low genetic diversity associated with low prevalence of anaplasma marginale in water buffaloes in marajc3b3 island brazil

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TicksandTick-borneDiseases5(2014)801–804

ContentslistsavailableatScienceDirect

Ticks

and

Tick-borne

Diseases

jo u r n al hom ep a g e :ww w . e l s e v i e r . c o m / l o c a t e / t t b d i s

Short

communication

Low

genetic

diversity

associated

with

low

prevalence

of

Anaplasma

marginale

in

water

buffaloes

in

Marajó

Island,

Brazil

Jenevaldo

B. Silva

a

_,

_Adivaldo

_H.

_Fonseca

b

_,

_José

_D.

_Barbosa

c

_,

_Alejandro

_Cabezas-Cruz

d,e

_,

José

de

la

Fuente

d,f,∗

a_Departamento_de_Patologia_{Veterinária,}_Universidade_Estadual_Paulista,_Jaboticabal,_São_Paulo,_Brazil b_Departamento_de_Clínica_{Veterinária,}_Universidade_Federal_do_Pará,_Castanhal,_Pará,_Brazil

c_Departamento_de_{Parasitologia,}_Universidade_Federal_Rural_do_Rio_de_Janeiro,_Seropédica,_Rio_de_Janeiro,_Brazil

d_SaBio_Instituto_de_{Investigación}_en_Recursos_Cinegéticos_IREC,_{CSIC-UCLM-JCCM,}_Ronda_de_Toledo_s/n,₁₃₀₀₅_Ciudad_Real,_Spain

e_Center_for_Infection_and_Immunity_of_Lille_(CIIL),_INSERM_U1019_–_CNRS_UMR_8204,_Université_Lille_Nord_de_France,_Institut_Pasteur_de_Lille,_Lille,_France f_Department_of_Veterinary_{Pathobiology,}_Center_for_Veterinary_Health_Sciences,_Oklahoma_State_University,_Stillwater,_OK_74078,_USA

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1April2014

Receivedinrevisedform21May2014 Accepted4June2014

Availableonline22July2014

Keywords: Anaplasmamarginale

Buffalo MSP1a Genetics Anaplasmosis

a

b

s

t

r

a

c

t

TherickettsiaAnaplasmamarginaleistheetiologicagentofbovineanaplasmosis,animportant tick-bornediseaseaffectingcattleintropicalandsubtropicalregionsoftheworld.Inendemicregions,the geneticdiversityofthispathogenisusuallyrelatedtothehighprevalenceofthediseaseincattle.The majorsurfaceprotein1alpha(MSP1a)hasbeenusedasamarkertocharacterizethegeneticdiversity andforgeographicalidentificationofA.marginalestrains.Thepresentstudyreportsthe characteriza-tionofA.marginaleMSP1adiversityinwaterbuffaloes.Bloodsampleswerecollectedfrom200water buffaloesonMarajóIsland,BrazilwherethelargestbuffaloherdislocatedintheWesternhemisphere. Fifteenbuffaloes(7.5%)werepositiveforA.marginalemsp1˛byPCR.FourdifferentstrainsofA.marginale withMSP1atandemrepeatstructures(4-63-27),(162-63-27),(78-24-24-25-31)and(␶-10-10-15)were

found,being(4-63-27)themostcommon.MSP1atandemrepeatscompositioninbuffalosand phyloge-neticanalysisusingmsp1˛geneshowedthattheA.marginalestrainsidentifiedinbuffaloesareclosely relatedtoA.marginalestrainsfromcattle.TheresultsdemonstratedlowgeneticdiversityofA.marginale associatedwithlowbacterialprevalenceinbuffaloesandsuggestedthatbuffaloesmaybereservoirsof thispathogenforcattlelivinginthesamearea.Theresultsalsosuggestedthatmechanicaltransmission andnotbiologicaltransmissionbyticksmightbeplayingthemajorroleforpathogencirculationamong waterbuffaloesinMarajóIsland,Brazil.

