Detection and phylogenetic characterization of Theileria spp. and Anaplasma marginale in Rhipicephalus bursa in Portugal

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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

Detection

and

phylogenetic

characterization

of

Theileria

spp.

and

Anaplasma

marginale

in

Rhipicephalus

bursa

in

Portugal

Joana

Ferrolho

a

_,

_Sandra

_Antunes

a

_,

_Ana

_S.

_Santos

b

_,

_Rita

_Velez

b

_,

_Ludovina

_Padre

c

_,

Alejandro

Cabezas-Cruz

d

_,

_Maria

_Margarida

_Santos-Silva

b

_,

_Ana

_Domingos

a,∗

a_Instituto_de_Higiene_e_Medicina_Tropical_–_Global_Health_and_Tropical_Medicine,_Rua_da_Junqueira_100,_1349-008_Lisboa,_Portugal

b_Centro_de_Estudos_de_Vetores_e_Doenc¸_as_Infeciosas_Dr._Francisco_Cambournac,_Instituto_Nacional_de_Saúde_Dr._Ricardo_Jorge_I.P.,_Águas_de_Moura,_Portugal

c_Universidade_de_Évora,_Évora,_Portugal

d_Center_for_Infection_and_Immunity_of_Lille_(CIIL),_INSERM_U1019_–_CNRS_UMR_8204,_Université_de_Lille,_Institut_Pasteur_de_Lille,₁_rue_Professeur_Calmette,

59019LilleCedex,France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25August2015

Receivedinrevisedform9December2015

Accepted10January2016 Availableonlinexxx Keywords: Rhipicephalusbursa Tick-bornepathogens Theileriaannulata Theileriaequi Anaplasmamarginale Portugal

a

b

s

t

r

a

c

t

Ticksareobligatoryblood-suckingarthropod(Acari:Ixodida)ectoparasitesofdomesticandwildanimals

aswellashumans.Theincidenceoftick-bornediseasesisrisingworldwide,challengingourapproach

towarddiagnosis,treatmentandcontroloptions.RhipicephalusbursaCanestriniandFanzago,1877,a

two-hosttickwidelydistributedinthePalearcticMediterraneanregion,isconsideredamulti-hosttick

thatcanbecommonlyfoundonsheep,goatsandcattle,andoccasionallyonhorses,dogs,deerand

humans.R.bursaisaspeciesinvolvedinthetransmissionofseveraltick-bornepathogenswithaknown

impactonanimalhealthandproduction.TheaimofthisstudywastoestimateR.bursaprevalencein

PortugalMainlandandcirculatingpathogensinordertocontributetoabetterknowledgeoftheimpactof

thistickspecies.AnaplasmamarginaleandTheileriaspp.weredetectedandclassiﬁedusingphylogenetic

analysis.ThisistheﬁrstreportofTheileriaannulataandTheileriaequidetectioninR.bursaticksfeedingon

cattleandhorses,respectively,inPortugal.Thisstudycontributestowardtheidentiﬁcationofcurrently

circulatingpathogensinthistickspeciesasaprerequisitefordevelopingfutureeffectiveanti-tickcontrol

measures.

1. Introduction

Duringthelasttwodecades,agrowinginterestintick-borne diseasesfromveterinary,medical,andpublichealthperspectives has emerged (Jongejan and Uilenberg, 2004). The worldwide prevalenceofthesediseasesissteadilyrising,challenginghowwe approachdiagnosis,treatmentandpreventativecontrolmeasures, andunderliningtheimportanceoftheOneHealthconcept(

Dantas-Torresetal.,2012).Ticksareknowntohaveasigniﬁcantimpact

onhostspeciesthroughtheirfeedingbehavior,causingdirectskin and sub-cutaneous tissue damage and blood depletion, whilst actingasvectorsofdifferentpathogens,suchasviruses,bacteria,

∗ Correspondingauthor.

