Page 1 帯広畜産大学学術情報リポジトリOAK:Obihiro university

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Title
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Characterization of Epitopes on an 18 kDa
Piroplasm Surface Protein of Babesia equi
Author(s)
Ali, Shuja, Sugimoto, Chihiro, Kanemaru, Takumi,
Kamada, Masanobu, Onuma, Misao
Citation
The Journal of Protozoology Research, 5(2): 4757
Issue Date
URL
Rights
1995-04
http://ir.obihiro.ac.jp/dspace/handle/10322/225
The Research Center for Protozoan Molecular
Immunology
帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge
J．戸川J♂ZククJ．月eJリブ，イ7−∫7／J995J
C叩γ′fg血◎J9労．T力ど伽∫どd化力Cビ几‘gr♪rJ’roJoz88〝〟oJgc山αrJ汀m〟〃βわgッ
CharaeteriヱationofEpitopesonan18kDa Piroplasm
Surfa亡eProteinof月〟bg5fα叩〟l
SHUJA ALTl′CHIHrROSUGIMOTOl，TAKUMIKANEMARU2，
MASANOBU KAMADA2′MISAO ONUMAl
廟叩眈呵肌闇路卿山地加摘血相肋血町触柚．〃d九九加
仇血r∫如∫耶｝Orロ0∂0，ノ叩d〃8血2且押通蝕瑠rC力′心〟加ピノ叩〟〃伽cf〃g
心∫ロビ血rわ几，乃c力igij29−イ，ノ叩〟〃
Rec亡ivedlnAugⅥS【1995／AccepteJ200し：l（−kr1995
Keywords：B，equL，PlrC・plasm，epltOpe
ARSTRACT
Mor）OCIonalantibodies were produced agalnSt Babesia equlPlrOplasms．
ThreeMoAbsreactjngwithan18kDasurfacemembraneprotein（P18）ofB．
equiinimmunoblotanalysIS，WereuSedtocharacterizetheepltOPeSOnp18．All
thethreeMoAbsrecognizedthesameepitopeonp18asindicatedbycompetitive
ELISA・Negative resultsintwo−Site ELISA suggest absence of repetitive
epltOPeSOnP18・TritonX−114phasepar町10nlngCOnfimedthat18kDaantlgen
is anintegralmembrane protein oflB．equlPlrOPlasms．Asthese MoAbs
identifiedaslngleproteinandshowednocrossreactionwithB．cabalLic・requlne
erythrocyte proteins，these can be a candidate to be usedinlhe differen
diagnosisofmixedequlneplrOPlasrnainfectiorlS．
INTRODUCTION
Equinebabesiosisisoneofthemajorproblemsinhorsetradeespecia11ydue
tothedangerofdiseasetransmissiondufingthemovementofracehorsesfrom
COuntrytO COurltry・TbereisaneedtoimprovetheexistlngSerOloglCalassays
used to detectthe equlneplrOPlasmosis as no slngle reliabletestis avail
Most researchers propose thata combination ofatleast two different assa
Should t光uSed toincrease the diagnostic Teliability of the serodiagnosis of
Babesklinfections（「陀nter and Friedhoff1986；Vhiiand1986；Weiland and
Reiter1988）．
47
SUmCE PROTEIN OFβAβ且∫掴ge〃J
Efforts have been made to demonstrate parasite−SPeCific antlgenS by
generationofmonoclonalamibodies（MoAbs）・AcompetitiveTinl1ibitionenzyme−
1inkedimmunosorbent assay（ELISA）using a MoAb recognizing a
geographicallyconservedepitopehasbeendescribed（Knowlesetal・1991aand
b）．None ofthe antigens diagnostic forEuropeanisolates ofB・equiwere
recognizedbysera＆omneld−infectedhorsesfromBrazil（BoseandHentrich
1994）．Previously，Weidentified siximmunodominant B・equipiroplasm
protejns・Outofthese，Mr18・30and32werefoundtobemembranebound
proteins（Alietal．1993）．Ttisnecessarytodeterminewhethertheseproteins
areinvoIvedinreactionsinvariousserologlCaltestsforequlnebabesiosis・In
thisexperimentweproduced MoAbs agalnStBr equiplrOplasms and fuTther
characterized18kDasurfaceproteinuslngtheseMoAbs・