Introduction

Anaplasma marginale (Rickettsiales: Anaplasmataceae) is the most prevalent pathogen transmitted by ticks worldwide, dis-tributedonthesixcontinentsandresponsibleforhighmorbidity and mortality in cattle in temperate, subtropical, and tropi-cal regions (Vidotto et al., 1998; Kocan et al., 2010). Bacteria of the genus Anaplasma are obligate intracellular pathogens that can be transmitted biologically by ticks, mechanically by hematophagousinsectsandblood-contaminatedfomitesandless frequentlytransplacentally(Kocanetal.,2010).

TheglobaldistributionandhighpathogenicityofA.marginale isduetothediversityandgeneticvariabilityofthisbacterium(de

∗Correspondingauthorat:SaBio,InstitutodeInvestigaciónenRecursos Cinegéti-cos,RondadeToledos/n,13005CiudadReal,SpainTel.:+34926295450.

E-mailaddress:Josedelafuente@yahoo.com(J.delaFuente).

laFuenteetal.,2007).Thispathogenhasover20proteins capa-bleofinducingprotectiveimmunity(SuarezandNoh,2011)from whichmajorsurfaceproteins(MSPs)havebeenextensively char-acterized(Kocanetal.,2010).Amongthemajorsurfaceproteins (MSPs),specialattentionhasbeendirectedtoMSP1abecauseitis involvedintheinteractionofthebacteriumwithvertebrateand invertebratehostcells(delaFuenteetal.,2010).Severalstrains ofA.marginalehavebeenidentifiedworldwideandthesestrains differintheirmorphology,MSP1aaminoacidsequence,antigenic characteristics,andabilitytobetransmittedbyticks(delaFuente etal.,2007;Estrada-Pe ˜naetal.,2009;Cabezas-Cruzetal.,2013).

TheprimaryhostforA.marginaleiscattle,butotherruminants suchasdeerandbuffaloescanalsobeinfected(Kocanetal.,2010). Approximately300,000buffaloes aregeographicallyisolated on MarajóIsland,Brazil,representingthelargestbuffaloherdinthe Westernhemisphere,andtheseanimalshavebeenusedasa pri-marysourceofmeat,milk,andleather,besidesbeingusedtoplow thelandandtotransportpeopleandcrops(IBGE,2012).Serological

http://dx.doi.org/10.1016/j.ttbdis.2014.06.003

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802 J.B.Silvaetal./TicksandTick-borneDiseases5(2014)801–804

andmoleculardetectionofA.marginaleinwaterbuffaloesinBrazil haveshownaprevalenceof49.0%and5.4%,respectively(Silvaetal., 2014).However,althoughtheA.marginalemsp1˛geneticdiversity

hasbeencharacterizedinBraziliancattle(delaFuenteetal.,2007; Estrada-Pe ˜naetal., 2009;Cabezas-Cruzet al.,2013;Pohl etal., 2013),asimilarstudyhasnotbeenconductedinbuffaloes.

Inthisstudy,wecharacterizedtheA.marginalemsp1˛genetic

diversityinnaturallyinfectedwater buffaloesonMarajó Island, Brazil. The results demonstrated low genetic diversity of A. marginaleassociated tolow prevalenceofthebacteriain water buffaloes and suggested that buffaloes may be a reservoir of thispathogenforcattlelivinginthesamearea.Theresultsalso suggestedthatmechanicaltransmissionandnotbiological trans-missionbyticksmightbeplayinganessentialroleforpathogen circulationamongwaterbuffaloesinMarajóIsland,Brazil.

