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THE EFFECTS OF PYRANTEL TARTRATE (STRONGIDG-C)
ON SARC‘OC YST/S NEURONA

presented by

ELIZABETH ANN KRUTTLIN, DVM

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in COMPARATIVE MEDICINE
AND INTEGRATIVE BIOLOGY

 

 

 
 

I

Major rofessor’s Signat re

/ 21/2 [/03
Date

 

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6/01 cJCIRC/DateDuepBS-p. I 5

THE EFFECTS OF PYRANTEL TARTRATE (STRONGID®-C) ON SARCOCYSTIS
NE URONA

By

Elizabeth Ann Kruttlin, DVM

A THESIS

Submitted to
Michigan State University
In partial fulﬁllment of the requirements
For the degree of

MASTER OF SCIENCE
Comparative Medicine and Integrative Biology

2003

ABSTRACT
THE EFFECTS OF PYRANTEL TARTRATE (STRONGID®-C) ON SARCOCYSTIS
NEURONA
By

Elizabeth Ann Kruttlin, DVM

This thesis describes the effects of pyrantel tartrate (Strongid®-C) on Sarcocystis
neurona merozoites and sporozoites both in vitro and in a gamma interferon knock-out
mouse model of the disease equine protozoal myeloencephalitis (EPM). Anecdotal
reports from private practicing veterinarinans suggest that horses fed daily Strongid®-C
showed a lower incidence of clinical EPM than those not fed the dewormer, leading us to
formulate the hypothesis that pyrantel tartrate affects the viability of S. neurona.

The results of this study show pyrantel tartrate, at concentrations greater than 2.5
mM, is 100% lethal to S. neurona merozoites. Pyrantel tartrate at concentrations greater
than 5 mM is lethal to S. neurona sporozoites. Pyrantel tartrate given orally to gamma
interferon knock-out mice at the dose of 2.5 mM increased the mean survival time of
mice. Acetylcholine (ACh) chloride, at concentrations similar to that of pyrantel tartrate,
did not produce the same lethal consequences in S. neurona merozoites, indicating that
the drug is unlikely to work at cholinergic receptors in this parasite.

From these studies, we concluded that pyrantel tartrate has the potential to be
effective against the merozoite and sporozoite stages of S. neurona, if high enough
concentrations of the drug can be delivered effectively in vivo. Efﬁcacy against the

parasite in vitro was demonstrated to be dose dependent and repeatable in cell culture.

ACKNOWLEDGEMENTS

There are many people who made this study possible. I would speciﬁcally like to
thank my family and ﬁ'iends for their support and for listening to me ramble for hours and
hours about parasites.

I thank Dr. Linda Mansﬁeld for her endless encouragement, enthusiasm, and
patience. You have been a most wonderful mentor. Thanks to Dr. John Kaneene for
always having a reason to smile and for trying to turn me into an “Epi Star”. I would like
to thank Dr. Hal Schott for always “challenging” me and for showing me how to use my
graduate work to be come a better clinician. A special thanks to Dr. Jon Patterson for
trying to make sense of all the samples provided to him and for his teaching and advice
throughout vet school. You have all contributed immensely to both of my degrees, and I
will always be grateful.

I would also like to thank Ruth Vrable, Alice Murphy, Nicole Grosjean, and Mary
Rossano for giving me a home away from home. Without you, this project would not
have been possible. Thank you for listening all of these years.

For the ﬁnancial support of these studies, I thank and acknowledge Pﬁzer, Inc.

Thank you to Robert Templeman for statistical support.

iii

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... vii
LIST OF FIGURES ................................................................................ viii
CHAPTER 1
LITERATURE REVIEW AND STUDY OBJECTIVES ....................................... 1
EQUINE PROTOZOAL MYELOENCEPHALITIS ................................... 1
Introduction and History ........................................................... 1
Clinical Signs ........................................................................ 1
Causative Agent ..................................................................... 2
Pathogenesis ......................................................................... 4
Pathology ............................................................................ 4
Diagnosis ............................................................................. 5
Treatment ............................................................................ 6
Epidemiology ........................................................................ 7
RATIONALE AND JUSTIFICATION FOR THE STUDY ........................... 9
STUDY OBJECTIVES AND HYPOTHESES ........................................ 11
LITERATURE CITED .................................................................... 12
CHAPTER 2
THE RELATIONSHIP BETWEEN SARCOC YST IS NE URONA SPOROCYSTS AND
STRONGYLE LARVAE ........................................................................... 17
INTRODUCTION ......................................................................... 17
MATERIALS AND METHODS ......................................................... 18
Strongyle Collection and Growth ............................................... 18
PCR ................................................................................. 19
RESULTS ................................................................................... 20
DISCUSSION .............................................................................. 21
CONCLUSIONS ........................................................................... 23
LITERATURE CITED .................................................................... 24
CHAPTER 3
THE EFFECTS OF PYRANTEL TARTRATE ON SARCOCYSTIS NE URONA
MEROZOITE VIABILITY ........................................................................ 25
ABSTRACT ................................................................................. 25
INTRODUCTION ......................................................................... 26
MATERIALS AND METHODS ......................................................... 28
Preparation of Merozoites ........................................................ 28
Cell Culture Preparation .......................................................... 29
Preparation of Drug ............................................................... 29
Experimental Design .............................................................. 29
Plaque Number Determination .................................................. 30

iv

DNA Extraction and PCR ........................................................ 31

Drug Activity Control ............................................................ 32
RESULTS ................................................................................... 34
Plaque numbers .................................................................... 34
PCR ................................................................................. 34
Drug Activity Control ............................................................ 35
DISCUSSION .............................................................................. 36
CONCLUSION ............................................................................. 4O
LITERATURE CITED .................................................................... 41

CHAPTER 4
THE EFFECTS OF PYRANTEL TARTRATE ON SARCOCYSTIS NE URONA
SPOROZOITES IN CELL CUTLURE AND IN GAMMA INTERFERONE KNOCK-

OUT MICE .......................................................................................... 50
INTRODUCTION ......................................................................... 50
MATERIALS AND METHODS: CELL CULTURE ................................. 52

Cell Culture ........................................................................ 52
Sporocysts .......................................................................... 52
Preparation of Sporozoites ....................................................... 52
Preparation of Drug for Use in Cell Culture ................................... 53
Experimental Procedure .......................................................... 53
Plaque Number Determination .................................................. 54
Statistical Analysis ................................................................ 54
MATERIALS AND METHODS: MICE ................................................ 55
Mice ................................................................................. 55
Sporocysts .......................................................................... 55
Experimental Procedure .......................................................... 55
Histopathology ..................................................................... 56
Statistical Analysis ................................................................ 56
RESULTS ................................................................................... 57
Cell Culture ........................................................................ 57
Mice ................................................................................. 57
DISCUSSION .............................................................................. 58
CONCLUSIONS ........................................................................... 59
LITERATURE CITED .................................................................... 60

CHAPTER 5

SARCOCYST IS NE URONA: IN VITRO EXPOSURE OF MEROZOITES TO

ACETYLCHOLINE AND ATROPINE ......................................................... 63
ABSTRACT ................................................................................. 63
INTRODUCTION ......................................................................... 63
MATERIALS AND METHODS ......................................................... 66

Preparation of Merozoites ........................................................ 66
Cell Culture Preparation ......................................................... 67
Preparation of Drug ............................................................... 67
Experimental Design .............................................................. 67

Plaque Number Determination .................................................. 69

Western Blot ....................................................................... 69

RESULTS ................................................................................... 71

Plaque Numbers .................................................................... 71

Western Blot ....................................................................... 71

DISCUSSION .............................................................................. 72

LITERATURE CITED .................................................................... 76
CHAPTER 6

MAJOR FINDINGS AND CONCLUSIONS ................................................... 81

vi

Table 3.1.

Table 3.2.

Table 3.3.

Table 3.4.

Table 5.1.

LIST OF TABLES

Plaque numbers for pyrantel tartrate concentrations. *P-value refers to
comparison between pyrantel tartrate dilutions and the DMEM control
(Wilcoxan rank sum test, two tailed, exact test) .............................. 43

Plaque numbers for sodium tartrate concentrations. *P-value refers to
comparison between sodium tartrate dilutions and the DMEM control
(Wilcoxan rank sum test, two tailed, exact test) ............................... 44

Plaque numbers for sodium tartrate (ST) and pyrantel tartrate (PT)
concentrations. * P value refers to comparison between sodium tartrate and
pyrantel tartrate numbers at the same molarities (Wilcoxan rank sum test,
two tailed, exact test) ............................................................. 45

Numbers of viable and non-viable strongyle larvae after exposure to
pyrantel tartrate. "' P value refers to comparisons between non-viable
larvae from pyrantel tartrate dilutions and the DMEM control (Fisher’s
exact test, 2-tailed) ................................................................ 46

Results of Dunnett’s t-tests comparing each dose group to DMEM control
at alpha = 0.05. All results are based on n = 4 plates per group, except for
atropine sulfate week 2 where n = 3 plates per group. (*) indicates a

signiﬁcant difference from the control at p _<_ 0.05 ........................... 78

vii

Figure 3.1.

Figure 3.2.

Figure 3.3.

Figure 4.1.

Figure 5.1.

Figure 5.2

LIST OF FIGURES

Plaque numbers (mean of four wells) one week after culture inoculation.
(*) indicates a signiﬁcant difference between plaque numbers in sodium
tartrate treatment groups and pyrantel tartrate treatment groups. I = one
standard deviation ................................................................. 47

Plaque numbers (mean of four wells) two weeks after culture inoculation.
(*) indicates a signiﬁcant difference between plaque numbers in sodium
tartrate treatment groups and pyrantel tartrate treatment groups. I = one
standard deviation ................................................................. 48

PCR of media removed from cell cultures infected with merozoites treated
with one of the following solutions: Lane 1) 100 bp size standard, A)
DMEM, B) 0.001M pyrantel tartrate (PT), C) 0.0025M PT, D) 0.005M
PT, E) 0.0075M PT, F) 0.01M PT, G) DMEM, H) 0.001M sodium tartrate
(ST), 1) 0.0025M ST, J) 0.005M ST, K) 0.0075M ST, L) 0.01M ST, M)
Equine dermal cell DNA control, N) S. neurona DNA (+ control) 0) PCR
solution only (- control) ........... - ............................................... 4 9

Pyrantel tartrate plaque assay, _Plaque numbers (mean of four wells) three
and four weeks after culture inoculation. (*) indicates a signiﬁcant
difference between plaque numbers in media control and pyrantel tartrate
treatment groups. I = one standard deviation ................................. 62

Acetvlcholine plaque assay results. Plaque numbers (mean of four wells 3;
standard deviation) one and two weeks post-inoculation. Concentrations
of 10'6M and 10'3 M were signiﬁcantly lower than the DMEM control after
one week of growth; however, no statistical difference was seen between
the media control and treatment groups at all concentrations after two
weeks of growth ................................................................... 79

Atropine plague assay results. Plaque numbers (mean of four wells 1
standard deviation) one and two weeks post-inoculation. No statistical
difference between media control and treatment groups at all
concentrations after two weeks of growth ..................................... 80

viii

CHAPTER 1
LITERATURE REVIEW AND STUDY OBJECTIVES
1.1 Equine Protozoal Myeloencephalitis (EPM)
1.1 I Introduction and History:

Equine protozoal myeloencephalitis (EPM) is a progressive neurologic disease of
equids. The clinical syndrome was ﬁrst described as “segmental myelitis” by Rooney et
al in 19701, but the name “equine protozoal myeloencephalitis”, coined by Mayhew et al.
in 19762, gained favor after Cusick et al. reported the presence of protozoa in lesions
from affected horses3 . First believed to be caused by T oxoplasma gondii3, conclusive
evidence that EPM was caused by a Sarcocystis species was provided by Simpson and

Mayhew in 19804.

1.12 Clinical signs:

EPM is described as a progressively debilitating disease affecting the central
nervous system (CNS) of horsess‘6‘7‘8’9. Clinical signs often range from acute to insidious
onset of focal or multifocal signs of neurologic disease. The brain, brainstem, spinal
cord, or any combination of these areas can be involved. Affected horses also may have
abnormal upper airway function, seizures, or unusual or atypical lameness. Gait
abnormalities are a result of damage to the spinal cord and can be variable depending on
severity and location of lesions. Physical examination of affected animals generally
shows vital signs to be normal; however, some horses may be thin and mildly depressed.

Most horses also have normal complete blood count and serum chemistry values.

Many other neurologic diseases can manifest clinical signs similar to those of
EPM"). Examples include cervical vertebral myelopathy (CVM), equine herpes virus
myelitis (EHV-l), equine motor neuron disease (EMND), rabies, tetanus, neoplasia,
leukoencephalomalacia (moldy corn poisoning), equine viral encephalomyelitis, trauma
to the CNS, or multi-focal bacterial encephalitis. One of the most useful clinical signs in
establishing the diagnosis of EPM is that horses with the disease often have asymmetric
gait deﬁcits with focal muscle atrophy5’6'7‘8’9. In order for clinicians to make the proper
diagnosis of EPM, all previously mentioned diseases should be considered and ruled out

based on clinical signs and diagnostic testing.

[.13 Causative agent:

Sarcocystis neurona is the primary etiologic agent of EPM“ 1"2’13’”. Neospora
hughesi has also been identiﬁed in four cases of EPM'5 “6'11”, even though the majority
of cases of EPM continue to result from S. neurona infection.

The lifecycle of Sarcocystis sp. is complex and involves two hosts. The sarcocyst
stage of the parasite is found within muscle tissue of the intermediate host and is
consumed by the deﬁnitive host. Upon digestion, the cyst wall is broken down and the
bradyzoite stage of the parasite is released. Bradyzoites penetrate the host’s intestinal
epithelium and develop into the sexual stages of the parasite, microgametes and
macrogametes. The microgarnete fertilizes the macrogamete, resulting in a zygote which
matures to become an oocyst containing two Sporocysts. The oocyst is released from its
host cell, travels down the intestine until the oocyst wall is ruptured, and Sporocysts are

excreted in the feces of the deﬁnitive host'q’zo.

The intermediate host becomes infected by ingesting the sporocyst stage of the
parasite from fecal matter or food or water contaminated with fecal matter. Upon
ingestion, Sporocysts release sporozoites within the gastrointestinal (GI) tract of the host.
Sporozoites penetrate the gut and migrate to endothelial cells of the host’s internal
organs. In most cases, sporozoites undergo two rounds of asexual schizogony and
become tachyzoites, then motile merozoites. Merozoites are released from schizonts and
travel in circulating lymphocytes to muscle tissue. In muscle, they undergo ﬁxed rounds
of merogony (asexual replication) and develop into bradyzoites in intramuscular cysts.
Sarcocysts contain large numbers of tightly packed bradyzoites that are infective to the
deﬁnitive host. The lifecycle is continued when a deﬁnitive host ingests the muscle
tissue as prey or carrion, and the bradyzoites emerge in the host’s intestine'g’zo.