MATERIALS AND METHOT）S
Pβr（7∫Jreα門dα肋ge叩′琴，α和才わ托∫
Non−SPlenectomizedhorseswereinoculatedwithBabesiaequiTinfectedblood
thathadbeenobtained丘omNationalVtterinary Services Laboratories（United
States DepartmentofAgriculture，Iowa，USA）・Piroplasms ofB・equiwere
purifiedfrominfectedhorseerythrocytesasdescribedbefore（Alietal・1993）・
For prcparation of enzyme−1inkedimmunosortxnt assay antlgen，Purified
plrOPlasms were treatedwith2％Nonidet P−40in phosphate−buffered saline
（PBS，PH7．2）at40Cfor3hrs・Aftercentrifugationat2，000gforlOmin，the
supematant WaS uSed for ELTSA according to the method ofShimizu et al・
（1988）．
SouthAfricanB・equlantlgenuSedintheWesternblotanalysISWaSkindly
supplied by Dr・E・Zweygarth，Onderstepoort VeterinaryInstitute，Onder−
stepoort，South Africa・Dr・V・T Zablotsky of AllRussiaInstitute of
Experimental＼屯terinaryMedicine（VTEV），Moscow，Russiahaskindlyprovided
theRussianB・equlantlgenuSedinthisexperirnenL
凸甲∬血加明仲川阻止肋山肌蘭側転
Six weeks old BALB／cmice were subcutaneouslyimmunizedwithlOO Llg
proteinP．1mi of B．equipiroplasms emulsined with the same volurne of
Freund’s complete adjuvant，rmeyWereboostedwiththesameamountofthe
antlgentWOWeeksa丘erthefirst叫eCtionr ′mespleenwasremovedthreedays
afterthesecondbooster．Spleencells（1xlO8cells）weTefusedwithlx107
myeloma cells（P3Ul）in the presence of ploye（hylene glycol（PEG1500，
BoehringerMannl1eim，GmbH，Germany）・HybridcellswereselectedbyHAT
medium（hypoxanthineaminopterine−thymidine）・Hybridomasupematantswere
screenedbyimmunoblotting・lneantibody−prOducinghybrjdomaswerecloned
48
SURFACE PROTEIN OF BABESIA EQU［
twicebythelimitlngdilutionmethodina96we11plasticmicroplate・
To determine the class，Subclass andlight chain type ofthe MoAbls，
ammoniumsulphatepreclpltatedandproteinGaffinitypurifiedMoAb，s were
examinedbyaMoAbisotypingkit（AmershamIntemationalplc．UK）．
P〟r折CdJわ〝α〃d加β血〆ゆ〃げ〟βA如
MoAbs were purified from hybridomas grownin serum−free medium and
fromasciticfluidsbyprecIPltationwith50％ammoniumsulphateandproteinG
afnnitychromatographyusingMAbTrapGIIkit（PhamlaCjaLKB，Sweden）．
Forbiotinylation ofMoAbs，Sulfosuccinimidobiotin（Rockford，Illinois，USA）
WaSuSedtocovalentlybindbiotintopurifiedMoAbs・Onemlofthepurified
MoAbs（1mg／ml）wasmixedataratioof250ugofester／mgofantibodyand
incubatedatroomtemperatureR）r4hrs・Incubationreactionwasstoppedby
theaddiLionof20匹loflMNH4CIper250LLgOfester・ThemixttlreWaS
dialyzedagainstPBSandO・1％thimersalwasaddedaspreservative．Labelled
MoAbswerestoredat40Ctilluse・ThespeCiflcityandoptlmalconcentrationof
the biotinylated MoAbs was determined by a standard ELIS＾，aS descrikd
txlow，uSlng Br equL plrOPlasm antlgen and avidin−COnJugatedperOXidase
（ZymedLaboratories，CA，USA）．
PJ∽∫叩dr血わ〝占ブ升加乃ズーノノヰ
PiroplasmproteinsweI℃SubjectedtoTritonX−‖4phasepa−1itionaccording
tothemethodofBordier（1981）．Purifiedpiroplasms weretreaLed withTrisT
buffer saline（TBS）containingl％（v／v）Triton X−114 andlmM
Phenylmethylsulfonylfluoride（PMSF）onice for30min．Thelysate was