E-mailaddresses:joana.ferrolho@ihmt.unl.pt

(J.Ferrolho),santunes@ihmt.unl.pt(S.Antunes),asps@vodafone.pt(A.S.Santos),

ritavlz@yahoo.co.uk(R.Velez),lpadre@ue.pt(L.Padre),

cabezasalejandrocruz@gmail.com(A.Cabezas-Cruz),

m.santos.silva@insa.min-saude.pt(M.M.Santos-Silva),adomingos@ihmt.unl.pt

(A.Domingos).

protozoa or fungi (Bell-Sakyi et al., 2007; Colebrook and Wall, 2004).Itisestimatedthatapproximately10%oftickspeciesexert anactiveroleasbiologicalvectorsinthetransmissionoftick-borne pathogens,includingseveralzoonoticagents(Heymanetal.,2010;

JongejanandUilenberg,2004;LabudaandNuttall,2004).Amongst

thesetickspeciesisRhipicephalusbursaCanestrini andFanzago, 1877, classiﬁed in the Ixodidae family (Walker et al., 2000). Epidemiologicalstudieshave identiﬁedR.bursaasbeingwidely distributedintheMediterraneanregionwheretheclimateis typi-callycharacterizedbylongdrysummersandcoldwinters(Walker

etal.,2000;Yeruhametal.,1985).Consideredamulti-hosttick,

theprimaryhostsofthisspeciesincludecattle,sheep,andgoats

(Santos-Silvaetal.,2011;Walkeretal.,2000).Thoughless

com-mon,thistickcanalsobefoundinotherdomesticanimals,aswell asinwildungulatesandsmall-mediumsizedmammalsand spo-radically,inhumans(delaFuenteetal.,2004b;Mihalcaetal.,2012;

Psaroulakietal.,2006;Santos-Silvaetal.,2011;Sattaetal.,2011;

Walkeretal.,2000).R.bursahasbeendescribedasbeinginvolved

inthetransmissionofagentsofthegenusAnaplasma(delaFuente

etal., 2004a),Babesia(Altayetal., 2008;M’Ghirbietal.,2010),

http://dx.doi.org/10.1016/j.ttbdis.2016.01.004

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Theileria(Garcia-Sanmartinet al.,2008), Rickettsia(Boudebouch

etal.,2009;deSousaetal.,2006;Ioannouetal.,2011;Toledoetal.,

2009)amongothers,withaknownimpactonanimalhealth.The mainobjectiveofthisstudywastoprovideup-datedinformation regardingthecurrentlycirculatingpathogensinR.bursaandtheir phylogeneticcharacterization,inPortugalMainland,forthefuture developmentandplanningofeffectivetickcontrolmeasures.

2. Materialsandmethods

2.1. Tickssamples

Atotalof266R.bursatickswereincludedinthisstudy.Ticks werecollectedfrom2007to2014in24localadministrativeunits– municipalities(LAUI)belongingto11outofthe28Mainland Por-tuguesesubregions(NomenclatureofTerritorialUnitsforStatistics regions–NUTSIII),includingtheintermunicipalcommunity(IMC) ofMinho-Lima,IMCCávado,IMCAve,AltoTrás-os-Montes,IMC Douro,Beira InteriorSul,IMC MédioTejo, AltoAlentejo, Penin-suladeSetúbal,IMCAlentejoLitoralandIMCBaixoAlentejo.Fig.1

showsthenumberofR.bursaspecimenscollectedandthe loca-tions,accordingtogeographicalcoordinatesandsubregions(QGIS 2.4.0. Chugiak). Ticks wereeither removed from domestic ani-malsbylocalveterinariansorcollectedbyflagging/draggingthe vegetationandfurtheridentifiedtospecieslevelusing morpho-logical keys,as previously described (Santos-Silva et al., 2011). Afteridentification, ticks werepreserved in 70% alcohol, sepa-ratedaccordingtoinstars,originandsiteofcollection,untilfurther manipulation.

2.2. DNAextraction,PCRscreeningandampliconsequencing Each tick was recovered from ethanol, rinsed in pH 7.4 phosphate-bufferedsaline(PBS),homogenizedandusedforDNA extractionusingTriReagent(Sigma–Aldrich,Lisbon,Portugal),as previously described (Antunes etal., 2015).DNA concentration andpuritywasaccessedbyspectrophotometry(ThermoScientiﬁc NanoDrop2000,Lisbon,Portugal).DNAwasstoredat−20◦_C_for

downstreamapplication.

