genetic diversity in trypanosoma theileri from sri lankan cattle and water buffaloes

(1)

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j ourn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Short

Communication

Genetic

diversity

in

Trypanosoma

theileri

from

Sri

Lankan

cattle

and

water

buffaloes

Naoaki

Yokoyama

a,∗

,

Thillaiampalam

Sivakumar

a,b

,

Shintaro

Fukushi

a

,

Muncharee

Tattiyapong

a

_,

_Bumduuren

_Tuvshintulga

a

_,

_Hemal

_Kothalawala

b

_,

Seekkuge

Susil

Priyantha

Silva

b

_,

_Ikuo

_Igarashi

a

_,

_Noboru

_Inoue

a

a_National_Research_Center_for_Protozoan_Diseases,_Obihiro_University_of_Agriculture_and_Veterinary_Medicine,_Hokkaido,_Japan b_Veterinary_Research_Institute,_Peradeniya,_Sri_Lanka

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received12June2014 Receivedinrevisedform 13November2014 Accepted11December2014

Keywords: Cattle

Geneticdiversity SriLanka

Trypanosomatheileri Waterbuffalo

a

b

s

t

r

a

c

t

Trypanosomatheileriisahemoprotozoanparasitethatinfectsvariousruminantspecies. Weinvestigatedtheepidemiologyofthisparasiteamongcattleandwaterbuffalo popula-tionsbredinSriLanka,usingadiagnosticPCRassaybasedonthecathepsinL-likeprotein (CATL)gene.BloodDNAsamplessourcedfromcattle(n=316)andwaterbuffaloes(n=320) bredindifferentgeographicalareasofSriLankawerePCRscreenedforT.theileri. Para-siteDNAwasdetectedincattleandwaterbuffaloesalikeinallthesamplinglocations. TheoverallT.theileri-positiveratewashigherinwaterbuffaloes(15.9%)thanincattle (7.6%).Subsequently,PCRampliconsweresequencedandthepartialCATLsequenceswere phylogeneticallyanalyzed.TheidentityvaluesfortheCATLgenewere89.6–99.7%among thecattle-derivedsequences,comparedwithvaluesof90.7–100%forthebuffalo-derived sequences.However,thecattle-derivedsequencesshared88.2–100%identityvalueswith thosefrombuffaloes.Inthephylogenetictree,theSriLankanCATLgenesequencesfellinto twomajorclades(TthIandTthII),bothofwhichcontainCATLsequencesfromseveralother countries.AlthoughmostoftheCATLsequencesfromSriLankancattleandbuffaloes clus-teredindependently,twobuffalo-derivedsequenceswereobservedtobecloselyrelatedto thoseoftheSriLankancattle.Furthermore,aSriLankanbuffalosequenceclusteredwith CATLgenesequencesfromBrazilianbuffaloandThaicattle.Inadditiontoreportingthe firstPCR-basedsurveyofT.theileriamongSriLankan-bredcattleandwaterbuffaloes,the presentstudyfoundthatsomeoftheCATLgenefragmentssourcedfromwaterbuffaloes sharedsimilaritywiththosedeterminedfromcattleinthiscountry.

1. Introduction

Variousspecies of Trypanosoma parasites are known toinfectseveralwildand domesticanimals worldwide.

∗ Correspondingauthorat:NationalResearchCenterforProtozoan Diseases, ObihiroUniversity ofAgricultureandVeterinaryMedicine, Inada-cho,Obihiro,Hokkaido080-8555,Japan.Tel.:+81155495649; fax:+81155495643.

E-mailaddress:[email protected](N.Yokoyama).