Centrifugedforlhra（100，000gandthepe11etwasdiscarded・7l−CSuPematant
WaSCarefu11ylayered overanequalvolumeofa6％（w／v）sucrose cushionin
TBScontainlnglmMPMSF・Af［erincubationat300Cfor15min，thesample
WaSCenthfugedatl，000g for15minatroomtempcrature・llletOPaqueOuS
Phaseandbottomdetergentphasewerecollected・Eachfrac−ionwaspreclpltated
With4volumesofacetoncandcentrifugedat6，000gforlOminat4QC．rIne
Pelletswerestoredat−80OC．
∫od∼〟椚血decγJ∫〟（わJ叩（ゆ〟Cり血れJdegeJeJβCJ叩んけ化．TJ∫α〝df′WZ〟〃∂hbJ上座
Sodiumdodecylsulfate−POlyacrylamidegelcIcctrophoresjs（SDS−PAGE）and
immunoblot were carried out as descrlbed（＾liet al．1993）．Briefly，One−
dimensionalSDS−PAGE was perbrmed uslng aI2・5％polyacrylamide gel
according to the method described by Laemmli（1970）．Samples were
SOlubilizedinTris−HClbuffer（0・0625M，pH6．8）containing2％（w／v）SDSand
5％（w／v）2−merCaptOethan01（Laemmlj1970）atlOO OC for2min．Af（er
SeParation by SDS−PAGE・the plrOplasm proteins were electrophoretically
transferredtopolyvinylidenedifluoride（PVDF）membrane sheets（rmmobilon
49
SURFACE PROTEIN OF BABESL4 EQtH
tranSftr membranes；Mjllipore，USA）according to the inmunoblotting
technique of Dunn（1986）・The sheets were blocked forlhr at room
temperatureinO・01MPBS（PH7・2），0・05％（V／v）Tween−20（PBST）containing
5％skimmilk（SM）．TTleSeWereWaShedthreetimeswithPBSTandincubated
forlhrat room temperaturewith MoAbs withl％SM・ne Sheets were
washedandincubatedforlhratroomtemperaturewithgoatanti−mOuSeIgG
perOXidaseconjugate（JacksonInc・，USA）dilutedl：1，000inPBSTwithl％SM・
Afterwashing，thesheetsweretreatedwithafreshlypreparedsubstrate
containingl・3 mM diaminobenzidine tetrahydrochloride（DAB；Nakarai
Chemicals，Ltd，Kyoto，Japan），1・3mM CoCl3and O・02％（v／v）hydrogen
PerOXideinPBST
助平me肋たどdJ椚椚〟〃0∫Orわe乃Jd∫∫αγ
ELISA was perfomled according to the method described previou
（ShiTr．izuetal．1988）withaminormodification・Briefly，Optimalconcentrations
ofantlgen，antibodyandenzymeCOTUugateWeredeterminedbycheckerboa
titration．Serialtwofolddilutionsof36けgpermlpiroplasmproteins／0・1mlin
O．05M caねonate／bicarbonate buffer（pH9．6）per we11，Were teSted through
ELISA，using serialtwo−foLd dilutions ofenzyme conjugate（froml：500to
l：2，000），Standardpositiveandnegative（Alietal・1993）horsesera（froml：20
tol：1，280）andunlaklledMoAbs（froml．25pg／mlto50ug／ml）・For color
development，0．1miof substrate（0・2rnM azino−di−［3−ethylbenzthiazoline］
sulfonicacid）wasused．Theplateswerereadat405nminaspectrophotometer
（CoronaElectric，rIbkyo，Japan）．TheendpointofELISAwasestimatedfrom
thedose−reSPOnSeCurVeandtheantibodytiterwasexpressedasthereclpr
thehighestdilutionofMoAbsshowlnganabsorbanceoverO・1・
Co〝p抽血抽d如d∫∫町わが伸ee〃〟0舶∫
rrYle aSSay WaS Pe血rmed according to the method described previously
（Zavalaetal．1983）．rnleWe11sofmicroplatewerecoatedwithO・1mlofB・
equipiroplasmlysate（protein concentration18トg／ml）in O・O5M carbonate，
bicarbonatebuffer（pH9．6）andkeptovemightat4OC・Non・SPeCificsiteswere
blockedwithO．5％SMinPBSTatroomtemperatureforlhL
Asanantjbodycompetitor，0・lmlofserial2−folddilutions ofunlabelled
