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The biology of Sarcocystis spp. of the Virginia Opossum (Didelphis virginiana) in Florida

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Title:
The biology of Sarcocystis spp. of the Virginia Opossum (Didelphis virginiana) in Florida
Creator:
Cheadle, Mark Andrew, 1973-
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Language:
English
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xi, 103 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Department of Veterinary Medical Sciences thesis Ph.D ( mesh )
Dissertations, Academic -- College of Veterinary Medicine -- Department of Veterinary Medical Sciences -- UF ( mesh )
Host-Parasite Relations ( mesh )
Opossums -- parasitology ( mesh )
Sarcocystis ( mesh )
Sarcocystosis -- prevention & control ( mesh )
City of Gainesville ( local )
Opossums ( jstor )
Sarcocysts ( jstor )
Skunks ( jstor )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 2001.
Bibliography:
Includes bibliographical references (leaves 95-102).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Cheadle, Mark Andrew.

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Full Text










THE BIOLOGY OF Sarcocystis SPP. OF THE VIRGINIA OPOSSUM (Didelphi virginiana) IN FLORIDA











By

MARK ANDREW CHEADLE















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2001














ACKNOWLEDGMENTS


I would like to thank my family and friends first. Without strong family support throughout my graduate program, the work would have been much more difficult. Secondly, I thank the animals that gave their lives for this research. Biologic evidence can only be acquired through the use of animals and their contribution to this study was invaluable. I would like to thank the following people, without whose help I would have been unable to complete such a complicated project. Ellis Greiner was chair of my committee and a friend. Without his mentorship, I would have been unable to properly conduct my studies and gain the knowledge needed for my degree. John Dame was original chair of my committee and a friend. Without his guidance through molecular biology, I would still be in the dark. Thanks to the other members of my committee, Pam Ginn, Don Forrester, and Mel Sunquist. All of the members were patient and helpful in suggesting ways to improve and expand my research. Appreciation is extended to my coauthors on papers that have been published from this data, Dr. Ellis Greiner, Dr. John Dame, Dr. Pam Ginn, Dr. Deb Sellon, Dr. Mellisa Hines, Dr. Susan Tanhauser, Dr. Tim Scase, Dr. Antoinette Marsh, Charles Yowell, and Dr. Robert Mackay. I thank Dr. Charles Courtney for his assistance performing statistical analysis and for fishing breaks on the weekends. Many student workers did much of the "less than exciting" work required for this project: Kristin Munsterman, Laura Dixon, Alysia Posey, Eric Seymour, Taj Ryland, Kimberly Baird, Randa Antar, Melissa Perez Velasco, Michelle Delucia,


ii









Britany Benson, Alexis Smith, Jessica Meliti, Persilla Medina, Summer Gustat, Jenny Freeman, Stephanie Badge, and Jennifer Willey. All performed animal collection and care, sporocyst detection, and sporocyst purification. I thank Siobhan Ellison for the use of the polyclonal antibody to S. neurona. I thank Richard Hill and Karen Scott for Greyhound acquisition and care. I thank Dr. Janet Yamamoto and Maki Arai Tanabe for cat acquisition and care. I thank Glenda Eldred for histopathologic processing. I thank the Electron Microscopy Core Laboratory of the Interdisciplinary Center for Biotechnology Research, University of Florida for help with electron microscopy. I thank Richard Truman (Louisiana State University) for assistance with technical information concerning the armadillo. These studies were supported in part by grants from the Florida Pari-Mutual Wagering Trust Fund, United States Department of Agriculture grant No. 98-35204-6487, the Florida Agricultural Experiment Station, and the Grayson-Jockey Club Research Foundation.























111
















TABLE OF CONTENTS

page
ACKNOWLEDGMENTS ................................................. ii

LIST OF TABLES ..................................................... vi

LIST OF FIGURES ....................................... .......... viii

ABSTRACT .......................................................... x

CHAPTERS

1 INTRODUCTION .................................................1

2 SPOROCYST SIZE OF ISOLATES OF Sarcocystis SHED BY THE VIRGINIA
OPOSSUM (Didelphis virginiana) .................................. 16
Introduction ........................................16
Materials and Methods ............................................ 17
Sporocyst Collection and Storage .............................. 17
Sporocyst Typing ........................................... 17
Observation and Measurement ................................ 17
Data Analysis ............................................ 18
Results ........................................ 18
Discussion ..................................................... 19

3 VIABILITY OF Sarcocystis neurona SPOROCYSTS AND DOSE TITRATION
IN GAMMA-INTERFERON KNOCKOUT MICE .................... 26
Introduction ..... ......... ...................................26
Materials and Methods ........................................ 27
Collection of Sporocysts .................................... 27
Identification of Sporocyst Type ...................... ..... 28
Anim als ..................................................28
Inoculation Procedure ..................................... 28
Necropsy and Histopathology ................................. 29
Results ............. ............. ........ ................... 29
Discussion .....................................................30

4 Sarcocystis greineri N. SP. (PROTOZOA: SARCOCYSTIDAE) IN THE
VIRGINIA OPOSSUM (Didelphis virginiana) ......................... 40
Introduction .....................................................40

iv









Materials and Methods ............................................40
Opossum Collection and Processing ............................ 40
Light Microscopy ........................................... 41
Feeding Trials ............................................41
Transmission Electron Microscopy ............................. 42
Results ........................................ 43
Description .............................................. ...... ...43
Taxonomic Summary ............................................ 44
Discussion .................................................... 44

5 THE NINE-BANDED ARMADILLO (Dasypus novemcinctus) IS AN
INTERMEDIATE HOST FOR Sarcocystis neurona ................. ... 50

6 THE STRIPED SKUNK (Mephitis mephitis) IS AN INTERMEDIATE HOST
FOR Sarcocystis neurona ......... .................................62
Introduction ..................................................... 62
M aterials and M ethods ............................................. 63
Results ....................................................... 65
D iscussion ..................................................... 68

7 ATTEMPTS TO COMPLETE THE LIFE CYCLE OF VARIOUS Sarcocystis SP.
USING THE VIRGINIA OPOSSUM (Didelphis virginiana) AS A DEFINITIVE
H O ST .................. ....................................... 79
Introduction .....................................................79
Materials and Methods ............................................. 81
Sporocyst and Sarcocyst Identification .......................... 81
Inoculation of Anim als ....................................... 81
Oral Inoculation Trials .......... ......................... 81
Muscle Feeding Trials ...................................... 83
Results ...................................................... 84
Discussion .................................. .................84

8 CONCLUSIONS ................................................ 90

REFERENCES ....................................................... 95

BIOGRAPHICAL SKETCH ............................................ 103















LIST OF TABLES

Table page

1-1. Spectrum of animals found in the gut contents or feces of Virginia opossums.. 12 2-1. Comparison of Sarcocystis sporocyst sizes............................ 21

3-1. Dose and isolate/s of Sarcocystis neurona given to each gamma-interferon
knockout mouse ................................................ 35

3-2. Microscopic identification of protozoa in routinely and immunohistochemically
treated sections of cerebrum and cerebellum from gamma-interferon knockout
mice inoculated with Sarcocystis neurona sporocysts. ................... 36

3-3. Summary of disease in Sarcocystis neurona infected gamma-interferon knockout
mice.....................................................37

3-4. Histopathological lesions observed in gamma-interferon knockout mice orally
inoculated with Sarcocystis neurona sporocysts .......................... 38

4-1. Comparison of structural measurements of sarcocysts of Sarcocystis spp. found in
the muscle of opossums. ...........................................46

5-1. Armadillo inoculum fed to laboratory raised opossums and prepatent period of
Sarcocystis neurona sporocysts in faeces ............................ 59

5-2. Results of cytologic analysis and Western immunoblot of CSF samples from a
foal inoculated with 5 x 10' sporocysts of Sarcocystis neurona collected from
opossum DV22 fed armadillo muscle containing sarcocysts. ............... 60

6-1. Skunk inoculum fed to laboratory raised opossums and prepatent period of
Sarcocystis neurona sporocysts in faeces. .......................... 72

6-2. Inoculation of gamma-interferon knockout mice with sporocysts collected from
opossums fed Sarcocystis infected skunk muscle ....................... 73

6-3. Results of cytologic analysis and immunoblot of CSF and serum samples from a
foal inoculated with 5 x 105 sporocysts of Sarcocystis neurona collected from
opossums fed skunk muscle containing sarcocysts ....................... 74


vi









7-1. Summary of animals used to attempt the completion of Sarcocystis spp. that use
the opossum as a definitive host .................................... 87

7-2. Summary of animals orally inoculated with sporocysts collected from Virginia
opossum s ....................................................... 88


















































vii
















LIST OF FIGURES

Figure page 2-1. Sporocyst of Sarcocystis speeri (2079 isolate).. ........................ 22

2-2. Sporocyst of Sarcocystis neurona (3063 isolate)... .................. ... 22

2-3. Sporocyst of Sarcocystis falcatula (3106 isolate)......................... 23

2-4. Sporocysts of 1085 type (3013 isolate).. ........................... 23

2-5. Sporocyst of 3344 type (3344 isolate). ............................. 24

2-6. Detailed view of sporocyst of 3344 type (3344 isolate)..................... 24

2-7. Brightfield micrograph of 3344 type sporocyst ......................... 25

3-1. H & E stained section of cerebellum from mouse M8 containing a group of
m erozoites .......................................................39

3-2. Immunohistochemically stained schizont of S. neurona from mouse M8
cerebellum containing multiple merozoites... ................. ...... 39

4-1. Sarcocyst of Sarcocystis greineri n. sp. from the skeletal muscle of a Virginia
opossum.... ........................ .............................47

4-2. High magnification of an invagination in the sarcocyst wall................. 47

4-3. Sarcocyst in thigh muscle of an opossum. ............................... 48

4-4. TEM of a sarcocyst of Sarcocystis greineri n. sp. from the Virginia opossum......49 5-1. TEM of sarcocyst from armadillo DN42 fed to opossum DV02 ............ 61

6-1. Sarcocystis neurona stages isolated from skunks ......................... 75

6-2. TEM of Sarcocystis neurona isolated from skunks ..................... 76

6-3. Reactivity of serum collected from gamma-interferon knockout mice inoculated
with sporocysts collected from opossums fed S. neurona infected skunk viii









muscle ........ .. ... ................... ................... 77

6-4 Reactivity of serum collected from skunks before and after inoculation with S.
neurona sporocysts collected from a naturally infected opossum ............ 78


















































ix















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

THE BIOLOGY OF Sarcocystis SPP. OF THE VIRGINIA OPOSSUM (Didelphis virginiana) IN FLORIDA

By

Mark Andrew Cheadle

August 2001


Chairperson: Dr. Ellis C. Greiner
Major Department: Veterinary Medical Sciences

Virginia opossums (Didelphis virginiana) are definitive hosts for at least four

species of Sarcocystis. Only the life cycle of Sarcocystis falcatula had been completed at the inception of this dissertation research. Goals of this study were to establish the life cycles of one or more of the species whose life cycles were unknown. To this end, the author either: inoculated live candidate animals with sporocysts to determine if sarcocysts would form; or fed Virginia opossums sarcocyst infected muscles from various animals to determine if they would shed sporocysts. Additional studies were done to determine if other vertebrate species would become infected with S. falcatula. For intermediate host trials, we used New Zealand white rabbits, an eastern cottontail rabbit, mallards, a northern shoveler, a green-winged teal, boat-tailed grackles, a great blue heron, brown-headed cowbirds, red-winged blackbirds, house sparrows, a ringbilled gull, a sandhill crane, a great egret, a bald eagle, leopard frogs, gray squirrels, a


x









southern flying squirrel, striped skunks, nine-banded armadillos, horses, white-tailed deer, deer mice, rice rats, a cotton mouse, and Virginia opossums. Of the species examined, the nine-banded armadillo (Dasypus novemcinctus) and striped skunk (Mephitis mephitis) served as intermediate hosts for Sarcocystis neurona, the causative agent for the neuromuscular disease in horses called equine protozoal myeloencephalitis. The brown-headed cowbird, house sparrow, and great blue heron were intermediate hosts for S. falcatula. None of the life cycles of any Sarcocystis sp. tested was completed in any other animal from the aforementioned list. Light microscopy of sporocysts was a useful means of distinguishing Sarcocystis speeri from other taxa. Use of gammainterferon gene knockout mice for infection trials with S. neurona was expanded to determine if sporocysts being used for studies were viable. Sporocysts processed using bleach and stored at 40 C remained viable for 7 months. The finding of two intermediate hosts will allow investigators to produce large numbers of sporocysts of S. neurona, which can then be used for future studies. It will also allow veterinarians and horse owners to control the intermediate hosts on their farms as a method of prevention of infection with S. neurona.

















xi














CHAPTER 1
INTRODUCTION


Sarcocystis spp. are obligatory, intracellular protozoan parasites that use

mammals, birds, and reptiles as both intermediate and definitive hosts. The intermediate host contains asexual stages of the parasite and the definitive host contains the sexual stages (Dubey et al. 1989). There are at least 189 species included in this genus (Odening 1998). Sarcocystis spp. have a two-host cycle usually involving a carnivore definitive host and an herbivore and/or omnivore intermediate host. The life cycle begins with the ingestion of a sarcocyst containing bradyzoites in tissue fed upon by an appropriate definitive host. After degradation of the sarcocyst wall in the stomach, bradyzoites contained within the residual sarcocyst are released within the small intestine. These stages invade the intestinal epithelial cells and begin the sexual cycle of reproduction culminating in the production of two sporocysts contained within an oocyst. Sporocysts sporulate or mature in situ and are released within the feces into the environment to infect an intermediate host. Once sporocysts are ingested by the intermediate host, sporozoites that are contained within are released and invade the intestinal epithelial cells and begin a migration to the muscle tissue. In capillary endothelial cells, sporozoites undergo asexual reproduction leading to the formation of a schizont with merozoites contained within. Merozoites are then released and invade new host cells, forming another schizont. After a series of these stages, a terminal merozoite migrates to muscle tissue and forms a sarcocyst with metrocytes contained within. These

1







2

metrocytes eventually mature into bradyzoites within the cyst and the cycle continues. Bradyzoites are considered a hypobiotic stage (Dubey et al. 1989).

The name Sarcocystis is derived from the asexual stage of the parasite that

encysts in muscle. Although sarcocysts can be found in the CNS and smooth muscles, they are primarily found in striated muscles throughout the body (Dubey et al. 1989). The sarcocyst can range in size from microscopic to visible by the naked eye. Shapes of sarcocysts vary from spindle to globular. The bradyzoites contained within this stage are the only portion of the life cycle infective to the definitive host (Dubey et al. 1989).

The Virginia opossum (Didelphis virginiana) is a definitive host for several

species of Sarcocystis, some of which cause clinical disease in other animals. The firstnamed species of Sarcocystis that uses the opossum as a definitive host was originally named Sarcocystis debonei, however, it is currently named Sarcocvstis falcatula (Box et al. 1984). Sarcocystis is a major pathogen of psittaciform birds and can cause severe disease and death (Box and Smith 1982). Unlike most Sarcocystis spp., S. falcatula is not species-specific with reference to intermediate hosts. This species can naturally and experimentally infect a variety of avian species across the passeriform, psittaciform and columbiform orders (Box and Smith 1982). Natural intermediate hosts include the brown-headed cowbird (Molothrus ater), the boat-tailed grackle (Cassidix mexicanus), and the Patagonian conure (Cyanoliseus patagonus) (Bolon et al. 1989; Box et al. 1984). Experimental hosts include budgerigars (Melopsittacus undulatus), canaries (Serinus canarius), rock doves (Columba livia), English sparrows (Passer domesticus), zebra finches (Poephila guttata), and cockatiels (Nvmphicus hollandicus) (Box and Duszynski 1980; Box and Smith 1982; Hillyer et al. 1991). Although authors originally suggested







3

that S. neurona and S. falcatula were synonymous (Dame et al. 1995), subsequent studies found that S. neurona and S. falcatula can be differentiated using biological and molecular characteristics (Cutler et al. 1999; Dubey and Lindsay 1998a; Tanhauser et al. 1999).

Sarcocystis speeri (= 2079 isolate) originally was collected and differentiated

from other species shed by the opossum at the University of Florida. Although this was not named by the University of Florida, researchers Dubey and Lindsay (1999b) isolated the same organism and named it S. speeri. This species has been isolated also from Didelphis albiventris, the South American opossum (Dubey et al. 2000e). Dubey et al. (1998b) described the isolation of a third unidentified species of Sarcocystis, based on sarcocyst formation and structure in gamma-interferon gene knockout mice. After inoculation of gamma-interferon gene knockout mice with sporocysts of this unidentified species, sarcocysts were observed in the leg muscles at Days 50 and 54. There was some cross reactivity between the sarcocysts and an S. neurona antibody when the S. neurona antibody was applied to tissue sections. However, according to the authors, much less reactivity was seen than when the antibody was applied to S. neurona infected tissues. Nude mice inoculated with sporocysts from the same opossum did not form sarcocysts. In the paper by Dubey et al. (1999b) in which they described and named S. speeri, the authors found that one nude mouse did form sarcocysts when inoculated with 10-1 sporocysts of S. speeri. Interestingly, schizonts were seen in the brain of 14 of 34 (41%) of mice inoculated with sporocysts. This indicates either a mixed infection with S. neurona or the possibility that S. speeri will infect the brains of mice. This problem was never addressed in the paper and size differences of the merozoites were never compared







4

even though merozoites of S. speeri are much larger than those of S. neurona (Dubey et al. 1998b). Dubey et al. (2000d) also found that S. speeri would grow in cell culture using bovine monocytes and equine kidney cells.

A third species of Sarcocystis using the opossum as a definitive host is

Sarcocystis neurona. In the original description of S. neurona by Dubey et al. (1991), they reported the isolation of an "equine protozoal myeloencephalitis (EPM)-like" protozoal organism from the spinal cord of a naturally infected horse from Ithaca, New York. This description came after most researchers felt that the protozoal organism seen previously in horse CNS tissue diagnosed with EPM was Toxoplasma gondii. However, a description by Simpson and Mayhew (1980) identified a Sarcocvstis sp. in the spinal cord of a naturally infected horse that was diagnosed with EPM, thus indicating that a Sarcocystis sp. was the source of infection. Fenger et al. (1997) and Tanhauser et al. (1999) identified the definitive host of S. neurona as the Virginia opossum (Didelphis virginiana).

Sarcocystis neurona was thought to be synonymous with Sarcocystis falcatula

based on the similarity of the three segments of the small subunit ribosomal RNA (Dame et al. 1995). It now appears, based on extensive molecular and biological evidence, that S. falcatula and S. neurona are separate species. However, the use of the cowbird as an intermediate host for S. neurona is still in doubt (Cutler et al. 1999; Dubey and Lindsay 1998a; Tanhauser et al. 1999). Due to the fact that sarcocyst wall configuration is considered to be the differentiating characteristic for Sarcocystis spp. and that the configuration of the sarcocyst wall for S. neurona was unavailable, there was no way to definitively differentiate the two organisms. The method described by Tanhauser et al.







5

(1999) is considered the most appropriate method for determining the species.

Another animal that appears to be affected by S. neurona is the Alaskan sea otter (Enhydra lutris kenyoni) and the southern sea otter (Enhydra lutris nereis) (Lindsay et al. 2000; Miller et al. 2001: Rosonke et al. 1999). Lesions in the southern sea otter consisted of widely disseminated nonsupperative meningoencephalomyelitis with severe and diffuse inflammation in the cerebellar molecular layer and blood vessels in the cerebellum. Large numbers of macrophages and smaller numbers of lymphocytes and plasma cells were observed. Similar but less severe inflammation was observed in the midbrain, brainstem, and gray matter of the spinal cord. Other lesions associated with infection were pulmonary hemorrhage, lymphoid depletion, and edema in a tracheobronchial lymph node. Organisms from these isolations have been grown in culture and reacted positively to antibodies raised against S. neurona (Lindsay et al. 2000; Miller et al. 2001). Organisms collected by Lindsay et al. (2000) amplified using primer pairs JNB33 and JNB54 produced a product that cut with Dra I in a pattern similar to that of S. neurona. Lindsay et al. (2000) also found that four of four gamma-interferon knockout mice inoculated with merozoites collected from the study developed encephalitis. Miller et al. (2001) isolated merozoites in cell culture and sequenced segments of DNA (18s ribosomal DNA and the entire adjacent ITS-I sequence). Sequences from this study showed identity with comparable sequences from S. neurona isolated from horses. Although sarcocysts were documented by Rosonke et al. (1999), no definitive identification was made and only low-power micrographs were available making it difficult to identify the species observed in this study.

There have been cases of S. neurona-like protozoal organisms causing







6

encephalitis in other wildlife species including mink (Mustela vison), a striped skunk (Mephitis mephitis), a rhesus monkey (Macaca mulatta), Pacific harbor seals (Phoca vitulina richardsi), and raccoons (Procyon lotor) (Dubey et al. 1996a; Dubey and Hamir 2000a; Dubey and Hedstrom 1993; Klumpp et al. 1994; Lapointe et al. 1998; Stroffregen et al. 1991). Although these authors documented stages of Sarcocvstis that reacted positively with antibodies to S. neurona, sarcocyst stages that would enable the completion of the life cycle through the opossum were not found.

In horses, EPM is a severe, debilitating, neurologic disease. Horses with this

disease commonly have abnormalities of gait. Clinical signs range from a mild lameness to sudden recumbency. Gross lesions in the spinal cord can consist of hemorrhage. Microscopically, there may be multifocal areas of necrosis, hemorrhage, and nonsuppurative inflammation of the grey and white matter (Davis et al. 1991). The lesions associated with this disease in the United States were first recognized by Rooney in 1964. Similar lesions were observed by Macruz et al. (1975), in Brazil, during the same period. The protozoal organisms seen in these lesions were assumed to be Toxoplasma gondii at that time.

Since 1970, EPM has been reported in most of the lower 48 states and in Brazil, Canada, and Panama (Clark et al. 1981; Dubey et al. 2001; Granstrom et al. 1992; Masri et al. 1992). Most of horses affected by this disease are between 1 and 4 years of age, however other age groups are also represented (Fayer et al. 1990). Sarcocystis neurona has been isolated from naturally infected horses in various areas of North and South America and has been continuously grown in cell culture for an extended period of time (Davis et al. 1991). Neospora hughesi also has been isolated from the spinal cord of a







7

horse and has been grown continuously (Cheadle et al. 1999; Marsh et al. 1998).

The only protozoal organisms documented to be in the central nervous system (CNS) of horses are Sarcocystis sp. and Neospora sp. (Cheadle et al. 1999; Dubey et al. 1991; Marsh et al. 1996). These organisms usually are associated with a mixed inflammatory cellular response and destruction of nervous tissue. Various stages such as merozoites (Sarcocystis sp.), schizonts (Sarcocvstis sp.), and tachyzoites (Neospora sp.) can be seen in the cytoplasm of neurons or mononuclear macrophages. Cells that can be parasitized during the course of infection in the CNS include intravascular and tissue neutrophils and eosinophils as well as capillary endothelial cells and myelinated axons. Organisms are commonly associated with areas of focal necrosis. Merozoites and tachyzoites may be found intracellularly or extracellularly and in groups or as individuals. Sarcocysts of S. neurona do not form in muscle or any other organ. Therefore, it is assumed that the horse is a dead-end host of S. neurona due to the lack of formation of this stage in the muscle or other organs of this host (Dubey and Lindsay 1996b; MacKay 1997).

Nothing is known about the life cycle and pathogenicity of the 1085 isolate of Sarcocystis. Sporocysts from this isolate were collected from naturally infected opossums (Tanhauser et al., 1999). They have been shown to be different from S. neurona and S. falcatula by agarose gel electrophoresis of PCR products using different restriction enzymes (J.B. Dame, personal communication; Tanhauser et al. 1999). It is assumed that it will have a similar life cycle to that of S. neurona and S. falcatula, although this needs to be confirmed. There is also a possibility that this species has been named previously and that documenting the occurrence in the opossum will provide







8

scientists with information to more fully understand the life cycle of the organism. The work on this isolate began by molecularly characterizing it and will continue by attempting to identify intermediate hosts that can become infected by ingestion of sporocysts.

The objective of this study was to elucidate the intermediate hosts of one

or more of the following species: Sarcocystis neurona, Sarcocvstis strain 1085, and Sarcocystis speeri. The author either: inoculated live candidate animals with sporocysts to determine if sarcocysts would form; or fed Virginia opossums sarcocyst infected muscles from various animals to determine if they would shed sporocysts. It is hypothesized that the intermediate hosts for S. neurona, the 1085 Sarcocvstis isolate, and/or S. speeri are wild animals in Florida. The animals become infected with S. neurona, 1085, and/or S. speeri by ingestion of sporulated sporocysts and they form sarcocysts in muscle tissue that can then infect the common Virginia opossum and cause this animal to shed sporocysts. The major emphasis for this project was on S. neurona due to its devastating influence on horses.

The term opossum refers to members of Didelphidae. These animals are only found in the New World. Although there are relatives of opossums on the continent of Australia called possums, they are not considered for this discussion. The large, American opossums are marsupials that span much of the land mass of North, Central, and South America. Opossums also occur on some of the islands of the Lesser Antilles (Nowak and Paradiso 1983). Best known for their innate ability to get hit by cars and feed vultures, opossums are very complex creatures that are composed of 75 species and they fill many environmental niches. Opossums of 11 different genera range from







9

southern Argentina to southern Canada. Animals can be found in habitats ranging from those at or below sea level to upwards of 9,000 feet above sea level in the mountains of Central and South America. Specific habitats are generally associated with water and include tropical forest (scrub, deciduous, evergreen, rain, and cloud), savanna, thorn forest, temperate oak-pine forest, and prairie/mesquite grasslands (Gardner 1973).

The hair of Didelphis virginiana is unique to the Dildelphidae in that it consists of long, thick, soft woolly underfur of approximately 40 to 50 mm in length with an outer long coat of coarse white-tipped guard hairs that are approximately 60 to 80 mm long (Allen 1901). Guard hairs are rare or absent in other species of opossums (Nowak and Paradiso 1983). The tail is generally shorter (270 to 350 mm) than the combined length of the head and body, with the head and body being approximately 450 to 520 mm. The tail contains long body hair for approximately 2 inches starting at the base. The naked portion of the tail is a brown to gray color with flesh tones mixed in between. The base of the tail can be a darker, even blackish color (Allen 1901). Body color can range from black to white (mutant), with grey being the common body fur color. The black coloration is uncommon north of Georgia and the gulf coast states but is common in the southeastern US and southward to Central America (Gardner 1982). Ears are black with a broadly flesh color or narrowly edged with this color. Ears are smaller in the northern than the southern opossums. In tropical areas, the opossum's ear has a greater superficial area than in extreme northern specimens (Allen 1901). The larger ear size in tropical specimens may help dissipate heat which would be deleterious to northern animals. Lower legs are normally a lighter color, but feet can also be a black color in certain subspecies (Hall 1981).







10

Currently, the Virginia opossum can be found in most habitats from sea level to those above 9000 feet high, although they tend to favor moist woodlands and thick brush near water (Gardner 1982; Nowak and Paradiso 1983). Along the eastern seaboard, the opossum previously was found no farther north than the Hudson River Valley and reached into the Northeast United States in only the early 1900s (Guilday 1958). Opossums have been moving north, reaching as far as southern Ontario, Canada by 1956. Now, they occur in many parts of Canada. Northern limits of the population appear to be controlled only by winter conditions, availability of food, and prevalence of den sites. All populations of opossums from Canada and the United States that are located west of the Great Plaines are the result of transplants from the eastern United States, many of which were introduced via human intervention (Gardner 1982).

Adults of D. virginiana, both male and female, in the wild seldom live past 2 years of age with one author determining the life expectancy in one area to be a little more than 15 months. However, laboratory kept opossums can be maintained for upwards of 4 years, suggesting that predators and natural phenomena take a heavy toll on the wild opossum (Gardner 1982; Lee and Cockburn 1985).

Didelphis virginiana is a scavenger and there is no evidence that they compete with local North American placental mammals (Pough et al. 1999). However, many marsupials have similar niches to placentals, leading to the possibility of some competition, however small. Many studies of stomach and scat contents have been conducted and confirm the fact that opossums are opportunistic omnivorous feeders (Gardner 1982). D. virginiana often scavenges rotten flesh and many times even cannibalizes members of its own species. A larger portion of its diet, however, is







11

comprised of insects, earthworms, and vegetation. Although many have claimed otherwise, D. virginiana apparently is not a good predator and does not normally prey on chickens, rabbits, waterfowl, or other game (Gardner 1982). The food items found in the stomachs of opossums vary from region to region and from season to season, and appear to be based mainly on what food items are available in the local environment. To initiate the search for the intermediate hosts of the Sarcocystis spp. that parasitize the opossum, it is necessary to be familiar with the diet of the opossum. Knowing what species of animals are available to the opossum in its area narrows the field of intermediate host candidates. Unfortunately, the opossum consumes a wide variety of animals. Animal species found in opossums stomach/fecal contents are listed in Table 1-1.







12

Table 1-1. Spectrum of animals found in the gut contents or feces of Virginia
opossums

Animal Species Location Reference
Bufo americanus/Bufo sp. New York, Florida, Texas Pournelle 1950; Hamilton (Toad) 1951; Wood 1954 Rana pipiens/Rana sylvatica New York Hamilton 1951 (Frog)
Ambystoma New York, Michigan Taube 1947; Hamilton jeffersonianum/Ambystoma 1951 maculatum (Mole
salamander)
Hyla versicolor (Tree frog) New York Hamilton 1951 Plethodon New York Hamilton 1951 cinereus/Plethodon
glutinosus (Woodland
salamander)
Sylvilagus floridanus New York, Texas, Taube 1947; Hamilton (Eastern cottontail rabbit) Michigan 1951; Wood 1954 Glaucomys sabrinus Oregon Hopkins 1980 (Northern flying squirrel)
Sciurus rufiventer (Fox Michigan Taube 1947 squirrel)
Didelphis virginiana New York, Texas, Taube 1947; Hamilton (Virginia opossum) Michigan, Pennsylvania, 1951; Wood 1954; Oregon Blumenthal 1976; Hopkins 1980
Mephitis mephitis (Striped Michigan Taube 1947 skunk)
Spilogale gracilis (Spotted Oregon Hopkins 1980 skunk)
Felis catus (Domestic cat) Oregon Hopkins 1980 Microtus New York, Michigan, Taube 1947; Hamilton pennsylvanicus/Microtus sp. Pennsylvania 1951; Blumenthal 1976 (Meadow vole)
Scalopus aquaticus (Eastern Michigan Taube 1947 mole)
Blarina brevicauda New York, Michigan, Taube 1947; Hamilton (Northern short-tailed Pennsylvania 1951; Blumenthal 1976 shrew)







13

Table 1.1-continued

Animal Species Location Reference
Peromvscus New York, Texas, Hamilton 1951; Wood leucopus/Peromyscus sp. Pennsylvania 1954; Blumenthal 1976 (Deer and white-footed
mouse)
Mus musculus (House Pennsylvania, Oregon Blumenthal 1976; Hopkins mouse) 1980 Simodon sp. (Cotton rat) Texas Wood 1954 Tamias sp. (Chipmunk) New York Hamilton 1951 Sorex cinereus (Masked New York Hamilton 1951 shrew)
Ondatra zibethicus New York Hamilton 1951 (Muskrat)
Zapus sp. (Jumping mouse) New York Hamilton 1951 Condvlura sp. (Star-nosed New York Hamilton 1951 mole)
Rattus sp. (Old world rat) New York, Oregon Hamilton 1951; Hopkins 1980
Neurotrichus gibbsii (Shrew Oregon Hopkins 1980 mole)
Cryptotis sp. (Small-eared New York Hamilton 1951 shrew)
Lumbricus New York, Michigan, Taube 1947; Hamilton terrestris/Lumbricus sp. Pennsylvania, Oregon 1951; Blumenthal 1976; (Earthworm) Hopkins 1980 Triodopsis sp. (Snail) New York, Michigan Taube 1947; Hamilton 1951
Anguispira sp. (Snail) New York, Michigan Taube 1947; Hamilton 1951
Deroceras reticulatum (Slug) New York Hamilton 1951 Arion spp. (Slug) New York, Oregon Hamilton 1951; Hopkins 1980
Thamnophis sp. (Ribbon Texas Wood 1954 snake)
Thamnophis sirtalis New York Hamilton 1951 (Common garter snake)







14

Table 1.1-continued

Animal Species Location Reference Storeria occipitomaculata New York Hamilton 1951 (Red-bellied snake)

Diadophis punctatus (Ring- New York Hamilton 1951 necked snake)
Lampropeltis triangulum New York Hamilton 1951 (Milk snake)
Storeria dekavi (DeKay's New York Hamilton 1951 snake)
Thamnophis sp. (Garter Oregon Hopkins 1980 snake)
Sonora semiannulata sp. Texas Wood 1954 (Southern ground snake)
Agkistrodon contortrix Texas Wood 1954 (Copperhead snake)
Family Scincidae (Skink) Texas Wood 1954 Sceloporus sp.(Fence lizard) Texas Wood 1954 Natrix sipedon (Lake Erie New York Hamilton 1951 water snake)
Chelydra serpentina New York Hamilton 1951 (Snapping turtle)
Terrapene sp.(Box Turtle) New York Hamilton 1951 Phasianus colchicus (Ring- New York, Michigan Taube 1947; Hamilton necked pheasant) 1951 Turdus sp. (Robin) New York, Florida, Pournelle 1950; Hamilton Oregon 1951; Hopkins 1980 Corvus sp.(Crow) New York Hamilton 1951 Sturnella sp.(Meadowlark) New York Hamilton 1951 Quiscalus quiscula (Bronzed New York Hamilton 1951 Grackle)
Pooecetes gramineus New York Hamilton 1951 (Vesper Sparrow)
Family Anatidae (Duck) New York Hamilton 1951 Agelaius phoeniceus (Red- New York Hamilton 1951 winged blackbird)
Sphvrapicus varius (Yellow- Texas Wood 1954 bellied sapsucker)







15

Table 1.1-continued

Animal Species Location Reference Zenaida macroura Texas Wood 1954 (Mourning dove)
Thyromanes bewickii Oregon Hopkins 1980 (Bewick's Wren)
Pipilo sp. (Towhee) Texas, Oregon Wood 1954; Hopkins 1980 Gallus domesticus (Chicken) Texas, Michigan Taube 1947; Wood 1954














CHAPTER 2
SPOROCYST SIZE OF ISOLATES OF SARCOCYSTIS SHED BY THE VIRGINIA OPOSSUM (Didelphis virginiana)'


Introduction

Equine protozoal myeloencephalitis (EPM) is a severe neuromuscular disease affecting many horses in North and South America (Dubey et al. 1991; Granstrom et al. 1992; MacKay 1997; Masri et al. 1992). While the disease has been diagnosed for several years, only recently have causative agents been identified. Two different organisms have been implicated as causative agents of EPM: Sarcocystis neurona and Neospora hughesi (Cheadle et al. 1999; Dubey et al. 1991; Marsh et al. 1998). While information on N. hughesi is emerging, the contribution of this organism to EPM is unknown. The main focus of research regarding EPM centered on S. neurona. The definitive host of S. neurona, the Virginia opossum (Didelphis virginiana), sheds sporocysts of several types of Sarcocystis spp. in its feces. The current method used to differentiate these organisms is DNA-based marker analysis developed in this laboratory (Tanhauser et al. 1999). In our paper, we assess the potential to differentiate the five types of sporocysts based on light microscopy.






