Protozoan and bacterial pathogens in tick salivary glands in wild and domestic animal environments in South Africa

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glands in wild and domestic animal environments in

South Africa

M. Berggoetz, M. Schmid, D. Ston, V. Wyss, Christine Chevillon, A.-M

Pretorius, L. Gern

To cite this version:

M. Berggoetz, M. Schmid, D. Ston, V. Wyss, Christine Chevillon, et al.. Protozoan and bacterial

pathogens in tick salivary glands in wild and domestic animal environments in South Africa. Ticks and

Tick-borne Diseases, Elsevier, 2014, 5 (2), pp.176-185. �10.1016/j.ttbdis.2013.10.003�. �hal-02152195�

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Pleasecitethisarticleinpressas:Berggoetz,M.,etal.,Protozoanandbacterialpathogensinticksalivaryglandsinwildanddomesticanimal environmentsinSouthAfrica.TicksTick-borneDis.(2013),http://dx.doi.org/10.1016/j.ttbdis.2013.10.003

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TicksandTick-borneDiseasesxxx (2013) xxx–xxx

ContentslistsavailableatScienceDirect

Ticks

and

Tick-borne

Diseases

j ou rn 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 / t t b d i s

Original

article

Protozoan

and

bacterial

pathogens

in

tick

salivary

glands

in

wild

and

domestic

animal

environments

in

South

Africa

M. Berggoetz

a,∗

_,

_M.

_Schmid

a

_,

_D.

_Ston

a

_,

_V.

_Wyss

a

_,

_C.

_Chevillon

b

_,

_A.-M.

_Pretorius

c

_,

_L.

_Gern

a

a_Institut_de_Biologie,_Laboratoire_{d’Eco-Epidémiologie}_des_Parasites,_University_of_Neuchâtel,_Emile_Argand_11,₂₀₀₀_Neuchâtel,_Switzerland

b_Maladies_Infectieuses_et_Vecteurs:_Ecologie,_Génétique,_Evolution,_Contrôle_(MIVEGEC;_UMR₅₂₉₀_{CNRS-IRD-Universités}_Montpellier_I_et_II),_Montpellier,

911AvenueAgropolis,BP64501,34394Montpelliercedex5,France

c_Department_of_Health_Sciences,_Faculty_of_Health_and_{Environmental}_Sciences,_Central_University_of_Technology,_Free_State_Province,_Bloemfontein_9300,

SouthAfrica

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received10July2013

Receivedinrevisedform10October2013 Accepted14October2013 Available online xxx Keywords: Ticks Tick-bornepathogens Africanwildlife Livestock Coinfections Theileria Babesia Ehrlichia Anaplasma

a

b

s

t

r

a

c

t

Atotalof7364ticksbelongingto13specieswascollectedfrom64gameanimals(belongingto11species) andfrom64livestockanimals(cattleandsheep)livinginclosevicinityat6localitiesin3SouthAfrican Provinces(FreeState,Mpumalanga,andLimpopo).Thegeographicdistributionofalltickspecieswas congruentwiththeliteratureexceptforHaemaphysalissilacea.Fromeachinfestedhost,amaximum of10malesand10femalesofeachtickspeciesweredissectedtoisolatethesalivaryglands.Salivary glandswerescreenedfortick-bornepathogensusingpolymerasechainreactionfollowedbyreverse lineblottingandsequencing.Thisapproachallowedustoevaluatetheexposureofwildanddomestic hoststotick-bornepathogensintheirrespectiveenvironments.Amongthe2117examinedticks,329 (15.5%),belongingto8species,wereinfectedandharboured397infections.Amongthose,57.7%were identifiedtospecieslevelandwereassignedto23pathogenspeciesofthegeneraBabesia,Theileria, Anaplasma,andEhrlichia.In3outof6localities,salivaryglandsfromticksinfestingwildruminants displayedsignificantlyhigherinfectionprevalenceandpathogenmeandensitythansalivaryglandsfrom ticksinfestinglivestockanimals.Fourpiroplasmspecies[Theileriabicornis,Babesiasp.(sable),Theileriasp. (giraffe),andTheileriasp.(kudu)]weredetectedforthefirsttimeinticks.ThetickspeciesRhipicephalus evertsievertsi,Rhipicephalus(Boophilus)decoloratus,Hyalommarufipes,Rhipicephalusappendiculatus,and Amblyommahebraeumwereassociatedwithabroaderpathogenrangethanpreviouslyknown,andthus newvector–pathogencombinationsaredescribed.Inaddition,previouslyunknowncoinfectionpatterns inticksalivaryglandsarereported.

Introduction

Ticksarevectorsofagreatvarietyoftick-bornepathogens.In therecentdecades,thedevelopmentofmoleculartoolsincreased theirknown number and theirknown variety.The majority of tick-bornepathogenspeciesthatcirculatebetweenticksandboth gameandlivestockanimalshavebeenreportedinthevertebrate hosts theyinfect and less in theticks. Indeed, in earlystudies thataimedtodetecttick-bornepathogensinwildAfrican ungu-lates,animalserawerescreened(NeitzandDuToit,1932;Neitz,

1935;Löhrand Meyer, 1973;Löhret al.,1974;Carmichael and

Hobday, 1975). More recently, in studies reporting tick-borne

∗ Correspondingauthor.Tel.:+41327183043;fax:+41327183001. E-mailaddresses:mirko.berggoetz@unine.ch(M.Berggoetz),

melody.schmid@unine.ch(M.Schmid),daniel.ston@unine.ch(D.Ston),

virginie.wyss@unine.ch(V.Wyss),Christine.chevillon@ird.fr(C.Chevillon),

gnvramp@gmail.com(A.-M.Pretorius),lise.gern@unine.ch(L.Gern).

pathogensaffectinggameanimals,hostbloodwasexamined,but nottheassociatedtickspecies(Nijhofetal.,2003,2005;Spitalska

etal.,2005;Brothersetal.,2011;Oosthuizenetal.,2008,2009;

Pﬁtzeretal.,2011).Inaddition,severaltick-bornepathogenswith

knowntick vectorsdisplay abroader hostrange thanformerly thought.AnexampleisTheileriasp.(sable)thatinfectsthesable (Hippotragusniger)and roan(H. equinus) antelopeandthat has beennewly described in red hartebeest(Alcelaphus buselaphus)

(Spitalskaet al.,2005), cattle(Yusufmiaetal., 2010), andnyala

(Tragelaphusangassii)(Pﬁtzeretal.,2011).Forthesepathogens,a broaderhostrangecouldmeanthat additional,sofarunknown vectors, especially when associated with a wide host variety, mightbeinvolvedintheirtransmission.Fromthispointofview, the two-hosttick R. e. evertsi, thethree-host ticks Amblyomma hebraeum and R. appendiculatusas wellas the one-hosttick R. (Boophilus)decoloratusaregoodcandidates.Thesetickspecieswere recordedfroma greatvarietyofhostspeciesandare geograph-icallywidelydistributedinsouthern Africa(Walkeretal.,2000,

2003).