Recently, the lifecycle of S. neurona has been completed. The North American
opossum (Didelphis virginiana) is the deﬁnitive host of S. neuronamz. Additionally, the
South American opossum (Didelphis albiventris)also has been shown to act as a
deﬁnitive host”. The parasite has been shown to utilize a variety of intermediate hosts
including the nine-banded armadillo (Dasypus novemcinctus)23’24, the striped skunk
(Mephitis mephitis)”, the raccoon (Procyon Iotor)26, and the domestic cat (F elis

domesticus)27. Horses are considered aberrant hosts of S. neuronal "28

, and until recently,
it was believed that the parasite could not undergo its ﬁnal stage of cyst formation in this
host. However, a recent study by Mullaney et al. found both mature schizonts in the

brain and spinal cord and mature sarcocysts in the tongue of a young colt with neurologic

disease consistent with EPM”. Sarcocysts were found to have features identical to

published features of S. neurona on electron microscopy and produced Sarcocystis

speciﬁc PCR products. RFLP analysis of these products showed banding patterns
characteristic for S. neurona. While this evidence is highly suggestive that horses have
the potential to act as intermediate hosts, further studies are needed to demonstrate

Koch’s postulates before this claim can be made.

1.14 Pathogenesis:

The pathogenesis of EPM is not clear. It is assumed that horses are infected with
S. neurona by consuming Sporocysts in contaminated hay, grain, grass, or water7. Studies
in gamma interferon knock-out mice orally inoculated with S. neurona Sporocysts
indicate that the parasite initially replicates to a limited extent in visceral tissues and then
is transported to the CNS probably via leukocytes”. The development of a reliable
equine model is necessary to study the pathogenesis of this disease. It is suggested that
stress plays an important role in the pathogenesis of EPM, and inﬂicting a stressor, such
as dexamethasone or transport, on horses prior to oral inoculation with S. neurona
Sporocysts has been shown to produce EPM in these animals3 1. However, the small
number of test animals used this transport stress study does not provide solid evidence of
a reliable equine model. Further studies are needed to demonstrate the infection

dynamics of S. neurona in horses.

1.15 Pathology:
Sarcocystis neurona causes a non-suppurative inﬂammation of the CNS leading
to gross lesions in both gray and white matter and necrosis of affected neural

tissuel’7‘m‘32‘33 . Although the brainstem is more often involved than other brain areas,

lesions are more frequently seen in the spinal cordl’m’32’33. Perivascular cufﬁng by
mononuclear cells is evident in some affected areas, particularly the meningesl’lo. The
inﬂammatory response is highly variable and can consist of inﬁltrates of a mixture of
lymphocytes, neutrophils, eosinophils, macrophages, and multinucleate giant cells”.

Degeneration of neurons and axons is commonly seen].

1.16 Diagnosis:
EPM remains the most commonly diagnosed infectious equine neurologic disease

34,35

in America , and the Western blot test is the most commonly used diagnostic test for

- 9
EPM In current use ’36

. Western blot technique was developed in 1993; it detects
antibodies in serum or cerebrospinal ﬂuid”. In 2000, the test was enhanced by adding a
blocking step in which blots are exposed to bovine anti-Sarcocystis cruzi IgG38. This
treatment reduces false-positive results from antibody cross-reactivity to proteins
common to Sarcocystis species, and has been found to have >99% sensitivity and 98%
speciﬁcity”. A positive serological test indicates past or current infection with S.
neurona, but does not conﬁrm that the parasite has reached the CNS. For this reason, and
given the high seroprevalence in clinically normal animals, testing of CSF is preferred936.
A deﬁnitive diagnosis of EPM requires ﬁnding the parasite in neural tissue sections at
postmortem examination or culturing the parasite from neural tissue1 ”0’39.

Antemortem diagnosis of EPM should be made by combining the history and clinical

picture of the animal in question with results of testing techniques described above.

A PCR test would help to decrease the number of false positive diagnoses of EPM
because it can directly detect parasite DNA, rather than antibodies to the parasite that do
not always suggest clinical disease. However, in clinical use, the test appears to suffer
from low sensitivity because of the low number of merozoites present in the serum or
CSF of the horse“). Parasite DNA may be easily missed. Techniques to collect and
concentrate merozoites from large volumes of CSF (i.e. magnetic beads) would help to
reduce the number of false negatives found with PCR and help to improve this test for

clinical use.

1.1 7 Treatment:

Currently, one drug is FDA approved for the treatment of EPM in horses.
MarquisTM (15% w/w ponazuril) given orally once daily at the dose of 5 mg/kg for 28
days has been found have activity against S. neurona in both laboratory and ﬁeld
settings“~42. Ponazuril is an anticoccidial triazine-based compound and is microbicidal to
S. neurona. A similar drug, diclazuril, has been found to have anti-S. neurona activity in
cell culture and may have some use as a prophylactic agent against S. neurona infections
in horses43’44. Toltrazuril, an anti-coccidial drug used in several species, also has
potential efﬁcacy for the treatment of EPM45 .

Prior to approval of Marquis“, treatment was conﬁned to the use of dihydrofolate
reductase inhibitors such as sulfonamides and pyrimethamine”. Usual treatment
involves the use of sulfadiazine at a dose of 20 mg/kg in combination with
pyrimethamine at 1.0 mg/kg given orally once daily”. Treatment can be continued for

120 days or longer. A determination to discontinue treatment is based on either

signiﬁcant improvement of clinical signs or the horse returning to normal and Western
blot testing of CSF returning to negative”. This treatment regimen is not without risk.
Prolonged antifolate therapy has been known to result in undesirable side effects
including reduced spermatogenesis in stallions, harm to the fetus in pregnant mares, and
bone marrow suppression in adult horsesg.

Nitazoxanide has broad spectrum activity against bacteria, protozoa, and
helrninthes and is effective against S. neurona in cell culture“. It has also recently been
tested in a clinical ﬁeld trial for the treatment of horses with EPM. The safety study
indicated that, at a 2 X dose for 1 week, horses became lethargic, and at 4 X dosing,
horses showed illness and one horse died". The efﬁcacy study of 70 horses showed 63%
of horses as improving one grade or more or becoming negative on Western blot of
CSF".

In addition to anti-protozoal drugs, anti-inﬂammatory drugs such as
phenybutazone, dimethylsulfoxide and glucocorticosteroids may be used7. In situations
where EPM is considered due to a poor immune response of the horse, use of

immunostirnulants such as levamisole, alpha-interferon, mycobacterial wall extract or

killed Propionibacterium acne can also be considered7.

1.18 Epidemiology:
EPM is the most commonly diagnosed equine protozoal disease in the eastern
US“. It has not been shown to be contagious from horse to horse, and focal clusters of

cases are unusual33 . However, a study at The Ohio State University found that if EPM

had been diagnosed at a farm prior to diagnosis in one of the cases in the study, the risk
of EPM was >25 times higher than if EPM had never been diagnosed previously”.

EPM has been diagnosed in horses throughout North, Central, and South
American. Cases reported outside of this region were in horses imported from the
Western hemispheremz. Prevalence of EPM in horses is estimated to be 0.5-1% of the
horse population53 ; however, disease exposure is much higher with a 60% seroprevalence
in some states“. The distribution of EPM follows the distribution of opossums, and the
exact prevalence of S. neurona in opossums in unknown due to the absence of a simple
method with which to identify S. neurona in opossum fecesso. Based on completed
surveys in F lorida5 ' and Michigansz, the prevalence of S. neurona in opossums is
considered to be very high.

EPM has been diagnosed in horses ranging in age from 2 months to 24 years7’32.
More than 60% of cases in a retrospective study conducted in 1991 were reported in
horses 4 years and younger7. There does not appear to be a breed or sex predilection”.
Risk analysis has found that the risk for EPM was three times higher in spring and
summer and six times higher in the fall when compared to winter”. This is believed to
be due to climactic factors such as freezing temperatures affecting exposure and disease
rates. Transport stress in relation to annual athletic competitions in the fall of the year
may also contribute to the seasonal effect. A 2.5 X higher risk of EPM was present if
opossums were observed on the property when compared to those barns where opossums

were never seen”. A strong dose response relationship was seen between health events

(i.e., aging, exercise, transport, injury, surgery, or parturition) and the risk for EPM”.

These events may lead to immune suppression with subsequent development of clinical

signs of EPM.

1.19 Rationale and justification for the study:

EPM is a devastating disease that is costly and often unrewarding to treat.
Environmental exposure to S. neurona is difficult to prevent and occurs frequently based
on seroprevalence. A logical means of controlling disease would be to focus on
prevention. Currently, a killed, whole organism vaccine is available from Fort Dodge;
however, it has limited approval, and safety and efficacy of the vaccine are questionable.
Additionally, a vaccinated horse is indistinguishable from a non-vaccinated, exposed
horse on serum Western blot, making disease diagnosis difficult.

Strongid®-C (pyrantel tartrate) is a daily anthelmintic feed additive used to
prevent Strongyle infestation in horses. Pyrantel tartrate is an anthelmintic compound that
possesses nicotine-like properties, acting similar to acetylcholine (ACh), and by
inhibiting acetylcholinesterase55 . These properties make the drug a depolarizing
neuromuscular-blocking agent in susceptible parasites. Theoretically, daily pyrantel
tartrate administration could prevent S. neurona infection in equids by killing sporozoites
as they are excysted in the gut. If ACh receptors were found to be present in S. neurona
sporozoites, it could be extrapolated that the drug works at a target similar to that of
helminth worms; however, the role of ACh in protozoa yet to be deﬁned.

Anecdotal reports from practitioners suggest horses fed Strongid®-C have a

decreased incidence of clinical EPM. If the drug could stop S. neurona from passing into

the bloodstream of the horse, it could be used as a means of preventing S. neurona

infection in equids.

10

1.2 Hypotheses:
It was hypothesized that:
> Strongyle larvae are able to attach to or ingest sporozoites and can serve as a
transport host into the bloodstream of the horse.
> Pyrantel tartrate is lethal to S. neurona merozoites and sporozoites in vitro.
> Pyrantel tartrate prevents clinical disease in gamma interferon knock-out mice
dosed orally with S. neurona Sporocysts.
> S. neurona merozoites possess acetylcholine receptors similar to those of

helminths, a possible drug target for pyrantel tartrate.

1.3 Study Objectives:
1. Determine if Strongyle larvae can attach to or ingest S. neurona sporozoites.
II. Develop an in vitro drug screening assay for S. neurona merozoites and
sporozoites.
III. Determine the effects of pyrantel tartrate on S. neurona merozoites in vitro.
IV. Determine the effects of pyrantel tartrate on S. neurona sporozoites in vitro.
V. Determine the effects of pyrantel tartrate on S. neurona sporocyst infection in
gamma interferon knock-out mice.

VI. Explore possible drug targets of pyrantel tartrate in S. neurona merozoites.

ll

1.3 LITERATURE CITED:

1. Rooney JR, Rrickett ME, Delaney FM, Crowe FW. 1970. Focal myelitis-
encephalitis in horses. Cornell Vet. 50, 494-501.

2. Meyhew IG, de Lahunta A, Whitlock RH, et a1. 1976. Equine protozoal
myeloencephalitis. In Proceedings of the 22ml Annual Convention of the
American Association of Equine Practitioners, Dallas, TX, Nov-Dec, pp107-114.

3. Cusick PK, Sells DM, Hamilton DP, et al. 1974. Toxoplasmosis in two horses. J.
Am. Vet. Med. Assoc. 164, 77-80.

4. Simpson, C.F., Mayhew, LG, 1980. Evidence for Sarcocystis as the etiologic
agent of equine protozoal myeloencephalitis. J. Protozool. 27, 288-292.

5. Beech J. 1974. Equine protozoal myeloencephalitis. Vet. Med. Small. Anim.
Clin. 69, 1562-1566.

6. Brewer BD and Mayhew IG. 1988. Multifocal neurologic disease in a horse.
Equine Vet. Sci. 8, 302-304.

7. MacKay RJ, Davis SW, Dubey JP. 1992. Equine protozoal myeloencephalitis.
Comp. Cont. Educ. Pract. Vet. 14, 1359-1367.

8. Granstrom DE and Reed SM. 1994. Equine protozoal myeloencephalitis. Equine
Pract. 16, 23-26.

9. MacKay RJ. 1997. Equine protozoal myeloencephalitis. Vet Clin N. Am. Equine
Pract. 13, 79-96.

10. Meyhew IG, de Lahunta A, Whitlock RH, et al. 1978. Spinal cord disease in the
horse. Cornell Vet 68, (Suppl 6) 148-160.

11. Bowman DD, Cummings JF, Davis SW, et al. 1992. Characterization of
Sarcocystis neurona from a throroughbred with equine protozoal
myeloencephalitis. Cornell Vet 82, 41-52.

12. Dubey JP, Lindsay DS. 1998. Isolation in immunodeﬁcient mice of Sarcocystis
neurona from opossum (Didelphis virginiana) feces, and its differentiation from
Sarcocystisfalcatula. J Parasitol 28, 1823-1828.

13. Dubey JP, Speer CA, Lindsay DS. 1998. Isolation of a third species of
Sarcocystis in immunodeﬁcient mice fed feces from opossums (Didelphis

virginiana) and its differentiation from Sarcocystisfalcatula and Sarcocystis
neurona. J Parasitol 84, 1158-1164.

14. Fenger CK, Granstrom DE, Gajadhar AA, et a1. 1997. Experimental induction of
equine protozoal myeloencephalitis in horses using Sarcocystis sp. spororcysts
from the opossum (Didelphis virginiana). Vet Parasitol 68, 199-213.

15. Marsh AE, Barr BC, Madigan J, et al. 1996. Neosporosis as a cause of equine
protozoal myeloencephalitis. J. Am. Vet. Med. Assoc. 209, 1907-1913.

16. Daft BM, Carr BC, Collins N, et al. 1996. Neospora encephalomyelitis and
polyradiculoneuritis in an aged mare with Cushing’s disease. Equine Vet. J. 28,
240-243.

17. Harnir AN, Tomquist SJ, Gerros TC, et al. 1998. Neospora caninum-associated
equine protozoal myeloencephalitis. Vet Parasitol. 79, 269-274.