'Paper published with coauthors under the citation "Cheadle, M.A., Dame, J.B., and E.C. Greiner. 2001. Sporocyst size of isolates of Sarcocystis shed by the Virginia opossum (Didelphis virginiana). Veterinary Parasitology 95: 305-311."

16







17

Materials and Methods

Sporocyst Collection and Storage

Automobile-killed opossums were collected and feces and/or gut scrapings were examined for the presence of Sarcocystis sporocysts using sugar flotation with centrifugation. For samples found to be positive for the presence of sporocysts, intestines were removed and the mucosa was scraped using the edge of a glass slide to collect sporocysts contained within the mucosa. The collection was then placed into a solution containing 50% bleach and water, mixed thoroughly, and incubated on ice for 30 min. After filtration through gauze, the sporocysts were washed by repetitive centrifugation and resuspension in distilled water to remove residual amounts of bleach. Once the bleach was removed, we placed storage media (Hanks Balanced Salt Solution, 500 mL) plus antibiotics [10 x 104 I.U. penicillin and 10 x 104 lag streptomycin (Mediatech, Herndon, Virginia)] and fungicide [250 ig amphotericin (Mediatech, Herndon, Virginia)] in the tube containing the sporocyst mixture. The sporocysts were stored at 40 C until use.

Sporocyst Typing

Sporocyst type was elucidated using restriction enzyme analysis as described by Tanhauser et al. (1999). Sarcocvstis speeri was differentiated based on sequence analysis (J. B. Dame, personal communication).

Observation and Measurement

Sporocyst isolates used from each taxa are as follows: S. neurona (3027, 3113,

3120, 3063); 1085 type (3013, 1114, 1086, 1085); S. falcatula (3114, 3106, 3105, 3101, 3021); S. speeri (2226, 2079, 2046); 3344 type (3344). Tubes containing stored







18

sporocysts of each isolate were removed from refrigeration and an aliquot of approximately 10 pL was placed on the center of a glass slide. To prevent deformation of the sporocysts caused by the weight of the coverslip, four small dots of glycerine jelly (Fisher, Fair Lawn, New Jersey) were applied to each corner of a glass coverslip. The coverslip was then placed over the 10 tL of media forming a hanging drop. The slide was then observed on a light microscope under oil at 1000X magnification. Twenty sporocysts of each isolate from the five taxa were measured using a calibrated ocular micrometer. Sporocysts also were observed for differences in internal structure. Data Analysis

Because length and width data were not normally distributed (P <0.001 by the Kolmogorov-Smirnov test for normality), we analyzed our findings by the KruskalWallis one-way analysis of variance on rank (P < 0.05). Where a significant difference was found, pairwise multiple comparisons were made using Dunn's method. All statistical calculations were carried out using SigmaPlot@ and SigmaStat@ software for windows version 2.03 (SPSS, Inc., Illinois).

Results

Results of sporocyst measurements are presented in Table 2-1. The length and

width of S. speeri (Fig. 2-1) was statistically different (P < 0.05) from the other sporocyst isolates. The length of S. neurona (Fig. 2-2) and S. falcatula (Fig. 2-3) sporocysts were statistically different (P < 0.05) from each other and the width of S. falcatula and 1085 type (Fig. 2-4) differed (P < 0.05).

Due to the obvious size difference of the 3344 type (Fig. 2-5) sporocyst, statistical data were not compared between it and the other isolates. This type has been found in







19

the feces of 10 opossums. This sporocyst is morphologically different from other isolates because it is much larger and has more pointed ends with plug-like structures (Fig. 2-6). Although other sporocyst isolates have a more rounded shape and are devoid of the pluglike structures (Figs. 2-1 thru 2-4), they do exhibit a similar refractory quality when observed using light microscopy. Diagnosis and collection were similar to diagnosis and collection of other taxa. However, this sporocyst type was very difficult to store because of the trait of collapsing after collection. As seen in Fig. 2-7, the sporocyst collapses within minutes of processing as compared to other taxa that maintain wall integrity throughout processing and storage. After collection and storage, intact sporocysts were not observed and thus the isolate has yet to be characterized molecularly.

The internal morphology of the sporocysts was observed. All sporocysts

contained four sporozoites and a residuum. Although there was no consistent difference between internal morphology of isolates, there appeared to be many forms. Forms ranged from a single, large, ball-like residual body approximately one-third the size of the sporocyst to upwards of 20 small, ball-like residual bodies. Multiple, small bodies were found in compact areas and diffuse throughout the inside of the sporocyst. There did not appear to be any trend that would allow the differentiation of type based on the makeup of the sporocyst residuum.

Discussion

Based on gross visual observations by light microscopy, only S. speeri and 3344 type sporocysts were different from the other similar isolates found in the feces of the opossum. Single-dimension differences such as S. falcatula vs 1085 (width) and S. falcatula vs S. neurona (length), were not considered sufficient to differentiate between







20

the species/types. The 3344 type of sporocyst was originally thought not to be a sporocyst, but after close examination, 4 sporozoites and a distinct residuum were observed (Fig. 2-6). Dubey et al. (1989) did not describe sporocysts of this size being found in the feces of the opossum or any other definitive host. This type composed <5% of infections with sporocysts found in the feces and/or gut scrapings. Because this sporocyst collapses soon after flotation, fecal flotations of this preparation should be read immediately after the flotation procedure is finished. We were unable to determine why this sporocyst type exhibits this characteristic and whether this type uses the opossum as a host or simply is contained within a food item that is being passed through the gut during the time of sampling.

The isolates in this study cannot be proven to be pure isolates. There is no way to insure that there is only a single sporocyst species/type present in the primary isolates collected from opossums used in this study. However, based on molecular data showing that the isolate is of one species, the authors feel that if there is a mixed infection in the opossum, the number of sporocysts of the second species/type would be low and should not influence the outcome of this analysis.







21

Table 2-1. Comparison of Sarcocystis sporocyst sizes Taxa Length(Am) Width (um) Mean(SE) Range Mean(SE) Range Sarcocystis 10.65 (0.09)a 9.7-11.4 7.03 (0.09)ab 6.2-8.4 neurona
1085 type 10.85 (0.07)ab 9.7-11.9 6.81 (0.07)a 6.2-7.9 Sarcocystis 10.95 (0.13)b 8.8-11.9 7.05 (0.10)b 6.6-7.9 falcatula

Sarcocystis 12.23 (0.14)c 11.0-13.2 8.81 (0.09)c 7.5-9.7 speeri
3344 type 19.43 (0.20)' 17.6-20.7 10.49 (0.20)' 9.2-11.9 SE = Standard error of the mean a, b, c = Isolates with the same superscript within columns designate no significant differences between isolates
* = Statistical analysis not performed on this isolate








22


















Fig. 2-1. Sporocyst of Sarcocystis speeri (2079 isolate). Note the residual body
(arrowhead). Bar = 6 pm.




















Fig. 2-2. Sporocyst of Sarcocystis neurona (3063 isolate). Note the sporozoites (arrow)
and residual body (arrowhead). Bar = 5.5 utm.








23

















Fig. 2-3. Sporocyst of Sarcocystisfalcatula (3106 isolate). Note the sporozoites
(arrowhead). Bar = 5.5 [pm.



















Fig. 2-4. Sporocysts of 1085 type (3013 isolate). Note the sporozoite (arrow) and
residual body (arrowhead). Bar = 5 upm.








24















Fig. 2-5. Sporocyst of 3344 type (3344 isolate). Note the sporozoite (arrowhead). Bar =
4.8 jim
























Fig. 2-6. Detailed view of sporocyst of 3344 type (3344 isolate). Note the polar plug-like
structures (double arrowhead), sporozoite (arrow), and residual body
(arrowhead). Bar = 4.8 um.







25





















Fig. 2-7. Brightfield micrograph of 3344 type sporocyst. Collapsed sporocyst of 3344
type (isolate 3395). Photographed 10 minutes post flotation. Bar = 6.5 im.














CHAPTER 3
VIABILITY OF Sarcocystis neurona SPOROCYSTS AND DOSE TITRATION IN GAMMA-INTERFERON KNOCKOUT MICE2


Introduction

Sarcocystis neurona is a causative agent of the neuromuscular disease equine protozoal myeloencephalitis (EPM) in horses and CNS disease in southern sea otters (Dubey et al. 1991; Lindsay et al. 2000; MacKay 1997). S. neurona has been isolated from horses in North and South America (Dubey et al. 1991; Granstrom et al. 1992). Neospora hughesi has also been shown to be a causative agent of EPM, however, data concerning its involvement in the disease is still emerging (Cheadle et al. 1999). While the occurrence of EPM in horses is low, the prevalence of antibodies to S. neurona in the United States is approximately 50% and approximately 35% in South America (Dubey et al. 1999a; Dubey et al. 1999c; MacKay 1997). The definitive host of S. neurona has been identified as the Virginia opossum (Didelphis virginiana) (Fenger et al. 1995 and 1997). Currently, there appear to be five different taxa of Sarcocystis sporocysts being shed in the feces of opossums (Cheadle et al. 2001 a). The current methods used to differentiate Sarcocvstis sporocysts being released in the feces of opossums are DNA-based marker analysis and morphological examination developed in this laboratory (Cheadle et al. 2001 a; Tanhauser et al. 1999).


2Paper published with coauthors under the citation "Cheadle, M.A., Tanhauser, S.M., Scase, T.J., Dame, J.B., MacKay, R.J., Ginn, P.E., and E.C. Greiner. 2001. Viability of
Sarcocvstis neurona sporocysts and dose titration in gamma-interferon knockout mice. Veterinary Parasitology 95: 223-232."
26







27

Until recently, no model for the study of the EPM was readily available. Dubey and Lindsay (1998a) found that gamma-interferon knockout mice inoculated with uncharacterized sporocysts collected from the feces of the opossum developed clinical encephalitis. The tissues from these animals were found to contain stages of Sarcocystis neurona as determined by immunohistochemical staining of tissue sections and an immunofluoresent antibody test (IFAT) of cell cultured organisms. In order to ensure quality control in our laboratory, it was necessary to test the viability of S. neurona sporocysts in a banked collection and to also determine a low dose of sporocysts that can cause clinical signs in gamma-interferon knockout mice. Thus, in the present study, the use of gamma-interferon knockout mice as a model for S. neurona is expanded by determining the viability of molecularly characterized S. neurona sporocysts and the dose of sporocysts required to cause a clinical infection in these mice. We also correlate molecular identification of sporocysts with clinical disease in knockout mice.

Materials and Methods

Collection of Sporocysts

Sporocyst collection was similar to that described by Cheadle et al. (2001a).

Opossums were collected and feces and/or gut scrapings were checked for the presence of Sarcocystis sporocysts using sugar flotation with centrifugation. Intestines of opossums found to be positive for the presence of sporocysts were removed and scraped. The collection was then cleaned using NaOCI, washed to remove bleach, and stored in storage media at 40 C. Sodium dodecyl sulphate (SDS) was substituted for NaOCI when isolate number 3227 was treated to determine if differences in viability would exist.







28

Identification of Sporocyst Type

Sporocyst type was elucidated using restriction enzyme analysis and

morphological analysis as described by Tanhauser et al. (1999) and Cheadle et al. (2001a).

Animals

Eighteen gamma-interferon knockout mice (BALB/c-Ifngm"TS) were obtained

from Jackson Laboratories (Bar Harbor, ME). These mice lack the gene that encodes the production of gamma-interferon, a component of the cellular immune response, which is critical to host defense against protozoal organisms. All study mice were 46 days old and male. Mice were contained within isolation boxes and housed at the Department of Animal Resources, University of Florida. Sterilized commercial mouse ration and water were fed ad libitum during the course of the study. Inoculation Procedure

Each mouse was inoculated with Sarcocystis neurona sporocysts via gastric

gavage. Doses given to each mouse are listed in Table 3-1. Each mouse was observed daily for the development of clinical signs. To reduce consumption of a single isolate, four similar-aged isolates (3048, 6 months old; 3063, 6 months old; 3113, 4 months old; 3120, 4 months old) were mixed in equal proportion and inoculated at five different doses: 500 sporocysts, 1,000 sporocysts, 5,000 sporocysts, 20,000 sporocysts, and 50,000 sporocysts. Sporocyst isolates 3027 (7 months old) and 3227 (2 months old) were inoculated in a similar manner at doses of 500 sporocysts, 5,000 sporocysts, and 50,000 sporocysts. Mouse M17 received 5,000 sporocysts of an equal mix of isolates 1067, 1112, 2011, 2033, 2043, 2103, 2052A, and 2052B, which were 16 months to 28 months







29

old. Inoculum consisted of sporocysts in 0.1 mL of a solution that contained Hanks Balanced Salt Solution (HBSS) (500 mL) plus antibiotics (10 x 10 I.U. penicillin and 10 x 104 4g streptomycin (Mediatech, Herndon, VA)) and fungicide (250 ,g amphotericin (Mediatech, Herndon, VA)). Mouse M18 was inoculated with sterile HBSS. Necropsy and Histopathology

A necropsy was performed on all mice that died or were euthanized. The

following tissues were removed: lung, liver, kidney, muscle, stomach, intestine, brain, heart, testicle, pancreas, and spleen. Portions of these organs were kept fresh and fixed in 10% buffered formalin. Fixed tissues were submitted to the Department of Pathology, University of Florida for embedding, sectioning, and staining using hematoxylin and eosin (H & E). Sections were observed for the presence of lesions and Sarcocystis organisms using light microscopy at a power of 100 and 400X. Immunohistochemistry was performed using a polyclonal antibody to S. neurona to facilitate visualization of organisms (Hamir et al. 1993).

Results

Clinical signs observed in the gamma-interferon knockout mice were similar to the neurologic signs observed to occur in horses diagnosed with EPM (MacKay 1997) (Table 3-3). These included paralysis, rough coat, huddling, eye squinting, mild to severe ataxia in limbs, mild to severe head tilt, and circling. The clinical signs observed varied depending on the mouse and the dose. During the course of the 90-day observation period, mice M11 and M 17 never showed clinical signs consistent with neurologic disease. M 11 did appear to have a roughened coat during various days of the study, but clinical signs never progressed beyond that point. Mouse M 17 and M 18







30

appeared normal through the entire course of observation.

Organisms were observed in tissue sections of the cerebrum and/or cerebellum in 13 of 17 (77%) knockout mice inoculated with sporocysts (Table 3-2). No other organ was observed to contain organisms. Fewer numbers of organisms were observed in H&E sections compared to the number of organisms observed using immunohistochemistry. Of the 15 mice that were examined using immunohistochemical techniques, 12 (80%) had protozoal organisms present in one or more portions of the brain. Two mice were not processed using immunohistochemistry. Protozoal merozoites were frequently observed in groups (Figs. 3-1 and 3-2). Mouse M18 had no lesions.

Lesions observed in mice were closely associated with, but not confined to, the presence of organisms. The lesions and number of mice affected is presented in Table 3-4. The brain and lungs were the two areas of parasite focus and lesions. In the brain, large areas of necrotizing meningoencephalitis with associated lymphocytic and neutrophilic infiltrates were observed. Similarly, the lung contained large numbers of peribronchiolar infiltrates of neutrophils and lymphocytes associated with a necrotizing interstitial pneumonia. The lesions in other organs were less consistent and not as severe.

Discussion

The visualization of protozoal organisms in this study was facilitated by the use of immunohistochemistry. The results are similar to the observations made by Dubey and Lindsay (1998a), where more organisms were identified using immunohistochemistry compared to H & E stained sections. In the same study, the brain and liver were deemed to be the areas of primary infection in the knockout mice. In this study, however, the constant and severe hepatitis documented by Dubey and Lindsay







31

(1998a) was not observed. Clinical signs that can be attributed to brain infection were consistently observed and therefore the brain was the organ on which our investigation was focused. Although the spleen also consistently contained lesions, parasites were never observed in this organ. Based on the author's experience, lesions in the spinal cord of horses are very difficult to find. Conversely, lesions in the brains of gamma-interferon knockout mice have been consistently observed by others, therefore the spinal cord was not used in this study (Dubey and Lindsay 1998a).

Mouse M 11 did not show clinical signs after receiving a low dose (500) of

sporocysts of an isolate that was at the age and dose limit used in this study. Because other isolates inoculated at this dose also cause disease in mice and that higher doses of this isolate did cause disease in mice, it is likely that few of the remaining sporocysts were viable and that age was the factor that limited the ability of the sporocysts of this isolate to cause clinical disease. All mice inoculated with higher doses of this isolate (3027) had an increased time to clinical signs and death/euthanasia as compared to other isolates. The mouse (M17) inoculated with isolates 1067, 1112, 2011, 2033, 2043, 2103, 2052A, and 2052B never showed clinical signs during the course of the 56 dpi observation period. The age of these isolates ranged from 16 to 28 months old at time of inoculation into mice, indicating that, in our laboratory, sporocysts that have been processed and stored for more than 16 months are not viable. The authors show that a base dose of 500 sporocysts of inoculum that is <:7 months old at time of inoculation can be used for experiments using the gamma-interferon knockout mice (BALB/c-Ifngmlrs) as a model for neurologic infection with Sarcocystis neurona.

This laboratory (ECG) has recently changed its method of storing sporocysts to







32

one in which the mixture is not bleached until an infection trial is about to begin. Although the authors in this paper indicates that sporocysts that have been bleached and stored (< 7 months) are still infectious, long-term storage after bleach treatment does decrease the number of viable sporocysts remaining in the inoculum. We have adjusted our procedure to store sporocysts in a Hanks solution similar to that used to suspend sporocysts during inoculation as described previously in this paper and bleach only directly prior to use.

Although the brain of mouse (M 13) did contain lesions, no organisms were observed. The reason for this lack of organisms is unknown. However, the mouse regurgitated a small portion of the sporocyst mixture during the inoculation procedure. The portion was approximately one tenth of the entire inoculum. It is likely that the mouse received a decreased number of sporocysts that, while they caused clinical signs, were to low in numbers to be detected by light microscopy. Also, the 3027 isolate is at the threshold of age limits used in this study and viability is likely decreased. The course of the disease was also delayed in onset as compared to other mice. These factors lead to the question of the possibility of some other factor such as a toxic substance being produced by the organism which may be a cause of disease rather than simple physical damage to the host cells.

The authors have confirmed that sporocysts determined to be Sarcocystis neurona by molecular characterization were are able to infect and cause disease in a susceptible host. The link between the molecular and biological data strengthens the ability of researchers to differentiate S. neurona sporocysts from other similar sporocysts being shed in the feces of the opossum. This study validates the use of molecular







33

characterization of sporocysts collected from the opossum as compared to the time consuming and expensive use of mice to type isolates.

Based on data by Dubey and Lindsay (1999) and Dubey et al. (2000d),

Sarcocystis speeri can induce sarcocyst formation in skeletal muscle and encephalitis in the brains of knockout mice. Although encephalitis induced by S. speeri has been documented, no clinical signs were reported (Dubey et al. 2000d). Also, studies by Dubey and Lindsay (1998a), have shown that Sarcocystis falcatula does not infect gamma-interferon knockout mice. Based on sporocyst measurements made before inoculation, the sporocysts in this inoculum were smaller (- 11 x 7 kim) than those of S. seri (-12 x 9 pm). Furthermore, all mice in the study by Dubey and Lindsay (1998a) lacked sarcocysts and/or organisms in their skeletal muscle. Therefore, it is assumed that S. neurona sporocysts were the infectious agent in this study and that they are the only Sarcocystis sp. in the 11 x 7 /m range that are shed in the feces of the opossum that can cause clinical signs consistent with neurological disease in gamma-interferon knockout mice. Further studies comparing the clinical signs caused by S. neurona with those caused by S. speeri and other species of Sarcocvstis being shed by the opossum need to be conducted to further define the mouse model.

Ensuring the viability of sporocysts to infect a host is critical. While excystation data derived in the laboratory will provide information on the ability of sporozoites to be released from a sporocyst and infect a cell monolayer, it provides no information about the ability of those sporozoites to properly infect a host and cause disease. In order to study the life cycle of S. neurona, viable sporocysts must be available that can infect intermediate host candidate species. Without first ensuring the capacity of sporocysts to







34

cause disease in a model animal, there is no way to determine whether or not the intermediate host candidate received a reliable challenge.

High expense and large amounts of time are required to house and maintain an

experimental animal such as a horse. The use of the gamma-interferon knockout mice as an experimental model for EPM in horses is an effective means of studying the disease in a laboratory setting. The clinical signs exhibited are similar in severity and duration. The use of this model allows the testing of chemotherapeutic drugs and vaccine candidates before implementation of expensive trials in horses.







35

Table 3-1. Dose and isolate/s of Sarcocystis neurona given to each gamma-interferon
knockout mouse
Mouse Number of Isolate/s Age of sporocysts number sporocysts counted at time of mouse in inoculum inoculation (months)
M1 500 3048,3063,3113,3120 4 to 6 M2 500 3048,3063,3113,3120 4to 6 M3 1,000 3048,3063,3113,3120 4to 6 M4 1,000 3048,3063,3113,3120 4to 6 M5 5,000 3048,3063,3113,3120 4to 6 M6 5,000 3048,3063,3113,3120 4 to 6 M7 20,000 3048, 3063, 3113, 3120 4 to 6 M8 20,000 3048, 3063, 3113, 3120 4 to 6 M9 50,000 3048, 3063, 3113, 3120 4 to 6 M10 50,000 3048,3063,3113,3120 4 to 6 M11 500 3027 7 M12 5,000 3027 7 M13 3027 7 50,000
M14 500 3227 2 M15 5,000 3227 2 M16 50,000 3227 2 M17 5,000 1067,1112,2011,2033, 16to 28 2043,2103,2052A, 2052B
M18 control not applicable not applicable







36

Table 3-2. Microscopic identification of protozoa in routinely and
immunohistochemically treated sections of cerebrum and cerebellum from
gamma-interferon knockout mice inoculated with Sarcocystis neurona
sporocysts
Mouse number Protozoa observed using Protozoa observed using H & E light microscopy immunohistochemistry
M1 + Cerebrum + Cerebrum M2 + Cerebrum + Cerebrum M3 + Cerebellum, cerebrum + Cerebellum, cerebrum M4 + Cerebrum + Cerebrum M5 + Cerebrum n/a M6 + Cerebrum + Cerebrum M7 n/a M8 + Cerebellum + Cerebellum M9 + Cerebrum + Cerebellum, cerebrum M10 + Cerebellum, cerebrum Mll
M12 + Cerebellum, cerebrum M13
M14 + Cerebellum, cerebrum + Cerebellum, cerebrum M15 + Cerebrum + Cerebellum, cerebrum M16 + Cerebellum, cerebrum + Cerebellum, cerebrum M17
M18*
n/a = sample not available
* = M18 is a negative control
- = no protozoa observed
+ = protozoa consistent with schizonts of Sarcocystis observed in tissues







37

Table 3-3. Summary of disease in Sarcocystis neurona infected gamma-interferon
knockout mice
Mouse First clinical signs Animal dead or Clinic signs observed during number observed (dpi) euthanized (dpi) course of infection
M1 22 33 rough coat, paralysis in rear limbs
M2 28 31 rough coat, huddling, mild ataxia in limbs
M3 28 32 rough coat, huddling, severe ataxia in limbs
M4 28 32 rough coat, huddling, eye squinting, severe ataxia in limbs M5 10 25 rough coat, severe head tilt M6 20 24 rough coat, mild to severe head tilt
M7 22 28 rough coat, huddling, severe head tilt, circling
M8 24 28 rough coat, moderate ataxia in limbs
M9 19 28 rough coat, mild head tilt M10 21 22 rough coat, slight to severe head tilt, circling
Mll n/a 90 none M12 10 35 rough coat, huddling, eye squinting, high stepping, mild head tilt, slight to severe ataxia in limbs
M13 28 33 rough coat, huddling, slight to severe head tilt, circling
M14 28 31 rough coat, huddling, slight to severe ataxia in limbs
M15 26 31 rough coat, huddling, mild ataxia M16 20 28 rough coat, huddling, slight head tilt, paralysis in rear limbs
M17 none 68 none M18 none 68 none dpi = days post inoculation







38

Table 3-4. Histopathological lesions observed in gamma-interferon knockout mice orally
inoculated with Sarcocystis neurona sporocysts
Number of
mice
Organ with lesion/s Lesion/s Lung 15 Multifocal to extensive necrotizing interstitial pneumonia, peribronchiolar infiltrates of neutrophils and lymphocytes
Liver 1 Aggregates of neutrophils and hemosiderophages scattered throughout hepatic sinusoids. Stomach 8 Multifocal to extensive neutrophilic gastritis Skeletal 1 Perivascular infiltrates of neutrophils in supporting muscle fibrovascular connective tissue stroma Heart 3 Accumulations of mineralized material present in subepicardial fibrovascular connective tissue stroma Spleen 14 Periarteriolar follicular hyperplasia Cerebrum 12 Multifocal necrotizing meningoencephalitis, lymphocytic and neutrophilic infiltrates Cerebellum 5 Multifocal to extensive histiocytic and neutrophilic meningoencephalitis.
Pancreas No lesions Intestine No lesions Kidney No lesions Testicle No lesions
- = no lesions observed








39



















Fig. 3-1. H & E stained section of cerebellum from mouse M8 containing a group of
merozoites (arrowhead). Bar = 8.8 pm.






















Fig. 3-2. Immunohistochemically stained schizont of S. neurona from mouse M8
cerebellum containing multiple merozoites (arrowhead). Bar = 8.8 ,m.














CHAPTER 4
Sarcocystis greineri N. SP. (PROTOZOA: SARCOCYSTIDAE) IN THE VIRGINIA OPOSSUM (Didelphis virginiana)


Introduction

The Virginia opossum (Didelphis virginiana) has been implicated as the definitive host for at least four species of Sarcocystis (Box et al. 1984; Dubey et al. 1991; Dubey and Lindsay 1999b; Tanhauser et al. 1999). Little information has been reported about opossums being intermediate hosts of Sarcocystis spp. Sarcocysts were found in a D. virginiana by Scholtyseck et al. (1982), but not named. There are three other named species of Sarcocystis in other opossum species, Sarcocystis garnhami (Didelphis marsupialis, Philander sp.), Sarcocystis marmosae (Marmosa murina), and Sarcocystis didelphidis (Didelphis marsupialis) (reviewed by Odening 1998). During the course of routine necropsy of road-killed and live opossums for other research projects, sarcocysts were observed in skeletal muscle of D. virginiana. The purpose of the present paper is to describe and name this previously unnamed species of Sarcocystis that uses the Virginia opossum as an intermediate host.

Materials and Methods

Opossum Collection and Processing

Opossums were collected either as road-kills or were live trapped and killed (Beuthanasia-D Special, Schering Plough Animal Health, Kenilworth, New Jersey). Each animal was assigned an identification number and data concerning the date and


40







41

location of collection were recorded. Samples of muscle were removed from the tongue, abdomen, and rear thigh muscle. Small portions of muscle were removed from these samples and processed for light and electron microscopy. The remaining portion of the muscle sample was stored at 40 C for use in feeding trials. Light Microscopy

A small portion of muscle was fixed for histopathologic examination using 10% buffered formalin for a period of 24 to 48 hr. The tissue was processed, embedded, sectioned at 5 pm, and stained with hematoxylin and eosin. Sections of tongue and thigh skeletal muscle were scanned for the presence of sarcocysts using a dissecting microscope at a magnification of 25X. When a sarcocyst was detected, the cyst was removed and observed at 40X magnification on a compound microscope for verification of a sarcocyst wall. Sarcocysts as well as the bradyzoites contained within were measured, photographed, and the sizes recorded. Feeding Trials

Approximately 112 g of skeletal and tongue muscle tissue from a single opossum observed to contain sarcocysts was fed to a greyhound dog (Canis familiaris) and a domestic short hair cat (Felis catus). Fecal samples were collected at 3-day intervals during the course of the trial from Days 0 (dog) and -5 (cat) post inoculation (PI) through Day 30. Samples were refrigerated at 40 C until processed. Sugar flotation was used to detect the presence of Sarcocystis sp. sporocysts in all fecal samples. The cat was killed and the intestine removed. The mucosal epithelium was scraped with a glass slide. The scrapings were collected and observed using light microscopy for the presence of Sarcocystis sp. sporocysts. Dog intestine and tissues were not available at the end of the







42

trial. Sections of liver, lung, kidney, brain, heart, spleen, and muscle were removed from the cat and observed by light microscopy for stages of Sarcocystis.

A wild-caught opossum determined to be negative for Sarcocystis by fecal

flotation was fed 76 samples of opossum tongue and skeletal muscle from 76 different opossums. Seven of these opossum samples contained sarcocysts as visualized by light microscopy. Fecal samples were collected at 3-day intervals during the course of the trial from Day -30 through Day 74 when the opossum was killed. Fecal samples and the intestine were treated as above. Sections of liver, lung, kidney, brain, heart, spleen, and muscle were removed from the opossum and observed by light microscopy for stages of Sarcocvstis.

Transmission Electron Microscopy

Muscle samples containing sarcocysts were removed from an opossum and

immediately immersion-fixed in a 1.5 mL plastic vial containing approximately 1 ml of cold Trumps fixative for at least 24 h. The tissue was then removed, placed in phosphate buffered saline (pH 7.3), and stored at 40 C until processed. Sarcocysts were observed and removed from muscles using a dissecting microscope. The cysts were washed two times for 10 min each in PBS. Samples were then postfixed in 1% osmium tetroxide (w/v) for 1 h and washed three times for a period of 10 min in double deionized water. Samples were then dehydrated in ethanol (25 and 50%) for 10 min, followed by a mixture of 75% ethanol and 2% uranyl acetate overnight at 40 C, and dehydrated further with 95 and 100% ethanol at 10 min each and 100% acetone two times at 10 min each. Flat embedding in EmBed epoxy resin (Ted Pella, Redding, California) included dilution steps using acetone (1:2, 1:1, 2:1) into a final concentration of 100% EmBed. Fresh







43

100% EmBed was added and allowed to polymerize at 600 C for 2 days. Thin (70-80 nm) sections were placed on formvar coated 50 mesh copper grids and double stained with 2% aqueous uranyl acetate followed by Reynolds lead citrate. Stained sections were then examined with a Hitachi H-7000 TEM operating at a voltage of 75 kv and images were taken with a Gatan Bioscan ccd camera.

Results

The cat, dog, and opossum fed infected opossum tissue did not shed sporocysts of Sarcocystis sp. during the course of this study. Scrapings from the opossum and cat intestines were also negative for the presence of sporocysts. No stages of Sarcocystis were observed in the tissue sections of the opossum and cat fed muscle tissue containing sarcocysts.

Description

Sarcocystis greineri n. sp.

Diagnosis. Sarcocysts occur in skeletal muscle of the tongue, thigh, and

abdomen. The sarcocysts visible using a dissecting microscope (25X) and average 3.8 mm (2.0 to 6.0 mm; n = 5) x 154.6 tm (108.0 to 189.0 [Lm; n = 10). The outer cyst wall with moderate to deep invaginations throughout the entire length of the cyst to the point of bisecting the entire cyst (Fig. 4-1 thru 4-2). Invaginations occur in both fresh and fixed muscle specimens. The mature sarcocyst wall with stumpy, digit-like protrusions or villi, some being pedunculated near the cyst wall similar to the Type 9 protrusions of Sarcocystis campestris as described by Dubey et al. (1989) (Figs. 4-3, 4-4A). The protrusions differ from those of S. campestris in that they do not contain electron-dense granules. All protrusions with large numbers of fibrillar elements which extend from the







44

interior portion of the sarcocyst wall through the villi. By TEM, protrusions approximately 3.4 gm (2.8 to 4.0 jim; n =10) x 1.5 (1.3 to 2.0 gtm; n =10). The interior portion of the sarcocyst with thick septa (0.2 to 4.5 jpm) separating pockets of bradyzoites or metrocytes. By light microscopy, live bradyzoites freed from the sarcocyst measure 11.0 x 4.4 Lm. Using electron microscopy, bradyzoites with structures typical of Sarcocystis spp. (Dubey et al. 1989), a posteriorly arranged nucleus, many anterior micronemes, dense bodies, and amylopectin granules (Fig. 4-4B). Rhoptries rarely seen and occurred as a single unit and not in pairs. Taxonomic Summary

Type intermediate host. Virginia opossum (Didelphis virginiana).

