' Title Author(s) Citation Issue Date URL Rights ' Anemia in Experimental African Trypanosomiasis Ogbadoyi, Emmanuel O., Ukoha, Agwu I., Kyewalabe, Elizabeth K. The Journal of Protozoology Research, 9(2): 5563 1999-04 http://ir.obihiro.ac.jp/dspace/handle/10322/317 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.Prot()ZOOI.Rcs.,9,55−6ユ(1朔) C叩γrfg加⑥Jタワ夕.mど月ど∫gd′dC〝眈rβrPr〃加㈹柁〟bJど川血rJmm〟〃ロわgγ AnemiainExperimentalAfricaLnTiypaJ10SOmiasis EMMANUEL O.OGBADOYIl),AGWUI.UKOHA2)ANr) ELIZAl∃ETH K.KYEWALA13E3) /舶那加鋸肋止血椚叫′Z〟γれ好打d】川〟5上月fg′ⅣJgビrれ2D叩招rfmど”′げ別oJ咽fc〟J5c椚−Cど5′ Fビ加r〟川′∼加r5和好花eJ川OJpgy′旭〃闇′Nfgどγれヱ翫pf亡〟JDf5ビ〟5ビS尺ど5ビβrr7−Cピ〃恒ノ P.0.月8エ7コ7占g′N由れZ爪肌あ㍍ Rccej−−edlM町Ch】9りウノAl℃ePledlユAprilL9粥 Kcywords:丁わp【1〃0∫∂〝MリルdJ.■anemia;meChanjsm=r)’panOSOmiasi5. ABSTRACT Anemiaisarecognizedfeatureofbothanimalandhumantrypanosomiasis・ Themechanismoftrypanosomalanemiaishoweverunknown.Understandingthe TrleChanism of anemiaisimportant for the effeetjve management of trypanosomiasispatients,eSpeCiallylnanimalswhereanemiacouldbeextremely SeVere,Anemia was monitoredin five goats experimenta]lyinfectedwith Tbpanosoma vivax by measuring the packed ce11volume(PCV)and the hemoglobinlevel(Hb)upto26dayspostinfection.Parasitemiaandserum free fattyacid(FFA)1evelswerealsoestimated.Massiveparasitemiawasobservedon days5and12postinfection・Therewa545−59%decreaseinthePCVand33−63% decreaseinthe Hblevel.Theserum Fl仏1evelproBreSSivelylnCreaSedthrough Outthecourseofinfection,Anemiaintheearlystagesoftheinfectionisinitiated andmaintainedbylivlngtryPanOSOmeS,the severltyOftheanemiadependingon thelevelofparasitemia.Persistent anemiaasthe disease processprogressesis CauSedbyfactor(S)otherthanlivingtrypanosomes.Itissuggestedthaterythrocyte haemolysIS and erythrophagocytosis are the underlylng CauSeS Of trypanosomal anem】a. INTRODUCTION Africantrypanosomiasesare aspectrum ofdiseasesin mananddomestic animalscausedbythetsetsetransmittedprotozoanparasite,tryPanOSOme.Huma African trypanosomiasis(Sleeping sickness)is caused by TbpanoL”ma bruceE ま∫ ANEMlA 1N AFRlCAN TRYPANOSOMlASlS gambienseandT.b・rhodesiense・Animaltrypanosomiasis(naganaincattle)onthe Other handiscausedbythreespeciesoftrypanosomes,namely T.b.brucei,T. COngOZenseandT.vivax.Ofthesethreetrypanosomespecjes,T.vivaxisthemos important trypanosome of veterinary importance in West Africa. Animal trypanosomiasissti11constitutesamaJOrCOnStraintinthegrowthanddevelopment Of thelivestockindustryin many eountries of sub−Saharan Africa,thereby limiting10talfoodproduction.AnimaltrypanosomiasisandEastCoastfeverare believed toaccountforthe deathof3mi11ion cattle ayearand oneor bothOf these diseases occur(S)across morethan one third of the African continent (ILRAD1987).The estimated annuallossinlivestock due to African trypanosomiasisisputatUS$5,000mi11ion(WHO1997). Anemiahassincebeenrecognizedasam叫OrpathologlCalfeatureofclinical trypanOSOmiasisintx)thmanandanimals(fiennes1954;1970)andhasbeenused as anindicator