18. Cheadle MA, Lindsay, DS, Rowe S, et al. 1999. Prevalence of antibodies to
Neospora sp. in horses from Alabama and characterization of an isolate recovered
from a naturally infected horse. Int. J. Parasitol. 29, 1537-1543.

19. Levine ND. 1985. Veterinary Protozoology. 1St ed. Iowa State University Press.
Pp 244-245.

20. Urquhart GM, Armour J, Duncan JL, et al. 1996. Veterinary Protozoology, in
Veterinary Parasitology, 2nd ed. Blackwell Science Ltd. Pp238-241.

21. F enger, CK, Granstrom, DE, Langemeier, J L et al. 1995. Identiﬁcation of
opossums (Didelphis virginiana) as the putative deﬁnitive host of Sarcocystis
neurona. J Parasitol. 81,199-21 3.

22. Dubey JP, Lindsay DS, Kerber CE, et a1. 2000. First isolation of Sarcocystis
neurona from the South American opossum, Didelphis albiventris, from Barzil.
Vet. Parasitol. 95, 295-304.

23. Cheadle MA, Tanhauser SM, Dame J B, et al. 2001. The nine-banded armadillo
(Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona. Int J
Parasitol 31(4): 330-335.

24. Tanhauser SM, Cheadle MA, Massey ET, et al. 2001. The nine-banded
armadillo (Dasypus novemcinctus) is naturally infected with Sarcocystis neurona.
Int J Parasitol. 31(4): 325-329.

25. Cheadle MA, Yowell CA, Sellon DC, et al. 2001. The striped skunk (Mephitis

mephitis) is an intermediate host for Sarcocystis neurona. Int J Parasitol 31(8):
843-849.

13

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Stanek JF, Dubey JP, Oglesbee MJ, et a1. 2002. Life cycle of Sarcocystis neurona
in its natural intermediate host, the raccoon, Procyon lotor. J Parastiol. 88(6):
1151-1158.

Dubey JP, WJA Saville, DS Lindsay, et a1. 2000. Completion of the life cycle of
Sarcocystis neurona. J Parasitol 86(6): 1276-1280.

Dubey JP, Davis SW, Speer CA, et al. 1991. Sarcocystis neurona n. sp.
(Protozoa: Apicomplexa), the etiologic agent of equine protozoal
myeloencephalitis. J Parasitol 77: 212-218.

Mullaney TP, Kiupal M, Murphy AJ, et a1. 2003. Evidence that horses are natural
intermediate hosts for Sarcocystis neurona. American Association of Veterinary
Parasitologists 48th Annual Meeting. Denver, CO.

Dubey JP. 2001 . Migration and development of Sarcocystis neurona in tissues of
mice fed Sporocysts from a naturally infected opossum. Vet Parasitol. 95:341-
351.

Saville WJ, Stich RW, Reed SM, et a1. 2001. Utilization of stress in the
development of an equine model for equine protozoal myeloencephalitis. Vet.
Parasitol. 95, 211-222.

Fayer R, Mayhew IG, Baird JD, et a1. 1990. Epidemiology of equine protozoal
myeloencephalitis in North America based on histologically conﬁrmed cases. J
Vet Intern Med. 4, 54-57.

Madigan JE, Higgins RJ. 1987. Equine Protozoa] Myeloencephalitis. Veterinary
Clinics of North America: Equine Practice. 3, 397-403.

Boy MG, Galligan DT, Divers TJ. 1990. Protozoa] encephalomyelitis in horses:
82 cases (1972-1986). J Am Vet Med Assoc. 196, 632-634.

Harnir AN, Moser G, Rupprecht CE. 1992. A ﬁve year (1985-1989)
retrospective study of equine neurological diseases with special reference to
rabies. J. Comp. Pathol. 106, 411-421.

Fenger CK. 1997. Equine protozoal meyloencephalitis. Comprend Contin Educ
Prac Vet 19, 513-523.

Granstrom DE, Dubey JP, Davis SW, et al. 1993. Equine protozoal

myeloencephalitis: antigen analysis of cultured Sarcocystis neurona merozoites. J
Vet Diagn Invest 5, 88-90.

14

38. Rossano MG, Mansﬁeld LS, Kaneene J B, et al. 2000. Improvement of Western
blot test speciﬁcity for detecting equine serum antibodies to Sarcocystis neurona.
J. Vet Diag Invest. 12, 28-32.

39. Hamir AN, Moser G, Galligan DT, et al. 1993. Immunohistochemical study to
demostrate Sarcocystis neurona in equine protozoa] myeloencephalitis. J Vet
Diagn Invest 5, 418-422.

40. Marsh AE, Barr BC, Madigan J, et a]. 1996. Sequence analysis and polymerase
chain reaction ampliﬁcation of small subunit ribosomal DNA from Sarcocystis
neurona. Am J Vet Res. 57, 975-981.

41. Lindsay DA, Dubey JP, Kennedy T. 2000. Determination of the activity of
ponazuril against Sarcocystis neurona in cell culture. Vet Parasitol. 92, 165-169.

42. Furr M, Kennedy T, MacKay R, et al. 2001.Efﬁcacy of ponazm'i] 15% oral paste
as a treatment for equine protozoal myeloencephalitis. Vet Therapetics. 2, 215-
222.

43. Lindsay DS and Dubey JP. 2000. Determination of the activity of diclazuril

against Sarcocystis neurona and Sarcocystisfalcatula in cell cultures. J Parasitol.
86, 164-166.

44. Dubey JP, Fritz D, Lindsay DS, et al. 2001. Diclazuril preventive therapy of
gamma interferon knockout mice fed Sarcocystis neurona sporocysts. Vet
Parasitol. 94, 257-263.

45. Furr, M. 2000. Treatment and management of equine protozoa]
myeloencephalitis. In: Proceedings of the North American Veterinary
Conference, Vol. 14. Orlando, FL. Ppl37-l38.

46. Lindsay DS, Zhang Y, Dubey JP, et al. 1998. Determination of the activity of
nitazoxanide against Sarcocystis neurona in cell cultures. In: Proceedings of the

American Association of Veterinary Parasitologists Annual Meeting, Baltimore,
MD. p 44.

47. McClure SR, Palma KG. 1999. Treatment of equine protozoa] myeloencephalitis
with nitazoxanide. J Eq Vet Sci. 19, 639-641.

48. Meyhew IG and Greiner Ec. 1986. Protozoa] diseases. Veterinary Clinics of
North America: Equine Practice. 2, 4439-459.

49. Saville WJA, Reed SM, Morley PS> 2000. Analysis of risk factors for the

development of equine protozoa] myeloencephalitis in horses. J Am Vet Med
Assoc. 217, 1174-1180.

15

50. Dubey JP, Lindsay DS, Saville WJA, et a]. 2001. A review of Sarcocystis

51.

52.

53.

54.

55.

neruona and equine protzoal myeloencephalitis (EPM). Vet Parasitol. 95, 89-131.

Tanhauser SM, Yowell CA, Cutler TJ, eta]: Multiple DNA markers differentiate
Sarcocystis neurona and Sarcocystis falcatula. J Parasit 85(2): 221-228, 1999.

Murphy AJ, Mansﬁeld LS. 1999. Simpliﬁed technique for isolation, excystation,
and culture of Sarcocystis species from opossums. J Parasitol. 85(5): 979-981.

Granstrom, DE. 1997. Equine protzoal myeloencephalitis: parasite biology,
experimental disease, and laboratory diagnosis. In: Proceedings of the
International Equine Neurology Conference, Ithaca, NY. p 4.

Rossano MJ, Kaneene JB, Marteniuk JV, et a]: The seroprevalence of antibodies
to Sarcocystis neurona in Michigan equids. Prev Vet Med 48: 113-128, 2001.

Plumb, DO, 1999. Veterinary Drug Handbook. Iowa State University Press,
Ames, IA

CHAPTER 2
THE RELATIONSHIP BETWEEN SARCOCYSTIS NE URONA SPOROCYSTS
AND STRONGYLE LARVAE
2.1 INTRODUCTION
The purpose of this series of experiments was to determine if S. neurona

Sporocysts can attach to or be ingested by ﬁrst, second, or third stage Strongyle larvae.
The pathogenesis of EPM is unclear. The route of migration of the parasite from the time
of ingestion of Sporocysts to the presence of merozoites in the CNS is unknown. It has
been shown in gamma interferon knock-out mice fed S. neurona Sporocysts that the
parasite initially replicates to a limited extent in visceral tissues and is then transported to
the CNS probably via leukocytes]. However, the possibility exists that Sporocysts
penetrate the GI tract and enter the bloodstream while attached to or while inside of
Strongyle larvae and that pyrantel tartrate causes a decrease in the number of horses with
EPM by killing the carrier of S. neurona (Strongyle larvae) and not by a direct effect on

Sarcocystis.

2.2 MATERIALS AND METHODS
2.21 Strangle collection and growth:

Fresh feces was collected from horses with natural Strangle infections. The
samples were collected directly after voidance from the animal, before hitting the ground,
to prevent contamination with larvae from soil nematodes. A sucrose fecal ﬂoatation was
performed to conﬁrm the presence of healthy strongyle-type eggs. The fecal sample was
then mixed in a 50:50 solution with autoclaved wood shavings in a metal bucket and
distilled water added to the mixture until it became moist. Four subsamples were taken
from this bucket (described later) and the bucket was covered with aluminum foil and left
to develop and grow to the third larval stage for 10 days. After this time, larvae were
collected using the Baermann technique and washed three times by repeated
centrifugation for 15 minutes at 1,000 RPM and rinsing with tap water. Larvae were then
placed in a petri dish of tap water with 0.250 ml Hank’s Balanced Salt Solution (HBSS)
containing 8 X 103 S. neurona sporocysts (collected from an adult male opossum and
shown by western blot, polymerase chain reaction (PCR), and growth pattern in cell
culture to be S. neurona). The petri dish was placed in a cool, dark environment for 5
days after which larvae were collected by centrifugation and washed two times with tap
water. Larvae collected prior to sporocyst exposure were used as negative controls.

In order to test the ability of ﬁrst and second stage strongyle larvae to
ingest/attach to sporocysts, the four subsamples taken from the culture described above
were placed in four separate petri dishes. Weights of the dry matter in each dish ranged
from 66.6 g to 90.5 g. A solution containing 12 m] of Hank’s Balanced Salt Solution

with 6 X 105 S. neurona sporocysts was split into 3 ml aliquots and spread evenly over

18

the fecal matter in each petri dish. Dishes were covered in aluminum foil and placed in a
cool dark enviromnent for 10 days. After 10 days, larvae were collected from each
sample using the Baermann technique and washed two times by repeated centrifugation

for 15 minutes at 1,000 RPM and rinsing with tap water.

2.22 PCR:

Prior to DNA extraction, each sample was frozen with dry ice and then thawed
with warm water 10X to help facilitate the extraction process. DNA was extracted from
each sample with Qiagen DNeasyTM Tissue Kit (Qiagen, Inc., Valencia, CA) according to
the manufacturer’s instructions. PCR using oligonucleotide primers JNB25 and JD396
was used to determine the presence or absence of a 334 base pair product of S. neurona
DNA in each sample.2 Brieﬂy, PCR was performed in standard reaction conditions (50
nM KCL, 10 mM tris-HC], pH 9.0 at 25 °C. 0.1% Triton X-100, 1.5 mM MgC12, 0.2 mM
each deoxynucleotide triphosphate) with a GeneAmp® PCR System 9600 (Perkin Elmer,
Foster City, CA) thermocycler with the following conditions: 72 °C, 3 min; 35 cycles of
(94 °C, 30 sec; 37 0C, 1 min; 72 °C, 45 sec); 72 0C, 6 min; 4 °C, hold. PCR products were
electrophoresed, visualized, and compared on a 1.8% agarose gel with ethidium bromide
staining. These samples were tested on two separate occasions. A positive Sarcocystis

neurona control was run along with the sample wells.

2.3 RESULTS:
A Sarcocystis PCR product was not detected in DNA extracted from any of the
Strangle groups. Positive controls were positive, untreated Strangle larvae did not

produce a Sarcocystis band.

20

2.4 DISCUSSION:

Strangle L1 and L2 larvae feed in their environment prior to being ingested by the
horse. It is reasonable to think that if S. neurona sporocysts were ingested by these
larvae, it would occur during these phases of the lifecycle. Similar situations occur in
nature. For example, Anaplacephala perfaliata, the equine tapeworm, infects horses alter
being ingested by the oribatid mite.

In this experiment, S. neurona DNA was not detected by PCR, indicating that the
parasite was not present or present at levels below detection within the larvae. It is
unlikely, but possible, that a small number of sporocysts could have gone undetected.
The PCR technique used in this study has been previously found to be very sensitive and
able to detect a DNA product of S. neurona with as little as 10 merozoites per 0.5 g of
tissue (Keith Nelson, personal communication, 2001) making sporozoites unlikely to
miss. There is the possibility that in the extraction process we were unable to excyst
sporocysts within larvae and these went undetected. However, freezing and thawing
prior to extraction decreases the chance of this occurrence.

Because this study did not take place under natural conditions (i.e. in an outdoor
environment) the possibility cannot be ruled out that these larvae did not feed as they
would in the soil. However, it is also unlikely that the larvae would come into contact
with as large a number of sporocysts as were used in this study, so if ingestion does occur
in nature, we should have been able to recreate a similar scenario in vitro.

Third-stage Strangle larvae are covered with a protective sheath and are the stage
of the parasite that penetrates the GI tract. It is possible that S. neurona sporocysts

become entrapped in the sheath or become mechanically attached to this stage of the

21

parasite due to surface interactions and penetrate the GI tract along with the larvae. In
this study, S. neurona DNA was not detected by PCR in third-stage larvae exposed to
sporocysts for a 5-day period. This indicates that attachment either does not occur at this
stage, does not occur in a liquid medium, or a longer period of exposure is needed for
attachment. GI transit time in the horse does not exceed 5 days, making longer exposure

times in viva highly unlikely.

22

2.5 CONCLUSIONS:

We found no evidence from in vitra studies to support the hypothesis that
Strangle larvae can ingest or attract S. neurona sporocysts to transport them across the
GI tract. This discredits the theory that Strongid®-C decreases the incidence of clinical

EPM by an indirect method (i.e. decreasing the number of available transport hosts).

23

2.6 LITERATURE CITED:

1. Dubey JP. 2001. Migration and development of Sarcocystis neurona in tissues of
mice fed sporocysts from a naturally infected opossum. Vet Parasitol. 95:341-
3 5 1 .

