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Contents lists available atScienceDirect

Veterinary

Parasitology

j o u r n a l h o m e p a 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

Research

paper

Population

genetic

analysis

of

Theileria

parva

isolated

in

cattle

and

buffaloes

in

Tanzania

using

minisatellite

and

microsatellite

markers

Elpidius

Rukambile

a,∗

,

Eunice

Machuka

b

,

Moses

Njahira

b

,

Martina

Kyalo

b

,

Robert

Skilton

b

_,

_Elisa

_Mwega

c

_,

_Andrew

_Chota

a

_,

_Mkama

_Mathias

a

_,

_Raphael

_Sallu

a

_,

Diaeldin

Salih

d

a_Tanzania_Veterinary_Laboratory_Agency_(TVLA),_P.O._Box_9254,_Dar_es_Salaam,_Tanzania

b_Biosciences_Eastern_and_Central_{Africa—International}_Livestock_Research_Institute_Hub_(BecA-ILRI_Hub),_P.O._Box_30709-00100,_Nairobi,_Kenya c_Faculty_of_Veterinary_Medicine,_Genome_Science_Center,_Sokoine_University_of_Agriculture_(SUA),_P.O._Box_3019,_Morogoro,_Tanzania d_Veterinary_Research_Institute_(VRI),_P.O._Box_8067,_Khartoum,_Sudan

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18December2015 Receivedinrevisedform22April2016 Accepted24April2016

Keywords: Buffalo Cattle Diversity Populationgenetic Tanzania Theileriaparva

a

b

s

t

r

a

c

t

ApopulationgeneticstudyofTheileriaparvawasconductedon103cattleand30buffaloisolatesfrom Kibaha,Lushoto,NjombeDistrictsandselectedNationalparksinTanzania.Bovinebloodsampleswere collectedfromthesestudyareasandcategorizedinto5populations;Buffalo,Cattlewhichgrazecloseto buffalo,Kibaha,LushotoandNjombe.SamplesweretestedbynestedPCRforT.parvaDNAandpositives werecomparedforgeneticdiversitytotheT.parvaMugugavaccinereferencestrain,using3microand 11minisatellitemarkersselectedfromall4chromosomesoftheparasitegenome.Thediversityacross populationswasdeterminedbythemeannumberofdifferentalleles,meannumberofeffective alle-les,meannumberofprivatealleleandexpectedheterozygosity.Themeannumberofalleleuniqueto populationsforCattleclosetobuffalo,Muguga,Njombe,Kibaha,LushotoandBuffalopopulationswere 0.18,0.24,0.63,0.71,1.63and3.37,respectively.Themeannumberofdifferentallelesrangedfrom6.97 (Buffalo)to0.07(Muguga).Meannumberofeffectiveallelesrangedfrom4.49(Buffalo)to0.29(Muguga). Themeanexpectedheterozygositywere0.070.29,0.45,0.48,0.59and0.64forMuguga,cattlecloseto buffalo,Kibaha,Njombe,LushotoandBuffalopopulations,respectively.TheBuffaloandLushotoisolates possessedaclosedegreeofdiversityintermsofmeannumberofdifferentalleles,effectivealleles, pri-vateallelesandexpectedheterozygosity.Thestudyrevealedmorediversityinbuffaloisolatesandfurther studiesarerecommendedtoestablishifthereissharingofparasitesbetweencattleandbuffaloeswhich mayaffecttheeffectivenessofthecontrolmethodscurrentlyinuse.

1. Introduction

TheileriosisintheformofEastCoastfever(ECF)isavery dev-astatingtick-bornediseaseofcattlewhich ishighlyendemic in 11countriesintheEastern,Central andSouthern Africa(ECSA) regionwhereitisamajorhindrancetocattleproduction(Mukhebi etal.,1992).EastCoastfeveriscausedbythetick-borne haemo-protozoanparasiteTheileriaparvatransmittedbybrowneartick,

Rhipicephalusappendiculatus.EconomiclossesresultingfromECF

∗Correspondingauthor.

