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Ticks and Tick-borne Diseases
jo u r n al h om epa 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
Tick-borne pathogens in the blood of wild and domestic ungulates in South Africa: Interplay of game and livestock
M. Berggoetz
a,∗, M. Schmid
a, D. Ston
a, V. Wyss
a, C. Chevillon
b, A.-M. Pretorius
c, L. Gern
aaInstitutdeBiologie,Laboratoired’Eco-EpidémiologiedesParasites,UniversityofNeuchâtel,EmileArgand11,2000Neuchâtel,Switzerland
bMaladiesInfectieusesetVecteurs:Ecologie,Génétique,Evolution,Contrôle(MIVEGEC;UMR5290CNRS-IRD-UniversitésMontpellierIetII),911Avenue AgropolisBP64501,34394MontpellierCedex5,France
cDepartmentofHealthSciences,FacultyofHealthandEnvironmentalSciences,CentralUniversityofTechnology,FreeStateProvince,Bloemfontein9300, SouthAfrica
a r t i c l e i n f o
Articlehistory:
Received9July2013
Receivedinrevisedform9October2013 Accepted14October2013
Available online 11 January 2014
Keywords:
Tick-bornepathogens Africanwildlife Livestock Coinfections Theileria Babesia Ehrlichia Anaplasma
a b s t r a c t
Wescreenedfortick-bornepathogensbloodsamplesfrom181wildanddomesticungulatesbelonging to18hostspeciesin4SouthAfricanProvinces.Polymerasechainreactionfollowedbyreverselineblot- tingandsequencingalloweddetecting16tick-bornepathogenspeciesbelongingtothegeneraBabesia, Theileria,Anaplasma,andEhrlichia.Tenpathogenspecieswereinvolvedin29newhost–pathogencombi- nations.Mostinfections(77.9%)involvedmorethanonepathogenspecies.Principalcomponentanalysis (PCA)assignedthe163infections,identifiedtospecieslevel,to4groups.Threegroupswereassociated withsheep,cattle,andhorseandtheirrespectivewildcounterparts.Eachgroupwascharacterisedbyhigh homogeneityinpathogenassemblageandhostphylogeneticstatus.Thesegroupscharacterisedthemost privilegedtransmissionroutesbetweenandamongwildanddomesticungulates.The4thgroupshowed highheterogeneityinpathogenassemblageandhostphylogeneticstatus.Thisgroupseemstoindicate frequentspillovereventsinimpalaofpathogensthatusuallycirculateamongcattle-orsheep-related species.Within6localities,wesampledanequalnumberofwildanddomesticanimals(n=128).Onthis datasetoncehavingcontrolledforthesignificantvariationamonglocalities,theinfectionprevalenceand intensityofinfectiondidnotdiffersignificantlybetweenwildanddomestichosts.Thissuggeststhatboth animaltypes,domesticandwildhosts,couldactasevenlyefficientsourcesofinfectionforthemselves andforeachother.Overall,thisstudyshednewlightonthepathogencirculationnaturallyachievedat theinterplaybetweenwildanddomesticungulates.
© 2013 Elsevier GmbH. All rights reserved.
Introduction
Theknowledgeontheepidemiologicalrelationsbetweenwild anddomesticungulatesinthecontextoftick-bornepathogensof thegeneraBabesia,Theileria,Anaplasma,andEhrlichia in south- ernAfricaremainsincomplete.Someofthesepathogensevolved togetherwithwildungulatesover manymillionyearsresulting, usually, in a stateof equilibrium due toreciprocal adaptations (JongejanandUilenberg,2004).Livestockanimalsarrivedonthe currentSouthAfricanterritoryabout2000yearsagorepresentedby
∗Correspondingauthorat:InstituteofBiology,LaboratoryofEco-Epidemiology ofParasites,UniversityofNeuchâtel,EmileArgand11,2000Neuchâtel,Switzerland.
Tel.:+41327183043;fax:+41327183001.
E-mailaddresses:[email protected](M.Berggoetz), [email protected](M.Schmid),[email protected](D.Ston), [email protected](V.Wyss),[email protected](C.Chevillon), [email protected](A.-M.Pretorius),[email protected](L.Gern).
fat-tailedsheepfollowedbySanga-typecattleapproximately1500 yearsago(DuToit,2008).Europeanlivestockbreedsintroduced since1706weremoresusceptibletotick-bornepathogens,which challengedtheirestablishmentinSouthAfrica(Bigalke,1994).
InSouthAfrica,themainindigenoustick-bornediseasesaffect- ingcattlearecorridordisease,heartwater,Africanredwater,and gallsicknesswhich arecausedbyTheileria parva,Ehrlichia rumi- nantium,Babesiabigemina,andAnaplasmamarginale,respectively (Bigalke,1994;Uilenberg,1995).LesspathogenicorganismslikeT.
mutansandT.veliferaalsoaffectcattle.Allthesepathogenspecies werefirst describedin cattle.However,theancestral hostofT.
parva,T.mutans,andT.veliferaistheAfricanbuffalo(Synceruscaf- fer)(Bigalke,1994;Mansetal.,2011).Moreover,it seemslikely thatE.ruminantium,B.bigemina,andA.marginalefirstcirculated amongwildAfricanruminants(Bigalke,1994).AccordingtoYin etal.(2007),T.separatathatisgeneticallyrelatedtothesableante- lopeparasiteTheileriasp.(sable)probablyarosefromaspillover fromAfricansableantelope(Hippotragusniger)tosheep.Zebras lookedalsoasthemostlikelyancestralhostsofT.equi,theagent 1877-959X/$–seefrontmatter© 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.ttbdis.2013.10.007
M.Berggoetzetal./TicksandTick-borneDiseases5 (2014) 166–175 167
ofequinepiroplasmosis,sinceauniquegenotypeseemstooccur intheseanimals(Bhooraetal.,2009).Moreover,theintroduction ofexoticcattlebreedsmayalsohavepromotedtheintroductionof newpathogensspeciesinSouthAfrica.Forinstance,thecausative agentofAsiaticredwater,B.bovisisgenerallythoughttobeanAsian parasiteandhasprobablybeenintroducedintoSouthAfricaatthe beginningofthe19thcentury(Theiler,1962).Similarly,thecattle parasiteT.buffeliismostlikelyanAsiaticwaterbuffalo(Bubalus bubalis)-derivedparasite(Gubbelsetal.,2000).