Species of mammalian trypanosomes were divided by

Hoare(1964)intotwomajorsections,Stercorariaand Sali-varia.TrypanosomesoftheSalivariasection,whichinclude

Trypanosomavivax,T.congelense,T.brucei,andT.evansi,are

transmittedtotheirvertebratehostduringthebloodmeal ofaparasite-infectedinsectvector,inwhichparasite devel-opmentisterminatedinthesalivaryglands(Haagetal., 1998;VanDenAbbeeleetal.,2010;Vickermanetal.,1998). Incontrast,forthetrypanosomesoftheStercorariasection, whichincludeT.cruzi,T.melophagium,andT.theileri,the finalstagesofparasitedevelopmenttakeplaceinthelower

(2)

digestivetractofthevectorsand,therefore,theseparasite speciesaretransmittedbycontaminationwith parasite-infectedinsectvectorexcreta(SchusterandSchaub,2000). AmongtheTrypanosomaparasites,T.vivax,T.congelense,

andT.bruceiareconsideredtobethemostpathogenicto

susceptibleanimals(Steverding,2008).However,T.evansi

alsocausesaseverewastingdiseaseininfectedcattle(Brun etal.,1998;Desquesnesetal.,2013).Ontheotherhand,T.

theilerihasalsobeenimplicatedinsporadicclinicalcases

indomesticanimalsinvariouscountries(Dohertyetal., 1993;Soodetal.,2011;Villaetal.,2008).Therefore,the epidemiologyofT.theilerishouldbestudied.Trypanosoma

theileriisalargeTrypanosomaparasitevectoredbytabanid

flies(Böseetal.,1987)andticks(Latifetal.,2004;Shastri andDeshpande,1981).

Severalgeneticmarkergenes,suchasthesmall sub-unitrRNA(ssrRNA),internaltranscribedspacer1(ITS1), cytochromeb(Cytb),splicedleader(SL),glycosomal glyc-eraldehyde 3-phosphate dehydrogenase (gGAPDH), and cathepsinL-likeprotein(CATL)genes,havebeenusedto analyzegeneticvariationinT.theileriparasitesfrom sev-eral host animals, including cattle, buffaloes, and deer (Fisheretal.,2013;Garciaetal.,2011a,b;Rodriguesetal., 2006,2010).Inthesephylogeneticanalyses,theT.theileri

genesequences alwaysclusteredindependently accord-ingtothehostspecies (Fisheretal.,2013;Garciaetal., 2011a,b;Rodriguesetal.,2006,2010).Thesefindingsled theresearchersinthesestudiestoconcludethatT. thei-lerigenotypes might be host-specific.However, genetic diversityinT.theilerihasonlybeeninvestigatedinasmall numberofcountries,includingBrazil,Venezuela,Thailand, Vietnam,Philippines,andtheUnitedStates(Fisheretal., 2013;Garciaetal.,2011a,b;Rodriguesetal.,2006,2010; Sivakumaretal.,2013;Yba ˜nezetal.,2013).Therefore, fur-theranalysesofT.theileripopulationsinotherendemic countries are required to gain a more comprehensive understandingofthegeneticdiversityandhostspecificity ofthisparasite.

ThepresenceofT.theileriinSriLankanwaterbuffaloes, basedonmorphologicalandindirectimmunofluorescence antibodytests,haspreviously beenreported(Weilgama etal.,1989).However,moleculardetectionmethodshave notbeenusedtodetectT.theileriinSriLanka.Therefore,the aimofthepresentstudywastodeterminetheprevalence

ofT.theileriincattleand waterbuffaloesbredin

differ-entgeographicallocationsofSriLanka,usingadiagnostic PCRassaybasedontheCATLgene,andtoanalyzegenetic diversityin theparasites. Our findings indicatedthat T.

theileriisacommonparasiteamongthecattleandwater

buffaloesbredin SriLanka, and thattheparasite popu-lationsare genetically diverse. Additionally, our results indicatedthatsomeofthewaterbuffalo-derivedT. thei-leriCATLnucleotidesequencesdeterminedinthepresent studysharedhighidentityscoreswiththosefromcattle.