Natural definitive host. Unknown.

Type location. North-central Florida (290 to 300N, 82o to 830W).

Site of infection Skeletal muscle.

Prevalence. 10% (2 opossums infected/20 opossums examined)

Specimens deposited. Histologic sections of sarcocysts in the U. S. National Parasite Collection, Beltsville, Maryland as USNPC nos. 90685 and 90686.

Etymology. Sarcocystis greineri is named in honor of Ellis C. Greiner,

Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, who has made major contributions to the diagnosis and study of coccidia and other parasites of veterinary importance.

Discussion

According to Odening (1998), there are three valid species of Sarcocystis in opossums. Sarcocvstis didelphidis (Scorza et al. 1957) occurs in the South American







45

opossum (Didelphis marsupialis), Sarcocystis garnhami (Mandour 1965) in the skeletal muscle of Didelphis marsupialis found in Honduras, and Sarcocystis marmosae (Shaw and Lainson 1969) found in the skeletal muscle of Marmosa murina in Brazil. None of these species has been documented in the Virginia opossum. Structural measurements were compared to determine similarities and/or differences (Table 4-1).

Microscopically, sarcocysts and bradyzoites from S. greineri are larger than those for the other valid species described from opossums. The bradyzoites are much closer in size to those described by Scholtyseck et al. (1982), who found that fixed merozoites within the cyst measured approximately 7 to 10 x 2.5 to 3 pm. Using transmission electron microscopy, the cyst wall protrusions/villi for S. reineri are approximately half the size of the other two species that have been described. Sarcocystis garnhami also varies from the other species because protrusions from its cyst wall are very sharply pointed as compared to those of S. greineri and S. marmosae, which have finger-like projections that are rounded off at the distal end. Also, because TEM data with regards to S. didelphidis, S. marmosae, and S. garnhami are lacking in the original descriptions, determination of the presence of pedunculated protrusions in these species is impossible.







46

Table 4-1. Comparison of structural measurements of sarcocysts of Sarcocystis spp.
found in the muscle of opossums
Parasite Intermediate Sarcocyst (jpm) Bradyzoite Sarcocyst wall host length x width (jPm) protrusions/villi (state of sample) (jPm)

Sarcocystis Didelphis 0.9mm 6.5 x 1.5 pm 5.2 [lm didelphidis marsupialis x (fixed) 345 pm

Sarcocystis Didelphis 310 plm-3.3mm 5.3-6.9 pm x 6-8 [im x 1.5-2.0 gamhami marsupialis x 1.3-1.9 jpm jPm 110-250 pm (fixed) acidophilic sharply pointed
spines

Sarcocystis Marmosa _2 mm x 800 6.2-9.0 pm x 11.5-13.0 pm x marmosae murina [Im 1.8-3.0 jlm 2.6 jtm (dried smear) finger-like with rounded tips

Sarcocystis Didelphis 2.0-6.0 mm 11.0 x 4.4 pLm 2.8-4.0 jim x greineri virginiana x (live) 1.3-2.0 pm 108.0-189.0jm stumpy, digitlike protrusions,
some
pedunculated
near cyst wall


















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49


















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protrtisions (a Toc i hciadd i.nsaied. Bar- :::: 4 rnm.








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dens2 bodies ODB,d a a rhoprie (Rh), Bar = 2Ltmn 011.) Sarcocysi. .al showing '..e 4'urs cudiike villar protr'usions (VP), hi'>a eienems
(arrowhead) ca In be obser-ved running throughout the vi exreudmg into the eranular ULver ((i.) of tbe cya. Villa protrusion s contnai.i.uag pedunculnation:s
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(small a-row) of the vjhj. Rau 2 pm.














CHAPTER 5
THE NINE-BANDED ARMADILLO (Dasvpus novemcinctus) IS AN INTERMEDIATE HOST FOR Sarcocvstis neurona3


Introduction

Sarcocystis neurona is a protozoan parasite known to be associated with the neurologic disease equine protozoal myeloencephalitis (EPM). This disease has been considered one of the most important neurologic diseases of horses in the United States (MacKay 1997). Fenger et al. (1997) and Tanhauser et al. (1999) identified the definitive host of S. neurona as the Virginia opossum (Didelphis virginiana). Dubey et al. (2000c), found that an immunosuppressed cat (Felis domesticus) fed sporocysts identified as S. neurona collected from naturally infected opossums formed mature sarcocysts that infected an opossum, thus completing the life cycle of S. neurona in a laboratory setting. However, Dubey et al. (2000c) proposed that the cat is a laboratory host, but that completion of the life cycle of S. neurona using the domestic cat in a natural setting is not likely. Our goal was to identify natural intermediate hosts that allow completion of the life cycle in nature. Identifying natural intermediate hosts may provide veterinarians and horse owners with a method of controlling the intermediate host and thus S. neurona infected opossums on horse farms.




3Paper published with coauthors under the citation "Cheadle, M.A., Tanhauser S.M., Dame, J.B., Sellon, D.C., Hines, M., Ginn, P.E., MacKay, R.J., and E.C. Greiner. 2001.
The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona. International Journal for Parasitology 31: 330-335."
50







51

During the course of studies in our laboratory (ECG), the opossum has been

found to shed at least four types of sporocysts in addition to S. neurona (Cheadle et al. 2001a; Tanhauser et al. 1999). Shedding of multiple species of Sarcocystis by the opossum makes collection and differentiation of S. neurona a complicated process. Our search for possible intermediate hosts has been extensive. When wildlife carcasses had sarcocysts in muscles, as determined by microscopic examination, these sarcocysts were excised and characterised molecularly for comparison with S. neurona. At the same time, portions of remaining tissues were fed to opossums to determine if sporocysts are subsequently shed.

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host of at least three species of Sarcocystis: Sarcocystis dasypi, Sarcocystis diminuta, and an unidentified species (Lindsay et al. 1996). Armadillos are frequently killed by automobiles making them a readily available food source for opportunistic feeders such as D. virginiana. Muscle from road-killed armadillos containing sarcocysts was fed to opossums and excised sarcocysts were characterised molecularly (Tanhauser et al. 2001). Sporocysts shed by experimentally infected opossums were collected and characterised by sequencing species-specific regions of the DNA following PCR amplification (Tanhauser et al. 1999). Here and in a companion paper (Tanhauser et al. 2001), we combine molecular and biological evidence demonstrating that the nine-banded armadillo (D. novemcinctus) is an intermediate host to S. neurona.

Armadillos were collected either as automobile-kills or were live trapped and killed (Beuthanasia-D Special, Schering Plough Animal Health). Each animal was assigned an identification number and data concerning the date and location of collection







52

recorded. Skeletal muscle from the tongue and legs were removed. Portions of each muscle were kept at 40 C for feeding to laboratory-reared opossums and fixed for light and TEM.

Tissues were processed for light and TEM as described previously (Cheadle et al. 2001c; Luznar et at. 2001). Muscle was fixed using 10% buffered formalin, processed, embedded, sectioned at 5 plm, and stained with H & E. Sections of skeletal muscle were scanned for the presence of sarcocysts using a dissecting microscope at a magnification of 25X. For TEM, fresh muscle samples containing sarcocysts were excised and immediately immersion-fixed in cold Trumps fixative. Samples were postfixed in 1% osmium tetroxide (w/v), dehydrated in ethanol, embedded in EmBed epoxy resin, and thin (70 to 80 nm) sectioned. Sections were double stained with 2% aqueous uranyl acetate followed by Reynolds lead citrate. Stained sections were then examined with a Hitachi H-7000 TEM operating at a voltage of 75 kv and images were taken with a Gatan Bioscan CCD camera.

Four wild-caught and eight laboratory-reared opossums were housed at the

Department of Animal Resources, University of Florida during the course of this study. Fecal samples were collected and screened for the presence of Sarcocystis sp. sporocysts before the start of the feeding trial. Feces were processed using Sheather's sugar flotation centrifugation method and observed for the presence of Sarcocystis sp. sporocysts using light microscopy (100X). If an opossum shed sporocysts in its feces during the preinfection trial period, it was removed from the study. Portions of skeletal muscle from armadillos were stored at 40 C until fed to non-sporocyst shedding opossums. Muscle sections from the armadillo(s) fed to each opossum were observed







53

using a dissecting microscope to ensure that sarcocysts were present. Muscles found to contain sarcocysts were fed to opossums over a 2- to 4-day period. Concurrent with feeding trials, armadillo sarcocysts were excised and DNA was extracted (Tanhauser et al. 2001). DNA was analysed using PCR amplification, restriction enzyme analysis, and DNA sequencing techniques (Tanhauser et al. 1999). Banked plasma samples were obtained from wild caught and laboratory raised armadillos and observed for the presence of antibodies to S. neurona by immunoblot (Tanhauser et al. 2001).

Experimentally infected opossums were killed approximately 45 days p.i. and

intestines were removed. A portion of the small intestine was scraped using a glass slide and a portion of the scraping was then processed using sugar flotation. The direct scraping and flotation preparations were observed by light microscopy (100X) for the presence of oocysts/sporocysts. The remaining portion of the scraping was then homogenized in a blender for 30 to 60 s and stirred for 4 to 6 h in 500 mL distilled water containing one-three drops of Tween 80. After filtration through a tea strainer, the mixture was centrifuged for 10 min. After removal of the supernatant, storage media (Hanks Balanced Salt Solution (500 mL) plus antibiotics (10 x 104 I.U. penicillin and 10 x 104 Cpg streptomycin (Mediatech) and fungicide (250 tg amphotericin (Mediatech) was placed in the tube containing the oocyst/sporocyst mixture.

Sporocysts from opossums DV02, DV14, and DV 22 were analyzed using

techniques as described by Tanhauser et al. (1999) with one exception. Excystation was induced by a freeze-thaw technique rather than by incubation in equine bile. Sporocysts were pelleted by centrifugation. The storage media was removed and replaced with 100 pL lysis buffer plus 5 pL Proteinase K. Sporocysts were then frozen in liquid nitrogen







54

for 1 min. After freezing, the sporocysts were heated to 37' C for 1 min. This procedure was repeated twice. The PCR primer pair JNB25/JD396 were used for amplification. PCR products were sequenced and a 256 bp segment of product was compared with the same segment of DNA from S. neurona isolate UCD-1 as described by Tanhauser et al. (1999).

A 2-month-old, immune competent, Arabian colt was given 5 x 10' S. neurona

sporocysts collected from opossum DV22 via intragastric inoculation. Two days prior to inoculation, the foal was determined to be healthy on the basis of physical examination, neurological examination, complete blood count and serum biochemical profile, and was weaned from the mare. The foal was anaesthetized with xylazine (1.1 mg/kg IV) and ketamine (2.2 mg/kg IV) and a sample of CSF was obtained from the atlanto-occipital space for cytological evaluation. An aliquot of CSF was submitted to a commercial laboratory (Equine Biodiagnostics, Inc., Lexington, KY) for immunoblot for the detection of antibodies against S. neurona. Temperature, heart rate, and respiratory rate were monitored daily after infection; a neurological examination was performed biweekly. At post-infection Weeks 2, 4, 6, and 10, the foal was anaesthetized and a sample of CSF was obtained from the atlanto-occipital space for cytological evaluation and immunoblot. No treatment for EPM was given during the course of this study.

Four of four (100%) wild-caught and 5 of 8 (63%) laboratory-raised opossums contained sporocysts in their intestinal scrapings after being fed armadillo muscle. The mean dimensions of sporocysts collected from these opossums were 11.0 x 7.5 Pm (n = 10 per opossum, range = 11.0 to 11.4 x 7.0 to 7.9 pm). Prepatent period of shedding ranged from 18 to 46 days post feeding (Table 5-1). Total numbers of sporocysts







55

collected from each opossum are listed in Table 5-1. Total numbers of sporocysts collected ranged between 0 to 4.1 x 107. Sporocyst production shed in feces was approximately two-four sporocysts per 4 grams of feces per day as determined by sugar flotation techniques.

Ultrastructural examination of sarcocysts revealed a sarcocyst wall similar to that of S. dasypi (Lindsay et al. 1996) (Fig. 5-1). Approximately 50% of armadillos observed contained sarcocysts determined to be S. dasypi by light microscopy, while less than 10% contained sarcocysts of S. diminuta. The probability that S. dasypi and S. neurona are synonymous is high, however further studies need to be conducted.

The 256 bp segment of sporocyst DNA collected from opossums DV02 and

DV14 was 100% homologous with the same segment of UCD-1. The 256 bp segment of sporocyst DNA collected from opossum DV22 was 99.6% homologous with the same segment of UCD-1 with a single bp difference.

The infected foal maintained a normal temperature, heart rate, and respiratory rate throughout the evaluation period. The foal was seronegative and CSF negative for antibodies to S. neurona prior to infection. Both CSF and serum were positive for antibodies by 4 weeks p.i. (Table 5-2). CSF cytology, including red blood cell count, was within normal limits at every time point (Table 5-2). At 6 weeks p.i., the foal developed an abnormal gait with proprioceptive deficits and ataxia of all four limbs. Neurologic deficits gradually improved over a 3 week period; at 10 weeks p.i., the foal was considered neurologically normal except for the right hind limb. No necropsy was performed.

By infecting both wild caught and laboratory raised opossums via armadillo tissue







56

infected with sarcocysts, we have replicated our results. It was important to verify that different opossums will shed sporocysts after being fed muscle from different armadillos. This will ensure that the phenomenon of shedding can be reliably duplicated in different individuals and that completion of the life cycle was not simply due to an abnormality in an individual opossum, such as compromised immune status, that would allow it to aberrantly complete the life cycle.

Sporocysts were never detected in the feces of opossum DV 17 (Table 5-1), but at the end of the trial, gut scrapings were found to contain low numbers of sporocysts. Therefore, it is possible that even if wild caught opossums feces are monitored for an extended period of time and sporocysts are not detected, the gut may still contain latent stages. Therefore, coupling infection trials with DNA analysis of sarcocysts is very useful.

Opossums DV 18 and DV24 did not shed sporocysts in their feces and upon euthanasia and necropsy, the intestinal scrapings did not contain sporocysts. The muscles fed to these opossums were 61 and 104 days old, possibly allowing time for sarcocyst degradation. It is also possible that sarcocysts from a Sarcocvstis sp. other than S. neurona may have been detected or that the muscle contained a low density of sarcocysts (approx. one sarcocyst per 10 mm2 section of muscle).

Opossum DV21 was observed to shed sporocysts in its feces on days 23 and 37. Counts were minimal and only one to three sporocysts were observed per 1 to 2 g of feces floated. The authors cannot explain the lack of sporocysts observed in intestinal scrapings. Observation of muscle sections of the armadillo fed to DV21 indicated that while few sarcocysts were present, numbers were sufficient to produce a patent infection.







57

Also, the muscle tissue was only 11 days old at the time of inoculation, therefore sarcocyst degradation should have been minimal.

Sequence analysis of the PCR products from sporocysts indicates identity with S. neurona. Although sample DV22 differed from UCD-I by one base (0.4%), it is within the normal range of variability among isolates of S. neurona (Tanhauser et al. 1999). Further DNA characterisation will need to be performed to determine the amount of sequence variability among armadillo S. neurona isolates.

The host range of the nine-banded armadillo in the United States is limited. Data indicate that the range extends from Florida to New Mexico in the south and as far north as South Carolina to Kansas and parts of Nebraska (Hall 1981). In Central and South America, the range encompasses most of Mexico south to Peru, northern Argentina and Uruguay as well as Grenada and Trinidad and Tobago (Nowak and Paradiso 1983; Nowak 1999). The geographic range of the armadillo in the United States does not extend sufficiently north to encompass all areas that have endemic cases of EPM. The simplest explanation for this phenomenon is that there is more than one natural intermediate host of S. neurona. Another explanation for the widespread antibody prevalence in horses is the use of commercially available food stuffs that have not been heat treated and thus may be contaminated with sporocysts. Food items could be contaminated with sporocysts at harvesting and the sporocysts remain infectious through the processing procedure.

Wild caught armadillos were not used for infection trials to complete the

reciprocal infectious nature of the parasite due to the high prevalence of Sarcocystis infected animals in the wild (Lindsay et al. 1996). Armadillos are very difficult to raise







58

in captivity. Though the animals occasionally became pregnant, researchers have seen only 14 successful rearings among more than 700 females housed indoors (Truman and Sanchez 1993).







59

Table 5-1. Armadillo inoculum fed to laboratory raised opossums and prepatent period of Sarcocystis neurona sporocysts in feces
Opossum number Age of Armadillo muscle Prepatent Total samples at consumed period sporocysts feeding (days) collected (days) from opossum
DV02' n/a DN35-DN55 (21) 25 2.6 x 105
DV09' n/a DN 58, DN 59, 46 DN63 (3)
DV1O' n/a DN58, DN59, 44 1.1 x 105 DN64 (3)

DV14' n/a DN60, DN64 (2) 40 3.4 x 10' DV172 66 DN66 (1) ns 2.4 x 105
DV182 61 DN67 (1) ns 0
DV 192 45 DN69 (1) 18 1.6 x 106 DV202 41 DN70 (1) 18 1.1 X 106
DV212 11 DN71 (1) 23 0
DV222 7 DN72 (1) 18 4.1 x 10' DV232 6 DN73 (1) 18 1.5 x 107
DV242 104 DN64 (1) ns 0
n/a = not available; ns = no sporocysts observed in faeces; *Sporocysts observed in scraping but numbers deemed not sufficient to count; Wild caught opossum; 2Laboratory raised opossum; () Total number different armadillos from which muscle was consumed







60

Table 5-2. Results of cytologic analysis and Western immunoblot of CSF samples from a foal inoculated with 5 x 10' sporocysts of Sarcocystis neurona collected from opossum DV22 fed armadillo muscle containing sarcocysts
Week Post- RBC WBC Protein Western
infection (per pL) (per jtL) (mg/dL) immunoblot
Pre-infection 6 0 33.5 Negative
2 1102 2 24.9 Negative
4 0 1 34.2 Low positive
6 1 3 36.7 Positive
10 0 4 35.7 Strong positive















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CHAPTER 6
THE STRIPED SKUNK (Mephitis mephitis) IS AN INTERMEDIATE HOST FOR Sarcocvstis neurona4


Introduction

Sarcocystis neurona is a protozoan parasite known to be a causative agent of the neurologic disease equine protozoal myeloencephalitis (EPM). This disease has been considered one of the most common neurologic diseases of horses in the United States (MacKay 1997). Fenger et al. (1997), Dubey and Lindsay (1998a), and Tanhauser et al. (1999) identified the definitive host of S. neurona as the Virginia opossum (Didelphis virginiana). Dubey et al. (2000c) found that domestic cats (Felis domesticus) can be induced to become infected with S. neurona in the laboratory and serve as an experimental intermediate host. We recently found that opossums fed muscle from ninebanded armadillos (Dasypus novemcinctus) naturally-infected with sarcocysts would shed sporocysts that were molecularly and biologically identified as S. neurona, implicating the armadillo as a natural intermediate host for S. neurona (Cheadle et al. 2001c).

The striped skunk (Mephitis mephitis) is a definitive host for one species of

Sarcocystis, Sarcocystis rilevi, and an intermediate host for one unnamed Sarcocystis sp. (Cawthorn and Rainnie 1981; Erdman 1978; Wicht 1981). Skunks have a range that 4Paper published with coauthors under the citation "Cheadle, M.A., Yowell, C.A., Sellon, D.C., Hines, M., Ginn, P.E., Marsh, A.E., Dame, J.B., and E.C. Greiner. 2001. The
striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona. International Journal for Parasitology 31: 843-849."
62







63

encompasses the majority of the United States, Canada, and northern Mexico, providing a coverage of areas in which EPM is endemic (Hall 1981). Muscles from skunks containing sarcocysts were fed to opossums to determine if the opossum would produce Sarcocystis oocysts/sporocysts. Sarcocysts that were excised from skunks and sporocysts that were collected from opossums fed skunk muscle were compared with S. neurona using PCR followed by DNA sequence analysis of PCR products. In the present paper, we provide evidence that the striped skunk is an intermediate host for S. neurona using laboratory-reared opossums.

Materials and Methods

Three 10-month-old striped skunks (SK01, SK02, and SK03) were obtained from a commercial supplier (Ruby Fur Farm Inc.). Skunks SKOI and SK02 were orally inoculated with 5 x 10' and 5 x 104 sporocysts, respectively, of an S. neurona isolate collected from a naturally infected opossum (isolate # 4180). These sporocysts were identified as S. neurona based on techniques described by Tanhauser et al. 1999 and Cheadle et al. 2001 a. Skunk SK03 was inoculated with sterile Hanks balanced salt solution (HBSS). Skunk SKOI was found dead 90 d.p.i. and skunks SK02 and SK03 were killed 92 d.p.i. (Beuthanasia-D Special, Schering Plough Animal Health). Skeletal muscles were removed and portions were kept at 4oC for feeding to laboratory-reared opossums. Tissues including liver, lung, kidney, muscle, stomach, intestine, brain, heart, and spleen were removed and processed for light microscopy, immunohistochemistry, and TEM as described previously (Cheadle et al. 2001b; Cheadle et al. 2001c). Two additional mature skunks were obtained from the same rearing facility, killed, and muscles were observed using histologic sections and gross observation to determine if







64

natural infection was present in the facilities population. No sarcocysts were observed in the muscles of these animals using these methods.

Laboratory-reared opossums were maintained as described by Cheadle et al. (2001b). Fecal samples were collected and processed as described by Cheadle et al. (2001b) to detect the presence of Sarcocystis sporocysts from day 0 p.i. and 3 days a week thereafter throughout the course of the study. Portions of skeletal muscle from skunks were stored at 40 C for 1-6 days until fed to non-sporocyst shedding opossums. Before the feeding trial, muscle sections from skunks were observed using a dissecting microscope at 15X to ensure that sarcocysts were present. Sarcocysts were visible only with the aid of a dissecting microscope. Approximately 80 g of sarcocyst positive skeletal muscle was fed to each opossum over the course of 2 to 3 days. The total numbers of sarcocysts fed was not determined. Opossums readily consumed all sarcocyst muscle that was presented. Feces were collected and processed as described by Cheadle et al. (2001b) for a period of 4 weeks after opossums began the feeding trial. Concurrent with the feeding trial, skunk sarcocysts were excised and DNA was extracted. DNA was analysed and compared to the UCD- I1 isolate of S. neurona using primer set JNB25/JD396 for PCR amplification and sequencing techniques (Tanhauser et al. 1999).

Opossums were killed (Beuthanasia-D Special, Schering Plough Animal Health) approximately 30 days p.i. and intestines were removed. Collection and measurement of sporocysts was conducted as described by Cheadle et al. 2001a and 2001 c. Sporocyst DNA was extracted. DNA was analysed and compared to the UCD-1 isolate of S. neurona using primer set JNB25/JD396 for PCR amplification and sequencing techniques (Tanhauser et al. 1999). A 2-month-old, clinically normal pony was given 5 x 105 S.







65

neurona sporocysts collected from opossum DV37 via intragastric inoculation and monitored as described by Cheadle et al. (2001 b). Two gamma-interferon gene knockout mice (BALB/c-Ifng"'ITs) (Jackson Laboratories) per opossum isolate were inoculated with 5 x 103 sporocysts, monitored, necropsied, and tissues processed as described by Cheadle et al. (2001c).

Serum samples from sporocyst inoculated mice and skunks were tested for the presence of S. neurona reacting antibodies by Western blotting. Skunk serum samples were collected at 16 days preinoculation and 28, 41, 63, 78, 90, and/or 92 days p.i. Sera from mice were obtained at necropsy. Sera obtained from a naive mouse and laboratoryreared opossum prior to and after inoculation with culture-derived S. neurona merozoites served as negative and positive controls. Immunoblots were performed as previously described (Lapointe et al. 1998) with slight modifications. Specifically, the antigen preparation was non-reduced (Marsh et al. 2001) and different secondary antibodies, antimouse IgG Fc (Jackson ImmunoResearch Laboratories, Inc.) or anti-raccoon IgG (H+L) (Kirkegaard & Perry Laboratories Inc.), were used according to manufacturer's recommendations. These detection antibodies were labelled with peroxidase for use with chemiluminescence signal development (Amersham Life Science, Inc.).

Results

All skunks contained Sarcocystis sarcocysts in skeletal muscles. TEM and light microscopy revealed homology among the sarcocysts from all skunks. Six of six laboratory-reared opossums shed Sarcocystis sporocysts in their feces after being fed infected skunk muscle. Mean sporocyst dimensions were 11.0 x 7.5 Pm (n = 10 per opossum, range = 10.6 to 11.0 x 7.0 to 7.5 jim) (Fig. 1). Diameter of sarcocysts in fixed,







66

histologic sections of muscle averaged 27.7 pm (n = 15, S.E.M. = 2.4). Prepatent period of sporocyst shedding ranged from 12 to 20 days post feeding (Table 6-1). Numbers of sporocysts collected were calculated using a hemocytometer. Total numbers ranged from

6.0 x 10' to 7.6 x 10' (Table 6-1).

Ultrastructural examination of sarcocysts revealed a type 11 sarcocyst wall

(Dubey et al. 1989) (Figs 6-1 and 6-2). In longitudinal section, villi are perpendicular or at an approximate 450 angle to the cyst wall and measure 3.0 ptm (n = 12, S.E.M. = 0.3) x

0.4 gim (n = 12, S.E.M. = 0.03) (Fig 6-1). No electron dense granules were observed in villi.

Sequence analysis of PCR products using the JNB25/JD396 primer set from

sarcocysts and sporocysts indicates identity with S. neurona. The sequence of the 280 bp segment of DNA amplified from skunk SKO 1 and SK03 sarcocysts and from sporocysts collected from opossums DV34, DV36, DV37, and DV39 was 100% homologous to that amplified from UCD-1. This sequence was only 96% homologous to that of the comparable amplicon from Sarcocystis falcatula.

Twelve of 12 gamma-interferon gene knockout mice inoculated with sporocysts collected from opossums that were fed skunk muscle developed clinical neurologic signs requiring euthanasia and/or died. The day mice were killed or died ranged from 26 to 34 (Table 6-2). Clinical signs and histologic lesions were similar to those observed previously (Cheadle et al. 2001c; Dubey and Lindsay 1998a). Grossly, infected mice had splenomegaly and circular white foci on the lungs. Histologically, white foci in lungs were associated with a lymphohistocytic and neutrophilic chronic interstitial pneumonia with diffuse merozoites throughout. Immunohistochemistry, using polyclonal S. neurona







67

antibodies, stained merozoites in the cerebrum, cerebellum, and lung (Table 6-2).

The inoculated pony maintained a normal temperature, heart rate, and respiratory rate throughout the evaluation period. The pony was seronegative and CSF negative for antibodies to S. neurona prior to infection (Table 6-3). CSF was low positive and serum was positive for antibodies by 4 weeks p.i. and both were strongly positive by 8 weeks p.i. (Table 6-3). CSF cytology, including erythrocyte count, was within normal limits at every time point (Table 6-3). No necropsy was performed and the pony remains clinically normal.

Gamma-interferon gene knockout mice sera showed similar reactivity to S. neurona antigens when compared with hyperimmune serum obtained from a mouse inoculated with culture-derived S. neurona merozoites (UCD-1 isolate) (Fig 6-3). Reactivity to a S. neurona protein running below the 14 kDa marker was very similar among all inoculated mice and no reactivity was seen with the negative controls or preinoculation samples. Reactivity to the antigen between the 28 and 17 kDa marker was much less intense in sporocyst inoculated mice as compared to the merozoite inoculated mice.

All skunks were serum antibody negative to S. neurona at the beginning of the trial (Fig. 6-4). By 28 days p.i., skunks seroconverted to S. neurona based on immunoblot analysis (Fig. 6-4). Reactivity increased over time with all skunks except skunk SKOI whose serum reactivity decreased at days 78 and 92 p.i. (Fig. 6-4). The reason for this reduced activity is not known. Anti-raccoon IgG was found useful for detection of opossum and skunk IgG reactivity. Serum from skunks showed similar reactivity to S. neurona antigens when compared with the merozoite inoculated control







68

opossum.

Discussion

All animals orally inoculated with sporocysts produced antibodies to S. neurona or formed sarcocysts. Although the pony did not develop clinical neurologic signs, it did develop antibodies to S. neurona in serum and CSF, indicating infection. Gammainterferon gene knockout mice developed clinical neurologic signs consistent with infection of S. neurona. The course of infection with the sporocysts collected from opossums that were fed skunk muscle was similar to that of mice inoculated with sporocysts collected from naturally infected opossums or opossums that had been fed armadillo muscle infected with S. neurona (Cheadle et al. 2001b; Cheadle et al. 2001c).

Although skunks SK02 and SK03 appeared clinically normal throughout the trial, histopathologic lesions were noted at necropsy. Skunk SKO02 had a nonsuppurative, interstitial pneumonia, and severe, hepatic vacuolar degeneration. Skunk SK03 had a lymphoplasmacytic interstitial nephritis, severe, hepatic vacuolar degeneration, and cerebral lymphoplasmacytic meningitis, and multifocal necrotizing nonsuppurative encephalitis. Sarcocysts observed in muscle sections were not associated with a host response. Protozoan organisms were observed only as sarcocysts in muscle tissues although non-specific binding of antibody was observed in the spleen. Sarcocysts were not stained by polyclonal antibodies to S. neurona in this study.

Skunk SK01 died on Day 90 of the study. Gross lesions consisted of hemorrhage in the lungs and kidneys and frank blood in the GI tract. Histopathologic lesions consisted of severe, diffuse necrotising alveolitis with marked bacterial colonization, severe lymphoplasmacytic interstitial nephritis, mild hepatic lipidosis, contracted spleen,







69

necrosuppurative interstitial myositis, cerebellar white matter spongiosis, and lymphoplasmacytic meningitis. The animal was found to be emaciated. No protozoan organisms were observed in the tissues other than sarcocysts in muscle. A wasting syndrome similar to that observed in SK01 has been observed in gamma-interferon knockout mice orally inoculated with S. neurona sporocysts (Cheadle, unpublished), however the cause of the wasting is unknown.

Opossum DV 38 never shed sufficient sporocysts in its feces to detect by flotation (Table 6-1). At the end of the trial, gut scrapings were observed and found to contain low numbers of sporocysts. Therefore, wild caught opossums are not suited for use in feeding trials. Even if feces are monitored for an extended period of time and sporocysts are not detected, the gut may still contain stages.

Although skunk SK03 was not inoculated with sporocysts, muscle sarcocysts were found at necropsy. Infection of this skunk likely occurred in our facility based on the negative antibody titer found at the inception of the study and the lack of sarcocysts in non-inoculated skunks examined from the commercial supplier. Similar contamination has been documented previously with Sarcocystis species (Clubb and Frenkel 1992; Hillyer et al. 1991; Smith and Frenkel 1978). Inoculated skunks were housed separately from the non-inoculated skunk, but were maintained in the same room in cages approximately 2 m apart. The control skunk was inoculated using a sterile inoculation needle, syringe, and HBSS media. Gloves were changed after handling infected animals. No portion of the inoculum was spit out or spilled. No opossums were housed in the room at any time. The exact route of infection to this skunk is unknown.

The villi (Figs. 6-1 and 6-2) of the sarcocyst wall of S. neurona collected from







70

skunks is similar to that of Sarcocystis dasvpi (Cheadle et al. 2001b; Howells et al. 1975; Lindsay et al. 1996), Sarcocystis kirkpatricki (Snyder et al. 1990), Sarcocystis sp. from a mink (Ramos-Vara et al. 1997), S. falcatula (Box et al. 1984), Sarcocystis fayeri (Tinling et al. 1980), and S. neurona from an experimentally infected cat (Dubey et al. 2000c). The wall has type 11 villar protrusions as indicated by Dubey et al. (1989), although many of the microtubules terminate in the granular layer and do not extend to the plasmalemma of bradyzoites. Comparison of villi widths between species reveals homology. Widths range from approximately 0.2 pim to 0.7 lm. Similarities among the villar protrusions of these species makes it impossible to differentiate them based on TEM alone. A need for further investigation into the role of the raccoon and mink in the life cycle of S. neurona should to be addressed. Although molecular data show that S. neurona and S. falcatula are closely related, S. falcatula has been shown to be biologically and molecularly different (Dubey and Lindsay 1998a; Marsh et al. 1997; Tanhauser et al. 1999). Although hobnails were observed on villi of skunk sarcocysts, they were not as prominent as those reported for S. falcatula in a budgerigar at 24 weeks (Box et al. 1984).

There is a single report of Sarcocystis sarcocysts found in naturally infected

skunks (Erdman et al. 1978). In that study, dogs (Canis familiaris) were found to be a definitive host, shedding sporocysts that measured 10 X 12 pm.

Natural infection causing an encephalitis has been reported in skunks, but no sarcocysts were observed (Dubey et al. 1996a; Dubey and Hamir 2000a). Although neurologic signs were never observed in skunks inoculated with S. neurona sporocysts in the present study, nor were protozoan parasites observed in neural tissues using light







71

microscopy and immunohistochemical techniques, a mild meningitis and necrotizing nonsuppurative encephalitis was observed in SK03.

Combined with our prior discovery that the nine-banded or long-nosed armadillo (Dasypus novemcinctus) is an intermediate host for S. neurona, the information demonstrated in this paper that the striped skunk is also an intermediate host for S. neurona provides critical new information to explain the full geographic distribution of endemic EPM. The host range of the striped skunk encompasses much of the United States, Canada and northern Mexico, while nine-banded armadillos range from the southeastern United States to Peru and northern Argentina (Nowak and Paradiso 1983).