of trypanotolerancein animalsinftcted with trypaT10SOmeS (Akinbamijoetal.1998;drIterenetal.1998).However,tOdate,themechanism{)f the anemiais unknown.Rather,hypo血eses whichhave failed t0月nd common grollndofacceptablespecincityhavebeenputforward.Theseinclllde:hypoplasia Ofthe bonemarrow,hydraemia,immunologlCalreactions,erythrophagocytosis, hemorrhagesyndromeandseverehaemolysis(reviewedbyMurrayandI)exter 1988).AmorerecentadditiontothislistofhypothesesisthereportofIgbokweet al.(1994)thatperOXidativeipjury to erythrocytes may play a rolein the pathogenesis of anemia in trypanosomiasis. A good understanding of the PathogenesisoftheanemiaisimportantforabettermanagementofthepatieIltS, espeCla11ylnanimaltrypanosomiasiswheretheanemiacouldbeverysevere・This Studyis part ofour contribution to the efforts towardsthe elucidation ofthe mechanismofanemiaintrypanosomiasis・ MATERIALS AND METHODS 什り柚町、・岬‥、− Tbpanosomavivaxstabilate,StOCkKabam/84/NITR/7・4wasobtainedfrom the NigerianInstitute for Trypanosomiasis Research,Vom,Nigeria.This was inoculatedinsheep■sbloodandkeptlnliquidnitrogenat−196bcbeforebeingused ingoats. (JIMJ∫.・ GoatswerepurchasedfromlocalmarketsinZaria,NorthernNigeriaand keptinfly−prOOf perISin the facu]ty of veterinary medici11e,Ahmadu Bello Universlty,Zaria・ApproprlateStepSWeretakentoensurethatallthegoatswere ingoodhealthandapparentlyfreefromtrypanosomiasisandanyotherformof ぎ首 ANEMlA IN AFRlCAN TRYPANOSOMlASlS ParaSiticinfectionbeforetheexperimentalinfection・ Cんβ椚fc(】血.・ Unless otherwise stated,a]1chemicals used were obtained from Sigma, England,andwereallofanaJytlCalgrade. 均匹雨脚舶用抑血肌 Tengoatsweredividedinto2groupsof5each・Onegroupof5wereeach infectedwithlO7T.vivaxce11swhilethesecondgroupof5wereuninfectedand servedascontrol. PdJⅥ∫fJg椚Jdα〃dク〟CたどdcβJJt,OJ〟用ビrPCVノご Parasitemia and PCV were deterrnined usJng the haematocrit centrifuge technique(W〔)0】97())・ 〃g椚〃gJぴぁf〃“ノ〃Jピ〃J.■ ThehemoglobincontentwasestimatedbysimplytakingalittlequantltyOf bloodsampleandthenreadingoffthehemoglobinlevelinaCounter haemoglobinometer. J、、〃〃J・い用い、い‥、州、川・声い・い.トJJl・∫・・バ・ FFAlevelswereestimatedasdescribedbyFalhoILetal.(1973)・Allthe parametersweremeasuredessentiallytwiceaweek,beginnlngfromdayOtoday 26postinfection・ REStTLTS PりJ・り∫情,川扇.、 Therewerecyclicalnuctuationsinthenumberofparasitesincirculation whichresultedin3successivewavesofparasitemiawithpeaksondays5,12and 22postinfection(Fig.1).Between days5and8,the number oIlparasitesin Circulation decreasedsharply,reaChingaverylowlevel(1essthan halfthepeak Value)by day8・The second phase ofdeclining parasitemia whjch was rnore drasticthanthefirstocc11rred betweendays12and19postinfection・Byday19, thelevelofparasitemiawasthesameasthatonday2(6timeslessthanthepeak Value on day12).Thepeak valueson days5and12were the sal一一e(about4 trypanosomes per microscopefield),While the value for day22was aboutl lrypanosomeperfield.Theva]uesg】VenareanaVerageOfthevaluesforthefive gOatS・ ANEMtA TN AFRICAN TRYfnNOSOMIASIS Figurel・DeveJopmentofparasitemiathrough 山e course orin托ction.Ani11uslmtわn of 仇e CyClical fluctuatJOnSin parasitemia duriTlg the VeJticalbars represent standard errors of mean つL 吋叫EO︼叫S已dh COurSeOftheex匹hmen血infecltonwhich】a$ted 払ー払days.