Table Of ContentDEVELOPMENT OF A RECOMBINANT PROTEIN FOR THE IDENTIFICATION
OFSARCOCYSTFSNEURONA INFECTIONS IN HORSES
By
SIOBHAN PATRICIAELLISON
ADISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OFTHE UNIVERSITYOF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FORTHE DEGREE OF
DOCTOROF PHILOSOPHY
UNIVERSITY OF FLORIDA
2001
DEDICTEDTO VAUNTED VAMP
ACKNOWLEDGMENTS
I wish to thank the peoplewho contributedto thework described inthis
dissertation and whose help was invaluable First, Dr. Dameforboth theopportunityto
do thework in his laboratory and his advice. I thank Dr. Nancy Denslowfor scientific
guidanceas well as servingas arole model. I thank Dr. Ellis Greinerfor serving asa
mentorand providingendlessguidance. I thank Dr. Jack Gaskin for his scientificaswell
asclinical insight. 1 thank Dr Rob MacKayforall his support.
1 wishto acknowledgethe help and experienceofMarjorieChow, LindaGreen,
Jamie Kelso, Sevita Shanker, Bill Farmarie, Levi Omara, and Scott Whittaker. They each
had significant and unique input into thedirection ofmy project aswell as spendingextra
time so mywork wasexpedited.
A significant contributionto this work wasthroughthegenerosityofDr.
Antoinette Marsh Shedonated important and precious materials, without which the
project would have faltered. Shealso servesasa role model and I thank herforher
advice. I thank Dr. Larry Ritzhaupt forthe European connection resultingin needed
samples. 1 wish tothank Dr. Mike Black, Dr. David Granstrom, Dr. J. Boothroyd, Dr. J.
Dubey, Dr. D. Howe, and Dr. T. Jaekel foranswering questionsby phone oremail and
reviewing photographs.
Finally, 1 wish to thank Bill Killeen foremotional support and keepingthe home
firesburning while I pursued a dream.
TABLE OF CONTENTS
ACKNOWLEDGMENTS
ii
ABSTRACT v
INTRODUCTION
1
Equine Protozoal Myeloencephalitis 1
Sarcocystis 1
Sarcocystisneurona Infection in Horses 2
Sarcocystisneurona 3
Equine Responseto Sarcocystosis 5
Parasite Antigens 8
Diagnostic Antigensinthe Horse 8
Hypothesis 10
Summary 11
GROWTH, PRODUCTION, AND PURIFICATION OF S NEURONA
MEROZOITES 12
Introduction 12
Materialsand Methods 13
In Vitro Cultureof S. neuronaMerozoitsand Host Cell Lines 13
Preparation ofSporocystsforIn Vitro Culture 14
lonophore A23187-stimulated Release and Parasite Purification 16
MolecularAnalysis ofParasiteCultures 17
Mono Specific Polyclonal Rabbit Antiseraand Monoclonal Antibodies 17
Staining 18
Results 19
Discussion 24
IDENTIFICATION OF SPECIES SPECICIC PROTEINS OFS. NEURONA
THAT HAVE DIAGNOSTIC VALUE 32
Introduction 32
Materialsand methods 40
In Vitro CultureofParasites, Antigen Preparation 40
Antibody Production 41
Western Blotting 41
Periodateand PhospholipaseC Treatment ofS. neurona 42
Elisa Protocols 43
1FA 44
Immunoprecipitation and 2D Electrophoresis 44
Results 45
Discussion 52
CLONING AND EXPRESSION IN eSCHERICHIA COL1OF cDNAs
ENCODING SURFACE ANTIGENS ofSARCOCYSTISNEURONA 73
Introduction 73
Materials and Mthods 74
Growth and Preparation ofS. neurona 74
LibraryConstruction 75
Library Screening 77
Expression ofFusion Protein 79
Preparation ofrSnMSA1 Protein 80
Production ofPolyclonal Anti-serain Rabbitsand Mice 80
Proteolytic Removal ofthe HisTagfromthe Rusion Protein 81
Southern andNorthernBlot Hybridizations 82
Nucleic Acid Blottingand Analysis 83
Immunoblot Analysis 84
Production ofPolyclonal Anti-serain Mice 84
Capture ELISAwasused to Quantitate S. neurona Antigens 84
DNA Sequencing 85
Protein Analysis 85
Results 85
Discussion 88
CONCLUSIONS 98
LIST OF REFERENCES 107
BIOGRAPHICAL SKETCH 115
Abstract ofDissertation Presented to the Graduate School ofthe UniversityofFloridain
Partial Fulfillment ofthe Requirementsforthe DegreeofDoctorofPhilosophy
DEVELOPMENT OF A RECOMBINANT PROTEIN FORTHE IDENTIFICATION
OFSARCOCYSTISNEURONA INFECTIONS IN HORSES
By
Siobhan PatriciaEllison
May2001
Chairman: John Dame Ph.D.