72

Table 6-1. Skunk inoculum fed to laboratory raised opossums and prepatent period of
Sarcocystis neurona sporocysts in feces
Age of Skunk Prepatent Total sporocysts Opossum samples at muscle period collected from number feeding (days) consumed (days) opossum DV33 1 SK02 20 1.3 x 106 DV34 1 SK03 12 1.2 x 107 DV36 5 SK02 13 3.4 x 106 DV37 5 SK03 14 7.6 x 107 DV38 6 SK01 ns 6.0 x 10' DV39 6 SKO1 14 2.6 x 106 ns = no sporocysts observed in feces







73

Table 6-2. Inoculation of gamma-interferon knockout mice with sporocysts collected
from opossums fed Sarcocvstis infected skunk muscle
Mouse Sporocysts in Opossum from Day Tissues Immunoblot
number inoculum which inoculum killed or positive for S. results
number
was collected dead neurona

MIS 5,000 DV33 34 Cb, Cl, Lu pos M2S 5,000 DV33 32 Cb, Cl pos M3S 5,000 DV34 32 Cb, Cl, Lu pos M4S 5,000 DV34 26 Cb, Cl, Lu pos M5S 5,000 DV36 28 Cb, Cl, Lu pos M6S 5,000 DV36 29 Cb, Cl, Lu pos M7S 5,000 DV37 30 Cb, Cl, Lu pos M8S 5,000 DV37 32 Cb, Cl pos M9S 5,000 DV38 26 Cb, Cl pos MI1S 5,000 DV38 26 Cb, Cl, Lu pos MiIS 5,000 DV39 33 Cb, Cl pos M12S 5,000 DV39 34 Cb, Cl, Lu pos M13S negative n/a 36* neg
control

M14S negative n/a 36* neg
control
* = Mice killed due to end of study, no clinical signs were observed; n/a = not available; Cb = Cerebrum; Cl = Cerebellum; Lu = Lung; = no parasites observed







74

Table 6-3. Results of cytologic analysis and immunoblot of CSF and serum samples
from a foal inoculated with 5 x 105 sporocysts of Sarcocystis neurona
collected from opossums fed skunk muscle containing sarcocysts
Week post- Serum CSF RBC WBC Protein infection immunoblot immunoblot (per pL) (per 1iL) (mg/dL) Pre-infection negative negative 0 0 47.3 2 negative negative 1 1 58.6
4 positive low positive 0 5 56 6 positive positive 67 4 59
8 strong positive strong positive 0 1 59.8
















































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arc Loca ed along the ent ire length of the pIaasitphiorou vascua membrane (PVM) and eletron-dense layr IEL) Scale' bar === N, Jun p(b)1 Sarcocyst in cross section. Note the vilr protrusions \small arrowhad} nd bradyzoites
(large arrowheadd. Scale ba =3. int (c) Sporuiated nocysts from opossumfl
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~ili~i! p n roc. te f. a'A Sale Iicj vl. I;cl zP .~~










+ 1- 12 neg




49












Fig 6-3, Rcactivi ty of scrunm colected from ganuai-nterferon knockout mice inoculated
dwith sporocysts collected from opossunlS i i ne W infected skunk mtuscie,
tImnunoblokt from SDS.-PAIAC separated non-reduced ,S naroni, proteins
(UCD1 isolate grown i n equine dnnal ceIs probed with hyperinrmune mouse scra ( -- pre-irculation control seo (-., spor'cyst inoculated mice sera (Lanes
1-12 corre spond to mice MS-MII2S), and sham inoculatedI miice ne gave
cont-rol sera e'g M.iS, M .'14S All samples :ere diluted at 1:10 exc ept Ip'.i".mm'ne positive control sera which was run at 1:40 dilution due to the hi;g antibod titer of this immunocompetenn t Ialbic mouse which received 3
itraperitoneal injccti.ons of 2 xl 0 me re .rozoites C .C)- isolate of' S n 'ur' a on
days 25, and 57 prior to bleeding. Approximate molecular s a'es are given at
the mar tin in kDa.











1-6 7-12 13-18 -+








28


14






Fig 6-4. Reacivity of serurm co ecd from sekunks before ad aft-ieor irocu.:ion I w".ith .:;
ane Nuroa sporocy sts collected fromr a na turally in ected oPOSIsi. fImmunobIots
from S:IS-i:PAGE separatIed on-reduced S uRin proteins ((UCD-. isolate
g-rown ir eqine dermai cells) probed with skunk sra. (pre and post-i roculsion
samples). vanes 6: skunk SK03 sera from dayvs -16. 28. 4 1, 63 78, and 9
respectively; iies 7-12: .skunk SK.02 sera from das -16. 2, 41, 63, 78, and 92 respeccivdely; Lanes 13-18: skunk SKI1 sera rom daiys -16, 28, 4, 63, 78
and 90 respectively. The negative sample, (-), was pre-i ocuiatio serum ir om a
laboratory reared opossum and the positive sample. (+). was the same ropossum,
that had rieciei live culture-derived x ;nra merozOit (C-I isoae.
grown in equine dermal cells) on day 109 (primary inocut.ior) and day 3 0 prior
to bleedin.i date. Approximate molecular sizes ar. e given at margin in k.a.














CHAPTER 7
ATTEMPTS TO COMPLETE THE LIFE CYCLE OF VARIOUS Sarcocystis SP.
USING THE VIRGINIA OPOSSUM (Didelphis virginiana) AS A DEFINITIVE HOST Introduction

The Virginia opossum (Didelphis virginiana) sheds at least 4 species of Sarcocystis sporocysts in its feces (Cheadle et al. 2001a). A fifth taxa has been identified, but whether the species uses the opossum as a definitive host is still in doubt (Cheadle et al. 2001a). The life cycles of only Sarcocystis neurona and Sarcocystis falcatula are known (Box et al. 1984; Cheadle et al. 2001b; Cheadle et al. 2001d). Little is known about the other two species.

Sarcocystis neurona is a protozoan parasite known to be a causative agent of the neurologic disease equine protozoal myeloencephalitis (EPM). This disease has been considered one of the most common neurologic diseases of horses in the United States (MacKay 1997). Dubey and Lindsay (1998a), Fenger et al. (1997), and Tanhauser et al. (1999) identified the definitive host of S. neurona as the Virginia opossum. Dubey et al. (2000c) found that domestic cats (Felis domesticus) can be induced to become infected with S. neurona in the laboratory and serve as an experimental intermediate host. We recently found that nine-banded armadillos (Dasypus novemcinctus) and striped skunks (Mephitis mephitis) are intermediate hosts for S. neurona (Cheadle et al. 2001b; Cheadle et al. 2001 d).

Sarcocystis falcatula can naturally and experimentally infect various bird species


79







80

among the passeriform, psittaciform and columbiform orders (Box and Smith 1982). Natural intermediate hosts include the brown-headed cowbird (Molothrus ater), Patagonian conure (Cyanoliseus patagonus), and the boat-tailed grackle (Cassidix mexicanus) (Bolon et al. 1989; Box et al. 1984). Experimental hosts that can become infected include budgerigars (Melopsittacus undulatus), canaries (Serinus canarius), pigeons (Columba livia), English sparrows (Passer domesticus), zebra finches (Poephila guttata), and cockatiels (Nymphicus hollandicus) (Box and Duszynski 1980; Box and Smith 1982; Hillyer et al. 1991). If S. falcatula is introduced into aviaries, severe infection and mortality will likely occur (Bolon et al. 1989; Hillyer et al. 1991).

Sarcocystis speeri sporocysts collected from the feces of a Virginia opossum can be differentiated from other species being shed in the feces based on morphologic and biologic characteristics (Cheadle et al. 2001a; Dubey and Lindsay 1999b). This species has also been isolated from Didelphis albiventris, the South American opossum (Dubey et al. 2000e). Originally, Dubey et al. (1998b) described the isolation of this species of Sarcocystis from the feces of an opossum. After inoculation of gamma-interferon gene knockout mice with sporocysts of this unidentified species, sarcocysts were observed in the leg muscles at Days 50 and 54. Dubey et al. (1999b) determined that one nude mouse formed sarcocysts when inoculated with 10-' sporocysts of S. speeri.

Nothing is known about the life cycle and pathogenicity of the 1085 isolate of Sarcocystis other than that it uses the Virginia opossum as a definitive host. The 1085 taxa has been shown to be different from S. neurona and S. falcatula by agarose gel electrophoresis of PCR products using different restriction enzymes (Tanhauser et al. 1999; J.B. Dame, personal communication). Dubey et al. (2000b), examine a Sarcocystis







81

falcatula-like isolate of Sarcocystis from a Didelphis albiventris in Brazil. Based on the data presented, it appears that the species described in that paper is most likely the same as 1085, indicating that 1085 has a wide range.

In an attempt to determine the intermediate hosts of the two unknown cycles, two approaches were taken. Opossums were fed muscle that was known/suspected to be infected with sarcocysts and candidate animals were inoculated with sporocysts from S. neurona, S. falcatula, S. speeri, and the 1085 taxa.

Materials and Methods

Sporocyst and Sarcocyst Identification

Identification of sporocysts and sarcocysts was conducted as previously described (Cheadle et al. 2001c; Tanhauser et al. 1999). Inoculation of Animals

Inoculation of animals with sporocysts was performed via gavage as previously described (Cheadle et al. 2001b).

Oral Inoculation Trials

Wild gray squirrels (n = 2) (Sciurus carolinensis) were trapped in Alachua county and inoculated with a 5 x 103 sporocysts of S. neurona, S. falcatula, S. speeri, and 1085 species each (total inoculation mixture = 2 x 104 sporocysts; maximum age of sporocysts = 1 year since collection). A wild flying squirrel (Glaucomys volans) was trapped in Alachua county and inoculated with a 5 x 103 sporocysts of S. neurona, S. falcatula, S. seeri, and 1085 species each (total inoculation mixture = 2 x 10' sporocysts; maximum age of sporocysts = 1 year since collection). Captive raised deer mice (n = 10) (Peromyscus maniculatus) were inoculated with 5 x 10' sporocysts of S. neurona, S.







82

falcatula, S. speeri, and 1085 species (maximum age of sporocysts = 1 year since collection). Two mice were inoculated per isolate and two mice served as negative controls. Eleven (13 day old) mallard (Anas platyrhynchos) ducklings were obtained from a local supplier (Alachua county feed and seed, Gainesville, FL). Ducklings were inoculated with 5 x 10' sporocysts of S. neurona, S. falcatula, S. speeri, and 1085 species (maximum age of sporocysts = 1 year since collection). Two ducklings were inoculated per isolate and three ducklings served as negative controls. Ten wild caught, adult boattailed grackles (Quiscalus major), brown headed cowbirds (Molothrus ater), red winged blackbirds (Agelaius phoeniceus), and house sparrows (Passer domesticus) obtained from the United States Department of Agriculture (Gainesville, FL) were inoculated with 5 x 103 sporocysts of S. neurona, S. falcatula, S. speeri, and 1085 species (maximum age of sporocysts = 1 year since collection). Two birds of each species were inoculated per isolate and two birds of each species served as negative controls. Ten adult New Zealand white rabbits (Oryctolagus cuniculus) were obtained from a commercial supplier (via Animal Resources, Gainesville, FL) and were inoculated with 5 x 103 sporocysts of S. neurona, S. falcatula, S. speeri, and 1085 species (maximum age of sporocysts = 1 year since collection). Two rabbits were inoculated per isolate and two rabbits served as negative controls. Leopard frogs (Rana utricularia) (n = 8) were obtained as tadpoles (wild-caught in Alachua county, FL) and raised in the laboratory to adulthood. Frogs were inoculated with 5 x 103 sporocysts of S. neurona, S. falcatula, and S. speeri (maximum age of sporocysts = 1 year since collection). Two frogs were inoculated per isolate and two frogs served as negative controls. All animals were observed for approximately 90 days and then were euthanized. A necropsy was performed on all







83

animals that died or were euthanized. Sections of the following tissues were removed and fixed in 10% buffered formalin: lung, liver, kidney, muscle, stomach, intestine, brain, heart, and spleen. Fixed tissues were submitted to the Department of Pathobiology, University of Florida for embedding, sectioning, and staining using H & E. The remaining tissues were collected and fed to captive opossums (one opossum per isolate). Fecal samples were collected from the opossums for a period of at least 30 days post feeding and examined for the presence of sporocysts. Muscle Feeding Trials

Horse (Equus caballus) (H01-H49) (n = 49) skeletal muscles were collected from the Anatomic Pathology Necropsy Service, University of Florida over a period of five months. Muscles of at least 10 horses contained sarcocysts. White-tailed deer (Odocoileus virginianus) (WTDOI-WTD27) tongues (n = 27) positive for sarcocysts were collected from hunter killed animals in Florida for a period of two months. Gray squirrel (n = 2) (Sciurus carolinensis) (S02 and S03) skeletal muscles positive for sarcocysts were collected in Florida from road-killed animals. Sarcocyst positive skeletal muscle from a hunter killed northern-shoveler (Anas clypeata) (NS01) and green-winged teal (Anas crecca) (GWTO 1) was collected in Florida. Wild trapped rice rats (Oryzomyvs plaustris) (RR03-RR06) (n = 3) and a cotton mouse (Peromyscus gossvpinus) (PG02) (n = 1) positive for sarcocysts were collected in Florida. Great blue heron (Adrea herodias) (GBHO1), ring-billed gull (Larus delawarensis) (RBG 01), sandhill crane (Grus canadensis) (SHOI), great egret (Casmerodius albus) (GE02), and bald eagle (Haliaeetus leucocephalus) (BEO1) (n = 1 per species) muscle positive for sarcocysts was collected from necropsy at the University of Floida. A wild eastern cottontail (Sylvilagus







84

floridanus) (CR01) with muscle infected with sarcocysts was collected in Florida from a single road-killed animal. Virginia opossum (D. virginiana)(OP 1 -OP7) (n = 7) muscle was collected from road-killed animals. All muscles were obtained, given an identification number, and fed to an individual (wild caught or lab reared) opossum. Fecal samples from these opossums were examined for the presence of Sarcocystis sporocysts approximately once per week during the feeding process for a period of 60 days after the last piece of muscle was fed. Muscle was fixed and submitted to the Department of Pathobiology, University of Florida for embedding, sectioning, and staining using H & E. The remaining tissues were collected and fed to a wild caught or lab reared opossum. Sarcocysts were observed in H & E sections of the muscle tissue submitted. Opossums were observed to ensure consumption of muscles.

Results

Results of feeding and inoculation trials are summarized in Tables 7-1 and 7-2. None of the naturally infected muscle tissue fed to opossums caused infection in the opossums. All of the Sarcocystis sp. that completed their life cycles through the opossum in this trial were from birds and were identified as S. falcatula.

Discussion

Sarcocystis falcatula infects a wide range of birds and completes its life cycle

through the opossum (Box and Smith 1982; Clubb and Frenkel 1992; Duszynski and Box 1978; Hillyer et al. 1991). In this study, S. falcatula infected great blue herons, cowbirds, and English house sparrows. The lack of infectivity to grackles was surprising based on a previous study that found grackles were intermediate hosts for S. falcatula (Box et al. 1984). Because the birds used in this study were wild caught, it is possible that the







85

grackle was naturally infected, prior to this study, with another Sarcocystis sp. that does not complete its life cycle through the opossum.

Although no attempt was made to identify the Sarcocystis spp. found in the

muscles that were fed to the opossums, the author did observe the sarcocysts found in the northern shoveler and cottontail rabbit using TEM (unpublished). Based on villar structure of the sarcocyst wall viewed in micrographs, the duck was infected with Sarcocystis rilevi and the rabbit was Sarcocystis leporum (Dubey et al. 1989; Elwasila, et al. 1984). The finding that S. rileyvi would not infect the opossum is not surprising based on a previous study by Duszynski and Box (1978) in which they found that teal and shovler muscle heavily infected with sarcocysts fed to multiple opossums would not cause infection. Although the species of Sarcocystis used in the study by Duszynski and Box (1978) was not identified as S. rilevi, it was collected from a northern shoveler and is most likely S. rilevi based on species specificity. Of further interest in the study by Duszynski and Box (1978), was that an opossum did shed sporocysts in its feces after being fed muscle from a northern pintail duck (Anas acuta), indicating that a different species of Sarcocvstis, possibly S. falcatula, likely infects the pintail. Previous authors attempted to complete the life cycle of S. leporum using cats (Felis domestica) and dogs (Canis familiaris) (Fayer and Kradel 1977). In that study, cats would produce sporocysts thereby completing the life cycle of S. leporum, while dogs would not. The current study indicates that S. leporum does not use the Virginia opossum as a definitive host.

More than one species of Sarcocystis could infect the animals used in this study, allowing the possible completion of life cycles through those animals. However, only the great blue heron completed the life cycle of any of the Sarcocystis spp. using the







86

opossum as a definitive host. Although none of the life cycles, except the previously documented cycle of S. falcatula, were completed, valuable information on the scope definitive/intermediate hosts for the Sarcocystis sp. used in this study was obtained.







87

Table 7-1. Summary of sarcocyst infected animal muscle used in and results of attempts
to complete the life cycle of Sarcocystis spp. using the opossum as a
definitive host
Number of Sporocysts
positive produced in
Sarcocysts samples opossum Identification observed fed to feces of sporocysts Animal (yes or no) opossum (yes or no)

Horse (Equus caballus) yes 49 no n/a

White-tailed deer yes 27 no n/a (Odocoileus virginianus)

Grey squirrel (Sciurus yes 2 no n/a carolinensis)

Northern shoveler (Anas yes 1 no n/a clypeata)

Green-winged teal (Anas yes 1 no n/a crecca)

Great Blue Heron (Adrea yes 1 yes S. falcatula herodias)

Ring-billed gull (Larus yes 1 no n/a delawarensis)

Sandhill crane (Grus yes 1 no n/a canadensis)

Great egret (Casmerodius yes 1 no n/a albus)

Bald eagle (Haliaeetus yes 1 no n/a leucocephalus)

Eastern cottontail yes 1 no n/a (Sylvilagus floridanus)

Rice rat (Oryzomys yes 3 no n/a plaustris)

Cotton mouse yes 1 no n/a (Peromvscus gossvpinus)

Virginia opossum yes 7 no n/a (Didelphis virginiana)
n/a = not applicable







88

Table 7-2. Summary of animals orally inoculated with sporocysts collected from
Virginia opossums
Sporocysts Identification produced of sporocysts Sample Sarcocystis Sarcocysts in opossum collected Animal size sp. used in observed feces (yes from
inoculum (yes or no) or no) opossum

Grey squirrel 2 SN, SF, no no n/a (Sciurus SS, and carolinensis) 1085

Deer mouse 10 SN, SF, no no n/a (Peromyscus SS, and maniculatus) 1085

Flying squirrel 1 SN, SF, no no n/a (Glaucomyvs SS, and volans) 1085

Mallard (Anas 11 SN, SF, no no n/a platyrhynchos) SS, and 1085

Boat-tailed 10 SN, SF, yes no n/a grackle SS, and (Quiscalus major) 1085

Brown-headed 10 SN, SF, yes yes S. falcatula cowbird SS, and (Molothrus ater) 1085

Red-winged 10 SN, SF, yes no n/a blackbird SS, and (Agelaius 1085 phoeniceus)

House sparrow 10 SN, SF, yes yes S. falcatula (Passer SS, and domesticus) 1085

New Zealand 10 SN, SF, no no n/a white rabbit SS, and (Oryctolagus 1085 cuniculus)

Leopard frog 8 SN, SF, no no n/a (Rana utricularia) SS, and 1085







89

n/a = not available
SN = 5 x 103 sporocysts of Sarcocystis neurona SF = 5 x 103 sporocysts of Sarcocystis falcatula SS = 5 x 103 sporocysts of Sarcocystis speeri 1085 = 5 x 103 sporocysts of a 1085-like taxa




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THE BIOLOGY OF Sarcocvstis SPP. OF THE VIRGINIA OPOSSUM (Didelphis virginiana ) IN FLORIDA By MARK ANDREW CHEADLE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2001

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ACKNOWLEDGMENTS I would like to thank my family and friends first. Without strong family support throughout my graduate program, the work would have been much more difficult. Secondly, I thank the animals that gave their lives for this research. Biologic evidence can only be acquired through the use of animals and their contribution to this study was invaluable. I would like to thank the following people, without whose help I would have been unable to complete such a complicated project. Ellis Greiner was chair of my committee and a friend. Without his mentorship, I would have been unable to properly conduct my studies and gain the knowledge needed for my degree. John Dame was original chair of my committee and a friend. Without his guidance through molecular biology, I would still be in the dark. Thanks to the other members of my committee, Pam Girm, Don Forrester, and Mel Sunquist. All of the members were patient and helpful in suggesting ways to improve and expand my research. Appreciation is extended to my coauthors on papers that have been published from this data. Dr. Ellis Greiner, Dr. John Dame, Dr. Pam Ginn, Dr. Deb Sellon, Dr. Mellisa Hines, Dr. Susan Tanhauser, Dr. Tim Scase, Dr. Antoinette Marsh, Charles Yowell, and Dr. Robert Mackay. I thank Dr. Charles Courtney for his assistance performing statistical analysis and for fishing breaks on the weekends. Many student workers did much of the "less than exciting" work required for this project: Kristin Munsterman, Laura Dixon, Alysia Posey, Eric Seymour, Taj Ryland, Kimberly Baird, Randa Antar, Melissa Perez Velasco, Michelle Delucia, ii

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Britany Benson, Alexis Smith, Jessica Meliti, Persilla Medina, Summer Gustat, Jenny Freeman, Stephanie Badge, and Jennifer Willey. All performed animal collection and care, sporocyst detection, and sporocyst purification. I thank Siobhan Ellison for the use of the polyclonal antibody to S. neurona I thank Richard Hill and Karen Scott for Greyhound acquisition and care. I thank Dr. Janet Yamamoto and Maki Aral Tanabe for cat acquisition and care. I thank Glenda Eldred for histopathologic processing. I thank the Electron Microscopy Core Laboratory of the Interdisciplinary Center for Biotechnology Research, University of Florida for help with electron microscopy. I thank Richard Truman (Louisiana State University) for assistance with technical information concerning the armadillo. These studies were supported in part by grants from the Florida Pari-Mutual Wagering Trust Fund, United States Department of Agriculture grant No. 98-35204-6487, the Florida Agricultural Experiment Station, and the GraysonJockey Club Research Foundation. iii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ii LIST OF TABLES vi LIST OF FIGURES viii ABSTRACT x CHAPTERS 1 INTRODUCTION 1 2 SPOROCYST SIZE OF ISOLATES OF Sarcocvstis SHED BY THE VIRGINIA OPOSSUM ( Didelphis virginiana ') 16 Introduction 16 Materials and Methods 17 Sporocyst Collection and Storage 17 Sporocyst Typing 17 Observation and Measurement 17 Data Analysis 18 Results 18 Discussion 19 3 VIABILITY OF Sarcocvstis neurona SPOROC YSTS AND DOSE TITRATION IN GAMMA-INTERFERON KNOCKOUT MICE 26 Introduction 26 Materials and Methods 27 Collection of Sporocysts 27 Identification of Sporocyst Type 28 Animals 28 Inoculation Procedure 28 Necropsy and Histopathology 29 Results 29 Discussion 30 4 Sarcocvstis greineri N. SP. (PROTOZOA: SARCOCYSTIDAE) IN THE VIRGINIA OPOSSUM (Didelphis virginiana ) 40 Introduction 40 iv

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Materials and Methods 40 Opossum Collection and Processing 40 Light Microscopy 41 Feeding Trials 41 Transmission Electron Microscopy 42 Results 43 Description 43 Taxonomic Summary 44 Discussion 44 5 THE NINE-BANDED ARMADILLO (Dasvpus novemcinctus ) IS AN INTERMEDIATE HOST FOR Sarcocvstis neurona 50 6 THE STRIPED SKUNK ( Mephitis mephitis ) IS AN INTERMEDIATE HOST FOR Sarcocvstis neurona 62 Introduction 62 Materials and Methods 63 Results 65 Discussion 68 7 ATTEMPTS TO COMPLETE THE LIFE CYCLE OF VARIOUS Sarcocvstis SP. USING THE VIRGINIA OPOSSUM (Didelphis virginiana ) AS A DEFINITIVE HOST 79 Introduction 79 Materials and Methods 81 Sporocyst and Sarcocyst Identification 81 Inoculation of Animals 81 Oral Inoculation Trials 81 Muscle Feeding Trials 83 Results 84 Discussion 84 8 CONCLUSIONS 90 REFERENCES 95 BIOGRAPHICAL SKETCH 103 V

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LIST OF TABLES Table page 1L Spectrum of animals found in the gut contents or feces of Virginia opossums 12 21 Comparison of Sarcocystis sporocyst sizes 21 31 Dose and isolate/s of Sarcocystis neurona given to each gamma-interferon knockout mouse 35 3-2. Microscopic identification of protozoa in routinely and immunohistochemically treated sections of cerebrum and cerebellum from gamma-interferon knockout mice inoculated with Sarcocystis neurona sporocysts 36 3-3. Summary of disease in Sarcocystis neurona infected gamma-interferon knockout mice 37 34. Histopathological lesions observed in gamma-interferon knockout mice orally inoculated with Sarcocystis neurona sporocysts 38 41 Comparison of structural measurements of sarcocysts of Sarcocystis spp. found in the muscle of opossums 46 51 Armadillo inoculum fed to laboratory raised opossums and prepatent period of Sarcocystis neurona sporocysts in faeces 59 52. Results of cytologic analysis and Western immunoblot of CSF samples from a foal inoculated with 5x10^ sporocysts of Sarcocystis neurona collected from opossum DV22 fed armadillo muscle containing sarcocysts 60 61 Skunk inoculum fed to laboratory raised opossums and prepatent period of Sarcocystis neurona sporocysts in faeces 72 6-2. Inoculation of gamma-interferon knockout mice with sporocysts collected from opossums fed Sarcocystis infected skunk muscle 73 6-3. Results of cytologic analysis and immunoblot of CSF and serum samples from a foal inoculated with 5x10' sporocysts of Sarcocystis neurona collected from opossums fed skunk muscle containing sarcocysts 74 vi

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7-1 Summary of animals used to attempt the completion of Sarcocystis spp. that use the opossum as a definitive host 87 7-2. Summary of animals orally inoculated with sporocysts collected from Virginia opossums 88 vii

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LIST OF FIGURES Figure page 2-1 Sporocyst of Sarcocvstis speeri (2079 isolate) 22 2-2. Sporocyst of Sarcocvstis neurona (3063 isolate) 22 2-3. Sporocyst of Sarcocystis falcatula (3 1 06 isolate) 23 2-4. Sporocy sts of 1 085 type (3013 isolate) 23 2-5. Sporocyst of 3344 type (3344 isolate) 24 2-6. Detailed view of sporocyst of 3344 type (3344 isolate) 24 27. Brightfield micrograph of 3344 type sporocyst 25 31 H & E stained section of cerebellum from mouse M8 containing a group of merozoites 39 32. Immunohistochemically stained schizont of S. neurona from mouse MS cerebellum containing multiple merozoites 39 41 Sarcocyst of Sarcocystis greineri n. sp. from the skeletal muscle of a Virginia opossum 47 4-2. High magnification of an invagination in the sarcocyst wall 47 4-3. Sarcocyst in thigh muscle of an opossum 48 44. TEM of a sarcocyst of Sarcocystis greineri n. sp. from the Virginia opossum 49 51. TEM of sarcocyst from armadillo DN42 fed to opossum DV02 61 61 Sarcocystis neurona stages isolated from skunks 75 6-2. TEM of Sarcocystis neurona isolated from skunks 76 6-3. Reactivity of serum collected from gamma-interferon knockout mice inoculated with sporocysts collected from opossums fed S. neurona infected skunk viii

PAGE 9

muscle 77 6-4 Reactivity of serum collected from skunks before and after inoculation with S. neurona sporocysts collected from a naturally infected opossum 78 ix

PAGE 10

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE BIOLOGY OF Sarcocvstis SPP. OF THE VIRGINIA OPOSSUM (Didelphis virginiana ) IN FLORIDA By Mark Andrew Cheadle August 2001 Chairperson: Dr. Ellis C. Greiner Major Department: Veterinary Medical Sciences Virginia opossums ( Didelphis virginiana ) are definitive hosts for at least four species of Sarcocystis Only the life cycle of Sarcocystis falcatula had been completed at the inception of this dissertation research. Goals of this study were to establish the life cycles of one or more of the species whose life cycles were unknown. To this end, the author either: inoculated live candidate animals with sporocysts to determine if sarcocysts would form; or fed Virginia opossums sarcocyst infected muscles from various animals to determine if they would shed sporocysts. Additional studies were done to determine if other vertebrate species would become infected with S. falcatula For intermediate host trials, we used New Zealand white rabbits, an eastern cottontail rabbit, mallards, a northern shoveler, a green-winged teal, boat-tailed grackles, a great blue heron, brown-headed cowbirds, red-winged blackbirds, house sparrows, a ringbilled gull, a sandhill crane, a great egret, a bald eagle, leopard frogs, gray squirrels, a X

PAGE 11

southern flying squirrel, striped skunks, nine-banded armadillos, horses, white-tailed deer, deer mice, rice rats, a cotton mouse, and Virginia opossums. Of the species examined, the nine-banded armadillo ( Dasypus novemcinctus ) and striped skunk ( Mephitis mephitis ) served as intermediate hosts for Sarcocystis neurona the causative agent for the neuromuscular disease in horses called equine protozoal myeloencephalitis. The brown-headed cowbird, house sparrow, and great blue heron were intermediate hosts for S. falcatula None of the life cycles of any Sarcocystis sp. tested was completed in any other animal from the aforementioned list. Light microscopy of sporocysts was a useftil means of distinguishing Sarcocystis speeri from other taxa. Use of gammainterferon gene knockout mice for infection trials with S. neurona was expanded to determine if sporocysts being used for studies were viable. Sporocysts processed using bleach and stored at 4 C remained viable for <7 months. The finding of two intermediate hosts will allow investigators to produce large numbers of sporocysts of S. neurona which can then be used for future studies. It will also allow veterinarians and horse owners to control the intermediate hosts on their farms as a method of prevention of infection with S. neurona. xi

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CHAPTER 1 INTRODUCTION Sarcocystis spp. are obligatory, intracellular protozoan parasites that use mammals, birds, and reptiles as both intermediate and definitive hosts. The intermediate host contains asexual stages of the parasite and the definitive host contains the sexual stages (Dubey et al. 1989). There are at least 1 89 species included in this genus (Odening 1998). Sarcocystis spp. have a two-host cycle usually involving a carnivore definitive host and an herbivore and/or omnivore intermediate host. The life cycle begins with the ingestion of a sarcocyst containing bradyzoites in tissue fed upon by an appropriate definitive host. After degradation of the sarcocyst wall in the stomach, bradyzoites contained within the residual sarcocyst are released within the small intestine. These stages invade the intestinal epithelial cells and begin the sexual cycle of reproduction culminating in the production of two sporocysts contained within an oocyst. Sporocysts sporulate or mature in situ and are released within the feces into the environment to infect an intermediate host. Once sporocysts are ingested by the intermediate host, sporozoites that are contained within are released and invade the intestinal epithelial cells and begin a migration to the muscle tissue. In capillary endothelial cells, sporozoites undergo asexual reproduction leading to the formation of a schizont with merozoites contained within. Merozoites are then released and invade new host cells, forming another schizont. After a series of these stages, a terminal merozoite migrates to muscle tissue and forms a sarcocyst with metrocytes contained within. These 1

PAGE 13

metrocytes eventually mature into bradyzoites within the cyst and the cycle continues. Bradyzoites are considered a hypobiotic stage (Dubey et al. 1989). The name Sarcocystis is derived from the asexual stage of the parasite that encysts in muscle. Although sarcocysts can be found in the CNS and smooth muscles, they are primarily found in striated muscles throughout the body (Dubey et al. 1989). The sarcocyst can range in size from microscopic to visible by the naked eye. Shapes of sarcocysts vary from spindle to globular. The bradyzoites contained within this stage are the only portion of the life cycle infective to the definitive host (Dubey et al. 1989). The Virginia opossum ( Didelphis virginiana l is a definitive host for several species of Sarcocystis some of which cause clinical disease in other animals. The firstnamed species of Sarcocvstis that uses the opossum as a definitive host was originally named Sarcocystis debonei however, it is currently named Sarcocystis falcatula (Box et al. 1984). Sarcocystis is a major pathogen of psittaciform birds and can cause severe disease and death (Box and Smith 1982). Unlike most Sarcocystis spp., S. falcatula is not species-specific with reference to intermediate hosts. This species can naturally and experimentally infect a variety of avian species across the passeriform, psittaciform and columbiform orders (Box and Smith 1982). Natural intermediate hosts include the brown-headed cowbird ( Molothrus ater ). the boat-tailed grackle ( Cassidix mexicanus ). and the Patagonian conure ( Cyanoliseus patagonus ) (Bolon et al. 1989; Box et al. 1984). Experimental hosts include budgerigars ( Melopsittacus undulatus ). canaries ( Serinus canarius ). rock doves ( Columba livia ). English sparrows ( Passer domesticus ). zebra finches ( Poephila guttata ), and cockatiels ( Nymphicus hollandicus ) (Box and Duszynski 1980; Box and Smith 1982; Hillyer et al. 1991). Although authors originally suggested

PAGE 14

that S. neurona and S. falcatula were synonymous (Dame et al. 1995), subsequent studies found that S. neurona and S. falcatula can be differentiated using biological and molecular characteristics (Cutler et al. 1999; Dubey and Lindsay 1998a; Tanhauser et al. 1999). Sarcocystis speed (= 2079 isolate) originally was collected and differentiated from other species shed by the opossum at the University of Florida. Although this was not named by the University of Florida, researchers Dubey and Lindsay (1999b) isolated the same organism and named it S. speeri This species has been isolated also from Didelphis albiventris the South American opossum (Dubey et al. 2000e). Dubey et al. (1998b) described the isolation of a third unidentified species of Sarcocystis based on sarcocyst formation and structure in gamma-interferon gene knockout mice. After inoculation of gamma-interferon gene knockout mice with sporocysts of this unidentified species, sarcocysts were observed in the leg muscles at Days 50 and 54. There was some cross reactivity between the sarcocysts and an S. neurona antibody when the S. neurona antibody was applied to tissue sections. However, according to the authors, much less reactivity was seen than when the antibody was applied to S. neurona infected tissues. Nude mice inoculated with sporocysts from the same opossum did not form sarcocysts. In the paper by Dubey et al. (1999b) in which they described and named S. speeri the authors found that one nude mouse did form sarcocysts when inoculated with 10"' sporocysts of S. speeri Interestingly, schizonts were seen in the brain of 14 of 34 (41%) of mice inoculated with sporocysts. This indicates either a mixed infection with S. neurona or the possibility that S. speeri will infect the brains of mice. This problem was never addressed in the paper and size differences of the merozoites were never compared

PAGE 15

even though merozoites of S. speeri are much larger than those of S. neurona (Dubey et al. 1998b). Dubey et al. (2000d) also found that S. speeri would grow in cell culture using bovine monocytes and equine kidney cells. A third species of Sarcocystis using the opossum as a definitive host is Sarcocystis neurona In the original description of S. neurona by Dubey et al. (1991), they reported the isolation of an "equine protozoal myeloencephalitis (EPM)-lik;e" protozoal organism from the spinal cord of a naturally infected horse from Ithaca, New York. This description came after most researchers felt that the protozoal organism seen previously in horse CNS tissue diagnosed with EPM was Toxoplasma gondii However, a description by Simpson and Mayhew (1980) identified a Sarcocystis sp. in the spinal cord of a naturally infected horse that was diagnosed with EPM, thus indicating that a Sarcocystis sp. was the source of infection. Fenger et al. (1997) and Tanhauser et al. (1999) identified the definitive host of S. neurona as the Virginia opossum (" Didelphis virginiana ). Sarcocystis neurona was thought to be synonymous with Sarcocystis falcatula based on the similarity of the three segments of the small subunit ribosomal RNA (Dame et al. 1995). It now appears, based on extensive molecular and biological evidence, that S. falcatula and S. neurona are separate species. However, the use of the cowbird as an intermediate host for S. neurona is still in doubt (Cutler et al. 1999; Dubey and Lindsay 1998a; Tanhauser et al. 1999). Due to the fact that sarcocyst wall configuration is considered to be the differentiating characteristic for Sarcocystis spp. and that the configuration of the sarcocyst wall for S. neurona was unavailable, there was no way to definifively differentiate the two organisms. The method described by Tanhauser et al.