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2 M.Berggoetzetal./TicksandTick-borneDiseasesxxx (2013) xxx–xxx

Therefore,inthisstudy,wefocusedonthepresenceofBabesia, Theileria,Anaplasma,andEhrlichiainthesalivaryglandsof differ-enttickspeciescollectedfromwildanddomestic ruminantsin SouthAfrica. Detectionofpathogens in salivaryglands of ticks allowedustodistinguishpathogensthatinfectedtheticksbefore theyattachedtothehostsfromthose takenupbyticksduring theirbloodmeal.Thus,thisapproachallowstoevaluatethe expo-sureofwildanddomesticruminantstotick-bornepathogensin theirrespectiveenvironments.Thepresenceofapathogenintick salivaryglands stronglysuggests thatit may betransmittedto thehost,butthisisnotsufﬁcienttoproveitsvectorrole,which wouldrequiretransmissionexperiments.Thepurposeherewas(i) toevaluatethepossibleroleofticksinthetransmissionofrecently describedpathogenspecies,(ii)toverifywhetherpathogenswith a broadhostrange have a broadervector rangethan currently known,(iii)toinvestigatetheexposureofgameandlivestockto tick-bornepathogensintheirrespectiveenvironments,and(iv)to obtaininformationoncoinfectionsinticks.

Materialsandmethods

Studyareas

Tickswerecollectedin 2009(May toJuly),2010(Januaryto May),and2011(ApriltoJune)at6localitiesin3SouthAfrican Provinces.IntheFreeState,3provincialnaturereserves, Tüssen-Die-Riviere, Willem Pretorius, and Sandveld, as wellas several livestockfarmsattheirsurroundingswereinvestigated(highveld). IntheMpumalangaProvince,onegamefarmandonelivestockfarm wereinvestigatednearBethal(highveld).Finally,5gamefarmsand 5livestockfarmswereinvestigatedintheLimpopoProvinceinthe ThabazimbiandLephalaleareas(lowveld).

Ticksampling

Ateachlocality,ticksamplingswereperformedondomesticand wildanimalswithinthesameweek.Ticksweresampledfroman equalnumberoflivestockanimalslivingonfarmssharinga com-monborderwiththereservesorgamefarmsorlocatedintheclose surroundings(within the range of40km).Ticks werecollected fromgameanimalsduringgamecapture,cullingoperations,and hunts.Cattleweremaintainedinholdingfacilitiesduringsampling, andsheepwereimmobilisedbyhandinsmallcamps.Hostswere visuallyexaminedforticks;palpationhelpedtolocalisespecimens attachedonﬂanks,back,belly,neck,andlegs.Allthefoundticks wereremovedwithtweezers.Tickidentiﬁcationwasperformed accordingtoMatthysseandColbo(1987)andWalkeretal.(2000,

2003). Tickswere pooled per species, host,and developmental

stageandstockedinalcoholin50-mllabelledtubes. Detectionandidentiﬁcationofpathogenspeciesinticksalivary glands

Fromeach individual host,a maximum of 10 males and 10 females from each tick species was analysed. To distinguish pathogensthathadinfectedticksbeforetheyattachedtothehost fromthosetakenupbyticksduringtheircurrentbloodmeal,each tickwasdissectedandthesalivaryglandsofdissectedtickswere analysedfor pathogens.Salivaryglandswerecarefullyremoved withtweezers–specialattentionwaspaidtoavoid contamina-tionwiththemidgut,andwashedtwiceinPBSin96-wellplates (Milian®_,_Geneva,_{Switzerland).}_Instruments_were_sterilised_for_a fewsecondsin5MHCland5MNaOH(Aktasetal.,2009)anddried withsterilewipesbetweeneachdissection.

DNAfromticksalivaryglands wasextractedusingQIAamp® DNAMicrokit(Qiagen,Hombrechtikon,Switzerland)followingthe

manufacturer’sinstructionswithmodiﬁcations.Tissuelysisbuffer andproteinaseKwereaddedinthe1.5-mltubesbeforethesalivary glands.DNAwasstoredat−20◦_C.

Anapproximately500-basepair(bp)fragmentofthe16S ribo-somalRNA(rRNA)genespanningthehypervariableV1regionofthe generaAnaplasmaandEhrlichiaandanapproximately400-bp frag-mentofthe18SrRNAgenespanningtheV4hypervariableregion ofthegeneraBabesiaandTheileriawereamplifiedbyPCR(Tonetti etal.,2009).Positivecontrol,includedineachrun,consistedof DNAofA.phagocytophilum(providedbyAnaSofiaSantos, Insti-tutoNacionaldeSaude,Lisboa,Portugal)andB.divergens(provided bySimonaCasati,InstitutCantonaldeMicrobiologie,Bellinzone, Switzerland).PCRproductswereanalysedusingreverseline blot-ting (RLB)(Tonetti et al., 2009). In addition to the original 15 oligonucleotideprobes,whicharelistedinTonettietal.(2009)(2 genus-specificBabesia/TheileriaandAnaplasma/Ehrlichia,and13 species-specific),26probeswereadded:onegenus-specific

Theile-riaspp.(Nagoreetal.,2004)and25species-speciﬁcprobes,B.ovis,

B.crassa(Schnittgeretal.,2004),B.major(Georgesetal.,2001), Babesiasp.(sable)(Oosthuizenetal.,2008),B.caballi(Butleretal., 2008),B.occultans(Ros-Garciaetal.,2011),B.orientalis(Heaetal., 2011),B.gibsoni(fromPﬁtzer,2009),B.rossi(Matjilaetal.,2004),B. bicornis(Nijhofetal.,2003),B.motasi,Theileriasp.(greaterkudu), Theileriasp.(sable)(Nijhofetal.,2005),T.separata,T.lestoquardi,T.

ovis(Schnittgeretal.,2004),T.buffeli(Gubbelsetal.,1999),T.

bicor-nis(Nijhofetal.,2003),Theileriasp.(buffalo)(Ouraetal.,2004),T.

equi(Butleretal.,2008),T.annulata(Georgesetal.,2001),Ehrlichia

sp.(Omatjenne)(Bekkeretal.,2002),and4A.phagocytophilum (fromPfitzer,2009)thatreplacedtheoriginaldegeneratedprobe. SamplesreactingonlywiththeBabesia/Theileriaprobewere con-sidered Babesia spp. since a genus-specific probe wasincluded forthegenusTheileria.Totestfortheoreticalspecificity, oligonu-cleotideprobeswerealignedwithvarioussequencesoftargeted speciesavailablefromtheNationalCentreforBiotechnology Infor-mation(NCBI)usingasoftwarepackage:CLCSequenceViewer6 (CLCbio,Aarhus,Denmark).