Earlyinvestigationsoftick-bornepathogensinwildruminants mainlyaimedatevaluatingtheirroleintheepidemiologyoflive- stockdiseases (Neitzand Du Toit, 1932; Neitz,1935; Löhr and Meyer, 1973;Löhr etal., 1974;Carmichael and Hobday, 1975).
Wildruminantscanneverthelesssufferfromtick-bornepathogens.
Forinstance,naturalinfectionbyT.taurotragicanbefatalinthe commoneland(Tragelaphusoryx)(Lawrenceetal.,1994b).Never- theless,mostofthefatalcasesingameanimalsareassociatedwith translocations,whennaiveanimalsorpathogensareintroduced intonewregions(Oosthuizenetal.,2009).Forinstance,deathof translocatedblackrhinoceroses(Dicerosbicornis)duetoBabesia bicorniswasreportedaftertranslocation(Nijhofetal.,2003).Sim- ilarly,fatal infectioncasesaftertranslocation werereportedfor infectionbyTheileriasp.(sable)inroanantelope(H.equinus)(Nijhof et al., 2005), a parasite known to cause mortalityin roan and sable antelopes (Steylet al., 2012), threatening thesedeclining hostspecies (http://www.iucnredlist.org). Allthis suggests that relationsbetweenwildanddomesticungulatesaretightandthat pathogensofdomesticanimalsmaythreatenhealthofwildanimals andviceversa.
Recentstudies,basedonlarge-scalemoleculardetectionmeth- ods,allowedidentifyingabroaderhostrangeingameandlivestock animalsfor severaltick-borne pathogenspeciesthan suspected (Pfitzeretal.,2011;Tonettietal.,2009;Muhanguzietal.,2010;
Yusufmiaetal.,2010;amongothers).Weusedtheadvantagesof reverselineblot(RLB)hybridisationtoscreenbloodsamplesfrom wildanddomesticanimalsfortick-bornepathogens.Inthisstudy, weaimedat(i)evaluatingthetick-bornepathogenloadinwildand domesticungulateslivinginclosevicinity,(ii)studyingassociations betweenhostandpathogenspecies,(iii)examiningcoinfectionpat- ternsofpathogenspecies,and(iv)highlightingaspectsofthenature oftheinterplayexistingbetweenthepathogensandtheirwildand domestichosts.
Materialsandmethods Studyareasanddatasets
Sampling was undertaken during 3 fieldwork sessions (May–July 2009, January–May 2010, and April–June 2011) in 4SouthAfricanprovinces(Fig.1).Afirstdatasetof128animals corresponds to a paired-sampling of wild and domestic hosts within each of 6 localities (Fig. 1). Wild animal samples were collectedat Tüssen-Die-Riviere,Willem Pretorius,and Sandveld intheFreeStateProvince(3provincial reserves),nearBethalin theMpumalanga Province (one privategame farm) and in the LephalaleandThabazimbiareasintheLimpopoProvince(5private gamefarms).Domesticanimalsampleswerecollectedwithinthe sameweekastheirwildcounterpartsfrom severalcommercial livestockfarmslocatedintheclosevicinityofthese6sites(com- monborder orwithin a rangeof 40km). Thisfirstdatasetwas extendedbytheadditionof53animals.Theseanimalsoriginated fromTüssen-Die-Riviere,WillemPretorius,andSandveldandwere sampledfrom3additionalsitesatthesametimeasanimalsfrom thefirst sampling:atSterkfontein,Seekoeivleiin theFreeState
Province(2naturereserves)andPretoriaintheGautengProvince (2livestockfarms).
Bloodsampling
Bloodwascollectedfromgameanimalsduringgamecapture, cullingoperations,andhunts.Cattleweredrivenintokraalswhere holdingfacilitieswereavailable.Sheepwerebroughtintosmaller campsandimmobilisedbyhand.Bloodwaswithdrawnfromthe jugularorthecoccygealveinfromliveanimalsorcollectedfromthe throatofhuntedanimals,and200lwereplacedonFTAClassic Cards(Whatman®,Buckinghamshire,UK).Oncedried,thecards werestoredasindicatedbythemanufacturer.Allsampledanimals appearedingoodhealthconditions,exceptonecow(seebelow).
Detectionandidentificationofpathogenspecies
To obtain template DNA for PCR, 1.2mm diameter discs were punched into FTA card blood spot with the use of the HarrisMicro-Punch® (Whatman®,Buckinghamshire,UK).Before each sample,the tipof theHarris Micro-Punch® was rinsedin bleachand 70%alcohol anddriedwithsterilewipe. Discswere washed3 timesin200lFTA purificationreagent(Whatman®, Buckinghamshire, UK) and twice in 200l house-made TE- 1 buffer (10mM Tris–HCL, 0.1mM EDTA, pH 8.0) in 300l tubes as recommended by the manufacturer. Discs were dried at 56◦C for 20min in a stove, followed by PCR amplification.
Primers16S8FE(5-GGAATTCAGAGTTGGATCMTGGYTCAG-3)and B-GA1B-new(5-Biotin-CGGGATCCCGAGTTTGCCGGGACTTYTTCT- 3)(Schoulsetal.,1999,modifiedbyBekkeretal.,2002)wereused toamplifyanapproximately500-base-pair(bp)fragmentofthe16S ribosomalRNA(rRNA)genespanningthehypervariableV1region ofthegeneraAnaplasmaandEhrlichia.TodetectthegeneraBabesia andTheileria,anapproximately400-bpfragmentofthe18SrRNA genespanningtheV4hypervariableregionwasamplifiedwiththe useoftheprimersetRLB-F2(5-GACACAGGGAGGTAGTGACAAG-3) andRLB-R2(5-Biotin-CTAAGAATTTCACCTCTGACAGT-3)(Georges etal.,2001).PrimerswereobtainedfromMicrosynthAG(Balgach, Switzerland).AmplificationreactionswereperformedinaMas- terMixvolumeof25l(Bekkeretal.,2002).PCRreactionstook placeinaWhatmanBiometra®TprofessionalBasicGradient(Göt- tingen,Germany)byusingatouchdownPCRprogramme(Bekker etal.,2002),withanannealingtemperatureloweredby1◦Cinstead of2◦C (Tonettiet al.,2009).Positivecontrolsconsistingin FTA cardswithcattlebloodfromtheIvoryCoastinfectedbyA.cen- trale or A. marginale and B.bovis (kindly provided byC. Rühle, UniversityofNeuchâtel)andnegativecontrolconsistinginaclean FTAcard wereincludedin eachPCR run.Toidentifypathogens atspecieslevel,theobtainedPCRproductswereanalysedbyRLB hybridisation(Gubbelsetal.,1999,modifiedbyTonettietal.,2009) using41oligonucleotideprobes,including3genus-specificprobes Babesia/Theileria,Anaplasma/Ehrlichia,andTheileria(Supplemen- taryButleretal.,2008;Heaetal.,2011;Matjilaetal.,2004;Nagore etal.,2004;Oosthuizenetal.,2008;Ouraetal.,2004;Pfitzer,2009;
Ros-Garciaetal.,2011;TableS1).Samplesreactingonlywiththe Babesia/Theileriaprobe wereconsideredasBabesiaspp.since a genus-specificprobewasincludedforthegenusTheileria.Inorder totestfortheoreticalspecificity,theoligonucleotideprobesusedin thisstudywerealignedwithvarioussequencesoftargetedspecies availablefromtheNationalCentreforBiotechnologyInformation (NCBI)usingasoftwarepackage:CLCSequenceViewer6(CLCbio, Aarhus,Denmark).
Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.ttbdis.2013.10.007.
Fig.1.HostsamplinginstudyareasinSouthAfrica.(A)Tüssen-Die-Riviere(cattlen=17;sheepn=10;commonelandn=8;springbokn=6;greaterkudun=5;impalan=5;
blackwildebeestn=2;Africanbuffalon=1;additionalhosts:warthogn=3;commonelandn=4;greaterkudun=1).(B)WillemPretorius(cattlen=7;Africanbuffalon=7;
additionalhost:Africanbuffalon=1).(C)Sterkfontein(additionalhosts:blackwildebeestn=4).(D)Seekoeivlei(additionalhosts:blackwildebeestn=11;redhartebeest n=15;plainzebran=6;Africanbuffalon=1).(E)Sandveld(cattlen=12;sheepn=4;Africanbuffalon=7;bluewildebeestn=4;greaterkudun=3;commonelandn=1;
gemsbokn=1;additionalhosts:roanantelopen=5).(F)Bethal(cattlen=4;blesbokn=3;blackwildebeestn=1).(G)Thabazimbi(cattlen=3;impalan=2;bluewildebeest n=1).(H)Lephalale(cattlen=7;Southerngiraffen=5;sableantelopen=2).(I)Pretoria(additionalhosts:horsen=2).
Modifiedfromhttp://home.global.co.za/∼mercon/map.htm
Sequencing
Prior sequencing, PCR products that reacted only with genus-specific probes Babesia/Theileria, Theileria, or Anaplasma/Ehrlichia and not with species-specific probes were purifiedusingacommercialavailablekit(Wizard®SVGelandPCR Clean-Up System,Promega, Madison, USA). Themanufacturer’s instructions were followed, except that we eluted in 35l of nuclease-free water. Sequencing wasperformed by Microsynth AG(Balgach,Switzerland).Theobtainedsequenceswerecorrected andcomparedbyusingsoftwarepackagesCLCSequenceViewer 6 (CLC bio, Aarhus, Denmark) and Bioedit (Tom Hall Ibis Bio- sciences,Carlsbad,USA).Sequenceswerecomparedwithavailable sequencesfromtheNCBIusingtheBasicLocalAlignmentSearch Tool(BLAST).
Dataanalysis
Datawereanalysedwith“R”2.14forWindows(RDevelopment CoreTeam, 2012.R: Alanguageandenvironment forstatistical computing.RFoundationforStatisticalComputing,Vienna,Austria.
ISBN3-900051-07-0,URLhttp://www.R-project.org/),usingsoft- warepackages(Skaugetal.,2010;Hussonetal.,2012).
Twogeneralisedlinearmodels(GLM)wereusedonthepair- sampledatasetinvolvingwildanddomesticanimalsthathadbeen sampled withineach of 6 localities. In each case, we assessed thesignificanceofthefactorsLOCATION(6localities)and HOST TYPE(wild vs. domestic) as wellas theLOCATION–HOST TYPE interaction.ThefirstGLM,withbinomialerrors,evaluatedinfec- tion prevalence of pathogens. The second GLM, with negative
binomialerrors,evaluatedtheintensityofinfection(numberof infections/numberinfectedanimals).
Principalcomponentanalysis(PCA)wasemployedasordina- tiontechniqueinordertogroupthehostanimalswithregardto thepathogenspeciesinfectingthem.Pvalueswereconsideredsig- nificantwhenbelow0.05.Thepermutationtestproposedinitially byRaupandCrick(1979)anddevelopedbyCluaetal.(2010)was constrainedtolocationsforevaluatingwhethertheadjustedasso- ciationsbetweenpathogenspeciesincoinfectionsweresignificant.
Inthislatercase,thePvaluewasconsideredsignificantwhenbelow 0.0004(Bonferronicorrected).
Results
Bloodsampleswerecollectedfrom181animalsbelongingto18 species(Fig.2).Amongthose,128sampleswerepairedinorder tocomparethepathogenprevalenceandtheintensityofinfection betweenwildanddomesticanimalsforagivenlocality.Fifty-three animalswere thenadded toevaluatewhetheranyassociations betweenhostandpathogenspeciesoccur.
Allsamples thatwere identified tospecies level wereiden- tified by RLB except 7 samplesthat were identified to species levelaftersequencing.Thefirstone(GenBankaccessionnumber KF414713)wasisolatedfromsheepandshowed99%homology with A. platys (GenBank accession number AY040853.1). Three identicalsequences(GenBankaccession numberKF414712)iso- latedfromcattleandAfricanbuffaloshowed100%homologywith A. marginale(GenBankaccession number FJ155998.1).Finally,3 identicalsequences(GenBankaccessionnumberKF414721)were
M.Berggoetzetal./TicksandTick-borneDiseases5 (2014) 166–175 169
Fig.2. Sampledhosts(n=181)assignedto18species,andtheirtaxonomicrelations,accordingtoMattheeandDavis(2001)andFernandezandVrba(2005).
isolatedfromsheepand displayed 100%homology withA. ovis (GenBankaccessionnumberEU191231.1).
Comparisonbetweenwildanddomesticanimalswithinsix localities
Atotal of 40 wildand 43 domestic animals (83/128,64.8%) werefoundinfected and161infections weredetected inthese animals (Table 1). The intensity of infection (number of infec- tions/numberofinfectedanimals)reached1.94(161/83)pathogen.
Thirty-threeinfections(20.5%)wereidentifiedatgenuslevelonly:
25 Anaplasmaspp. and/or Ehrlichia spp., 5 Theileria spp., and 3 Babesiaspp.(Table1).Sequencingofthesesamplesdidnotallow identificationtospecies.Theremaining128infections(79.5%)were identifiedatspecieslevel;theybelongedto15pathogenspecies.