E-mailaddresses:rukambile@yahoo.com(E.Rukambile),enidm32@gmail.com (E.Machuka),wanjahira@gmail.com(M.Njahira),M.Kyalo@cgiar.org(M.Kyalo), rskilton@icipe.org(R.Skilton),elisamwega@yahoo.co.uk(E.Mwega), chotaandrew@gmail.com(A.Chota),matsonics2000@yahoo.co.uk(M.Mathias), raphaelsallu@yahoo.com(R.Sallu),diaeldin2000@hotmail.com(D.Salih).

areattributedtohighmorbidityandmortalityinlocalandexotic cattleandreducedmilkproductioninlactatingcows(Mbwambo

etal.,2002;Kivariaetal.,2007).

T.parvainfectioninsusceptible cattleresultsinarapid pro-liferationofinfectedlymphocytesanddeathwithintwotothree weeksifnotreatmentiscommenced(IrvinandMwamachi,1983). Duringthecourseofinfection,lymphocytesandantigen present-ingmacrophagesoftheMphilineagearetransformed,resultingin alossofphenotypicmarkersandconsequently,alossoffunction

(Morrison,2009;Jeongetal.,2009).InTanzaniacontrolofT.parva

involvesapplicationofacaricidesonceortwiceperweektocontrol thevector,useofcommerciallyavailablechemotherapyand immu-nizationofanimalsusinginfectionandtreatmentmethod(ITM). ITMcomprisesinoculationofcattlewithahomogenateofinfected tickssimultaneouslywithasingledoseofalong-actingformulation ofoxy-tetracyclinetocurtailtheinfection.Thisresultsinlongterm immunity againstthe homologousparasite strainsbut variable

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protectionagainst challengewith heterologous parasite strains

(Irvinetal.,1983).PopulationgeneticstudiesofT.parvaare

impor-tanttodeterminethegeneticexchangeamongtheparasiteisolates. Thelevelsofrecombinationamongparasitesdeterminetheextent ofgeneralpopulationdiversitywhichhavegreatimplicationinthe successand/orfailureofITM(Katzeretal.,2006;McKeever,2007;

KuoandKissinger,2008;Katzeretal.,2010).Recombinationunder

selectioncansupportrapiddisseminationofanallelewithina pop-ulation.InTheileriaspp.,recombinationcanplayanimportantrole ingeneratingdiversityatlocithatarerelevantformanipulating hostimmunityduringevasion(Katzeretal.,2011).

Miniand microsatelliteshave highrates ofmutation dueto replicationslippageandrepair.Thehighratesofmutationresult inhighlevelsofpolymorphism atthevariable numbertandem repeat(VNTR)locimakingthemidealmarkersforpathogen diver-sitystudy(Ouraetal.,2003).Miniandmicrosatelliteshavebeen successfullyusedtodeterminepopulationdiversityofT.parvain differentstudies.Miniand microsatellitemarkerswereusedto characterizethethreestrainswhicharepresentascomponents oftheMugugaT.parvavaccinecocktail,todeterminetheabilityof eachcomponenttoinducecarrierstate(Ouraetal.,2004)andthe possibilityofbeingtransmittedfromvaccinatedanimalstoother susceptibleanimals(Ouraetal.,2007).Fourminiand microsatel-litemarkerswereusedtostudydiversityofthethreestockswhich arethecomponentsoftheMugugacocktailvaccinewhereby14 differentgenotypeswererevealed(Pateletal.,2011).Ontheother hand,9minisatelliteswereusedtostudypopulationgeneticsand sub-structureofT.parvapopulationinthesamplescollectedfrom twodistrictsinZambiawithsuccess(Muleyaetal.,2012).The cur-rentstudyfocusedonthepopulationstructureofT.parvaisolated fromcattleintwodistrictsintheEasternzoneandonedistrictinthe SouthernhighlandzoneandbuffaloesfromselectedNationalParks ofTanzaniausing11miniand3microsatellitemarkers. Determi-nationofparasitediversityinbuffaloisequallyimportantbecause thebuffalo-derivedparasitescanbemaintainedbetweencattleand tickpassageandyettheparasitepopulationinbuffaloisoftenmore diversethanincattle(Bishopetal.,1994;Potgieteretal.,1988;

CollinsandAllsopp,1998;Sibekoetal.,2010).