AninitialscreeningtovalidateDNAextractionwasperformed inagroupofsamplesrandomlyselected,representing20%ofall extractedticks.UsingtheprimerpairT1B/T2Athattargetsa360bp fragmentoftickmitochondrial12SrDNA,aPCRwasperformedas previouslydescribed(BeatiandKeirans,2001).

Toamplify Anaplasma spp. and Ehrlichia spp. a broad range PCRscreenwiththeprimersEHR16sD/EHR16sRwasconducted asreportedbefore(Inokumaetal.,2000).Thisprimerset ampli-ﬁesa345bpfragmentofthe16SrRNAgeneofbacteriawithinthe familyAnaplasmataceae,includingthegeneraAnaplasma,Ehrlichia, Neorickettsia,and Wolbachia. For piroplasms, a PCR targeting a 408bpfragmentfromthesmallsubunitof18SrDNAofBabesiaspp. andTheileriaspp.wasconductedusingtheprimersetPiro-Aand Piro-Basdescribedelsewhere(Harrusetal.,2011).Todetect Cox-iellaburnetiiDNA,anested-touchdownPCRwasdoneusingthe primerpairsTrans1/2followedbyTrans3/4thatamplifya243bp fragmentoftherepetitiveinsertionelementIS1111(Lorenzetal., 1998).PrimerswereobtainedfromStabVida(Lisbon,Portugal).PCR wereperformedin25␮lreactionswithSupremeNZYTaq2×Green MasterMix(NZYTech,Lisbon,Portugal),1␮Mprimersandupto 5␮loftemplateDNA.Nuclease-freewaterwasusedasnegative control.Aspositivecontrols,DNAextractedfromreferencestrains was used: Anaplasma marginale Va-48 strain, Babesia bigemina Israelstrain,C.burnetiiNineMilestrain(Vircell,Spain)andTheileria annulata(Uzbekstrain).AmpliﬁcationswereperformedinaT100 thermalcycler(Biorad,Amadora,Portugal)accordingtoreferences

Fig.1. GeographicallocationsandnumberofRhipicephalusbursafoundaccordingto

NUTSIIISubregions.MapwasperformedusingtheQGIS2.4.0.Chugiakprogram.

Cir-cles–Correspondstotheexactcoordinatesofcollectionsitesinpreviouslyreported

municipalities;Triangles–Correspondstotheexactcoordinatesofcollectionsites

innewmunicipalities;I–Intermunicipalcommunity(IMC)ofMinho-Lima,II–IMC

Cávado,III–IMCAve,IV–AltoTrás-os-Montes,V–IMCDouro,VI–BeiraInterior

Sul,VII–IMCMédioTejo,VIII–AltoAlentejo,IX–PeninsuladeSetúbal,X–IMC

AlentejoLitoral,XI–IMCBaixoAlentejo.

(Harrusetal.,2011;Inokumaetal.,2000;Lorenzetal.,1998).

Pos-itiveampliconswerepuriﬁedusingtheNZYGelpurekit(NZYtech, Lisbon,Portugal)andsentforSangersequencingatStabVida (Lis-bon,Portugal).Theobtainedsequenceswerealigned,comparedto thosealreadydepositedintheNCBInucleotidedatabase(http://

blast.ncbi.nlm.nih.gov/Blast).

2.3. Phylogeneticanalysis

ThephylogeneticanalyseswereconductedwithA.marginale, Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Ehrlichiachaffeensis,Ehrlichiaewingii,Ehrlichiacanis16SrDNA (fam-ilyAnaplasmataceae)andTheileriaspp.18SrDNA(Apicomplexan) nucleotidesequencesalignedwithMAFFT(v7)conﬁguredforthe highestaccuracy(KatohandStandley,2013).Afteralignment,the sequenceswerecuredusingGblocks(Castresana,2000)withthe followingparameters:minimumlengthofablockaftergap clean-ing:10,nogappositionswereallowedintheﬁnalalignmentand allsegmentswithcontiguousnonconservedpositionsbiggerthan

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Table1

ResultsofPCRscreeningfortick-bornepathogensinRhipicephalusbursafromMainlandPortugal.

