A molecular and parasitological survey of Hepatozoon canis in domestic dogs in Turkey

VeterinaryParasitology209(2015)264–267

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j o ur na l h o me p a g e:w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Short

Communication

A

molecular

and

parasitological

survey

of

Hepatozoon

canis

in

domestic

dogs

in

Turkey

Munir

Aktas

_,

_Sezayi

_Özübek

_,

_Kürs¸

_at

_Altay

_{, ˙Ibrahim}

_Balkaya

_,

Armagan

Erdem

Utuk

_,

_Akın

_Kırbas

_,

_Sami

_S¸

_imsek

_,

_Nazir

_Dumanlı

a_{DepartmentofParasitology,CollegeofVeterinaryMedicine,FiratUniversity,23119,Elazig,Turkey}

b_{DepartmentofParasitology,CollegeofVeterinaryMedicine,CumhuriyetUniversity,Sivas,Turkey}

c_{DepartmentofParasitology,CollegeofVeterinaryMedicine,AtatürkUniversity,Erzurum,Turkey}

d_{DepartmentofParasitology,CeyhanVeterinaryMedicine,CukurovaUniversity,Adana,Turkey}

e_{DepartmentofInternalMedicine,CollegeofVeterinaryMedicine,AtatürkUniversity,Erzurum,Turkey}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13November2014

Receivedinrevisedform10February2015

Accepted13February2015 Keywords: Hepatozooncanis Dog PCR

a

b

s

t

r

a

c

t

Inthisstudy,asymptomaticdogsinnineprovincesofTurkeyweresurveyedto investi-gatetheprevalenceandintensityofHepatozooncanisinfection.DNAobtainedfromblood samplescollectedfrom694domesticdogs(243stray,288shelter,and163pets)ofboth gendersandvaryingageswereevaluatedbypolymerasechainreaction(PCR).In addi-tion,285thinbloodsmearspreparedfromthesebloodsampleswerealsoevaluatedfor microscopicexamination.DirectmicroscopyrevealedHepatozoongamontsinthe periph-eralbloodofthreeof285(1.0%;95%confidenceinterval(CI):0.21–3.04)tested.UsingPCR, 155ofthe694(22.3%;95%CI:19.28–25.61)werefoundtobepositiveforthepresenceofH. canisDNA.Theprevalenceofinfectionwashigherinadultdogs(26.2%;95%CI:22.1–30.7) thanyounganimals(16.4%;95%CI:12.2–21.3).Althoughtheprevalencedeterminedby PCRwashigherinmaledogs(24.5%;95%CI:19.6–29.9)thaninfemaledogs(20.8%;95% CI:16.9–25.1),genderdifferenceswerenotsignificant.Petdogshadalowerprevalenceof infection(10.4%;95%CI:6.2–16.2)comparedtostray(26.3%;95%CI:20.9–32.3)andshelter dogs(25.7%;95%CI:20.7–31.1),butnosignificantassociationbetweenstrayandshelter dogswasfoundforthepresenceoftheparasite.Partialsequencesofthe18SribosomalRNA (rRNA)geneshared99–100%similaritywiththecorrespondingH.canisisolates.This epi-demiologicalsurveyrevealedahighprevalenceofH.canisindogsfromseveralprovinces inTurkey,anditsuggeststhattheageandoriginareassociatedwiththeparasite.

©2015ElsevierB.V.Allrightsreserved.

1. Introduction

Hepatozoaareprotozoanparasitesthatinfectawide rangeofdomesticandwildcarnivores,birds,reptiles,and amphibians(Littleetal.,2009).Hepatozooncanisand Hepa-tozoonamericanumarefoundindomesticandwildcanids,

∗ Correspondingauthor.Tel.:+904242370000;fax:+904242388173.