2. Materialsandmethods

2.1. Studysites

Thestudywascarried outin 3 districtsnamely, Kibaha and LushotointheEasternandNjombeintheSouthernHighland agro-ecologicalzonesofTanzania.KibahaandNjombeare675kmapart, Kibaha andLushotoare419km apart,Lushotoand Njombeare 847kmapart.EastCoastfeverhasbeenmorefrequentlyreported inKibahadistrictandimmunizationhasbeenpracticed.Previously therewerefewerreportsofthediseaseinNjombeand Lushoto highlandsbutinrecenttimes,ECFcaseshavebeenreportedand thevectors arefrequently seen (Personalcommunications). No vaccinationhadbeenreportedinthesiteswheresamplingwas con-ductedinNjombeandLushotoandthecattlesampledatKibaha, LushotoandNjombehadnohistoryofco-grazingwithbuffaloes. Ruahaand Mikumi National parks are along theroad towards Njombeandtheyareabout247kmapart,theclosestNationalpark toNjombebeingRuahawhichis161kmfromNjombe.Mkomazi NationalParkislocatedintheNorthEasternTanzania(Fig.1)

2.2. Bloodsampling

Atotalof150bloodsampleswererandomlycollectedfrom cat-tlewhich havenohistoryofco-grazingwithbuffaloinNjombe, Kibaha and Lushotodistricts, 50 blood samplesbeing collected

Fig.1.AMapofTanzaniashowingsamplingareas.

Table1

Locationsandhostswherethesampleswerecollectfrom.

Location Numberofsample Host

fromeachdistrict.Tenmillimeterofbloodsamplefromeach ani-malwascollectedinEDTA Vacutainertubes (BectonDickinson, Oxford,UK)throughjugularveinpuncture,keptincoolboxand laterwerefrozenat−20◦_C_at_Central_Veterinary_Laboratory,_Dar

EsSalaam,Tanzania.Thebloodsamplesfrombuffaloandthe cat-tlesharingpasturewithbuffalowereobtainedfromthesample archiveatCentreforInfectiousdiseaseandBiotechnology,DarEs Salaam,Tanzania(Table1).

2.3. ScreeningforT.parvapositivesamples

DNA was extracted from thawed blood using a commer-cial extractionkit according tothe manufacturer’s instructions (PureLink® _Genomic_DNA_Mini_Kit,_Invitrogen,_USA)._All_samples

werescreenedforT.parvausinganestedpolymerasechain reac-tion(PCR)assaytargetingthe104kDaantigen(p104)gene(Skilton etal.,2002).TheassaywasrunintwoPCRrounds;theprimaryand secondaryPCR;usingouterprimersforprimaryPCR(For15′_-ATT

TAAGGAACCTGACGTGACTGC-3′_and_Rev1₅′_-TAA_GAT_GCC_GAC

TATTAATGACACC-3′_),_and_inner_primers_for_secondary_(nested)

PCR(For25-GGCCAAGGTCTCCTTCAGAATACG-3andRev25′_-TGG

GTGTGTTTCCTCGTCATCTGC-3′_)._Primers_were_designed_based

onp104antigenicgene(GenebankM29954).Inboth,primaryand secondaryPCR,BioneerAccuPower®_PCR_Pre-mixes_(Seoul,_South

Korea)wasused.TheprimaryPCRcomposedof17␮lnucleasefree

water,10pmoleofeachoftheprimers(ForandRev)and2␮lof

gDNAat 20ng/␮ltohavea ﬁnal volumeof 20␮l.Thereaction

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Table2

PanelofminiandmicrosatellitemarkersusedtogenotypeT.parvasamplesusedinthisstudywiththeirrespectivechromosomes,outernestedprimers(Ouraetal.,2003) andinnernestedprimers(Salihetal.unpublisheddata).