Subregions/no municipalitiesa

Noofpositives/testedticksb _Sequencing

results

Overall B.taurus C.familiaris C.hircus E.caballus O.aries S.scrofa Vegetation

IMCMinho-Lima 4 33 – – – – – – 15m;18f

IMCCávado 1 3 3m – – – – – –

IMCAve 1 2/23 – – – 2/9m;14f – – – 2T.equi

AltoTrás-os-Montes 2 6 3m;2f 1m – – – – –

IMCDouro 1 3 – 3f – – – – –

BeiraInteriorSul 1 1/4 – – – – 1/4f – – 1A.marginale

IMCMédioTejo 1 1 – – – – – – 1m

PeninsuladeSetúbal 1 2/6 – – – – – – 3m;2/3f 1A.marginale

AltoAlentejo 7 1/61 1f;43n;3l – 3m;1/7f 1m;1f;1n – 1n – 1A.marginale

IMCAlentejoLitoral 2 4/120 2/65m;2/49f 2m;3f – – 1m – – 3c_A._marginale;

1T.annulata

IMCBaixoAlentejo 3 6 3f – – – 3f – –

Total 24 10/266 2/71m;2/55f; 43n;3l 3m;6f 3m;1/7f 10males, 15females, 1nymph 1m;1/7f 1n 19m;2/21f

a_Portuguese_Mainland_according_to_Nomenclature_of_Territorial_Units_for_Statistics_regions_–_NUTS_III_and_local_{administrative}_units_or_{municipalities}_(LAU_I).

b_According_to_tick_instars:_m-male;_f-female;_n-nymph;_l-larva.

c _One_male_and_two_females_with_Anaplasma_marginale.

4wererejected.Twophylogenetictreeswerebuilt,onefor mem-bersofAnaplasmataceaeandtheotherforApicomplexanparasites. Phylogenetictreeswerereconstructedusingneighborjoining(NJ) based on Kimura 2-parameters genetic distances. Phylogenetic inference and model selection were performed using MEGA 6

(Tamuraetal.,2013).Thereliabilityfortheinternalbranchesof

NJwasassessedusingthebootstrappingmethod(1000bootstrap replicates).Graphicalrepresentationandeditingofthe phyloge-netictreeswereperformedwithEvolView(Zhangetal.,2012).The accessionnumbersandgeographicoriginsoftheisolatesusedin thephylogeneticanalysisareshowninthephylogenetictrees.The sequencesobtainedinthisstudyweresubmittedtoGenBankand identiﬁedwiththerespectiveaccessionnumber.

3. Results

Fromthe266R.bursastudied,226werefoundfeedingon ani-mals(88males,90females,45nymphsand3larvae)and40were questingticks(19malesand21females).Aspreviouslymentioned, ticks wereobtained from 24 municipalities that correspondto 11PortugueseMainland subregions(NUTSIII).Fig.1showsthe numberandgeographicallocationofR.bursaaccordingtothose subregions.MostofthetickswereobtainedfromAlentejoLitoral (45.1%),followedbyAltoAlentejo(22.9%),Minho-Lima(12.4%)and Ave(8.6%).Asmallernumberoftickswerefoundintheremaining subregions,rangingfrom2.3to0.4%(Table1).Ofnoteisthefact thattwoofthesevenmunicipalitiesfromAltoAlentejorepresent newdatarecordsforcurrentR.bursadistribution(AlterdoChão andCrato)astheoccurrenceofthistickspecieswasnotpreviously reportedinthoseplaces(Fig.1).Regardingseasonality,adultswere mostlyfoundduringspringandsummermonths,contrastingwith immaturesthatwereobtainedfromfalltowinter.Considering tick-hostassociations,R.bursawereremovedfromcattle(Bostaurus; N=172),followedbyhorses(Equuscaballus;N=26),goats(Capra hircus;N=10),sheep(Ovisaries;N=8),dogs(Canisfamiliaris;N=9) andpigs(Susscrofa;N=1)(Table1).Tickswereobservedinbody locationsasvariedasthehead,namelyintheinnersurfaceofthe ears;abdominalarea,includingscrotumandudder;perineum,tail andlimbs.