2. Tanhauser SM, Yowell CA, Cutler TJ, et a]. 1999. Multiple DNA markers

differentiate Sarcocystis neurona and Sarcacystisfalcatula. J Parasit 85(2): 221-
228.

24

CHAPTER 3

THE EFFECTS OF PYRANTEL TARTRATE ON SARCOC YS T IS NE URONA
MEROZOITE VIABILITY

3.1 ABSTRACT:

Sarcocystis neurona is the etiological agent of equine protozoal myeloencephalitis
(EPM), a neurological disease of horses. In this study, we tested the hypothesis that the
anthelmintic pyrantel tartrate can kill S. neurona merozoites growing in equine dermal
cell culture. S. neurona merozoites were exposed to a range of concentrations of pyrantel
tartrate or sodium tartrate between 1 mM and 10 mM. Merozoites were then placed onto
equine dermal cell cultures and incubated for two weeks to check for viability. At one
and two weeks post inoculation, plaques were counted and the counts compared between
treatment groups and controls using the Wilcoxon rank-sum test. Merozoites exposed to
concentrations of pyrantel tartrate higher than 2.5 mM (8.91X10'4 g/ml) did not produce
plaques in equine dermal cells while those exposed to similar concentrations of the
tartrate salt or medium alone produced signiﬁcant numbers of plaques. These results
demonstrate that pyrantel tartrate has activity against S. neurona merozoites in vitra and
suggest that it may have activity against the sporozoite stage of the parasite found in the

horse’s intestinal tract.

25

3.2 INTRODUCTION:

Sarcocystis neurona is the etiological agent of equine protozoal myeloencephalitis
(EPM), a neurological disease of horses.I Disease results from infection of the central
nervous system (CNS) of equids with this protozoan parasite.l The lifecycle of S.
neurona is complex and requires two hosts. The parasite replicates asexually in an
intermediate host, forming tissue cysts in muscle that are infective to a carnivorous
deﬁnitive host after ingestion. The nine-banded armadillo (Dasypus navemcinctus) has
been found to be a natural intermediate host for S. neurona.“3 Cats have also been shown
to act as intermediate hosts when experimentally infected with large numbers of S.
neurona sporocysts.4 A number of other intermediate hosts have been discovered
recently and were described in Chapter One. Sexual replication of S. neurona occurs in
the intestine of the deﬁnitive host, the opossum (Didelphis virginiana).5 Infective
sporocysts are shed into the environment in the feces of the opossum where they are
consumed by an appropriate intermediate host to continue the life cycle.’5 Horses and
ponies become infected when they ingest sporocysts from opossum feces, but S. neurona
does not form tissue cysts in these species. Thus, equids are aberrant hosts.l It is likely
that sporozoites are excysted from sporocysts in the digestive tract of the horse, penetrate
the gut wall, and travel to the CNS of some horses where they are found as merozoites.
The merozoite stage of the parasite replicates asexually in the neurons of the brain and
spinal cord in equids, leading to the clinical signs of EPM.7’8‘9

EPM is considered to be the most important protozoal disease of horses in the
Americas.10 Exposure to S. neurona is high, with a seroprevalence in horses in some

areas exceeding 50%.1 ”2’13 This high exposure rate, in addition to the seriousness of the

26

disease, supports the need for prevention, rather than treatment, as a method of
controlling the disease. Daily feeding of an effective pharmaceutical preventative would
be a possible means of protecting horses from infection with S. neurona and subsequent
development of EPM. It has been speculated that pyrantel tartrate, the active ingredient
of Strongid® C (Pﬁzer, Inc., New York City, NY), may have activity against S. neurona.
Strongid® C is a daily anthelmintic feed supplement used to protect horses from strongyle
larvae. Theoretically, daily pyrantel tartrate administration could prevent S. neurona
infection in equids by killing sporozoites as they are excysted in the gut.

The purpose of this study was to determine if pyrantel tartrate has activity against
Sarcocystis neurona merozoites. The merozoite stage of the parasite used in this study
replicates in the central nervous system of horses and is not likely to be exposed to
pyrantel tartrate, a drug that has low absorption and remains primarily within the
digestive tract. However, it is believed that results attained from merozoite studies may
be useful to model how the drug will work against the sporozoite stage of S. neurona
found in the digestive tract. Positive results would lead to consideration of ﬁtrther studies
investigating the possibility of using Strongid® C as a preventative drug for EPM in

horses.

27

3.3 MATERIALS AND METHODS:
3.3] Preparation of Merozoites:

S. neurona merozoites (MI horse #1)'4 were grown and maintained on low
passage (1-19) equine dermal cells (American Type Culture Collection CCL57, strain
NBL-6) with Dulbecco’s modiﬁed Eagle’s medium (GIBCO, Rockville, MD)
supplemented with 6% heat-inactivated fetal bovine serum, penicillin (100 U/ml),
amikacin (100ug/ml), and amphotericin B (1.25 ug/ml) (solution hereafter referred to as
DMEM). Medium containing asexually replicating stages of the parasite was removed
ham 4 heavily infected ﬂasks (~100 ml). This solution was not ﬁltered and, therefore,
contained both the free merozoite stage of the parasite and any free equine dermal cells
containing other asexually replicating stages of the parasite (early and late schizonts).
The solution was placed in two 50 m1 conical tubes and centrifuged for 40 minutes at 209
x g. After centrifugation, the supernatant was removed, and the pellet from each tube
was resuspended in 1.5 ml of DMEM. The suspension from each tube was combined and
stirred to evenly mix the parasite. Merozoites from ﬁve ~50 u] subsamples of this
solution were counted using a Bright-Line hemacytometer (Hausser Scientiﬁc, Horsham,
PA). The mean and standard deviation of these counts were determined. The merozoite
count of the stock solution was 2.77i0.13 x 106 merozoites per ml. A 200 pl aliquot of
this solution was placed in each of twelve 15 ml conical tubes making the ﬁnal number of

merozoites per tube approximately 5.54 x 105.

28

3.32 Cell Culture Preparation:

The aforementioned equine dermal cells (passage 19) were grown to conﬂuency
as determined by visual examination using an Olympus CK2 inverted microscope
(Olympus Optical Co., Ltd., Tokyo, Japan) in eight 6-well cell culture plates (Corning,

Inc., Corning, NY).

3. 33 Preparation of Drug:

The activity of two chemical compounds was tested against S. neurona
merozoites and all cellular stages of the asexually replicating parasite. Pyrantel tartrate
(Sigma, Inc., St. Louis, MO) was tested as a possible preventative drug for EPM, and
sodium tartrate (Sigma, Inc., St. Louis, MO) was used as a salt control. A 100 mM
solution (3.564X10'2 g/ml) of pyrantel tartrate (FW 356.4) in DMEM (adjusted to pH
7.98 with 5.0 N NaOH) was ﬁltered through a 0.22 pm ﬁlter to remove contaminants and
diluted in DMEM to concentrations of 10 mM, 7.5 mM, 5 mM, 2.5 mM, and 1 mM. A
100 mM solution (2.30X10’2 g/ml) of sodium tartrate (FW 230.1) and DMEM (solution
pH 7.78) was ﬁltered through a 0.22 pm ﬁlter and diluted as above. For each drug, 1.0
m] of each dilution was added to a 15 ml conical tube containing merozoites (see above).
One ml of DMEM alone (pH 7.88) was added back to two additional tubes as a negative

control.
3.34 Experimental Design:

Tubes prepared above (sec 3.33) were incubated at 37°C in 5% C02 for 24 hours.

After this time, the tubes were centrifuged for 10 minutes at 209 x g, the supernatant

29

carefully removed to prevent pellet disruption, and 1.0 ml of DMEM added back to each
tube as a washing step. Tubes were again centrifuged. This washing procedure was
repeated twice to remove any residual drug from the pellet. After the ﬁnal wash, the
supernatant was carefully removed and the pellet gently resuspended in 4 ml of DMEM.
The suspension ﬁ'om each tube was distributed between four wells (1.0 ml per well) of 6-
well cell culture plates with conﬂuent equine dermal cells. An additional 2 ml of DMEM
was added to each well. The plates were incubated at 37°C in 5%C02 for 24 hours, after
which all media was aspirated from all wells. Two m] of fresh DMEM was added back
to each well and aspirated as a washing step. Three ml of DMEM was then added to
maintain the cultures. DMEM in all wells was changed weekly for the duration of the

experiment.

3.35 Plaque Number Determination:

After one week, equine dermal cell monolayers were observed for plaques and the
plaque numbers counted with the inverted microscope (4X magniﬁcation). Wells were
scanned in a uniform pattern from side to side in such a way that no ﬁelds overlapped and
no plaque was counted twice. Plaque numbers for each pyrantel tartrate and sodium
tartrate dilution (4 wells per dilution) were compared to numbers from the DMEM
control (4 wells) using the Wilcoxon rank-sum test (exact test, two-sided). Plaque
numbers from each sodium tartrate dilution (4 wells per dilution) were also compared to
numbers from the DMEM control (4 wells) using the same statistical test. In addition, for
each pyrantel tartrate dilution plaque numbers were compared to those of the sodium

tartrate dilutions using the Wilcoxon rank-surns test (exact test, two-sided). A P-value of

30

_<_0.05 was used to determine signiﬁcance for all comparisons. Plates were returned to the
incubator after the ﬁrst count, and plaques from each drug dilution were again counted

one week later and analyzed using the same statistical methods.

3.36 DNA Extraction and PCR:

Cell culture supematants and cell monolayers were both tested for the presence of
S. neurona DNA using PCR. Medium was removed from all four wells of each drug
dilution after two weeks and combined in 15 ml conical tubes (one tube per dilution).
This media was centrifuged for 15 min at 1877 x g, the supernatant removed, and 0.5 ml
of phosphate buffered saline (PBS) added back to the pellet. The solution from each tube
was then transferred to separate 1.5 m1 Eppendorf tubes and frozen at —20°C for later
DNA extraction. One ml of alkaline chelating solution (ACS) was added back to the
monolayer of cells in each well. Gentle rocking of the plate caused the monolayers to
detach from the wells and become suspended in the ACS. The suspension from all four
wells of each dilution was removed and combined as above. The suspensions were then
centrifuged for 15 min at 1877 x g, the supematants removed, and 0.5 ml of phosphate
buffered saline (PBS) added back to the pellets. The suspensions from each tube were
transferred to separate 1.5 m] Eppendorf tubes and frozen at —20°C for later DNA
extraction.

Frozen samples were thawed in warm water and re-frozen with dry ice 3 times
prior to extraction. DNA was extracted from each sample with Qiagen DNeasyTM Tissue

Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions.

31

PCR, using oligonucleotide primers JNB25 and JD396, was used to determine the
presence or absence of a 334 base pair product of S. neurona DNA in each sample.”
Brieﬂy, PCR was performed in standard reaction conditions (50 nM KCL, 10 mM tris-
HCl, pH 9.0 at 25 °C. 0. 1% Triton X-100, 1.5 mM MgC12, 0.2 mM each deoxynucleotide
triphosphate) with a GeneAmp® PCR System 9600 (Perkin Elmer, Foster City, CA)
thermocycler with the following conditions: 72 °C, 3 min; 35 cycles of (94 °C, 30 sec; 37
oC, 1 min; 72 °C, 45 sec); 72 °C, 6 min; 4 °C, hold. PCR products were visualized and
compared on a 1.8% agarose gel with ethidium bromide staining. These samples were

tested on two separate occasions.

3. 3 7 Drug Activity C antral :

To ensure activity of the drug concentrations used, third-stage strongyle larvae
were grown in vitro from equine feces containing large quantities of embryonated
strongyle-type eggs. Larvae were collected after ten days of growth in a damp, room
temperature, aerated environment using the Baermann technique, separated into six 15 ml
conical tubes, and exposed to 1.0 ml solutions of pyrantel tartrate diluted with DMEM to
concentrations of 0.0001 mM, 0.001 mM, 0.0] mM, lmM, and 10 mM. One ml of
DMEM was added to one tube of larvae as a negative control. After incubation at room
temperature for 24 hours, larvae in each tube were assessed for viability based on motility
using a dissecting microscope. Larvae were prodded with a drawn glass Pasteur pipette
to distinguish non-motile from non-viable larvae. Larvae that did not move when
prodded with the pipette were considered to be non-viable, and motile larvae viable. The

number of non-viable larvae for each drug dilution was compared to the number of non-

32

viable larvae in the DMEM treated group using the Fisher’s exact test. A P-value of

50.05 was used as the maximum critical value for evaluating the results.

33

3.4 RESULTS:
3.4] Plaque Numbers:

All pyrantel tartrate dilutions produced signiﬁcantly lower plaque numbers when
compared to the DMEM control after one and two weeks (P 5 0.03) (Table 3.1).
Merozoites exposed to pyrantel tartrate concentrations of 5 mM, 7.5 mM, and 10 mM
produced no plaques after one and two weeks. Merozoites exposed to pyrantel tartrate at
the concentration of 2.5 mM produced no plaques after one week and only a small
number of plaques after two weeks. Merozoites exposed to pyrantel tartrate at the
concentration of 1 mM produced plaques, however the number of plaques was
signiﬁcantly lower than that of the media control (P 5 0.03) (Table 3.1). Plaque numbers
for all sodium tartrate dilutions were not signiﬁcantly different from the DMEM control
after one and two weeks (P >0.05) (Table 3.2).

All pyrantel tartrate dilutions produced signiﬁcantly lower ntunbers of plaques
than sodium tartrate dilutions of similar molarities (P 50.03) with the exception of 1 mM
after two weeks (P >0.03) (Figures 3.1, 3.2; Table 3.3). DMEM control group plaque
numbers for the sodium tartrate experiment and pyrantel tartrate experiment were not

statistically different after one and two weeks (P>0.05) (Table 3.3).

3. 42 PCR:

A strong Sarcocystis PCR product was only detected in medium removed from
the lowest concentration of pyrantel tartrate (1 mM). A weak Sarcocystis PCR product
was detected in medium removed from pyrantel tartrate concentrations of 2.5 mM and

greater. In contrast, a strong Sarcocystis PCR product was detected in the medium of all

34

sodium tartrate treatment groups and DMEM controls (Figure 3.3). Identical results were
obtained with PCR of cells removed from culture wells (data not shown). Although a
second PCR product of smaller size was seen in several wells, this product ampliﬁes
from equine dermal cell DNA, is not related, and should be disregarded in the

interpretation of the gel (Figure 3.3). '4

3.43 Drug Activity Control:
The number of viable third-stage strongyle larvae signiﬁcantly decreased with
exposure to all concentrations of pyrantel tartrate when compared to that of the DMEM

control (P < 0.0001) (Table 3.4).