Outernestedprimers Innernestedprimers

Chromosome Name Sequence Name Sequence

1 ms2ForO aagttagtatcaccaccaggctgg ms2ForN gcccaatgtaccgagaatcctcac ms2RevO ggctcatctaccactccaactcc ms2RevN attctccgcattctccaccacctc 1 ms5ForO aacacagtaactaacccaggcc ms5ForN aatcttccaatcccaaccacatac

ms5RevO aactccagcggaatcccgaaata ms5RevN cccgaaataaaaccaaattccacc 2 ms7ForO tgggtagagattggaatacgcgag ms7ForN ttaacttatctccctctccctccc

ms7RevO aataccgacgcgttccgaggaatc ms7RevN acactctcaacaactcactcttcc 1 MS3ForO cccgatctcactcacatacaacc MS3ForN ccaccgtaaccctctataccat

MS3RevO cagcaaatccaactcgtcgtcctg MS3RevN gacatctccctcaaatcagactc 1 MS7ForO ctcctcagcatcctgctgctcattg MS7ForN gttcagtcctatggcaattcag

MS7RevO gcgcatgactgcttttacattaaccc MS7RevN caaacctcttcaaattcactctagg 1 MS8ForO ggcgtgacggtaatacaccttcc MS8ForN gcctcctcaagcaattcagta

MS8RevO cctcctagacactcccgaagatg MS8RevN ctggttcaaacacatcaaggtaca 2 MS16ForO catggcattcctaggcatcacatc MS16ForN cctcctccatactactaacctacc

MS16RevO ccaagggaattaatactgttggag MS16RevN cagcgctcagattcacttgtact 2 MS19ForO ccgcgtacagactaaactcgccg MS19ForN ccagacacctcaaatcccaagta

MS19RevO cctccaactgatccaccctcgcag MS19RevN ccacactgccacctaatacaaa 3 MS21ForO gatgagcacaaggagttcctggtg MS21ForN ttctacccaacgccactctatgcg

MS21RevO gacggcgtctgagatggcgatgac MS21RevN tgactcccgtcttatccaaattcg 3 MS25ForO ccagagatctcggacacaactcc MS25ForN acacacccatcaacgtagtaac

MS25RevO taaggtgccaaacggcggacac MS25RevN caccatcacactcttaaccat 3 MS27ForO ccgccacctcagtctcgaga MS27ForN cctgcgatacatttctaatcc MS27RevO cacaactcacaccggaatctcac MS27RevN gtaataccattcccacctctac 4 MS33ForO ctcattaccactctacccatcgc MS33ForN cttctcaaggtaccgtaaacc MS33RevO catggtccatcttcatctggtcc MS33RevN cctcactactccaatagttcttc 4 MS34ForO gtgcctagagagggaacggatatg MS34ForN gattacgccactgtcataaccacc

MS34RevO cctccggctgagattagtggcagg MS34RevN aacactccacgctccacattcacc 4 MS40ForO cggtggaggctcggatgtgtgc MS40ForN catatcacctcatggtacacac

MS40RevO gcaggatcaacatcgccaaccac MS40RevN ccagccctaatacacaaatc ms—Microsatellite.

MS—Minisatellite.

ForO—Forwardouternestedprimer. RevO—Reverseouternestedprimer. ForN—Forwardinnernestedprimer. RevN—Reverseinnernestedprimer.

control.TheampliﬁcationconditionsforprimaryPCRwere94◦_C

for1minfollowedby35cyclesof94◦_C_for₁_min,₆₀◦_C_for₁_min,

72◦_C_for₁_min_with_additional₁₀_min_at₇₂◦_C_as_ﬁnal_extension.

ThecomponentsofthesecondaryPCRremainedthesameasforthe primaryPCRexceptthetemplatewhichwasprimaryPCRproducts dilutedat1:10.Theampliﬁcationconditionswerethesameexcept theannealingtemperatureandthenumberofcycleswasreduced to55◦_C_and₃₀_cycles,_{respectively.}_All_{ampliﬁcations}_were_done

usingaprogrammablethermalcycler(MJResearch,Watertown, MA,USA).ThesecondaryroundPCRproductswereanalyzedby electrophoresisrunat100Vfor40minin2%agarosegelstained withGelRed(BiotiumInc.USA).