PAGE 16

5 (1999) is considered the most appropriate method for determining the species. Another animal that appears to be affected by S. neurona is the Alaskan sea otter ( Enhydra lutris kenyoni ) and the southern sea otter (Enhydra lutris nereis ) (Lindsay et al. 2000; Miller et al. 2001 : Rosonke et al. 1999). Lesions in the southern sea otter consisted of widely disseminated nonsupperative meningoencephalomyelitis with severe and diffiase inflammation in the cerebellar molecular layer and blood vessels in the cerebellum. Large numbers of macrophages and smaller numbers of lymphocytes and plasma cells were observed. Similar but less severe inflammation was observed in the midbrain, brainstem, and gray matter of the spinal cord. Other lesions associated with infection were pulmonary hemorrhage, lymphoid depletion, and edema in a tracheobronchial lymph node. Organisms from these isolations have been grown in culture and reacted positively to antibodies raised against S. neurona (Lindsay et al. 2000; Miller et al. 2001). Organisms collected by Lindsay et al. (2000) amplified using primer pairs JNB33 and JNB54 produced a product that cut with Dra I in a pattern similar to that of S. neurona Lindsay et al. (2000) also found that four of four gamma-interferon knockout mice inoculated with merozoites collected from the study developed encephalitis. Miller et al. (2001) isolated merozoites in cell culture and sequenced segments of DNA (18s ribosomal DNA and the entire adjacent ITS-1 sequence). Sequences from this study showed identity with comparable sequences from S. neurona isolated from horses. Although sarcocysts were documented by Rosonke et al. (1999), no definitive identification was made and only low-power micrographs were available making it difficult to idenfify the species observed in this study. There have been cases of S. neurona -like protozoal organisms causing

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encephalitis in other wildlife species including mink ( Mustela vison ). a striped skunk ( Mephitis mephitis '), a rhesus monkey ( Macaca mulatta ). Pacific harbor seals (Phoca vitulina richardsi ), and raccoons ( Procyon lotor ) (Dubey et al. 1996a; Dubey and Hamir 2000a; Dubey and Hedstrom 1993; Klumpp et al. 1994; Lapointe et al. 1998; Stroffregen et al. 1991). Although these authors documented stages of Sarcocystis that reacted positively with antibodies to S. neurona sarcocyst stages that would enable the completion of the life cycle through the opossum were not found. In horses, EPM is a severe, debilitating, neurologic disease. Horses with this disease commonly have abnormalities of gait. Clinical signs range from a mild lameness to sudden recumbency. Gross lesions in the spinal cord can consist of hemorrhage. Microscopically, there may be multifocal areas of necrosis, hemorrhage, and nonsuppurative inflammation of the grey and white matter (Davis et al. 1991). The lesions associated with this disease in the United States were first recognized by Rooney in 1 964. Similar lesions were observed by Macruz et al. (1975), in Brazil, during the same period. The protozoal organisms seen in these lesions were assumed to be Toxoplasma gondii at that time. Since 1970, EPM has been reported in most of the lower 48 states and in Brazil, Canada, and Panama (Clark et al. 1981; Dubey et al. 2001; Granstrom et al. 1992; Masri et al. 1992). Most of horses affected by this disease are between 1 and 4 years of age, however other age groups are also represented (Payer et al. 1990). Sarcocvstis neurona has been isolated from naturally infected horses in various areas of North and South America and has been continuously grown in cell culture for an extended period of time (Davis et al. 1991). Neospora hughesi also has been isolated from the spinal cord of a

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horse and has been grown continuously (Cheadle et al. 1999; Marsh et al. 1998). The only protozoal organisms documented to be in the central nervous system (CNS) of horses are Sarcocystis sp. and Neospora sp. (Cheadle et al. 1999; Dubey et al. 1991; Marsh et al. 1996). These organisms usually are associated with a mixed inflammatory cellular response and destruction of nervous tissue. Various stages such as merozoites ( Sarcocystis sp.), schizonts ( Sarcocystis sp.), and tachyzoites (Neospora sp.) can be seen in the cytoplasm of neurons or mononuclear macrophages. Cells that can be parasitized during the course of infection in the CNS include intravascular and tissue neutrophils and eosinophils as well as capillary endothelial cells and myelinated axons. Organisms are commonly associated with areas of focal necrosis. Merozoites and tachyzoites may be found intracellularly or extracellularly and in groups or as individuals. Sarcocysts of S. neurona do not form in muscle or any other organ. Therefore, it is assumed that the horse is a dead-end host of S. neurona due to the lack of formation of this stage in the muscle or other organs of this host (Dubey and Lindsay 1996b; MacKay 1997). Nothing is known about the life cycle and pathogenicity of the 1085 isolate of Sarcocystis Sporocysts from this isolate were collected from naturally infected opossums (Tanhauser et al., 1999). They have been shown to be different from S, neurona and S. falcatula by agarose gel electrophoresis of PCR products using different restriction enzymes (J.B. Dame, personal communication; Tanhauser et al. 1999). It is assumed that it will have a similar life cycle to that of S. neurona and S. falcatula although this needs to be confirmed. There is also a possibility that this species has been named previously and that documenting the occurrence in the opossum will provide

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scientists with information to more fully understand the life cycle of the organism. The work on this isolate began by molecularly characterizing it and will continue by attempting to identify intermediate hosts that can become infected by ingestion of sporocysts. The objective of this study was to elucidate the intermediate hosts of one or more of the following species: Sarcocystis neurona Sarcocystis strain 1085, and Sarcocystis speeri The author either: inoculated live candidate animals with sporocysts to determine if sarcocysts would form; or fed Virginia opossums sarcocyst infected muscles from various animals to determine if they would shed sporocysts. It is hypothesized that the intermediate hosts for S. neurona the 1085 Sarcocystis isolate, and/or S. speeri are wild animals in Florida. The animals become infected with S. neurona 1085, and/or S. speeri by ingestion of sporulated sporocysts and they form sarcocysts in muscle tissue that can then infect the common Virginia opossum and cause this animal to shed sporocysts. The major emphasis for this project was on S. neurona due to its devastating influence on horses. The term opossum refers to members of Didelphidae. These animals are only found in the New World. Although there are relatives of opossums on the continent of Australia called possums, they are not considered for this discussion. The large, American opossums are marsupials that span much of the land mass of North, Central, and South America. Opossums also occur on some of the islands of the Lesser Antilles (Nowak and Paradiso 1 983). Best known for their innate ability to get hit by cars and feed vultures, opossums are very complex creatures that are composed of 75 species and they fill many environmental niches. Opossums of 1 1 different genera range from

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southern Argentina to southern Canada. Animals can be found in habitats ranging from those at or below sea level to upwards of 9,000 feet above sea level in the mountains of Central and South America. Specific habitats are generally associated with water and include tropical forest (scrub, deciduous, evergreen, rain, and cloud), savarma, thorn forest, temperate oak-pine forest, and prairie/mesquite grasslands (Gardner 1973). The hair of Didelphis virginiana is unique to the Dildelphidae in that it consists of long, thick, soft woolly underfur of approximately 40 to 50 mm in length with an outer long coat of coarse white-tipped guard hairs that are approximately 60 to 80 mm long (Allen 1901). Guard hairs are rare or absent in other species of opossums (Nowak and Paradiso 1983). The tail is generally shorter (270 to 350 mm) than the combined length of the head and body, with the head and body being approximately 450 to 520 mm. The tail contains long body hair for approximately 2 inches starting at the base. The naked portion of the tail is a brown to gray color with flesh tones mixed in between. The base of the tail can be a darker, even blackish color (Allen 1901). Body color can range from black to white (mutant), with grey being the common body fur color. The black coloration is uncommon north of Georgia and the gulf coast states but is common in the southeastern US and southward to Central America (Gardner 1982). Ears are black with a broadly flesh color or narrowly edged with this color. Ears are smaller in the northern than the southern opossums. In tropical areas, the opossum's ear has a greater superficial area than in extreme northern specimens (Allen 1901). The larger ear size in tropical specimens may help dissipate heat which would be deleterious to northern animals. Lower legs are normally a lighter color, but feet can also be a black color in certain subspecies (Hall 1981).

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Currently, the Virginia opossum can be found in most habitats from sea level to those above 9000 feet high, although they tend to favor moist woodlands and thick brush near water (Gardner 1982; Nowak and Paradiso 1983). Along the eastern seaboard, the opossum previously was found no farther north than the Hudson River Valley and reached into the Northeast United States in only the early 1900s (Guilday 1958). Opossums have been moving north, reaching as far as southern Ontario, Canada by 1956. Now, they occur in many parts of Canada. Northern limits of the population appear to be controlled only by winter conditions, availability of food, and prevalence of den sites. All populations of opossums from Canada and the United States that are located west of the Great Plaines are the result of transplants from the eastern United States, many of which were introduced via human intervention (Gardner 1982). Adults of D. virginiana both male and female, in the wild seldom live past 2 years of age with one author determining the life expectancy in one area to be a little more than 1 5 months. However, laboratory kept opossums can be maintained for upwards of 4 years, suggesting that predators and natural phenomena take a heavy toll on the wild opossum (Gardner 1982; Lee and Cockbum 1985). Didelphis virginiana is a scavenger and there is no evidence that they compete with local North American placental mammals (Pough et al. 1999). However, many marsupials have similar niches to placentals, leading to the possibility of some competition, however small. Many studies of stomach and scat contents have been conducted and confirm the fact that opossums are opportunistic omnivorous feeders (Gardner 1 982). D. virginiana often scavenges rotten flesh and many times even cannibalizes members of its own species. A larger portion of its diet, however, is

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11 comprised of insects, earthworms, and vegetation. Although many have claimed otherwise, D. virginiana apparently is not a good predator and does not normally prey on chickens, rabbits, waterfowl, or other game (Gardner 1982). The food items found in the stomachs of opossums vary from region to region and from season to season, and appear to be based mainly on what food items are available in the local environment. To initiate the search for the intermediate hosts of the Sarcocystis spp. that parasitize the opossum, it is necessary to be familiar with the diet of the opossum. Knowing what species of animals are available to the opossum in its area narrows the field of intermediate host candidates. Unfortunately, the opossum consumes a wide variety of animals. Animal species found in opossums stomach/fecal contents are listed in Table 1-1.

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Table 1-1. Spectrum of animals found in the gut contents or feces of Virginia opossums Animal Species Location Reference Bufo americanus/Bufo sp. New York, Florida, Texas Poumelle 1950; Hamilton IQSl • Wnnd 1954 Rana pipiens/Rana sylvatica New York Hamilton IVM (Frog) Ambvstoma New York, Michigan Taube 1947; Hamilton j effersonianum/Ambv stoma 1951 maculatum (Mole oaicUIlallUCI ) Hvla versicolor ( 1 ree rrog ) New York Hamilton 1951 Plethodon New York Hamilton 1951 cinereus/Plethodon (j|iitinn 1 7ou skunk) Felis catus (Domestic cat) Oregon Hopkins 1980 Microtus New York, Michigan, Taube 1947; Hamilton permsvlvanicus/Microtus sp. Pennsylvania 1951; Blumenthal 1976 (Meadow vole) Scalopus aquaticus (Eastern Michigan Taube 1947 mole) Blarina brevicauda New York, Michigan, Taube 1947; Hamilton (Northern short-tailed Pennsylvania 1951; Blumenthal 1976 shrew)

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13 Table 1 1 -continued Animal Species Location Reference Peromvscus leuconus/Peromvscus sn (Deer and white-footed New York, Texas, Pennsylvania TT '1a ^ f\C ^ W 7 J Hamilton 1951; Wood 1954Blumenthal 1976 mouse) Mus musculus (House mouse) Pennsylvania, Oregon Blumenthal 1 976; Hopkins 1980 Sigmodon sp. (Cotton rat) Texas Wood 1954 Tamias sp. (Chipmunk) New York Hamilton 1951 shrew) Npw York Hamilton 1951 v/llUaird ZlDcullCUb (Muskrat) J.NCW 1 urK T-Tnmiltnn ^Q'^^ ndJlllXLUXl 1 J 1 Mpw York Hamilton 1951 Condvlura sp. (Star-nosed New York Hamilton 1951 Rattus sp. (Old world rat) New York, Oregon Hamilton 1951; Hopkins 1980 Neurotrichus gibbsu (Shrew mole) Oregon Hopkms 1980 Crvptotis sp. (Small-eared shrew) New York Hamilton 1951 Lumbricus tprrp
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14 Table 1 1 -continued Animal Species Location Reference Storeria occipitomaculata (Red-bellied snake) New York Hamilton 1951 Diadophis punctatus (Ringnecked snake) New York Hamilton 1951 Lampropeltis triangulum fMilk snaked New York Hamilton 1951 Storeria dekavi (DeKav's snaked New York Hamilton 1951 snake) Orppon Hookins 1980 Sonora semiannulata sp. (Southern ground snake) Texas Wood 1954 Agkistrodon contortrix (Copperhead snake) Texas Wood 1954 Family Scincidae (Skink) Texas Wood 1954 Sceloporus sp. (Fence lizard) Texas Wood 1954 water snake) 1 t W 1 \Jl rk. Hamilton 1 9S1 Chelvdra sementina \^ I Aw A T VIA VA W A !_/ w A A L A A At.4. (Snapping turtle) New York A ^ W VT X \Jl. AV Hamilton 1951 Terrapene sp.(Box Turtle) New York Hamilton 1951 Phasianus colchicus (Ringnecked pheasant) New York, Michigan Taube 1947; Hamilton 1951 Turdus sp. (Robin) New York, Florida, Oregon Poumelle 1950; Hamilton 1951; Hopkins 1980 Corvus sp.(Crow) New York Hamilton 1951 Sturnella sp.(Meadowlark) New York Hamilton 1951 Ouiscalus Quiscula (Rrnn7pd Grackle) TsIpw YnrW Pooecetes gramineus (Vesper Sparrow) New York A 1 W V T A \j A A V Hamilton 1951 Family Anatidae (Duck) New York Hamilton 1951 Agelaius phoeniceus (Redwinged blackbird) New York Hamilton 1951 Sphvrapicus varius (Yellowbellied sapsucker) Texas Wood 1954

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15 Table 1 1 -continued Animal Species Location Reference Zenaida macroura Texas Wood 1954 (Mourning dove) Thvromanes bewickii Oregon Hopkins 1980 (Bewick's Wren) Pipilo sp. (Towhee) Texas, Oregon Wood 1954; Hopkins 1980 Gallus domesticus (Chicken) Texas, Michigan Taube 1947; Wood 1954

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CHAPTER 2 SPOROCYST SIZE OF ISOLATES OF SARCOCYSTIS SHED BY THE VIRGINIA OPOSSUM ( Didelphis vireiniana V Introduction Equine protozoal myeloencephalitis (EPM) is a severe neuromuscular disease affecting many horses in North and South America (Dubey et al. 1991; Granstrom et al. 1992; MacKay 1997; Masri et al. 1992). While the disease has been diagnosed for several years, only recently have causative agents been identified. Two different organisms have been implicated as causative agents of EPM: Sarcocystis neurona and Neospora hughesi (Cheadle et al. 1999; Dubey et al. 1991; Marsh et al. 1998). While information on N. hughesi is emerging, the contribution of this organism to EPM is unknown. The main focus of research regarding EPM centered on S. neurona The definitive host of S. neurona the Virginia opossum ( Didelphis virginiana ). sheds sporocysts of several types of Sarcocystis spp. in its feces. The current method used to differentiate these organisms is DNA-based marker analysis developed in this laboratory (Tanhauser et al. 1999). In our paper, we assess the potential to differentiate the five types of sporocysts based on light microscopy. 'Paper published with coauthors under the citation "Cheadle, M.A., Dame, J.B., and E.C. Greiner. 2001 Sporocyst size of isolates of Sarcocystis shed by the Virginia opossum ( Didelphis virginiana ). Veterinary Parasitology 95: 305-31 1." 16

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Materials and Methods i Sporocyst Collection and Storage Automobile-killed opossums were collected and feces and/or gut scrapings were examined for the presence of Sarcocystis sporocysts using sugar flotation with centrifugation. For samples found to be positive for the presence of sporocysts, intestines were removed and the mucosa was scraped using the edge of a glass slide to collect sporocysts contained within the mucosa. The collection was then placed into a solution containing 50% bleach and water, mixed thoroughly, and incubated on ice for 30 min. After filtration through gauze, the sporocysts were washed by repetitive centriftigation and resuspension in distilled water to remove residual amounts of bleach. Once the bleach was removed, we placed storage media (Hanks Balanced Salt Solution, 500 mL) plus antibiotics [10 x lO^'l.U. penicillin and 10 x lO'' fig streptomycin (Mediatech, Hemdon, Virginia)] and fungicide [250 |ig amphotericin (Mediatech, Hemdon, Virginia)] in the tube containing the sporocyst mixture. The sporocysts were stored at 4 C until use. Sporocyst Typing Sporocyst type was elucidated using restriction enzyme analysis as described by Tanhauser et al. (1999). Sarcocvstis speed was differentiated based on sequence analysis (J. B. Dame, personal communication). Observation and Measurement Sporocyst isolates used from each taxa are as follows: S. neurona (3027, 3113, 3120,3063); 1085 type (3013, 1 114, 1086, 1085); SJalcatula (31 14, 3106, 3105, 3101, 3021); S^jpeeri (2226, 2079, 2046); 3344 type (3344). Tubes containing stored

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18 sporocysts of each isolate were removed from refrigeration and an aliquot of approximately 10 was placed on the center of a glass slide. To prevent deformation of the sporocysts caused by the weight of the coverslip, four small dots of glycerine jelly (Fisher, Fair Lawn, New Jersey) were applied to each comer of a glass coverslip. The coverslip was then placed over the 1 0 |iL of media forming a hanging drop. The slide was then observed on a light microscope under oil at lOOOX magnification. Twenty sporocysts of each isolate from the five taxa were measured using a calibrated ocular micrometer. Sporocysts also were observed for differences in internal structure. Data Analysis Because length and width data were not normally distributed (P <0.001 by the Kolmogorov-Smimov test for normality), we analyzed our findings by the KruskalWallis one-way analysis of variance on rank (P < 0.05). Where a significant difference was found, pairwise multiple comparisons were made using Dunn's method. All statistical calculations were carried out using SigmaPlot and SigmaStat software for windows version 2.03 (SPSS, Inc., Illinois). Results Results of sporocyst measurements are presented in Table 2-1. The length and width of S. speeri (Fig. 2-1) was statistically different (P < 0.05) from the other sporocyst isolates. The length of S. neurona (Fig. 2-2) and S. falcatula (Fig. 2-3) sporocysts were statistically different (P < 0.05) from each other and the width of S. falcatula and 1085 type (Fig. 2-4) differed (P < 0.05). Due to the obvious size difference of the 3344 type (Fig. 2-5) sporocyst, statistical data were not compared between it and the other isolates. This type has been found in

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19 the feces of 10 opossums. This sporocyst is morphologically different from other isolates because it is much larger and has more pointed ends with plug-like structures (Fig. 2-6). Although other sporocyst isolates have a more rounded shape and are devoid of the pluglike structures (Figs. 2-1 thru 2-4), they do exhibit a similar refractory quality when observed using light microscopy. Diagnosis and collection were similar to diagnosis and collection of other taxa. However, this sporocyst type was very difficult to store because of the trait of collapsing after collection. As seen in Fig. 2-7, the sporocyst collapses within minutes of processing as compared to other taxa that maintain wall integrity throughout processing and storage. After collection and storage, intact sporocysts were not observed and thus the isolate has yet to be characterized molecularly. The internal morphology of the sporocysts was observed. All sporocysts contained four sporozoites and a residuum. Although there was no consistent difference between internal morphology of isolates, there appeared to be many forms. Forms ranged from a single, large, ball-like residual body approximately one-third the size of the sporocyst to upwards of 20 small, ball-like residual bodies. Multiple, small bodies were found in compact areas and diffuse throughout the inside of the sporocyst. There did not appear to be any trend that would allow the differentiation of type based on the makeup of the sporocyst residuum. Discussion Based on gross visual observations by light microscopy, only S. speeri and 3344 type sporocysts were different from the other similar isolates found in the feces of the opossum. Single-dimension differences such as S. falcatula vs 1085 (width) and falcatula vs S. neurona (length), were not considered sufficient to differentiate between

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20 the species/types. The 3344 type of sporocyst was originally thought not to be a sporocyst, but after close examination, 4 sporozoites and a distinct residuum were observed (Fig. 2-6). Dubey et al. (1989) did not describe sporocysts of this size being found in the feces of the opossum or any other definitive host. This type composed <5% of infections with sporocysts found in the feces and/or gut scrapings. Because this sporocyst collapses soon after flotation, fecal flotations of this preparation should be read immediately after the flotation procedure is finished. We were unable to determine why this sporocyst type exhibits this characteristic and whether this type uses the opossum as a host or simply is contained within a food item that is being passed through the gut during the time of sampling. The isolates in this study cannot be proven to be pure isolates. There is no way to insure that there is only a single sporocyst species/type present in the primary isolates collected from opossums used in this study. However, based on molecular data showing that the isolate is of one species, the authors feel that if there is a mixed infection in the opossum, the number of sporocysts of the second species/type would be low and should not influence the outcome of this analysis.

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21 Table 2-1 Comparison of Sarcocystis sporocyst sizes Taxa Mean(SE) Length([im) Range Mean(SE) Width f ^m) Range Sarcocvstis 10.65 (0.09)" 9.7-11.4 7.03 (0.09)"" 6.2-8.4 LAX unci 1085 type 10.85 (0.07)"9.7-11.9 6.81 (0.07)' 6.2-7.9 Sarcocvstis falcatula 10.95 (0.13)^ 8.8-11.9 7.05 (0.10)" 6.6-7.9 Sarcocvstis 12.23 (0.14)^ 11.0-13.2 8.81 (0.09)= 7.5-9.7 speeri 3344 type 19.43 (0.20)* 17.6-20.7 10.49 (0.20)* 9.2-11.9 SE = Standard error of the mean Isolates with the same superscript within columns designate no significant differences between isolates = Statistical analysis not performed on this isolate

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22 Fig. 2-2. Sporocyst of Sarcocystis newona (3063 isolate). Note the sporozoites (arrow) and residual body (arrowhead). Bar = 5.5 |im.

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23 Fig. 2-3. Sporocyst of Sarcocystis falcatula (3106 isolate). Note the sporozoites (arrowhead). Bar = 5.5|im. Fig. 2-4. Sporocysts of 1085 type (3013 isolate). Note the sporozoite (arrow) and residual body (arrowhead). Bar = 5 |im.

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Fig. 2-6. Detailed view of sporocyst of 3344 type (3344 isolate). Note the polar plug-like structures (double arrowhead), sporozoite (arrow), and residual body (arrowhead). Bar = 4.8 |am.

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25 Fig. 2-7. Brightfield micrograph of 3344 type sporocyst. Collapsed sporocyst of 3344 type (isolate 3395). Photographed 10 minutes post flotation. Bar = 6.5 \im.

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CHAPTER 3 VIABILITY OF Sarcocvstis neurona SPOROCYSTS AND DOSE TITRATION IN GAMMA-INTERFERON KNOCKOUT MICE" Introduction Sarcocvstis neurona is a causative agent of the neuromuscular disease equine protozoal myeloencephalitis (EPM) in horses and CNS disease in southern sea otters (Dubey et al. 1991; Lindsay et al. 2000; MacKay 1997). S. neurona has been isolated from horses in North and South America (Dubey et al. 1991; Granstrom et al. 1992). Neospora hughesi has also been shown to be a causative agent of EPM, however, data concerning its involvement in the disease is still emerging (Cheadle et al. 1999). While the occurrence of EPM in horses is low, the prevalence of antibodies to S. neurona in the United States is approximately 50% and approximately 35% in South America (Dubey et al. 1999a; Dubey et al. 1999c; MacKay 1997). The definitive host of S. neurona has been identified as the Virginia opossum ( Didelphis virginiana ) (Fenger et al. 1995 and 1997). Currently, there appear to be five different taxa of Sarcocystis sporocysts being shed in the feces of opossums (Cheadle et al. 2001a). The current methods used to differentiate Sarcocvstis sporocysts being released in the feces of opossums are DNA-based marker analysis and morphological examination developed in this laboratory (Cheadle et al. 2001a; Tanhauser et al. 1999). ^Paper published with coauthors under the citation "Cheadle, M.A., Tanhauser, S.M., Scase, T.J., Dame, J.B., MacKay, R.J., Ginn, P.E., and E.C. Greiner. 2001. Viability of Sarcocvsti s neurona sporocysts and dose titration in gamma-interferon knockout mice. Veterinary Parasitology 95: 223-232." 26

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Until recently, no model for the study of the EPM was readily available. Dubey and Lindsay (1998a) found that gamma-interferon knockout mice inoculated with uncharacterized sporocysts collected from the feces of the opossum developed clinical encephalitis. The tissues from these animals were found to contain stages of Sarcocystis neurona as determined by immunohistochemical staining of tissue sections and an immunofluoresent antibody test (IFAT) of cell cultured organisms. In order to ensure quality control in our laboratory, it was necessary to test the viability of S. neurona sporocysts in a banked collection and to also determine a low dose of sporocysts that can cause clinical signs in gamma-interferon knockout mice. Thus, in the present study, the use of gamma-interferon knockout mice as a model for S. neurona is expanded by determining the viability of molecularly characterized S. neurona sporocysts and the dose of sporocysts required to cause a clinical infection in these mice. We also correlate molecular identification of sporocysts with clinical disease in knockout mice. Materials and Methods Collection of Sporocysts Sporocyst collection was similar to that described by Cheadle et al. (2001a). Opossums were collected and feces and/or gut scrapings were checked for the presence of Sarcocystis sporocysts using sugar flotation with centrifiigation. Intestines of opossums found to be positive for the presence of sporocysts were removed and scraped. The collection was then cleaned using NaOCl, washed to remove bleach, and stored in storage media at 4 C. Sodium dodecyl sulphate (SDS) was substituted for NaOCl when isolate number 3227 was treated to determine if differences in viability would exist.