臨chpointon仙eve摘録1axisisthe m組nOf也eYalueso一也efiveh触短dgoats.The (SEM). Ul ∈) れ ⊂〉 ■・→r−M レ「 r】 Q { Day5(pDStimfecLion) J,油・Å.・.J.・lJ/い巾〃Iけり一(l−Jご Ⅶth the exception of goat number3808which showed no apparent decreaseinPCVbyday2postinfection,thePCVofalltheinfectedgoatsshowed Signi鮎antdecreasesbyday2,withasmuchas25%decreaseingoatnumber3845 (Fig.2).Thedecreaseattheendoftheexperimentranged between45−59%.In general,therewasaprogressivedecreaseinPCVofa11theinfectedgoatsuntilthe terminationoftheexperiment. Figure2.The packedcelJvolum芦Ofdle im飴ctedgoatsdurlngthepeddofinfection This shows the decliningPCV duringthe 26rday period of expenmentalinfection. Eachpo)ntOntheverticalaxisistheneanof 仙e valu8SOfthe flveinfヒc便d go且暮SWhile the SEM are represented by the ve鵬cal bars. C >「 【=) r【†・・」N Ln 「】 く=〉 rl D往)・S(POStinf¢C暮ipn) 〃g椚¢gわぁ血「励ノ励gJ.・ LikethePCV,theHblevelsofa11theinfectedgoatsdecreasedsignificantly 朗 ANEMIA TN AFRTCAN TRYPANOSOMTASTS duringthecourseofinfectionwithanaverageof48%reductionexceptforgoat 3844whichhaditshemoglobinlevelreducedbya5muChas63%beforeitfinal1y died(Fig.3). Figure 3.The hemoglobinlevels ofthe infected goatsduTlr[gtheperiodofinfeetion. Thisillustrates(he decreasein Hb]evels with VeJticalbarsrepresentSEM. ムり ︵一pも︶芯已t上H progressiorlthroughthediseaseproces5■7hch poJntOr)dle Vertic且1axisIsthe mean ofthe Ya山es of山e fjYeinfected goats while the く︶ ⊂〉 ln ⊂⊃ レ1 ⊂コ ー・{−・{M 「】 r′ ロays(pos【infec【i8n〉 ∫gr〟椚/佗gβrJγ〟Cfdr錯弓りJgveJニ TherewasaprogressiveincreaseintheserumFFAlevelofalltheinfected goats(Fig・4)・Thevaluesofalltheparametersmeasuredina11thecontrolanimals did not show any appreciable change but remained essentially constantwitll ∝CaSionaldroporrisebylunit(datanotincluded). Figure4・Serumfreel加yacid(FFA)1evels Of血ein托ctedgoats.Ser11mFEAlevelsoflhe infected goats measured over the 26−day PerlOdofinfection・EachpoJn10ntheverticaI axisis the mean of the va]ues of the nve ex匹nmenta11yinfected goats,Vellicalbars r叩reSentSEM・ ⊂⊃l( (つレ1 く⊃ 「」 lハ ⊂⊃ 「l r’ ̄ D8yS(postinfec【ion〉 且9 ANEMlA 1N AFRlCAN TRYPANOSOMlASlS DISCIJSSION AnemiaintrypanosomiasisproceedsintwodistinctbutoverlapplngPhases, eachphasebeing mediatedbydifferentmechanisms(Murray andDexter1988)・ Thefirst phaseis believedto bemediated byfactors releasedby degeneratln trypanOSOmeS(Tizzardetal.1978b;ILRAD1987)andredcelldamagebyliving trypanosomes(Banks1979;Esievo1983).Ourobservationsinthisstudyarein SuppOtt Oflivlng tryPanOSOmeS Playlng a key rolein theinitiation and maintenanceoftheprogressivedevelopment ofanemiaintrypanosomiasis・The onset ofanemia andthe severltyimthe nrst phase strongly correlate witht appearance,intensity.and durationofparasitemia・Theobserved considerable declineinthePCVandtheHbcontentbetweendaysOand5postinfectionandthe