E-mailaddress:maktas@firat.edu.tr(M.Aktas).

and H. canis has long been recognized as the cause of hepatozoonosisindogsinAsia,Europe,Africa,andLatin America.Dogsbecomeinfectedwiththeparasiteby ingest-ing ticks or tick parts containing mature oocysts with infectivesporozoites.H.canisisconsideredless virulent thanH.americanum,andcausesseldomclinicalsigns.The mainvectoristhebrowndogtick,Rhipicephalussanguineus, although severalother species have been suggested as potentialvectorsforH.canis(Aktas,2014;Giannellietal., 2013).InEurope,thegeographicaldistributionofH.canisis

http://dx.doi.org/10.1016/j.vetpar.2015.02.015

M.Aktasetal./VeterinaryParasitology209(2015)264–267 265

restrictedtotheMediterraneanregion,andtheBalkanand IberianpeninsulaswherethetickR.sanguineusisfrequent (Estrada-Pe ˜naetal.,2013).Thediseasehasbeendetected inthefollowingsoutheasternEuropeancountries:Croatia (Dezdeketal.,2010),Italy(Gabriellietal.,2010),Bulgaria (Tsachevetal.,2008),andGreece(Mylonakisetal.,2004). Tick-borne pathogens such as Theileria, Babesia, and Anaplasmahavebeendocumentedindomesticruminants andtickvectorsinTurkey,butthereislimited informa-tionregardingtheepidemiologyofcaninehepatozoonosis. Arecentmolecularstudyusingpolymerasechainreaction (PCR)amplificationandDNAsequencingwasconductedto identifyHepatozoonspeciesandtodescribe developmen-talstages ofR.sanguineus feedingondogs(Aktasetal., 2013).Theobjectiveofthissurveywastoinvestigatethe frequency and distributionof H. canis in asymptomatic domesticdogsfromnineprovincesofTurkey.

2. Materialsandmethods

2.1. Areaofstudyandcollectionofsamples

Atotalof694domesticdogbloodsampleswere col-lectedfromfivecoastalprovinces(Sakarya,Kocaeli,Mersin, Giresun,and ˙Izmir)andfourinlandprovinces(Elazig, Erzu-rum,Ankara,andNevs¸ehir)ofTurkey(Fig.1).Thefieldwork wasundertaken incollaborationwithmunicipalshelter officers,privateveterinaryclinics,andtheFiratUniversity VeterinaryTeachingHospital(FUVTH).Ofthetotal num-berofdogs,288werefrommunicipalshelters,243from theFUVTH,and163fromseveralprivateveterinaryclinics wherestraydogsaresurgicallyneuteredandreleasedback totheiroriginallocationinthescopeofofficialanimal con-trolprograms.BloodsamplescollectedfromFUVTHwere fromtheElazigProvince,whereit islocated.The samp-lingwasconductedfromJune2010toOctober2012.The dogswereofvariousbreeds andageandofboth sexes. Gender,age,andoriginandbodyconditionwererecorded. Agewasestimatedbasedondentitionandbodysize,and dogsweredesignatedasyoung(6monthsto1yearold)or adult(1–7yearsold).Thedogswereclassifiedas asymp-tomaticbasedontheirbehavioratthetimeofsampling, butadetailedclinicalexaminationwasnotperformed.A 3-mLbloodsamplewastakenfromthecephalicveinintoa tubecoatedwithethylenediaminetetraaceticacid(EDTA).

2.2. Microscopicexamination

A total of 285 thin blood smears (150 from shelter and135fromstray)wereimmediatelypreparedfromthe blood samplesformicroscopicexamination.Thesmears were fixed with methanol for 5min and stained with 5%May–GrunwaldGiemsafor30min.Thestainedslides wereexaminedat1000×magnificationforthepresence of Hepatozoon gamonts.The parasitemiaoftheinfected dogswasdeterminedbycounting500leukocytesunderoil immersion.

2.3. DNAextractionandPCRamplification

DNA was extracted using the QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. For the detection of H. canis, a PCR assay wasperformed using primers Hep-F (5-ATACATGAGCAAAATCTCAAC-3) and Hep-R (5 -CTTATTATTCCATGCTGCAG-3),whichamplifyafragment of 666bp of the 18S ribosomal RNA (rRNA) gene of Hepatozoonspecies (Inokumaetal.,2002).ThePCRwas performedinatotalreactionvolumeof25␮Lcontaining 2.5␮Lof10×PCRbuffer(100mMTris–HCl(pH9),500mM KCl,and1%TritonX-100),2.5␮LofMgCl2,250␮Mofeach

