported to be a single
dose of 100 (Sofaly et al., 2002). Thus, 100 sporocysts/day was chosen as the dose for
this study. For the first 28 days horses were given 100 sporocysts/day. When weekly
western blot testing showed no seroconversion, the dose was increased to 500

sporocysts/day for an additional 28 days. When that dose failed to produce

47

seroconversion, the horses were given 1,000 sporocysts/day for 56 days, after

seroconversion had been detected in some horses at day 14 of this dose.

 

2.2.5 Biosecurity
Efforts were made to prevent the spread of sporocysts from experimental horses
to the on-farm controls and other horses at the Animal Resources Unit. Foot baths were

placed outside the experimental pens, and persons exiting the pens used them to wash

 

o

n
‘-
I.
.t
S
1

contaminants from their shoes or boots. When experimental horses were fed, their hay
was thrown into the rack from the adjacent pen, thus preventing daily truck traffic in the
pens. Also, the experimental pens were the last ones cleaned on the farm, and the
equipment was immediately hosed afterwards (The pen layout and cleaning protocol is

shown in Appendix II).

2.2.5 Pyrantel tartrate treatment and placebo:

Upon arrival at the study site, the horses were randomized into treatment groups
A and B. Group A received Strongid® C 2x (Pfizer, Inc) and group B received a look-
alike placebo pellet provided by Pfizer, Inc. The two treatments were put into buckets
marked A or B prior to shipment to WSU, and all persons associated with the daily
conduct of the study were blinded to the identity of the treatments.

Treatments were administered to the horses daily, starting 4 days prior to the start
of sporocyst dosing and continued until the last day of the study. When both sporocysts
and treatments were given, the sporocysts were administered first, immediately followed

by the treatment. Doses of treatments were determined according to the weight of each

48

horse, with each horse receiving 1 oz of pellets per 250 lbs of body weight. Horses were
weighed monthly and doses were adjusted as they grew. Doses were measured using the
cup provided with the Strongid® C 2x. Pellets were delivered orally in a 60cc syringe
with the end cutoff, then the horse was observed while it chewed and swallowed the
pellets. More pellets were given if a horse dropped any. If a horse was reluctant to eat
the pellets, a small amount of corn syrup was squeezed into the syringe to improve

palatability. The complete protocol used for horse dosing is presented in Appendix III.

2.2.6 Physical assessment:

Horses were examined for neurological deficits, conformational defects and any
gait abnormalities on day 0 of the study. These examinations were performed loose, as
the horses were not yet trained to lead. Subsequent examinations took place in hand 2
weeks later, then on a weekly basis thereafter until the end of the study. These physical
examinations were made by investigators who were blind to identity of the treatments the
horses were receiving and results were recorded on a neurological evaluation form
(Appendix IV). Briefly, the horses were assessed for cranial nerve deficits, abnormal
stances and their ability to circle, back and trot in both directions. Horses were also

observed daily for general health when treatments were being administered.

2. 2. 7 Diagnostic tests:
Serum and cerebrospinal fluid (CSF) were tested for antibodies against S. neurona
using the western blot test offered by the Diagnostic Center for Population and Animal

Health at Michigan State University (MSU). The test was performed using previously

49

wild ‘- ' irei“.
I

described methods (Rossano et al. 2000) and antibody reactivity to both the 30- and 16-
kDa antigens was the criterion for a positive test result. Serum and CSF samples were
stored in a refrigerator and tested as soon as possible upon aniving at the laboratory.
They were not frozen for long-term storage until after a final western blot result had been

obtained, approximately one month in most cases.

2.2.8 Specimen collection:

Blood samples were collected from experimental and control horses by jugular
venipuncture on day 0 and weekly thereafter until the conclusion of the study. The serum
fraction was removed from the blood clot, stored in a refiigerator, and shipped with cold
packs overnight to MSU for testing.

CSF was first collected from the 24 experimental horses on days 11 and 12 of the
study, then approximately monthly thereafter. There were 5 collections in total. The
CSF collections from the horses were performed over 2 days (12 horses per day), starting
with group A the first month, group B the second month and alternating groups
thereafter. CSF was collected from the atlanto-occipital site while the horses were under
general anesthesia. Horses were kept in stalls for observation for several hours following
the procedure, then returned to their group pens at the end of the day. The complete
protocol for CSF collection is presented in Appendix V.

Samples of CSF in EDTA were immediately submitted to the Clinical Pathology
Laboratory at WSU for cytological evaluation. Red blood cells and nucleated cells were .

counted and any cytological abnormalities were reported. In order to assure that positive

50

'w-“w

T"

CSF antibody test results were not false-positives due to blood contamination, samples
exceeding 50 RBC/ul were excluded from the analysis (Furr et al., 2002).

Feces were collected fi'om each horse at the time of CSF collection. Three-gram
samples of feces from each sample were tested by sucrose flotation for the presence of
unexcysted sporocysts.

Four horses were euthanized, using pentobarbitone sodium (Beuthanasia—D,
Schering Animal Health) to effect, at the end of the study and necropsies were performed.
Three showed no neurological abnormalities, but were selected because they were
deemed unadoptable due to conformational defects. One of the 4 horses was selected
because it developed neurological disease that coincided with the development of serum
antibodies to S. neurona. Neurological tissues (brain, brain stem and cervical, thoracic
and lumbar sections of spinal cord) were collected and shipped overnight to MSU to
attempt to culture S. neurona using a previously published technique (Mansfield et al.,
2001). Samples of the same tissues also were fixed in formalin and examined for
histopathologic evidence of EPM. Additionally, fresh and fonnalin-fixed samples of
tongue, diaphragm, upper thigh muscle, masseter muscle, heart, liver, small intestine and
lung were collected and shipped to MSU for histopathologic examination. The complete

protocol for the collection of tissues is presented in Appendix V].

2.2.9 Statistical analysis of results:
Test results for each horse were recorded individually in a spreadsheet. The
numbers of animals testing positive and negative by the Western blot test at the end of the

study were compared by treatment group for serum and CSF samples in 2 x 2 tables using

51

-'-.—'-
" Ii. .
I

the Fisher’s exact test, two-tailed (SAS). The Fisher’s exact test was used because it is an
appropriate test for discrete data, and low expected values in some cells made the chi-
square test an inappropriate choice.

The days to seroconversion for serum results also were compared by treatment
group and gender, using the Cox Proportional Hazards model (EGRET). Due to the
small number of observations, treatment and gender were examined separately in
univariable models. The Cox Proportional Hazards model was selected for this analysis
because there were horses in the study that did not seroconvert, so in order to preserve
their contribution to the results they were classified as “right censored” observations in
the models.

In all analyses, 3 p-value of 5 0.05 was deemed significant.

2.3 RESULTS:

Seroconversions were initially detected in two horses (one from group A and one
from group B) on day 77 (day 21 of receiving the 1,000 sporocysts/day dose). At the
conclusion of the study, there were 10/12 horses in each group that were seropositive.
There was no statistically significant difference between groups in the number of horses
that were seropositive (P 5 1.00). There was also no significant difference in days to
seroconversion between treatment groups (P _<_ 0.71) or by gender (P 5 0.21). All on-
farm control horses remained seronegative throughout the study. One off-farm control
horse developed a positive serum western blot test on day 35 of the study, and continued

to test positive thereafter. The other 5 off-fann control horses remained seronegative

52

Pm” .. mini-«In

throughout the study. A summary of the course of seroconversions by treatment group
can be viewed in Table 2.1.

The first positive CSF western blot antibody test was detected at the 4th sampling
date (104-105 days since the beginning of the study). At the 5th and final sampling date
(137-140 days since the beginning of the study) 4/12 horses in group A tested positive for
CSF antibodies and 3/ 12 horses in group B tested positive. There was no statistically
significant difference between groups (P 5 1.00).

At the last CSF collection, which was used for the final analysis, red blood cell
counts ranged from 0 to 29 (mean = 2.58, median = 0) for group A and from 0 to 2 (mean
= 0.25, median = 0) for group B. All CSF samples were below 50 RBC/ul and were,
therefore, included in the data analysis. Nucleated cell counts ranged ham 0 to 5 for
group A (mean = 1.83, median = 1.5) and from 0 to 5 for group B (mean = 1.58, median
= 1). No cytological abnormalities were seen. A complete summary of blood
contamination from all CSF collections during the study is shown in Table 2.2.

No sporocysts were found in fecal samples collected from experimental horses
during the course of the study.

Neurological examinations revealed one colt to be a probable subtle wobbler on
day 49 of the study, 42 days before seroconversion. Its condition did not change
appreciably thereafter. One filly (#27) developed mild asymmetric neurological signs
that coincided with its seroconversion. It was selected for necropsy. No other horses in
the study exhibited neurological signs during the study.

Postmortem examination of the 4 horses chosen for necropsy did not reveal any

gross CNS lesions. S. neurona was not cultured from brain, brain stem or spinal cord

53

from any of the horses. One myofiber in the section of upper thigh muscle from horse
#27 contained one protozoal cyst suggestive of Sarcocystis species, but further
investigation of adjacent tissue did not reveal any additional cysts. Lack of material
precluded additional work to identify the species of the cyst. No CNS lesions were found
in horse #27. In a section of tongue fi'om horse #154, a small, discrete aggregate of
lymphocytes, macrophages and plasma cells was found in deep muscle but no protozoal
organisms were identified. A longitudinal section of cervical spinal cord from horse
#154 showed one thin, linear area of Wallerian degeneration. No significant histological
findings were seen in a cross-section of cervical spinal cord taken fi'om the same area,
and no lesions were seen in any other tissue from that horse. No histopathologic
abnormalities were found in the tissues of the remaining 2 horses that were necropsied
(#33 and #42). All of the horses that were examined were seropositive, however, only
horse #154 tested positive for CSF antibodies at the final collection. A summary of post-

mortem findings and western blot test results are presented in Table 2.3.

2.4 DISCUSSION:

The results of this study clearly demonstrate that when given according to the
label dose for Strongid C 2X, daily pyrantel tartrate did not prevent or delay infection of
' the experimental horses with S. neurona, as reflected by the production of antibodies in
the serum. In addition, there was no significant difference between treatment groups
when the proportion of CSF-positive horses were compared, suggesting that pyrantel
tartrate did not have an effect on the ability of the parasite to gain access to the central

nervous system. Horse gender was also not a significant factor in time to seroconversion,

although gender was a stronger association than treatment group, with males being the
group that seroconverted sooner.