Sequencing

PCR products that reacted only with genus-specific probes Babesia/Theileria,Theileriaspp.,orAnaplasma/Ehrlichiaand did not hybridise with the panel of species-specific probes were sequenced.Prior sequencing,PCR products werepurified using Wizard®_SV_and_PCR_Clean-Up_System_(Promega,_Madison,_USA) followingthemanufacturer’sinstructions exceptthatweeluted with35␮lratherthan70␮lNuclease-FreeWater.Sequencingwas performedbyMicrosynthAG (Balgach,Switzerland).Sequences werecomparedandcorrectedwithCLCSequenceViewer6(CLCbio, Aarhus,Denmark)andBioedit(TomHallIbisBiosciences,Carlsbad). Corrected sequences were compared with available sequences retrievedfromGenBankusingNCBIBasicLocalAlignmentSearch Tool(BLAST).

Dataanalysis

Datawereanalysedwith“R”2.14forWindows(RDevelopment Core Team, 2012. R: A language and environment for statisti-cal computing. RFoundation for Statistical Computing,Vienna, Austria. ISBN 3-900051-07-0, URL: http://www.R-project.org/), usingsoftwarepackages(Skaugetal.,2010;Hussonetal.,2012).A generalisedlinearmixedmodel(GLMM)withnegativebinominal errors was used to evaluate tick salivary gland infection (tak-ingintoaccountthe4maininfectedtickspecies)collectedfrom wildanddomesticruminantsoriginatingfrom6localities.Ineach locality,thesigniﬁcanceofthefactorsLOCATIONandHOSTTYPE (i.e.ticksinfesting wildvs. domestic ruminants)as wellasthe

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LOCATION–HOSTTYPEinteractionwasassessed,Pvalueswere con-sideredsigniﬁcantwhenbelow0.05.Inordertocomputewhether associationsbetweenpathogenspeciesincoinfectionswere signiﬁ-cantapermutationtestproposedbyRaupandCrick(1979),adapted

toRbyCluaetal.(2010)constrainedtolocationswasused.Here,P

valueswereconsideredsigniﬁcantwhenbelow0.0001(Bonferroni corrected).

Results

Ticksampling

Atotalof7364ticksbelongingto13specieswascollectedfrom 13ruminantspecies(Table1).Fivetickspeciescontributed96.5% of thetotal sampling: R. (B.) decoloratus (n=2611), R.e. evertsi (n=2225),Margaropuswinthemi(n=1085),A.hebraeum(n=767), andHyalommaruﬁpes(n=417),and71%ofthemwerecollected fromwildanimals(Table1).Alltickspeciesweredetectedinareas that were congruentwith theirknown geographic distribution exceptHaemaphysalissilacea,whichwasrecordedatWillem Pre-toriusgamereserve(centralFreeState)(SupplementaryTableS1). The2mostabundanttickspecieswerecollectedateachofthe6 samplingsites,R.(B.)decoloratusinfested9ruminantspeciesand R.e.evertsiallhostspecies.

Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.ttbdis.2013.10.003.

Infectionofticksalivaryglands

Amongthe2117examinedticks,329(15.5%)wereinfected,and theyharboured397infections(Table2).Themajority(95%)ofthe infectionswereobservedin4tickspecies:R.e.evertsiharboured 48.6%oftheinfections(193/397),R.(B.)decoloratus24.7%(98/397), A.hebraeum11.8%(47/397),andHy.rufipes9.6%(38/397)(Table2). Fivetickspecies(Ixodesrubicundus,M.winthemi,Hae.silacea, Oto-biusmegnini, and R.zambeziensis) were free of pathogens. One hundredsixty-eightinfectionswereidentifiedatgenuslevelonly: 69Babesiaspp.,56 Theileriaspp.,and 43AnaplasmaorEhrlichia spp.Sequencingofallthesesamplesdidnotallowfurther identi-fication.Theremaininginfections(n=229)(57.7%)wereidentified tospecies level and were assigned to 23 pathogen species, all knowntooccurinSouthAfrica,exceptT.annulata(Table2).Among them were 46 identified by sequencing (Table 3). Theileria sp. (sable)(n=38;9.6%),T.buffeli(n=37;9.3%),T.bicornis(n=27;6.8%), Ehrlichiasp. (EU191229.1)(n=23;5.8%),and T.separata (n=18; 4.5%)werethemostfrequent(Table2).

Pathogendistributionamongtickspecies

Ninepathogenspeciesweredetectedinonetickspeciesonly. Theileriaequi,T.separata,A.ovis,andA.platysweredetectedinR.e. evertsi;B.occultansandBabesiasp.(sable)wereonlydetectedinHy. rufipes,andTheileriasp.(kudu),Anaplasmacentrale,andEhrlichia ruminantiuminR. appendiculatus,R.gertrudae,and A.hebraeum, respectively(Table2).Sevenpathogensweredetectedin 2tick species.Babesiabigemina,B.caballi,A.bovis,T.ovis,andTheileriasp. (giraffe)weredetectedinR.(B.)decoloratusandR.e.evertsi.Theileria bicorniswasdetectedinR.e.evertsiandR.appendiculatus.Finally,T. mutanswasdetectedinR.e.evertsiandA.hebraeum.Fourpathogen specieswereidentifiedin3tickspecies.Theileriasp.(sable)was detectedinR.e.evertsi,R.(B.)decoloratus,andA.hebraeum. Thei-leriataurotragiwasdetectedinR.appendiculatus,R.e.evertsi,and Hy.rufipes.AnaplasmamarginalewasdetectedinR.gertrudae,R.(B.) decoloratus,andR.e.evertsi.Ehrlichiasp.(EU191229.1)wasdetected inR.(B.)decoloratus,R.e.evertsi,andHy.rufipes.Finally,T.buffeli andE.ovinaweredetectedin4tickspecies:Theileriabuffeliwas

detectedinR.(B.)decoloratus,R.e.evertsi,Hy.ruﬁpes,andR. appen-diculatus,whileE.ovina wasdetectedinR.(B.)decoloratus, R.e. evertsi,Hy.ruﬁpes,andR.gertrudae.

Salivaryglandinfectionofticksattachedtowildversusdomestic ruminants

Ticksthatwereattachedtowildruminantsshowedan infec-tionprevalenceof23.3%(227/973)andharboured284infections, while ticksattachedtodomestic ruminantsdisplayedan infec-tionprevalenceof11.5%(102/885)andharboured113infections

(Tables4aand4b).ThedifferencesweremainlyduetoTheileria

spp.(16.1%vs.8.4%),Theileriasp.(sable)(12.6%vs.1.7%),T.buffeli (10.2%vs.6.7%),T.bicornis(7.4%vs.5%),andT.separata(4.9%vs. 3.4%)(Tables4aand4b).

Takingintoaccountthe4maininfectedtickspecies[R.(B.) decol-oratus,R.e.evertsi, A. hebraeum,and Hy.rufipes],salivarygland infection(prevalenceofinfectionandmeandensityofpathogens) variedsignificantlyamongthesamplinglocalities(Table5).The infectionpatternwasimpactedbythefactorsLOCATIONandHOST TYPEaswellasbytheinteractionofbothfactors(Glmmanalyses P<0.001forthefactorsLOCATION,HOSTTYPE,andtheir interac-tions).Incontrast,theintensitiesofinfectiondidnotsignificantly differamonghosttypesand/orlocationsatthe5%risk(datanot shown).InSandveld,Thabazimbi,andLephalale,infection preva-lenceandmeandensityofpathogensweresignificantlyhigherin salivaryglandsofticksinfestingwildthandomestichosts. Simi-larly,thetickmeandensitywassignificantlyhigheronwildanimals thanondomesticanimalsinSandveldandThabazimbi(datanot shown).