ofthefourdeoxynucleotidetriphosphates,2UTaqDNA polymerase (Promega, Madison, WI, USA), and 20pmol of each primer. The cycling conditions were 94◦C for 5minfollowed by30 cyclesat 94◦C for 30s, annealing at55◦C for 30s, extensionat 72◦C for 60s, and a final extension step at 72◦C for 5min.Positive control DNA previouslyisolatedfromadognaturallyinfectedwithH. canis (Gen-Bank accession no. JQ867390) and negative controlDNA(uninfectedcanineblood DNAanddistilled water)wereincludedineachPCRassay.Theamplification productswerevisualizedon1%agarosegelsstainedwith ethidiumbromideandobserved underUV illumination. DNA amplicons were purified with a PCR purification kit(Qiagen, Hilden, Germany).DNA sequencesobtained wereevaluatedwithChromasLitesoftware,version2.01 (Technelysium Pty, Ltd),and compared forsimilarity to sequencesdepositedinGenBank.

2.4. Dataanalysis

The prevalence (%) and 95% binomial exact confi-denceintervals(CIs)werecalculatedforthemicroscopy and PCR results for H. canis using Sourceforge. net® (http://sampsize.sourceforge.net/iface/index.html). The association between the prevalenceof H. canis by PCR andhostfactors(gender,age,andorigin)wascompared. Pearson’schi-squaredtestwasused,andp-valuesof≤0.05 wereconsideredstatisticallysignificant.

2.5. Ethicalapproval

Thisstudywasapproved byFiratUniversity’s exper-imental animalethic committee (approvedprotocol no. 16.02.2010-15).

3. Results

3.1. Thinbloodsmears

Thin blood smears revealed the number of para-sites in infected dogs ranging from one to seven. H. canis gamonts were observed to be ellipsoidal, sur-rounded by a capsule within the leukocyte cytoplasm. The standard deviation of gamonts was determined as 9.28–11.56␮m×4.22–6.70␮m.

266 M.Aktasetal./VeterinaryParasitology209(2015)264–267

Fig.1.ShowstheprovinceboundariesandthefrequencyofHepatozooninfectioninnaturallyinfecteddogsusingPCRbylocationinnineprovincesof

Turkey.

Table1

ComparisonofH.canispositivityinnaturallyinfecteddogsbyPCRinrelationtogender,age,andorigin(stray,shelter,andpets).

Gender Age Origin

Female Male Young Adult Stray Shelter Pet

No.samples 408 286 275 419 243 288 163

Positive 85(20.8%) 70(24.5%) 45(16.4%) 110(26.2%) 64(26.3%) 74(25.7%) 17(10.4%)

95%CI 17.0–25.1 19.6–29.9 12.2–21.3 22.1–30.7 20.9–32.3 20.7–31.1 6.2–16.2

p-value* _{>0.05 <0.05 >0.05 <0.05}

95%CI,95%confidenceintervals.

* _{Pearson’schi-squaredtest.}

3.2. Prevalenceofhepatozoonosisindogs

Threeof285thinblood smearswerepositiveforthe presenceofHepatozoon gametocytes. Allof thesamples positivebymicroscopywereconfirmedtobeinfectedwith H.canisbyPCRandsequencing.WithPCR,155ofthe694 dogswerefoundtobeinfectedwithH.canis.H.canis infec-tionswerefoundinallprovinces,withthepercentageof positivedogsineachprovincerangingfrom3.9%(95%CI: 0.5–13.4)to42.8%(95%CI:34.1–52)(Fig.1).Comparison ofH.canispositivityinnaturallyinfecteddogsbyPCRin relationtogender,age,andorigin(stray,shelter,andpets) ispresented inTable1.Althoughpositivity obtainedby PCRwashigherin maledogs(24.5%;19.6–29.9)thanin femaledogs(20.8%; 95%CI:17.0–25.1),significant gen-derdifferenceswerenotfound(p>0.05).Thefrequency of H.canis infections washigher in adultdogs than in youngerdogs(p<0.05).TheprevalenceofH.canis infec-tionwas26.3%(95%CI:20.9–32.3)amongstraydogs,25.7% (95%CI:20.7–31.1)amongshelterdogs,and10.4%(95%CI: 6.2–16.2)amongpetdogs.Petdogshadalowerprevalence comparedtostrayandshelterdogs(p<0.05);instead,no

significantassociationbetweenstrayandshelterdogswas foundforthepresenceoftheparasite(p>0.05)(Table1).