relativelystablevaluesbetweendays5and8(Figs・2and3)whichcoincidedwith increasinganddecreasingparasitemiarespeCtively(Fig.1)couldnothavebeena merecoincidence.ThePCVandHbcontentagaindecreasedsharplybetweendays 8and12which wasanOther period ofincreaslng paraSitemia・ThesefindiTlgS SuggeStthatlivlngtrypanOSOmeSareeSSentialfortheprogressivedevelopmen10f trypanosomalanemiaininfectedamimals・Ikedeetal・(1977)reportedtheabsence OfhaemolyslnintheplasmaofT.congolenseinfectedmiceatleastupto15days POStinfectionbutobservedmarkedreductioninerythrocytehalf−1ifeandmarked dropin PCVwithin the same period.In cattleinfected with T・COngOlense increasedred cellbreakdownisbelievedtocommence withthedevelopmentof parasitemia(HoImes1976;Prestonetal・1979)・Thereforecontrarytothewidely heldviewthatanemiaintrypanosomiasisisinducedbyfactorsreleasedbydying trypanosomes,Webelievethattheonsetofanemiaanditsprogression,atleastin the early stages ofinfectioT),dependslargely on the presence oflivlng trypanosomesincirculation・ ThepersistentanemiaevenwhenparasitesarebarelydetectabIeisthephase IIoftheanemia・Webelievethatthisstageismediatedbyfactorsotherthanthose producedbylivlngtryPanOSOmeS・FFAsandLysophospholipidsarebelievedtobe hemolytlC factors released by dying trypanosomes and are responsible fo trypanosomalanemia(Tizzardetal・1978a;Noketal・1993)・Weobservedsteady andsharplnCreaSeSintheFFAlevelsofalltheinfectedgoatsthrough outthe period ofinfection(Fig・4)and this could have played a key roIeiTlthe pathogenesisoftheperSistentanemia・Althoughserumalbuminisknowntobind toFFAstoneutralizetheirhemolyticeffects(StarinskyandShafrir1970;Tizzard etal.1978b),itispossiblethatthema5Siveparasitemiathatcharacterizedthefirst twosuccessivewavesofparasitemiaproducedsufficientFFAstosaturateavai1abl serumalbumin and someleft unbound to exert their hemolytlC effect on erythrocytes・ 甜 ANEMIAIN AFRICAN TRYP4NOSOMTASIS Elucidationofthemechanismofanemiaintrypanosomiasishasremainedan unsoIvedproblem・Jenningsetal,(1974)suggestedthatanemiaintrypanosomiasis WaSmainlyduetoextravascularhaemolysIS andruledoutirnmunemechanisms Sinee(heonsetofanemiawasevidentwithinafewdaysofinfection・Murrayand Dexter(1988)explainedthaLimmunologicalcompetencewasnotessentialforthe developmentofanemia.DargieetaJ.(1979a;1979b)andDargie(1980)ruledout haemodilution as a contributory factor to the development of anemia・ Hemorrhagic syndrome whichis characteristic of acuteinfections caused by Certainstrains ofT・Vivaxinfections wasnotevidentin thepresent study.The markeddropinPCVandthemarkedreductjonintheHbconten(Observedin[his WOrkaregoodindicatorsofhaemolysis・Wethereforebelievethatthefirstphase Ofanemiaislargelyduetoerythrocytehaemolysis.Erythrophagocytosis resultin frominducedalterationsinerythrocytesurfacestructure(Durocheretal.1975; Esievo et al・1982;Esievo1983)is alsoapossibility as acauseofanemiain trypanosomiasis. 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