Theileriasp.(sable)(21.9%)andT.separata(14.1%)werethemost frequentspeciesfollowedbyT.buffeli(9.4%)andA.marginale(9.4%).
The infection prevalence and intensity of infection differed significantly withthe LOCATION factor (P<0.001 and P=0.021, respectively),butnot withtheHOSTTYPEfactor(P=0.547and P=0.378,respectively)orwithLOCATION–HOSTTYPEinteraction (P=0.137andP=0.828,respectively).
Associationsofpathogenspecieswithhostspecies
Tobetterunderscorepossibleassociationsbetweenhostand pathogenspecies,weanalysedbloodsamplesfrom53additional hosts(Table2).Forty-nineinfectionsweredetectedintheseaddi- tionalhosts.Amongthose,35infections(71.4%)wereidentifiedto specieslevel,theremaining14infectionswereidentifiedtogenus levelonly(8Anaplasmaspp.and/orEhrlichiaspp.,5Theileriaspp., andone Babesiaspp.)(Table2).Mergingthis seconddatasetto thepreviousoneledto 163infectionsidentified at thespecies level, involving 16 pathogen species. This dataset (Hellinger- transformed)wasusedtoperformPCAinordertohighlightpossible associationsbetweenpathogenandhostspeciesthatwerenotdue tosamplinglocation(datanotshown).Fig.3adisplaysthegroupsof hostspeciesderivingfrompathogenassemblage.Fig.3bpresents therelativecontributionofagivenpathogenspeciestothedef- initionofhostgroupsviapathogenassemblage.Group1wasthe largesthostgroup.Itincluded9hostspecies(springbok,bluewilde- beest,black wildebeest,redhartebeest,blesbok, sableantelope, roanantelope,gemsbok,andsheep)thatshowedarelativelyhigh proportionofinfectionswithT.bicornis,T.separate,andTheileria sp.(sable),andtoalesserdegreewithT.ovisandA. ovis.These 5pathogenspecieswerestronglycorrelatedwitheachother,with
eachofthembeingsignificantlycorrelatedwiththefirstaxis(Pval- ues<0.001)(Fig.3b).Theileriabuffeli,A.platys,andA.centralewere alsocorrelatedwiththepathogenscharacterisingGroup1,butwere notsignificantlycorrelatedwiththefirstaxis(Fig.3b).Inthisgroup, T.buffeli(n=17)wasdetected11times,A.platysonce,andA.cen- trale4timesoutofatotalof6detectioncases(Tables1and2).
Attheoppositesidealongthefirstaxis,Group2consistedofbuf- falo,commoneland,greaterkudu,andcattle(Fig.3a).T.taurotragi, T.velifera,T.mutans,andA.marginalewereexclusivelydetected inthesehostspecies.BabesiabigeminaandB.boviswerecorre- latedwiththeotherpathogensofGroup2andwiththefirstaxis, althoughnotsignificantly(P=0.45)(Fig.3b).Babesiabigeminawas detectedin3outofthe4hostspeciesconstitutingGroup2whileB.
boviswasdetectedonlyonceincattle(Table1).Horsesandzebras formedGroup3;thisthirdgroupbeingcharacterisedbythepres- enceofT.equi(Fig.3a).Finally,impalaandgiraffeformedGroup 4whichisinterestinglydefinedbyamixofthepathogenassem- blagesdefiningtheGroups1and2(Fig.3a).Impalawasinfected with4pathogenspeciesthatwereabundantinGroup1(T.bicornis, T.sp.sable,T.buffeli,andA.centrale)aswellaswithB.bigemina occurringinGroup2.Theonlypathogenidentifiedtospecieslevel ingiraffewasEhrlichiasp.(Omatjenne)(n=1),whichalsooccurred inGroups1and2.
Coinfections
Amongthe163infectionsidentifiedatthespecies level,127 (77.9%) were coinfections involving more than one pathogen species(SupplementaryTableS2).Coinfectionsweredetectedin 22.1%(40/181)individualhosts[17/64domesticanimals(26.6%) and 23/117 wildanimals (20%)]. Among these coinfections, 22 (17.3%) involved 2 species, 47 (37%) were triple infections, 33 (26%) involved 4 pathogen species, and 25 (19.7%) involved 5 pathogenspecies.Associationsamongthe16pathogenspeciesare showninTable3.Significanceofcoinfectionswascomputedusing statisticalanalysisproposedbyRaupandCrick(1979)anddevel- opedbyCluaetal.(2010)andconstrainedtolocationstodecide whethertheadjusted associationsbetweenpathogenspeciesin coinfectionsweresignificant.Sixpathogenassociations,involving 5Theileriaspecies,weresignificant:Theileriasp.(sable)-T.separata (n=19);Theileriasp.(sable)-T.buffeli(n=15);Theileriasp.(sable)- T.bicornis(n=14);T.separata-T.bicornis(n=7);T.separata-T.ovis (n=6);T.bicornis-T.ovis(n=4)(Table3).Thesecoinfectionswere observedinanimalsbelongingtoGroup1aswellasinimpalasfrom Group4.
M.Berggoetzetal./TicksandTick-borneDiseases5 (2014) 166–175
Table1
Tick-bornepathogensbelongingtothegeneraBabesia,Theileria,Anaplasma,andEhrlichiainwild(n=64)andindomesticanimals(n=64)livinginclosevicinity(firstdataset).
Hosts/pathogens No.infected/
tested
B/Tgenus B.bovis B.bigemina Theileria genus
T.mutans T.taurotragi T.velifera T.bicornis T.separata T.buffeli T.ovis T.sp.(sable) A/Egenus A.centrale A.marginale A.ovis A.platys E.sp.