2.4. PCRamplificationandanalysisofminiandmicrosatelliteloci

ThenestedPCRdeployingtheouterandinnerprimersdesigned

byOuraetal.(2003)andSalihetal.(unpublisheddata)

respec-tively(Table2)wasperformedtoamplifyeachof11miniand3 microsatellitesusedinthestudyforeachsamplethatwas posi-tivebyp104nestedPCRampliﬁcation.Theinnerforwardprimer ofeachmarkerwasﬂuorescentlylabeledatthe5′ _end_with_one

ofthefollowingfourstandarddyes;6-FAM(Blue),NED(Yellow), PET(Red)andVIC(Green)(Bioneer,Korea),toenabledetectionon anABIgeneticanalyser.TheprimaryPCRampliﬁcationwasdone in10␮lcomprising of2␮lof 20ng/␮lgenomicDNA,0.08␮lof

0.5U/␮lDreamTaqpolymerase(ThermoScientiﬁc,Lithuania,EU),

0.2␮lof10mMdNTPs,0.4␮lofeachouterprimersat10pmoleand

1␮lof10XDreamTaqbufferwith20mMMgCl2(ThermoScientiﬁc,

Lithuania,EU).Thenucleasefreewaterwasusedasnegativecontrol andtheDNAsampleknowntobeT.parvapositivefromBecA-ILRI Hubsamplerepositorywasusedaspositivecontrol.Thecycling

conditionsfortheprimaryPCRwereasfollows;Initial denatura-tionat95◦_C_for₅_min_followed_by₃₅_cycles_of_denaturation_at₉₄◦_C

for30s,annealingat55◦_C_for₁_min,_extension_at₇₂◦_C_for₁_min

plusaﬁnalextensionat72◦_C_for₁₀_min._For_the_secondary_PCR,

allotherreagentsremainedthesameexceptthat0.5␮lofthe

pri-maryPCRwasusedasthetemplateandthevolumeofwaterwas increasedaccordinglytogiveatotalof10␮lreactionvolume.The

cyclingconditionsforthesecondaryPCRwereasfollows;Initial denaturationat95◦_C_for₅_min_followed_by₂₅_cycles_of

denatura-tionat94◦_C_for₃₀_s,_annealing_at₅₈◦_C_for₁_min,_extension_at₇₂◦_C

for1minplusaﬁnalextensionat72◦_C_for₂₀_min._Three_microliter

oftheampliconswereanalyzedona1.5%agarosegelstainedwith GelRed(BiotiumInc.USA)tocheckforampliﬁcationsuccess.One andhalftotwo␮lof2−4PCRproductsofthesamesample

ampli-ﬁedbyforwardprimerslabeledwithdifferentdyeswereco-loaded onthesamewellof96wellsplatewith8␮lmixtureofHiDiand

LIZ500mixedat1mlLIZ500and15␮lHiDi.Thesamplesonthe

plateweredenatureat95◦_C_by_thermocycler_for₃_min,_fast_cooled

onice for10minthentheplates wereloadedinanABI3730xl geneticanalyser(Appliedbiosystem)forcapillaryelectrophoresis

(Mwegaetal.,2014;Muleyaetal.,2012).Capillary

electrophore-siswascarriedoutatBioscienceeasternandcentralAfrica(BecA) sequencingandgenotypingunit.DNAfragmentsizeswere ana-lyzedbyLIZ500standardsizemarker(AppliedBiosystems)using GeneMappersoftware(AppliedBiosystems).

2.5. Categoriesofthepopulationusedinstudy

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Table3

Majorallelefrequency,genediversity,numberofallelesandpolymorphic informa-tioncontent(PIC)oftheminiandmicrosatellitemarkersusedtocharacterizing5 populationsandMugugaisolateusedinthisstudy.

Marker Majorallelefrequency Numberofallele Genediversity PIC

ms2 0.40 26.00 0.79 0.78

withbuffalo.Samplesfromtheseareasweretreatedasindividual populationsnamedasKibaha,NjombeandLushotopopulations. Samplesfrom buffaloes werecategorized as buffalo population regardlessoftheiroriginandthesamplesfromcattlewhichgraze closeorwithintheareaswherebuffaloesaregrazingwere catego-rizedascattleclosetobuffalopopulation.Mugugavaccinestrain wasalsoincludedinthestudyandwasreferredasMugugaisolate.