The fact that all of the on-farm control horses and 5/6 of the off-farm control
horses remained seronegative suggests that the experimental horses seroconverted as a
result of the sporocyst doses they were given not from sporocysts in feed from central
and eastern Washington. The off-farm control horse that tested positive, did so 42 days
before any experimental horse did, during which time they received daily doses of S.
neurona. The horse, an aged Quarter Horse gelding, had lived in Colorado as recently as
1999, and had been donated to WSU in June of 2000 because he was narcoleptic. No
other history was available on the horse, such as other places it may have resided in its
lifetime. It is possible that the horse had an existing'latent or subclinical infection that
had been reactivated to the extent that he produced antibodies in response. In following
naturally infected horses in Michigan, our research group has observed horses convert
from seronegative to seropositive back to seronegative status (Chapter 3 of this
dissertation). Perhaps the off-farm control horse exhibited a similar pattern of antibody
waxing and waning.

The difficulty we experienced in producing seroconversion in these horses was
markedly different from the experience of other researchers who used the same strain of
S. neurona. In a study that utilized long distance transport as a stressor prior to
experimental infection of horses, seroconversion was achieved using doses as low as 100
sporocysts (Sofaly et al., 2002). In the present study, daily doses of 100 sporocysts/day
for 28 days and daily doses of 500 sporocysts/day for 28 days failed to induce

seroconversion in any of the 24 experimental horses. It was not until 2] days after

55

receiving 1,000 sporocysts/day that the first seroconversions were seen. The disparity in
results in these studies could be due to a number of factors. The immunosuppression
induced by long distance transport may have made the horses in the previous study
(Sofaly et al., 2002) more susceptible to infection, or the horses may have had minimal
gut fill when they were dosed with sporocysts upon arrival at the study site. In contrast,
the horses in the present study were given over 2 weeks to recover flom transport and
acclimate to their new surroundings before beginning the study, and, thus, were probably
not irnmunosuppressed when sporocyst dosing began. They also were given flee choice
hay, and most likely had consistent gut fill during the study. This may have hindered the
ability of S. neurona sporozoites to reach and penetrate the intestinal epithelial cells of
the horses, due to physical barriers and the constant flow of ingesta. Another possible
explanation for the difference in results is that in the previous study (Sofaly et al., 2002),
horses were dosed with nasogastric tubes and the horses in the present study were dosed
with oral syringes. While the horses dosed by nasogastric tubes were more likely to
receive the full dose of sporocysts intended, it is unlikely that this difference would not
have been overcome by the fact that the horses in the present study were dosed on a daily
basis for a long period of time before seroconversion occurred. Finally, the previous
study (Sofaly et al., 2002) may have had greater success in infecting horses because their
sporocysts were flesher than those used in the present study, which had been stored for 1
year in Hank’s balanced salt solution. While it is not possible to make a direct
comparison between inoculums to determine this, the pathogenicity of the sporocysts

used in the present study was demonstrated by the fact that they produced neurological

56

disease in gamma-interferon knock-out mice at 1 tenth the standard infective mouse dose
of 1,000 sporocysts (Dubey et al., 2001d).

The fact that S. neurona was not cultured or identified in experimental horses at
postmortem examination was not surprising, considering that only 1 of the 4 horses
selected was found to have CSF antibodies to S. neurona at the time of euthanasia. Other
studies, in which larger doses of sporocysts were used, have failed to recover S. neurona
postmortem flom experimentally infected horses (F enger et al., 1997; Cutler et al., 2001;
Saville et al., 2001; Sofaly et al., 2002).

The proportion of seropositive horses in the present study that also tested positive
for CSF antibodies was 29% (7/24). In a recent study of 181 paired diagnostic samples
flom horses suspected of having EPM, 71% of the seropositive horses had CSF
antibodies to S. neurona (Rossano et al., 2003). This difference in proportions could be
explained by the fact that the diagnostic samples came flom horses more likely to have
active central nervous system infections with S. neurona, as opposed to the mostly
neurologically normal study population described here, and that in the present study, no
CSF sample exceeded the recommended limit of 50 RBC/u] (F urr et a], 2002), thus the
chance of false positive test results due to blood contamination was low. Information
regarding blood contamination of the clinical samples was not available, but it is likely
that some samples were contaminated, thus biasing the results (Rossano et al., 2003).
The success seen in the present study with CSF collection flom the atlanto-occipital site
demonstrates the advantage of using this method when general anesthesia is deemed safe.
Only 4/ 120 (3.3%) of CSF samples collected this way were unusable due to blood

contamination.

57

The response of the immunocompetent experimental horses to low, daily dosing
of sporocysts suggests that the high antibody seroprevalence seen in US. horses may be
the result of a different exposure scenario. Perhaps horses receive larger doses of
sporocysts under natural conditions by consmning opossum feces directly, rather than via
low-level contamination of feed or water. Perhaps horses are made vulnerable by certain
circumstances, such as an irnmunosuppressive event or fasting to the point of having an
empty gut when sporocysts are ingested. Further studies will be required to determine

the most likely conditions under which horses are naturally infected with S. neurona.

2.5 CONCLUSIONS:
Daily administration of pyrantel tartrate does not prevent or delay infection of
horses with S. neurona, and male horses did not seroconvert significantly faster than

females when given low daily doses of sporocysts.

58

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59

Table 2.2. Summary of red and white blood cell counts flom all CSF collections.

 

 

138 2nd 3rd 4tF 5!
Variable Collection Collection Collection Collection collection
RBC/ul
range 0-4 0-1512 0-6750 0-20 0-29
RBC/ul
median 0 0 0-1 0 0
WBC/ul
range 0-6 0-4 0-14 0-5 0-5
WBC/ul
median 1 0 1 1 1
# Positive
WB tests 0/24 0/24 0/24 1/24 7/24
# > 50 ,
RBC/ul 0 3 1 0 0

 

60

Table 2.3. Postmortem examination results for the four horses euthanized at the end of
the study. Only horse #27 exhibited neurological signs, grade 2.0-2.5.

 

 

27

33
42

Final western blot F inal western blot

Horse ID number Pathological findings antibody test — serum antibody test - CSF

One sarcocyst of Positive Negative

undefined species in

thigh muscle

None Positive Negative

None Positive Negative

One thin area of Positive Positive

Wallerian

154

degeneration in
cervical spinal cord

61

Figure 2.1 Washington Gap Analysis Project's Predicted Distribution Map published by
the University of Washington depicting the opossum distribution in the state. Opossums
were not reported to be in the southeast portion of the state, where the study was
conducted.

Virginia Opossum (Didelphis virginiana)

 

 

Legend:

* = Study site
. = Core Habitat

. Breeding Range Map
The green area shows the predicted habitats for breeding only. The habitats were
identified using 1991 satellite imagery, other datasets and experts throughout the
state, as part of the Washington Gap Analysis Project.

62

REFERENCES:

Blythe L, Granstrom DE, Hansen DE, Walker LL, Bartlett J, Stamper S, 1997.
Seroprevalence of antibodies to Sarcocystis neurona in horses residing in Oregon. I Am
Vet Med Assoc 210:525-527.

Cheadle MA, Tanhauser SM, Dame JB, Sellon DC, Hines MT, Ginn PE, MacKay RJ,
Greiner EC. 2001a. The nine-banded armadillo (Dasypus novemcinctus) is an
intermediate host for Sarcocystis neurona. Int J Parasitol 31(4):330-5.

Cheadle MA, Yowell CA, Sellon DC, Hines MT, Ginn PE, Marsh AB, MacKay R.J,
Dame JB, Greiner EC. 2001b. The striped skunk (Mephitis mephitis) is an intermediate
host for Sarcocystis neurona. Int J Parasitol 31(8):843-9.

Cutler TJ, MacKay RJ, Ginn PE, Gillis K, Tanhauser SM, LeRay EV, Dame JB, Greiner
EC. 2001. Immunoconversion against Sarcocystis neurona in normal and
dexamethasone-treated horses challenged with S. neurona sporocysts. Vet Parasitol
95(2-4):197-210.

Dubey JP, Davis SW, Speer CA, Bowman DD, de Lahunta A, Granstrom DE, Topper
MJ, Hamir AN, Cummings JF, Suter MM. 1991. Sarcocystis neurona n. sp. (Protozoa:
Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis. J Parasitol
77:212-217.

Dubey JP, Lindsay DS, Saville WJA, Reed SM, Granstrom DE, Speer CA. 2001a A
review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet
Parasitol 95(2-4):89-13 l .

Dubey JP, Rosypal AC, Rosenthal BM, Thomas NJ, Lindsay DS, Stanek JF, Reed SM,
Saville WJA. 2001b. Sarcocystis neurona infections in sea otter (Enhydra lutris):
evidence for natural infections with sarcocysts and transmission of infection to opossums
(Didelphis virginiana). J Parasitol 87(6):]387-93.

Dubey JP, Saville WJ A, Stanek JF, Lindsay DS, Rosenthal BM, Oglesbee MJ, Rosypal
AC, Njoku CJ, Stich RW, Kwok OC, Sheri SK, Hamir AN, Reed SM. 20010.

Sarcocystis neurona infections in raccoons (Procyon lotor): evidence for natural infection
with sarcocysts, transmission of infection to opossums (Didelphis virginiana), and
experimental induction of neurologic disease in raccoons. Vet Parasitol 100(3-4):117-29.

Dubey JP, Lindsay DS, Kwok OC, Shen SK. 2001d. The gamma interferon knockout

mouse model for Sarcocystis neurona: comparison of infectivity of sporocysts and
merozoites and routes of inoculation. J Parasitol 87(5):]171-3.

63

Dubey JP, Saville WJA, Lindsay DS, Stich RW, Stanek JF, Speer CA, Rosenthal BM,
Njoku CJ, Kwok OC, Shen SK, Reed SM. 2000. Completion of the life cycle of
Sarcocystis neurona. J Parasit0186(6):1276-80.

Elsheikha H.M., Murphy A.J., Fitzgerald S.D., Mansfield L.S., Massey J .P. and Saeed
MA. 2003. Purification of Sarcocystis neurona sporocysts flom opossum (Didelphis

virginiana) using potassium bromide discontinuous density gradient centrifugation.
Parasitol. Res. 90: 104-109.

Fenger CK, Granstrom DE, Gajadhar AA, Williams NM, McCrillis SA, Stamper S,
Langemeier JL, Dubey JP, 1997. Experimental induction of equine protozoal
myeloencephlitis in horses using Sarcocystis sp. sporocysts flom the opossum (Didelphis
virginiana). Vet Parasitol 68:199-213.

Furr M, MacKay R, Granstrom D, Schott H 2nd, Andrews F. 2002. Clinical diagnosis of
equine protozoal myeloencephalitis (EPM). J Vet Intern Med. Sep-Oct;l6(5):6l 8-21.

Kruttlin EA, Rossano MG, Murphy AJ, Vrable RA, Kaneene JB, Schott 1] HC, Mansfield
LS. 2001. The effects of pyrantel tartrate on Sarcocystis neurona merozoite viability.
Vet Therapeutics Vol 2, No 3.

Kruttlin EA, Rossano MG, Murphy AJ, Vrable RA, Kaneene JB, Schott H HC, Mansfield
LS. 2003. Activity of pyrantel tartrate on percoll-treated Sarcocystis neurona
sporozoites. 48°‘Annual Meeting of the American Association of Veterinary
Parasitologists, July 19-22, 2003, Denver, Colorado.