(Omatjenne) No.of infections
Cattle 31/50(62%) 1 1d 2c,e 0 10e,f 2d,f 6e,f 0 0 3e,f,b 0 5e,f 16 0 2a,c 0 0 3b,c 51
Sheep 12/14(85.7%) 0 0 0 0 0 0 0 6a,c 9a,c 0 7a,c 9a,c 0 4a 0 3a,c 1a 0 39
PrevalenceD 43/64(67.2%) 1(1.6%) 1(1.6%) 2(3.1%) 0 10(15.6%) 2(3.1%) 6(9.4%) 6(9.4%) 9(14.1%) 3(4.7%) 7(10.9%)14(21.9%) 16(25%) 4(6.3%) 2(3.1%) 3(4.7%)1(1.6%) 3(4.7%) 90
Bluewildebeest 5/5(100%) 0 0 0 0 0 0 0 0 4c,e 3c 1e 4c 0 0 0 0 0 0 12
Blackwildebeest 2/3(66.7%) 0 0 0 0 0 0 0 0 2a,d 1d 0 2a,d 0 0 0 0 0 0 5
Blesbok 3/3(100%) 0 0 0 0 0 0 0 0 0 0 1d 2d 0 0 0 0 0 0 3
Springbok 2/6(33.1%) 0 0 0 0 0 0 0 0 1a 0 0 1a 0 0 0 0 0 0 2
Sableantelope 2/2(100%) 0 0 0 0 0 0 0 2f 2f 2f 2f 2f 0 0 0 0 0 0 10
Gemsbok 1/1(100%) 0 0 0 0 0 0 0 0 0 0 0 1c 1 0 0 0 0 0 2
Buffalo 9/15(60%) 0 0 0 0 0 0 0 0 0 0 0 0 4 1c 5b,c 0 0 0 10
Commoneland 5/9(55.6%) 0 0 0 0 0 0 0 0 0 0 0 0 2 0 2a 0 0 0 4
Greaterkudu 3/8(37.5%) 1 0 1c 0 0 0 0 0 0 1c 0 0 0 0 3a,c 0 0 0 6
Impala 3/7(42.9%) 0 0 1a 0 0 0 0 2e 0 2e 0 2e 0 1e 0 0 0 0 8
Giraffe 5/5(100%) 1 0 0 5 0 0 0 0 0 0 0 0 2 0 0 0 0 1f 9
PrevalenceW 40/64(62.5%) 2(3.1%) 0 2(3.1%) 5(7.8%) 0 0 0 4(6.3%) 9(14.1%) 9(14.1%) 4(6.3%)14(21.9%) 9(14.1%) 2(3.1%) 10(15.6%) 0 0 1(1.6%) 71
Total 83/128(64.8%) 3(2.3%) 1(0.8%) 4(3.1%) 5(3.9%) 10(7.8%) 2(1.6%) 6(4.7%) 10(7.8%) 18(14.1%) 12(9.4%) 11(8.6%)28(21.9%) 25(19.5%) 6(4.7%) 12(9.4%) 3(2.3%) 1(0.8%) 4(3.1%) 161
B/T:Includesinfectionsreactingwiththisprobeonly;consideredasbelongingtothegenusBabesia.
D,domesticanimals;W,wildanimals.Locationsoftheinfectionsidentifiedatspecieslevel:
aTüssen-Die-Riviere.
b WillemPretorius.
c Sandveld.
d Bethal.
eThabazimbi.
f Lephalale.
Table2
Tick-bornepathogensbelongingtothegeneraBabesia,Theileria,Anaplasma,andEhrlichiainfectingthe53additionalhosts(seconddataset).
Host/pathogens No.infected/tested B/Tgenus Theileriagenus T.bicornis T.separata T.buffeli T.sp.(sable) T.equi A/Egenus A.marginale E.sp.(Omatjenne) No.ofinfections
Blackwildebeest 7/15(46.7%) 0 0 1c 6b,c 0 5b,c 0 0 0 0 12
Redhartebeest 1/15(6.7%) 0 0 0 1c 0 1c 0 0 0 0 2
Roanantelope 5/5(100%) 0 0 5d 0 5d 5d 0 3 0 2d 20
Buffalo 1/2(50%) 1 0 0 0 0 0 0 0 0 0 1
Commoneland 3/4(75%) 0 0 0 0 0 0 0 3 1a 0 4
Greaterkudu 1/1(100%) 0 0 0 0 0 0 0 1 0 0 1
Plainzebra 6/6(100%) 0 5 0 0 0 0 1c 0 0 0 6
Domestichorse 2/2(100%) 0 0 0 0 0 0 2e 0 0 0 2
Warthog 1/3(33.3%) 0 0 0 0 0 0 0 1 0 0 1
Total 27/53(50.9%) 1(1.9%) 5(9.4%) 6(11.3%) 7(13.2%) 5(9.4%) 11(20.8%) 3(5.7%) 8(15.1%) 1(1.9%) 2(3.8%) 49
B/T:Includesinfectionsreactingwiththisprobeonly;consideredasbelongingtothegenusBabesia.
Locationsoftheinfectionsidentifiedatspecieslevel:
aTüssen-Die-Riviere.
b Sterkfontein.
c Seekoeivlei.
d Sandveld.
ePretoria.
M.Berggoetzetal./TicksandTick-borneDiseases5 (2014) 166–175 171
Table3 Coinfections,constrainedtolocations,ofpathogenspeciesbelongingtothegeneraBabesia,Theileria,Anaplasma,andEhrlichiainvolvedincoinfectionsobservedamong181wildanddomesticruminants(datasets1and2). PathogensT.sp.(sable)T.separataT.ovisT.bicornisA.ovisA.centraleA.platysT.mutansT.veliferaT.taurotragiT.buffeliA.marginaleB.bigeminaE.sp. (Omatjenne)No.of infections/speciesa T.sp.(sable)/19*814*34155115*01239 T.separata19*/6*7*311000400025 T.ovis86*/4*230000000011 T.bicornis14*7*4*/210000500216 A.ovis3322/0000000003 A.centrale41310/000001006 A.platys110000/00000001 T.mutans5000000/61201010 T.velifera50000006/120106 T.taurotragi100000011/00002 T.buffeli15*405000220/12217 A.marginale00000100001/2013 B.bigemina100000011022/04 E.sp.(Omatjenne)200200000020006 No.ofinfections/speciesa392511163611062171346159 aObservedamongthe181ruminants. *Significantassociations(BonferronicorrectedPvalues<0.0004)givenbythepermutationtest(RaupandCrick,1979;modifiedbyCluaetal.,2010).
Fig.3. Principalcomponentanalysis(PCA)onHellinger-transformeddata:Infec- tionsby16pathogenspecies(163infections)of181hostsbelongingto18species.