2. Materialsandmethods

2.1. BloodsamplingandDNAextraction

The blood samples collected from cattle and water buffaloes in Sri Lanka have been described previously

(Sivakumaretal.,2012b,2014).Briefly,316cattlereared in four different districts (Nuwara Eliya, Polonnaruwa, Ampara,andJaffna),and320buffaloesbredinthree dif-ferent districts (Polonnaruwa, Mannar, and Mullaitivu) were sampled in April and June 2011 and in February 2013,respectively.Alltheanimalswereover 1year-old and apparently healthyduring thesampling.DNA sam-pleswereextractedfrom200␮lofwholeblood usinga

commercialkitaccordingtothemanufacturer’s instruc-tions(Qiagen,Hilden,Germany).TheDNAsampleswere storedat–20◦_C_until_further_use._These_DNA_samples_were

previouslyusedforinvestigationsonbovineBabesiaand

Theileriaparasites(Sivakumaretal.,2012b,2014).

2.2. DetectionofT.theileriusingadiagnosticPCRassay

ApreviouslyestablishedT.theileri-specificPCRassay, basedontheCATLgene,wasemployedtoscreentheDNA samples(Rodrigueset al.,2010).ThePCRreaction mix-turesandcyclingconditionsdescribedbyRodriguesetal. (2010) wereused withmodifications.Briefly, PCR reac-tionscontained1␮lof10× PCRreactionbuffer,200␮M

dNTPs(AppliedBiosystems,Branchburg,NJ),0.5␮Mof

for-ward(5′_{-CGTCTCTGGCTCCGGTCAAAC-3}′₎_and_reverse₍₅′

-TTAAAGCTTCCACGAGTTCTTGATGATCCAGTA-3′₎ _primers,

0.5 units of Taq polymerase (Applied Biosystems), and 5.9␮lofdoubledistilledwater.Aftertheadditionof1␮lof

eachDNAsample,themixturewassubjectedtoanenzyme activationstepat95◦_C_for₅_min,_followed_by₄₅_cycles_each

includingadenaturingstepat95◦_C_for₃₀_s,_an_annealing

stepat55◦_C_for₁_min,_and_an_extension_step_at₇₂◦_C_for

1min.Afterfinalelongationat72◦_C _for₁₀_min,_the_PCR

productswereanalyzedbyagarosegelelectrophoresis.PCR ampliconscloseinsizetothepositivecontrolwere

consid-eredT.theileri-positive.

2.3. Cloning,sequencing,andphylogeneticanalysis

PCR amplicons with high band-intensities, detected from both cattle and water buffaloes, werecloned into plasmid vectorsand sequenced as described previously (Sivakumar et al.,2012a).For each cloned product,the bothstrandsofasinglecloneweresequenced.Resultant sequences werethen analyzedby basiclocal alignment tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Identity values among the CATL gene sequences were calculated using the Emboss needle online program (http://emboss.bioinformatics.nl/cgi-bin/emboss/needle). Singlenucleotidepolymorphisms amongtheSri Lankan

(3)

Table1

PCRdetectionofT.theileriinSriLankancattleandwaterbuffaloes.

Districts Cattle Waterbuffaloes

No.sample No.positive %(CIb₎ _No._sample _No._positive _%_(CI)

NuwaraEliya 83 6 7.2(3.4–14.9) ND – –

Polonnaruwa 84 8 9.5(4.9–17.7) 118 8 6.8(3.5–12.8)

Ampara 88 7 8.0(3.9–15.5) ND – –

Jaffna 61 3 4.9(1.7–13.5) ND – –

Mannar NDa _– _– ₁₄₆ ₃₅ _24.0_{(17.8–31.5)}

Mullaitivu ND – – 56 8 14.3(7.4–25.7)

Total 316 24 7.6(5.2–11.1) 320 51 15.9(12.3–20.4)

a_ND,_sampling_was_not_done. b_95%_confidence_interval.

2.4. Statisticalanalysis

The 95% confidence intervals for the positive rates werecomputedusingtheopenepionlineprogram(http:// www.openepi.com/v37/Proportion/Proportion.htm), basedonthemethoddescribedbyWilson(1927).