PCRscreeningforthepresenceoftick-bornepathogensrevealed an overall prevalenceof infection in R. bursaof approximately 3.8%(10/266). Seventicks(2.6%) werepositive for 16SrDNAof Anaplasma/Ehrlichiaspp. and three were positive for 18S rDNA

subunitofBabesia/Theileriaspp.(1.1%).Ampliconsequencing con-ﬁrmedthepresenceofA.marginale,T.annulataandTheileriaequiin R.bursaticks.A.marginaleinfectedtickswereobtainedfrom cat-tle(N=3,1maleand2females),sheep(N=1,female),goat(N=1, female),andvegetation(N=2,females),inthefoursubregionsIMC AlentejoLitoral,BeiraInteriorSul,AltoAlentejoandPeninsulade Setúbal,respectively.T.annulatawasfoundinonemalefeedingin cattlefromIMCAlentejoLitoral;whereasT.equiintwomales feed-inginhorsesfromIMCAve.NoC.burnetiiDNAwasdetectednor thepresenceofco-infections.

Phylogenetic analysis using 16S rDNA from different world-wideisolatesofA.marginaleandotherpathogenscloselyrelated asA.platys,A.phagocytophilum,E.canis,Ehrlichiaruminantium,E. ewingii and E.chaffeensisconﬁrmedinfection withA. marginale (Fig.2).Blastanalysisshowedthat thefoursequencesobtained bothfromquestingandcattleparasitizingticks(withthe acces-sionno.A.marginalePortugalKT004409,KT004411andKT004412 inFig.2)presentedbetween98and100%identifytothebovine strainKNP/M8/a (labeledasA.marginale SouthAfricaaccession no.KC189852inFig.2).Theremainingsequencesfromticks col-lectedfromsmallruminantsandvegetation(withtheaccession no.A.marginalePortugalKT004414,KT004410andlabeledasA. marginalePortugal.2inFig.2)presentedbetween97and99% iden-tifytothecloneLuzon(labeledasA.marginalePhilippinesaccession no.LC007100inFig.2).ThetwosequencesfromT.equidetectedin aninfectedhorse(withtheaccessionno.T.equiPortugalKT004407 andKT004408inFig.3)clusteredtogetherwithT.equiisolatesfrom BrazilandSouthAfrica.Finally,anisolatedlabeledasTheileriasp. PortugalKT004406(Fig.3)didnotshowclearphylogenetic rela-tionshipwithanyoftheTheileria spp.usedinthephylogenetic analysis.However,thisisolateshowed96%similaritytoasequence detectedin Hyalommaanatolicum fromTajikistan(labeled asT. annulataTajikistanaccessionno.KM288519inFig.3).

4. Discussion

R.bursaiswidelydistributedacrosstheMediterraneanregion, includingPortugal. InPortugal thepresenceof this tickspecies wasﬁrstrecognizedin1943byAboim-Inglês(Tendeiro,1963),and subsequentlyreportedinseveralareasandondifferentvertebrate hosts(deSousaetal.,2011;Santos-Silvaetal.,2011,2012,2014).In thepresentstudy,R.bursawerecollectedfromsixspeciesof domes-ticanimals(B.taurus,E.caballus,C.hircus,O.aries,C.familiarisand

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Fig.2.PhylogenetictreeofAnaplasmataceaeusing16SribosomalRNAgene sequences.TheevolutionaryhistorywasinferredbyusingtheNJmethodbased

ontheKimura2-parametermodel.16SrDNAgenenucleotidesequencesfromA.

marginale,A.phagocytophilum,A.platys,E.canis,E.chaffeensis,E.ewingii,E.canis

(familyAnaplasmataceae)wereused.Accessionnumbersofsequencesareshown

andisolatesfromPortugalreportedinthisstudyaremarkedwith“*”.Numeralson

branchesarebootstrapvalues(1000replicates).R.prowazekiiwasusedasoutgroup

toAnaplasmataceae.Anaccessionno.wasnotattributedtotheisolateA.marginale.