Coinfections

Amonginfectionsidentified atspecieslevel,93(44.7%) were coinfections:57(61.3%)involved 2pathogens,24(25.8%)3,and 12(12.9%)4(datanotshown).Rhipicephaluse.evertsishowedthe highestprevalenceofcoinfections(n=67;72%),followedbyR.(B.) decoloratus(n=21;22.6%),R.appendiculatus(n=3;3.2%),andHy. rufipes(n=2;2.2%)(datanotshown).Sixassociations involving 5pathogenspeciesweresignificant:Theileriasp.(sable)withT. separata;Theileria sp.(sable)withT.bicornis;T.separatawithT. bicornis;T.separatawithT.buffeli;T.bicorniswithT.buffeli,and T.buffeliwithB.caballi[P<0.0004,permutationtestconstrained tolocations(RaupandCrick,1979,modifiedbyCluaetal.,2010)] (SupplementaryTableS2).

Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.ttbdis.2013.10.003.

Discussion

Inthisstudy,13tickspeciescollectedfromwildanddomestic ruminantswereanalysedbyRLBhybridisationusingprobes allow-ingidentificationoftick-bornepathogensatthegenusandspecies level.Themajority(n=229)ofthe397infectionswereidentified atspecieslevel.Amongthose,46infectionsassignedto7species wereidentifiedbysequencing:Ehrlichiasp.(EU191229.1),E.ovina, Theileriasp.(giraffe),A.platys,A.ovis,A.marginale,andA.bovis.No species-specificprobeswereincludedintheassayfortheformer4 species.Thelatter3speciesdidnotreactwiththeirspecific oligonu-cleotideprobes.ForA.ovisandA.marginale,thiswasprobablydue totheforwardpositionoftheprobesontheamplifiedsequence, while for A. bovis,it wasprobably due toone basepair differ-enceintheamplifiedsequences.Theremaining168infectionswere onlyidentifiedatthegenuslevel,evenaftersequencing,probably becauseofthepresenceofmultiplepathogens(Microsynth,pers. communication)forwhichnoprobeswereavailable.

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Table1

Hostinfestation(n=128)by13tickspeciesinsixlocalitiesinSouthAfrica.

Tick(bold)andhostspecies Hosts Numberofticks

No.infested/examined Larvae Nymphs Males Females Total

Rhipicephalus(B.)decoloratus

Commoneland(Tragelaphusoryx) 1/9(11%) 9 91 135 97 332 Greaterkudu(T.strepsiceros) 3/8(38%) 1 59 35 35 130 Impala(Aepycerosmelampus) 2/7(29%) 213 483 434 207 1337 Southerngiraffe(Giraffacamelopardalis) 5/5(100%) 0 0 6 23 29 Bluewildebeest(Connochaetestaurinus) 4/5(80%) 8 10 40 53 111 Sableantelope(Hippotragusniger) 2/2(100%) 0 14 37 45 96 Gemsbok(Oryxgazellagazella) 1/1(100%) 33 52 72 56 213 Cattle(Bosprimigeniustaurus/B.p.taurus/indicus) 17/50(34%) 0 21 136 204 361

Sheep(Ovisaries) 2/14(14%) 0 0 0 2 2

Total 37/101(37%) 264 730 895 722 2611

R.e.evertsi

Africanbuffalo(Synceruscaffer) 14/15(93%) 0 0 330 112 442 Commoneland(T.oryx) 9/9(100%) 0 2 262 27 291 Greaterkudu(T.strepsiceros) 4/8(50%) 7 24 7 0 38

Impala(A.melampus) 3/7(43%) 0 49 11 2 62

Bluewildebeest(C.taurinus) 5/5(100%) 0 0 48 38 86 Springbok(Antidorcasmarsupialis) 4/6(67%) 0 0 6 5 11 Southerngiraffe(G.camelopardalis) 4/5(80%) 0 0 15 12 27 Blesbok(Damaliscuspygarusphillipsi) 1/3(33%) 3 5 0 0 8 Blackwildebeest(C.gnou) 2/3(67%) 0 0 3 3 6 Sableantelope(H.niger) 2/2(100%) 0 0 9 1 10 Gemsbok(O.g.gazella) 1/1(100%) 0 0 26 5 31 Cattle(B.p.taurus/B.p.t./indicus) 49/50(98%) 189 424 431 153 1197

Sheep(O.aries) 5/14(36%) 0 0 14 2 16

Total 101/128(79%) 199 504 1162 360 2225

Margaropuswinthemi

Commoneland(T.oryx) 6/9(67%) 11 505 287 234 1037 Greaterkudu(T.strepsiceros) 1/8(13%) 0 0 0 9 9 Gemsbok(O.g.gazella) 1/1(100%) 1 0 0 27 28 Cattle(B.p.taurus/B.p.t./indicus) 3/50(6%) 0 0 0 11 11

Total 11/68(16%) 12 505 287 281 1085

Amblyommahebraeum

Southerngiraffe(G.camelopardalis) 5/5(100%) 0 0 307 117 424 Cattle(B.p.taurus/B.p.t./indicus) 10/50(20%) 0 0 229 114 343

Total 15/55(27%) 0 0 536 231 767

Hyalommam.ruﬁpes

Buffalo(S.caffer) 8/15(53%) 0 0 13 8 21

Commoneland(T.oryx) 9/9(100%) 0 0 215 14 229 Southerngiraffe(G.camelopardalis) 4/5(80%) 0 0 19 5 24 Bluewildebeest(C.taurinus) 3/5(60%) 0 0 4 7 11 Sableantelope(H.niger) 1/2(50%) 0 0 1 0 1 Cattle(B.p.taurus/B.p.t./indicus) 28/50(56%) 0 0 80 49 129

Sheep(O.aries) 2/14(14%) 0 0 1 1 2

Total 55/100(55%) 0 0 333 84 417

Ixodesrubicundus

Commoneland(T.oryx) 4/9(44%) 0 0 22 32 54 Greaterkudu(T.strepsiceros) 5/8(63%) 0 0 16 34 50 Springbok(A.marsupialis) 5/6(83%) 0 0 9 11 20 Cattle(B.p.taurus/B.p.t./indicus) 4/50(8%) 0 0 7 21 28

Sheep(O.aries) 2/14(14%) 0 0 1 1 2

Total 20/87(23%) 0 0 55 99 154

R.appendiculatus

Southerngiraffe(G.camelopardalis) 1/5(20%) 0 0 9 4 13 Cattle(B.p.taurus/B.p.t./indicus) 3/50(6%) 0 0 19 2 21