purifiedmonoclonalantibodies（0−20pg）wereadded・Afterincubationatroom
temperaturebrlhr・dleplateswerewashedwithPBSTandO・1mlofbiotin−
1abe11edmonoclonalantibodies（20いgrgG／ml）wereadded・Followingincuba−
tionatroomtemperatureforlhrandwashingwithPBST・0・1mlofavidin−
peroxidase（ZymedLaboratories，CA，USA）dilutedl：1，OOOinO・2％SM−PBST
was addedandtheplates werejncubatedat roomtemperatureforlhr・The
wellswithoutB・equlantJgenfollowedbybiotin−1abelledantibodywereusedas
50
SURFACE PROTEIN OF BABESIA EeUI
negativecontroIs．Forcolordevelopment，0．1mlofsubstrate（0．2mMazin0−
di−［3qethylbenzthiazoline］sulphonicacid）wasused．TTleabsorbanceofthewells
WaSreadat405nminaspectrophotometer（CoronaElectric，rIbkyo，Japan）．
rwの血e一肌
Two−Site ELISA wasperformed by the method descri反d previously by
ZavalaetalL（1983）andBu血tetal．（1984）．TYlemicrophtewascoatedwith
O・1miofpurified unlabelled MoAb C2，C5and D3（20llghnl）and】e托
OVemightat40C・AfterwashingwidlPBST，nOn−SpeCificbindingsiteswere
blocked as described above．Plates were washedand O．1mlof seria12＿fbld
dilutionsofl町ghnlsolubilizedmerozoiteantigenwereadded．Afterincubation
atroomtemperatllreforlhr，0・1mlofbiotin−1abelledl−OmOlogousMoAb（20
Pghnl）wasadded・Plateswereincubatedatroomtemperatureforlhr，and
PrOCeSSedasdescribedabove・Antibody−COatedwe11sincubatedwithoutantlgen
followedbybiotin−1at光11edantibodywereusedasnegativecontroIs．
罫EStJI∬S
（I厄r仙、Jt・rh〟ん・〃一再毎川棚■・−・J■一…Jl川曲■・．／it・、†
7rnmunoblotlingwascarriedoutforthedeterminationofthespecincityof
MoÅbsandfortheidentincationofthemoleculesrecognizedbytheseMoAbs．
A totalof54clones secretlngantibodies agalnSt B・equlplrOPlasms were
Obtainedin5fusionexperiments．SupematantSOfthree（i．e．C2，C5andD3）out
Of54hybridomas，thatreactedwiththe18kDaprotein（p18）ofB．equiin
imm皿Oblotting，WereSelectedforfurtherstudies．Noneofthethree MoAbs
reacted either withBtcabaZlior equlne erythrocyte proteinesin Westemb）
analysis（Fig・1＝Lanes3to5）・Thesemonoclonalantibodiesalsorecognizedan
18kDaproteinintheWestemblotofaB・equistraincollectedfromRussia（Fig．
2：Laneslto3）・OtherdominantproteinsoftheRussianstrainwereMr40，5，
28and16・5kDa・However，theseMoAbsdidnotrecognlZep18inWestemblot
OfB．equistraincollectedfromSou血Afhca（Fig．2：Lane4）．Determinationof
immunoglobulinisotypeshowedthatallofthethreemonoclonalantibodieswere
IgGl（K）．
n巾…ト＼−／／JJ，ノ刷り，…、J吊＝〃イ／小り由†J…べ〃ゞ川
rrYleTritonX−114phasepartitionme【hodwasusedtodeterminewhether18
kDa antlgenis anintegralmembrane prOtein・When仇e extracted materials
WereelectroblottedandreactedwithMoAbs，allofwhichrecognizeda18kDa
moleculeoftheB・equlplrOPlasm，thismoleculewaspartitionedexclusivelyinto
thedetergentphase（Fig，1：LaJle2）．ThishnmunoblotwasprotN：dwithMoAb
C5andwasrepresentativeofal1thethreeMoAbsusedinthisstudy．
SURFACE PROTEIN OF BABESL4 EQ乙〝
1
ヱ
3
4
5
MWM
ぎポ ■−■1（I‘
一柳
1
1
■4，．5
■32．5
㍍く
■■■27．5
‡Fく‡−18．き
一冊−− 一−
Fig．1Recognitibno往純血BbbesiaequEpiroplasmsurfaceproteinbyMoAbs．B．equian岬．
Cahm piroplasmproteins weresubjected toTriton X−114phasepartitionlng，Separa（edbY