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Identification of Sporocyst Type Sporocyst type was elucidated using restriction enzyme analysis and morphological analysis as described by Tanhauser et al. (1999) and Cheadle et al. (2001a). Animals Eighteen gamma-interferon knockout mice (BALB/c-Ifng""'^') were obtained from Jackson Laboratories (Bar Harbor, ME). These mice lack the gene that encodes the production of gamma-interferon, a component of the cellular immune response, which is critical to host defense against protozoal organisms. All study mice were 46 days old and male. Mice were contained within isolation boxes and housed at the Department of Animal Resources, University of Florida. Sterilized commercial mouse ration and water were fed ad libitum during the course of the study. Inoculation Procedure Each mouse was inoculated with Sarcocystis neurona sporocysts via gastric gavage. Doses given to each mouse are listed in Table 3-1 Each mouse was observed daily for the development of clinical signs. To reduce consumption of a single isolate, four similar-aged isolates (3048, 6 months old; 3063, 6 months old; 31 13, 4 months old; 3120, 4 months old) were mixed in equal proportion and inoculated at five different doses: 500 sporocysts, 1,000 sporocysts, 5,000 sporocysts, 20,000 sporocysts, and 50,000 sporocysts. Sporocyst isolates 3027 (7 months old) and 3227 (2 months old) were inoculated in a similar manner at doses of 500 sporocysts, 5,000 sporocysts, and 50,000 sporocysts. Mouse Ml 7 received 5,000 sporocysts of an equal mix of isolates 1067, 1 112, 2011, 2033, 2043, 2103, 2052A, and 2052B, which were 16 months to 28 months

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29 old. Inoculum consisted of sporocysts in 0.1 mL of a solution that contained Hanks Balanced Salt Solution (HBSS) (500 mL) plus antibiotics (10 x 10''I.U. penicillin and 10 xlO'* /ug streptomycin (Mediatech, Hemdon, VA)) and fungicide (250 /Ug amphotericin (Mediatech, Hemdon, VA)). Mouse Ml 8 was inoculated with sterile HBSS. Necropsy and Histopathology A necropsy was performed on all mice that died or were euthanized. The following tissues were removed: lung, liver, kidney, muscle, stomach, intestine, brain, heart, testicle, pancreas, and spleen. Portions of these organs were kept fresh and fixed in 10% buffered formalin. Fixed tissues were submitted to the Department of Pathology, University of Florida for embedding, sectioning, and staining using hematoxylin and eosin (H & E). Sections were observed for the presence of lesions and Sarcocystis organisms using light microscopy at a power of 100 and 400X. Immunohistochemistry was performed using a polyclonal antibody to S. neurona to facilitate visualization of organisms (Hamir et al. 1993). Results Clinical signs observed in the gamma-interferon knockout mice were similar to the neurologic signs observed to occur in horses diagnosed with EPM (MacKay 1997) (Table 3-3). These included paralysis, rough coat, huddling, eye squinting, mild to severe ataxia in limbs, mild to severe head tilt, and circling. The clinical signs observed varied depending on the mouse and the dose. During the course of the 90-day observation period, mice Ml 1 and Ml 7 never showed clinical signs consistent with neurologic disease. Mil did appear to have a roughened coat during various days of the study, but clinical signs never progressed beyond that point. Mouse Ml 7 and Ml 8

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appeared normal through the entire course of observation. Organisms were observed in tissue sections of the cerebrum and/or cerebellum in 13 of 17 (77%) knockout mice inoculated with sporocysts (Table 3-2). No other organ was observed to contain organisms. Fewer numbers of organisms were observed in H&E sections compared to the number of organisms observed using immunohistochemistry. Of the 1 5 mice that were examined using immunohistochemical techniques, 1 2 (80%) had protozoal organisms present in one or more portions of the brain. Two mice were not processed using immunohistochemistry. Protozoal merozoites were frequently observed in groups (Figs. 3-1 and 3-2). Mouse Ml 8 had no lesions. Lesions observed in mice were closely associated with, but not confined to, the presence of organisms. The lesions and number of mice affected is presented in Table 3-4. The brain and lungs were the two areas of parasite focus and lesions. In the brain, large areas of necrotizing meningoencephalitis with associated lymphocytic and neutrophilic infiltrates were observed. Similarly, the lung contained large numbers of peribronchiolar infiltrates of neutrophils and lymphocytes associated with a necrotizing interstitial pneumonia. The lesions in other organs were less consistent and not as severe. Discussion The visualization of protozoal organisms in this study was facilitated by the use of immunohistochemistry. The results are similar to the observations made by Dubey and Lindsay (1998a), where more organisms were identified using immunohistochemistry compared to H & E stained sections. In the same study, the brain and liver were deemed to be the areas of primary infection in the knockout mice. In this study, however, the constant and severe hepatitis documented by Dubey and Lindsay

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31 (1998a) was not observed. Clinical signs that can be attributed to brain infection were consistently observed and therefore the brain was the organ on which our investigation was focused. Although the spleen also consistently contained lesions, parasites were never observed in this organ. Based on the author's experience, lesions in the spinal cord of horses are very difficult to find. Conversely, lesions in the brains of gamma-interferon knockout mice have been consistently observed by others, therefore the spinal cord was not used in this study (Dubey and Lindsay 1998a). Mouse Ml 1 did not show clinical signs after receiving a low dose (500) of sporocysts of an isolate that was at the age and dose limit used in this study. Because other isolates inoculated at this dose also cause disease in mice and that higher doses of this isolate did cause disease in mice, it is likely that few of the remaining sporocysts were viable and that age was the factor that limited the ability of the sporocysts of this isolate to cause clinical disease. All mice inoculated with higher doses of this isolate (3027) had an increased time to clinical signs and death/euthanasia as compared to other isolates. The mouse (M17) inoculated with isolates 1067, 1 1 12, 201 1, 2033, 2043, 2103, 2052A, and 2052B never showed clinical signs during the course of the 56 dpi observation period. The age of these isolates ranged from 16 to 28 months old at time of inoculation into mice, indicating that, in our laboratory, sporocysts that have been processed and stored for more than 16 months are not viable. The authors show that a base dose of 500 sporocysts of inoculum that is <7 months old at time of inoculation can be used for experiments using the gamma-interferon knockout mice (BALB/c-lfng""'^') as a model for neurologic infection with Sarcocystis neurona This laboratory (ECG) has recently changed its method of storing sporocysts to

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one in which the mixture is not bleached until an infection trial is about to begin. Although the authors in this paper indicates that sporocysts that have been bleached and stored (< 7 months) are still infectious, long-term storage after bleach treatment does decrease the number of viable sporocysts remaining in the inoculum. We have adjusted our procedure to store sporocysts in a Hanks solution similar to that used to suspend sporocysts during inoculation as described previously in this paper and bleach only directly prior to use. Although the brain of mouse (Ml 3) did contain lesions, no organisms were observed. The reason for this lack of organisms is unknown. However, the mouse regurgitated a small portion of the sporocyst mixture during the inoculation procedure. The portion was approximately one tenth of the entire inoculum. It is likely that the mouse received a decreased number of sporocysts that, while they caused clinical signs, were to low in numbers to be detected by light microscopy. Also, the 3027 isolate is at the threshold of age limits used in this study and viability is likely decreased. The course of the disease was also delayed in onset as compared to other mice. These factors lead to the question of the possibility of some other factor such as a toxic substance being produced by the organism which may be a cause of disease rather than simple physical damage to the host cells. The authors have confirmed that sporocysts determined to be Sarcocystis neurona by molecular characterization were are able to infect and cause disease in a susceptible host. The link between the molecular and biological data strengthens the ability of researchers to differentiate S. neurona sporocysts from other similar sporocysts being shed in the feces of the opossum. This study validates the use of molecular

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characterization of sporocysts collected from the opossum as compared to the time consuming and expensive use of mice to type isolates. Based on data by Dubey and Lindsay (1999) and Dubey et al. (2000d), Sarcocystis speeri can induce sarcocyst formation in skeletal muscle and encephalitis in the brains of knockout mice. Although encephalitis induced by S. speeri has been documented, no clinical signs were reported (Dubey et al. 2000d). Also, studies by Dubey and Lindsay (1998a), have shown that Sarcocystis falcatula does not infect gamma-interferon knockout mice. Based on sporocyst measurements made before inoculation, the sporocysts in this inoculum were smaller (-11 x 7 /um) than those of S. speeri (-12x9 /urn). Furthermore, all mice in the study by Dubey and Lindsay (1 998a) lacked sarcocysts and/or organisms in their skeletal muscle. Therefore, it is assumed that S. neurona sporocysts were the infectious agent in this study and that they are the only Sarcocystis sp. in the 1 1 x 7 fxm range that are shed in the feces of the opossum that can cause clinical signs consistent with neurological disease in gamma-interferon knockout mice. Further studies comparing the clinical signs caused by S. neurona with those caused by S. speeri and other species of Sarcocystis being shed by the opossum need to be conducted to further define the mouse model. Ensuring the viability of sporocysts to infect a host is critical. While excystation data derived in the laboratory will provide information on the ability of sporozoites to be released from a sporocyst and infect a cell monolayer, it provides no information about the ability of those sporozoites to properly infect a host and cause disease. In order to study the life cycle of S. neurona viable sporocysts must be available that can infect intermediate host candidate species. Without first ensuring the capacity of sporocysts to

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34 cause disease in a model animal, there is no way to determine whether or not the intermediate host candidate received a reliable challenge. High expense and large amounts of time are required to house and maintain an experimental animal such as a horse. The use of the gamma-interferon knockout mice as an experimental model for EPM in horses is an effective means of studying the disease in a laboratory setting. The clinical signs exhibited are similar in severity and duration. The use of this model allows the testing of chemotherapeutic drugs and vaccine candidates before implementation of expensive trials in horses.

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35 Table 3-1. Dose and isolate/s of Sarcocvstis neurona given to each gamma-interferon knockout mouse Mouse niimhpr Number of sporocysts counted In mocuium Isolate/s Age of sporocysts at time of mouse mocuidiion (months) Ml 500 3048,3063,3113,3120 4 to 6 M2 500 3048,3063,3113,3120 4 to 6 M3 1,000 3048,3063,3113,3120 4 to 6 M4 1,000 3048,3063,3113,3120 4 to 6 M5 5,000 3048,3063,3113,3120 4 to 6 M6 5,000 3048,3063,3113,3120 4 to 6 M7 20,000 3048,3063,3113,3120 4 to 6 M8 20,000 3048,3063,3113,3120 4 to 6 M9 50,000 3048,3063,3113,3120 4 to 6 IVllU 50,000 3048,3063,3113,3120 4 to 6 \A^ 1 500 3027 7 IVll/ 5,000 3027 7 IVl 1 J jU,UUU 3027 7 M14 500 3227 2 M15 5,000 3227 2 M16 50,000 3227 2 M17 5,000 1067, 1112, 2011,2033, 2043, 2103, 2052A, 2052B 16 to 28 M18 control not applicable not applicable

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Table 3-2. Microscopic identification of protozoa in routinely and immunohistochemically treated sections of cerebrum and cerebellum from gamma-interferon knockout mice inoculated with Sarcocystis neurona sporocysts Mouse number Protozoa observed using H & E light microscopy Protozoa observed using immunohistochemistry Ml + Cerebrum + Cerebrum M2 + Cerebrum + Cerebrum M3 + Cerebellum, cerebrum + Cerebellum, cerebrum M4 + Cerebrum + Cerebrum M5 + Cerebrum n/a M6 + Cerebrum + Cerebrum M7 n/a M8 + Cerebellum + Cerebellum M9 + Cerebrum + Cerebellum, cerebrum MIO + Cerebellum, cerebrum Mil M12 + Cerebellum, cerebrum M13 M14 + Cerebellum, cerebrum + Cerebellum, cerebrum M15 + Cerebrum + Cerebellum, cerebrum M16 + Cerebellum, cerebrum + Cerebellum, cerebrum M17 M18* n/a = sample not available = Ml 8 is a negative control = no protozoa observed + = protozoa consistent with schizonts of Sarcocystis observed in tissues

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Table 3-3. Summary of disease in Sarcocystis neurona infected gamma-interferon knockout mice Mouse First clinical signs Animal dead or Clinic signs observed during number observed (dpi) euthanized (dpi) course of infection Ml 22 33 rough coat, paralysis in rear limbs M2 28 31 rough coat, huddling, mild ataxia in limbs M3 28 32 rough coat, huddling, severe ataxia in limbs M4 28 32 rough coat, huddling, eye squinting, severe ataxia in limbs M5 10 25 rough coat, severe head tilt M6 20 24 rough coat, mild to severe head tilt M7 22 28 rough coat, huddling, severe head tilt, circling M8 24 28 rough coat, moderate ataxia in limbs M9 19 28 rough coat, mild head tilt Ml n 91 zz rougn coai, sugni lo severe neau tilt, circling Ml 1 n/a on y\) none M12 10 35 rough coat, huddling, eye squinting, high stepping, mild head tilt, slight to severe ataxia in limbs M13 28 33 rough coat, huddling, slight to severe head tilt, circling M14 28 31 rough coat, huddling, slight to severe ataxia in limbs M15 M16 26 20 31 28 rough coat, huddling, mild ataxia rough coat, huddling, slight head tilt, paralysis in rear limbs M17 none 68 none M18 none 68 none dpi = days post inoculation

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38 Table 3-4. Histopathological lesions observed in gamma-interferon knockout mice orally inoculated with Sarcocvstis neurona sporocysts Number of Organ mice with lesion/s Lesion/s Lung 15 Multifocal to extensive necrotizing interstitial pneumonia, peribronchiolar infiltrates of neutrophils and lymphocytes Liver 1 Aggregates of neutrophils and hemosiderophages scattered throughout hepatic sinusoids. Stomach 8 Multifocal to extensive neutrophilic gastritis Skeletal muscle 1 Perivascular infiltrates of neutrophils in supporting fibrovascular connective tissue stroma Heart 3 Accumulations of mineralized material present in subepicardial fibrovascular connective tissue stroma Spleen 14 Periarteriolar follicular hyperplasia Cerebrum 12 Multifocal necrotizing meningoencephalitis, Ivmnhnrvtip anrl npiitronhilir infiltrjitp
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Fig. 3-1 H & E stained section of cerebellum from mouse M8 containing a group of merozoites (arrowhead). Bar = 8.8 ^im. Fig. 3-2. Immunohistochemically stained schizont of S. neiironu from mouse M8 cerebellum containing multiple merozoites (arrowhead). Bar = 8.8 //m.

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CHAPTER 4 Sarcocvstis greineri N. SP. (PROTOZOA: SARCOCYSTIDAE) IN THE VIRGINIA OPOSSUM r Pidelphis virginiana ) Introduction The Virginia opossum ( Didelphis virginiana ') has been implicated as the definitive host for at least four species of Sarcocvstis (Box et al. 1984; Dubey et al. 1991; Dubey and Lindsay 1999b; Tanhauser et al. 1999). Little information has been reported about opossums being intermediate hosts of Sarcocvstis spp. Sarcocysts were found in a D. virginiana by Scholtyseck et al. (1982), but not named. There are three other named species of Sarcocvstis in other opossum species, Sarcocvstis gamhami (Didelphis marsupialis Philander sp.), Sarcocvstis marmosae (Marmosa murina ), and Sarcocvstis didelphidis ( Didelphis marsupialis ) (reviewed by Odening 1998). During the course of routine necropsy of road-killed and live opossums for other research projects, sarcocysts were observed in skeletal muscle of D. virginiana The purpose of the present paper is to describe and name this previously unnamed species of Sarcocvstis that uses the Virginia opossum as an intermediate host. Materials and Methods Opossum Collection and Processing Opossums were collected either as road-kills or were live trapped and killed (Beuthanasia-D Special, Schering Plough Animal Health, Kenilworth, New Jersey). Each animal was assigned an identification number and data concerning the date and 40

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location of collection were recorded. Samples of muscle were removed from the tongue, abdomen, and rear thigh muscle. Small portions of muscle were removed from these samples and processed for light and electron microscopy. The remaining portion of the muscle sample was stored at 4 C for use in feeding trials. Light Microscopy A small portion of muscle was fixed for histopathologic examination using 1 0% buffered formalin for a period of 24 to 48 hr. The tissue was processed, embedded, sectioned at 5 fim, and stained with hematoxylin and eosin. Sections of tongue and thigh skeletal muscle were scanned for the presence of sarcocysts using a dissecting microscope at a magnification of 25X. When a sarcocyst was detected, the cyst was removed and observed at 40X magnification on a compound microscope for verification of a sarcocyst wall. Sarcocysts as well as the bradyzoites contained within were measured, photographed, and the sizes recorded. Feeding Trials Approximately 1 12 g of skeletal and tongue muscle tissue from a single opossum observed to contain sarcocysts was fed to a greyhound dog (Canis familiaris ) and a domestic short hair cat (Feliscatus). Fecal samples were collected at 3-day intervals during the course of the trial from Days 0 (dog) and -5 (cat) post inoculation (PI) through Day 30. Samples were refrigerated at 4 C until processed. Sugar flotation was used to detect the presence of Sarcocystis sp. sporocysts in all fecal samples. The cat was killed and the intestine removed. The mucosal epithelium was scraped with a glass slide. The scrapings were collected and observed using light microscopy for the presence of Sarcocystis sp. sporocysts. Dog intestine and tissues were not available at the end of the

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trial. Sections of liver, lung, kidney, brain, heart, spleen, and muscle were removed from the cat and observed by light microscopy for stages of Sarcocystis A wild-caught opossum determined to be negative for Sarcocystis by fecal flotation was fed 76 samples of opossum tongue and skeletal muscle from 76 different opossums. Seven of these opossum samples contained sarcocysts as visualized by light microscopy. Fecal samples were collected at 3-day intervals during the course of the trial j&om Day -30 through Day 74 when the opossum was killed. Fecal samples and the intestine were treated as above. Sections of liver, lung, kidney, brain, heart, spleen, and muscle were removed from the opossum and observed by light microscopy for stages of Sarcocystis Transmission Electron Microscopy Muscle samples containing sarcocysts were removed from an opossum and immediately immersion-fixed in a 1.5 mL plastic vial containing approximately 1 ml of cold Trumps fixative for at least 24 h. The tissue was then removed, placed in phosphate buffered saline (pH 7.3), and stored at 4 C until processed. Sarcocysts were observed and removed from muscles using a dissecting microscope. The cysts were washed two times for 10 min each in PBS. Samples were then postfixed in 1% osmium tetroxide (w/v) for 1 h and washed three times for a period of 1 0 min in double deionized water. Samples were then dehydrated in ethanol (25 and 50%) for 10 min, followed by a mixture of 75% ethanol and 2% uranyl acetate overnight at 4 C, and dehydrated further with 95 and 100% ethanol at 10 min each and 100% acetone two times at 10 min each. Flat embedding in EmBed epoxy resin (Ted Pella, Redding, California) included dilution steps using acetone (1 :2, 1 : 1 2: 1 ) into a final concentration of 1 00% EmBed. Fresh

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43 100% EmBed was added and allowed to polymerize at 60 C for 2 days. Thin (70-80 nm) sections were placed on formvar coated 50 mesh copper grids and double stained with 2% aqueous uranyl acetate followed by Reynolds lead citrate. Stained sections were then examined with a Hitachi H-7000 TEM operating at a voltage of 75 kv and images were taken with a Gatan Bioscan ccd camera. Results The cat, dog, and opossum fed infected opossum tissue did not shed sporocysts of Sarcocystis sp. during the course of this study. Scrapings from the opossum and cat intestines were also negative for the presence of sporocysts. No stages of Sarcocystis were observed in the tissue sections of the opossum and cat fed muscle tissue containing sarcocysts. Description Sarcocystis greineri n. sp. Diagnosis. Sarcocysts occur in skeletal muscle of the tongue, thigh, and abdomen. The sarcocysts visible using a dissecting microscope (25X) and average 3.8 mm (2.0 to 6.0 mm; n = 5) X 154.6 |im (108.0 to 189.0 |am;n= 10). The outer cyst wall with moderate to deep invaginations throughout the entire length of the cyst to the point of bisecting the entire cyst (Fig. 4-1 thru 4-2). Invaginations occur in both fresh and fixed muscle specimens. The mature sarcocyst wall with stumpy, digit-like protrusions or villi, some being pedunculated near the cyst wall similar to the Type 9 protrusions of Sarcocvstis campestris as described by Dubey et al. (1989) (Figs. 4-3, 4-4A). The protrusions differ from those of S. campestris in that they do not contain electron-dense granules. All protrusions with large numbers of fibrillar elements which extend from the

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interior portion of the sarcocyst wall through the villi. By TEM, protrusions approximately 3.4 fim (2.8 to 4.0 |im; n =10) x 1.5 (1.3 to 2.0 [im; n =10). The interior portion of the sarcocyst with thick septa (0.2 to 4.5 nm) separating pockets of bradyzoites or metrocytes. By light microscopy, live bradyzoites freed from the sarcocyst measure 1 1 .0 X 4.4 |am. Using electron microscopy, bradyzoites with structures typical of Sarcocystis spp. (Dubey et al. 1989), a posteriorly arranged nucleus, many anterior micronemes, dense bodies, and amylopectin granules (Fig. 4-4B). Rhoptries rarely seen and occurred as a single unit and not in pairs. Taxonomic Summary Type intermediate host. Virginia opossum (Didelphis virginiana ). Natural deflnitive host. Unknown. Type location. North-central Florida (29 to 30N, 82 to 83W). Site of infection Skeletal muscle. Prevalence. 1 0% (2 opossums infected/20 opossums examined) Specimens deposited. Histologic sections of sarcocysts in the U. S. National Parasite Collection, Beltsville, Maryland as USNPC nos. 90685 and 90686. Etymology. Sarcocystis greineri is named in honor of Ellis C. Greiner, Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, who has made major contributions to the diagnosis and study of coccidia and other parasites of veterinary importance. Discussion According to Odening (1998), there are three valid species of Sarcocystis in opossums. Sarcocystis didelphidis (Scorza et al. 1957) occurs in the South American

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opossum ( Didelphis marsupialis ). Sarcocystis gamhami (Mandour 1 965) in the skeletal muscle of Didelphis marsupialis found in Honduras, and Sarcocystis marmosae (Shaw and Lainson 1969) found in the skeletal muscle of Marmosa murina in Brazil. None of these species has been documented in the Virginia opossum. Structural measurements were compared to determine similarities and/or differences (Table 4-1). Microscopically, sarcocysts and bradyzoites from S. greineri are larger than those for the other valid species described from opossums. The bradyzoites are much closer in size to those described by Scholtyseck et al. (1982), who found that fixed merozoites within the cyst measured approximately 7 to 10 x 2.5 to 3 |am. Using transmission electron microscopy, the cyst wall protrusions/villi for S. greineri are approximately half the size of the other two species that have been described. Sarcocystis gamhami also varies from the other species because protrusions from its cyst wall are very sharply pointed as compared to those of S. greineri and S. marmosae which have finger-like projections that are rounded off at the distal end. Also, because TEM data with regards to S. didelphidis S. marmosae. and S. garnhami are lacking in the original descriptions, determination of the presence of pedunculated protrusions in these species is impossible.

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46 Table 4-1 Comparison of structural measurements of sarcocysts of Sarcocystis spp. found in the muscle of opossums Parasite Intermediate Sarcocyst (|iim) Bradyzoite Sarcocyst wall host length X width (^m) protrusions/villi (state of sample) (|am) Sarcocystis didelphidis Didelphis marsupialis 0.9mm X "3 /I ^ II rvt jhj |i.m 6.5 X 1.5 |im (fixed) 5.2 fim Sarcocystis gamhami Didelphis marsupialis 310 |im-3.3mm X 110-250 ^m 5.3-6.9 nm x 1.3-1.9 ^im (fixed) 6-8 nmx 1.5-2.0 fim acidophilic sharply pointed spines Sarcocystis marmosae Marmosa murina <2 mm X 800 |im 6.2-9.0 nm X 1.8-3.0 ^m (dried smear) 11 5-13 0 umx 2.6 nm finger-like with rounded tips Sarcocystis greineri Didelphis yirginiana 2.0-6.0 mm X 108.0-189.0|im 1 1.0 X 4.4 ^m (live) 2.8-4.0 nm X 1.3-2.0 ^m stumpy, digitlike protrusions, some pedunculated near cyst wall

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Fig, 42, H igh njagi uflcauoii of an mvaginatioii (arrow) xn the sarcocyst wall. Note the viiiar protrusions (arrowhead). MematO-Kvlin arsd eosm stained. Bar ~ 15 jnn.

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ig. 4-3. S v o^\'m opoN -am Note the thick, iuiger-like viHar

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49 Fig. 4-4. TEM of a sai-cocyst of Sarcocysfis gremeri n. sp, from the Virginia oposs-unt (A.) Micrograph of bradyxoites. Note the apical rings (arrowheads), conoid (arrow), iiunierous raicronenies (M). amylopectin granules (Am), numerous dense bodies (DB). arid a rhoptrie (Rh). Bar = 2 um. (1:1.) Sarcocysi wall showing the stumpy, digit-like villar protrusions (VP), Fibrillar elenjcms i'arro.whe.-^Ji c ibc L'runuiar l.tscr (Id ; i^l'xhc cy 4. Vdlar pr.)ir!iM4)n'-; com-innn^ pedussculauc^ris darifc arrtsH ;• a^c ihuvd througluaii Ihe \'>aH. N'utc the i^nvAl nnaginudons small arrow} of the villi. Bar 2 um.

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CHAPTER 5 THE NINE-BANDED ARMADILLO (Dasvpus novemcinctus ) IS AN INTERMEDIATE HOST FOR Sarcocystis neurona ^ Introduction Sarcocystis neurona is a protozoan parasite known to be associated with the neurologic disease equine protozoal myeloencephalitis (EPM). This disease has been considered one of the most important neurologic diseases of horses in the United States (MacKay 1997). Fenger et al. (1997) and Tanhauser et al. (1999) identified the definitive host of S. neurona as the Virginia opossum ( Didelphis virginiana ). Dubey et al. (2000c), found that an immunosuppressed cat ( Felis domesticus ) fed sporocysts identified as S. neurona collected from naturally infected opossums formed mature sarcocysts that infected an opossum, thus completing the life cycle of S. neurona in a laboratory setting. However, Dubey et al. (2000c) proposed that the cat is a laboratory host, but that completion of the life cycle of S. neurona using the domestic cat in a natural setting is not likely. Our goal was to identify natural intermediate hosts that allow completion of the life cycle in nature. Identifying natural intermediate hosts may provide veterinarians and horse owners with a method of controlling the intermediate host and thus S. neurona infected opossums on horse farms. ^Paper published with coauthors under the citation "Cheadle, M.A., Tanhauser S.M., Dame,J.B., Sellon, D.C., Hines, M., Ginn,P.E., MacKay, R. J., and E.G. Greiner. 2001. The nine-banded armadillo ( Dasypus novemcinctus ) is an intermediate host for Sarcocvstis neurona International Journal for Parasitology 31: 330-335." 50

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51 During the course of studies in our laboratory (ECG), the opossum has been found to shed at least four types of sporocysts in addition to S. neurona (Cheadle et al. 2001a; Tanhauser et al. 1999). Shedding of multiple species of Sarcocystis by the opossum makes collection and differentiation of S. neurona a complicated process. Our search for possible intermediate hosts has been extensive. When wildlife carcasses had sarcocysts in muscles, as determined by microscopic examination, these sarcocysts were excised and characterised molecularly for comparison with S. neurona At the same time, portions of remaining tissues were fed to opossums to determine if sporocysts are subsequently shed. The nine-banded armadillo ( Dasypus novemcinctus ) is an intermediate host of at least three species of Sarcocystis : Sarcocystis dasypi Sarcocystis diminuta and an unidentified species (Lindsay et al. 1996). Armadillos are frequently killed by automobiles making them a readily available food source for opportunistic feeders such as D. virginiana Muscle from road-killed armadillos containing sarcocysts was fed to opossums and excised sarcocysts were characterised molecularly (Tanhauser et al. 2001). Sporocysts shed by experimentally infected opossums were collected and characterised by sequencing species-specific regions of the DNA following PGR amplification (Tanhauser et al. 1999). Here and in a companion paper (Tanhauser et al. 2001), we combine molecular and biological evidence demonstrating that the nine-banded armadillo ( D. novemcinctus ) is an intermediate host to S. neurona Armadillos were collected either as automobile-kills or were live trapped and killed (Beuthanasia-D Special, Schering Plough Animal Health). Each animal was assigned an identification number and data concerning the date and location of collection

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recorded. Skeletal muscle from the tongue and legs were removed. Portions of each muscle were kept at 4 C for feeding to laboratory-reared opossums and fixed for light and TEM. Tissues were processed for light and TEM as described previously (Cheadle et al. 2001c; Luznar et at. 2001). Muscle was fixed using 10% buffered formalin, processed, embedded, sectioned at 5 ^m, and stained with H & E. Sections of skeletal muscle were scanned for the presence of sarcocysts using a dissecting microscope at a magnification of 25X. For TEM, fresh muscle samples containing sarcocysts were excised and immediately immersion-fixed in cold Trumps fixative. Samples were postfixed in 1% osmium tetroxide (w/v), dehydrated in ethanol, embedded in EmBed epoxy resin, and thin (70 to 80 nm) sectioned. Sections were double stained with 2% aqueous uranyl acetate followed by Reynolds lead citrate. Stained sections were then examined with a Hitachi H-7000 TEM operating at a voltage of 75 kv and images were taken with a Gatan Bioscan CCD camera. Four wild-caught and eight laboratory-reared opossums were housed at the Department of Animal Resources, University of Florida during the course of this study. Fecal samples were collected and screened for the presence of Sarcocystis sp. sporocysts before the start of the feeding trial. Feces were processed using Sheather's sugar flotation centrifugation method and observed for the presence of Sarcocvstis sp. sporocysts using light microscopy (lOOX). If an opossum shed sporocysts in its feces during the preinfection trial period, it was removed from the study. Portions of skeletal muscle from armadillos were stored at 4 C until fed to non-sporocyst shedding opossums. Muscle sections from the armadillo(s) fed to each opossum were observed

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using a dissecting microscope to ensure that sarcocysts were present. Muscles found to contain sarcocysts were fed to opossums over a 2to 4-day period. Concurrent with feeding trials, armadillo sarcocysts were excised and DNA was extracted (Tanhauser et al. 2001). DNA was analysed using PCR amplification, restriction enzyme analysis, and DNA sequencing techniques (Tanhauser et al. 1 999). Banked plasma samples were obtained from wild caught and laboratory raised armadillos and observed for the presence of antibodies to S. neurona by immunoblot (Tanhauser et al. 2001). Experimentally infected opossums were killed approximately 45 days p.i. and intestines were removed. A portion of the small intestine was scraped using a glass slide and a portion of the scraping was then processed using sugar flotation. The direct scraping and flotation preparations were observed by light microscopy (lOOX) for the presence of oocysts/sporocysts. The remaining portion of the scraping was then homogenized in a blender for 30 to 60 s and stirred for 4 to 6 h in 500 mL distilled water containing one-three drops of Tween 80. After filtration through a tea strainer, the mixture was centrifuged for 10 min. After removal of the supernatant, storage media (Hanks Balanced Salt Solution (500 mL) plus antibiotics (10 x 10''I.U. penicillin and 10 x lO"* fig streptomycin (Mediatech) and fungicide (250 amphotericin (Mediatech) was placed in the tube containing the oocyst/sporocyst mixture. Sporocysts from opossums DV02, DV14, and DV 22 were analyzed using techniques as described by Tanhauser et al. (1999) with one exception. Excystation was induced by a fi-eeze-thaw technique rather than by incubation in equine bile. Sporocysts were pelleted by centrifugation. The storage media was removed and replaced with 100 lysis buffer plus 5 Proteinase K. Sporocysts were then frozen in liquid nitrogen

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for 1 min. After fi-eezing, the sporocysts were heated to 37 C for 1 min. This procedure was repeated twice. The PCR primer pair JNB25/JD396 were used for amplification. PCR products were sequenced and a 256 bp segment of product was compared with the same segment of DNA from S. neurona isolate UCD-1 as described by Tanhauser et al. (1999). A 2-month-old, immune competent, Arabian colt was given 5x10' S. neurona sporocysts collected from opossum DV22 via intragastric inoculation. Two days prior to inoculation, the foal was determined to be healthy on the basis of physical examination, neurological examination, complete blood count and serum biochemical profile, and was weaned from the mare. The foal was anaesthetized with xylazine (1.1 mg/kg IV) and ketamine (2.2 mg/kg IV) and a sample of CSF was obtained from the atlanto-occipital space for cytological evaluation. An aliquot of CSF was submitted to a commercial laboratory (Equine Biodiagnostics, Inc., Lexington, KY) for immunoblot for the detection of antibodies against S. neurona Temperature, heart rate, and respiratory rate were monitored daily after infection; a neurological examination was performed biweekly. At post-infection Weeks 2, 4, 6, and 10, the foal was anaesthetized and a sample of CSF was obtained from the atlanto-occipital space for cytological evaluation and immunoblot. No treatment for EPM was given during the course of this study. Four of four (100%) wild-caught and 5 of 8 (63%) laboratory -raised opossums contained sporocysts in their intestinal scrapings after being fed armadillo muscle. The mean dimensions of sporocysts collected from these opossums were 1 1.0 x 7.5 |am (n = 10 per opossum, range = 1 1.0 to 1 1.4 x 7.0 to 7.9 |im). Prepatent period of shedding ranged from 18 to 46 days post feeding (Table 5-1). Total numbers of sporocysts

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55 collected from each opossum are listed in Table 5-1 Total numbers of sporocysts collected ranged between 0 to 4.1 x 10^. Sporocyst production shed in feces was approximately two-four sporocysts per 4 grams of feces per day as determined by sugar flotation techniques. Ultrastructural examination of sarcocysts revealed a sarcocyst wall similar to that of S. dasypi (Lindsay et al. 1996) (Fig. 5-1). Approximately 50% of armadillos observed contained sarcocysts determined to be S. dasypi by light microscopy, while less than 10% contained sarcocysts of S. diminuta The probability that S. dasypi and S. neurona are synonymous is high, however further studies need to be conducted. The 256 bp segment of sporocyst DNA collected from opossums DV02 and DV14 was 100% homologous with the same segment of UCD-1. The 256 bp segment of sporocyst DNA collected from opossum DV22 was 99.6% homologous with the same segment of UCD-1 with a single bp difference. The infected foal maintained a normal temperature, heart rate, and respiratory rate throughout the evaluation period. The foal was seronegative and CSF negative for antibodies to S. neurona prior to infection. Both CSF and serum were positive for antibodies by 4 weeks p.i. (Table 5-2). CSF cytology, including red blood cell count, was within normal limits at every time point (Table 5-2). At 6 weeks p.i., the foal developed an abnormal gait with proprioceptive deficits and ataxia of all four limbs. Neurologic deficits gradually improved over a 3 week period; at 10 weeks p.i., the foal was considered neurologically normal except for the right hind limb. No necropsy was performed. By infecting both wild caught and laboratory raised opossums via armadillo tissue

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infected with sarcocysts, we have repHcated our results. It was important to verify that different opossums will shed sporocysts after being fed muscle from different armadillos. This will ensure that the phenomenon of shedding can be reliably duplicated in different individuals and that completion of the life cycle was not simply due to an abnormality in an individual opossum, such as compromised immune status, that would allow it to aberrantly complete the life cycle. Sporocysts were never detected in the feces of opossum DV 17 (Table 5-1), but at the end of the trial, gut scrapings were found to contain low numbers of sporocysts. Therefore, it is possible that even if wild caught opossums feces are monitored for an extended period of time and sporocysts are not detected, the gut may still contain latent stages. Therefore, coupling infection trials with DNA analysis of sarcocysts is very useful. Opossums DV18 and DV24 did not shed sporocysts in their feces and upon euthanasia and necropsy, the intestinal scrapings did not contain sporocysts. The muscles fed to these opossums were 61 and 104 days old, possibly allowing time for sarcocyst degradation. It is also possible that sarcocysts from a Sarcocystis sp. other than S. neurona may have been detected or that the muscle contained a low density of sarcocysts (approx. one sarcocyst per 10 mm^ section of muscle). Opossum DV21 was observed to shed sporocysts in its feces on days 23 and 37. Counts were minimal and only one to three sporocysts were observed per 1 to 2 g of feces floated. The authors cannot explain the lack of sporocysts observed in intestinal scrapings. Observation of muscle sections of the armadillo fed to DV21 indicated that while few sarcocysts were present, numbers were sufficient to produce a patent infection.

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Also, the muscle tissue was only 1 1 days old at the time of inoculation, therefore sarcocyst degradation should have been minimal. Sequence analysis of the PCR products from sporocysts indicates identity with S, neurona Although sample DV22 differed from UCD-1 by one base (0.4%), it is within the normal range of variability among isolates of S. neurona (Tanhauser et al. 1999). Further DNA characterisation will need to be performed to determine the amount of sequence variability among armadillo S. neurona isolates. The host range of the nine-banded armadillo in the United States is limited. Data indicate that the range extends from Florida to New Mexico in the south and as far north as South Carolina to Kansas and parts of Nebraska (Hall 1981). In Central and South America, the range encompasses most of Mexico south to Peru, northern Argentina and Uruguay as well as Grenada and Trinidad and Tobago (Nowak and Paradiso 1983; Nowak 1999). The geographic range of the armadillo in the United States does not extend sufficiently north to encompass all areas that have endemic cases of EPM. The simplest explanation for this phenomenon is that there is more than one natural intermediate host of S. neurona Another explanation for the widespread antibody prevalence in horses is the use of commercially available food stuffs that have not been heat treated and thus may be contaminated with sporocysts. Food items could be contaminated with sporocysts at harvesting and the sporocysts remain infectious through the processing procedure. Wild caught armadillos were not used for infection trials to complete the reciprocal infectious nature of the parasite due to the high prevalence of Sarcocystis infected animals in the wild (Lindsay et al. 1996). Armadillos are very difficult to raise

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58 in captivity. Though the animals occasionally became pregnant, researchers have seen only 14 successful rearings among more than 700 females housed indoors (Truman and Sanchez 1993).