Total 5/62(8%) 0 0 32 6 38

R.gertrudae

Africanbuffalo(S.caffer) 1/15(7%) 0 0 21 1 22

Commoneland(T.oryx) 1/9(11%) 0 0 8 0 8

Total 2/24(8%) 0 0 29 1 30

R.warburtoni

AfricanBuffalo(S.caffer) 1/15(7%) 0 0 1 0 1

Commoneland(T.oryx) 2/9(22%) 0 0 13 1 14

Total 3/24(12%) 0 0 14 1 15

Haemaphysalissilacea

Africanbuffalo(S.caffer) 2/15(13%) 0 0 0 8 8

Total 2/15(13%) 0 0 0 8 8

R.(B.)microplus

Cattle(B.p.taurus/B.p.t./indicus) 1/50(2%) 0 3 1 3 7

Otobiusmegnini

Cattle(B.p.taurus/B.p.t./indicus) 2/50(4%) 0 5 0 0 5

Total 2/50(4%) 0 5 0 0 5

R.zambeziensis

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Please cite this article in press as: Berggoetz, M., et al., Protozoan and bacterial pathogens in tick salivary glands in wild and domestic animal environments in South Africa. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.10.003

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G Model TTBDIS-266; No. of Pages 10 M. Berggoetz et al. / Ticks and Tick-borne Diseases xxx (2013) xxx – xxx 5 Table2

Infectionof8outof13tickspeciesby23pathogenspeciescollectedfrom64wildand64domesticruminantsinSouthAfrica.

Pathogens/ticks R.(B.) decoloratus R.e. evertsi A. hebraeum H.m. ruﬁpes R. appendiculatus R. gertrudae R.(B.) microplus R. warburtoni I. rubicundus M. winthemi H. silacea O. megnini R. zambeziensis %ofpathogen speciesD B/Tgenusg ₂₄ ₃₀ ₆ ₆ ₃ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₆₉_(17.4%) B.occultans 0 0 0 2a,f ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₂_(0.5%) B.bigemina 5b,c,e,f ₁f ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₆_(1.5%) B.caballi 7b,c,e ₅b,c ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₁₂_(3%) B.sp.Sable 0 0 0 1b ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₁_(0.3%) T.spp. 15 23 13 2 3 0 0 0 0 0 0 0 0 56(14.1%) T.bicornis 0 26a,b,c,e ₀ ₀ ₁e ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₂₇_(6.8%) T.ovis 2e ₂a,c ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₄_(1%) T.equi 0 14a,b,c,e,f ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₁₄_(3.5%) T.separata 0 18b,c,d,e ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₁₈_(4.5%)

T.sp.(sable) 9c,e,f ₂₄b,c,d,e ₅e,f ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₃₈_(9.6%)

T.buffeli 17b,c,e,f ₁₇b,c,e,f ₀ ₂a,f ₁e ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₃₇_(9.3%)

T.taurotragi 0 1f ₀ ₁b ₂e ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₄_(1%)

T.annulata 4b,e ₂b,e ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₆_(1.5%)

T.sp.(kudu) 0 0 0 0 1e ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₁_(0.3%) T.mutans 0 1f ₁f ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₂_(0.5%) T.sp.(giraffe) 1f ₁f ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₂_(0.5%) A/Egenus 3 7 18 11 0 0 1 3 0 0 0 0 0 43(10.8%) A.bovis 1b ₁₁a,b,c ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₁₂_(3%) A.centrale 0 0 0 0 0 1a ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₁_(0.3%) A.marginale 1d ₁c ₀ ₀ ₀ ₄a ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₆_(1.5%) A.ovis 0 3c,f ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₃_(0.8%)

E.sp.(EU191229.1) 8b,e ₃c,f ₀ ₁₂a,c ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₂₃_(5.8%)

E.ovina 1c ₂a,c ₀ ₁a ₀ ₁a ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₅_(1.3%) E.ruminantium 0 0 4f ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₄_(1%) A.platys 0 1a ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₀ ₁_(0.3%) Prevalenceof infectionA 84/395 (21.3%) 147/909 (16.2%) 42/233 (18%) 37/259 (14.3%) 9/26(34.6%) 6/18 (33.3%) 1/4(25%) 3/14 (21.4%) 0/131 0/116 0/7 0/4 0/1 329/2117 (15.5%) Pathogenmean densityB 98/395(0.25) 193/909 (0.21) 47/233 (0.20) 38/259 (0.15) 11/26(0.42) 6/18 (0.33) 1/4(0.25) 3/14(0.21) 0/131 0/116 0/7 0/4 0/1 397/2117 (0.19) Intensityof infectionC 98/84(1.17) 193/147 (1.31) 47/42 (1.12) 38/37 (1.03) 11/9(1.22) 6/6(1) 1/1(1) 3/3(1) 0 0 0 0 0 397/329(1.21)

A_No._of_infected_ticks_(data_not_shown)/no._of_tested_ticks. B _No._of_{infections/no.}_of_tested_ticks.

C _No._of_{infections/no.}_of_infected_ticks.

D _%_of_the_given_pathogen_species_among_the₃₉₇_infections.

Locationsoftheinfectionsidentiﬁedatspecieslevel:a,Tüssen-Die-Riviere;b,WillemPretorius;c,Sandveld;d,Bethal;e,Thabazimbi;f,Lephalale. Inbold:newvector–pathogencombinations.

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Table3

Pathogenspeciesidentiﬁedfromticksalivaryglandsbysequencing.

Identifiedsequence Homologywith %ofhomology Speciesname Identifiedfrom KF414714 EU191229.1 100% Unnamed(E.sp.) H.m.rufipes(n=12) KF414714 EU191229.1 100% Unnamed(E.sp.) R.(B.)decoloratus(n=8) KF414714 EU191229.1 100% Unnamed(E.sp.) R.e.evertsi(n=3) KF414715 AF318946.1 99% E.ovina R.e.evertsi(n=2) KF414715 AF318946.1 99% E.ovina H.m.rufipes(n=1) KF414715 AF318946.1 99% E.ovina R.gertrudae(n=1) KF414715 AF318946.1 99% E.ovina R.(B.)decoloratus(n=1) KF414716 FJ155997.1 100% A.bovis R.e.evertsi(n=11) KF414717 EU191231.1 100% A.ovis R.e.evertsi(n=3) KF414718 FJ155998.1 99% A.marginale R.gertrudae(n=1) KF414719 AY040853.1 99% A.platys R.e.evertsi(n=1) KF414720 FJ213583.1 100% T.sp.(giraffe) R.e.evertsi(n=1) KF414720 FJ213583.1 100% T.sp.(giraffe) R.(B.)decoloratus(n=1)

Thegeographic distributionof alltick and pathogen species wascongruentwiththeirknowngeographicdistributionexcept forHae. silacea,T.annulata, andEhrlichia sp.(EU191229.1). The tickHae.silaceawascollectedatWillemPretoriusgamereserve inthecentreoftheFreeState.Itsknowngeographicdistribution isthenorth-easternKwaZulu-NatalandtheEasternCapeProvince

(Norval,1975;Walker,1991;Horaketal.,1991,2007).InAfrica,

T.annulataoccursonlyinnorthernAfrica(Pipano,1994;Jacquiet etal.,1990).Here,T.annulatawasdetectedinthesalivaryglands of4R.(B.)decoloratusand2R.e.evertsiatThabazimbiandinthe FreeState(WillemPretorius)wheretheyweresharingthesame individualhosts.AllT.annulatainfectionswereidentifiedthrough hybridisationwithRLB-probesdesignedbyGeorgesetal.(2001). Crossreactivity with pathogens identified in this studycan be excluded.Whethercrossreactivity occurredwithanalternative orunknownTheileriaspeciesremainsapossibility.Unfortunately, sequencingofthesesampleswasnotsuccessful.Thus,thefinding

ofthiseconomicallyimportantcattleparasiteinSouthAfricamust beconsideredwithcaution.Thispathogenisnotdiscussed fur-ther,andEhrlichiasp.(EU191229.1)willbediscussedelsewhere (Berggoetzetal.,unpublished).