Major Department: Veterinary Medicine
ABSTRACT
Equine protozoal myeloencephalitisisan infectious parasitic infection ofhorses
caused bySurcocystisneuronathat results in devastating neurologic disease. Factors
surroundingthe pathogenesisofinfection inthe horseareunclear, but it iscurrently
believed thehorse isan aberrant host. Thediagnosisofequine protozoal
myeloencephalitis hasbeen based on serum and cerebral spinal fluid antibodiesto
Sarcocystisneuronaderived antigensdemonstrated byimmunoblot. Theantigens used in
thediagnosisofequine protozoal myeloencephalitis have not been demonstrated to be
species specificordiagnostic.
Sarcocystisneuronamerozoitesweregrownin cultured cellsand anovel method
forparasite release wasdeveloped. Parasitederived antigenswere separated by
electrophoresisand used in immunoassaysto identifyantigensrecognized byantibodies
from infected horses, but not recognized bythosefrom uninfected horses. A major
surfaceantigen was characterized. The nucleotide sequence ofagene encodinga major
surface antigen ofS. neuronawascloned and sequenced. Oligonucleotideswere
developed to amplifyan RT-PCR product fromSarcocystisneuronaRNA.
A cDNA librarywas prepared from the mRNA ofA', neuronaand cloned into an
expression vector A cDNA cloneencodinga putative surfaceantigen was identified
among 96 clonescharacterized by preparing sequencetags. The sequencetag encoded a
peptidewith significant sequence similarityto a segment ofthemajorsurface antigen ofS.
maris. ThecDNA librarywas screened withthe partial nucleotide sequence asa probe,
and a clonecontainingthe sequenceofthefull coding region ofSnMSA-1 wasobtained.
Southern and Northern analysisofthe SnMSA-1 gene revealed a single copy in the
genomic DNA ofS. neuronacultured merozoitesand an abundance ofmRNAin the
merozoite. A recombinant form was produced, rSnMSA-1, and purified usingan affinity
column. Monoclonal antibodiesraised against merozoites recognized the recombinant
protein overexpressed in pET 14b. Studiesofthe nativeand recombinant protein
confirmed that theantigen wasa 29 kDa protein with a pi of7,3 located on the surfaceof
the parasite. Immunoblotted rSnMSA-1 confirmed thepresenceofantibodies to this
antigen in serum and cerebrospinal fluidsfrom horseswith clinical EPM. Antibodies
raised against rSNMSA-1 wereuseful forthe quantitation ofantigen in preparative
samples and in the identification ofparasites in clinical tissues.
CHAPTER
1
INTRODUCTION
Equine Protozoal Myeloencephalitis
Equineprotozoal myeloencephalitis(EPM) isaparasitic infectionofhorsescaused
bySarcocystisneuronathat results in devastatingneurologic disease. All sectorsofthe
equinecommunity are influenced with what isperceived asanepidemic ofEPM. The
apprehension ofthehorse industryisdueto serological datathat estimatetheprevalence
ofinfection in theequine population isat least fiftypercent.' It isunclearthat fifty per
cent ofthe animals havebeen exposed toS. neurona. Lowtest specificitywiththe
commercial immunoblot fosters anxietyand misunderstanding ofthisdisease. Thefirst
rational step tothedevelopment ofa sero-diagnostictest istheidentification and
characterization ofspecies-specificantigensthat inducean immuneresponse inthehorse.