3. Resultsanddiscussion

The diagnostic PCR assay revealed the presence of

T. theileri in the cattle and water buffaloes bred in all

the surveyed districts of Sri Lanka (Table 1). The

over-allT.theileri-positiveratewasrelativelyhigherinwater

buffaloes (15.9%) than in cattle (7.6%). However, the prevalences ofT. theileriamongwater buffaloesbred in Polonnaruwa and Mullaitivu didnot significantly differ fromthosedetermined forthecattlepopulationsinthe surveyeddistricts.On theotherhand,T.theileri-positive rateamongthewaterbuffaloesinMannarwassignificantly higher than those reported for the cattleand has con-tributedtothehigheroverallprevalenceinwaterbuffaloes incomparisontocattle.Therefore,itseemsthatT.theileri -positive rates in Sri Lanka might be influenced by the geographicallocation.TheMannardistrictischaracterized byhotandhumidclimate,andfurtherstudiesto investi-gatethepossibleeffectofdifferentclimaticconditionson thedistributionoftabanidfliesmightshedanadditional lightonthediscrepanciesbetweentheprevalencesofT.

theileriindifferentgeographicallocationswithinSriLanka.

AlthoughT.theileriisknowntoberelativelyless viru-lent,theparasitesometimesbecomestroublesomewhen presentinco-infectionswithotherpathogens(Mansfield, 1977;Wells,1976).Therefore,weanalyzedforpossible co-infectionsbasedontheBabesia(B.bovisandB.bigemina)

andTheileria(T.annulataandT.orientalis)infectiondata

obtainedfromthesameDNAsamplesinprevious inves-tigations(Sivakumaretal.,2012b,2014).In thepresent study,83.3%oftheT.theileri-positivecattleand86.3%ofthe

T.theileri-positivewaterbuffaloeswereco-infectedwith

Babesiaand/orTheileriaparasites(Table2).AmongtheT.

theileri-infectedcattle,10,5,4,and1wereco-infectedwith

one,two,three,andfourBabesiaand/orTheileriaspecies, respectively.Incontrast,outof44T.theileri-positivewater buffaloes that had co-infections, 43 had dual infections

with T. orientalis, while the remaining animal was

co-infectedwithB.bigeminaandT.orientalis.Thediscrepancies relatingtothedifferentco-infectionprofilesofthecattle

Table2

Co-infectionsofT.theileriwithBabesiaandTheileriaparasitesinSriLankan cattleandwaterbuffaloes. T.theileri+B.bigemina+T.orientalis 1 1 T.theileri+T.annulata+T.orientalis 1 ND 2parasites

T.theileri+T.orientalis 6 43

T.theileri+B.bigemina 4 ND

Total(%c₎ ₂₀_(83.3) ₄₄_(86.3)

a_{Co-infections} _were _determined _based _on _the _Babesia _(B. _bovis

andB.bigemina)andTheileria(T.annulataandT.orientalis)infection dataobtainedfromthesameDNAsamplesinpreviousinvestigations (Sivakumaretal.,2012b,2014).

b _ND,_not_detected.

c _Expressed_as_a_percentage_of_T._{theileri-infected}_animals.

andwaterbuffaloesarecausedbydifferencesinthe posi-tiveratesofBabesiaandTheileriaspeciesbetweenthecattle andwaterbuffaloesinSriLanka(Sivakumaretal.,2012b, 2014).Nevertheless,thehighT.theilerico-infectionrates areofconcerninSriLanka.Therefore, further investiga-tionontheclinicalstatusofco-infectedanimalsisnowa priorityinthiscountry.

A total of 15 and 9 PCR amplicons from cattle and waterbuffaloes,respectively,wereclonedandsequenced. All the CATL gene fragments amplified in the present studywere289-bpin length,but highgeneticvariation wasfoundamongtheCATLsequences.Singlenucleotide polymorphisms were frequently detected, as shown in

Fig.1.Theidentityvaluesamongthecattle-derivedCATL

sequences(AB930145–AB930159)were89.6–99.7%,while the buffalo-derived sequences (AB930160–AB930168) exhibited 90.7–100% identity values. In contrast, the buffalo-derivedCATLsequenceshad88.2–100%sequence identity with those of cattle. Two cattle-derived CATL

(4)