35

3.5 DISCUSSION:

In this study, the free merozoite stage of S. neurona and equine dermal cells
containing asexually replicating stages of S. neurona (early and late schzonts) were
exposed to various concentrations of pyrantel tartrate for 24 hours to determine if the
drug had activity against the parasite. It was found that merozoites exposed to
concentrations of pyrantel tartrate greater than 2.5 mM showed no growth when added to
cell cultures. Pyrantel tartrate appears to be 100% lethal to merozoites at these
concentrations. When merozoites were exposed to pyrantel tartrate at the concentration
of 2.5 mM, no growth occurred after one week in culture, and limited growth was seen
after two weeks. The drug at this concentration appeared to inactivate a large proportion
of merozoites and may still be effective as a preventative.

Despite the absence of plaques with pyrantel tartrate concentrations greater than
2.5 mM, a faint Sarcocystis PCR product was detected in medium and cells from these
wells. The PCR technique used in this study had been previously found to be quite
sensitive: a DNA product can be detected with as little as 10 merozoites per 0.5 g of
tissue (Keith Nelson, personal communication). The weak bands produced from PCR of
medium and cells removed from wells with zero or few plaques may be due to small
amounts of residual DNA from non-viable organisms trapped in the equine dermal cell
monolayer. The fact that PCR products from wells with large plaque numbers showed a
greater ampliﬁcation of DNA indicated a larger amount of S. neurona DNA was present
in those cultures due to parasite grth and replication. It is unlikely that viable

organism remained in wells with no plaque growth; however, we are unable to

36

distinguish whether the remaining organisms causing the positive PCR reaction were
viable or dead with this PCR technique.

All S. neurona merozoites exposed to sodium tartrate at molarities similar to that
of pyrantel tartrate showed growth in culture similar to that of the DMEM control, and
PCR detected a strong Sarcocystis PCR product in all treatment groups. These results
lead to the conclusion that merozoite death was due to the presence of the tartrate
compound and not due to the presence of DMEM alone or the tartrate salt alone.
Additionally, the decrease in viability of third-stage strongyle larvae after exposure to
concentrations of pyrantel tartate at and below that which inactivated merozoites shows
that the drug was active at all concentrations used in this study.

The statistical comparison of the number of plaques produced in pyrantel tartrate
dilutions and sodium tartrate dilutions indicates that in all groups, with the exception of 1
mM at two weeks, the number of plaques produced by the sodium tartrate dilutions was
signiﬁcantly greater than that of the pyrantel tartrate dilutions. The 1 mM pyrantel
tartrate dilution produced a signiﬁcantly lower number of plaques than the DMEM
control; however, this group produced a much larger number of plaques than the numbers
found at higher concentrations of pyrantel tartrate. This indicates that at the dose of 1
mM, the lowest dose tested in this study, pyrantel tartrate may not be effective against S.
neurona merozoites.

The bioequivalence of Strongid® C and generic pyrantel tartrate against
gastrointestinal parasites in horses has been previously demonstrated.“5 The
concentration of pyrantel tartrate given daily to equids in the form of Strongid® C is 1.2

mg per lb. of body weight making the daily dose 1.2 g for a 1000 lb. horse. This

37

concentration greatly exceeds the amount of pyrantel tartrate found to kill merozoites in
this study on a gram for gram basis, ﬁrrther evidence supporting the possibility that
Strongid® C could be used as a preventative for EPM. However, drug dilution and
distribution in the gastrointestinal tract of the horse may vary, making it difﬁcult to
compare an in viva gram per volume dose to the dosage tested in this study. Pyrantel
tartrate was found to be ineffective against S. neurona infection in gamma-interferon
gene knockout mice”; however, the digestive tracts and gastrointestinal transit times of
mice and equids vary greatly. Future in viva studies will be conducted to determine if
Strongid® C protects horses from infection with S. neurona.

Pyrantel tartrate is an anthelminthic compound that acts in two ways: 1) it
possesses nicotine-like properties and acts similarly to acetylcholine (ACh), and 2) it
inhibits acetylcholinesterase. '8 These properties make the drug a depolarizing
neuromuscular blocking agent in susceptible parasites. Exposure to pyrantel tartrate leads
to paralysis of the susceptible organism.’8 The presence of ACh has been detected in
bacteria and primitive organisms, such as the blue-green algae, yeast, fungi, tubellaria,
protozoa (T typanasama rhadesiense, Paramecium), nematodes, and spongesm’20 In
studied organisms, ACh occurs in both neuronal and non-neuronal tissues. Non-neuronal
ACh appears to be involved in the regulation of basic cell ﬁmctions, such as proliferation,
differentiation, cell-cell contact, immune functions, secretion, and absorption. 20It has
been speculated that ACh may be involved in the rapidly moving protozoa like
trypanosomes but not in sluggish arneboid movement.21 The fact that ACh has been
found in a wide variety of organisms leads us to consider the possibility that S. neurona

may also synthesize ACh. If ACh is present in S. neurona, pyrantel tartrate could affect

38

the parasite by disrupting basic cell functions regulated by ACh or by disrupting the
movement of S. neurona related to the ACh molecule. The presence and possible roles of
ACh in S. neurona need to be thoroughly explored before any conclusions can be made
about the mechanism of action of pyrantel tartrate in this organism.

The merozoite stage of S. neurona is found in the brain and spinal cord of horses
and is not exposed to pyrantel tartrate, a drug that remains primarily in the digestive tract.
However, because the merozoite stage has some similarities to the sporozoite stage found
in the gut of the horse and is readily available for in vitro studies, we elected to initially
investigate the effects of pyrantel tartrate on this stage of the parasite. The fact that
pyrantel tartrate appears to kill both free merozoites and stages of S. neurona asexually
replicating within cells is encouraging and provides further evidence that the drug may be

able to kill more than one stage of the parasite.

39

3.6 CONCLUSION:

Pyrantel tartrate at concentrations greater than 2.5 mM (8.91X10‘4 g/ml) kills both
free Sarcocystis neurona merozoites and stages of S. neurona asexually replicating within
equine dermal cells. This result has prompted further investigations into the possibility of

using Strongid® C as a preventative for EPM in horses.

40

3.7 LITERATURE CITED:

1.

10.

11.

12.

13.

Dubey JP, Davis SW, Speer CA, et al: Sarcocystis neurona n. sp. (Protozoa:
Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis. J Parasitol
77: 212—218, 1991.

Cheadle MA, Tanhauser SM, Dame JB, et al: The nine-banded armadillo (Dasypus
navemcinctus) is an intermediate host for Sarcocystis neurona. Int J Parasitol
31(4):330-335, 2001.

Tanhauser SM, Cheadle MA, Massey ET, et a]: The nine-banded armadillo (Dasypus
novemcinctus) is naturally infected with Sarcocystis neurona. Int J Parasitol 3 1 (4):
325-329, 2001.

Dubey JP, WJA Saville, DS Lindsay, et al: Completion of the life cycle of
Sarcocystis neurona. J Parasitol 86(6): 1276-1280, 2000.

Fenger CK, Granstrom DE, Langemeier J L, et al: Identiﬁcation of opossums
(Didelphis virginiana) as the putative deﬁnitive host of Sarcocystis neurona. J
Parasitol 81 : 199-213, 1992.

Levine ND: The taxonomy of Sarcocystis (Protozoa, Apicomplexa) species. J
Parasitol 72:372-382, 1986.

Simpson CF and Mayhew IG: Evidence for Sarcocystis as the etiologic agent of
equine protozoal myeloencephalitis. J Protozaal 27:288-292, 1980.

Davis SW, Speer CA, Dubey JP: In vitro cultivation of Sarcocystis neurona from the
spinal cord of a horse with equine protozoal myelitis. J Parasitol 77:792-796, 1991.

Bowman DD, Cummings JF, Davis SW, et a]: Characterization of Sarcocystis
neurona from a thoroughbred with equine protozoal myeloencephalitis. Cornell Vet
82:41-52, 1992.

Dubey JP, Lindsay DS, Saville WJA, et al: A review of Sarcocystis neurona and
equine protozoal nyeloencephalitis (EPM). Vet Parasitol 95:89-131, 2001.

Rossano MJ, Kaneene J B, Marteniuk JV, et al: The seroprevalence of antibodies to
Sarcocystis neurona in Michigan equids. Prev Vet Med 48: 113-128, 2001.

Granstrom DE: Diagnosis of equine protozoal myeloencephalitis: western blot
analysis, in Proceedings. Am Coll Vet Intern Med Forum 587-590, 1993.

Saville WJ, Reed SM, Granstrom DE, et al: Seroprevalence of antibodies to
Sarcocystis neurona in horses residing in Ohio. J Am Vet Med Assoc 210:519-524,
1997.

41

14. Mansﬁeld LS, Schott II HG, Murphy AJ, et a]: Comparison of Sarcocystis neurona
isolates derived from horse neural tissue. Vet Parasitol 95:167-178, 2001.

15. Tanhauser SM, Yowell CA, Cutler TJ, eta]: Multiple DNA markers differentiate
Sarcocystis neurona and Sarcocystis falcatula. J Parasit 85(2): 221-228, 1999.

16. Valdez RA, Dipietro JA, Paul AJ, et a]: Controlled efficacy study of the
bioequivalence of Strongid®-C and generic pyrantel tartrate in horses. Vet Parasitol
60(1-2): 83-102, 1995.

17. Lindsay DS, Dubey JP: Determination of the activity of pyrantel tartrate against
Sarcocystis neurona in gamma-interferon gene knockout mice. Vet Parasitol 97:141-
144, 2001.

18. Plumb DC: Plumb ’s Veterinary Drug Handbook, Third Edition. White Bear Lake,
Pharrna Vet Publishing, 1999, pp 640-642.

19. Bulbring E, Lourie EM, and Pardoe U: Presence of acetylcholine in T rypanasama
rhadesiense and its absence from Plasmadium gallinaceum. Brit J Pharmacol 4:290-
294, 1949.

20. Wessler I, Kirkpatrick CJ, and Racke K: The Cholinergic 'Pitfall': Acetylcholine, a
Universal Cell Molecule in Biological Systems, Including Humans. Clin Exp

PharmacaIPhysi0126(3): 198-205, 1999.

21. Sastry BV and Sadavongvivad C: Cholinergic systems in non-nervous tissues.
Pharmacol Rev 30(1): 65-132, 1978.

42

 

Treatment Group Plaque Numbers Standard Deviation P value“

 

(mean)

Week 1 Week 2 Week 1 Week 2 Week 1 Week 2
DMEM 86.75 143.25 7.09 7.62 --- --
0.001M 64.50 120.50 8.06 8.96 0.03 0.03
0.0025M 0 5.25 0 2.63 0.03 0.03
0.005M 0 0 0 0 0.03 0.03
0.0075M 0 0 0 0 0.03 0.03
0.01M 0 0 0 O 0.03 0 03

 

Table 3.1. Plaque numbers for pyrantel tartrate concentrations. *P-value refers to
comparison between pyrantel tartrate dilutions and the DMEM control (Wilcoxan rank
sum test, two tailed, exact test).

43

 

Treatment Group Plaque Numbers Standard Deviation P value"
(mean)

Week 1 Week 2 Week 1 Week 2 Week 1 Week 2

DMEM 89.75 141.25 3.10 20.0 --- --
0.001 M 96.25 133.00 10.8 13.0 0.49 0.69
0.0025M 82.25 140.50 1 1.4 18.2 0.69 0.97
0.005M 93.50 137.75 1.92 14.0 0.14 0.89
0.0075M 94.50 135.00 4.20 15.5 0.14 0.69
0.01M 79.50 1 19.75 7.33 21.0 0.06 0.20

 

Table 3.2. Plaque numbers for soditun tartrate concentrations. *P-value refers to
comparison between sodium tartrate dilutions and the DMEM control (Wilcoxan rank
sum test, two tailed, exact test).

44

 

 

Plaque Numbers— Plaque Numbers— P value“
Week 1 Week 2
(mean : SD) (mean : SIB
E El S_T [1: Week 1 Week 2
DMEM 89.75:3.10 86.75:7.1 141.3:20.0 143.25:7.6 0.31 0.68
0.001 M 96.25:10.8 64.50:8.1 133: 1 3.0 120.50:9.0 0.03 0.20
0.0025M 82.25:] 1.4 0.00:0 140.5:18.2 5.25:2.6 0.03 0.03
0.005M 93.50:] .92 0:0 137.8: 14.0 0:0 0.03 0.03
0.0075M 94.50:4.20 0:0 135:] 5.5 0:0 0.03 0.03
0.01M 79.50:7.33 0:0 1 19.8:21 .0 0:0 0.03 0.03

 

Table 3.3. Plaque numbers for sodium tartrate (ST) and pyrantel tartrate (PT)
concentrations. *P value refers to comparison between sodium tartrate and pyrantel
tartrate numbers at the same molarities (Wilcoxan rank sum test, two tailed, exact test).

45

 

 

Number of Number of Number of
Treatment Viable Viable Larvae Non-Viable Percent Non- P-value“
Group Larvae After Pyrantel Larvae After Viable
- Before Exposure Pyrantel
Pyrantel Exposure
Exposure
DMEM 8 7 1 12.5 ---
0.000001M 18 1 17 94.4 < 0.0001
0.00001M 13 0 13 100 < 0.0001
0.0001M 16 0 16 100 < 0.0001
0.001M 27 1 26 96.3 < 0.0001
0.01M 14 0 14 100 < 0.0001

 

Table 3.4. Numbers of viable and non-viable strongyle larvae after exposure to pyrantel
tartrate. *P value refers to comparisons between non-viable larvae from pyrantel tartrate

dilutions and the DMEM control (Fisher’s exact test, 2-tailed).

46

 

 

0.01

0.0075

 

 

 

 

120
100 *—

Sau-E Lo Lon—.52

one standard deviation.

indicates a signiﬁcant difference between plaque numbers in sodium tartrate treatment

Figure 3.1. Plaque numbers (mean of four wells) one week after culture inoculation. (*)

groups and pyrantel tartrate treatment groups. I

47

um Tamale
Pymd Tartrate

I Sodi
Iii

 

 

 

 

0.01

 

0.0075

0.1135

__A

_ V
L

E , A

are
Drug Concmtrdion (M)

0.0025

1

 

 

manner...

Baa-E .0 conEaz

one standard deviation.