Current moleculartechniques maketheidentification and production ofrecombinant
protein antigenspossible. These recombinant antigenswill formthebasis ofa specifictest
EPM
for
Sarcocystis
Coccidia are obligatory intracellularapicomplexan protozoawhoselifecycles
notablyconsist ofmerogony, gametogony, and sporogony.2The life cycleiscompleted in
eitheroneortwo hosts Ifintwo hosts, theasexual phase iscompleted intheintermediate
host and the sexual phase in thedefinitivehost. Thetaxonomiccriteriaused in identifying
coccidia togenus havebeen cyst and oocyst morphologyand life cycle. Uncertaintywith
thetraditional criteria used to distinguish species arisesbecause some morphological
featuresare subject to changeduringthe development oftheparasiteandthecomplete life
cyclesofmany organisms currently classified ascoccidiaare still unknown. The recent
development ofmolecular methodsto recognizevarious speciesofcoccidiaprovides
powerful toolsto reevaluatethe acceptedtaxonomy. Few moleculardataareavailablefor
the named 189 species however, and littleis known at present about thegenetic
divergence among speciesofSarcocystis. In the natural hosts, sarcocystosisis usually
benign causing littlepathologyin eitherhost. However, disease hasbeen recognized with
several Sarcocystis species, generally involvingmerogonyoftheparasite in the
intermediate host.
SarcocystisNeurona Infection in Horses
Equine sarcocystosiscausedbyS. neuronacauses a neurologicdisease ofhorsesin
the Americas, found only in the Western hemisphere.' The most typical presentation isa
lame horse, but animals may alternativelypresent with primarybrain diseaseora
combination. Thedisease has no defining signsbecausetheparasitecan inhabit anyarea
ofthe central nervous system (CNS) ofthe horse. Clinically localizingCNS signsarea
direct reflection ofthe siteofpathology and arequite specific. Sarcocystisneurona
infectionscan rangefrom subtleto severe involvingonlythebrain, spinal cord, orboth.
The disease isusually progressive. A prototypical lesion is a mixed inflammatory cell
responsewith neuronal destruction and protozoa mayormay not befound in the lesion.
'
Thetypical spinal cord lesionswerereported inhorsesin Kentuckyby Rooney and co-
workersin 1964 and recognized intheCNS ofBrazilian horsesby Macruzet al. at about
u
the sametime. ' Protozoawerefirst identified in EPM lesions in 1974.6"8 Theetiology
wasoriginallythought to be Toxoplasmagondii, however, with improved diagnosticsthe
parasitewasrecognized asaSarcocystisspecies.8"12 Theorganismthat causesEPM in
horseswas namedSarcocystisneuronain 1991 based on structural similarityto schizonts
of SarcocystisandFrenkelia.
Sarcocystisneurona
Dubeyet al. notedthat merozoitesofS. neuronalack rhoptries, lacka
parasitophorousvacuoleinthetissuesexamined, and dividebyendopolygeny. The
absenceofrhoptriesin merozoitesdistinguishesS. neurona, otherspeciesofSarcocystis,
andFrenkeliafrom any othercyst-formingcoccidian parasitefound in domestic animals.
In addition to structural data, Dubeyet al. reported antigenicdifferences amongS.
neuronaand T. gondii, Neosporacaninum, Hammondiahammondi, and Caryospora
bigettetica.b Dubeyet al. reported positivecross-reactivityofS. neuronamerozoiteswith
S. cruzi antiserum interpretingthesedataasindicativeofa closerelationship with other
species ofSarcocystis.'' Thecross-reactivenature ofantigensofSarcocystishasbeen
reported and can not serve asatool to differentiate specieswithinthegenus. Sarcocystis
neuronaiscloselyrelatedtoS.falcatula andS. muris based on rRNA sequenceanalysis
and shares cross-reactiveantigenswith otherSarocystissp. 7 Thehistorical criterionfor
differentiation ofspeciesofSarcocystishasbeenthe structureofthesarcocystwall inthe
intermediatehost. Inthe caseofS. neurona, no cystshavebeen identified and the