Fig.1.PolymorphicsiteswithinT.theileriCATLgenesequencesisolatedfromcattleandwaterbuffaloesinSriLanka.TheCATLsequencesgeneratedinthe presentstudywereanalyzedbyDnaSPv5software(LibradoandRozas,2009)usingacattle-derivedsequence(AB930146)asareferencefordetermining thepolymorphicsiteswithinthe289nucleotidesamplifiedbythediagnosticPCRassay.CladesrefertothecladenamesinFig.2.Dotsdenotenucleotides thatareidenticaltothereferencesequence.NotethatthereishighgeneticdiversityamongtheSriLankanT.theileriCATLsequencesandthattwo buffalo-derivedsequences(AB930167andAB930168)sharehighsequenceidentityscoreswithtwocattle-derivedsequences(AB930146andAB930147)inclade ID.

99.3% identity values with a buffalo-derived sequence (AB930167), and these cattle-derived sequences also shared99.7%identitywithanothersequence(AB960168) derived from a Sri Lankan water buffalo. Similarly, a sequencefromcattle(AB930151)shareda highidentity score(97.9%)withbuffalo-derivedsequences(AB930161, AB930162,andAB930166).Geneticdiversitylevelsamong

theT.theileriCATLgenesequencesdetectedinSriLanka

areinagreementwiththoseofpreviousstudies,whichalso foundthatCATLsequencesarehighlydiverseamongT. thei-leriisolatesfromdifferentcountries(Garciaetal.,2011a,b; Rodriguesetal.,2006,2010).Severalpaststudieshave sug-gestedthattheT.theilerigenotypesthatinfectcattleand waterbuffaloesmightdifferfromeachother(Fisheretal., 2013;Garciaetal.,2011a,b;Rodriguesetal.,2006,2010). BasedonssrRNAandITSnucleotidesequences,Rodrigues et al. (2006) alsoshowed that the T. theileri genotypes fromBraziliancattleandwaterbuffaloesdifferedfromeach other.Inarecentstudy,whichusedseveralmarkergenes,

theT.theilerigenotypesobtainedweredescribedasbeing

host-specificinBrazilandVenezuela(Garciaetal.,2011b). Tothebestofourknowledge,therefore,ourstudyisthefirst toreporthighlevelsofsequenceidentityamongdirectly PCR-amplifiedCATLDNAsequencesofT.theilerifromcattle andwaterbuffaloes.

In the phylogenetic tree, the CATL sequences deter-mined in the present study were found in multiple clades(Fig.2).Althoughmostofthecattle-and buffalo-derived sequences clustered independently, a few CATL

sequences from water buffaloes (AB930167, AB930168, and AB930162) clustered together with the cattle sequences.Thesefindingsareconsistentwiththe sequenc-ingdataanalysisdescribed above.Wealsofoundthata Braziliansequence(HQ664735)fromwaterbuffaloshared 100 and 99.6% identity values with a buffalo-derived

sequence (AB930162) from Sri Lanka and with two cattle-derivedsequences(HQ543073andHQ543074)from Thailand,respectively.Althoughtheseresultsquestionthe hostspecificityofT.theileriCATLvariantsincattleand buf-faloesfromSriLanka,thefindingsmightbeinconclusive asonly thepartialCATLgene fragmentswereanalyzed. Ontheotherhand,researchershaveusedseveraldifferent markergenes toinvestigategenetic diversityin T. thei-leri inthe recentpast (Fisher etal., 2013;Garcia et al., 2011a,b; Rodrigues et al., 2006, 2010). In an extensive studyusinglargeDNAdatasetgeneratedfromcultured

T.theileriisolates,Garciaetal.(2011b)demonstratedthe

highconcordancebetweenthegenotypesofseveral geno-typicmakers,suchasthessrRNA,ITS1,Cytb,SL,gGAPDH, andCATLgenes,andfoundthatthegenotypeswere host-specific.However,ourpresentstudyexaminedonlythe DNAsamplesextractedfromthehostbloodandanalyzed the geneticvariations based onpartialCATL sequences. Therefore,thepresentfindingsmaynotallowustoreacha conclusionregardingthehost-specificityofT.theileri geno-typesinSriLanka.