Lindsay DS, Dubey JP. 2001. Determination of the activity of pyrantel tartrate against
Sarcocystis neurona in gamma-interferon gene knockout mice. Vet Parasitol 97(2): 141-
4.

MacKay RJ. 1997. Equine protozoal myeloencephalitis. Vet Clin North Am Equine
Pract l3(1):79-96.

Mansfield LS, Schott II HC, Murphy AJ, Rossano MG, Tanhauser SM, Patterson J S,
Nelson K, Ewart SL, Marteniuk JV, Bowman DD, Kaneene JB. 2001. Comparison of
Sarcocystis neurona isolates derived flom horse neural tissue. Vet Parasitol 95 167-178.

Mullaney TP, Kiupal M, Murphy AJ, Bell J, Mansfield LS. 2003. Evidence that horses
are natural intermediate hosts for Sarcocystis neurona. American Association of
Veterinary Parasitologists 48th Annual Meeting. July 19-22, 2003, Denver, CO.

Murphy AJ and Mansfield LS. 1999. Simplified technique for isolation, excystation, and
culture of Sarcocystis species flom opossums. J Parasitol 85(5):979-81.

64

NAHMS. 2001. Equine Protozoa] Myeloencephalitis (EPM) in the US.
USDA:APHIS:VS, CEAH, National Animal Health Monitoring System. Fort Collins,
CO. #N312.0501.

Rossano MG, Mansfield LS, Kaneene JB, Murphy AJ, Brown CM, Schott HC II, Fox J C.
2000. Improvement of western blot test specificity for detecting equine serum antibodies
to Sarcocystis neurona. J Vet Diagn Invest 12(1):28-32.

Rossano MG, Kaneene JB, Marteniuk JV, Banks BD, Schott II HC, Mansfield LS. 2001.
The seroprevalence of antibodies to Sarcocystis neurona in Michigan equids. Prev Vet
Med 48:113-128.

Rossano MG, Kaneene JB, Schott II HC, Sheline KD, Mansfield LS. 2003. Assessing
the agreement of Western blot test results for paired serum and cerebrospinal fluid

samples flom horses tested for antibodies to Sarcocystis neurona. Vet Parasitol
115(3):233-8.

Saville WJA, Stich RW, Reed SM, Njoku CJ, Oglesbee MJ, Wunschmann A, Grover DL,
Larew—Naugle AL, Stanek JF, Granstrom DE, Dubey JP. 2001. Utilization of stress in

the development of an equine model for equine protozoal myeloencephalitis. Vet
Parasitol 95(2-4):21 1-22.

Sofaly CD, Reed SM., Gordon J C, Dubey JP, Ogleebee MJ, Njoku CJ, Grover DL,
Saville WJ A. 2002. Experimental induction of equine protozoan myeloencephalitis
(EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the
development of clinical neurologic disease. J Parasitol 88(6):]164-70.

Tanhauser SM. Yowell CA, Cutler TJ, Greiner EC, MacKay RJ, Dame JB. 1999.
Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystisfalcatula.
J Parasitol 85(2):221-8.

Tillotson K, McCue PM, Granstrom DE, Dargatz DA, Smith MO, Traub-Dargatz J L.

1999. Seroprevalence of antibodies to Sarcocystis neurona in horses residing in northern
Colorado. J Equine Vet Science 19(2):]22-126.

65

 

CHAPTER 3

DOES DAILY ADMIN STRATION OF PYRANTEL TARTRATE PREVENT S.
NEURONA INFECTION IN HORSES? A CONTROLLED FIELD
INTERVENTION STUDY.

3.0 ABSTRACT:

Equine protozoal myeloencephalitis (EPM) is primarily caused by the protozoan
parasite Sarcocystis neurona. In vitro studies have shown that pyrantel tartrate is lethal
to S. neurona merozoites and sporozoites. We hypothesized that daily administration of
pyrantel tartrate prevents S. neurona infection in horses, as determined by the presence of
antibodies in the serum. This hypothesis was tested by conducting a double-blind
controlled field intervention study on three Michigan horse farms. Thirty-four yearling
horses were identified as either seronegative or suspect positive by the western blot
antibody test for S. neurona. These horses were randomized into two groups: group A
received pyrantel tartrate (Strongid C 2x) at the label dose and group B received a
placebo pellet. Pellets were fed daily, mixed with grain by farm staff according to label
instructions for Strongid C 2x. After receiving treatments for approximately 2 weeks, the
horses were tested again for serum antibodies to finalize entry into the study. After this
final screening test, 11 horses on 2 farms were available for follow-up. One of these
horses was subsequently sold. Horses who completed the study were tested for serum
antibodies approximately bi-monthly. Test results for each treatment group were
summarized in a 2 x 2 table and tested for independence by the two-tailed Fisher’s exact

test.

66

Six months later, at the conclusion of the study, 1/3 horses receiving daily pyrantel
tartrate (group A) was seronegative and 2/4 horses receiving the placebo (group B) were
seronegative. No statistically significant difference was found between groups A and B
for serum antibody tests (P < 1.00). We conclude that daily administration of pyrantel
tartrate does not prevent infection with S. neurona in horses when given according to

label instructions under field conditions.

67

3.1 INTRODUCTION:

Equine protozoal myeloencephalitis (EPM) is a potentially serious neurological
disease affecting horses and ponies of the Americas. Clinical signs of EPM may include
weakness, incoordination, loss of proprioception, behavioral changes, muscle wasting,
recumbancy and/or death (Dubey et al., 2001a; MacKay, 1997).

The primary causative agent of EPM is the protozoan parasite, Sarcocystis
neurona (Dubey et al. 1991). S. neurona requires a definitive and intermediate host to
complete its life cycle. Opossums serve as the only known definitive host of S. neurona
(Dubey et al., 2001a). Intermediate hosts of S. neurona identified to date include the
domestic cat (Dubey et al., 2000), the nine-banded armadillo (Cheadle et al., 2001a), the
striped skunk (Cheadle et al., 2001b), the raccoon (Dubey et al., 2001c) and the sea otter
(Dubey et al., 2001b). Horses and ponies are infected when they ingest sporocysts of S.
neurona that are present in feed or water that have been contaminated with opossum
feces (Dubey et al., 2001a). To date, no published accounts of mature sarcocysts cf S.
neurona detected in equids exist, therefore equids are generally considered to be dead end
hosts (Dubey et al., 2001a). Recently, however, there was a report at the 2003 annual
meeting of the American Association of Veterinary Parasitologists of a young colt with
neurological disease in which both mature schizonts in the brain and spinal cord and
mature sarcocysts in the tongue were found (Mullany et al., 2003). Electron microscopy
revealed that the sarcocysts had features identical to published features of S. neurona.
Testing by PCR with RFLP showed that the sarcocysts produced Sarcocystis specific
PCR products and these products exhibited banding patterns characteristic of S. neurona.

Further studies will be required to determine whether horses are true intermediate hosts to

68

S .neurona or if this colt was uniquely susceptible due to his young age or some form of
immunosuppression.

Once S. neurona invades the central nervous system (CNS) of an equid,
neurological disease may develop. The US. Department of Agriculture estimated the
" annual incidence of EPM cases in 1998 to be 0.014%, or 14 cases per 1000 horses
(N .A.H.M.S.). Cross-sectional studies conducted in various geographic areas of the
United States reported that in regions of western Oregon and southern Michigan,
seroprevalence was as high as 63-65% (Blythe, et al., 1997; Rossano et al., 2001) and as
low as 22-34% in regions of eastern Oregon and northern Colorado (Blythe et al., 1997;
Tillstone et al., 1999). Collectively, these data suggest that many horses are infected with
S. neurona at some time in their lives, but few of them go on to develop EPM. Despite
the low incidence of EPM, there is great interest among members of the horse industry to
find effective preventive strategies. This great interest for effective preventive strategies
‘ can be attributed to the high cost of treating the disease, and the poor recovery seen in
some treated horses. In 2001, a whole-cell killed vaccine of S. neurona merozoites was
granted a conditional license for the prevention of EPM (Fort Dodge Animal Health).
The efficacy of the vaccine in horses has yet to be demonstrated. One disadvantage of
. using the vaccine is that vaccinated animals will test positive for antibodies to S. neurona,
thus confusing the diagnosis of vaccinated horses presenting neurological signs.

The drug pyrantel tartrate has shown promise as a preventive agent in an in vitro
study against the merozoite stage of S. neurona (Kruttlin et al., 2001). In that study, the
drug was lethal to the parasite at concentrations greater than 0.0025M (8.91X104 g/ml).

In another in vitro study, not yet published but presented at the 2003 annual meeting of

69

the American Association of Veterinary Parasitologists, pyrantel tartrate was tested on
the sporozoite state of the parasite (which is the stage of S. neurona that infects horses
intestines and would be most likely to be exposed to the drug). There, the drug was
found to be effective at concentrations of 0.005M or greater. Based on these encouraging
results, we hypothesized daily administration of pyrantel tartrate (Strongid C 2x®) could
prevent infection by S. neurona in horses when used by horse farm operators according to
label instructions. The purpose of this study was to conduct a field intervention trial to

test the hypothesis.

3.2 MATERIALS AND METHODS:

3.2.1 Study design:

The study was a double-blind field intervention trial. Persons administering the
treatments and the person analyzing the data were “blinded” to the identity of the
treatment groups. When preliminary results were given to the primary investigators who
knew the treatment identities, the treatments were described by yet another set of names

in order to prevent bias in the interpretation of results by the principal investigators.

3.2.2 Study population:
The study focused on yearling horses in order to have the greatest chance to enrol]
a population of seronegative animals. Horses could not have been vaccinated for EPM to

be enrolled in the study or while participating in the study.

70

3.2.3 Sample size calculations:

A seroprevalence study conducted in Michigan revealed that 48% of horses in that
state tested positive for antibodies to S. neurona by the time they were yearlings
(Rossano et al., 2001). Therefore, it was expected that at least 50% of the horses
receiving the placebo would be seropositive by the end of the observation period.
Assuming 95% significance (p<0.05) and 80% power, two-tailed hypothesis testing, and
50% rate of seroconversion among horses on the placebo, it was determined that there
should be at least 55 foals in each treatment group to detect a risk ratio of at least 1.5 for
horses on the placebo (Thrusfield, 1995). We proposed to enroll 60 foals in each cohort,
to accommodate for potential losses to follow-up, thus we attempted to total of 120
horses in the study. Due to a lack of available farms with large numbers of young horses,
it was decided that the study would take place in two rounds, on successive foal crops

over the course of 2 years.