To determine the relationship of tick burden with the intensityof H. canis infection,dogs sampledin the DiyarbakırProvinceininlandTurkeywereexaminedfor thepresenceofixodidticks.Visualexaminationrevealed 41.26%ofthedogstobeinfestedwithadultandnymphal R.sanguineus,andPCRpositivityforH.canisinfectionwas correlatedwiththepresenceofticksondogs(Aktasetal., 2013).

ToconfirmthePCR-positiveresults,randomlyselected representative PCR positive samplesweresequenced. A BLASTsearchperformedwiththe18SrRNAgenesequences of H.canisisolatesshared99–100%similaritytovarious GenBanksequences.

4. Discussion

Usingdirectmicroscopyofstainedbloodsmears,this studyobserved circulatinggamonts ofH.canisin 3/285 (1.0%;95%CI:0.2–3)ofdogssampled.Thefindingwasnot surprising,asnodogshowedevidenceofclinicalsignsof

M.Aktasetal./VeterinaryParasitology209(2015)264–267 267

infection.Similarpositivityrateshavebeenreportedina previousstudy(Amolietal.,2012).In thepresentstudy covering9Turkishprovinces,H.canisinfectionswerefound inall,withthepercentageofpositivedogsineachsampled provincerangingfrom3.9(95%CI:0.5–13.4)to42.8%(95% CI:34.1–52).WesuggestthatH.canisispresentthroughout Turkey.

TheoverallprevalenceofHepatozooninfectionrevealed byPCRinthepresentstudywassimilartopreviousreports indomesticdogsfromtheAegeancoastofTurkey(22.3%) (Karagencetal.,2006),butlowerthanthatobservedinItaly (57.8%)(Otrantoetal.,2011).Ithasbeensuggestedthatthe prevalenceofH.canismayberelatedtothedistribution andpopulationdensityofthevector(Otrantoetal.,2011), thesamplingmethodology,andthecharacteristicsofthe targeteddogpopulation(Gomesetal.,2010).

Inthisstudy,H.canisinfectionwasmorefrequentin adultdogsthaninyoungerdogssimilartoapreviousstudy (Gomesetal.,2010).Thismaybeattributedto cumula-tiveexposureof olderanimalstotheparasite.Itiswell documentedthatcertaintick-bornepathogens,including H.canis,maybeassociated withanimallong-term sub-clinicalinfections(Ranietal.,2011).Theincreasedcontact withthevectormayalsobethereasonforahigher preva-lenceinadultscomparedwithyoungerdogs.However,it hasbeenassertedthatthedifferencesinprevalenceofH. canisinfectionin adultandyoungdogswasnot signifi-cant(Rojaset al.,2014),suggesting thatfactorssuchas vectordensity,geographicdistribution,andhostimmune statusmayplayaroleintheprevalenceofH.canis infec-tion.Strayandshelterdogsshowedasignificantlyhigher prevalenceof H.canisinfection incomparison withpet dogs.

Althoughtheprevalencewashigherinmalesthanin females, gender differences were not significant in the present study. The finding is consistent with previous reportsofnocorrelationofgenderwiththepresenceof infection(Gomesetal.,2010).Inconclusion,this epidemi-ological survey revealsthe highprevalence of H. canis infectionindogsinTurkey,anditsuggeststhatinfectionis associatedwithorigin,butnotwithage.

Acknowledgments

Thisworkwassupportedfinanciallybyagrant(110O 870)fromtheScientificandTechnicalResearchCouncilof Turkey(TUBITAK).Wethankallveterinarians,technicians,

andanimalbreedersintheregionfortheirassistancein samplecollection.

References

Aktas,M.,Ozübek,S.,Ipek,D.N.,2013.Molecularinvestigationsof

Hep-atozoonspeciesindogsanddevelopmentalstagesofRhipicephalus sanguineus.Parasitol.Res.112,2381–2385.

Aktas,M.,2014.Asurveyofixodidtickspeciesandmolecular

identifica-tionoftick-bornepathogens.Vet.Parasitol.200,276–283.

Amoli,A.A.R.,Khoshnegah,J.,Razmi,G.R.,2012.Apreliminary

parasito-logicalsurveyofHepatozoonspp.infectionindogsinMashhad,Iran. Iran.J.Parasitol.7,99–103.