Amongtheobservedtick-pathogencombinations,17were con-gruent with the literature. Among those, 10 were recorded in thesalivaryglandsofR.e.evertsi:B.bigemina(Büscher,1988),B. caballi(DeWaalandvanHeerden,1994),T.ovis(JansenandNeitz,

1956),T.equi(DeWaalandPotgieter,1987),T.separata(Jansen

andNeitz, 1956), Theileriasp.(sable)(Steyletal.,2012), T.

tau-rotragi(Lawrenceetal.,1994a),A.marginale(Potgieter,1981),A.

ovis(Kaufmann,1996),andE.ovina(Neitz,1956).Theremaining7

knownvector–pathogencombinationswererecordedinthe sali-varyglandsof4tickspecies:B.bigemina(PotgieterandEls,1977),

T.ovis(JansenandNeitz,1956),andA.marginale(Potgieter,1981)

inR.(B.)decoloratus;T.mutans(Lawrenceetal.,1994a)andE. rumi-nantium(Bezuidenhoutetal.,1994)inA.hebraeum;B.occultans

Table4a

Pathogensidentiﬁedinseventickspeciescollectedfrom64wildruminants.

Pathogens/ticks R.(B.)decoloratus R.e.evertsi A.hebraeum H.m.ruﬁpes R.appendiculatus R.gertrudae R.warburtoni %ofpathogen speciesd B/Tgenuse ₁₆ ₂₁ ₂ ₁ ₃ ₀ ₀ ₄₃_(15.1%) B.occultans 0 0 0 1 0 0 0 1(0.4%) B.bigemina 2 0 0 0 0 0 0 2(0.7%) B.caballi 6 5 0 0 0 0 0 11(3.9%) T.spp. 14 19 9 1 3 0 0 46(16.1%) T.bicornis 0 20 0 0 1 0 0 21(7.4%) T.ovis 2 0 0 0 0 0 0 2(0.7%) T.equi 0 14 0 0 0 0 0 14(4.9%) T.separata 0 14 0 0 0 0 0 14(4.9%) T.sp.(sable) 9 23 4 0 0 0 0 36(12.6%) T.buffeli 14 14 0 1 0 0 0 29(10.2%) T.taurotragi 0 1 0 1 0 0 0 2(0.7%) T.annulata 3 1 0 0 0 0 0 4(1.4%) T.sp.(giraffe) 1 1 0 0 0 0 0 2(0.7%) A/Egenus 2 7 9 5 0 0 3 26(9.1%) A.bovis 0 8 0 0 0 0 0 8(2.8%) A.centrale 0 0 0 0 0 1 0 1(0.4%) A.marginale 0 0 0 0 0 4 0 4(1.4%) E.sp.(EU191229.1) 2 1 0 11 0 0 0 14(4.9%) E.ovina 1 0 0 0 0 1 0 2(0.7%) E.ruminantium 0 0 2 0 0 0 0 2(0.7%) Prevalenceof infectiona 61/235(26%) 108/474(22.78%) 22/91(24.18%) 20/129(15.5%) 7/12(58.33%) 6/18(33.33%) 3/14(21.43%) 227/973(23.33%) Pathogenmean densityb 72/235(0.31) 149/474(0.31) 26/91(0.29) 21/129(0.16) 7/12(0.58) 6/18(0.33) 3/14(0.21) 284/973(0.29) Intensityof infectionc 72/61(1.18) 149/108(1.38) 26/22(1.18) 21/20(1.05) 7/7(1) 6/6(1) 3/3(1) 284/227(1.25)

a_No._of_infected_ticks_(data_not_shown)/no._of_tested_ticks. b _No._of_{infections/no.}_of_tested_ticks.

c _No._of_{infections/no.}_of_infected_ticks.

d _%_of_the_given_pathogen_species_among_the₂₈₄_infections.

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GModel

Table4b

Pathogensidentiﬁedinsixtickspeciescollectedfrom64domesticruminants.

Pathogens/ticks R.(B.)decoloratus R.e.evertsi A.hebraeum H.m.ruﬁpes R.appendiculatus R.(B.)microplus %ofpathogen speciesd B/Tgenuse ₈ ₉ ₄ ₅ ₀ ₀ ₂₆_(21.8%) B.occultans 0 0 0 1 0 0 1(0.8%) B.bigemina 3 1 0 0 0 0 4(3.4%) B.caballi 1 0 0 0 0 0 1(0.8%) B.sp.(sable) 0 0 0 1 0 0 1(0.8%) T.spp. 1 4 4 1 0 0 10(8.4%) T.bicornis 0 6 0 0 0 0 6(5%) T.ovis 0 2 0 0 0 0 2(1.7%) T.separata 0 4 0 0 0 0 4(3.4%) T.sp.(sable) 0 1 1 0 0 0 2(1.7%) T.buffeli 3 3 0 1 1 0 8(6.7%) T.taurotragi 0 0 0 0 2 0 2(1.7%) T.annulata 1 1 0 0 0 0 2(1.7%) T.sp.(kudu) 0 0 0 0 1 0 1(0.8%) T.mutans 0 1 1 0 0 0 2(1.7%) A/Egenus 1 0 9 6 0 1 17(14.3%) A.bovis 1 3 0 0 0 0 4(3.4%) A.marginale 1 1 0 0 0 0 2(1.7%) A.ovis 0 3 0 0 0 0 3(2.5%) E.sp.(EU191229.1) 6 2 0 1 0 0 9(7.6%) E.ovina 0 2 0 1 0 0 3(2.5%) E.ruminantium 0 0 2 0 0 0 2(1.7%) A.platys 0 1 0 0 0 0 1(0.8%) Prevalenceofinfectiona _23/160_(14.4%) _39/435_(9%) _20/142_(14.1%) _17/130_(13.1%) _2/14_(14.3%) _1/4_(25%) _102/885_(11.5%)

Pathogenmeandensityb _26/160_(0.16) _44/435_(0.10) _21/142_(0.15) _17/130_(0.13) _4/14_(0.29) _1/4_(0.25) _113/885_(0.13)

Intensityofinfectionc _26/23_(1.13) _44/39_(1.13) _21/20_(1.05) _17/17₍₁₎ _4/2₍₂₎ _1/1₍₁₎ _113/102_(1.11)

a_No._of_infected_ticks_(data_not_shown)/no._of_tested_ticks. b_No._of_{infections/no.}_of_tested_ticks.

c _No._of_{infections/no.}_of_infected_ticks.

d _%_of_the_given_pathogen_species_among_the₁₁₃_infections.

e_B/T:_includes_infections_reacting_with_this_probe_only;_considered_as_belonging_to_the_genus_Babesia.