3.2.4 Enrollment of horses:

A press release was issued to Michigan equine-related publications to recruit large
breeding farms into the study (Appendix _). Although participation in the study would
provide flee deworming products and blood test results to participants, the response to the
solicitation was lower than expected. Three equine breeding farms were enrolled in the
study. They were the Michigan State University Horse Teaching and Research Center
(MSU HTRC) in East Lansing, Michigan, which bred Arabian horses; the MSU Merillat
Equine Center in Adrian, Michigan, which bred American Quarter Horses; and a smaller

private Arabian farm in Davison, Michigan. In fall, weanling horses were screened for

71

enrolhnent using the western blot antibody to S. neurona test on serum collected by
jugular venipuncture (Rossano et al., 2000). The criterion for a positive test was
simultaneous reactivity to the 30- and 16-kDa S. neurona antigens. Horses that had only
produced antibodies to the 30-kDa antigen were classified as “suspects” who might be in
the process of seroconverting. Horses that had no reactivity to either antigen were
classified as negative. Horses that tested positive were excluded flom participation in the
study; horses that tested suspect or negative were enrolled in the study.

Managers of participating farms filled out questionnaires detailing the feeding,
housing and preventive health management of each horse in the study. Initial
questionnaires (Appendix VIII) were administered at the start of the study and follow-up

questionnaires (Appendix IX) were administered at every blood collection date thereafter.

2. 2.5 Allocation into treatment groups:

For each farm, horses in the negative and suspect test groups were given ID
numbers and a computer was used to generate random numbers to allocate them into
treatment groups. In order to minimize between- group variation, negative testers and
suspect testers were randomized separately, so that an equal number flom each group
would be divided between treatments. Horses in group A would received the active drug,
pyrantel tartrate in the form of Strongid C 2x®, and horses in group B received a look-
alike placebo pellet. To insure that horses receiving the placebo would not suffer ill
effects flom parasites, all horses in the study received ivermectin at the start of the study

and every 2 months after.

72

3. 2. 6 Analysis of data:

For purposes of analysis, only horses that tested fully positive on the western blot
test were classified as positives. Horses testing suspect or negative were classified as
negatives. Results for the 3 herds were combined in a 2 x 2 table and the independence
of results by treatment group was tested by the Fisher’s exact test (2-tailed). A p-value of
5 0.05 was deemed to be significant.

If there had been a sufficient number of observations for a multivariable logistic
regression model, one would have been developed to evaluate potential risk factors
identified in the horse management data, and the analysis would have been conducted at
the individual animal level. Because horses in the study were in herds, and therefore
would not represent independent observations, the model would have included a random

effects term grouped by herd.

3.2.7 Study timeline:

Weanling horses were screened for enrollment flom October to December of
2001. The study protocol required starting the horses on their treatments 2 weeks prior to
being retested to assure their serological status upon starting the study. This was done to
avoid misclassification error due to seroconversions that occurred due to infections with
S. neurona that preceded the treatments. Upon testing negative or remaining suspect at
the final screening, horses were retested on a bi-monthly basis until they tested positive.
Because of a lack of pasture space and staff, the two largest farms, (the MSU HTRC and

MSU Merillat) were not able to start feeding treatments until February and March, 2002,

73

respectively. In order to keep the farms on approximately the same tirneline, the small
private Arabian farm started the study in February.

Weights were estimated using a weight tape, and doses were determined
according to label instructions for Strongid C 2x®. While enrolled in the study, the
horses received their treatments daily. On the two largest farms the horses were group-
housed by treatment group and fed their treatments mixed in the grain according to label
instructions for Strongid C 2x®. There, the doses were based on mean weights per group
and the grain with pellets were provided in group feeders. On the smaller farm, horses
were group housed during the day, but individually fed their treatments in stalls in the
evening. The study was not continued for a second year, and the final blood collection

took place in August 2002.

3.3 RESULTS:
3.3.1 Enrollment of horses:

Thirty-four horses on the 3 farms were eligible for the study after testing negative
or suspect for antibodies to S. neurona on the screening test. Unfortunately,
seroconversions that occurred during the period between screening and sampling 2 weeks
after starting the treatments resulted in the loss of many horses. This was an unexpected
complication for the time of the year (late winter), when opossums are less active. All of
the horses (8/8) in Group A and 3 of 7 horses in Group B at the MSU HTRC tested
positive for antibodies to S. neurona after 2 weeks of treatment. Because there were no
horses in Group A to continue the study, the study was stopped there in April, 2002. The

first blood collection following 2 weeks of treatment at MSU Merillat showed that 5 of 7

74

horses in Group A bad seroconverted, and 3 of 8 horses in Group B had seroconverted,
reducing the sample size at MSU Merillat to 7 horses, one of which was subsequently lost
to the study when it was sold. At smaller Arabian farm, 2 of the 6 horses on the trial
refused to eat their treatment (1 was in Group A, l was in Group B), however the
remaining 4 horses did test negative for enrollment in the study. Thus, a total of only 10

horses were available for follow-up.

3.3.2 Statistical analysis:

By August, 2002, only 3 of the 10 horses enrolled had remained seronegative for
the duration of the study, 1 in the pyrantel tartrate group and 2 in the placebo group
(Table 3.1). No significant difference was found between groups (P 5 1.0). The 4 horses
at the small Arabian were all tested on the final day of the study (as opposed to only the 1
remaining negative horse) and over the course of the final 2 months, 2 of 3 seropositive
horses (1 flom each treatment group) had reverted to seronegative status. Because those
horses had already been formally dropped flom the study, the changes in status were not
included in the final summary table.

Sample size was not sufficient for a multivariable logistic mode], so this analysis

was not performed.

3.4 DISCUSSION:
The results of this study did not support they hypothesis that daily administration
of pyrantel tartrate prevents infection of horses with S. neurona, and are inconclusive.

The sample size of 10 horses did not provide adequate statistical power to detect a

75

difference between treatment groups, and this fact should be taken into consideration
when interpreting the results.

The lack of ability to obtain timely cooperation flom the farm managers when
separating horses into treatment groups and scheduling blood collections was a major
impediment to the success of the study. This was largely due to a shortage of personnel
with time to assist the investigators with catching and identifying horses in pastures.
Better results could probably have been obtained if the majority of the horses in the study
had been stalled at some point during the day, rather than pastured continuously. That
would have allowed for easier identification of the horses by investigators, who could
have proceeded to collect blood without assistance flom the farm employees. This is not
a typical way of housing growing horses on large breeding farms in Michigan, however,
so finding herds to fit that study criteria would most likely have been difficult.

It was interesting to see that 2 horses reverted to seronegative status after testing
positive 2 months before. If time and funding had allowed, follow-up testing of other
seropositive horses in the study might have revealed a pattern of waxing and waning of
antibody titers that could have reflected either successful elimination of the parasite
followed by re-infection, or the suppression and re-emergence of the parasite due to the
existence of a latent or dormant stage in the horse.

The difficulties experienced in this study demonstrate some of the challenges that
can be encountered when conducting a controlled field intervention involving horse
farms. Future investigators should be aware of how busy horse farm managers are, and
try to plan studies that require minimal involvement flom them, or provide sufficient

compensation to provide a motivation for better compliance with the study protocol.

76

Table 3.1. Summary of results of antibody tests for horses by the end of the
study. Totals for positive horses are for blood samples drawn on the day they were
dropped flom the study, totals for negative horses are for the last samples drawn.

 

 

Positive Test Negative Test Totals
Treatment Group
A (Strongid C°) 3 1 4
B (placebo) 4 2 6
Totals 7 3 10

 

77

REFERENCES:

Blythe L, Granstrom DE, Hansen DE, Walker LL, Bartlett J, Stamper S, 1997.
Seroprevalence of antibodies to Sarcocystis neurona in horses residing in Oregon. J Am
Vet Med Assoc 210:525-527.

Cheadle MA, Tanhauser SM, Dame JB, Sellon DC, Hines MT, Ginn PE, MacKay RJ,
Greiner EC. 2001a. The nine-banded armadillo (Dasypus novemcinctus) is an
intermediate host for Sarcocystis neurona. Int J Parasitol. 31(4):330-5.

Cheadle M.A., Yowell CA, Sellon D.C., Hines M.T., Ginn P.E., Marsh A.E., MacKay
R.J., Dame J .B., Greiner E.C. 2001b. The striped skunk (Mephitis mephitis) is an
intermediate host for Sarcocystis neurona. Int. J. Parasitol. 31(8):843-9.

Dubey JP, Davis SW, Speer CA, Bowman DD, de Lahunta A, Granstrom DE, Topper
MJ, Hamir AN, Cummings JF, Suter MM, 1991. Sarcocystis neurona n. sp. (Protozoa:
Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis. J Parasitol
77:212-217.

Dubey JP, Lindsay DS, Saville WJA, Reed SM, Granstrom DE, Speer CA. 2001a. A
review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet
Parasitol 95(2-4):89-13 1 .

Dubey JP, Rosypal AC, Rosenthal BM, Thomas NJ, Lindsay DS, Stanek JF, Reed SM,
Saville WJA. 2001b. Sarcocystis neurona infections in sea otter (Enhydra lutris):
evidence for natural infections with sarcocysts and transmission of infection to opossums
(Didelphis virginiana). J Parasitol 87(6):1387-93.

Dubey JP, Saville WJ A, Stanek JF, Lindsay DS, Rosenthal BM, Oglesbee MJ, Rosypal
AC, Njoku CJ, Stich RW, Kwok 0C, Shen SK, Hamir AN, Reed SM. 2001c.

Sarcocystis neurona infections in raccoons (Procyon lotor): evidence for natural infection
with sarcocysts, transmission of infection to opossums (Didelphis virginiana), and
experimental induction of neurologic disease in raccoons. Vet Parasitol 100(3-4):117-29.

Dubey JP, Saville WJA, Lindsay DS, Stich RW, Stanek JF, Speer CA, Rosenthal BM,
Njoku CJ, Kwok 0C, Shen SK, Reed SM. 2000. Completion of the life cycle of
Sarcocystis neurona. J Parasitol 86(6):1276-80.

Fenger CK, Granstrom DE, Gajadhar AA, Williams NM, McCrillis SA, Stamper S,
Langemeier J L, Dubey JP, 1997. Experimental induction of equine protozoal
myeloencephlitis in horses using Sarcocystis sp. sporocysts flom the opossum (Didelphis
virginiana). Vet Parasitol 68:199-213.

78

Kruttlin EA, Rossano MG, Murphy AJ, Vrable RA, Kaneene JB, Schott II HC, Mansfield
LS. 2001. The effects of pyrantel tartrate on Sarcocystis neurona merozoite viability.
Vet. Therapeutics V012, No 3.

Kruttlin EA, Rossano MG, Murphy AJ, Vrable RA, Kaneene JB, Schott II HC, Mansfield
LS. 2003. Activity of pyrantel tartrate on percoll-treated Sarcocystis neurona
sporozoites. 48"‘Annual Meeting of the American Association of Veterinary
Parasitologists, July 19-22, 2003, Denver, Colorado.