Dezdek,D.,Vojta,L.,Curkovi ´c,S.,Lipej,Z.,Mihaljevi ´c,Z.,Cvetni ´c,Z.,

Beck,R.,2010.MoleculardetectionofTheileriaannaeand

Hepato-zooncanisinfoxes(Vulpesvulpes)inCroatia.Vet.Parasitol.172, 333–336.

Estrada-Pe ˜na,A.,Jaenson,T.G.T.,Farkas,R.,Pascucci,I.,2013.Mapsof

reportedoccurrenceofticks.In:Salman,M.,Tarres-Call,J.(Eds.),In TicksandTick-BorneDiseases:Geographicaldistributionandcontrol strategiesintheEuro-Asiaregion.CABI(CABInternational), Walling-ford,UK,pp.89–97.

Gabrielli,S.,Kumlien,S.,Calderini,P.,Brozzi,A.,Iori,A.,Cancrini,G.,2010.

ThefirstreportofHepatozooncanisidentifiedinVulpesvulpesand ticksfromItaly.VectorBorneZoonoticDis.10,855–859.

Giannelli,A.,Ramos,R.A.,DiPaola,G.,Mencke,N.,Dantas-Torres,F.,

Baneth,G.,Otranto,D.,2013.TransstadialtransmissionofHepatozoon

canisfromlarvaetonymphsofRhipicephalussanguineus.Vet.Parasitol. 196,1–5.

Gomes,P.V.,Mundim,M.J.S.,Mundim,A.V.,Ávila,D.F.,Guimarães,E.C.,

Cury,M.C.,2010.OccurrenceofHepatozoonsp.indogsintheurban

areaoriginatingfromamunicipalityinsoutheasternBrazil.Vet. Para-sitol.174,155–161.

Inokuma,H.,Okuda,M.,Ohno,K.,Shimoda,K.,Onishi,T.,2002.Analysisof

the18SrRNAgenesequenceofaHepatozoondetectedintwoJapanese dogs.Vet.Parasitol.106,265–271.

Karagenc,T.I.,Pasa,S.,Kirli,G.,Hosgor,M.,Bilgic,H.B.,Ozon,Y.H.,Atasoy,

A.,Eren,H.,2006.Aparasitological,molecularandserological

sur-veyofHepatozooncanisinfectionindogsaroundtheAegeancoastof Turkey.Vet.Parasitol.135,113–119.

Little,S.E.,Allen,K.E.,Johnson,E.M.,Panciera,R.J.,Reichard,M.V.,Ewing,

S.A.,2009.NewdevelopmentsincaninehepatozoonosisinNorth

America:areview.Parasit.Vectors2(Suppl.1),S5.

Mylonakis,M.E.,Koutinas,A.F.,Baneth,G.,Polizopoulou,Z.,Fytianou,

A.,2004.MixedEhrlichiacanis,Hepatozooncanis,andpresumptive

Anaplasmaphagocytophiluminfectioninadog.Vet.Clin.Pathol.33 (4),249–251.

Otranto,D.,Dantas-Torres,F.,Weigl,S.,Latrofa,M.S.,Stanneck,D.,de

Caprariis,D.,Capelli,G.,Baneth,G.,2011.DiagnosisofHepatozoon

canisinyoungdogsbycytologyandPCR.Parasit.Vectors4,55.

Rani,P.A.M.A.,Irwin,P.J.,Coleman,G.T.,Gatne,M.,Traub,R.J.,2011.A

surveyofcaninetick-bornediseasesinIndia.Parasit.Vectors4,141.

Rojas,A.,Rojas,D.,Montenegro,V.,Gutiérrez,R.,Yasur-Landau,D.,Baneth,

G.,2014.Vector-bornepathogensindogsfromCostaRica:first

molec-ulardescriptionofBabesiavogeliandHepatozooncanisinfectionswith ahighprevalenceofmonocyticehrlichiosisandthemanifestationsof co-infection.Vet.Parasitol.199,121–128.

Tsachev,I.,Ivanov,A.,Dinev,I.,Simeonova,G.,Kanakov,D.,2008.Clinical

EhrlichiacanisandHepatozooncaniscoinfectioninadoginBulgaria. Rev.Med.Vet.159,68–73.