(*) indicates a signiﬁcant difference between plaque numbers in sodium tartrate treatment
48

Figure 3.2. Plaque numbers (mean of four wells) two weeks after culture inoculation.

groups and pyrantel tartrate treatment groups. I

 

Figure 3.3. PCR of media removed from cell cultures infected with merozoites treated
with the following solutions:

Lane 1) 100 bp size standard

A) DMEM

B) 0.001M pyrantel tartrate (PT)
C) 0.0025M PT

D) 0.005M PT

E) 0.0075M PT

F) 0.01M PT

G) DMEM

H) 0.001M sodium tartrate (ST)

1) 0.0025M ST

J) 0.005M ST

K) 0.0075M ST

L) 0.01M ST

M) Equine dermal cell DNA control
N) S. neurona DNA (+ control)

0) PCR solution only (- control)

CHAPTER 4

49

THE EFFECTS OF PYRANTEL TARTRATE ON SARCOCYSTIS NE URONA
SPOROZOITES IN CELL CULTURE AND IN GAMMA INTERFERON
KNOCK-OUT MICE
4.1 INTRODUCTION:

Equine protozoal myeloencephalitis (EPM), a neurologic disease of horses, is
caused by the protozoan parasite Sarcocystis neuronal. The lifecycle of S. neurona is
complex and involves the deﬁnitive host, the opossum (Didelphis virginiana)2, and a
variety of intermediate hosts including the nine-banded armadillo (Dasypus
navemcinctus)3’4, the striped skunk (Mephitis mephitis)5, the raccoon (Pracyan Iatar)’,
and the domestic cat (F elis damesticus)7. Horses are considered aberrant hosts and
become infected with S. neurona after accidentally ingesting the sporocyst stage of the
parasite passed in opossum feces. It is hypothesized that sporozoites contained within the
sporocyst are excysted in the gastrointestinal tract of the horse, then penetrate the gut
wall and enter the bloodstreams. From the bloodstream they travel to the central nervous
system and replicate asexually in the brain and spinal cord as merozoites leading to the
clinical signs of EPM.

Exposure to S. neurona is high, with the seroprevalence in horses in some areas
exceeding 50%9. However, the number of horses with clinical signs of disease is much
lower. This high exposure rate, combined with the serious consequences of the disease,
indicates a form of prevention, rather than treatment, would be a logical way of
controlling disease. Pyrantel tartrate at concentrations greater than 2.5 mM has been
found to be lethal to S. neurona merozoites in cell culture"). This drug is available in a

pelleted form, Strongid®-C (Pﬁzer, Inc., New York, NY), that is fed daily to horses to

help prevent Strongyle infection. If pyrantel tartrate were also found to be effective

50

against the sporozoite stage of the parasite, daily pyrantel administration could
theoretically prevent S. neurona infection by killing sporozoites as they are excysted in
the GI tract.

The purpose of the present study was to determine if pyrantel tartrate has efﬁcacy
against the sporozoite stage of S. neurona in either cell culture or in a gamma interferon

knock-out mouse model of EPMl 1.

5]

4.2 MATERIALS AND METHODS: CELL CULTURE:
4.21 Cell culture:

Low passage (p22) equine dermal cells (American Type Culture Collection
CCL57, strain NBL-6) were grown to conﬂuency as determined by visual examination
using an Olympus CK2 inverted microscope in four, 6-well cell culture plates (Corning,

Inc., Corning, NY).

4. 22 Sporocysts:
Sporocysts were harvested from intestines of laboratory reared, speciﬁc pathogen
free opossums, which were euthanized 14 days after oral inoculation with Sarcocystis

neurona-infected cat tissue7. Sporocysts were cleaned and stored using the technique

described by Murphy and Mansﬁeld”.

4.23 Preparation afsparazaites:

Approximately 100,000 S. neurona sporozoites were excysted using methods
described previously”. Excysted sporozoites were conﬁrmed present by visually
assessing a sample of the solution under an Olympus CK2 inverted microscope (Olympus
Optical Co., Ltd., Tokyo, Japan). Solution containing intact sporocysts, remnants of
cysts, and sporozoites was placed onto a 60% PercollTM (Amersham Biosciences,
Piscataway, NJ) and phosphate buffered saline (PBS) gradient. An additional tube
containing the same 60% PercollTM and PBS solution plus PercollT" density marker beads
and the tube containing sporocysts and sporozoites were centrifuged for 60 minutes at

12,000 RPM. After this time the sporozoite solution below the density marker bead line

52

representing 1.05 g/ml was removed and added to 30 ml of DMEM in a 50 m1 conical
tube. This tube was centrifuged for 30 minutes at 2,000 RPM as a washing step. The
supernatant was removed and 1.2 ml DMEM added back to the pellet. This solution was

split evenly between six, 15 ml conical tubes.

4.24 Preparation of drug for use in cell culture:

A 100 mM solution (3.564X10'2 g/ml) of pyrantel tartrate (FW 356.4) in DMEM
(Dulbecco’s modiﬁed Eagle’s medium (GIBCO, Rockville, MD) supplemented with 6%
heat-inactivated fetal bovine serum, penicillin (100 U/ml), amikacin(100ug/ml), and
amphotericin B (1.25 ug/ml) was pH adjusted with 5.0 N NaOH, ﬁltered through a 0.22
pm ﬁlter to remove contaminants, and diluted in DMEM to concentrations of 10 mM, 5
mM, 1 mM, 0.5 mM, and 0.1 mM. One ml of each dilution was added to a 15 ml conical
tube containing sporozoites (sec 4.23). One ml of DMEM alone was added back to one

additional tube as a negative control.

4.25 Experimental procedure:

Tubes prepared above were incubated at 37°C in 5% CO2 for 24 hours. After this
time, the tubes were centrifuged for 20 minutes at 209 x g, the supernatant carefully
removed to prevent pellet disruption, and 1.0 ml of DMEM added back to each tube as a
washing step. Tubes were centrifuged in the same manner. This washing procedure was
repeated twice to remove any residual drug from the pellet. After the ﬁnal wash, the
supernatant was carefully removed and the pellet gently resuspended in 2 ml of DMEM.

The suspension from each tube was distributed between four wells (0.5 ml per well) of 6-

53

well cell culture plates with conﬂuent equine dermal cells. An additional 2.5 ml of
DMEM was added to each well. The plates were incubated at 37°C in 5% CO2 for three
weeks with weekly media changes. Plates were observed weekly for plaque growth with

an Olympus CK2 inverted microscope (Olympus Optical Co., Ltd., Tokyo, Japan).

4.26 Plaque Number Determination:

After three and four weeks, equine dermal cell monolayers were observed for
plaques, and the plaque numbers counted with the microscope described previously (sec
4.21). Wells were scanned in a uniform pattern from side to side in such a way that no

ﬁelds overlapped and no plaque was counted twice.

4. 2 7 Statistical analysis:

Plaque numbers for each pyrantel tartrate dilution (4 wells per dilution) were
compared to numbers from the DMEM control (4 wells) using the Wilcoxon rank-sum
test (exact test, two-sided). A P-value of 50.05 was used to determine signiﬁcance for all
comparisons. Plates were returned to the incubator after the ﬁrst count, and plaques from
each drug dilution were again counted one week later and analyzed using the same

statistical methods.

54

4.3 MATERIALS AND METHODS: MICE:
4.31 Mice:

Fifteen female, 12 week old, IFN-y-knockout mice (BALB/c-Ifng’mm) were
acquired ﬁ‘om Jackson Laboratories, Bar Harbor, ME. Mice were housed in ﬁltered
cages in a laminar ﬂow rack, fed sterilized food and water, and kept on autoclaved

bedding. All mice weighed approximately 20 grams.

4.32 Sporocysts:

Sporocysts were harvested from intestines of laboratory reared, speciﬁc pathogen
free opossums, which were euthanized 14 days after oral inoculation with Sarcocystis
neurona strain SN-37R raccoon tissue°. Sporocysts were cleaned and stored using the

technique described by Murphy and Mansﬁeld”.

4.33 Experimental procedure:

Mice were randomly assigned to three treatment groups of 5 mice each. Mice in
Group 1 were dosed once daily for six days with 0.2 m] sterilized distilled water through
a 5 French rubber urinary catheter. On day 3 of dosing, these mice were given 1000 S.
neurona sporocysts via a 22 ga gavage needle. Mice in Group 2 were dosed once daily
for six days with 0.2 m1 of 5 mM pyrantel tartrate (Sigma, Inc., St. Louis, MO, F W
356.4) solution in sterilized distilled water. On day 3 of dosing, mice in this group
received 1000 S. neurona sporocysts via a 22 ga gavage needle. Mice in Group 3
received six days of 0.2m] 5 mM pyrantel tartrate solution in distilled water via a 5F

rubber urinary catheter. Group 3 remained unchallenged.

55

Mice were observed daily for abnorrna] clinical signs. Any mouse unable to
ambulate or consume food or water or in respiratory distress was euthanized with
gradually increasing doses of carbon dioxide in an approved chamber according to the
guidelines of the AVMA". Mice in Group 3 were euthanized 14 days after the death of
the last infected mouse. A complete necropsy was performed on each mouse, and
samples of brain, liver, kidney, lung, and spleen were collected and ﬁxed in 10% neutral
buffered formalin. A sample of brain tissue from each mouse was also prepared for cell

culture.

4.34 Histapathalag:

Following ﬁxation in formalin, tissues were dehydrated through a graded series of
alcohol to xylol, embedded in parafﬁn, sectioned at 5 microns, and processed routinely.
All tissue sections were stained with hematoxylin and eosin (HE), and were examined via
light microscopy by a board-certiﬁed veterinary anatomic pathologist (J SP), who was

blinded to treatment group.
4.35 Statistical analysis:

Mean (: standard deviation) post survival time was compared between Group 1

and Group 2 using the student’s t-test (P5005).

56

4.4 RESULTS:
4.4] Cell culture:

Zero plaque growth was seen in the 5 mM and 10 mM pyrantel tartrate treated
groups after 3 weeks of growth. Zero plaque growth was seen in the 10 mM treated
group and minimal plaque growth was seen in the 5 mM treated group after 4 weeks of
incubation. One mM, 5 mM, and 10 mM pyrantel tartrate treated groups had plaque
numbers signiﬁcantly lower than the media control (P< 0.03) after both 3 and 4 weeks of

growth (Figure 4.1).

4. 42 Mice:

Untreated mice (Group I) survived a mean of 3 1 .4 : 2.3 days. Treated mice
(Group 2) survived a mean of 35 : 1.9 days. This was signiﬁcant increase in mean
survival time (P=0.03).

Additionally, 5/5 mice in Group 1 had evidence of both encephalitis and
pneumonia when examined histopathologically while only one mouse in Group 2 had
histologic lesions consistent with encephalitis and pneumonia. Three additional mice in
this group showed lesions of pneumonia only, and one mouse had no histologic lesions.
No protozoal organisms were seen in the brains of mice in Group 2 while 3/5 of the mice
in Group 1 contained protozoa. Protozoa were also seen in the lungs of 1/5 mice in
Group 1 and 3 out of the four mice with lesions in Group 2. Merozoites were cultured
from the brain tissue of all ﬁve mice in Group 1 and all ﬁve mice in Group 2. Mice in

Group 3 had no histologic lesions. No merozoites were cultured from any mice in Group

3.

57

4.5 DISCUSSION:

In this study, the action of pyrantel tartrate was tested against the sporozoite stage
of S. neurona in two different ways. First, the effects of the drug directly on sporozoites
were observed using a previously published cell culture drug screening technique"). The
drug was found to be 100% lethal to sporozoites at pyrantel tartrate concentrations of 5
mM and higher. Pyrantel at the concentration of 1 mM also caused a signiﬁcantly lower
number of plaques than the media control. These concentrations are identical to those
that have been shown to cause merozoite death in a previous study”. Results of this
study show that pyrantel tartrate has a direct effect on sporozoite numbers and their
ability to reproduce in cell culture.

The second part of this study tested the in viva effects of pyrantel tartrate on S.
neurona sporocysts using a mouse model. Mice treated with pyrantel prior to being
dosed with sporocysts lived signiﬁcantly longer than those that were dosed with sterile
water. This indicates that the drug may decrease the number of infective sporozoites,
making the number insufﬁcient enough to penetrate the central nervous system (CNS) or
small enough that they are able to be cleared by the host’s immune system before they
can cause CNS damage. This hypothesis is supported by a study completed by Dubey
that established a time course of infection in gamma interferon knock-out mice to
understand the pathogenesis of this parasite”. The study found that infection and

replication occur in the lungs prior to the CNS.

58

4.6 CONCLUSIONS:

From this study we conclude that pyrantel tartrate is lethal to S. neurona
sporozoites at concentrations of 5 mM and greater. The drug also affects sporozoite
infection in gamma interferon knock-out mice causing treated mice to live a signiﬁcantly
longer duration of time following S. neurona sporozoite infection and to have different
organs affected with similar histologic lesions than mice that did not receive pyrantel

tartrate.

59

4.7 LITERATURE CITED:

1.

Dubey JP, Davis SW, Speer CA, et a]. 1991. Sarcocystis neurona n. sp.
(Protozoa: Apicomplexa), the etiologic agent of equine protozoal
myeloencephalitis. J Parasitol 77: 212-218.

F enger CK, Granstrom DE, Langemeier J L, et a]: 1992. Identiﬁcation of

opossums (Didelphis virginiana) as the putative deﬁnitive host of Sarcocystis
neurona. J Parasitol 81 : 199-213.

. Cheadle MA, Tanhauser SM, Dame J B, et al. 2001. The nine-banded armadillo

(Dasypus navemcinctus) is an intermediate host for Sarcocystis neurona. Int J
Parasitol 31(4): 330-335.

Tanhauser SM, Cheadle MA, Massey ET, et a]. 2001. The nine-banded
armadillo (Dasypus navemcinctus) is naturally infected with Sarcocystis neurona.
Int J Parasitol. 31(4): 325-329.

Cheadle MA, Yowell CA, Sellon DC, et a]. 2001. The striped skunk (Mephitis
mephitis) is an intermediate host for Sarcocystis neurona. Int J Parasitol 31(8):
843-849.

Stanek JF, Dubey JP, Oglesbee MJ, et a]. 2002. Life cycle of Sarcocystis neurona
in its natural intermediate host, the raccoon, Procyon lotor. J Parastiol. 88(6):
1151-1158.

Dubey JP, WJA Saville, DS Lindsay, et a]. 2000. Completion of the life cycle of
Sarcocystis neurona. J Parasitol 86(6): 1276-1280.

Simpson CF and Mayhew IG: Evidence for Sarcocystis as the etiologic agent of
equine protozoa] myeloencephalitis. J Protozoa! 27:288-292, 1980.