MacKay RJ. 1997. Equine protozoal myeloencephalitis. Vet Clin North Am Equine
Pract l3(1):79-96.

Mullaney TP, Kiupal M, Murphy AJ, Bell J, Mansfield LS. 2003. Evidence that horses
are natural intermediate hosts for Sarcocystis neurona. American Association of
Veterinary Parasitologists 48°1 Annual Meeting. July 19-22, 2003, Denver, CO.

NAHMS. 2001. Equine Protozoa] Myeloencephalitis (EPM) in the US.
USDA:APHIS:VS, CEAH, National Animal Health Monitoring System. Fort Collins,
CO. #N312.0501.

Rossano MG, Mansfield LS, Kaneene JB, Murphy AJ, Brown CM, Schott HC H, Fox J C.
2000. Improvement of western blot test specificity for detecting equine serum antibodies
to Sarcocystis neurona. J Vet Diagn Invest 12(1):28-32.

Rossano MG, Kaneene JB, Marteniuk JV, Banks BD, Schott II HC, Mansfield LS. 2001.
The seroprevalence of antibodies to Sarcocystis neurona in Michigan equids. Prev Vet
Med 48:113-128.

Thrusfield M. Veterinary epidemiology. 2nd edition. 1995. Blackwell Science, Oxford,
England, p. 235.

Tillotson K, McCue PM, Granstrom DE, Dargatz DA, Smith MO, Traub-Dargatz JL.

1999. Seroprevalence of antibodies to Sarcocystis neurona in horses residing in northern
Colorado. J Equine Vet Science 19(2):]22-126.

79

CHAPTER 4

PARASITEMIA IN AN IMMUNOCOMPETENT HORSE
EXPERIMENTALLY CHALLENGED WITH SARCOC YS T IS NE URONA

SPOROCYSTS

4.0 ABSTRACT:

Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of
horses in the Americas. Most cases are attributed to infection of the central nervous
system with Sarcocystis neurona. Parasitemia has been documented in one
immunocompromised foal, but has not been demonstrated in immunocompetent horses.
The objective of this study was to isolate S. neurona flom the blood of immunocompetent
horses.

Horses used in this study had received S. neurona sporocysts administered at the
following doses and time periods: 100/day for 28 days, followed by 500/day for 28 days,
followed by 1,000/day for 42 days. Six yearling colts were selected for attempted culture
flom blood, 2 testing positive, 2 testing suspect and 2 testing negative for antibodies to S.
neurona, 2 weeks prior to participating in this study. Two 10 ml tubes with EDTA
additive were filled by jugular venipuncture flom each horse and allowed to settle at
room temperature for approximately 2 hours. For each horse, 3-3 ‘/2 mls of plasma was
pipetted flom the tops of the tubes and combined in a 15 ml conical centrifuge tube. The
centrifuge tubes were subsequently spun for 10 minutes at 2,500 RPM and the plasma

was decanted flom the tubes and discarded. The pellet was resuspended in plain DMEM

80

and spun again for 10 minutes at 2,500 RPM. The wash solution was removed and the
pellet was resuspended in a medium of 10 ml DMEM, plus 10% fetal bovine serum,
HEPES, PenStrepFungizone, L-glutarnine and sodium pyruvate. The tubes were stored at
room temperature while they were being transported to our laboratory (approximately
three days) and were subsequently pipetted onto confluent equine dermal cell cultures
and incubated at 37°C with 5% C02. The medium was removed the next day and
replaced with DMEM with 6% fetal bovine serum with HEPES, PenStrepFungizone, L-
glutamine and sodium pyruvate. The cultures were monitored weekly for parasite growth
for 12 weeks. Any merozoites gown florn cultures were harvested and tested by PCR
with RFLP to confirm species identity, and PCR products were sequenced for
comparison to known strains of S. neurona.

At the conclusion of the study, a culture was isolated flom one horse, which was
confirmed to be S. neurona. Gene sequence analysis showed that the isolate was identical
to the strain with which the horse inoculated, and differed flom the known strains. To
our knowledge this is the first report of parasitemia with S. neurona in an

immunocompetent horse.

81

4.1 INTRODUCTION:

Equine protozoa] myeloencephalitis (EPM) is a serious neurological disease of
horses in the Americas. Most cases are attributed to infection of the CNS with
Sarcocystis neurona, although several cases have been due to infection with Neospora sp
(Dubey et al., 20013). The definitive host of S. neurona in the North America is the
Virginia opossum (Didelphis virginiana), and intermediate hosts of S. neurona identified
to date include the domestic cat (Dubey et al., 2000), the nine-banded armadillo (Cheadle
et 3]., 20013), the striped skunk (Cheadle et al., 2001b), the raccoon (Dubey et al., 2001c)
and the sea otter (Enhydra lutris) (Dubey et al., 2001b). Horses are believed to be
aberrant hosts to S. neurona, and despite the fact that a large proportion (SO-60%)of
horses that live in areas where there are opossums are seropositive for antibodies to S.
neurona (Dubey et al., 20013), the incidence of EPM appears to be low (0.15%
nationally) (NAHMS, 2001).

A number of researchers have attempted to experimentally infect horses with S.
neurona sporocysts, but none have succeeded in recovering the parasite flom tissues
examined at necropsy (Fenger et al., 1997; Cutler et al., 2001; Saville et al., 2001; Sofaly
et al., 2002). One exception has been the demonstration of parasitemia in an Arabian foal
with severe combined immunodeficiency (SCID) that was fed 500,000 sporocysts in a
single dose (Long et al., 2002). 801]) foals lack the ability to mount specific B and T cell
immune responses because of a genetic mutation (Shin et al., 1997). Sarcocystis neurona
was cultured flom blood drawn flom the foal 21 days post-inoculation (Long et al.,

2002).

82

We hypothesized that S. neurona could be cultured from the blood of
immunocompetent horses. The purpose of this study was demonstrate parasitemia in

immunocompetent horses receiving low daily doses of S. neurona sporocysts.

4.2 MATERIALS AND METHODS:

Horses used in the present study were part of a larger study that tested the ability
of daily administration of pyrantel tartrate to prevent infection with S. neurona. The
details of the study has been described elsewhere (Chapter 3 of this dissertation). Briefly,
the horses were given oral doses of S. neurona sporocysts (strain SN37-R, kindly
provided by J .P. Dubey) every day, and this dose was immediately followed by feeding
either pyrantel tartrate pellets or a placebo pellet. The treatment was not found to have an
effect on seroconversion by treatment group, indicating that it did not kill S. neurona at
the dose given. Sporocysts were administered at the following doses and time periods:
100/day for 28 days, followed by 500/day for 28 days, followed by 1,000/day for 56
days. Serum was tested weekly and CSF was tested monthly for antibodies to S. neurona
by western blot at Michigan State University (Rossano et al., 2000). Seroconversions
were first seen in 2 colts on day 77 of the study, 14 days afier starting the
1,000/sporocysts per day dose. When the present study was conducted, the horses had
been receiving 1,000 sporocysts/day for 42 days, and had received their most recent dose
of sporocysts approximately 18 hours before blood was collected.

Six yearling colts (ID numbers 7, 33, 42, 64, 105 and 163) were selected for
attempted culture fi'om blood, and the selection was made based on western blot test

results for serum drawn 2 weeks previously. (Test results fiom 1 week prior to the study

83

were not yet available.) Since little is known about the staging of S. neurona infection in
horses, the goal of the sample selection was to choose horses at different stages of
seroconversion in order to increase the chances of detecting parasitemia. Two of the
horses had tested negative for serum antibodies to S. neurona, 2 horses were in the
process of seroconversion and had only produced antibodies to the 30-kDa antigen, and 2
horses had tested positive (producing antibodies to both the 30- and 16-kDa antigens) for
serum antibodies to S. neurona. The seropositive horses had seroconverted approximately
4 weeks (#163) and 2 weeks (#64) prior to the day culture was attempted. Western blot
test results were later obtained for serum and CSF samples taken at the time of culture.
Two 10 ml- tubes with EDTA additive were filled by jugular venipuncture from
each horse and allowed to settle at room temperature for approximately 2 hours. Three to
3 ‘/2 mls of plasma was gently pipetted from the tops of the tubes and transferred to a 15
ml conical centrifuge tube. This procedure was repeated for the samples from each horse.
The centrifuge tubes were then spun for 10 minutes at 2,500 RPM and the clear plasma
was decanted from the tubes and discarded. The pellet was resuspended in plain DMEM
and spun again for 10 minutes at 2,500 RPM. The wash solution was removed and the
pellet was resuspended in a medium of 10 ml DMEM, plus 10% fetal bovine serum,
HEPES, PenStrepFungizone, L-glutamine and sodium pyruvate. The tubes were stored at
room temperature while they were being transported to MSU (approximately three days)
and were subsequently pipetted onto confluent equine dermal cell cultures and incubated
at 37°C with 5% C02. The medium was removed the next day and replaced with DMEM
with 6% fetal bovine serum with HEPES, PenStrepFungizone, L-glutamine and sodium

pyruvate. The cultures were monitored weekly for parasite growth for 12 weeks.

84

Merozoites grown from the cultures were harvested on a weekly basis. First the
cell culture medium, including merozoites was pipetted off the culture flask and the
medium was spun for 30 minutes at 3,000 RPM. The medium was subsequently pipetted
from the tubes and the pellet was resuspended in sterile phosphate buffered saline (PBS)
solution. The tubes were spun again for 30 minutes at 3,000 RPM and the PBS was
decanted from the pellet. The pellets were then resuspended in 1.5 ml of sterile PBS and
transferred to microfuge tubes and spun for 2 minutes at 14,000 RPM. The PBS was
decanted from the pellets, and the process was repeated. The PBS was removed from the
pellets and the tubes were stored at —80°C for future use.

DNA was extracted fiom merozoites harvested from cell culture using the
QIAGEN Dneasy kit for tissue samples. PCR was performed using primers designed for
a Sarcocystis species 334 bp product, JN25/JD396, using an annealing temperature of
55°C (Tanhauser et al., 1999). Samples from culture were tested using MIHl as a
positive control and a no-DNA water control. PCR products were cut using restriction
enzymes (Tanhauser et al., 1999) to confirm the identity of S. neurona.

PCR products confirmed to be S. neurona were purified using the QIAquick PCR
purification kit and were submitted to the MSU Genomics Technology Support Facility
for sequencing. PCR products fiom a single amplification reaction using DNA isolated
from the cultured S. neurona isolate were sequenced in both directions. This was
performed twice. The Pretty routine of the Wisconsin CGC Squeb program was used
to produce consensus sequences for each PCR product. The culture sequence was then
compared with known strain UCDl (Genebank #AF093158), MIHl (Mansfield et al.,

2001), and SN3 7-R. The Squeb PileUp routine was used to align the consensus

85

sequences with sequences from GenBank and MSU. The sequences used for comparison

spanned from 201 bp to 450 bp of the PCR target from JNB25/JD396.