Apreviousstudydemonstratedthatthetabanidflies canharbordiversegeneticvariantsofT.theileriatagiven time(Rodriguesetal.,2006).Therefore,theT.theileri geno-typesincattlecouldhavebeentransmittedtothewater buffaloesinSriLanka,asboththehostanimalsareusedto beinclosecontactwitheachother,especiallyduring graz-ing,inthesampledlocations.However,althoughdifferent

T.theilerigenotypesaretransmittedtothehostanimals,

(5)

(6)

etal.,2008).Therefore,thepresentfindings,whichshowed that someof thewater buffalo-derived CATLsequences sharedsimilarity to cattle-derived sequences,might be duetoyetunestablishedinfectionswithdifferent geno-typesinthesehostanimals.Sothat,thedetectionofCATL

sequencesthatsharesimilaritybetweencattleandwater buffaloesinthepresentstudydoesnotnecessarilymean thattheseCATL variantslack host-specificity.Therefore, futurestudiesin SriLankashouldanalyzea large num-berofsequencesbelongingtoseveralgenotypic marker genesusingculturedT.theileriisolatesfromdifferenthost animals.Nevertheless,thepresentfindingsareusefulfor furtheringourunderstandingoftherelationshipbetween

T.theilerigenotypesandhost-specificity.Inconclusion,in

additiontoreportingthefirstPCR-basedsurveyofT. thei-leri,thepresentstudyfoundevidencethatsomeT.theileri

CATLDNAsequencesobtainedfromnaturallyinfected cat-tleandwaterbuffaloesinSriLankamaybesharedamong thesehosts.

Conflictofintereststatement

Theauthorshavenoconflictsofinterestconcerningthe workreportedinthispaper.

Acknowledgments

WethankthestaffoftheSriLankancattleandwater buffalo farms and at the Veterinary Research Institute, DepartmentofAnimalProductionandHealth,Peradeniya, SriLankafortheirkindcooperation.We alsothankMs. Hiroko Yamamoto (National Research Center for Pro-tozoan Diseases, Obihiro University of Agriculture and VeterinaryMedicine), Mr.H. Maithripala and Mr.H. A. Wijithasiri(VeterinaryResearchInstitute,Departmentof AnimalProductionandHealth,Peradeniya,SriLanka)for theirexcellenttechnicalassistance.Thisstudywas sup-portedbya grantfromtheJapanSocietyfor Promotion ofScience(JSPS)Grant-in-AidforScientificResearch(JSPS KAKENHINumber26257417).

References

Böse,R.,Friedhoff,K.T.,Olbrich,S.,Büscher,G.,Domeyer,I.,1987. Trans-missionofTrypanosomatheileritocattlebyTabanidae.Parasitol.Res. 73,421–424.

Brooks,D.R.,McLennan,D.A.,2002.TheNatureofDiversity:An Evolu-tionaryVoyageofDiscovery.UniversityofChicagoPress,Chicago, IL.

Brun,R.,Hecker,H.,Lun,Z.,1998.TrypanosomaevansiandT. equiper-dum:distribution,biology,treatmentandphylogeneticrelationship (areview).Vet.Parasitol.79,95–107.

Desquesnes,M.,Holzmuller,P.,Lai, D.H.,Dargantes,A.,Lun,Z.R., Jit-taplapong,S.,2013.Trypanosoma evansiandsurra:areviewand perspectivesonorigin,history,distribution,taxonomy,morphology, hosts,andpathogeniceffects.Biomed.Res.Int.2013,194176. Doherty,M.L.,Windle,H.,Voorheis,H.P.,Larkin,H.,Casey,M.,Clery,D.,

Murray,M.,1993.ClinicaldiseaseassociatedwithTrypanosomatheileri infectioninacalfinIreland.Vet.Rec.132,653–656.