electrophoresison12．5％SDS−POlyacrylamideandtransferredtoPVDFmembranesheet．The
b10tWaSprObedwithMoAbC5andisrepresentativeofallth巳threeMoAbsusedinthisstudy．
Lanesl．B．equL wholepiroplasmantlgeTl；2．B．equEdetergentphase；3．B，Caballiwh
Piroplasm
．
Fig．2ReactionofMoAbswith
且q画Wholeplmplasm
mtlgCnSObtalnedfTOmSou山
＾fricaandRussia．Piroplasm
PrOteinswereseparatedby
electrophoresison12，5％SDS−
l氾1yac巧Iamidegelandwe托
transferred to PVDF membrane
Sheet．Russianβ．甲山踊bgcn
PrObedwi（hMoAbsC2匝e
l），C5（Lane2）andD3（1ane3）；
La肥4．Sou【hA飢caれanいgen
pmbedwithC2．Positionof18
kDaproteinisshownonthe
le丘（MWMa∫kerxl（P）．
ー
8
nr
52
SURFÅCE PROTEIN OFβAβE封A geこ〃
0
5
10 15 ヱ0 ヱ5
Ab（帽／m】）
Fig・3DeteminationofsensitiYltyOfbiodn−labelledMoAbs．
Se血12一ぬ1ddiludonsdbiodn−1akll由MoAbs（2町l如11）
Wereincubatedinmicr9Platewe11scoatedwithsolubilized
β・g叩plmplasmanugen・Anlihdiesbundtotbeanbgen−
absort光dwellweredetectedbyELISA．We11swithoutantlgen
WereuSedasneg如iveconmls．Al】Yalucs即℃themeanor
tnplicated5amPles．一口ーC2；p◆一C5rro−D3；
一A−COnt和1（withoutamdgen）
ぬ扉軸わげ地由一Jα占g鮎d〟仇亜∫
Checkerbardlitrationsl10Wedtha＝地0．1mlofB．equipiroplasmantlgen
（18pgh11）perWellcouldproduce a optimum reactioninEuSA．nerefore，
thisamountofthe antlgenWaSuSedforthedetemlinationofreactivltyOfthe
biotinlat地dMoAbs・Fromtheresults（ShoninFig・3），20pg血1biotin−
1abeledMoAbwasusedinthefollowlngteSt，SlnCethisamountofeachMoAb
COuldgivetheoptlmuml℃aCtion．Thespecificityofthebiotin−1abelledMoÅbs
WaSdemonstratedbythelackofreactivltylnnegativecontroIwells．
C（フ〝甲gJfrルビ鳳ばA∂ピタwge〃〟∂舶∫
CompetitiveELISAwasperfbrmedtodeterminewhetheranyoftheMoAbs
recognizedthe same epitopes on the18kDa protein，MoAbs at different
COnCentration（0−20pg）wereusedincompetitionwiththeheterologousbiotin−
1abelledMoAb（20pghTll）．ResultswereexpressedasperCenlageofinllibition
Ofbinding ofeach MoAb by the others．Asshownin Fig．4，eaCh MoAb
inl1ibited binding ofhomologous as wellas heterologous biotinylated MoAb．
MoAb C5andD3producedareductionof83・3％and84・6％inthebindingof
53
SURFACE PROTEIN OF BABESLA EQtH
爪V
只V
∧U
‘U
一八り
uO叩一叫q芸ul一∈む‖こむd
V
∧U nV
史爪U
／b
4’一
∈○叫l叫貞‘亡︼一亡面白Lむd
0
5 10 15
ヱ0
25
ComT）etitor（ug／ml）
O
鑓U
∧U
O
／hV
O
∈○叫l叫q叫‘亡︻
几U
′〇
．q
l亡むUJむら
∧U
4
已うで叫一てー∈−−uむ・、こむh
つ‘
几U
’一
0
5 10 15 20 25
（I
5 10 15 ヱ0 25
Competitor（l鳩／m））
Competitor（ug／mll
Fig．4Compc（i（iveELISAassayt光tWeenMoAbs・Unlat光IedMoAbsascompetitors（0−20pg）
Wereincuba（edinmicroplate
．
COmpetewithbiotinlabelledMoAbC5andthejrTeCiprocalassay・（b）MoAbC2［ocompetewith
bio【in−1at杷1edMoAbD3andtheirreciprocalassay．（c）MoAbC5tocompetewi［hbio［in−1abeled
D3aLldtheirreciprocalassay．（d）HomoIogousun］abeledMoAbasacompetitorofbiotin−labeled
MoAb．AllvaluesglVenarethemeanoftTiplicatedsarnp］es・
54
SURFACE PROTEIN OF BABESL4 EQUI
biotinlabe11edC2at20llghllconcentration．Thereciprocal甫gureswere32．6％
and37・8％for biotynilated C5and D3，reSpeCtively．TYLeperCentage Of
inhibitionofbiotinlabeuedD3byC5was55・7％at20トLg／mllevel．ne頁gure
inrecIPrOCalassaywas69・0％・rmeinhibitoryefftctsincompetitionbindingof