4.3 RESULTS:

The culture from horse #64 developed a small plaque in the cell monolayer that
was observed to have motile merozoites in it 38 days after being placed on equine dermal
cells. The culture continued to grow well and develop multiple plaques. No other flask
produced a positive result, and they were discarded after 12 weeks of observation.

When tested by PCR, the #64 isolate produced a 334 bp band that was cut by Hinf
I and not by Hind III (Figure 1). This was consistent with results for the MIHl positive
control, a known strain of S. neurona. When the PCR products were sequenced, they
showed 100% agreement between SN37—R and #64. The SN37-R and #64 isolates
differed from UCDl at one position and from MIHl at 3 positions (Figure 2).

Western blot antibody tests for serum samples drawn the same day as culture was
attempted revealed that when the blood was drawn, 5 horses were seropositive and 1

horse was classified as suspect (Table 1).

4.4 DISCUSSION:

The results obtained in this study suggest that the parasite cultured from horse #64
was the same strain as SN37-R, the strain used to inoculate horses in the study. The PCR
with RF LP supports that the isolate is S. neurona, and the sequence analysis revealed that
while SN37-R and #64 were identical in the region examined, they were distinct from

UCDl and MIHI. To our knowledge, these results represent the second report of

86

parasitemia in a horse experimentally infected with S. neurona (Long et al., 2002) and the
first report describing parasitemia in an immunocompetent horse.

The serum antibody test results for the colt demonstrate that viable S. neurona can
be present in the bloodstream 4 weeks after a horse has mounted a detectable IgG
response. Because a daily dosing scheme was employed in the study, it is not possible to
determine the time to parasitemia from a single dose of sporocysts. Further studies
should pursue this important question. The present study does, however, demonstrate
that chronic low doses of sporocysts can be used to produce parasitemia under controlled
conditions, and this technique may be useful for future studies of horses’ immune

responses to S. neurona.

87

Table 4.1. Summary of test results for horses in the study. Treatment groups are (PT) =

pyrantel tartrate and (P) = placebo.

 

Serum antibody

test result 14
Horse ID# and days prior to
treatment grouL culture

Serum antibody CSF antibody
test result for test result for
Culture result day of culture day of culture

 

7 (P) Negative Negative Positive Negative
33 (PT) Negative Negative Suspect Negative
42 (PT) Suspect Negative Positive Suspect
64 (PT) Positive Positive Positive Suspect
105 (P) Suspect Negative Positive Suspect
163 (P) Positive Negative Positive Positive

 

88

Figure 1. Picture of ethidium bromide-stained 1.8% agarose gel showing Sarcocystis
neurona-specific PCR products and RFLP analysis of MIHl and #64. Lane lcontains a
100 bp DNA ladder, lane 2 contains MIHl, lane 3 contains MIHl digested with Hinf 1,
lane 4 contains MIHl digested with Hind III, lane 5 contains #64, lane 6 contains #64

digested with Hinf 1, lane 7 contains #64 digested with Hind III, and lane 8 contains the

negative control.

 

89

Figure 2. Sequence alignments for data spanning from 201 bp to 450 bp of the PCR
target from JNB25/JD396. Row 1 contains the sequence for UCDl (GenBank #

AF 093158), row 2 contains the sequence for SN37-R (the isolate administered to
experimentally infected horses), row 3 contains the sequence from S. neurona cultured
fi'om horse #64 and row 4 contains the sequence of MIHI. The sequences for SN3 7-R
and #64 were identical, but differed from UCDl and MIHl at the 249 bp position. The
rest of the sequences for SN37-R, #64 and UCDl were identical, and differed from MIHl

at the 267-268 positions and the 419 position.

AF093158
SN37-R
#64

MIHl

AF093158
SN37-R
#64

MIHl

AFO93158
SN37-R
#64

MIHl

AFO93158
SN37—R
#64

MIHl

AF093158
SN37-R
#64

MIHl

201 bp

catcaggagg
CATCAGGAGG
CATCAGGAGG
CATCAGGAGG

251 bp

ctgggcgcct
CTGGGCGCCT
CTGGGCGCCT
CTGGGCGCCT

301 bp

ggactaagag
GGACTAAGAG
GGACTAAGAG
GGACTAAGAG

351 bp

ccgctgcgac
CCGCTGCGAC
CCGCTGCGAC
CCGCTGCGAC

401 bp

gatttcctct
GATTTCCTCT
GATTTCCTCT
GATTTCCTCT

aactagtttg
AACTAGTTTG
AACTAGTTTG
AACTAGTTTG

H

gacactc..t
GACACTC..T
GACACTC..T
GACACTCTAT

tcgtgcaagt
TCGTGCAAGT
TCGTGCAAGT
TCGTGCAAGT

ttaagaccta
TTAAGACCTA
TTAAGACCTA
TTAAGACCTA

ttgtcaataa
TTGTCAATAA
TTGTCAATAA
TTGTCAAT.A

1.

tcatggtgcc
TCATGGTGCC
TCATGGTGCC
TCATGGTGCC

agcagagagt
AGCAGAGAGT
AGCAGAGAGT
AGCAGAGAGT

ttcattcgga
TTCATTCGGA
TTCATTCGGA
TTCATTCGGA

agtagagaag
AGTAGAGAAG
AGTAGAGAAG
AGTAGAGAAG

cacaggcagc
CACAGGCAGC
CACAGGCAGC
CACAGGCAGC

90

cctacagaac
CCTACAGAAC
CCTACAGAAC
CCTACAGAAC

gacgggtgga
GACGGGTGGA
GACGGGTGGA
GACGGGTGGA

gccaggagct
GCCAGGAGCT
GCCAGGAGCT
GCCAGGAGCT

ctggcggagg
CTGGCGGAGG
CTGGCGGAGG
CTGGCGGAGG

aatcacaaat
AATCACAAAT
AATCACAAAT
AATCACAAAT

250 bp
ccgattctgc
CCGATTCTTC
CCGATTCTTC
CCGATTCTGC

f.

300 bp
gcaactaaaa
GCAACTAAAA
GCAACTAAAA
GCAACTAAAA

350 bp
tcaatggaca
TCAATGGACA
TCAATGGACA
TCAATGGACA

400 bp
cgaaacagta
CGAAACAGTA
CGAAACAGTA
CGAAACAGTA

450 bp
gtaattatcg
GTAATTATCG
GTAATTATCG
GTAATTATCG

REFERENCES:

Cheadle MA, Tanhauser SM, Dame JB, Sellon DC, Hines MT, Ginn PE, MacKay RJ,
Greiner EC. 20013. The nine-banded armadillo (Dasypus novemcinctus) is an
intermediate host for Sarcocystis neurona. Int J Parasitol 31(4):330—5.

Cheadle MA, Yowell CA, Sellon DC, Hines MT, Ginn PE, Marsh AE, MacKay RJ,
Dame JB, Greiner EC. 2001b. The striped skunk (Mephitis mephitis) is an intermediate
host for Sarcocystis neurona. Int J Parasitol 31(8):843-9.

Cutler TJ, MacKay RJ, Ginn PE, Gillis K, Tanhauser SM, LeRay EV, Dame JB, Greiner
EC. 2001. Immunoconversion against Sarcocystis neurona in normal and

dexamethasone-treated horses challenged with S. neurona sporocysts. Vet Parasitol
95(2-4):197—210.

Dubey JP, Saville WJ A, Lindsay DS, Stich RW, Stanek JF, Speer CA, Rosenthal BM,
Njoku CJ, Kwok OC, Shen SK, Reed SM. 2000. Completion of the life cycle of
Sarcocystis neurona. J Parasitol 86(6):]276-80.

Dubey JP, Lindsay DS, Saville WJA, Reed SM, Granstrom DE, Speer CA. 2001a. A
review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet
Parasitol 95(2-4):89-131.

Dubey JP, Saville W] A, Stanek JF, Lindsay DS, Rosenthal BM, Oglesbee MJ, Rosypal
AC, Njoku CJ, Stich RW, Kwok OC, Shen SK, Hamir AN, Reed SM. 2001c.

Sarcocystis neurona infections in raccoons (Procyon lotor): evidence for natural infection
with sarcocysts, transmission of infection to opossums (Didelphis virginiana), and
experimental induction of neurologic disease in raccoons. Vet Parasitol 100(3-4):l 17-29.

Dubey JP, Rosypal AC, Rosenthal BM, Thomas NJ, Lindsay DS, Stanek JF, Reed SM,
Saville W] A. 2001b. Sarcogzstis neurona infections in sea otter (Enhydra lutris):
evidence for natural infections with sarcocysts and transmission of infection to opossums
(Didelphis virginiana). J Parasitol 87(6):1387-93.

Fenger CK, Granstrom, DE, Langemeier JL, Stamper S, Donahue JM, Patterson J S,

Gadj ahar AA, Marteniuk JV, Xiaomin Z, Dubey JP. 1995. Identification of the opossum
(Didelphis virginiana) as the putative definitive host of Sarcocystis neurona. J Parasitol
81 :916-919.

Fenger CK, Granstrom DE, Gajadhar AA, Williams NM, McCrillis SA, Stamper S,
Langemeier J L, Dubey JP. 1997a. Experimental induction of equine protozoal
myeloencephlitis in horses using Sarcocystis sp. sporocysts from the opossum (Didelphis
virginiana). Vet Parasitol 68:199-213.

91

Long MT, Mines MT, Knowles DP, Tanhauser SM, Dame JB, Cutler TJ, MacKay RJ,
Sellon DC. 2002. Sarcocystis neurona: parasitemia in a severe combined
immunodeficient (SCID) horse fed sporocysts. Exp Parasitol 100(3):150-4.

Mansfield LS, Schott II HC, Murphy AJ, Rossano MG, Tanhauser SM, Patterson J S,
Nelson K, Ewart SL, Marteniuk JV, Bowman DD, Kaneene JB. 2001. Comparison of
Sarcocystis neurona isolates derived from horse neural tissue. Vet Parasitol 95 167-178.

NAHMS. 2001. Equine Protozoa] Myeloencephalitis (EPM) in the US.
USDA:APHIS:VS, CEAH, National Animal Health Monitoring System. Fort Collins,
CO. #N312.0501.

Rossano MG, Mansfield LS, Kaneene JB, Murphy AJ, Brown CM, Schott II HC, Fox JC,
2000. Improvement of western blot test specificity for detecting equine serum antibodies
to Sarcocystis neurona. J Vet Diag Invest 12:28-32.

Saville WJA, Stich RW, Reed SM, Njoku CJ, Oglesbee MJ, Wunschmann A, Grover DL,
Larew-Naugle AL, Stanek JF, Granstrom DE, Dubey JP. 2001. Utilization of stress in
the development of an equine model for equine protozoal myeloencephalitis. Vet
Parasitol 95(2-4):21 1-22.