3aGroupsofhostspeciesderivingfrompathogenassemblage.Group1(inblack):
sk,springbok(n=6);bw,bluewildebeest(n=5);bkw,blackwildebeest(n=18);
rh,redhartebeest(n=15);bk,blesbok(n=3);sa,sableantelope(n=2);ro,roan antelope(n=5);ge,gemsbok(n=1);sh,sheep(n=14).Group2(inred):b,buffalo (n=17);e,commoneland(n=13);gk,greaterkudu(n=9);c,cattle(n=50).Group 3(inblue):Z,zebra(n=6);hor,horse(n=2).Group4(ingreen):I,Impala(n=7);
g,giraffe(n=5).3bContributionofthepathogenspeciestothedefinitionofhost groupsviapathogenassemblage.T.buffeli(n=17),Theileriasp.(sable)(n=39),T.
bicornis(n=16),T.separata(n=25),T.ovis(n=11),A.ovis(n=3),A.platys(n=1),T.
velifera(n=6),T.mutans(n=10),T.taurotragi(n=2),B.bigemina(n=4),A.marginale (n=13),B.bovis(n=1),Ehrlichiasp.(Omatjenne)(n=6),A.centrale(n=6),T.equi (n=3).(Forinterpretationofthereferencestocolorinthisfigurelegend,thereader isreferredtothewebversionofthearticle.)
Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.ttbdis.2013.10.007.
Discussion
In this study, tick-borne pathogens were detected by RLB hybridisation using probes allowingidentification at thegenus and atthe species level.Most infections wereidentified atthe specieslevel,neverthelesssomeofthem(n=47)wereidentified atthe genuslevel only evenafter sequencing,due probablyto the presence of multiple sequences of different species and/or variants (Microsynth, pers. communication) belonging mainly toAnaplasmaspp. and Ehrlichia spp.for which noprobeswere
available.Nevertheless,sequencingof7samplesallowedspecies identification:oneA.platys,3A.ovis,and3A.marginale.Absenceof hybridisationofthelast6sampleswiththespecificoligonucleotide probesincludedintheassaywasduetotheforwardpositionof theprobesontheamplifiedsequence.
Thegreatmajorityofinfectionsidentifiedatthespecieslevel (77.9%)involvedmorethanonepathogenspecies,withthemost frequentcombinationinvolving3species(37%).Thiscorroborates previousobservationsincattleinSouthAfricawheremostindi- vidualscarriedcoinfectionswithmainly3speciesofpiroplasms (Yusufmia etal.,2010).Similarly,themajorityof theinfections observedinnyalabyPfitzeretal.(2011)werecoinfections,with combinationof4haemoparasitesbeingthemostfrequent.Inthe presentstudy,coinfectionswith6Theileriaspecies,mainlyasso- ciated with sheep-related host species and with impala, were significant.Howevercoinfectionsinvolvingpathogensofdifferent generawerealsodetected(e.g.withA.marginaleandB.bigemina).
Yusufmiaetal. (2010)reportedcoinfectioninvolvingT. velifera, Theileriasp.(sable),andT.mutans(butnofurtherdetailsofspecies associationsareavailable).Pfitzer etal.(2011)observedcombi- nationswithTheileriasp.(kudu),T.buffeli,T.bicornis,Theileriasp.
(sable),Ehrlichiasp.(Omatjenne),andA.marginale.Stoltsz(1989) reportedthatT.veliferaisrarelyfoundinsingleinfection,butmore frequentlyassociated withother piroplasmspecies. Bintaet al.
(1998)observedhighcattlemorbidityresultingfromdualinfec- tionsofT.mutanswithT.taurotragi,whilesingleinfectionsbyeither oneofthesespeciesgenerallycausemildsymptoms.Theseauthors suggestedthatthesynergisticeffectsofthe2infectionsincreased pathologyandmightinduceimmunodepressiveeffects, enhanc- inginfectionbyadditionalpathogenspecies.Thismightexplain coinfectionwithupto4pathogenspecies,asobservedhere.
Among the 16 identified pathogen species, 9 were Theileria speciesand4(T.taurotragi,T.mutans,T.velifera,andT.equi)dis- playedahostrangecongruentwiththeliterature.Inthepresent study,T.taurotragi,T.mutans,andT.velifera,whichdifferintheir virulencetocattle,wereonlydetectedincattle.Theileriataurotragi isgenerallybenign,butcanoccasionallycausecerebraltheileriosis (DeVosetal.,1981),southernAfricanT.mutansstrainsinducea mildformofthediseasesincontrasttosomeoftheeasternAfrican strains(Lawrenceetal.,1994c), andnoclinicalsignsaregener- allyrecordedforT.velifera(UilenbergandSchreuder,1976).Other studiesdetectedthese3pathogenspeciesinwildBovinae:T.tauro- tragiinthecommoneland(MartinandBrocklesby,1960),T.mutans (Uilenberg,1981)andT.velifera(Uilenberg,1970)intheAfrican buffalo.Finally,T.equitheagentofequinepiroplasmosis(DeWaal andvanHeerden,1994)isknowntoinfecthorses,donkeys,mules, andzebras(Alsaadetal.,2012)andwasdetectedinhorsesand zebrasinthepresentstudy.
Screeningalargevarietyofwildanimals,wedetectedabroader hostrangethanreportedintheliterature.Thisconcerned5Thei- leriaspecies.Theileriasp.(sable)wasthemostfrequentlydetected pathogen.It was observed in 5new hostspecies (sheep, black wildebeest,springbok,gemsbok,andimpala).Itwasalsodetected in6knownhostspeciessuchascattle(Yusufmiaetal.,2010),blue wildebeest,blesbok(Steyletal.,2012),sableantelope,roanante- lope(Nijhofetal.,2005),andredhartebeest(Spitalskaetal.,2005).
Theileria buffeli,a worldwide pathogenin cattle(Gubbels etal., 2000)usuallyconsideredasbenign,althoughpathogenicvariants mayexist(Kamauetal.,2011),wasalsodescribedinAfricanbuf- falos(Allsoppetal.,1999)andnyala(Pfitzeretal.,2011).Inthe presentstudy,wedetectedT.buffeliincattleaswellasin6newhost species(greaterkudu,impala,bluewildebeest,blackwildebeest, sableantelope,androanantelope).Accordingtotheliterature,T.
bicornismainlyinfectswhiteandblackrhinoceroses(Nijhofetal., 2003;Govenderetal.,2011),butalsocattle(Muhanguzietal.,2010) andnyala(Pfitzeretal.,2011).Inthepresentstudy,itwasdetected
in5additionalanimalspecies(sheep,sableantelope,roanantelope, blackwildebeest,andimpala).ThemainhostsforT.separata,the agentofbenignovineandcaprinetheileriosis,aresmalldomestic ruminants(Lawrenceetal.,1994a).However,T.separatawasalso reportedinthecommongreyduiker(Nijhof etal.,2005)andin tsessebe(Brothersetal.,2011).Tonettietal.(2009)suggestedthat wildlifecouldbeasourceofT.separatainfectionforsheep.Inthe presentstudy,wedetectedT.separatanotonlyinsheep,butalso in5newwildanimalspecies(bluewildebeest,blackwildebeest, springbok,sableantelope,andredhartebeest).Thiscorroborates thepreviousobservationsofthisparasiteinwildlife.Overall,T.sep- arataappearsthusasacommoninfectionofwildruminantspecies closelyrelatedtoCaprinae.Finally,T.ovis,theotheragentofbenign ovineand caprinetheileriosis,wasidentified inits knownhost (sheep)aswellasin3newhostspecies(blesbok,bluewildebeest, andsableantelope).