2.6. Dataanalysis

ThepowerMarkersoftware(LiuandMuse,2005)wasusedto calculatethediversityparametersofthemarkersusedinthisstudy wherebythemajorallelefrequency,genediversity,numberof alle-lesandpolymorphicinformationcomponent(PIC)ofeachmarker weregenerated.Allelicrichnessasthemeanacrossalllociforeach populationandAnalysisofMolecularvariance(AMOVA)was esti-matedusingGenAlEXsoftwareversion6.5(PeakallandSmouse 2012).The samesoftwarewasusedtocalculate Principle com-ponentanalysis(PCoA).DARwinVersion5software(Perrierand

Jacquemoud-Collet,2006)wasusedtoconstructthedendrogramto

determinetherelationshipamong5populationsusedinthestudy andtheMugugaisolate.

3. Results

3.1. ScreeningforT.parvaandPCRamplificationofminiand microsatellitemarkers

Atotalof105(70%)cattle(Lushoto41,Njombe32,Kibaha23and cattleclosetobuffalo9)and30(49%)buffalosampleswerepositive byp104amplification.Samplespositiveforp104PCRamplification wereamplifiedforminiandmicrosatellitemarkers.Outof135 sam-plessubjectedtominiandmicrosatellitemarkersamplification, 133samplesweresuccessivelyamplified,2sampleswerepoorly amplifiedbymostofthemarkersusedandwereexcludedfromthe study.

3.2. Markersandallelicvariation

Themajorallelefrequency,genediversity,numberofalleleand polymorphicinformationcomponentacrossall14makersranged from0.24(MS19)to0.64(MS21)withmeanof0.41,from0.57(MS 21)to0.92(MS19)withameanof0.79,from22(ms7)to91(MS 19)withmeanof48.4andfrom0.57(MS21)to0.93(MS19)with meanof0.79,respectively(Table3).MS19makerwasmorediverse

andinformativeandMS21waslessdiverseandinformativethan othermarkersusedinthisstudy.

3.3. T.parvapopulationdiversity

Thediversityacrosspopulationswasdeterminedbythemean numberofdifferentalleles,meannumberofeffectivealleles,mean numberofprivatealleleandexpectedheterozygosity.Themean numberofalleleuniquetopopulationsforCattleclosetobuffalo, Muguga,Njombe,Kibaha,Lushotoand Buffalopopulationswere 0.18,0.24,0.63,0.71,1.63and3.37,respectively.Themean num-berofdifferentallelesrangedfrom6.97(Buffalo)to0.07(Muguga). Mean number ofeffective alleles rangedfrom4.49(Buffalo) to 0.29(Muguga).Themeanexpectedheterozygositywere0.07,0.29, 0.45,0.48,0.59and0.64forMuguga,cattleclosetobuffalo,Kibaha, Njombe,LushotoandBuffalopopulations,respectively(Fig.2).The populationwhichwasclosetoBuffaloinmeannumberofdifferent alleles,effectivealleles,privateallelesandexpectedheterozygosity wasLushotoandthevalueswere6.71,4.17,1.63and0.59 respec-tively.

The results from constructed principle component analysis (PCA)showedthreepatternsofclustering,thebuffalogenotypes whichclusteredonthetoprightquadrantcomprisingthebuffalo individualsandfewgenotypesfromNjombe,KibahaandLushoto; thebottomrightquadrantcomprisingmostofLushoto,Kibahaand Njombeandfewbuffalogenotypes.Thelefttopanddownquadrant wastheadmixgroupcomprisingthegenotypefromall popula-tionsincludingMugugaisolate(Fig.3).Itisinthisquadrantonly wherethecattleclosetobuffalopopulationgenotypeswerefound. Analysisofmolecularvariance(AMOVA)showedhighwithin popu-lationvariation(90%)comparetoamongpopulationvariation(10%) whereasamongindividualvariationwas0%(Table4).