Materialsandmethods

Experimentaldesignandstudysite

A cross-sectional molecular study was conducted sampling buffalo herds in fourprovinces of Marajó Island, Brazil (Soure, Salvaterra,Muaná, andChaves)betweenJanuaryand December 2012.TheMarajóIslandhoststhelargestwaterbuffalopopulation intheWesternhemisphere.Thevegetationonthisislandis pre-dominantlyprovidedbytheAmazontropicalrainforest(Furtado etal.,2009).Thebuffaloesarevaccinatedagainstbrucellosisand foot-and-mouthdisease,butendoandectoparasitecontrolisrarely used.Largeareasofbogandgrasslandalongthefloodplainsofrivers arefoundonMarajóIsland(Furtadoetal.,2009).Theseanimals arerearedusinganextensivesystem.Themaintickspeciesfound onanimalsareAmblyommacajennense,Rhipicephalus(Boophilus) microplus,DermacentornitensandA.maculatum.Thesetickspecies canbefoundonbuffaloeswithlowinfestationratesthroughout theyear(Silvaetal.,2014).

SamplecollectionandDNAextraction

Twohundred female water buffaloes with approximately 3 yearsofagewererandomlyselectedinatleastthreefarmsfrom eachprovinceincludedinthestudy.Bloodsampleswerecollected fromthecaudalorjugularveinsofindividualanimals.DNA extrac-tionwasperformedusingtheQIAampDNABloodMiniKit(Qiagen, Valencia,CA,USA)followingmanufacturersrecommendations.

A.marginalemsp1˛PCRandDNAsequencing

Theprimers1733F(5′_{TGTGCTTATGGCAGACATTTCC}₃′_),_3134R

(5′_{TCACGGTCAAAACCTTTGCTTACC}₃′_),_and_2957R₍₅′

AAACCTTG-TAGCCCCAACTTATCC3′₎_were_used_to_amplify_A._marginale_msp1_˛

asreportedpreviously(Lewetal.,2002).Briefly,primers1733F and3134RwereusedinthefirstPCRamplification,while1733F and2957Rwereusedinanested-PCRreaction.Forbothreactions, 12.5␮lPCRMasterMix(Qiagen,Valencia,CA,USA),20pmolofeach primerand5␮lgenomicDNA(firstreaction)wereusedinafinal volumeof25␮l.Forthesecondreaction1␮loftheDNAamplified inthefirstreactionwasusedastemplate.Controlreactionswere performedinasimilarwaybutwithoutDNAaddedtoit.Afterthe PCRreaction,ampliconswerepurifiedwiththeSilicaBeadDNAGel ExtractionKit(FermentasLifeSciences,SaoPaulo,Brazil) follow-ingmanufacturer’sinstructionsandsequenced.TheA.marginale msp1˛sequencesobtainedinthisstudyfromwaterbuffaloesare

availableinGenBankwithaccessionnumbersKJ575588–KJ575602.

A.marginalemsp1˛sequenceanalysis

A microsatellite is located at the msp1˛ 5′ untranslated

region(UTR)betweentheputativeShine-Dalgarno(SD)sequence (GTAGG)and the start codon (ATG). The generalmicrosatellite

structureisaspreviouslyreportedGTAGG(G/ATTT)m(GT)nTATG (Estrada-Pe ˜naetal.,2009)wheremicrosatellitesequenceisinbold letters.TheSD-ATGdistancewascalculatedaccordingtothe equa-tion(4×m)+(2×n)+1.Basedonthestructureofthismicrosatellite

elevengenotypes(namedwithLatinalphabetlettersfromAtoK)of A.marginalemsp1˛havebeenpreviouslyidentified(Estrada-Pe ˜na

etal.,2009;Cabezas-Cruzetal.,2013).Theoreticaltranslationof msp1˛DNAintoaminoacidsequenceswasperformedusingthe

Expasy TranslationTool(http://expasy.hcuge.ch/tools/dna.html). Tandem repeats were identified and named according to the nomenclatureproposedbydelaFuenteetal.(2007)andupdated by Cabezas-Cruz et al. (2013). Tandem repeat sequences were alignedusing MUSCLE(v3.7) (Edgar,2004).Codonbased align-mentwasperformedusingthecodonsuiteserver(Schneideretal., 2007).Detectionofselectionpressureonindividualcodonswas calculatedusingtwomethods,singlelikelihoodancestorcounting (SLAC)andfixedeffectslikelihood(FEL)implementedin Datamon-keywebserver(Delportetal.,2010).Positiveornegativeselection wasassignedtocodonswhereω=dN(non-synonymous

substi-tutions)/dS(synonymoussubstitutions)ratiowashigherorlower than1,respectively.AsrecommendedinDatamonkeywebserver (Delportetal.,2010),onlysiteswithp-value<0.25wereconsidered tobeunderselection.