(ThomasandMason,1981)andT.taurotragi(Lawrenceetal.,1994b)

inHy.ruﬁpesandR.appendiculatus,respectively.Rhipicephaluse. evertsiappearsasaveryimportantvectorforT.separata,T.equi, andTheileriasp.(sable)inSouthAfrica.Interestingly,B.bigemina wasdetectedinthesalivaryglandsofoneR.e.evertsiadult.The exactroleofR.e.evertsiintheepidemiologyofredwaterremains unclear(DeVosandPotgieter,1994).AccordingtoBüscher(1988), onlynymphstransmitB.bigemina,andtransovarialtransmission doesnotoccur.Furtherstudiesareneededtoevaluatetheroleof R.e.evertsiadultsinthecirculationofB.bigemina.

Toourknowledge,theremaining23vector–pathogen combi-nationswereneverdescribed before.Amongthose,11 involved combinations with multiple records at different sites. Three vector–pathogencombinationsinvolvedatickspecies,R.gertrudae, thatwasneverreportedasavector(Walkeretal.,2000).Onethird ofR.gertrudaespecimenscarriedbacterialpathogens,allidentiﬁed

atspecieslevel.Fouroutof6A.marginaleinfectionsweredetected inthistickspeciessuggestingapossiblevectorroleofR.gertrudaein theepidemiologyofgallsickness.Inaddition,E.ovinaandA.centrale wereeachidentifiedinoneR.gertrudae.Onevector–pathogen com-binationinvolvedT.bicornisdetectedforthefirsttimeinticksand previouslyreportedinrhinoceroses(Nijhofetal.,2003;Govender etal.,2011),cattle(Muhanguzietal.,2010),andnyala(Pfitzeretal., 2011).Itwasidentifiedinthesalivaryglandsof26R.e.evertsiat 4localitiesinthelowveldandinthehighveld.Thisstrongly sug-geststhatR.e.evertsitransmitsT.bicornisonalargearea.Three vector–pathogencombinationsinvolvedT.buffeliwhichhas rec-ognizedvectorsinsomepartsoftheworld(Gubbelsetal.,2000), buttoourknowledge,noidentifiedvectorinSouthAfrica. Theile-riabuffeliinfections(n=34)weredetectedinR.e.evertsiandR.(B.) decoloratus,aswellasin2Hy.rufipesatsitessituatedinthelowveld andhighveld.ThissuggeststhatR.e.evertsiandR.(B.)decoloratus Table5

Within-localitiescomparisonoftickinfectionprevalencesandtickinfectionmeandensitiesconsideringthefourmaininfectedtickspecies[R.(B.)decoloratus,R.e.evertsi,A. hebraeum,andHy.ruﬁpes].

Location Tickinfectionprevalence Tickpathogenmeandensity

No.infected/tested z-Values P-Values Meaninfection/tick z-Values P-Values Sandveld(w) 77/343(22.45%) 4.02 <0.0001 110/343(0.32) 5.09 <0.0001 Sandveld(d) 24/251(9.56%) 25/251(0.10) WillemPretorius(w) 14/137(10.22%) 0.1 0.91 18/137(0.13) 0.28 0.78 WillemPretorius(d) 25/254(9.84%) 29/254(0.11) Tüssen-Die-Riviere(w) 32/183(17.49%) -0.55 0.58 33/183(0.18) -0.09 0.93 Tüssen-Die-Riviere(d) 21/108(19.44%) 20/108(0.19) Bethal(w) 2/3(66.67%) 1.63 0.1 2/3(0.67) 2.41 0.016 Bethal(d) 3/62(4.84%) 4/62(0.06) Thabazimbi(w) 41/78(52.56%) 4.51 <0.0001 50/78(0.64) 4.01 <0.0001 Thabazimbi(d) 10/80(12.50%) 14/80(0.18) Lephalale(w) 59/228(25.88%) 2.43 0.01 71/228(0.31) 2.54 0.01 Lephalale(d) 21/131(16.3%) 21/131(0.16)

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playaroleinthemaintenanceofthisparasiteinSouthAfrica.Three vector–pathogencombinationsinvolvedA.bovisandTheileriasp. (sable)identiﬁedinthesalivaryglandsoftickspeciesthatwere suspectedvectors.AnaplasmaboviswasreportedbyTonettietal.

(2009)inentireR.e.evertsiticks.Here,A.boviswasdetectedinthe

salivaryglandsof11R.e.evertsicollectedat3localities.Our obser-vationcorroboratespreviousreportsandsupportsthatR.e.evertsi mayactasavectorforA.bovis.ThedetectionofTheileriasp.(sable) inthesalivaryglandsof9R.(B.)decoloratusat3sitesandof5A. hebraeumat2ofthesesitesindicatesthattheseticksmayactas vectorsinadditiontoR.e.evertsiandR.appendiculatus(Steyletal., 2012).TheobservationofTheileriasp.(sable)inthese2additional tickscontributestoexplainthebroadhostrangeofTheileriasp. (sable)(Spitalskaetal.,2005;Yusufmiaetal.,2010;Pﬁtzeretal.,

2011;Steyletal.,2012).BabesiacaballihasaknownvectorinSouth

Africa,butitwasobservedin2newvector–pathogen combina-tions,involvingatickspeciesknowntotransmitotherpathogens. WedetectedB.caballiinthesalivaryglandsof7R.(B.)decoloratus at3locationssuggestingthatthistickmaybeanadditionalvector ofR.e.evertsi(DeWaaletal.,1988).

Theremaining12vector–pathogencombinationsinvolvedonly oneor2records.Amongthose,5werepreviouslysuspected.This wasthecaseforT.mutansinR.e.evertsi(DeVosandRoos,1981), E.ovina in R. (B.) decoloratus (Schulz, 1940) and in Hy. rufipes (Hyalommaspp.,Schulz,1940),andT.taurotragiinH.rufipes(Binta etal.,1998).Inaddition,Babesiasp.(sable)(Oosthuizenetal.,2008) thatwasidentifiedinthesalivaryglandsofHy.rufipesis geneti-callyveryclosetoB.occultans(Oosthuizenetal.,2008)whichis transmittedbyHy.rufipes(ThomasandMason,1981).Thus,our observationisnotsosurprising.Theremaining7vector–pathogen combinationswereunknown.Theileriabicorniswasdetectedinone R.appendiculatus.AnaplasmaboviswasdetectedinoneR.(B.) decol-oratus;itsvectorinSouthAfricaisR.appendiculatus(Scott,1994). Anaplasmaplatyswhichisgenerallyassociatedwithcaninehosts

(Huangetal.,2005)andR.sanguineus(Hoskinsetal.,1991)was

detectedinoneR.e.evertsi.Theileriasp.(giraffe)whichwasonly knowningiraffe(Oosthuizenetal.,2009)wasdetectedinoneR. e.evertsiandoneR.(B)decoloratus.Finally,Theileriasp.(kudu),a pathogenofgreaterkudu(Nijhofetal.,2005)andnyala(T.

angas-sii)(Pﬁtzeretal.,2011),andT.buffeliwereidentiﬁedinthesalivary

glandsofR.appendiculatus. These12 vector–pathogen combina-tionsneedfurtherrecordsforconﬁrmationofthevectorroleof theseticks.