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59 Table 5-1 Armadillo inoculum fed to laboratory raised opossums and prepatent period of Sarcocvstis neurona sporocysts in feces Opossum number Age 01 samples at feeding (days) Armadillo muscle consumed Prepatent period (days) total sporocysts collected from opossum DV02' n/a DN35-DN55 (21) 25 2.6 X 10' DV09' n/a DN 58, DN 59, DN63 (3) 46 DVIO' n/a DN58 DN59 DN64 (3) 44 1.1 X 10' DV14' n/a DN60, DN64 (2) 40 3.4 X 10' DV17^ 66 DN66(1) ns 2.4 X 10' DV1861 DN67 (1) ns 0 DV 1945 DN69 (1) 18 1.6 X 10' DV202 41 DN70 (1) 18 1.1 X 10' 11 DN71 (1) 23 0 DV227 DN72(1) 18 4.1 X 10' DV23^ 6 DN73 (1) 18 1.5 X 10' DV24104 DN64(1) ns 0 n/a = not available; ns = no sporocysts observed in faeces; *Sporocysts observed in scraping but numbers deemed not sufficient to count; Wild caught opossum; Laboratory raised opossum; ( ) Total number different armadillos from which muscle was consumed

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60 Table 5-2. Results of cytologic analysis and Western immunoblot of CSF samples from a foal inoculated with 5x10^ sporocysts of Sarcocystis neurona collected from opossum DV22 fed armadillo muscle containing sarcocysts Week PostRBC WBC Protein Western infection (per uL) (per uL) (mg/dL) immunoblot Pre-infection 6 0 33.5 Negative 2 1102 2 24.9 Negative 4 0 1 34.2 Low positive 6 1 3 36.7 Positive 10 0 4 35.7 Strong positive

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Fig, 5-1, TEM of sarcocyst from annadiifo DH41 fed to opossvmi DV02, (A) !:sra.dy7.oites (B) are Cviniained wiih'm ihc graniihir layer (GL) of the s.;^"Ci>cyst, Villar protrussions (V) are throughout the sa.rcocyst wall arui appear lo he cut in serial sections exier^ding from the base nCiitest the sarcocyst lo the poriion closest to the host muscle (M), Note the apical rmg (arrow). Scale bar 2 |im. (B) High oiagoilication of a hradyzoite (Br) showing nucroneraes (sjrtall arri>'-AT!ead), a kirge posterior nucleus (N). asiJ coj'ioid (large arrovvheady Note the rliCiptries sopco. arrowhead") and smylopccii;-! granules { Am). A sepia (S) ean be observed separatusg the bradyzoites. Scale bar \ uru.

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CHAPTER 6 THE STRIPED SKUNK ( Mephitis mephitis ) IS AN INTERMEDIATE HOST FOR Sarcocystis neurona '* Introduction Sarcocystis neurona is a protozoan parasite known to be a causative agent of the neurologic disease equine protozoal myeloencephalitis (EPM). This disease has been considered one of the most common neurologic diseases of horses in the United States (MacKay 1997). Fenger et al. (1997), Dubey and Lindsay (1998a), and Tanhauser et al. (1999) identified the definitive host of S. neurona as the Virginia opossum ( Didelphis virginiana ). Dubey et al. (2000c) found that domestic cats (Felis domesticus ) can be induced to become infected with S. neurona in the laboratory and serve as an experimental intermediate host. We recently found that opossums fed muscle from ninebanded armadillos ( Dasvpus novemcinctus ) naturally-infected with sarcocysts would shed sporocysts that were molecularly and biologically identified as S. neurona implicating the armadillo as a natural intermediate host for S. neurona (Cheadle et al. 2001c). The striped skunk ( Mephitis mephitis ) is a definitive host for one species of Sarcocystis Sarcocystis rileyi and an intermediate host for one unnamed Sarcocystis sp. (Cawthom and Rainnie 1981; Erdman 1978; Wicht 1981). Skunks have a range that ''Paper published with coauthors under the citation "Cheadle, M.A., Yowell, C.A., Sellon, D.C., Hines, M., Ginn, P.E., Marsh, A.E., Dame, J.B., and E.C. Greiner. 2001. The striped skunk ( Mephitis mephitis ) is an intermediate host for Sarcocystis neurona International Journal for Parasitology 31: 843-849." 62

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encompasses the majority of the United States, Canada, and northern Mexico, providing a coverage of areas in which EPM is endemic (Hall 1981). Muscles from skunks containing sarcocysts were fed to opossums to determine if the opossum would produce Sarcocystis oocysts/sporocysts. Sarcocysts that were excised from skunks and sporocysts that were collected from opossums fed skunk muscle were compared with S. neurona using PCR followed by DNA sequence analysis of PCR products. In the present paper, we provide evidence that the striped skunk is an intermediate host for S. neurona using laboratory-reared opossums. Materials and Methods Three 10-month-old striped skunks (SKOl, SK02, and SK03) were obtained from a commercial supplier (Ruby Fur Farm Inc.). Skunks SKOl and SK02 were orally inoculated with 5x10^ and 5x10'* sporocysts, respectively, of an S. neurona isolate collected from a naturally infected opossum (isolate # 4180). These sporocysts were identified as S. neurona based on techniques described by Tanhauser et al. 1999 and Cheadle et al. 2001a. Skunk SK03 was inoculated with sterile Hanks balanced salt solution (HBSS). Skunk SKOl was found dead 90 d.p.i. and skunks SK02 and SK03 were killed 92 d.p.i. (Beuthanasia-D Special, Schering Plough Animal Health). Skeletal muscles were removed and portions were kept at 4C for feeding to laboratory-reared opossums. Tissues including liver, lung, kidney, muscle, stomach, intestine, brain, heart, and spleen were removed and processed for light microscopy, immunohistochemistry, and TEM as described previously (Cheadle et al. 2001b; Cheadle et al. 2001c). Two additional mature skunks were obtained from the same rearing facility, killed, and muscles were observed using histologic sections and gross observation to determine if

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64 natural infection was present in the facilities population. No sarcocysts were observed in the muscles of these animals using these methods. Laboratory-reared opossums were maintained as described by Cheadle et al. (2001b). Fecal samples were collected and processed as described by Cheadle et al. (2001b) to detect the presence of Sarcocystis sporocysts from day 0 p.i. and 3 days a week thereafter throughout the course of the study. Portions of skeletal muscle from skunks were stored at 4 C for 1 -6 days until fed to non-sporocyst shedding opossums. Before the feeding trial, muscle sections from skunks were observed using a dissecting microscope at 1 5X to ensure that sarcocysts were present. Sarcocysts were visible only with the aid of a dissecting microscope. Approximately 80 g of sarcocyst positive < skeletal muscle was fed to each opossum over the course of 2 to 3 days. The total numbers of sarcocysts fed was not determined. Opossums readily consumed all sarcocyst muscle that was presented. Feces were collected and processed as described by Cheadle et al. (2001b) for a period of 4 weeks after opossums began the feeding trial. Concurrent with the feeding trial, skunk sarcocysts were excised and DNA was extracted. DNA was analysed and compared to the UCD-1 isolate of S. neurona using primer set JNB25/JD396 for PCR amplification and sequencing techniques (Tanhauser et al. 1999). Opossums were killed (Beuthanasia-D Special, Schering Plough Animal Health) approximately 30 days p.i. and intestines were removed. Collection and measurement of sporocysts was conducted as described by Cheadle et al. 2001a and 2001c. Sporocyst DNA was extracted. DNA was analysed and compared to the UCD-1 isolate of S, neurona using primer set JNB25/JD396 for PCR amplification and sequencing techniques (Tanhauser et al. 1999). A 2-month-old, clinically normal pony was given 5 x 10' S,

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neurona sporocysts collected from opossum DV37 via intragastric inoculation and monitored as described by Cheadle et al. (2001b). Two gamma-interferon gene knockout mice (BALB/c-lfng""'^*) (Jackson Laboratories) per opossum isolate were inoculated with 5x10^ sporocysts, monitored, necropsied, and tissues processed as described by Cheadle et al. (2001c). Serum samples from sporocyst inoculated mice and skunks were tested for the presence of S. neurona reacting antibodies by Western blotting. Skunk serum samples were collected at 16 days preinoculation and 28, 41, 63, 78, 90, and/or 92 days p.i. Sera from mice were obtained at necropsy. Sera obtained from a naive mouse and laboratoryreared opossum prior to and after inoculation with culture-derived S. neurona merozoites served as negative and positive controls. Immunoblots were performed as previously described (Lapointe et al. 1998) with slight modifications. Specifically, the antigen preparation was non-reduced (Marsh et al. 2001) and different secondary antibodies, antimouse IgG Fc^ (Jackson ImmunoResearch Laboratories, Inc.) or anti-raccoon IgG (H+L) (Kirkegaard & Perry Laboratories Inc.), were used according to manufacturer's recommendations. These detection antibodies were labelled with peroxidase for use with chemiluminescence signal development (Amersham Life Science, Inc.). Results All skunks contained Sarcocystis sarcocysts in skeletal muscles. TEM and light microscopy revealed homology among the sarcocysts from all skunks. Six of six laboratory-reared opossums shed Sarcocystis sporocysts in their feces after being fed infected skunk muscle. Mean sporocyst dimensions were 1 1 .0 x 7.5 |im (n = 10 per opossum, range = 10.6 to 1 1 .0 x 7.0 to 7.5 |am) (Fig. 1). Diameter of sarcocysts in fixed,

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histologic sections of muscle averaged 27.7 (n = 15, S.E.M. = 2.4). Prepatent period of sporocyst shedding ranged from 12 to 20 days post feeding (Table 6-1). Numbers of sporocysts collected were calculated using a hemocytometer. Total numbers ranged from 6.0 X 10' to 7.6 X 10' (Table 6-1). Ultrastructural examination of sarcocysts revealed a type 1 1 sarcocyst wall (Dubey et al. 1989) (Figs 6-1 and 6-2). In longitudinal section, villi are perpendicular or at an approximate 45 angle to the cyst wall and measure 3.0 pm (n = 12, S.E.M. = 0.3) x 0.4 pm (n = 12, S.E.M. = 0.03) (Fig 6-1). No electron dense granules were observed in villi. Sequence analysis of PGR products using the JNB25/JD396 primer set from sarcocysts and sporocysts indicates identity with S. neurona The sequence of the 280 bp segment of DNA amplified from skunk SKOl and SK03 sarcocysts and from sporocysts collected from opossums DV34, DV36, DV37, and DV39 was 100% homologous to that amplified from UCD-1 This sequence was only 96% homologous to that of the comparable amplicon from Sarcocystis falcatula Twelve of 1 2 gamma-interferon gene knockout mice inoculated with sporocysts collected from opossums that were fed skunk muscle developed clinical neurologic signs requiring euthanasia and/or died. The day mice were killed or died ranged from 26 to 34 (Table 6-2). Clinical signs and histologic lesions were similar to those observed previously (Cheadle et al. 2001c; Dubey and Lindsay 1998a). Grossly, infected mice had splenomegaly and circular white foci on the lungs. Histologically, white foci in lungs were associated with a lymphohistocytic and neutrophilic chronic interstitial pneumonia with diffuse merozoites throughout. Immunohistochemistry, using polyclonal S. neurona

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67 antibodies, stained merozoites in the cerebrum, cerebellum, and lung (Table 6-2). The inoculated pony maintained a normal temperature, heart rate, and respiratory rate throughout the evaluation period. The pony was seronegative and CSF negative for antibodies to S. neurona prior to infection (Table 6-3). CSF was low positive and serum was positive for antibodies by 4 weeks p.i. and both were strongly positive by 8 weeks p.i. (Table 6-3). CSF cytology, including erythrocyte count, was within normal limits at every time point (Table 6-3). No necropsy was performed and the pony remains clinically normal. Gamma-interferon gene knockout mice sera showed similar reactivity to S^ neurona antigens when compared with hyperimmune serum obtained from a mouse inoculated with culture-derived S. neurona merozoites (UCD-1 isolate) (Fig 6-3). Reactivity to a S. neurona protein rurming below the 14 kDa marker was very similar among all inoculated mice and no reactivity was seen with the negative controls or preinoculation samples. Reactivity to the antigen between the 28 and 1 7 kDa marker was much less intense in sporocyst inoculated mice as compared to the merozoite inoculated mice. All skunks were serum antibody negative to S. neurona at the beginning of the trial (Fig. 6-4). By 28 days p.i., skunks seroconverted to S. neurona based on immunoblot analysis (Fig. 6-4). Reactivity increased over time with all skunks except skunk SKOl whose serum reactivity decreased at days 78 and 92 p.i. (Fig. 6-4). The reason for this reduced activity is not known. Anti-raccoon IgG was found useful for detection of opossum and skunk IgG reactivity. Serum from skunks showed similar reactivity to S. neurona antigens when compared with the merozoite inoculated control

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68 opossum. Discussion All animals orally inoculated with sporocysts produced antibodies to S. neurona or formed sarcocysts. Although the pony did not develop clinical neurologic signs, it did develop antibodies to S. neurona in serum and CSF, indicating infection. Gammainterferon gene knockout mice developed clinical neurologic signs consistent with infection of S. neurona The course of infection with the sporocysts collected from opossimis that were fed skunk muscle was similar to that of mice inoculated with sporocysts collected from naturally infected opossums or opossums that had been fed armadillo muscle infected with S. neurona (Cheadle et al. 2001b; Cheadle et al. 2001c). Although skunks SK02 and SK03 appeared clinically normal throughout the trial, histopathologic lesions were noted at necropsy. Skunk SK02 had a nonsuppurative, interstitial pneumonia, and severe, hepatic vacuolar degeneration. Skunk SK03 had a lymphoplasmacytic interstitial nephritis, severe, hepatic vacuolar degeneration, and cerebral lymphoplasmacytic meningitis, and multifocal necrotizing nonsuppurative encephalitis. Sarcocysts observed in muscle sections were not associated with a host response. Protozoan organisms were observed only as sarcocysts in muscle tissues although non-specific binding of antibody was observed in the spleen. Sarcocysts were not stained by polyclonal antibodies to S. neurona in this study. Skunk SKOl died on Day 90 of the study. Gross lesions consisted of hemorrhage in the lungs and kidneys and frank blood in the GI tract. Histopathologic lesions consisted of severe, diffuse necrotising alveolitis with marked bacterial colonization, severe lymphoplasmacytic interstitial nephritis, mild hepatic lipidosis, contracted spleen.

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necrosuppurative interstitial myositis, cerebellar white matter spongiosis, and lymphoplasmacytic meningitis. The animal was found to be emaciated. No protozoan organisms were observed in the tissues other than sarcocysts in muscle. A wasting syndrome similar to that observed in SKOl has been observed in gamma-interferon knockout mice orally inoculated with S. neurona sporocysts (Cheadle, unpublished), however the cause of the wasting is unknown. Opossum DV 38 never shed sufficient sporocysts in its feces to detect by flotation (Table 6-1). At the end of the trial, gut scrapings were observed and found to contain low numbers of sporocysts. Therefore, wild caught opossums are not suited for use in feeding trials. Even if feces are monitored for an extended period of time and sporocysts are not detected, the gut may still contain stages. Although skunk SK03 was not inoculated with sporocysts. muscle sarcocysts were found at necropsy. Infection of this skunk likely occurred in our facility based on the negative antibody titer found at the inception of the study and the lack of sarcocysts in non-inoculated skunks examined from the commercial supplier. Similar contamination has been documented previously with Sarcocystis species (Clubb and Frenkel 1992; Hillyer et al. 1991; Smith and Frenkel 1978). Inoculated skunks were housed separately from the non-inoculated skunk, but were maintained in the same room in cages approximately 2 m apart. The control skunk was inoculated using a sterile inoculation needle, syringe, and HBSS media. Gloves were changed after handling infected animals. No portion of the inoculum was spit out or spilled. No opossums were housed in the room at any time. The exact route of infection to this skunk is unknown. The villi (Figs. 6-1 and 6-2) of the sarcocyst wall of S. neurona collected from

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70 skunks is similar to that of Sarcocystis dasypi (Cheadle et al. 2001b; Howells et al. 1975; Lindsay et al. 1996), Sarcocystis kirkpatricki (Snyder et al. 1990), Sarcocystis sp. from a mink (Ramos-Vara et al. 1997), S. falcatula (Box et al. 1984), Sarcocystis fayeri (Tinling et al. 1980), and S. neurona from an experimentally infected cat (Dubey et al. 2000c). The wall has type 1 1 villar protrusions as indicated by Dubey et al. (1989), although many of the microtubules terminate in the granular layer and do not extend to the plasmalemma of bradyzoites. Comparison of villi widths between species reveals homology. Widths range from approximately 0.2 ^m to 0.7 |im. Similarities among the villar protrusions of these species makes it impossible to differentiate them based on i TEM alone. A need for further investigation into the role of the raccoon and mink in the life cycle of S. neurona should to be addressed. Although molecular data show that S. neurona and S. falcatula are closely related, S. falcatula has been shown to be biologically and molecularly different (Dubey and Lindsay 1998a; Marsh et al. 1997; Tanhauser et al. 1999). Although hobnails were observed on villi of skunk sarcocysts, they were not as prominent as those reported for S. falcatula in a budgerigar at 24 weeks (Boxetal. 1984). There is a single report of Sarcocystis sarcocysts found in naturally infected skunks (Erdman et al. 1978). In that study, dogs (Canis familiaris ) were found to be a definitive host, shedding sporocysts that measured 10X12 ^m. Natural infection causing an encephalitis has been reported in skunks, but no sarcocysts were observed (Dubey et al. 1 996a; Dubey and Hamir 2000a). Although neurologic signs were never observed in skunks inoculated with S. neurona sporocysts in the present study, nor were protozoan parasites observed in neural tissues using light

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microscopy and immunohistochemical techniques, a mild meningitis and necrotizing nonsuppurative encephalitis was observed in SK03. Combined with our prior discovery that the nine-banded or long-nosed armadillo ( Dasypus novemcinctus ) is an intermediate host for S. neurona the information demonstrated in this paper that the striped skunk is also an intermediate host for S. neurona provides critical new information to explain the full geographic distribution of endemic EPM. The host range of the striped skunk encompasses much of the United States, Canada and northern Mexico, while nine-banded armadillos range from the southeastern United States to Peru and northern Argentina (Nowak and Paradiso 1983).

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72 Table 6-1. Skunk inoculum fed to laboratory raised opossums and prepatent period of Sarcocvstis neurona sporocysts in feces Opossum niimhpr A OP c\\ samples at feeding (days) Skunk muscle consumed Prenatent period (days) Total snorocvsts collected from opossum 1 SK02 20 1.3 X 10* 1 SK03 12 1.2 X 10' U V JO 5 SK02 13 3.4 X 10* DV37 5 SK03 14 7.6 X 10' DV38 6 SKOl ns 6.0 X 10' DV39 6 SKOl 14 2.6 X 10* ns = no sporocysts observed in feces

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73 Table 6-2. Inoculation of gamma-interferon knockout mice with sporocysts collected from opossums fed Sarcocystis infected skunk muscle Mouse number Sporocysts in inoculum Opossum from which inoculum Day killed or Tissues positive for 5. Immunoblot results Ml S 5 000 DV33 34 Cb Cl Lu JLvLi DOS M?S S 000 DV33 32 rb ri r>n^ M3S S 000 nv34 32 Cb n I u V^L/, ^^^5 J_/U M4S '> 000 nV34 26 Cb ri T u -L/ V rb n T 11 L/\J& 000 j_/ V Ch C\ T 11 V^U, V^l, i-*U iVl / O S 000 10 S 000 Ch C\ M9S 5,000 DV38 26 Cb, Cl pos MIOS 5,000 DV38 26 Cb, Cl, Lu pos Mils 5,000 DV39 33 Cb, Cl pos M12S 5,000 DV39 34 Cb, Cl, Lu pos M13S negative control n/a 36* neg M14S negative control n/a 36* neg = Mice killed due to end of study, no clinical signs were observed; n/a = not available; Cb = Cerebrum; Cl = Cerebellum; Lu = Lung; = no parasites observed

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74 Table 6-3. Results of cytologic analysis and immunoblot of CSF and serum samples from a foal inoculated with 5x10' sporocysts of Sarcocystis neurona collected from opossums fed skunk muscle containing sarcocysts Week postinfection 111 J. WVf 11 Wll Serum immunoblot 1111111 VAi ivy \^ 1 L CSF immunoblot RBC foer uL) WBC (per ^L) Protein (mg/dL) Pre-infection negative negative 0 0 47.3 2 negative negative 1 1 58.6 4 positive low positive 0 5 56 6 positive positive 67 4 59 8 strong positive strong positive 0 • 1 59.8

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i\\"}J tig r?.^ t'lc i.\Ji .da? ia'>cs I p)' 'Jk' vuvoc)^. H-jhs-dJ' )

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Fig62. liX'ol ^ <' ^ ^"w, iv.or >d inn ->!tr^.-~ MM I Mir^k^s^ iiuo^^ { i-\u\ n < \w .U'-, drr hoih y ic-^ Bs 4.0 !tci>c>l ^vo sjr ^t>U'-pH r\t\) n ^ {he w > \mI '-^ ^ J aa hi V i 'uat eiK s*<.w Sivi.ca. uf

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Fig 6-3. Reactiviry of serum collected from ganmia-lnterteron knockout mice mocubted With sporocysts collected from opossiitns fed S. nenrona mfected skuak niuscle, ImmuRoblots from SDS-PAOE separated non-reduced S. neurona proteins (I 'CI")1 iholutc t'r<)v%'n w C'.)t:inc dcni'ai ccO^) p-oheJ x^nh h> jurinimiinc inousc i i. prc-s'V'CulaU.'';! ioi-smtl sera i-s spors-'C} i ''•„ ukncd mice ;! aae-1-1 ?! ..iirtx-spoiu! tt; mk.c M]S-\l!.?Si. and Niiaro invKuiuTcJ sr^i^e r:cuvii;\c routrol hCfo (ucj? M'i ^S. Mi^Sj AH --rjnipK.-N Ui.-ic diitUtd aS I '1" otcpl h\ pes inuviuae p^.-sitivt-^nnlutl scwi vAikh w.w r.m 'a>, I '4o dihjlitin Jt-e Ui th>.. l:ii.ds an5s!-sd> liic^ c4 ibis ajanurrisctiiapctem Basc uhkh rv;cc:\cd '> intrapcraoacai iaicciion's of 2 \ 10 !acH>/;>!fcf i '( s-i-^idtc (tLV >?t 'V;'' s'ln da>-^ '\ 25. afid ptioi to Nccdusi;, .\ppf (>>asnatc nir.]ci'uk.a' --j/c^. aa-ji'scsi the margin m kUa.

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Fig 6-4, ReacUvky of seruncollected iron! skunks before and after snocidauo?^ with S.. f7i ur'fua s|-(if?iK7 s^s coilci ic-d tu^sn a iKiiunOK infrcfeJ i>i;o-.Ninn linsviusiubh^ts {'\>u-. nI ?N-i*.\(.f[' '-cPij^aled iiij-sv',ii.;ccd ,V firnroia pn>W'u\s i\ C1)~i s-.^kik, j"twn in iXjiitnc dcnru'i cclKs probed wish skunk ^i„r<} (pre jskI pu--?-inirt;ul.:fj(>:s '-.li'npicsK 1 .i.acs I -6; skuj-^k >.kO^^ no?,j t'rons dass -lb. .?8 4) "k, ,'4nd v?. rc•^pccti^c^_v, kiiOc"> 7--ii. \kuak SKkC ^c!-\ -]<>, C^K. 41. .i-i-j *C fcspccuvch; I ancN 1 1 8: -^kunk SKisj sera tT<)nuk!\ -K,. 4j 7^. :sj'4'J rc^ ivctivcix I'hc iici^alivt; •^ajapic, '-}. u.jn pfc-hjiJcuiaikin ^nun;innn 'iaht'iraion -cared r'pi^.sam urai the positive sample, s • k vv:s Use ^ain^j <>pe>ssiins h.idreeeJUed c es.:Uuje-dcrj\ cd >' /K<'rr<'/;c/ n:erf>zcMtefl CIM !-<>kile gro>>.M! Si5 Ci-juinc d;.-r:?s {pvimury inoeulatiurs; aad d-sv 'i^i prior hi bleeda:;;, J^hcu <'J nua-glu in kDa

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CHAPTER 7 ATTEMPTS TO COMPLETE THE LIFE CYCLE OF VARIOUS Sarcocvstis SP. USING THE VIRGINIA OPOSSUM (Didelphis virginiana ) AS A DEFINITIVE HOST Introduction The Virginia opossum ( Didelphis virginiana ) sheds at least 4 species of Sarcocvstis sporocysts in its feces (Cheadle et al. 2001a). A fifth taxa has been identified, but whether the species uses the opossum as a definitive host is still in doubt (Cheadle et al. 2001a). The life cycles of only Sarcocystis neurona and Sarcocvstis falcatula are known (Box et al. 1984; Cheadle et al. 2001b; Cheadle et al. 200 Id). Little is known about the other two species. Sarcocystis neurona is a protozoan parasite known to be a causative agent of the neurologic disease equine protozoal myeloencephalitis (EPM). This disease has been considered one of the most common neurologic diseases of horses in the United States (MacKay 1997). Dubey and Lindsay (1998a), Fenger et al. (1997), and Tanhauser et al. (1 999) identified the definitive host of S. neurona as the Virginia opossum. Dubey et al. (2000c) found that domestic cats ( Felis domesticus ) can be induced to become infected with S. neurona in the laboratory and serve as an experimental intermediate host. We recently found that nine-banded armadillos (Dasvpus novemcinctus ) and striped skunks (Mephitis mephitis ) are intermediate hosts for S. neurona (Cheadle et al. 2001b; Cheadle etal. 200 Id). Sarcocvstis falcatula can naturally and experimentally infect various bird species 79

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among the passeriform, psittaciform and columbiform orders (Box and Smith 1982). Natural intermediate hosts include the brown-headed cowbird ( Molothrus ater ), Patagonian conure ( Cyanoliseus patagonus ). and the boat-tailed grackle (Cassidix mexicanus ) (Bolon et al. 1989; Box et al. 1984). Experimental hosts that can become infected include budgerigars ( Melopsittacus undulatus ). canaries ( Serinus canarius ). pigeons ( Columba livia ). English sparrows ( Passer domesticus ). zebra finches ( Poephila guttata ), and cockatiels rNymphicus hollandicus ) (Box and Duszynski 1980; Box and Smith 1982; Hillyer et al. 1991). If S. falcatula is introduced into aviaries, severe infection and mortality will likely occur (Bolon et al. 1989; Hillyer et al. 1991). Sarcocystis speeri sporocysts collected from the feces of a Virginia opossum can be differentiated from other species being shed in the feces based on morphologic and biologic characteristics (Cheadle et al. 2001a; Dubey and Lindsay 1999b). This species has also been isolated from Didelphis albiventris the South American opossum (Dubey et al. 2000e). Originally, Dubey et al. (1998b) described the isolation of this species of Sarcocystis from the feces of an opossum. After inoculation of gamma-interferon gene knockout mice with sporocysts of this unidentified species, sarcocysts were observed in the leg muscles at Days 50 and 54. Dubey et al. (1999b) determined that one nude mouse formed sarcocysts when inoculated with 10"' sporocysts of S. speeri Nothing is known about the life cycle and pathogenicity of the 1085 isolate of Sarcocvsfis other than that it uses the Virginia opossum as a definitive host. The 1 085 taxa has been shown to be different from S. neurona and S. falcatula by agarose gel electrophoresis of PCR products using different restriction enzymes (Tanhauser et al. 1999; J.B. Dame, personal communication). Dubey et al. (2000b), examine a Sarcocystis

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81 falcatula -like isolate of Sarcocystis from a Didelphis albiventris in Brazil. Based on the data presented, it appears that the species described in that paper is most likely the same as 1085, indicating that 1085 has a wide range. In an attempt to determine the intermediate hosts of the two unknown cycles, two approaches were taken. Opossums were fed muscle that was known/suspected to be infected with sarcocysts and candidate animals were inoculated with sporocysts from ^ neurona, S. falcatula S. speeri and the 1085 taxa. Materials and Methods Sporocyst and Sarcocyst Identification Identification of sporocysts and sarcocysts was conducted as previously described (Cheadle et al. 2001c; Tanhauser et al. 1999). Inoculation of Animals Inoculation of animals with sporocysts was performed via gavage as previously described (Cheadle et al. 2001b). Oral Inoculation Trials Wild gray squirrels (n = 2) ( Sciurus carolinensis ) were trapped in Alachua county and inoculated with a 5 x 10^ sporocysts of S. neurona S. falcatula S. speeri and 1085 species each (total inoculation mixture = 2 x 1 0'' sporocysts; maximum age of sporocysts = 1 year since collection). A wild flying squirrel (Glaucomys volans ) was trapped in Alachua county and inoculated with a 5 x 1 0^ sporocysts of S. neurona S. falcatula S, speeri and 1085 species each (total inoculation mixture = 2 x 10'' sporocysts; maximum age of sporocysts = 1 year since collection). Captive raised deer mice (n = 10) ( Peromyscus maniculatus ) were inoculated with 5x10^ sporocysts of S. neurona S,

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82 falcatula S. speeri and 1085 species (maximum age of sporocysts = 1 year since collection). Two mice were inoculated per isolate and two mice served as negative controls. Eleven (13 day old) mallard ( Anas platyrhynchos ) ducklings were obtained from a local supplier (Alachua county feed and seed, Gainesville, FL). Ducklings were inoculated with 5x10^ sporocysts of S. neurona S. falcatula S. speeri and 1085 species (maximum age of sporocysts = 1 year since collection). Two ducklings were inoculated per isolate and three ducklings served as negative controls. Ten wild caught, adult boattailed grackles ( Ouiscalus major ), brown headed cowbirds ( Molothrus ater ), red winged blackbirds ( Agelaius phoeniceus ). and house sparrows ( Passer domesticus ) obtained from the United States Department of Agriculture (Gainesville, FL) were inoculated with 5 x 10^ sporocysts of S. neurona S. falcatula S. speeri and 1085 species (maximum age of sporocysts = 1 year since collection). Two birds of each species were inoculated per isolate and two birds of each species served as negative controls. Ten adult New Zealand white rabbits ( Oryctolagus cuniculus ) were obtained from a commercial supplier (via Animal Resources, Gainesville, FL) and were inoculated with 5x10^ sporocysts of S. neurona S. falcatula S. speeri and 1085 species (maximum age of sporocysts = 1 year since collection). Two rabbits were inoculated per isolate and two rabbits served as negative controls. Leopard frogs (Rana utricularia ) (n = 8) were obtained as tadpoles (wild-caught in Alachua county, FL) and raised in the laboratory to adulthood. Frogs were inoculated with 5x10^ sporocysts of S. neurona S. falcatula and S. speeri (maximum age of sporocysts = 1 year since collection). Two frogs were inoculated per isolate and two frogs served as negative controls. All animals were observed for approximately 90 days and then were euthanized. A necropsy was performed on all

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animals that died or were euthanized. Sections of the following tissues were removed and fixed in 1 0% buffered formalin: lung, liver, kidney, muscle, stomach, intestine, brain, heart, and spleen. Fixed tissues were submitted to the Department of Pathobiology, University of Florida for embedding, sectioning, and staining using H & E. The remaining tissues were collected and fed to captive opossums (one opossum per isolate). Fecal samples were collected from the opossums for a period of at least 30 days post feeding and examined for the presence of sporocysts. Muscle Feeding Trials Horse ( Equus caballus ) (H01-H49) (n = 49) skeletal muscles were collected from the Anatomic Pathology Necropsy Service, University of Florida over a period of five months. Muscles of at least 1 0 horses contained sarcocysts. White-tailed deer ( Odocoileus virginianus ) (WTD01-WTD27) tongues (n = 27) positive for sarcocysts were collected from hunter killed animals in Florida for a period of two months. Gray squirrel (n = 2) ( Sciurus carolinensis ) (S02 and S03) skeletal muscles positive for sarcocysts were collected in Florida from road-killed animals. Sarcocyst positive skeletal muscle from a hunter killed northem-shoveler ( Anas clypeata ) (NSOl) and greenwinged teal (Anascrecca) (GWTOl) was collected in Florida. Wild trapped rice rats ( Oryzomys plaustris ) (RR03-RR06) (n = 3) and a cotton mouse ( Peromyscus gossvpinus ) (PG02) (n = 1) positive for sarcocysts were collected in Florida. Great blue heron (Adrea herodias ) (GBHOl), ring-billed gull (Larus delawarensis ^ (RBG 01), sandhill crane (Grus canadensis ) (SHOl), great egret (Casmerodius albus ) (GE02), and bald eagle (Haliaeetus leucocephalus ) (BEOl) (n = 1 per species) muscle positive for sarcocysts was collected from necropsy at the University of Floida. A wild eastern cottontail (Sylvilagus

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84 floridanus) (CROl) with muscle infected with sarcocysts was collected in Florida from a single road-killed animal. Virginia opossum (D,_virginiana)(0Pl-0P7) (n = 7) muscle was collected from road-killed animals. All muscles were obtained, given an identification number, and fed to an individual (wild caught or lab reared) opossum. Fecal samples from these opossums were examined for the presence of Sarcocystis sporocysts approximately once per week during the feeding process for a period of 60 days after the last piece of muscle was fed. Muscle was fixed and submitted to the Department of Pathobiology, University of Florida for embedding, sectioning, and staining using H & E. The remaining tissues were collected and fed to a wild caught or lab reared opossum. Sarcocysts were observed in H & E sections of the muscle tissue submitted. Opossums were observed to ensure consumption of muscles. Results Results of feeding and inoculation trials are summarized in Tables 7-1 and 7-2. None of the naturally infected muscle tissue fed to opossums caused infection in the opossums. All of the Sarcocystis sp. that completed their life cycles through the opossum in this trial were from birds and were identified as S. falcatula Discussion Sarcocystis falcatula infects a wide range of birds and completes its life cycle through the opossum (Box and Smith 1982; Clubb and Frenkel 1992; Duszynski and Box 1978; Hillyer et al. 1991). In this study, S. falcatula infected great blue herons, cowbirds, and English house sparrows. The lack of infectivity to grackles was surprising based on a previous study that found grackles were intermediate hosts for S. falcatula (Box et al. 1984). Because the birds used in this study were wild caught, it is possible that the

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85 grackle was naturally infected, prior to this study, with another Sarcocystis sp. that does not complete its life cycle through the opossum. Although no attempt was made to identify the Sarcocystis spp. found in the muscles that were fed to the opossums, the author did observe the sarcocysts found in the northern shoveler and cottontail rabbit using TEM (unpublished). Based on villar structure of the sarcocyst wall viewed in micrographs, the duck was infected with Sarcocystis rileyi and the rabbit was Sarcocystis leporum (Dubey et al. 1989; Elwasila, et al. 1984). The finding that S. rileyi would not infect the opossum is not surprising based on a previous study by Duszynski and Box (1978) in which they found that teal and shovler muscle heavily infected with sarcocysts fed to multiple opossums would not cause infection. Although the species of Sarcocystis used in the study by Duszynski and Box (1978) was not identified as S. rileyi it was collected from a northern shoveler and is most likely S. rileyi based on species specificity. Of further interest in the study by Duszynski and Box (1978), was that an opossum did shed sporocysts in its feces after being fed muscle from a northern pintail duck ( Anas acuta ), indicating that a different species of Sarcocystis possibly S. falcatula likely infects the pintail. Previous authors attempted to complete the life cycle of S. leporum using cats (Felis domestica ) and dogs ( Canis familiaris ) (Payer and Kradel 1977). In that study, cats would produce sporocysts thereby completing the life cycle of S. leporum while dogs would not. The current study indicates that S. leporum does not use the Virginia opossum as a definitive host. More than one species of Sarcocystis could infect the animals used in this study, allowing the possible completion of life cycles through those animals. However, only the great blue heron completed the life cycle of any of the Sarcocystis spp. using the

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opossum as a definitive host. Although none of the Hfe cycles, except the previously documented cycle of S. falcatula were completed, valuable information on the scope definitive/intermediate hosts for the Sarcocystis sp. used in this study was obtained.