FourAnaplasmaspecieswereidentified,namelyA.ovis,A.cen- trale, A. marginale, and A. platys, and 3 were involved in new host–pathogencombinations.Anaplasmaovis,theagentofovine andcaprineanaplasmosis,causessubclinicaltosevereillnesseven leadingtodeath,althoughrarelyinsouthernAfrica(Stoltsz,1994).
WedetectedA.ovisonlyinsheep,butitwaspreviouslyreported insableantelopes(Thomasetal.,1982).Anaplasmacentralethat usuallyproducesamildformofgallsicknesswasidentifiedinits knownhosts,sheep(PotgieterandVanRensburg,1987)andbuffalo (BrocklesbyandVidler,1966)aswellasinanewlyreportedwild animalspecies,theimpala.Anaplasmaspp.antigenswereprevi- ouslyreportedintheimpala(Löhretal.,1974),butitisnotknown whetherA.centralewasinvolved.IncontrasttotheotherAnaplasma species detectedhere,A. marginale, theagent ofgallsickness,is knowntoinfectvariouswildanimalspecies(NeitzandDuToit, 1932;Neitz,1935;Löhretal.,1974;Tonettietal.,2009)includ- ingAfricanbuffaloandcommoneland(Potgieter,1979;Ngeranwa etal.,1998).WedetectedA.marginale incattle,Africanbuffalo, commoneland,andgreaterkudu.Nopreviousreportforgreater kuduwasfoundintheliterature,butitwasdescribedintheclosely relatednyala(T.angassii)(Pfitzeretal.,2011).Theinfectionisusu- allysubclinicalin wildanimals,except ingiraffe,which isthus farthe only wildruminant seriouslyaffected bythis pathogen (AugustynandBigalke,1972).Finally,A.platys,previouslyE.platys (Dumler et al., 2001), responsible for canine infectious throm- bocytopenia (Huang et al., 2005)was surprisingly identified in onesheep.Thesequence(GenBankaccessionnumberKF414713) showed99% similarity toA. platys (GenBankaccession number AY040853.1)detectedinoneItaliandogbySparaganoetal.(2003).
However,Chochlakisetal.(2009)alsodetectedA.platysinCapri- nae(goats).Inokumaetal.(2005)showedthatA.platysisclosely relatedtoAnaplasmasp.(Omatjenne)(GenBankaccessionnumber U54806)fromsheepinSouthAfrica(Allsoppetal.,1997)andto Anaplasmasp.(BomPastor)(GenBankaccessionnumberAF318023) fromgoatinMozambique(Bekkeretal.,2001).Thus,ourfindingof A.platysinsheepisnotsosurprising,butneedsfurtherstudyingto clarifythesituation.
TwoBabesiaspecies,B.bovis,theaetiologicalagentsofAsiatic redwater,andB.bigeminaresponsibleforAfricanredwaterwere detectedinhostblood.Babesiaboviswasidentifiedonlyinonecow thathadsuccumbedtothedisease.Theextremelylowprevalenceof B.boviscanprobablybeexplainedbythefactthatbloodwastaken fromthejugularorthecoccygealveinandnotfromcapillaryblood forallsampledcattleexceptthecowthatwasinfectedwithB.bovis.
Inthisspecificcase,bloodwastakenfromthethroatofthedead animalprobablycontaminatedwithcapillaryblood.Themainvec- torofB.bovisinSouthAfrica,Rhipicephalus(Boophilus)microplus (Tonnesenetal.,2004),wasobservedonthisanimal(Berggoetz etal.,2013).Babesiabigeminawasdetectedinitsusualhosts(i.e.
cattleandimpala),whichconfirmspreviousfinding(Löhretal.,
M.Berggoetzetal./TicksandTick-borneDiseases5 (2014) 166–175 173
1974).However,wealsodetectedB.bigeminainonegreaterkudu.
Toourknowledge,thisisthefirstreportforthishostspecies.Usu- ally,clinicalsignsarerestrictedtocattle,andtheroleofwildlife intheepidemiologyofbovinebabesiosisneedstobedetermined (Geleta,2005).
TherecentlydescribedandstillfairlyunknownspeciesEhrlichia sp.(Omatjenne) was previously observed in cattle (Muhanguzi etal.,2010),sheep(DuPlessis,1990),goat(Bekkeretal.,2001), andnyala(Pfitzeretal.,2011).Weidentifiedthismicroorganismin cattleandforthefirsttimeinroanantelopeandSoutherngiraffe.
Ehrlichiasp.(Omatjenne)isconsideredasnon-pathogenic,butDu Plessis(1990)observedthatafterseveralpassagesthroughAmbly- ommaticks,thispathogenspeciesmightinducesevereclinicalsigns insheepsimilartothoseofheartwater.
Tosumup,amongthe16pathogenspeciesidentifiedin this study,10hada broaderhostrangethan previouslyknownand wereinvolvedin30newhost–pathogencombinations.Thisgives anewperspectiveofthecirculationofsomeofthesepathogensin nature.Forexample,Theileriasp.(sable),T.bicornis,andT.buffeli appearasmoregeneralistspeciesinthepresentstudythanusually recognisedintheliterature.