homologousantibodieswere付om65to75％．
0
10
5
15
ユO
Ag（帽／ml）
Fig・5Single−an（ibodytwo−SiteELISAforde［eCtlngrepeatedepi［opewi［hinthe18kDaprotein
OfB・equLmerOZOite・Piroplasmsolubilizedantigenswereincubatedinmicroplatewellscoated
withoneofthcMoAbs（C2，C5，D3）specific［o18kDaprotein・Afterwashing，homologous
bio血−1abeledMoAbwasusedtodetecttheantlgerlCaPturedonthemicroplaleSurface．Allvalues
glVenarethemeanoftriplicatedsamples．一口ーC2；−◆rC5；−○−D3
n仰−∫J紀比J朗
Two−Site ELISA was p已rfDrmed to detefmine the presence of repetltive
epltOpeShlthe18kDaproteinofB．equi，MoAbsirnmobilizedonmicroplate
WellswereincubatedwithB・equimerozoit＄1ysateandthenhomologousbiotin−
labelled MoAbwasaddedtotheplate．As showninFigure5，biotin−1at光11ed
MoAbsC2，C5andl）3didnotshowanypositivereactions．
DISCUSSION
ExposedepltOPeSOnthesporozoiteandmerozojLemembranesoroninfected
erythrocyte membranes arelikely to be the targets for a protectiveimmune
responseinBabesiainfbclions（McEIwain etal．1987）．rTlle COntamina【ion of
55
SURFACE PROTEIN OF BABESL4 EQU［
babesjalproteinswithlargeamountsofhostcellproductsisaproblemforthe
isolationofpurebat”SialantlgenS・TTleidentificationofsuchantlgenSisrequired
iftheirefficacyasimmunogenhastobeinvestlgated・GenerationofMoAbsthat
bindtoTrIerOZOitesurfaceproteinsisawaywhichleadstotheidentificatjonof
protectiveimmunc．gen・
The aims ofthe present study were to develop monoclonalantibodies
（MoAbs）againstthepreviouslyidentifiedirrlmunOdominantproteinsofB・equE
（Aliet al．1993）and to use these MoAbsin different serologicalassays and
cDNAscreenlngeXperiments・Asafirststep，apanelofMoAbswasdeveloped
agalnStB・equlplrOPlasms・Three MoAbs reactlng Withan18kDa surface
membrane protein（p18）of B・equlinimmunoblot analysis were used to
Characterizethcepl（OPeSOnp18・
rmeresultsofcompetitiveELISAindicatethatMoAbsrecognizedthesarne
epltOpeOIlthep18moleculeaseachMoAbinhibitedthebindingofhomoIogous
aswellasheterologousbiotinylatedMoAblCornparativelyhigherreductionin
bindingofbiotin−1at妃1ledC2irLCOmpeliLjonwithC5andD3ininl1ibitionassay
maytxduetothedifferencesinlheiraffinityfortheepltOPeOnLl−ep18・
In thi5eXPerhent，Triton X−114phasepartitionlngOfB・eqLilp．rOPlasms
proteinsconfirmedthat18kDaantlgenisaninLegralmembraneproteinofBr
equlplrOplasms・NoneofthethreeMoAbsmentionedinthjsstudyreactedeither
withB．cabaL［iorwithequmeerythrocyleprOteins・TheseMoAbsrecognizedthe
p18intheRussianstrainofB・equibutnotinthestrainobtainedfromSouth
Africa・This suggeststhatthereareantlgenic diffcrcncesbetweentheB・equL
strainsisolated from dirrerent parts ofthe world．Bose and Hentnch（1994），
demorlStratedthatnone ofthe antlgCnS diagnostic forEuropeanisolates ofB・
equiwere recognizcd by sera from field−infected horses rrom Brazil・Singlc
PrOteinidcntification and absence of crossreaction with B・Caba［lior equlne
erythrocyteproteinsrecommendlheuseoftheseMoAbsindiffcrentialdiagnosis
of mixed equine piroplasmosis infections.
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