The dendrogramgenerated showed three main clusters, the clusterwheremostofthebuffalogenotypeswerefoundandformed itsownsub-clusterwithonegenotypefromNjombe;thecluster comprisinghighpercentageoftheindividualsfromeach popula-tionexceptMuguga isolate.Inthisclusterthereisasub-cluster comprising only thegenotypesfrom all3 populations ofcattle whichhavenohistoryofco-grazingwithbuffaloes.Thelastcluster wassmallclustercomprisingonegenotypefrombuffaloandsmall numberofgenotypesfromallotherpopulations.Itisinthiscluster wheretheMugugaisolatewasfound(Fig.4).

4. Discussion

Thepopulationgeneticanalysisstudydeploying11miniand 3microsatelliteswasconductedtodetermineT.parvapopulation diversity.Inthisstudyatotalof103and30bloodsamplescollected fromclinicallyhealthcattleandbuffaloes,respectivelywereused. Thestudyrevealedtheprevalenceof70%and49%ofT.parvain cattleandbuffaloesrespectively.Theproblemwasbiggerthanit waspreviouslythoughtbeforepossiblybecauseofthelower sen-sitivityofthemethodswhichareroutinelyusedcomparedtothe p104nestedPCRassaywhich isabletodetectevensub-clinical andcarriercases(Skiltonetal.,2002).Thereishighpossibilitythat mostoftheanimalssampledwereincarrierstate,aphenomenon whichiscommonfollowingnaturalinfectioninbothhostsorpost immunizationincattle(Ouraetal.,2007;Ouraetal.,2011).

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Fig.2. Allelicpatternspresentingmeannumberofdifferentalleles,effectivealleles,privateallelesandexpectedheterozygosityacross5populationsandMugugaisolates usedinthisstudy.

Fig.3.Principalcomponentanalysis(PCA)ofT.parvaasgeneratedfrom5populationsandMugugaisolate.Theproportionofthevariationinthedatasetisindicatedineach axis.Theanalysispresentsthreemainclusters;righttopquadrant(purpleovalbordered)comprisingmostofthebuffaloindividuals,lefttopandbottomquadrants(redoval bordered)presentingadmixedgroupandtherightbottomquadrant(blueovalbordered)clustercomprisingmostofNjombe,LushotoandKibahapopulationgenotypes.

Table4

Analysisofmolecularvariance(AMOVA)calculatedfrom5populationsandMugugaisolate.

Sourceofvariation Degreeoffreedom Sumofsquare MeanSquare EstimatedVariation Percentagevariation

AmongPopulations 5 128.67 25.73 0.82 10

WithinPopulations 135 1006.11 7.45 7.45 90

Total 140 1134.79 8.27 100

than0.5(NgonoEmaetal.,2014;Ya-Boetal.,2006).Consequently,

theresultsobtainedinthisstudyareinformativeandshouldbe con-sideredreliableasanassessmentforT.parvapopulationgenetic diversityin theareas assessed.Furthermore thehigh polymor-phismoftheminiandmicrosatellitemarkersusedisrevealedby highmeangenediversity(0.79)andrelativelyhighermeanmajor allelesfrequency(0.41).

TheBuffalopopulationshowedahighpolymorphismcompared tootherpopulationsstudied.Thisissupportedbylargermean num-berofdifferentalleles,effectivealleles,privateallelesandexpected heterozygositythanotherpopulations,followedbyLushoto. Buf-faloesarebelievedtocarryaheterogeneouspopulationofparasites

(Sibekoetal.,2011),therefore;theseresultswerenotsurprising.

Thepresenceofuniqueallelesinallpopulationsindicatethatthere isa uniquesetofisolatesin thepopulationsusedin thisstudy aseachalleleis arepresentativeofadifferentisolate.Thismay compromiseITM,theonlyavailablecontrolmethodofthis par-asitethroughimmunizationsinceguaranteeforcrossprotection amongdifferentisolatesisverylimited(Pelléetal.,2011;Patel etal.,2011).Thevariationoftheindividualswithinpopulationwas furtherrevealedbyAMOVAwhichshowed90%withinindividual variations.