Morethan70%ofthecollectedtickspecieswereattachedtowild ruminants.Differencesininfestationbetweenwildanddomestic hostscanbeexplainedbyalowerexposuretoticksofdomestic animalsthatweretreatedwithacaricidesonaregularbasis. Fur-thermore,domesticanimalsgrazeinmanagedcampsrestrictedto severalhectareswhilewildanimalhabitatsaremuchlargerand non-managed.Salivaryglandsofticksthatwereattachedtowild ruminantsdisplayedsigniﬁcantlyhigherinfectionprevalencesand pathogenmeandensitiesthansalivaryglandsofticksattachedto domesticanimalsat3localities.Thepathogensthatmainly con-tributedtothesigniﬁcantdifferencebetweenwildanddomestic animalsweretheilerialspeciestransmittedonlytransstadiallyby thetwo-hosttickR.e.evertsi.Rhipicephaluse.evertsipopulationsin reserveshaveagreatervarietyofhoststhanthoselivingonfarms. Thisallowsthemtoaccumulatepathogenspeciesoriginatingfrom varioushosts.Thiscouldbeone explanationforthedifferences betweensalivaryglandinfectionsofticksassociatedwithwildvs. domesticruminantenvironments.Anotheronecouldbethat acari-cidetreatmentsareappliedondomesticanimals.Thiscouldaffect thetickpopulationstructure(e.g.proportionsbetweenlifestages) andthereforereduceverticaltransmissionandpathogenloadin tickpopulations.Finally,differencesinthesusceptibilityto aca-ricidesof infectedticksandnon-infectedtickscouldreducethe

proportionofinfectedticksintheenvironment.Thefrequenceof givenpathogenspeciesinwildordomesticruminantsisalsolinked tothehostpreferenceoftheirvectorticks.Forexample,B.bovis transmittedbyR.(B.)microplusinSouthAfrica(Tonnesenetal., 2004)isalmostexclusivelyfoundoncattleduetothefactthatthey representtheonlyeffectivehostsforR.(B.)microplus(Walkeretal.,

2003).

Here,72%ofthecoinfectionswereobservedinthetwo-hosttick R.e.evertsiandonly22.6%intheone-hosttickR.(B.)decoloratus, despitethefactthattheone-hosttickdisplayedaslightlyhigher infectionprevalenceandpathogenmeandensity.Ticklifecycles with2and 3 hosts increasethechance ofcoinfections. Among the529(23×23)possibilitiesofcoinfections,6pathogen associ-ationsinvolving4TheileriaspeciesandoneBabesiaspecieswere signiﬁcant.Ifcoinfections inIxodes ticksinvolvingBorreliaspp., Babesiaspp.,Anaplasmaspp.,and Rickettsiaspp. weredescribed inNorthAmerica(forexample,Swansonetal.,2006)andEurope (forexample,Lommanoetal.,2012),muchlessisknownon coin-fectionsinticksinsouthernAfrica.Here,signiﬁcantassociations involvedthemostfrequentpathogenspecies.Thiscouldindicate thatassociationsarelinkedtopathogenfrequency.However,the mostfrequentassociations[Theileriasp.(sable)withT.separata, Theileriasp.(sable)withT.bicornis,andT.separatawithT. bicor-nis]werethesameasthoseobservedinthebloodofhostsexposed totheseticks(Berggoetzetal.,2013).Thissuggeststhatthe asso-ciatedspeciestakeadvantageoftheirreciprocalpresence.Future studieswillhavetounderstandthemechanismsofcoinfectionsin ticksandevaluatetheirimpactonwildanddomesticruminants.

The present study shows that many examined tick species wereassociatedwithamuchbroaderpathogenrangethan pre-viouslyknown.Rhipicephaluse.evertsiandR.(B.)decoloratusare mainlyconcernedandappearasimportantvectorsinSouthAfrica. PathogenspecieslikeT.bicornis,Theileriasp.(giraffe),Theileriasp. (kudu),andBabesiasp.(sable)weredetectedfortheﬁrsttimein ticks,morepreciselyintheirsalivaryglands,whichsuggeststheir vectorrole.Furthermore,ourobservationsshowedthatticksfrom wildanimal environmentsgenerally harbouredmore infections thanticksfromdomesticanimalenvironments.Thisindicatesthat wildruminantsaremoreexposedtotick-bornepathogenswhich canprobablybeexplainedbythefactthattheyliveinan uncon-trolledenvironmentwithahigherhostspeciesrichnessthaninthe managedcampofdomesticruminants.Ourresultsalsoshedlight onthehighfrequencyofcoinfectionsinticks.

Acknowledgements

We particularlythank F. Marais,J. Watson, and P.Nel from theDepartmentofEconomicDevelopment,Tourismand Environ-mentalaffairs(Detea),FreeStateProvince,fortheirtechnicaland administrativesupport.Withouttheirhelpthisworkwouldnot havebeenpossible.WethanktheveterinariansE.Albertyn,F.Du Plessis,andN.Krielfortheirhelp.Wewouldliketothankallthe FreeStatereservemanagersfortheirsupport.Wearegratefulto theWebsterfamilymembersfortheirhelpandkindness.Wethank allfarmersforwelcomingusontheirfarmsfortickcollection.We addressspecialthankstoR.A.Slobodeanu(Instituteof Mathemat-ics,UniversityofNeuchâtel)forstatisticalanalysis.WethankO. Rais,V.Douet,andN.Tonettifortheirusefuladvicesin labora-torytechniques,andA.S.SantosandS.Casatiforpositivecontrols. Wewouldalsoliketothank«LeRéseauEcologiedesInteractions Durables»andthegroup«TiquesetMaladiesàTiques»aswellasthe «EcoleDoctorale»,UniversityofNeuchâtel.Thisstudyispartofthe PhDthesisofM.Berggoetz.TheSwissNationalScienceFoundation (SNSF)(FN31003A125492/1) fundedthis research.Inaddition, ﬁnancial support wasgiven by the Funds M. Wüthrich and A.

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ARTICLE IN PRESS

GModel

Mathey-Dupraz(UniversityofNeuchâtel)andtheSwissAcademy ofSciences(SCNAT).Wewouldliketothankthe2reviewersfor theirusefulcomments.

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