Rossano MJ, Kaneene J B, Marteniuk JV, eta]: The seroprevalence of antibodies
to Sarcocystis neurona in Michigan equids. Prev Vet Med 48: 113-128, 2001.

10. Kruttlin EA, Rossano MG, Murphy AM, et a]. 2001. The effects of pyrantel

tartrate on Sarcocystis neurona merozoite viability. Vet Therapetics. 2(3): 268-
276.

11. Dubey JP and Lindsay DS. 1998. Isolation in immunodeﬁcient mice of

Sarcocystis neurona from opossum (Didelphis virginiana) feces, and its
differentiation from Sarcocystis falcatula. Int. J. Parasitol. 28: 1311-1312.

12. Murphy AJ, Mansﬁeld LS. 1999. Simpliﬁed technique for isolation, excystation,

and culture of Sarcocystis species from opossums. J Parasitol. 85(5): 979-981.

60

13. Report of the AVMA Panel on Euthanasia. 2000. p 677.

14. Dubey JP. 2001. Migration and development of Sarcocystis neurona in tissues of
mice fed sporocysts from a naturally infected opossum. Vet Parasitol. 95:341-
35 1 .

61

 

 

 

lek 3
IWeek4

Number of Plaques

 

 

 

 

M 0.0001 0.0005 0.001 0.005 0.01
Drug Concentratlon (M)

Figure 4.1. Pyrantel tartrate plyue assay. _Plaque numbers (mean of four wells) three
and four weeks after culture inoculation. (*) indicates a signiﬁcant difference between
plaque numbers in media control and pyrantel tartrate treatment groups. I = one standard
deviation.

62

CHAPTER 5
SARCOCYST IS NE URONA: IN VIT R0 EXPOSURE OF MEROZOITES TO
ACETYLCHOLINE AND ATROPINE
5.1 ABSTRACT:

Sarcocystis neurona, a protozoan parasite, is the etiologic agent of equine
protozoal myeloencephalitis (EPM), a neurological disease of horses. In vitro studies
have shown that pyrantel tartrate, a depolarizing neuromuscular agent that works at
acetylcholine (ACh) receptors, has activity against the merozoite stage of this parasite'.
The following study was designed to test the hypothesis that S. neurona merozoites have
ACh receptors. Using the drug screening assay described by Kruttlin et al.], the activity
of acetylcholine chloride and atropine sulfate at various concentrations was tested against
S. neurona merozoites. Additionally, monoclonal antibodies against the ACh receptor or
protein and appropriate controls were obtained and used in Western blotting in an attempt
to detect this receptor protein in merozoites. Merozoites exposed to ACh showed no
decrease in viability after two weeks at all tested concentrations (0.001 mM to 10 mM)
while those exposed to atropine showed a decrease in viability at the concentration of 10
mM. The ACh receptor or protein was not detected with Western blot.

Preliminary results obtained by the previously described assays indicate that S.
neurona merozoites do not possess acetylcholine receptors and suggest that pyrantel
tartrate acts at a different target site in this parasite; however, the question remains as to
whether this assay is a valid technique for ACh screening in protozoa. Further studies
using gas chromatography and other proven techniques must be performed to support

described results.

63

5.2 INTRODUCTION:

Sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis
(EPM), a neurologic disease of horsesz. The coccidian parasite uses the opossum
(Didelphis virginiana) as a deﬁnitive host and a variety of mammals as intermediate
hosts4’5’6. Sexual replication of S. neurona occurs in the intestine of the deﬁnitive host,
after which infective sporocysts are shed into the environment to be consumed by an
appropriate intermediate or aberrant host°. The likely pathogenesis is that sporocysts
excyst releasing sporozoites the gastrointestinal tract of the horse that penetrate the gut
wall and travel to the central nervous system where they replicate asexually in neurons as
merozoites. The damage resulting from merozoite replication leads to the clinical signs
of EPM7.

Pyrantel tartrate is an anthelmintic compound that possesses nicotine-like
properties, acting similar to acetylcholine (ACh) and by inhibiting acetylcholinesterases.
These properties make the drug a depolarizing neuromuscular-blocking agent in
susceptible parasites. In vitro studies have found pyrantel tartrate to be lethal to S.
neurona merozoites at concentrations greater than 2.5 mM (8.91 X 104 g/ml)’; however,
the mechanism of action of the drug in this parasite is unknown. While the presence of
ACh has not been found in Sarcocystis species speciﬁcally, it has been detected in
several protozoal species (T rypanasama rhadesiense, Paramecium), as well as bacteria,
and primitive organisms such as blue-green algae, yeast, ﬁmgi, tubellaria, nematodes, and
sponges”. Because ACh has been found in a wide variety of organisms and because

pyrantel tartrate, a compound that acts similarly to ACh, causes lethality in merozoites,

we hypothesized that S. neurona merozoites possess ACh receptors. If ACh receptors are

64

present in S. neurona merozoites, pyrantel tartrate would be likely to affect the parasite
by disrupting basic cell functions regulated by ACh, as seen in Giardia lamblia“, or by
disrupting merozoite movement related to the ACh molecule, similar to that of helminth
larvae. In these studies, ACh drug screens against merozoites and testing for the ACh

receptor a protein showed no evidence that S. neurona merozoites have ACh receptors.

65

T

 

5.3 MATERIALS AND METHODS:
5. 31 Preparation of Merozoites:

S. neurona merozoites (MI horse #1) were grown and maintained on low passage
(1-19) equine dermal cells (American Type Culture Collection CCL57, strain NBL-6)
with Dulbecco’s modiﬁed Eagle’s medium (GIBCO, Rockville, MD) supplemented with
6% heat-inactivated fetal bovine serum, penicillin (100 U/ml), amikacin(100p.g/ml), and
amphotericin B (1.25 ug/ml) (solution hereafter referred to as DMEM) as described
previously”. Medium containing asexually replicating stages of the parasite was
removed from 7 heavily infected ﬂasks (~175 ml). This solution was not ﬁltered and,
therefore, contained both the free merozoite stage of the parasite and any free equine
dermal cells containing other asexually replicating stages of the parasite (early and late
schizonts). The solution was placed in a 250 ml ﬂask and centrifuged for 90 minutes at
209 x g. After centrifugation, the supernatant was removed, and the pellet from the ﬂask
resuspended with DMEM and transferred to a 50 ml conical tube. The tube was
centrifuged for 30 minutes at 209 x g. The supematent was removed and 4 ml DMEM
was added back to the pellet. Merozoites from four ~50 u] subsamples of this solution
were counted using a Bright-Line hemacytometer (Hausser Scientiﬁc, Horsham, PA).
The mean and standard deviation of these counts were determined. The merozoite count
of the stock solution was 9.08 : 0.72 x 106 merozoites per ml. A 200 u] aliquot of this
solution was placed in each of eighteen 15 ml conical tubes making the ﬁnal number of

merozoites per tube approximately 5.04 x 105.

66

5.32 Cell Culture Preparation:

Equine dermal cells described above (passage 25) were seeded in equal numbers
and grown to conﬂuency as determined by visual examination using an Olympus CK2
inverted microscope (Olympus Optical Co., Ltd., Tokyo, Japan) in twelve 6-well cell

culture plates (Corning, Inc., Corning, NY).

5. 33 Preparation of Drug:
The activity of acetylcholine chloride and atropine sulfate (Sigma, Inc., St. Louis,

MO) was tested against S. neurona merozoites and all cellular stages of the asexually

 

replicating parasite. A 0.1M solution (1.81X10'2 g/ml) of ACh (FW 181) in DMEM was
ﬁltered through a 0.22 pm ﬁlter to remove contaminants and diluted in DMEM to
concentrations of 10 mM, 1 mM, 0.1 mM, 0.0] mM, and 0.001 mM. A 0.1M solution
(6.76X10'2 g/ml) of sodium tartrate (FW 676) and DMEM was ﬁltered through a 0.22 pm
ﬁlter and diluted as above. For each drug, 1.0 ml of each dilution was added to a 15 m1
conical tube containing merozoites (see above). One ml of DMEM alone was added back
to two additional tubes as a negative control. Additionally, sodium sulfate (Sigma, Inc.,
St. Louis, MO) was tested as a sulfate control at concentrations identical to that of
atropine sulfate. A 5 mM solution of pyrantel tartrate ((Sigma, Inc., St. Louis, MO, FW

356.4) in DMEM was also prepared and ﬁltered as above.

5.34 Experimental Design:
Direct effects of ACh and atropine. Tubes prepared above were incubated at 37°C

in 5% CO2 for 24 hours. After this time, the tubes were centrifuged for 10 minutes at 209

67

x g, the supernatant carefully removed to prevent pellet disruption, and 1.0 ml of DMEM
added back to each tube as a washing step. Tubes were again centrifuged. This washing
procedure was repeated twice to remove any residual drug from the pellet. After the ﬁnal
wash, the supernatant was carefully removed and the pellet gently resuspended in 4 ml of
DMEM. The suspension from each tube was distributed between four wells (1.0 ml per
well) of 6-well cell culture plates with conﬂuent equine dermal cells. An additional 2 m1
of DMEM was added to each well. The plates were incubated at 37°C in 5%CO2 for 24
hours, after which all media was aspirated from all wells. Two ml of ﬂesh DMEM was
added back to each well and aspirated as a washing step. Three ml of DMEM was then
added to maintain the cultures. DMEM in all wells was changed weekly for the duration
of the experiment.

AbilitLof atropine to blocl_< the activity of pyrantel tm One ml of each
atropine solutions of 10 mM, 1 mM, 0.1 mM, and 0.01 mM was added back to separate
15 ml conical tubes. One ml of DMEM was added to a ﬁfth tube as a media control, and
one ml of 5 mM pyrantel tartrate was added to a sixth tube as a drug activity control.
Tubes prepared above were incubated at 37°C in 5% CO2 for one hour. After this time,
the tubes were centrifuged for 10 minutes at 209 x g, the supernatant carefully removed
to prevent pellet disruption. One ml of 5 mM pyrantel tartrate was added to each tube
with the exception of the DMEM control. Tubes were incubated at 37°C in 5% CO2 for

24 hours and then treated as above.

68

3. 35 Plaque Number Determination:

After one week, equine dermal cell monolayers were observed for plaques and the
plaque numbers counted with the inverted microscope described previously (4X
magniﬁcation). Wells were scanned in a uniform pattern from side to side in such a way
that no ﬁelds overlapped and no plaque was counted twice. Plaque numbers for each
ACh and atropine dilution (4 wells per dilution) were compared to numbers ﬁom the
DMEM control (4 wells) using the Dunnett’s t-test (two-sided). Plaque numbers from
each atropine+pyrantel tartrate dilution (4 wells per dilution) were also compared to
numbers from the DMEM control (4 wells) and pyrantel tartrate drug activity control
using the same statistical test. A p-value of 50.05 was used to determine signiﬁcance for
all comparisons. Plates were returned to the incubator after the ﬁrst count, and plaques

from each drug dilution were again counted one week later and analyzed using the same

statistical methods.

5.36 Western blot:

S. neurona merozoites were harvested from equine dermal cell culture,
centrifuged, and the pellet denatured in sample buffer (0.5 M Tris (pH 7.4) with 10%
sodium dodecyl sulfate, 20% glycerol and 5% B-mercaptoethanol) for 5 minutes at 95° C.
Proteins were loaded into wells of a lO-well 4% stacking gel with and separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 11-17%
linear gradient gels. For reference, biotinylated broad range molecular weight size
standard markers were run on each gel. Twenty microliters of AChR positive control

(BC3H1 Lysate) (BD Transduction Laboratories, San Diego, CA), was loaded on the gel

69

as a positive control for Western blotting experiments. Separated proteins were
electrophoretically transferred to polyvinylidene ﬂuoride (PVDF) membranes for 1.5
hours and blocked overnight in blocking buffer (1% bovine serum albumen in 0.5%
Tween-Tris Buffered Saline (TTBS)). Blots were dried and ﬁozen for later use.

For testing of samples, the blots were wetted in TTBS and clamped in a slot blot
apparatus. AChR antibody described above was diluted at 1:200 with blocking buffer
and 650p] placed on each of two wells run with positive control (BC3H1 Lysate) and
each of 4 wells containing blotted proteins from S. neurona merozoites. Serum samples
from S. neurona positive and negative horses were also added to two wells to serve as
transfer controls for the S. neurona merozoite preparation. Detection of two antigens of
approximately 30- and 16-kDa serves as the criterion for a positive test”. Solutions were
left to incubate overnight. Wells were then washed thoroughly with TTBS, and 650p]
Biotin-labeled anti-mouse (7.5 ul/ml BTTBS) was applied to all wells as the secondary
conjugate for 4 hours. Wells were washed again and 650p] of ExtrAvidin® (Sigma, St.
Louis, MO) added to each well for 45 minutes. After removal of ExtrAvidin® and

washing with TTBS, the blot was developed in an aminoethyl carbazole substrate.

70

5.4 RESULTS:
5.41 Plaque numbers:

ACh concentrations of 1 mM and 10 mM had signiﬁcantly decreased plaque
numbers from the DMEM control after one week of incubation (p < 0.05). The ACH
concentration of 10 mM had signiﬁcantly decreased plaque numbers compared to control
after 2 weeks of incubation (p < 0.05) (Table 5.1, Figure 5.1). All atropine dilutions,
with the exception of 10 mM, produced plaque numbers that were not signiﬁcantly
different from the DMEM control after both one and two weeks of growth (Table 5.1,
Figure 5.2). Atropine at the concentration of 10 mM caused a signiﬁcant decrease in
plaque numbers in both week 1 and week 2 (p < 0.05).

Merozoites exposed to 5 mM pyrantel tartrate alone produced zero plaques after
both one and two weeks. Merozoites exposed ﬁrst to atropine sulfate and then to pyrantel
tartrate produced zero plaques at all atropine concentrations. DMEM control for these
plates produced plaque growth similar to that of the DMEM controls for the ACh and

atropine experiments described above.

5.42 Western blot:

The Western blot results showed no evidence of an ACh receptor in S. neurona.
Monoclonal antibodies against the ACh receptor or protein did not produce a detectable
band when incubated with S. neurona merozoite proteins. A 49 kDa band was detected
with the BC3H1 Lysate positive control. The positive blot control produced bands at 30-
and 16-kDa as expected. The negative blot control (S. neurona merozoites incubated

with S. neurona negative horse sera) was negative.