Shin EK, Perryman LE, Meek K. 1997. A kinase-negative mutation of DNA-PK(CS) in
equine SCID results in defective coding and signal joint formation. J Irnmunol
158(8):3565-9.

Sofaly CD, Reed SM, Gordon J C, Dubey JP, Ogleebee MJ, Njoku CJ, Grover DL,
Saville W] A. 2002. Experimental induction of equine protozoan myeloencephalitis
(EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the
development of clinical neurologic disease. J Parasitol 88(6):1164-70.

Tanhauser SM, Yowell CA, Cutler TJ, Greiner EC, MacKay RJ, Dame JB. 1999.
Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystisfalcatula. J
Parasitol 85:221-228.

92

CHAPTER 5

SUMMARY AND CONCLUSIONS
5.1 SUMMARY:

These studies have investigated the ability of daily administration of pyrantel
tartrate to prevent infection with S. neurona in horses by two methods: by an
experimental challenge with sporocysts under controlled conditions and by a field
intervention to test the performance of the drug against natural infections in an on-farm
setting. Neither study supports the working hypothesis that pyrantel tartrate could be
used as a preventive agent for EPM.

The final study has demonstrated that S. neurona can be cultured fiom the blood
of an immunocompetent horse. If it proves repeatable, the technique described here may

have utility in future studies of the immune response of horses to S. neurona infection.

5.2 CONCLUSIONS:
The studies described here have addressed the hypotheses stated in Chapter 1:

H1: Daily pyrantel tartrate administration prevents infection with S. neurona in
horses.

H2: Daily pyrantel tartrate administration delays infection with S. neurona in
horses.

H3: Daily pyrantel tartrate administration prevents infection with S. neurona in
horses under field conditions.

H4: S. neurona can be detected in the blood of immunocompetent horses

93

The results of the experimental challenge study showed that the infection of
horses with S. neurona was neither prevented nor delayed by daily administration of
pyrantel tartrate, thus refuting H1 and H2_ The results of the field intervention study were
less conclusive, due to the small sample size and difficulties in conducting the study, but
they do no lend support to H3. As a whole, the studies suggest that if pyrantel tartrate is
to demonstrate efficacy in preventing infection with S. neurona in horses, it will most
likely not be at the label dose. It is possible that future studies could demonstrate a dose-
response if higher doses of pyrantel tartrate are given to horses, or if the drug is given
more frequently than once per day.

The results of the experimental challenge study suggest that horses may be less
susceptible to S. neurona infection than previously thought. This may be because the
experimental horses were not appreciably stressed and they had free-choice access to
forage, providing for constant gut fill and movement of ingesta. This leads to the
questions as to whether natural infections are the result of large doses of sporocysts or an
immunosuppressive event that makes some horses more vulnerable to infection, or the
practice of feeding horses at intervals rather than free choice.

The field intervention trial illustrates the potential pitfalls of working with horses
not owned or controlled by the investigators. There, it was learned that if staff is
unavailable to cooperate with the study schedule, the chances of following the protocol
are greatly diminished.

The results of the study in which S. neurona was cultured from the blood of an
immunocompetent horse supports H4, and has demonstrated that horses with detectable

IgG antibodies to the parasite may still not be able to control the infection. This may

94

have implications for the use of the EPM vaccine, which stimulates production of IgG
antibodies, but has not yet been shown to prevent infection with S. neurona.

No new horse management recommendations can be drawn from these studies,
other than ruling out daily pyrantel tartrate as an EPM preventative at the label dose for
Strongid C 2x, although some horse owners may derive comfort fiom knowing that

horses appear to be naturally resistant to the parasite.

95

APPENDICES

96

APPENDIX I

97

 

 

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can: Burpaag

 

 

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89.2. .23.. m
as... £95.80
5.8.7.8 was

 

 

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398m 0 Bacchm 2.32m 898..
mafifiaoh I < 35.0 I m 9.9.0 89.2. Om?
9mg .«uqofitonxm 3.58.593. Dm? .850 .850

Gas £95.80 E..&.....O
.8 N 33...... max—E Qummmq .m—uflwm flOmeOHH—H ~NwfloathQNm—H Snfim HON—rum

98

APPENDIX II

99

 

 

88m 838m

 

 

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u m m a
m. u. 5.58.. 58.8 5.... :88 m. m.
.5... :88 an... .m 3 . 8...: .888
m M .8.“ «8.8.80 8.» 08.8.80 m W
.358 8.. .880 8.. .880
m... a. .2.
8.9.8.. .8..8 m
2.... 8.8.5.88
58.-.... vi... 888.. «7.5 888.. m7...
88.8mm 0 Bugabm 2.88... .
8.8.8... I 4.. 98.0 I m 9.8.0 88.8..
Dm? 8888.....an 8888.....an 9w...» .850

 

 

 

 

.2888 58.8

88.8..

Dmg
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8.5m 85.....25. 8.. 88.. 9.8.8.0 8.. 888....

100

APPENDIX III

101

SPOROCY ST DOSING PROTOCOL FOR INFECTION STUDY

Materials needed:

Data sheet for recording treatments
Pipeting ball (rubber)

10 ml pipets

Sporocysts in solution, 3,000 sporocysts/m1 concentration
l-liter glass beaker

Corn syrup

Stir plate

Stirring bar

5cc oral syringes

4 oz specimen cups

Latex gloves

Rubber boots

Coveralls

Stock solution:

Gradually add 700 ml corn syrup to 300 ml distilled water in a glass beaker, while
stirring at speed #7 on the stir plate. Mix with stir bar until thoroughly blended. Makes 1
liter of 70% corn syrup solution. Store in refiigerator.

Prepare sporocyst inoculum for daily dosing immediately prior to dosing

Procedure:

Wear latex gloves

Add 19 ml of 70% corn syrup solution in specimen cup

Place stir bar in specimen cup and set stir plate to speed #4

Vortex sporocysts in solution at high speed for 30 seconds

Add 1 ml sporocyst solution to specimen cup and allow to stir for 1 minute
Gradually add 40 ml of corn syrup solution (10 ml at a time) to the specimen cup
Stir the sporocyst inoculum for an additional 2 minutes and remove the stir bar
Take the specimen cup to the horse paddocks

Draw 2 ml of sporocyst/syrup mixture into a syringe

Approach a horse that is being held for you

Place the syringe in the comer of the mouth, and quickly squirt the contents to the back of
the tongue

Observe the horse to insure that the dose is not spit out

Follow with dose of Strongid treatment (A or B)

When exiting the horse pen, wash your shoes in the footbath

When finished dosing all horses, remove gloves and wash hands

Record treatments on horses’ individual record sheets

102

APPENDIX IV

103

 

Neurological Exam Form

 

 

 

Strabisnars (III, N, VI)

Normal Abnormal L

Head Tilt (VII) Normal Abnonml L R

Pfizer/EPM Study

Date: Horse Number:
Examiner:
Behavior:
Mentation:
Cranial Nerves:
Olfactory (I) Normal Abnorrml L R Facial/Motor (VII)
Vision (II) Normal Abnormal L R Palpebral Normal Abnormal L R
Pupil size Normal Abnormal L R Ear Normal Abnormal L R
Pupil ligbtrci'lex (III) Normal Abnormal L R Lip Normal Abnormal L R

R

R

Muscle, Jaw m (V)

TrigerninallSensory (V):

Opthalnnc
Maxillary

Symmetry of necklbody: Normal Asymmetric (specify):

NornnlAbnormalL

Normal Abnormal L R
Normal Abnormal L R

Hearing
Nystagmus

Slap Test

NormalAbnormalLR

Normal Horizontal Positional

Swallow/Voice Nornnl Abnormal L R

Nornul AbnormalLR
Tongue(XII) Normal AbnormalLR

 

GaitaadPostare:(0-Nonml,4-Recrnnbent)

 

 

 

 

 

 

 

 

 

 

 

Paresis Ataxia Hyponretria Hypermetrla Description of gait:
LF
RF
LII
RH
Circling: Len Norm] Abnormal Backing: Normal Abnormal
Rigin Normal Abnormal
Describe drclng and/or backing:
Cntaneons sensation: Normal Abnormal Incline: Normal Abnormal
Tail pnll: Left Nornnl Weak Sweating: Normal Abnormal
Right Normal Weak Urination: Normal Abnormal
Anal sphincter reflex: Normal Abnormal Tail tone: Normal Abnormal

Case assessment:

 

 

 

104

APPENDIX V

105

Protocol for CSF Collection
Spinal tap procedure:

Preparation of horses:

Horses will be weighed on a livestock scale. Horses will then be clipped along
the jugular groove of the neck on both sides. The left side of the neck will be scrubbed
with Betadine solution and alcohol.

Placement of catheter:

The catheter site will be injected with 0.5 ml of 2% Lidocaine and allowed to
stand for several minutes. The catheter is then inserted in the jugular vein in a routine
manner, and an extension set is placed on the catheter.

Anesthesia:

Horses will be randomized into two groups receiving different anesthesia
protocols. The study uses a crossover design, and so the horses will receive the alternate
protocol for their second spinal tap. Doses of drugs used will be calculated according to
the horse’s weight.

All horses will be pre-medicated with Xylazine. One group will receive telazol,
ketamine and detomadine. The other group will receive ketamine and midazolam. When
the drugs take effect, the horse is eased down to the floor of the stall and placed in a
position to facilitate the collection of spinal fluid. Horses will be monitored by the
antesthesiologist, or a student under his supervision, during the spinal tap procedure and
during recovery. Time to recovery data will be collected by those personnel.

Blood Collection:

Once the horse is anesthetized blood can be collected from the catheter. The first
20 cc’s of blood are drawn and discarded. A new syringe is placed on the catheter and
approximately 90 ccs of blood are collected. 2 EDTA tubes, 2 yellow-topped tubes, 2
red-topped tubes and 3 serum separator tubes decapped, filled and capped shut. Hep
saline solution will be flushed through the extension set on the catheter and the stopcock
closed.

Spinal Fluid Collection:

The mane and hair coat immediately behind the ears, and over the first two
vertebrae of the spine will be clipped and scrubbed with Betadine solution and alcohol.
The horse’s head is flexed toward the chest. A veterinary clinician will supervise
students performing the procedure. Sterile gloves will be put on and a sterile spinal
needle will be placed on the midline of the spine at the atlanto-occipital site. The needle
will be slowly advanced until the dura mater is punctured and CSF flows from the needle
when the stilette is removed. Once the CSF is dripping from the needle, a 13 ml plastic
tube will be filled, followed by an EDTA tube and 6 1.8 ml cryo vials. When all the
tubes are filled the spinal needle is removed and the procedure is complete.

106

APPENDIX VI

107

Necropsy Protocol Pfizer Challenge Study

(L.S. Mansfield 2/17/03)

Purpose: Demonstrate and recover Sarcocystis neurona from infected horses to
demonstrate that neurological disease is associated with live parasite with identity to the
parasite we used for challenge.