Phylogeneticanalysis

For msp1˛ phylogeneticanalysis,nucleotidesequenceswere

alignedwithMUSCLE(v3.7)configuredforhighprecision(Edgar, 2004)followedbyremovaloftheambiguousregionswithGblocks (v0.91b)(Castresana,2000).Thephylogenetictreewasconstructed usingtheneighborjoiningmethodimplementedinNeighborfrom thePHYLIPpackage (v3.66) (Felsenstein,1989).Internalbranch confidencewasassessedbythebootstrappingmethodusing1000 bootstrapreplicates.SequencesofA.marginalemsp1˛previously

reportedin cattlefromBrazilandtheUSAwere obtainedfrom Genebankandusedasoutgroups.

Resultsanddiscussion

LowprevalenceofA.marginalewasrecentlyreportedin buf-faloesinMarajóIsland,Brazil,usingthemajorsurfaceantigen5 (msp5)genemarker(Silvaetal.,2014).Theresultsobtainedinthe presentstudyusingmsp1˛agreedwiththosereportedbySilva

etal.(2014)andshowed7.5%(15positivesamples)prevalenceofA. marginaleinwaterbuffaloesfromMarajóIsland,Brazil.This preva-lencecouldbeconsideredlowwhencomparedwiththeprevalence ofA. marginalein cattlefromBrazil.Forexample,usingmsp1˛,

arecentstudyshowed70%prevalenceofA.marginaleina herd ofBraziliancattle(Pohletal.,2013).Waterbuffaloes with clin-icalanaplasmosiswerenotregisteredinthepresentstudy.The pathogenicsignificanceofA.marginaleforwaterbuffaloesremains tobeelucidated,butthefactthatbuffaloescancarryA.marginale raiseconcernsregardingtheroleofthisspeciesasreservoirsofA. marginaleforcattlelivinginthesamearea(Silvaetal.,2014). Phylo-geneticanalysisusingmsp1˛showthatA.marginalestrainsfound

inbuffaloesarecloselyrelatedtostrainsisolatedpreviouslyfrom cattleinBrazil(Fig.1A),suggestingthatbuffaloescanbeinfected withthesamestrainsthatinfectcattleandthusbuffaloescould constitutereservoirhostsforA.marginaleincattle.Furtherresearch isneededtoelucidatetheroleofwaterbuffaloesasreservoirhosts forA.marginaleincattleinthisorotherregionswherebothspecies sharethesamespace.

Thegenemsp1˛hasbeenextensivelyusedforthe

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J.B.Silvaetal./TicksandTick-borneDiseases5(2014)801–804 803

Fig.1.CharacterizationofA.marginalemsp1˛sequences.(A)NeighborjoiningphylogenetictreeofA.marginalemsp1˛.Thetreewasconstructedusingtheneighborjoining methodwithA.marginalemsp1asequencesfromstrainsidentifiedinwaterbuffaloesandcattle.Bootstrapvaluesarerepresentedaspercentoninternalbranches(1000 replicates).TheGenBankaccessionnumbersoftherespectivesequencesusedforthephylogeneticanalysisareshown.ThefourdifferentA.marginalestrainsobtainedfrom waterbuffaloesinthisstudyareshown(togetherwithtandemrepeatstructureinparenthesis)asWaterbuffalo3(78,24,24,25,31)(blacktriangle);Waterbuffalo13(4, 63,27)(whitesquare);Waterbuffalo15(162,63,27)(blacksquare)andWaterbuffalo4(␶,10,10,15)(whitetriangle).(B)Aminoaciddifferencesbetweentandemrepeats