71

 

5.5 DISCUSSION:

Pyrantel acts as an agonist of acetylcholine receptors of nematodes and works by
depolarizing muscle membranes. While it has been shown to be lethal to S. neurona
merozoites, the mechanism of action in this species has yet to be explained. One step in
determining if the drug works by a mechanism similar to that in nematodes is to expose
merozoites to ACh and determine if the effect is similar to that of pyrantel exposure. The
ACh doses used in this study cause paralysis in parasitic helminth worms”; therefore
rendering them unable to counteract host GI parastaltic movements and leading to their
passage from the host GI tract. S. neurona merozoites rely on a circular “burrowing”
movement to penetrate neurons and other cells, and paralysis from ACh or pyrantel
exposure could block their ability to enter cells and, therefore, stop merozoite replication
and plaque growth. Pyrantel pamoate has been found to affect Giardia lamblia
trophozoites by decreasing ﬂagellar beating frequency and by causing severe changes in
the cytoplasm and peripheral vesicles15 . While merozoites do not possess ﬂagella, it is
possible that cytoplasmic or vesicle changes, similar to those seen in Giardia sp., could
occur in merozoites after exposure to pyrantel tartrate.

Exposure to a variety of concentrations of acetylcholine chloride did not decrease
plaque grth or affect S. neurona merozoite replication after two weeks of grth in
this study. If S. neurona merozoites possess ACh receptors, the application of ACh at the
concentrations in this study should theoretically have caused overstimulation of the
receptors and paralysis of the parasite or a similar observable phenomenon. The absence
of a signiﬁcant decrease in plaque growth after two weeks of growth seen with exposure

to ACh may indicate that S. neurona merozoites do not possess ACh receptors and that

72

pyrantel tartrate works at a different target site in this parasite. However, it cannot be
ruled out that the lack of change in viability after ACh exposure could have been a result
of ACh breakdown in solution over time or to parasite recovery prior to entering equine
dermal cells. A chloride control was not deemed necessary for this series of experiments
because the ion is found in DMEM media and has not previously affected S. neurona
viability. Additionally, completion of an experiment exposing Strongyle larvae to ACh
at concentrations used in this study and run parallel with merozoite exposure would have
strengthened the argument that merozoites do not possess the receptor. However, it may
also be possible that concentrations of ACh higher than those that affect parasitic
helminths may be needed to change the viability of S. neurona merozoites. Nevertheless,
in separate laboratory experiments we have found ACh concentrations of up to 100 mM
have been unable to cause a signiﬁcant decrease in S. neurona induced plaque numbers.
In hehninth worms, atropine is an antagonist at ACh receptors and stimulates
motility and contraction”. If S. neurona merozoites have ACh receptors it would be
logical to expect similar changes in motility followed by the inability of the parasite to
penetrate host cells and eventual death of the parasite. If the ACh molecule itself is vital
to merozoite survival and ACh receptors are present on merozoites, the addition of an
antagonist such as atropine could also cause parasite death. The possibility also exists
that AChR are present, but atropine does not affect viability of the parasite because it
cannot bind to the receptor for mechanical reasons due to merozoite anatomy. At the
highest concentration used in this study, 10 mM, atropine sulfate caused a statistically
signiﬁcant decrease in plaque numbers. However, complete absence of plaque growth, as

seen in merozoites exposed to concentrations of pyrantel tartrate greater than 2.5 mM',

73

did not occur. The decrease in plaque numbers does not appear to be a result of exposure
to the sulfate portion of the molecule, for numbers similar to that of the DMEM control
were seen with exposure to sodium sulfate. It cannot be ruled out that the decrease in
plaque numbers seen with atropine exposure may be a result of the drug working at the
ACh receptor; however, because of the lack of response to the ACh molecule itself, it is
most strongly suggested that atropine sulfate is effective against S. neurona merozoites
through an action separate from that of the AchR and that this action needs further
exploration.

If pyrantel tartrate did work at the AChR in S. neurona merozoites, theoretically,
a AChR antagonist like atropine could bind to the receptor and prevent the cascade of
events leading to merozoite death that follows exposure to pyrantel tartrate (or
acetylcholine). However, in order to test this theory, atropine molecule itself must not
affect parasite viability. In this study, atropine sulfate did not decrease viability at any
concentration with the exception of 10 mM. Exposure of merozoites to atropine sulfate
prior to pyrantel tartrate application did not cause a reversal of the lethal effects of the
drug on merozoites with any atropine concentration. The fact that this reversal did not
occur is ﬁirther evidence that ACh receptors are not present on S. neurona merozoites
and that pyrantel tartrate works at a novel drug target in this parasite. The possibility
does exist that merozoites were not exposed to atropine for a long enough period of time
for the drug to bind. Additional experiments using time of atropine exposure as a
variable should be completed before the possibility of using an antagonist to block

pyrantel activity is dismissed.

74

The acetylcholine receptor is a 250kDa pentameric complex of four
transmembrane subunits in a stoichiometery of (1207615. The ACh binding site is
primarily in the or subunit. Monoclonal antibodies against this protein did not produce a
49 kDa band on Western blot prepared with S. neurona merozoite lysate while a band
was seen at this molecular weight with the BC3H1 lysate control. The positive blot
control indicated the blot was prepared adequately. Because the protein of the AChR a
subunit was not detected with Western blot, it is likely that this protein, which acts as the
primary binding site for ACh, is not present in S. neurona merozoites. It is possible that
the monoclonal antibody used in this study, because it was prepared ﬁom the mammalian
AChR, is not compatible with the protozoal AChR or that the protozoal receptor differs
from the mammalian receptor to a degree that the antibody cannot sufﬁciently bind.
Even so, there is little evidence from this study that supports S. neurona merozoites
having acethycholine receptors, and the absence of a detectible AChR or subunit protein
in merozoites on Western blot adds to the theory that the receptor is simply not present in
this stage of the parasite.

Further biochemical and molecular testing is necessary to rule out the presence of
ACh receptors on S. neurona merozoites, as well as to determine the drug target of
pyrantel tartrate in this parasite. This initial study has found no evidence to support the

theory that S. neurona merozoites possess ACh receptors.

75

5.6 LITERATURE CITED:

1.

10.

Kruttlin, E.A., Rossano, M.G., Murphy, A.J., Vrable, R.A., Kaneene, J .B., Schott,
H.C., Mansﬁeld, LS, 2001. The effects of pyrantel tartrate on Sarcocystis
neurona merozoite viability. Veterinary Therapeutics. 2(3), 268-276.

Dubey JP, Davis SW, Speer CA, Bowman DD, de Lahunta A, Granstrom DE,
Topper MJ, Hamir AN, Cummings J F, Suter MM. 1991. Sarcocystis neurona n.
sp. (Protozoa: Apicomplexa), the etiologic agent of equine protozoal
myeloencephalitis. J Parasitol. 77, 212-218.

. Dubey, J .P., Saville, W.J., Lindsay, D.S., Stich, R.W., Stanek, J.F., Speert, C.A.,

Rosenthal, B.M., Njoku, C.J., Kwok, O.C., Shen, S.K., Reed, S.M., 2000.
Completion of the life cycle of Sarcocystis neurona. J. Parasitol. 86, 1276-1280.

Cheadle, M.A., Tanhauser, S.M., Dame, J.B., Sellon, D.C., Hines, M., Ginn, P.E.,
MacKay, R.J., Greiner, E.C., 2001a. The nine-banded armadillo (Dasypus

navemcinctus) is an intermediate host for Sarcocystis neurona. Int. J. Parasitol.
31, 330—335.

Cheadle, M.A., Yowell, C.A., Sellon, D.C., Hines, M., Ginn, P.E., Marsh, A.E.,
MacKay, R.J., Dame, J .B., Greiner, E.C., 2001b. The striped skunk (Mephitis

mephitis) is an intermediate host for Sarcocystis neurona. Int. J. Parasitol. 31,
843-849.

Fenger, C.K., Granstrom, D.E., Langemeier, J .L., Stamper, S., Donahue, J .M,
Patterson, J .S., Gajadhar, A.A., Marteniuk, J .V., Xiaomin, A., Dubey, J .P., 1995.
Identiﬁcation of opossums (Didelphis virginiana) as the putative deﬁnitive host of
Sarcocystis neurona. J Parasitol. 81,199-213.

Simpson, C.F., Mayhew, 1.0., 1980. Evidence for Sarcocystis as the etiologic
agent of equine protozoal myeloencephalitis. J. Protozool. 27, 288-292.

Plumb, DO, 1999. Veterinary Drug Handbook. Iowa State University Press,
Ames, IA

Bulbring, E., Lourie, B.M., Pardoe U., 1949. Presence of acetylcholine in

T typanasama rhadesiense and its absence from Plasmadium gallinaceum. Brit J
Pharmacol 4, 290-294.

Wessler, 1., Kirkpatrick, C.J., Racke, K, 1999. The Cholinergic 'Pitfall':

Acetylcholine, a Universal Cell Molecule in Biological Systems, Including
Humans. Clin Exp Pharmacol Physiol 26(3), 198-205.

76

 

ll.

12.

13.

14.

15.

Campanati, L., Gadelja, A.P., Monteiro-leal, L.H., 2001. Electron and video-light
microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia
lamblia. Exp Parasitol. 97(1), 9-14.

Mansﬁeld, L.S., Schott II HC, Murphy AJ, Rossano MG, Tanhauser SM,
Patterson J S, Nelson K, Ewart SL, Marteniuk JV, Bowman DD, Kaneene J B

2001. Comparison of Sarcocystis neurona isolates derived ﬁnm horse neural
tissue. Vet Parasitol 95, 167-178.

Rossano MG, Mansﬁeld LS, Kaneene JB, Murphy AJ, Browen CM, Schott 1] HC,
Fox JC, 2000. Improvement of Western blot test speciﬁcity for detecting equine
serum antibodies to Sarcocystis neurona. J. Vet. Diag. Invert. 12: 28-32.

Terada, M., Ishii, A.I., Kino, H., Sano, M., 1982. Studies on chemotherapy of
parasitic hehninths (V 11). Effects of various cholinergic agents on the motility of
Angiastranglus cantanensis. Jpn J Pharmacol. 32(4), 633-642.

Schroder, B., Reinhardt-Maelicke, S., Schrattenholz, A., McLane, K.E.,
Kretschmer, A., Conti-Tronconi, B.M., Maelicke, A., 1994. Monoclonal
antibodies PK] and WF6 deﬁne two neighboring ligand binding sites on Torpedo
acetylcholine receptor alpha-polypeptide. J Biol Chem, 269(14), 10407-10416.

77

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Simultaneous Simultaneous
95% 95% Conﬁdence
ACh chloride Conﬁdence Atropine sulfate Interval
Interval
Week 1 Difference Difference
between between means
means
DMEM - - - -
0.001 mM -9.50 -23.62, 4.62 -8.25 -26.36, 11.36
0.01 mM -l4.25 -28.37, -0.13* -10.00 -28.36, 8.36
0.1 mM -1.00 -15.12, 13,13 -7.00 -25.36, 11.36
1 mM -1.75 -15.87, 12.37 -12.00 -30.36, 6.36
10 mM -15.50 -29.62, -1.38* -54.25 -72.61, -35.89*
Week 2
DMEM - -
0.001 mM -34.50 -76.94, 7.94 -104.33 -139.98, -68.69*
0.01 mM -23.75 -66.19, 18.69 -10.67 -46.31, 24.98
0.1 mM -10.75 -53.19, 31.69 -4.67 -40.31, 30.98
1 mM -19.00 -61.44, 23.44 -3.67 -39.31, 31.98
10 mM -l8.75 -61.19, 23.69 -7.67 -43.31, 27.98

 

 

 

Table 5.1. Results of Dunnett’s t-tests comparing each dose group to the DMEM control

at alpha = 0.05. All results are based on n = 4 plates per group, except for atropine

sulfate week 2, where n = 3 plates per group. * Indicates a signiﬁcant difference from the

control at p 5 0.05.

78

 

 

 

 

 

 

 

5?

 

 

Number of Plequee
é

sass

 

 

 

O

DMEM 0.001 0.01 0.1 1 10

Ach Concentration (mM) lWeek 1IWeek 2

Figure 5.1. Ace lcholine l ue assa results. Plaque numbers (mean of four wells :
standard deviation) one and two weeks post-inoculation. Concentrations of 10'°M and
10'3 M were signiﬁcantly lower than the DMEM control after one week of growth;
however, no statistical difference was seen between the media control and treatment
groups at all concentrations after two weeks of growth.

 

79

120

NumberotPlequee
a a S

8

8

 

DMEM 0.001 0.01 0.1 1 10

Atropine concentration (mM) lWeek 1 :11ka 2

Figure 5.2. Atropine plaque assay results. Plaque numbers (mean of four wells :
standard deviation) one and two weeks post-inoculation. No statistical difference
between media control and treatment groups at all concentrations after two weeks of

grth

80

CHAPTER 6

MAJOR FINDINGS AND CONCLUSIONS

From the experiments performed in this study we have reached the following

conclusions:

1.

This study found no evidence that Strangle L1, L2, or L3 larvae ingest or attach to
Sarcocystis neurona sporocysts making it unlikely that a) the larvae transport
sporocysts into the bloodstream of the horse or b) pyrantel tartrate decreases the
incidence of EPM by reducing numbers of an indirect carrier (i.e. Strangle
larvae).

Pyrantel tartrate is 100% lethal to S. neurona merozoites at concentrations greater
than 2.5 mM. This indicates that the drug may work on the parasite directly to
prevent EPM; however, the merozoite stage of the parasite is found in the CNS of
horses, and pyrantel is not absorbed from the GI tract, making exposure of this
stage of the parasite to the drug highly unlikely in viva.

Pyrantel tartrate is lethal to S. neurona sporozoites at the concentration of 5 mM.
This stage of the drug is found in the GI tract and is the stage most likely to be
exposed to pyrantel; however, the concentration of 5 mM is much greater than the
actual drug concentration found in Strongid®-C. The possibility remains that the
drug is effective in preventing EPM because it is lethal to sporozoites. The next
step in proving this theory is to test the drug in a horse model.

Pyrantel tartrate increases the mean survival time of gamma interferon knock-out

mice when given before, during, and after oral S. neurona sporocyst dosing.

81

 

Additionally, oral dosing with the drug changes the infection pattern in these mice
by decreasing the incidence of encephalitis. Again, the potential for disease
prevention is present, but experiments using an equine model are necessary before
any conclusions can be made about the efﬁcacy of Strongid®-C in the prevention
of EPM.

. This study found no evidence that S. neurona merozoites possess acetylcholine
receptors. Further testing is required to determine the drug target of pyrantel

tartrate in S. neurona.

82

 

 

 

     
  
  

  
    

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MICHIGAN STATE UNIV
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3 1293 02504