Fisher,A.C.,Schuster,G.,Cobb, W.J.,James,A.M.,Cooper,S.M.,Peréz de León, A.A., Holman, P.J., 2013. Molecular characterization of Trypanosoma(Megatrypanum)spp.infectingcattle(Bostaurus), white-taileddeer(Odocoileusvirginianus),andelk(Cervuselaphuscanadensis) intheUnitedStates.Vet.Parasitol.197,29–42.

Garcia,H.A., Kamyingkird,K.,Rodrigues,A.C.,Jittapalapong, S., Teix-eira,M.M.,Desquesnes, M.,2011a.Highgeneticdiversityinfield

isolatesofTrypanosomatheileriassessedbyanalysisofcathepsin L-likesequencesdisclosedmultipleandnewgenotypesinfectingcattle inThailand.Vet.Parasitol.180,363–367.

Garcia,H.A.,Rodrigues,A.C.,Martinkovic,F.,Minervino,A.H.,Campaner, M.,Nunes,V.L.,Paiva,F.,Hamilton,P.B.,Teixeira,M.M.,2011b. Mul-tilocusphylogeographicalanalysisofTrypanosoma(Megatrypanum) genotypes from sympatric cattleand water buffalo populations supportsevolutionaryhostconstraintandclosephylogenetic rela-tionshipswithgenotypesfoundinotherruminants.Int.J.Parasitol. 41,1385–1396.

Haag,J.,O’hUigin,C.,Overath,P.,1998.Themolecularphylogenyof try-panosomes:evidenceforanearlydivergenceoftheSalivaria.Mol. Biochem.Parasitol.91,37–49.

Hellgren,O.,Bensch,S.,Malmqvist,B.,2008.Birdhosts,bloodparasites andtheirvectors-associationsuncoveredbymolecularanalysesof blackflybloodmeals.Mol.Ecol.17,1605–1613.

Hoare, C.A., 1964. Morphological and taxonomic studies on mam-maliantrypanosomes.X.Revisionofthesystematics.J.Protozool.11, 200–207.

Katoh,K.,Misawa,K.,Kuma,K.,Miyata,T.,2002.MAFFT:anovelmethod forrapidmultiplesequencealignmentbasedonfastFourier trans-form.NucleicAcidsRes.30,3059–3066.

Latif, A.A.,Bakheit, M.A., Mohamed, A.E., Zweygarth,E., 2004. High infectionratesofthetickHyalommaanatolicumanatolicumwith Try-panosomatheileri.OnderstepoortJ.Vet.Res.71,251–256.

Librado,P.,Rozas,J.,2009.DnaSPv5:asoftwareforcomprehensive anal-ysisofDNApolymorphismdata.Bioinformatics25,1451–1452. Mansfield,J.M.,1977. Nonpathogenictrypanosomesofmammals.In:

Kreier,J.P.(Ed.),ParasiticProtozoa,vol.1.AcademicPress,London, pp.297–327.

Rodrigues, A.C.,Garcia, H.A.,Ortiz, P.A.,Cortez,A.P., Martinkovic,F., Paiva, F.,Batista, J.S., Minervino, A.H.,Campaner, M., Pral, E.M., Alfieri,S.C.,Teixeira,M.M.,2010.CysteineproteasesofTrypanosoma (Megatrypanum)theileri:cathepsinL-likegenesequencesastargets forphylogeneticanalysis,genotypingdiagnosis.Parasitol.Int.59, 318–325.

Rodrigues,A.C.,Paiva,F.,Campaner,M.,Stevens,J.R.,Noyes,H.A., Teix-eira,M.M.,2006.PhylogenyofTrypanosoma(Megatrypanum)theileri andrelatedtrypanosomesrevealslineagesofisolatesassociatedwith artiodactylhostsdivergingonSSUandITSribosomalsequences. Para-sitology132,215–224.

Saitou,N.,Nei,M.,1987.Theneighbor-joiningmethod:anewmethodfor reconstructingphylogenetictrees.Mol.Biol.Evol.4,406–425. Schuster,J.P.,Schaub,G.A.,2000.Trypanosomacruzi:skin-penetration

kineticsofvector-derivedmetacyclictrypomastigotes.Int.J.Parasitol. 30,1475–1479.