Animals:

Horses with the following numbers from the Pfizer challenge study (27,33 42, 154).
Experimental design:

Groups:

Group #1: Uninfected none

Group #2: S. neurona challenged/Treated 1 horse

Group #3: S. neurona challenged/Treated 1 horse
Equipment needed:

From Laboratory

0 Syringes

o 60cc syringes - xx
0 3cc syringes - xx
0 Needles
o 18 gauge — 2 boxes
0 20 gauge — 2 boxes
0 Catheters
o 18 gauge - 4
0 extension set - 3
o Anesthetics
o Xylazine — X bottles
0 Fatal Plus — X bottles
0 Media
0 Sterile saline
0 DMEM with antibiotics
0 Instruments
0 Sterile scalpel blades and handles
0 Sterile forceps
o Sterile scissors
Heated transport container
Fixatives
o 3.7% Formaldehyde ~200mL
0 dry ice
0 Tubes
0 15cc tubes with labels
0 500C tubes with labels
0 cryovials

108

o Immunohistochemistry
0 Tissue molds
0 OCT compound
Cooler with ice packs
Parafilrn
Magic markers
Cultruing supplies
0 Sterile drapes for work surface for handling brain and spinal cord
0 Sterile instruments
0 50 cc conical plastic tubes

From Barn:
Halters
Twitch
Knives

Saw

Timing:
0 Horses will be necropsied on 3/3/03.
- CNS tissue samples will be mailed on 2/x/03 via Federal Express delivery service to:
Attention Alice Murphy _
A12 Veterinary Medical Center '
College of Veterinary medicine
Michigan State University
East Lansing, MI 48824
517-353-2296
0 Alice Murphy will be responsible for culture of CNS tissues when they arrive and
will place these on cells on 3/04/03.
0 Cells will be examined, brain slurry removed and fresh media placed on cells on
2/x/03.

Necropsy:
1) Schedule - On 3/3/03, 3 horses will be euthanatized and necropsied.
2) Anesthesia

a) Horses will be sedated with Xylazine (1M).

b) Once sedate, the horses will then be euthanized by intravenous overdose of
sodium pentobarbital (IV through the catheter). A catheter will be placed in the
right jugular vein and Fatal Plus given to effect, ~120 mLs per horse.

3) Areas to be examined during necropsy

a) After death, abdomen will be opened through a midline incision. The diaphragm
will be examined and tissues samples recovered.

b) The tongue, diaphragm, and heart will be examined and removed for tissue
samples.

0) The brain and spinal cord will be accessed and samples taken for culture,
fixation and snap freezing.

4) Processing of tissues samples

109

a)

b)

The spinal cord will be placed on a sterile drape on a stainless steel table, and full

thickness tissue sections taken from all regions of the cord leaving the dura mater

in place as protection to contamination.

The brain will be accessed through the foramen magnum, the cranium removed

and tissue samples taken from cerebrum, cerebellum, and brain stem.

Tools

ii) Tools must be sterile for cutting up the brain and spinal cord for culture.

iii) Tools must not cross contaminate from horses to horse on the other
samples

iv) Tools must not cross contaminate from tissue to tissue in a single horse.

v) Use you judgment on how to achieve these goals.

Tissue samples will be saved in:

a)

b)

(1)

Fresh samples to be sent to MSU on wet ice (Make sure to take the samples of
cord and brain that flank the formalin sample sites and that center on the
area predicted to be damaged based on the clinical examination. Use 50 cc

conical tubes for this.)
ii) Tongue
iii) Heart

iv) Diaphragm
v) Spinal cord
vi) Brain (sections of cerebrum, Cerebellum, brain stem)

10% formaldehyde solution (several sections of each for formalin with size

roughly 2 cm cubes)

ii) Tongue

iii) Heart

iv) Diaphragm

v) Muscles of mastication/chewing (jaw/cheek muscles)

vi) Muscles of the upper thigh (Semimembranosus, semitendinosus, muscles
down the back of the back leg)

vii) Spinal Cord

viii) Brain

ix) Lung

x) Liver

xi) Small intestine

Frozen slowly in O.C.T. compound at -20° for cryosectioning and
Immunohistochemistry (Take samples close to the lesion. These should be
very small thin sections ( 1 cm x 0.5 cm) placed in a small plastic bed and
covered with OCT compound, then freeze by putting these samples on a tray
in a -20 C freezer and letting them freeze until the next day. Then they can
be bundled up, put in boxes, and mail on dry ice. These must stay frozen in
transit.)

ii) Spinal Cord

iii) Brain

Snap frozen for later cytokine analysis for RT PCR (Take small samples and
freeze in liquid nitrogen or dry ice ASAP)

110

ii) Tongue

iii) Heart

iv) Diaphragm
v) Spinal Cord
vi) Brain

Follow-up Procedures Planned:

1)

2)

3)

4)

Culture and PCR to detect S. neurona

a) A tissue sample will be taken fi'om the spinal cord or brain after the horse is
euthanized

b) Half will go into Preston transport media and half into 50 CC tubes for culture

c) Samples will be ground using a Dounce homogenizer and plated onto ED cells by
standard technique (Mansfield et al., 2000).

d) Merozoites resulting from culture will be tested by a variety of S. neurona specific
PCR tests
ii) ITS Ribosomal targets (Marsh etal., 1998)
iii) Tanhauser targets and RFLP assays (Tanhauser et al, 1999)

Histopathology
a) Formalin (10%) fixed tissues will be paraffin embedded, sectioned, and stained
with hematoxylin and eosin.

Tissues collected and snap frozen for PCR

a) Samples will be snap frozen, ground in mortar and pestle, and RNA and DNA
recovered using a Qiagen Easy recovery kit.

b) Samples will be tested by PCR as described above.

Tissues collected and frozen at —20C for Immunohistochemistry

a) Samples will be taken as small snips, placed in small plastic IHC boats, covered
with OCT compound, and frozen slowly by incubation at —20 C.

b) Frozen sections will be shipped to MSU and given to Matti Kiupal for S. neurona
IHC.

Blocks will be stored long term in NFSTC floor #1 freezer.

111

APPENDIX VII

112'

Press Release
Contact: Dr. John Kaneene For Immediate Release
(517) 353-5941
or

Mary Rossano

(517) 355-1745
The Michigan State University EPM Research Team Seeks Volunteers for Strongid C
Study

EAST LANSING, Mich. -- The EPM research team at Michigan State
University is currently enrolling breeding farms in a new study that will examine whether
the daily deworrner Strongid C helps prevent infection with Sarcocystis neurona, the
parasite that causes EPM (equine protozoal myeloencephalitis).

The study will focus on weanlings, and will require individual feeding once per
day for approximately one year. Ivermectin will be provided for all enrolled horses every
eight weeks. Half of the weanlings will receive Strongid C and half will receive a look-
alike placebo.

Serum samples will be collected at the beginning of the study and every two
months thereafter for testing for antibodies against S. neurona. The deworrning
treatments, Strongid C and S. neurona testing will be provided to study participants at no
cost to them.

The researchers are hoping to recruit breeding farms that expect to have at least
12 weanlings this fall.

For more information about participating in this study contact Dr. John Kaneene,

Director of the Population Medicine Center at the Michigan State University College of

Veterinary Medicine at (517) 353-5941.

113

APPENDIX VIII

114

INITIAL QUESTIONNAIRE

Equine Protozoa! Myeloencephalitis (EPMyStrongid C Study
Michigan State University College of Veterinary Medicine

Farm ID: Date: / /

QENERAL INFORMATION:
1.) Horse name/ID: 2.) Breed:

 

3.) Birth date: / / 4.) Gender: 5.) Treament group:

 

HEAL INFORMATION:
6.) Date of last paste or liquid deworming: 7.) Type of dewormer used:

 

8.) What vaccinations has this horse received since January 1, 2001? (Check all that apply.)

Tetanus BEE/WEE Rabies Flu Potomac Horse Fever

W0 __ 3W (Wes) Othm:

 

 

9.) Has this horse ever been vaccinated for EPM? Yes /No 10.) If yes, date of vaccination:

 

MMAGEMEEI [N ERMATION:

ll.)Howmanyhorsesarekeptatthishorse'shorne/facility?

12.)Isthishorsehousedinabam? Yes/No l3.)Doesthishorsehaveaccesstopasture? Yes/No
14.)Doesdfishorsehaveaccessmadinlaaodreroutdoaexaciseuudutimtapasune? Yes/No

15.) IfumIedouLhowmanyotherhorsesonaveragearewiththishorae? 6 6-10 >10

 

 

 

l6.) Pleasedescnbethetypds) ofhay(s)thatis/are fedtothishorseandindicatewhatcomrtytheycornefiorn:

 

17.) Please describe the type(s) ofgrains tint is/are fed to thishorse (Checkanthat apply):
pelleted grains sweet feed other.

 

 

 

18.) Howmanytimesisthishorsefedperday? Grain Hay
19.) How is water provided to this horse? (Check aflthat apply)
individually/ bucket individuallyftank with other horses/ tank

 

individually/ automatic waterer with other horses/ automatic waterer

natural surface water Please describe (creek, pond, etc.):

 

20.) Have you ever seen an opossum (alive or dead) within 1 mile of this farm? (circle one) Yes / No

115

APPENDIX IX

116

FOLLOW-UP QUESTIONNAIRE

Equine Protozoal Myeloencephalitis (EPM)IStrongid C Study
Michigan State University College of Veterinary Medicine

 

 

Farm ID: Date: / /
GENERAL INFORMATION:
1.) Horse name/ID: 2.) Treament group:

 

 

HEALTH INFOMATION UPDATE:
6.) Date of last paste or liquid deworming: 7.) Type of dewormer used:

 

8.) What vaccinations has this horse received since the last blood collection? (Check all that apply.)

 

 

Tetanus BEE/WEE Rabies Flu Potomac Horse Fever
Rhino _ Strep (strangles) Others:
A E ON TE:

9.) Howmanyhorsesare kept at this horse's home/facility?
10.)isthishrnsehousedinabam? Yes/No ll.)DoesthishorsehaveaccesstopastIne? Yes/No
12.)Doesthisborsehaveaccesstoadirtlotorotheroutdoorexerciseareathatisn‘tapastm'e? Yes/No

13.)Ifmrnedorn,howmanyotherhorsesonaveragearewiththishorse? <5 6-10 >10

 

14.) Please describe the type(s) of hay(s) that is/are fed to this horse and indicate what county they come from:

 

15.) Please describe the type(s) of grains that is/are fed to this horse (Check all that apply):

pelleted grains sweet feed other:

 

 

16.) Please describe the type(s) of supplements that is/are fed to this horse:

 

 

17.) How many times is this horse fed per day? Grain Hay
18.) How is water provided to this horse? (Check all that apply)
individually/ bucket individually/tank with other horses/ tank

individually/ automatic waterer with other horses/ automatic waterer

 

naunl surface water Please describe (creek, pond, etc.):

117

   
   

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