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87 Table 7-1 Summary of sarcocyst infected animal muscle used in and results of attempts to complete the life cycle of Sarcocystis spp. using the opossum as a definitive host Animal Sarcocysts observed (yes or no) Number of positive samples fed to opossum Sporocysts produced in opossum feces (yes or no) Identification of sporocysts Horse (Equus caballus) yes 49 no n/a White-tailed deer fOdocoileus virginianus) yes 27 no n/a Grev squirrel (Sciurus carolinensis) yes 2 no n/a Northern shoveler ("Anas clvpeata) yes no n/a Green-winged teal fAnas crecca) yes no n/a Great Blue Heron f Adrea herodias) yes yes S. falcatula Ring-billed gull TLarus delawarensis) yes • no n/a Sandhill crane (Grus canadensis) yes no n/a Great egret TCasmerodius albus) yes > no n/a Bald eagle fHaliaeetus leucocephalus) yes no n/a Eastern cottontail (Svlvilagus floridanus) yes 1 no n/a Rice rat (Orvzomvs plaustris) yes 3 no n/a Cotton mouse fPeromvscus gossvpinus) yes 1 no n/a Virginia opossum (Didelphis vireiniana") yes 7 no n/a n/a not applicable

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88 Table 7-2. Summary of animals orally inoculated with sporocysts collected from Virginia opossums Animal Sample size Sarcocystis sp. used in inoculum Sarcocysts observed (yes or no) Sporocysts Ui \J\X UL/CLl in opossum feces (yes or no) Identification of* QT*\nrnr*VQtQ collected from opossum Grey squirrel fSciurus carolinensis) 2 SN, SF, SS, and 1085 no no n/a Deer mouse fPeromvscus maniculatus) 10 SN, SF, SS, and 1085 no no n/a Flying squirrel fGlaucomvs volans) 1 SN, SF, SS, and 1085 no no n/a Mallard ("Anas platvrhvnchos) 11 SN, SF, SS, and 1085 no no n/a Boat-tailed grackle fOuiscalus major) 10 SN, SF, SS, and 1085 yes no n/a Brown-headed cowbird (Molothrus ater) 1 f\ SS, and 1085 yes yes a. laicaiuia i\.eu-wmgeu blackbird CAgelaius phoeniceus) 1 n SS, and 1085 yes no n/a riouse sparrow (Passer domesticus) CXT CP SS,and 1085 yes yes S. falcatula XT-, "7 1 J New Zealand white rabbit fOrvctolagus cuniculus) 10 SN, SF, SS, and 1085 no no n/a Leopard frog CRana utricularia) 8 SN, SF, SS, and 1085 no no n/a

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89 n/a = not available SN 5 X 1 0^ sporocysts of Sarcocvstis neurona SF = 5 X 10^ sporocysts of Sarcocvstis falcatula SS = 5 X 10^ sporocysts of Sarcocvstis speeri 1085 = 5 X 10^ sporocysts of a 1085-like taxa f

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CHAPTER 8 CONCLUSIONS The progression of experiments in this dissertation uhimately culminated in the completion of the life cycle of the most important species of Sarcocystis that uses the opossum as a definitive host. Initial data concerning a new method of sporocyst identification were documented as well as a technique of determining if sporocysts were viable using an animal model. A new species of Sarcocystis that uses the opossum as an intermediate host was recognized and named. The culmination of these studies was the finding that the armadillo and the striped skunk are intermediate hosts for Sarcocystis neurona fulfilling the main goal of the study, which was to complete the life cycle of a least one of the species of Sarcocystis that use the opossum as a definitive host. The finding that the nine-banded armadillo ( Dasypus novemcinctus ) is an intermediate host for S. neurona was significant because it is the first and only naturally infected intermediate host that has been documented. However, due to the limited geographic range of the armadillo, a large gap of coverage was left between the range of the armadillo and that of endemic equine protozoal myeloencephalitis (EPM) (Dubey et al. 2001; Nowak and Paradiso 1983). Therefore, it was necessary to continue searching for an intermediate host whose range would help explain the presence of antibodies to S^ neurona and EPM in horses from the northern portion of the United States and southern Canada. Striped skunks ( Mephitis mephitis ) were examined because reports in the literature indicated that they could develop an encephalitis caused by infection with S, 90

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91 neurona that opossums used them as a food source, and because of a report of naturally infected skunks containing sarcocysts (Dubey et al. 1996b; Dubey and Hamir 2000a; Erdman, 1978; Taube, 1947). The host range of the skunk encompasses most of the United States and southern Canada expanding the area of intermediate hosts to cover areas were S. neurona or EPM is endemic. The finding that the skunk is an intermediate host explains how S. neurona infection in horses is so widespread. The finding of two intermediate hosts in which Sarcocvstis species have been reported represents a problem when attempting to determine the name of the species that causes EPM. The original description of S. neurona was based solely on the presence of asexual stages in the form of merozoites (Dubey et al. 1991). Naming of Sarcocvstis species is most commonly based on the structure of the sarcocyst wall, including the villi, and the host infected by the species (Dubey et al. 1989). Now that two species with sarcocysts are known, it would appear that the name S. neurona is a nomen nudum. There are at least two species of Sarcocvstis that infect the armadillo, however, the species that completes its life cycle through the opossum was not determined and it is therefore unknown (Cheadle et al. 2001b; Lindsay et al. 1996). The species that infects the skunk was named Sarcocystis erdmanae by Odening (1 998), however the information presented in the original paper by Erdman (1978) is not sufficient enough to differentiate that species from any other species of Sarcocystis that could infect the skunk, thus the name S. erdmanae should be considered a nomen nudum. Therefore, the true name of the Sarcocystis sp. that causes EPM is still unknown. The Sarcocystis sp. that uses the armadillo as an intermediate host that is found to be the causative agent of EPM will have naming priority because both species found in the armadillo were named before a

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92 Sarcocystis species was described in the striped skunk. The sporocysts size data provide a quick and simple method of differentiating sporocysts of Sarcocysts speed from the other species shed in the feces of the opossum. Without these data, lengthy and expensive mouse trials or DNA sequencing would be necessary to differentiate the species. A large type of sporocyst was observed during this study which has not been reported previously. This sporocyst type had a length that was at least 7 \im in length and 1.5 |im in width greater than that reported for the other species. Although it was not determine whether these sporocysts were using the opossum as a definitive host or not, these new sporocysts were described and structural details were given. A reliable method of finding out whether S. neurona sporocysts are viable or not was determined from the mouse trial. Determining viability was critical to intermediate host studies because parasites capable of infecting the host were necessary to determine if the candidate species would complete the life cycle or not. By using the gammainterferon gene knockout mice as a model for S. neurona viability of bleach treated sporocysts was determined and indicated that bleach treated sporocysts < 7 months old were necessary to conduct infection trials. This provided a time point at which it is reasonably certain that sporocysts would still be infectious. Because of the relatively short time period that sporocysts remained viable, the procedures for sporocyst collection and treatment were adjusted to exclude the bleaching procedure until just before inoculation. Another important result of the gamma-interferon gene knockout mouse trial was the determination of a dose that would reliably infect mice and cause disease. Before this study was completed, large quantities of sporocysts, 10^ were being used for

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inoculation trials. By determining that an infective working dose could be as small as 10\ the number of sporocysts used in trials could be reduced, thus preserving large numbers of sporocysts for use in other studies. Also, the use of molecular tools for identifying sporocyst types was defined. Sporocysts identified as being S. neurona by DNA extraction, PCR amplification, and restriction enzyme digestion were able to infect and cause clinical neurologic disease in the biological mouse model. By correlating molecular tools with biological evidence, the sole use of molecular tools for identification of sporocysts is strengthened. This provides an avenue for identification of sporocysts that does not require the use of laboratory animals. Identification of a new species of Sarcocvstis that uses the opossum as an intermediate host provides additional information to the database. Scholtyseck et al. (1982) documented the presence of sarcocysts in the opossum but did not provide detailed information concerning location, transmission, or prevalence. The data presented about Sarcocvstis greineri will provide specific characteristics of the species for future investigators who wish to document the presence of the species. Although attempts to complete the life cycle of Sarcocvstis greineri were unsuccessfiil, important information documenting that the cat and dog are not definitive hosts was collected. More importantly was the finding that this species would not complete its life cycle through the opossum, therefore excluding it as one of the species using the opossum as a definitive host. Although many of the food items from the list presented in the introduction were not investigated, the goal of this study was to use animals on the list until one was found to complete the life cycle of one of the species. The list was used as a guide to give a

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starting point for which to begin the investigation. Many of the attempts made were with related species and not the ones directly found in the previously documented studies. Many of the species documented in those studies are not found in the state of Florida so closely related species were used. Completion of the life cycle of S. neurona is a critical advancement for future studies on the organism. Production of large quantities of parasites for future infection trials and other studies can now be consistently produced in the laboratory setting. Veterinarians and horse owners can now control intermediate hosts on farms to decrease the likelihood of infective sporocysts contaminating pastures through the removal of skunks and armadillos, which are potential sources of infection to the opossums located in the vicinity of the farm. Although excluding intermediate hosts from a farm will not completely remove the problem of sporocysts on a farm, it will decrease the likelihood of infective opossums. Future studies that are critical to the advancement of research and control of EPM include: 1) development and production of a vaccine that will induce a cell mediated response in horses and protect them from clinical disease caused by S. neurona 2) development and use new chemotherapeutic compounds that will cause a reduction and/or eliminate the parasite population and clinical signs associated with EPM, 3) determine what causes some horses to develop clinical signs after being infected with S. neurona sporocysts while some don't, and 4) determine if species of Sarcocystis with similar villar structure to those sarcocysts of S. neurona in skunks and armadillos are additional intermediate hosts and define which species is the causative agent of EPM as well as determine the proper name for that species.

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REFERENCES Allen, J. A. 1901 A preliminary study of the North American opossums of the genus Didelphis Bulletin of the American Museum of Natural History 14: 149-193. Blumenthal, E.M. and G.L. Kirkland, Jr. 1976. The biology of the opossum, Didelphis virginiana in southcentral Pennsylvania. Proceedings of the Pennsylvania Academy of Science 50: 81-85. Bolon, B., E.C. Greiner, and M.B. Calderwood Mays 1989. Microscopic features of Sarcocystis falcatula in skeletal muscle from a Patagonian conure. Veterinary Pathology 26: 282-284. Box, E.D. and D.W. Duszynski. 1980. Sarcocystis of passerine birds: sexual stages in the opossum ( Didelphis virginiana ). Journal of Wildlife Diseases 16: 209-215. Box, E.D., J.L. Meier, and J.H. Smith. 1984. Description of Sarcocystis falcatula Stiles, 1893, a parasite of birds and opossums. Journal of Protozoology 31: 521-524. Box, E.D. and J.H. Smith. 1982. The intermediate host spectrum in a Sarcocystis species of birds. Journal of Parasitology 68: 668-673. Cawthom, R.J. and D. Rainnie 1981. Experimental transmission of Sarcocystis sp. (Protozoa: Sarcocystidae) between the sholver ( Anas clyeata ) duck and the striped skunk ( Mephitis mephitis ). Journal of Wildlife Diseases 17: 389-394. Cheadle, M.A., J.B. Dame, and E.C. Greiner. 2001a. Sporocyst size of isolates of Sarcocystis shed by the Virginia opossum (Didelphis virginiana ). Veterinary Parasitology 95: 305-311. Cheadle, M.A., D.S. Lindsay, S. Rowe, C.C. Dykstra, M.A. Williams, J.A. Spencer, M.A. Toivio-Kinnucan, S.D. Lenz, J.C. Newton, M.D. Rolsma, and B.L. Blagbum. 1999. Serosurvey of antibodies to Neospora hughesi in horses from Alabama and biological characterization of an isolate recovered from a naturally infected horse. International Journal for Parasitology 29: 1537-1543. Cheadle, M.A., S.M. Tanhauser, J.B. Dame, D.C. Sellon, M. Hines, P.E. Ginn, R.J. MacKay, and E.C. Greiner. 2001b. The nine-banded armadillo ( Dasypus novemcinctus ) is an intermediate host for Sarcocystis neurona International Journal for Parasitology 31: 330-335. 95

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96 Cheadle, M.A., S.M. Tanhauser, T.J. Scase, J.B. Dame, R.J. MacKay, P.E. Ginn, and E.C. Greiner. 2001c. Viability of Sarcocystis neurona sporocysts and dose titration in gamma-interferon knockout mice. Veterinary Parasitology 95: 223232. Cheadle, M.A., C.A. Yowell, D.C. Sellon, M. Hines, P.E. Ginn, A.E. Marsh, J.B. Dame, and E.C. Greiner. 200 Id. The striped skunk ( Mephitis mephitis ) is an intermediate host for Sarcocystis neurona International Journal for Parasitology 31: 843-849. Clark, E.G., H.G.G. Townsend, and N.T. McKenzie. 1981. Equine protozoal myeloencephalitis: A report of two cases from western Canada. Canadian Veterinary Journal 22:140-144. Clubb, S.L. and J.K. Frenkel. 1992. Sarcocystis falcatula of opossums: transmission by cockroaches with fatal pulmonary disease in psittacine birds. Journal of Parasitology 78(1): 116-124. Cutler, T.J., R.J. Mackay, P.E. Ginn, E.C. Greiner, R. Porter, C.A. Yowell, and J.B. Dame. 1999. Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge. Journal of Parasitology 85: 301-305. Dame, J.B., R.J. Mackay, C.A. Yowell, T.J. Cutler, A. Marsh, and E.C. Greiner. 1995. Sarcocystis falcatula from passerine and psittacine birds: synonymy with Sarcocystis neurona agent of equine protozoal myeloencephalitis. Journal of Parasitology 81: 930-935. Davis, S.W., B.N. Daft, and J.P. Dubey. 1991 Sarcocystis neurona cultured in vitro from a horse with equine protozoal myelitis. Equine Veterinary Journal 23: 315317. Dubey, J.P., S.W. Davis, C.A. Speer, D.D. Bowman, A. de Lahunta, D.E. Granstrom, M.J. Topper, A.N. Hamir, J.F. Cummings, and M.M. Suter. 1991. Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis. Journal of Parasitology 77: 212-218. Dubey, J.P., and A.N. Hamir. 2000a. Immunohistochemical confirmation of Sarcocystis neurona infections in raccoons, mink, cat, skunk, and pony. Journal of Parasitology 86: 1150-1152. Dubey, J.P., A.N. Hamir, M. Niezgoda, and C.E. Rupprecht. 1996a. A Sarcocystis. neurona-like organism associated with encephalitis in a striped skunk (Mephitis mephitis ). Journal of Parasitology 82: 172-174. Dubey, J.P. and O.R. Hedstrom. 1993. Meningoencephalitis in mink associated with a Sarcocystis neurona -like organism. Journal of Veterinary Diagnostic

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97 Investigation 5: 467-471. Dubey, J.P., C.E. Kerber, and D.E. Granstrom. 1999a. Serologic prevalence of Sarcocystis neurona Toxoplasma gondii and Neospora caninum in horses in Brazil. Journal of the American Veterinary Medical Association 215: 970-972. Dubey, J.P. and D.S. Lindsay. 1996b. A review of Neospora caninum and neosporosis. Veterinary Parasitology 67: 1-59. Dubey, J.P. and D.S. Lindsay. 1998a. Isolation in immunodeficient mice of Sarcocystis neurona from opossum ( Didelphis virginiana ) faeces, and its differentiation from Sarcocystis falcatula International Journal for Parasitology 28: 1823-1828. Dubey, J.P. and D.S. Lindsay. 1999b. Sarcocystis speeri n. sp. (Protozoa: Sarcocystidae) from the opossimi (Didelphis virginiana ). Journal of Parasitology 85: 903-909. Dubey, J. P., D.S. Lindsay, P.C.B. Rezende, and A.J. Costa. 2000b. Characterization of an unidentified Sarcocystis falcatula -like parasite from the South American opossum, Didelphis albiventris from Brazil. Journal of Eukaryotic Microbiology 47: 538-544. Dubey, J.P., D.S. Lindsay, W.J.A. Saville, S.M. Reed, D.E. Granstrom, and C.A. Speer. 2001 A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Veterinary Parasitology 95: 89-131. Dubey, J.P., W.J.A. Saville, D.S. Lindsay, R.W. Stich, J.P. Stanek, C.A. Speer, B.M. Rosenthal, C.J. Njoku, O.C.H. Kwok, S.K. Shen, and S.M. Reed. 2000d. Completion of the life cycle of Sarcocystis neurona Journal of Parasitology 86: 1276-1280. Dubey, J.P., C.A. Speer, and R. Payer. 1989. General biology. In Sarcocystosis of animals and man. CRC Press, Boca Raton, Florida, p. 1-91. Dubey, J.P., C.A. Speer, and D.S. Lindsay. 1998b. Isolation of a third species of Sarcocystis in immunodeficient mice fed feces from opossums ( Didelphis virginiana) and its differentiation from Sarcocystis falcatula and Sarcocystis neurona Journal of Parasitology 84: 1 158-1 164. Dubey, J.P., C.A. Speer, and D.S. Lindsay. 2000c. In vitro cuhivation of schizonts of Sarcocystis speeri Dubey and Lindsay, 1999. Journal of Parasitology 86: 671678. Dubey, J.P., M.C. Venturini, L. Venturini, J. Mckinney, and M. Pecoraro. 1999c. Prevalence of antibodies to Sarcocystis neurona Toxoplasma gondii and Neospora caninum in horses from Argentina. Veterinary Parasitology 86: 59-62.

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98 Dubey, J.P., L. Venturini, M.C. Venturini, and C.A. Speer. 2000e. Isolation of Sarcocystis speeri Dubey and Lindsay, 1999 from the South American opossum ( Didelphis albiventris ) from Argentina. Journal of Parasitology 86: 160-163. Duszynski, D.W. and E.D. Box. 1978. The opossum ( Didelphis virginiana ) as a host for Sarcocystis debonei from cowbirds ( Molothrus ater ) and grackles (Cassidix mexicanus. Quiscalus quiscula ). Journal of Parasitology 64: 326-329. Elwasila, M., R. Entzeroth, B. Chobotar, and E. Scholtyseck. 1984. Comparison of the structure of Sarcocystis cuniculi of the European rabbit ( Oryctolagus cuniculus ) and Sarcocystis leporum of the cottontail rabbit ( Sylvilagus floridanus ) by light and electron microscopy. Acta Veterinaria Hungarica 32: 71-78. Erdman, L.F. 1978. Sarcocystis in striped skunks. Iowa State University Veterinarian 40:112. Payer, R. and D. Kradel. 1977. Sarcocystis leporum in cottontail rabbits and its transmission to carnivores. Journal of Wildlife Diseases 13: 170-173. Payer, R., I.G. Mayhew, J.D. Baird, S.G. Dill, J.H. Foreman, J.C. Pox, R.J. Higgins, S.M. Reed, W.W. Ruoff, R.W. Sweeney, and P. Tuttle. 1990. Epidemiology of equine protozoal myeloencephalitis in North America based on histologically confirmed cases. Journal of Veterinary Internal Medicine 4: 54-57. Fenger, C.K., D.E. Granstrom, A.A. Gajadhar, N.M. Williams, S.A. McCrillis, S. Stamper, J.L. Langemeier, and J. P. Dubey. 1997. Experimental induction of equine protozoal myeloencephalitis in horses using Sarcocystis sp. sporocysts from the opossum ( Didelphis virginiana ). Veterinary Parasitology 68: 199-213. Fenger C.K., D.E. Granstrom, J.L. Langemeier, S. Stamper, J.M. Donahue, J.S. Patterson, A.A. Gajadher, J.V. Marteniuk, Z. Xiaomin, and J. P. Dubey. 1995. Identification of opossums ( Didelphis virginiana ) as the putative definitive host of Sarcocystis neurona Journal of Parasitology 81: 916-919. Gardner, A.L. 1973. The systematics of the genus Didelphis (Marsupialia: Didelphidae) in North and Middle America. Texas Tech Press, Lubbock, Texas, p. 1 -8 1 Gardner, A.L. 1982. Virginia Opossum. In Wild Mammals of North America, Biology, Management, and Economics, J.A. Chapman and G.A. Feldhamer (eds.). The Johns Hopkins University Press, Baltimore, Maryland, p. 3-26. Granstrom D.E., O. Alvarez Jr., J.P. Dubey, P.F. Comer, and N.M. Williams. 1992. Equine protozoal myelitis in Panamanian horses and isolation of Sarcocystis neurona Journal of Parasitology 78: 909-912.

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Guilday, J.E. 1958. The prehistoric distribution of the opossum. Journal of Mammalogy 39: 39-43. Hall, E. R. 1981. The mammals of North America. John Wiley & Sons, New York, 1: 2-1022. Hamilton Jr., W.J. 1951. The food of the opossum in New York state. The Journal of Wildlife Management 15: 258-264. Hamir, A.N., G. Moser, D.T. Galligan, S.W. Davis, D.E. Granstrom, and J.P. Dubey. 1993. Immunohistochemical study to demonstrate Sarcocystis neurona in equine protozoal myeloencephalitis. Journal of Veterinary Diagnostic Investigation 5: 418-422. Hillyer, E.V., M.P. Anderson, E.G. Greiner, C.T. Atkinson, and J.K. Frenkel. 1991. An outbreak of Sarcocystis in a collection of psittacines. Journal of Zoo and Wildlife Medicine 22: 434-445. Hopkins, D.D. and R.B. Forbes 1980. Dietary patterns of the Virginia opossiun in an urban environment. The Murrelet 61: 20-30. Howells, R.E., A.D.V. Carvalho, M.N. Mello, and N.M. Rangel. 1975. Morphological and histochemical observations on Sarcocystis from the nine-banded armadillo, Dasypus novemcinctus Annals of Tropical Medicine and Parasitology 69: 463474. Klumpp, S.A., D.C. Anderson, H.M. McClure, and J.P. Dubey. 1994. Encephalomyelitis due to a Sarcocystis neurona -like protozoan in a rhesus monkey ( Macaca mulatta ) infected with simian immunodeficiency virus. American Journal of Tropical Medicine and Hygiene 51: 332-338. Lapointe, J.M., P.J. Duignan, A.E. Marsh, P.M. Gulland, B.C. Barr, D.K. Naydan, D.P. King, C.A. Farman, K.A. Huntingdon, and L.J. Lowenstine. 1998. Meningoencephalitis due to a Sarcocystis neurona -like protozoan in Pacific harbor seals ( Phoca vitulina richardsi ). Journal of Parasitology 84: 1 184-1 189. Lee, A.K. and A. Cockbum. 1985. Life histories of the carnivorous marsupials. In Evolutionary ecology of marsupials. Cambridge University Press, New York, p. 86-119. Lindsay, D.S., R. McKown, S.J. Upton, C.T. McAllister, M.A. Toivio-Kinnucan, J.K. Veatch, and B.L. Blagbum. 1996. Prevalence and identity of Sarcocystis infections in armadillos ( Dasypus novemcinctus ) Journal of Parasitology 82: 518-520.

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100 Lindsay, D.S., N.J. Thomas, and J.P. Dubey. 2000. Biological characterisation of Sarcocvstis neurona isolated from a southern sea otter ( Enhydra lutris nereis ). International Journal for Parasitology 30: 617-624. Luznar, S.L., M.L. Avery, J.B. Dame, R.J. MacKay, and E.C. Greiner. 2001. Development of Sarcocvstis falcatula in its intermediate host, the Brown-headed cowbird ( Molothrus ater ). Veterinary Parasitology 95: 327-334. MacKay, R.J. 1997. Equine protozoal myeloencephalitis. Veterinary Clinics of North America: Equine Practice 13(1): 79-96. Macruz, R., O. Lenci, M.M. Ishizuka, O. Miguel, and R.A.F. daCunha. 1975. Toxoplasmosis in the equine: Serological evaluation. Revista do Faculdade de Medicinia Veterinaire Zootecnia Universidade de Sao Paulo 12: 277. Mandour, A. M. 1965. Sarcocvstis garnhami n. sp. in the skeletal muscle of an opossum, Didelphis marsupialis Journal of Protozoology 12: 606-609. Marsh, A. E., B.C. Barr, J. Madigan, J. Lakritz, R. Nordhausen, and P. A. Conrad. 1996. Neosporosis as a cause of equine protozoal myeloencephalitis. Journal of the American Veterinary Medical Association 209: 1907-1913. Marsh, A.E., B.C. Barr, A.E. Packham, and P. A. Conrad. 1998. Description of a new Neospora species (Protozoa: Apicomplexa: Sarcocystidae). Journal of Parasitology 84: 983-991. Marsh, A.E., B.C. Barr, L. Tell, M. Koski, E. Greiner, J. Dame, and P. A. Conrad. 1997. In vitro cultivation and experimental inoculation of Sarcocvstis falcatula and Sarcocvstis neurona merozoites into budgerigars (Melopsittacus undulatus ). Journal of Parasitology 83: 1189-1192. Marsh, A.E., P.J. Johnson, J. RamosVara, £md G.C. Johnson. 2001. Characterization of a Sarcocvstis neurona isolate from a Missouri horse with equine protozoal myeloencephalitis. Veterinary Parasitology 95: 143-154. Masri, M.D., J.L. Alda, and J.P. Dubey. 1992. Sarcocvstis neurona -associated ataxia in horses in Brazil. Veterinary Parasitology 44: 311-314. Miller, M.A., P.R. Crosbie, K. Sverlow, K. Hanni, B.C. Barr, N. Kock, M.J. Murray, L.J. Lowenstine, and P. A. Conrad. 2001. Isolation and characterization of Sarcocvstis from brain tissue of a free-living southern sea otter (Enhydra lutris nereis ) with fatal meningoencephalitis. Parasitology Research 87: 252-257. Nowak, R.M. 1999. Walker's mammals of the world. Sixth ed. The Johns Hopkins University Press, Baltimore, Maryland, p. 165-166.

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101 Nowak, R.M. and J.L. Paradiso. 1983. Walker's mammals of the world. Fourth ed. The Johns Hopkins University Press, Baltimore, Maryland, p. 10-467. Odening, K. 1998. The present state of species-systematics in Sarcocystis Lankester, 1882 (Protista, Sporozoa, Coccidia). Systematic Parasitology 41: 209-233. Pough, F.H., CM. Janis, and J.B. Reiser. 1999. Geography and ecology of the Cenozoic. In Vertebrate Life. Prentice-Hall, Inc., Simon & Schuster, Upper Saddle River, New Jersey, p. 581-598. Poumelle, G.H. 1950. Mammals of a north Florida swamp. Journal of Mammalogy 31:310-319. Ramos-Vara, J.A., J.P. Dubey, G.L. Watson, M. Winn-EUiot, J.S. Patterson, and B. Yamini. 1997. Sarcocystosis in mink (Mustela vison V Journal of Parasitology 83:1198-1201. Rooney, J.R., M.E. Prickett, F.M. Delaney, and M.W. Crowe. 1970. Focal myelitisencephalitis in horses. Cornell Veterinarian 60: 494. Rosonke, B.J., S.R. Brown, S.J. Tomquist, S.P. Snyder, M.M. Gamer, and L.L. BIythe. 1 999. Encephalomyelitis associated with a Sarcocystis neurona -like organism in a sea otter. Journal of the American Veterinary Medical Association 215: 18391842. Scholtyseck, E., R. Entzeroth, and B. Chobotar. 1982. Light and electron microscopy of Sarcocvstis sp. in the skeletal muscle of an opossum ( Didelphis virginiana ). Protistologica 18: 527-532. Scorza, J. V., J. F. Torrealba, and C. Dagert. 1957. Klossiella tejerai nov. sp. y Sarcocvstis didelphidis nov. sp. parasitos de un Didelphis marsupialis de Venezuela. Acta Biologica Venezuelica 2: 97-108. Shaw, J. J. and R. Lainson. 1969. Sarcocystis of rodents and marsupials in Brazil. Parasitology 59: 233-244. Simpson, C.F. and LG. Mayhew. 1980. Evidence for Sarcocystis as the etiologic agent of equine protozoal myeloencephalitis. Journal of Protozoology 27: 288-292. Smith, D.D. and J.K. Frenkel. 1978. Cockroaches as vectors of Sarcocvstis muris and of other coccidia in the laboratory. Journal of Parasitology 64: 3 1 5-3 1 9. Snyder, D.E., G.C. Sanderson, M. Toivio-Kinnucan, and B.L. Blagbum. 1990. Sarcocvstis kirkpatricki n. sp. (Apicomplexa: Sarcocystidae) in muscles of raccoons (Procyonjotor) from Illinois. Journal of Parasitology 76: 495-500.

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102 Stroffregen, D.A. and J.P. Dubey. 1991 A Sarcocystis sp.-like protozoan and concurrent canine distemper virus infection associated with encephalitis in a racoon ( Procyon lotor ). Journal of Wildlife Disease 27: 688-692. Tanhauser, S.M., M.A. Cheadle, E.T. Massey, B.A. Mayer, D.E. Schroedter, J.B. Dame, E.C. Greiner, and R.J. MacKay. 2001. The nine-banded armadillo ( Dasypus novemcinctus ) is naturally infected with Sarcocystis neurona International Journal for Parasitology 31: 325-329. Tanhauser, S.M., C.A. Yowell, T.J. Cutler, E.C. Greiner, R.J. Mackay, and J.B. Dame. 1 999. Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula Journal of Parasitology 85: 221-228. Taube, CM. 1947. Food habits of Michigan opossums. Journal of Wildlife Management 11: 97-103. Tinling, S.P., G.H. Cardinet, 111, L.L. Blythe, M. Cohen, and S.L. Vonderfecht. 1980. A light and electron microscopic study of sarcocysts in a horse. Journal of Parasitology 66: 458-465. Truman, R. and R. Sanchez. 1993. Armadillos: Models for leprosy. Lab Animal 22: 28-32. Wicht, R.J. 1 98 1 Transmission of Sarcocystis rileyi to the striped skunk (Mephitis mephitis ). Journal of Wildlife Diseases 17: 387-388. Wood, J.E. 1954. Food habits of fiirbearers of the upland post oak region in Texas. Journal of Mammalogy 35: 406-415.

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BIOGRAPHICAL SKETCH The author was bom in Atlanta, Georgia on July 26"", 1973. After attending R.D. Head Elementary School in Snellville, Georgia, the author moved to a 30 acre farm in Loganville, Georgia. Aspects of living on a farm sparked the authors interest in veterinary medicine. While attending middle school at George Walton Academy in Monroe, Georgia, he began working at a local veterinary clinic. He graduated with honors from high school at George Walton in 1991 and began attending Auburn University in Auburn, Alabama. In 1995, he completed his Bachelor of Science degree in Zoology and began working in the laboratory of Dr. Byron Blagburn. After graduation, he began a masters program in Veterinary Parasitology which he completed in 1998. The author is currently completing his doctorate in Veterinary Parasitology at the University of Florida under Dr. Ellis Greiner. The author was married in 1999 to Patricia Irby and now resides in Gainesville, Florida. Upon completion of his doctorate, the author plans on working in the pharmaceutical industry or in a university setting. Saltwater fishing consumes most of the author's free time. 103

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully ade^jKite. in scope and quality, as a dissertation for the degree of Doctor of Philosot Ellis C. Greiner, Chair Professor of Veterinary Medicine I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. John By Dame Professor of Veterinary Medicine I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Donald J. Forrester Professor of Veterinary Medicine I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of PhilcTsopJ 'amela E. Ginn Associate Professor of Veterinary Medicine I certify that 1 have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Melvin E. Sunquist Associate Professor of Wildlife Ecology and Conservation

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This dissertation was submitted to the Graduate Faculty of the College of Veterinary Medicine and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. Winfred Phillips, Dean Graduate School