Shastri,U.V.,Deshpande,P.D.,1981.Hyalommaanatolicumanatolicum (Koch1844)asapossiblevectorfortransmissionofTrypanosoma theileri,Laveran,1902incattle.Vet.Parasitol.9,151–155.

Sivakumar,T., Altangerel,K.,Battsetseg, B.,Battur, B.,AbouLaila, M., Munkhjargal, T., Yoshinari, T., Yokoyama, N., Igarashi, I., 2012a. GeneticdetectionofBabesiabigeminafromMongoliancattleusing apicalmembraneantigen-1genebasedPCRtechnique.Vet.Parasitol. 187,17–22.

Sivakumar, T., Kothalawala, H., Abeyratne, A.S., Vimalakumar, S.C., Meewawe, A.S., Hadirampela, D.T., Puvirajan, T., Sukumar, S., Kuleswarakumar,K.,Chandrasiri,A.D.N.,Igarashi,I.,Yokoyama,N., 2012b.APCR-basedsurveyofselectedBabesiaandTheileriaparasites incattleinSriLanka.Vet.Parasitol.190,263–267.

Sivakumar, T., Lan, D.T., Long, P.T., Yoshinari, T., Tattiyapong, M., Guswanto,A.,Okubo,K.,Igarashi,I.,Inoue,N.,Xuan,X.,Yokoyama,N., 2013.PCRdetectionandgeneticdiversityofbovinehemoprotozoan parasitesinVietnam.J.Vet.Med.Sci.75,1455–1462.

Sivakumar,T.,Tattiyapong,M.,Fukushi,S.,Hayashida,K.,Kothalawala, H.,Silva,S.S.P.,Vimalakumar,S.C.,Kanagaratnam,R.,Meewawe,A.S., Suthaharan,K.,Puvirajan,T.,deSilva,W.K.,Igarashi,I.,Yokoyama,N., 2014.GeneticcharacterizationofBabesiaandTheileriaparasitesin waterbuffaloesinSriLanka.Vet.Parasitol.200,24–30.

Sood,N.K.,Singla,L.D.,Singh,R.S.,Uppal,S.K.,2011.Associationof Try-panosomatheileriwithperitonitisinapregnantcross-bredcow:acase report.Vet.Med.56,82–84.

Steverding,D.,2008.ThehistoryofAfricantrypanosomiasis.Parasites Vectors12,3.

Tamura, K.,1992.Estimation ofthenumber ofnucleotide substitut-ionswhentherearestrongtransition–transversionandG+C-content biases.Mol.Biol.Evol.9,678–687.

(7)

VanDenAbbeele,J.,Caljon,G.,DeRidder,K.,DeBaetselier,P.,Coosemans, M.,2010.Trypanosomabruceimodifiesthetsetsesalivarycomposition, alteringtheflyfeedingbehaviorthatfavorsparasitetransmission. PLoSPathog.3(6).

Vickerman,K.,Tetley,L.,Hendry,K.A.,Turner,C.M.,1998.Biologyof Africantrypanosomesinthetsetsefly.Biol.Cell64,109–119. Villa,A.,Gutierrez,C.,Gracia,E.,Moreno,B.,Chacón,G.,Sanz,P.V.,Büscher,

P.,Touratier,L.,2008.PresenceofTrypanosomatheileriinSpanish cat-tle.Ann.N.Y.Acad.Sci.1149,352–354.

Weilgama,D.J.,Bahirathan,M.,Perera,P.S.G.,1989.Studies onsome protozoaninfectionsofbuffaloesinSriLanka.In:Proceedingsofa

SymposiumonBuffaloResearchinSriLanka,Kandy,SriLanka,7–10th March.

Wells,E.A.,1976.SubgenusMegatrypanum.In:Lumsden,W.H.R.,Evans, D.A.(Eds.),BiologyoftheKinetoplastida.AcademicPress,London,pp. 257–275.

Wilson,E.B.,1927.Probableinference,thelawofsuccession,andstatistical inference.J.Am.Stat.Assoc.22,209–212.