4and162andpositionevolvingundernegativeselection.Theonelettercodeisusedforthedifferentaminoacidsofthetandemrepeats.Conservedaminoacidpositions arehighlightedwithasterisks.Substitutionofglutamine(Q)intandemrepeat4byleucine(L)intandemrepeat162isshowwithanarrow.Aminoacidatposition10(-) evolvingundernegativeselection(p<0.25usingbothFELandSLACmethods)andresiduesoftheimmunodominantB-cellepitope(Garcia-Garciaetal.,2004)(box)arealso shown.

etal.,2009;Cabezas-Cruzetal.,2013;Pohletal.,2013)but lit-tleisknownaboutthegeneticdiversityofA.marginaleinother speciesofungulates,includingbuffaloes(delaFuenteetal.,2004). InordertodeterminethegeneticdiversityofA.marginaleinfecting buffaloes,wesequencedthe15msp1˛positivesamplesthatwere

obtainedinthepresentstudy(Table1).Theresultsshowedthatthe geneticdiversityofA.marginalemsp1˛inbuffaloesfromMarajó

Islandislow,withonlyfourdifferentstrainsidentified,showing themicrosatellitegenotypeE(Table1).Incontrast,theresultsby Pohletal. (2013)incattleshowed,in13sequencedsamples,8 differentstrainsofA.marginalewithfourdifferentmicrosatellite genotypes(B,D,EandG).Threepossibilitiescouldbeconsidered inordertoexplainthelowgeneticdiversityofA.marginalein buf-faloesinMarajóIsland:(a)inbovineanaplasmosisendemicregions, lowgeneticdiversityofA. marginalemsp1˛hasbeenrelatedto

Table1

OrganizationofMSP1atandemrepeatsinA.marginalestrainsidentifiedinwater buffaloes.

A.marginalestrainidentification No.ofanimalsinfected withthisstrain

Brazil/MarajóIsland/E–(4,63,27) 9 Brazil/MarajóIsland/E–(78,242_,_25,₃₁₎ ₃ Brazil/MarajóIsland/E–(␶,102_,₁₅₎ ₂ Brazil/MarajóIsland/E–(162,63,27) 1

Strainidentificationisbasedonmsp1˛andincludesCountry/Locality/microsatellite genotype–(tandemrepeatsstructure).Superscriptsrepresentthenumberoftimes thatatandemrepeatsarerepeated.ThenewMSP1atandemrepeat162wasnamed followingthesystemproposedbydelaFuenteetal.(2007)andupdatedby Cabezas-Cruzetal.(2013).

tickabsence(Ruybaletal.,2009).Mostofthesampledbuffaloesin thisstudywereraisedonsubmergedwetlands,wheretick attach-mentisrare(Silvaetal.,2014)andthustickinfestationratesare lowandtransmissionofA.marginaleisanunlikelyevent;(b)cattle movementhasbeenproposedasasourceofgeneticdiversityinA. marginaleworldwide(delaFuenteetal.,2007).InMarajóIsland,the entryofnewbuffaloesisprohibited,limitingthepossibilityofthe introductionofnewstrainsofA.marginaleandconsequently bacte-rialgeneticdiversityinthisarea.Thisphenomenonisinagreement withthefactthatcattlemovementislimitedinAustraliawhere onlyonestrainofA.marginalehassofarbeenidentifiedincattle (Lewetal.,2002);(c)finally,itcouldbearguedthatA.marginalewas justrecentlyintroducedinthisbuffaloherd,whichwillresultin lowgeneticdiversity.Lowgeneticdiversityofmsp1˛wasreported

inapreviouslyuninfectedcattleherdwhereonlyasinglemsp1˛

genotypewasfound(Palmeretal.,2001).