Portugal.2duetotheshortsizeofthesequence.

Fig.3. PhylogenetictreeofTheileriausing18SribosomalRNAgenesequences.

TheevolutionaryhistorywasinferredbyusingtheNJmethodbasedontheKimura

2-parametermodel.18SrDNAgenenucleotidesequencesfromT.buffeli,T.equi,T.

annulata,T.parva,T.ovis,T.capreoli,T.luwenshuni,T.sinensis,T.sergenti,T.

uilen-bergi(OrderPiroplasmida)wereused.Accessionnumbersofsequencesareshown

andisolatesfromPortugalreportedinthisstudyaremarkedwith“*”.Numeralson

branchesarebootstrapvalues(1000replicates).Babesiaoviswasusedasoutgroup

toTheileria.

S.scrofa),allpreviouslyincludedinthelistofhostsparasitizedby thistickspecies.Moreover,thisreportalsoconﬁrmstheoccurrence ofR.bursain22outofthe68priorrecordedmunicipalities,from atotalof278Mainland LAU(deSousaetal.,2011;Santos-Silva

etal.,2011,2012,2014).Newdataongeographicaldistribution

includestheidentiﬁcationofthisspeciesintwoadditional munic-ipalitiesfromthesubregionofAltoAlentejo.Interestingly,thisis alsotheﬁrstreportofR.bursalarvaedetectioninPortugal.The threelarvae werecollectedfromcattlefromAltoAlentejo; and togetherwithagroupof45nymphs,reinforcestheassociationof theimmatureinstarstoungulates,asdiscussedinpreviousstudies

(Santos-Silvaetal.,2011).ThepresenteddatashowsthatR.bursais

activeduringallyear,withamarkedseasonalbehavioraccording tothedevelopmentalstage,withadultsbeingpresentmostly dur-ingthespring-summermonths(MaytoSeptember)andimmature inautumn-winter(OctobertoJanuary–February).Thisisin agree-mentwiththeﬁndingsofotherstudies,althoughthemajorityof theavailabledatareferstoadultticks(deSousaetal.,2011;

Santos-Silvaetal.,2011,2012,2014).Althoughthehighestnumbersof

tickswerecollectedinthesummer(75.2%),thismightreﬂectnot onlyspeciesseasonaltendencybutalsothetickactivesurveillance duringcattlevaccinationcampaignsandotherlivestock manage-ment.FewsurveysconductedinPortugalhavefoundnymphsofR. bursa,andallspecimenswereobtainedoncattle,goats,sheepand

mice(Santos-Silvaetal.,2011).Nevertheless,thereduced

num-berofreportsregardingimmaturesislikelytobeduetoalimited screeningof ungulates duringcoldermonths.During ourstudy wehave foundanequivalentratioof male:female. Althougha similarratioofmale:femalehasbeenreported(Garcia-Sanmartin etal.,2008),moststudiesreportthatthenumberofmales gener-allyoutnumbersthenumberoffemales(Moshaveriniaetal.,2012;

Papadopoulosetal.,1996;Yeruhametal.,1996).Asmentioned,the

primaryhostsofthistickspeciesincludecattle,sheep,andgoats; andlessfrequentlydogsandsmallermammals,suchasrabbits(de

laFuenteetal.,2004a;Masalaetal.,2012;Mihalcaetal.,2012;

Psaroulakietal.,2006;Santos-Silvaetal.,2011;Sattaetal.,2011;