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Fig.4.Theneighbor-joiningdendrogrampresentingtherelationshipamong5populationsusedinthisstudyandtheMugugaisolate.Majorityofthebuffaloindividuals formedoneseparatesub-clusterofindividualswhileotherbuffaloindividualsbeingrandomlydistributedinothercattleindividuals.Mugugaformedtheclusterwithfew genotypesfromother5populations.

andonlyalimitedproportionofthebuffalo-derivedpopulationcan beadaptedandtransmittedbetweencattle.Thisstudyrevealeda buffalo-derivedT.parvasub-clusterdifferentfromcattleisolates, thismayaffecttheeffectivenessofITMcontrolmethodsifparasite sharingoccursbetweenthesetwohosts.Parasitesharingismore likelytooccursincepasturesharingiscommonunderthenomadic systemwhichisprevailinginthecountry.

Previouslythere were few casesof ECFreported in Lushoto highlandsandNjombebutthisstudyshowsthatthereisahigh prevalenceoftheparasiteintheseareas.Occurrenceofthe para-siteintheseareasmaybecausedbyextensivemovementofcattle totheseareasforstockestablishment,replacementand search-ingforpasturesfromthelowlandofLushotoandotherpartsof thecountrywherethevectorandparasiteisdominant.Thereisno clearexplanationtowhytheLushotopopulationismorediverse thantherestofcattlepopulationsbutextensiveinﬂuxofthe cat-tlemaybethereason.Kibahahasbeenpracticingimmunization byITMformanyyearshence;itwasexpectedtobemorediverse thanothercattleisolatesasimmunizationincreasediversityofthe parasite(Weiretal.,2011).Genotypesimilarityanalysisshowed Mugugavaccineisolateclusteringwithgenotypesfromall popu-lationsusedinthestudyasanadmixgroup.Thiswasunexpected sincethereisnoimmunizationwhichhasbeenconductedinthese areasexceptatKibaha.Genotypesharingamongthesepopulations maybecausedbyunrestrictedmovementofcattleamongthese areas.These resultsare concurrent withthose of Mwegaet al.

(2014)whichrevealedgenotypesharingamongMugugavaccine

isolate,EasternandSouthernzoneisolatesalthoughtherewasno vaccinationexercisewhichhadbeenconductedinSouthernzone. Thisstudyrevealed thegenotypic populationstructure of T. parvainareaswheretheisolateswerecollectedfrom.Inthisstudy buffalo-derivedisolateswerefoundtobemorediversethanthe cattle-derivedisolates.Thissituationposesmoreriskonthe sus-tainabilityofITMinthefeatureduetouncontrolledmovementof cattlewhichmayleadsub-populationsofT.parvafrombuffaloes toadapttocattlesinceparasitesharingbetweenthesetwohostsis

possible.Ontheotherhandtherewasnostrongevidenceshownby thisstudyonparasitesharingbetweencattleandbuffaloesgrazing inthesamearea.Itisstillpossiblethatsharingofisolatesmaybe occurringbutwasnotevidentgiventherelativelysmall popula-tionexaminedinthisstudy.Therefore,itissuggestedthatfuture studiesbeconductedincludingmoresamplesfromcattlethatare co-grazingwithbuffaloes.Thiswillhelptoestablishthepotential risksofcross-infectionbetweencattleandbuffaloesgrazinginthe samearea.

Financialsupport

We want toacknowledge the financial supportprovided to theBiosciencesEastern and Central AfricaHubat International Livestock Research Institute (BecA-ILRI Hub) by the Australian AgencyforInternationalDevelopment(AusAID)througha partner-shipbetweenAustralia’sCommonwealthScientificandIndustrial ResearchOrganisation(CSIRO)andtheBecA-ILRIHub;andbythe SyngentaFoundationforSustainableAgriculture(SFSA);theBill &MelindaGatesFoundation(BMGF);andtheSwedishMinistry ofForeignAffairsthroughtheSwedishInternationalDevelopment Agency (Sida). The financial support granted by Eastern Africa AgriculturalProductivityProgramme(EAAPP)onfieldsamples col-lectionishighlyappreciated.

Conflictofinterest

(7)

Acknowledgement

We send our sincere gratitudeto Joyce Njuguna and James Wainainafortheirvaluablesupportondataanalysis.

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