PCAshowedthatthe16detectedpathogenspeciesconstitute4 differentpathogenassemblagescorrespondingto4groupsofhost species.Theileriaseparata,T.ovis,A.ovis,T.bicornis,andTheileria sp.(sable)weresignificantlyassociatedwithGroup1,whichcon- tains9hostspecies,namely:bluewildebeest,blackwildebeest,red hartebeest,blesbok(subfamilyAlcelaphinae),sableantelope,roan antelope,gemsbok(subfamily Hippotraginae),sheep(subfamily Caprinae), and springbok (subfamily Antilopinae). Interestingly, thesehostspecies shareevolutionary relationships. Indeed,the subfamiliesAlcelaphinae and Hippotraginae are closely related toone another and constitutethe sister clade of theCaprinae (Matthee and Davis, 2001; Fernandez and Vrba, 2005). Some authorsplaced thespringbok inthe tribeAntilopini withinthe subfamilyAntilopinaetogetherwiththeAlcelaphini,Hippotragini, andCaprini(HassaninandDouzery,1999;RopiquetandHassanin, 2005),thussupportingtheinclusionofspringbokinGroup1.The affiliation of T. separata, T. ovis, and A. ovis to Group 1 is not surprisingasthesespeciesareknownaspathogens ofdomestic Caprinae.DespitetheirpreferenceforGroup1animals,Theileria sp.(sable)(Yusufmiaetal.,2010;Pfitzeretal.,2011)andT.bicornis (Muhanguzietal.,2010;Pfitzeretal.,2011)werereportedinBov- inae.WesuggestthatBovinaeareinfectedbyothergenotypesof thesepathogens,asreportedbyMansetal.(2011)whoobserved oneTheileria sp.(sable)genotype adapted to cattle.Whether a similarsituationoccursforA.centraleandA.platysremainstobe evaluated.ItissurprisingtoseeT.buffeli,aclassicparasiteofthe Bovinae,affiliatedtotheGroup1of pathogens.Furthergenetic studiesarerequiredtoclarifythesituationallthemoresosince Mansetal.(2011)reporteddifferentgenotypesinthisTheileria species.Thisprobablyhighlightsanongoingevolutionaryprocess ofthisspecies.
Theileriataurotragi,T.velifera,T.mutans,andA.marginaleare thepathogenspeciesdefiningtheGroup2,eachwithsignificant Pvalues. ThisgroupisalsosupportedbyB.bovis andB.bigem- ina,althoughnotsignificantly.Regardingthehostspecies,Group2 includedAfricanbuffalo,commoneland,greaterkudu,andcattle, i.e.membersofthesubfamilyBovinae(MattheeandDavis,2001;
FernandezandVrba,2005;RopiquetandHassanin,2005).Theile- riataurotragi,T.velifera,andT.mutansareknowntooccurinwild Bovinae,elandandbuffalo,respectively,withthegenotypesinfect- ingsuchwildhostsconsideredasrepresentingtheoriginofthe genotypesinfectingcattle(Mansetal.,2011).It ispossiblethat othergenotypesofA.marginaleareinvolvedintheinfectionsof non-bovinehostspeciessuchasblesbok,commonduiker,andblack wildebeest(NeitzandDuToit,1932;Neitz,1935).
Horsesandzebras,membersoftheEquidaefamily,formedthe Group3characterisedbythepresenceofT.equi.Bycontrastwith the3formergroups,theGroup4isnotwellcharacterised.This groupisneithercharacterisedbyacohortofpathogenspeciesnor byanycloserphylogeneticrelationshipsamongitshostspecies.
Group4 exhibitsveryheterogeneousinfection profilesencoun- tered in impala and giraffe. Giraffe is part of a distinct family referredtoasGiraffidae(FernandezandVrba,2005).Impalawas successivelyaffiliatedtotheAlcelaphinae(Vrba,1984),Antilopinae (Kingdon,1989),andtotheReduncinae(Murray,1984).Itiscur- rentlyrecognisedasadistinctsubfamily,Aepycerotinae(Matthee andDavis,2001;FernandezandVrba,2005;RopiquetandHassanin, 2005).Inthepresentstudy,impalasharedpathogensaffiliatedto hostsofGroups1and2.Thissuggestsafrequentspilloverofthe pathogensusuallyassociatedwithCaprinaeandBovinaeintothe impalaspecies.
Overall, the host–pathogen combinations detected in the presentstudy suggestthat transmissionoftick-bornepathogen species remains mainly restricted to genetically related host species,exceptforimpalaswhichmayrepresentabridgespecies between several transmission routes. Such importance of the phylogenetic status of the host has previously been observed in experimental infection of domestic animals with pathogens isolated from wild animals (Penzhorn, 2006). However, the present study is the first toour knowledge tocorroborate the host–pathogencompatibilitydependenceonhostphylogenyfrom a wide-scalesamplingdesign involving18 hostspecies and 16 pathogenspecies.Thishighlightsoneaspectofthenatureofthe interplayexistingbetweendomesticandwildanimals.Theobser- vationthat closelyrelated hostspecies, domesticor wild,were mainlyinfected bythesamepathogenspeciesisa crucialpoint tobearinmindingameandlivestockmanagement.
The common belief is that game animals harbour more pathogensthandomesticanimalsbecausetheyareseenasthemain infectionsourcesfordomesticanimals(Bigalke,1994).Inversely, pathogenscouldbemorefrequentindomesticanimalssincethey are known to be more susceptible to infections (Jongejan and Uilenberg,2004).Whenweevaluatedtheinfectionloadinwild (n=64)anddomestic(n=64)animalslivinginclosevicinity,within eachof6localities,weobservedsimilarrangesininfectionpreva- lenceand in intensityofinfectionin both hosttypesusingthe samedetectionmethod.Knowingthatgameusuallyhassubclinical infections(JongejanandUilenberg, 2004), whileremaining car- rier,wildanimalswithaverylowparasitaemiacouldhavebeen overlookedusingRLB.Fromanepidemiologicalpointofview,ani- malswithsuchalowparasitaemiaarenotveryrelevant.Indeed, those animalsare, most probably,not very infectivefor vector ticks.Takenintoaccountthatlivestockisprotectedbyacaricide treatments,vaccination,andfieldmanagementmeasuresandstill displaysimilarinfectionprofilesasgame,wildungulatesappear less susceptible totick-borneinfections than livestockanimals.
Ourresultsstronglysuggestthattheinterplaybetweenwildand domesticungulateslivinginclosevicinityisveryclose,withboth animal types consisting in efficient infection sources for ticks infectingthemselvesandeachother.Nevertheless,livestockani- malsshouldnotbeunderestimatedasinfectionsourceforgame animals.Löhr et al. (1974) reportedthat Theileria,Babesia, and Anaplasmaantibodiesweremuchmorefrequentinantelopesgraz- inginthevicinityofnon-dippedcattlethaninantelopesgrazingin areaswherecattlewereregularlydippedorwereabsent.
The present study revealed infection patterns among host groups,whichdefinedtransmissionpathwaysbetweengivenhost species,amongandbetweendomesticandwildungulates.Further- more,weshowedthatpathogenspecieslikeTheileriasp.(sable), T.buffeli,T.bicornis,T.separata,T.ovis,A.centrale,A.marginale,A.
platys,Ehrlichiasp.(Omatjenne),andB.bigeminahaveabroader