Walkeretal.,2000).Here,R.bursatickswereremovedmainlyfrom

largemammalssuchascattleandhorses;andinlessernumbers fromgoats,sheep,anddogs.Thisresultmaybelinkedtothefact thatthepresentsurveywasnotdesignedtocompareR.bursahost preference,buttoprimarilycontributetoabroaderinsightonthe tickR.bursadistribution.Nevertheless,ourresultsarevery simi-lartotheonesdescribedinastudyconductedinSardinia(Satta etal.,2011).Additionally,R.bursaisdescribedtohavepreference forparticularareasofthehostbody,accordingtotickinstarsand thehostspecies(Estrada-Penaetal.,2004;Yeruhametal.,1989). Indeed,inthisstudydistinctlocationswererecordedalthoughthe reducednumberofcollectedtickslimitsanystatisticalinference. Incattle,thehighestnumberofadulttickswascollectedfromthe abdominalandperinealareas,contrastingwiththeimmaturesthat weremainlyfoundontheheadanddorsalregions.Thesame obser-vationswererecordedbyotherauthors(Estrada-Penaetal.,2004;

Feldman-Muhsam,1953;Papadopoulosetal.,1996).More

speciﬁ-cally,inhorsesandgoatsmostoftheadultR.bursawerecollected fromtheposteriorregionsofthebody,includingperineumand tailandperineumandudder,respectively;insheepanddogs,ticks werefoundmostlyonthehead,mainlyfromtheinnersurfaceof theears,similarlytowhathavebeenreportedelsewhere(

Feldman-Muhsam,1953; Papadopoulosetal., 1996).No specimenswere

foundonotherlocationsalreadydescribedforthesehosts,suchas thetail,perianalandinguinalareas,interdigitalspacesandaxilla, forsheep(Moshaveriniaetal.,2012;Papadopoulosetal.,1996;

Yeruhametal.,1989);andneck,breastandtheinterdigitalspaces,

fordogs(Papadopoulosetal.,1996).

Molecularresultsconﬁrmed thepresence of A. marginale, T. annulataandT. equiinR.bursaticks.A.marginale wasfoundin

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ticksfeedingincattle,sheep,andgoats,andamongquestingticks. T.annulatawasfoundinatickfeedingincattle;andT.equiinticks feedinginhorses.Despitethedetectionofthesethreepathogens, itwasnotpossibletoconcludeaboutthevectorcapacityofthis tickspecies.Toaddressthisonewouldneedmoreevidence,ﬁrstly becausethebiologicmaterialwasobtainedfromthecompletetick andthepathogencouldbepresentonlyinthemidgutandnotin thesalivaryglandsfromwhereitwouldbetransmittedviasaliva; secondly,apositivePCRdoesnotconﬁrmthatthesepathogensare capableofmultiplicationinthistickandthustransmitted. Addi-tionally,oncethebloodofeachanimalwasnottestedtheoriginof eachpathogenwasnotdetermined.Twopossiblesourcesoftick infectioncouldbeimplicated: theblood mealonthehostfrom wherethetickwascollected,orthebloodmealfromtheprevious hostthroughtransstadialtransmission.

InPortugalandSpain,R.bursaisaprovenvectorofA.marginale andtheinfectionofcattle,knownasbovineanaplasmosis,caused bythispathogenhasbeenalreadyreported(Caeiro,1999;dela

Fuenteetal.,2004a;Kocanetal.,2004).Bovineanaplasmosisis

now known to beendemic in tropical and subtropical regions worldwide,causingsubstantialeconomiclossestocattleindustries

(Kocanetal.,2003).T.annulataismainlytransmittedbyticksof

thegenusHyalomma;however,similarlytoourstudy,R.bursawas alsoassociatedwiththetransmissionofthispathogeninTunisia