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804 J.B.Silvaetal./TicksandTick-borneDiseases5(2014)801–804

buffaloes. In agreement with this hypothesis, the phylogenetic analysisusingmsp1˛indicatedthatthestrainWaterbuffalo 15

(162,63,27)possiblyevolvedfromstrainWaterbuffalo13(4,63, 27)(Fig.1A).Inordertodeterminewhichselectivepressurescould betriggeringMSP1adiversificationinA.marginalefrombuffaloes, theratioωwascalculatedshowingthatcodonatposition10from

tandemrepeat4wasevolvingundernegativeselection(Fig.1B). Interestingly,this aminoacidposition ispresent in an immun-odominantB-cellepitopedescribedbeforeforA.marginaleMSP1a (Garcia-Garciaetal.,2004)(Fig.1B).Theseresultssuggestedthat thistandemrepeatwhichwaspresentinthemostcommonstrain ofA.marginalefoundinbuffaloes(Table1)maybeunderselective pressurebythehostimmunesystem(Garcia-Garciaetal.,2004).

Someofthetickspeciesfoundinfestingbuffaloessuchas Rhipi-cephalusandDermacentorspp.havebeenrecognizedasvectorsof A.marginale(Kocanetal.,2010).However,thelowtickinfestation ratesfoundinbuffaloesinthestudyareasuggestedthatmechanical and/ortransplacentaltransmissioncouldbeplayinganimportant roleinA. marginaletransmissioninthisbuffalo herd.The suck-inglice,HaematopinustuberculatuswasimplicatedrecentlyinA. marginaletransmissionandoutbreaksofthislicespecieshavebeen reportedinbuffaloes(DaSilvaetal.,2013).Differentialtick trans-missionfitnesshasbeenfoundamongdifferentA.marginalemsp1˛

genotypes(Palmeretal.,2004).Consideringthatticksmaynotbe playinganimportantroleintransmissionamongbuffaloesinthe studysite,themostcommonstrainfoundinwaterbuffaloesmay beadaptedtomechanicalortransplacentaltransmission.In agree-mentwiththesefindings,60%oftheA.marginaleMSP1atandem repeatsobtainedherepresentedtheaminoacidglycine(G)at posi-tion20andthisaminoacidwasinatleastoneoftheMSP1arepeats inalltheA.marginalestrains.Thenegativelychargedaminoacids asparticacid(D)andglutamicacid(E)atposition20wereshown tobeessentialforthebindingofMSP1atotickcellswhilewithaG atthispositionnobindingwasobserved(delaFuenteetal.,2003). TheseaminoacidsaffectMSP1aconformationandthese conforma-tionalchangesweresuggestedtoaffectA.marginaletransmission byticks(Cabezas-Cruzetal.,2013).

Conclusions

Inthisstudy,thegeneticdiversityofMSP1ainA.marginalewas characterizedinwaterbuffaloes.TheA.marginalegeneticdiversity waslowinbuffaloesandcorrelatedwiththelowbacterial preva-lenceinthisspecies.Onemajorfactorthatcouldbecontributingto thislowgeneticdiversityistheecologyofthestudiedarea,whichis notsuitableforticksthusreducingtheprobabilityforpathogen bio-logicaltransmission.Mechanicaltransmissionbyhematophagous Dipteracouldbeplayinga majorrole in thetransmissionof A. marginalein thestudysite. Evidence wasfoundtosupportthe hypothesis that MSP1a is under selective pressure by thehost immunesysteminbuffaloes.Finally,waterbuffaloesmayserveas reservoirhostsofA.marginaleforcattle.Theseresultsexpandedour knowledgeofA.marginalestrainsandprovidedadditionalsupport fortheroleofMSP1ainpathogenevolutionandtransmission.

Acknowledgments

WethanktheCoordinationfortheImprovementofHigher Edu-cationPersonnel(CAPES)foundationandtheNationalCouncilfor TechnologicalandScientificDevelopment(CNPq)fortheirfinancial support.

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