(M’Ghirbietal.,2010).Incattle,T.annulataisthecausativeagentof

tropicaltheileriosisresponsibleforseriousconstraintsonthe ani-malhealthandproductivityworldwideduetohighmorbidityand mortality(Brancoetal.,2010;Doliveiraetal.,1995).InPortugal, thepresenceofthistick-bornepathogenhasalreadybeenreported incattleduringanacutelethalinfectionofcalveshighlyinfested byticksofthegenusHyalomma(Brancoetal.,2010),andduring asurveyconductedinasymptomaticcattlefordetectionof piro-plasms(Gomeset al.,2013).Nevertheless, wewerenotableto identifyanystudyconductedinPortugalthathasisolatedT. annu-latafromR.bursaticks.T.equi(formerlyknownasBabesiaequi) canbenaturallytransmittedbyticksofthefamilyIxodidae(Stiller etal.,2002)andR.bursahasbeenimplicatedasthevectorof piro-plasmosiscausedbyT.equiinhorsesinSpainandIran(Abedietal.,

2014;Garcia-Sanmartinetal.,2008).Similarly,wehavefoundT.

equiinticksofthisspeciesinfestinghorses.Previousstudies con-ductedinPortugalreportthedetectionofthispathogeninsamples collectedfromhorses(Baptista etal.,2013;Ribeiroetal.,2013) but,todate,noreportswerefoundrelatingR.bursawithT.equi inourcountry. Equinepiroplasmosisisendemicinthenorthof Portugal(Ribeiroetal.,2013)anditisestimatedthat90%ofthe equinepopulationworldwidelivesinareaswhereequine piroplas-mosisispresent(Dewaal,1992).Nowadays,equinepiroplasmosis hasseriousimplicationsforincreasinginternationaltradeofhorses

(Ros-Garciaetal.,2013).Tothebestofourknowledge,ourreport

istheﬁrsttoidentifyT.annulataandT.equiinR.bursaticksfeeding indomesticcattleandhorses,respectively,inPortugal.

5. Conclusions

Ticksandtick-bornediseasesareacurrentandemergingglobal threatforhumanandanimalspecies.Theimpactofglobalclimate change, human activities, including land management, habitat destructionandcontrolstrategiesthatrelyontheuseofpesticides, might beresponsible for a changein tick biology and possibly contributetotherapidannualriseinthetickpopulation(Heyman

etal.,2010;Omeragic,2011;Santos-Silvaetal.,2011).Theresults

ofthisstudyindicatethatinPortugal,R.bursacanbefoundonhosts suchascattle,smallerruminants,suchassheepandgoats,horses, andoccasionallydogs.Also,thistickisactiveduringallyear,with amarkedseasonalbehavioraccordingtothedevelopmentalstage. Ourstudyalsodemonstrates thatseveralpathogensresponsible

fortick-bornediseases,particularlyA.marginale,T.annulataandT. equi,doexistinR.bursaticksandcirculateindeterminedareasof Portugal.Moreover,allpositiveticksonlyharboredasingle infec-tion.HerewereportforthefirsttimethedetectionofT.annulata andT.equiamongstR.bursaticksfeedingindomesticcattleand horses,respectively,inPortugal.Theconfirmationofthepresence ofAnaplasmaandTheileriainPortugalisofextremeimportance asbothpathogensexertgreatimpactinanimalhealth, influenc-inganimalproductionandtrading.Finally,informationaboutthe prevalenceofinfectioninticksisessentialtoprovideinformationin ordertodevelopfutureeffectivepreventiveandcontrolstrategies.

Conﬂictofinterest

Theauthorsdeclarenocompetingpersonalorﬁnancial inter-ests.

Acknowledgments

TheauthorswouldliketoacknowledgeVardaShkap(Kimron VeterinaryInstitute,Israel)forkindlyprovidingB.bigeminaand T.annulatacontrolsandalsoJosédelaFuente(SaBio,Institutode InvestigaciónenRecursosCinegéticos,Spain)fortheA.marginale control.AspecialacknowledgetoDanielBacellarfor his collab-orationintickcollection.WealsowouldliketorecognizeNuno Domingues(FaculdadedeCiênciasdaUniversidadedeLisboa)for thecontributionontickDNAextractionandAnaBuxo(Instituto SuperiorTécnicodeLisboa)forthehelpwithQGISsoftware.This researchwassupportedbytheprojectsPTDC/CVT-EPI/4339/2012 andPTDC/SAU-SAP/115266/2009,fundedbytheFundac¸ãoparaa CiênciaeTecnologia.

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