Molecular detection and characterization of Hepatozoon spp. in dogs from the Central part of Turkey

ContentslistsavailableatScienceDirect

Ticks

and

Tick-borne

Diseases

jo u r n al h om ep ag 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

Molecular

detection

and

characterization

of

Hepatozoon

spp.

in

dogs

from

the

Central

part

of

Turkey

夽

Mehmet

Fatih

Aydin

_,

_Ferda

_Sevinc

_,

_Mutlu

_Sevinc

a_{ResearchLaboratory,HigherHealthSchool,Karamano˘gluMehmetbeyUniversity,Karaman,Turkey} b_{DepartmentofParasitology,FacultyofVeterinaryMedicine,SelcukUniversity,Konya,Turkey} c_{DepartmentofInternalMedicine,FacultyofVeterinaryMedicine,SelcukUniversity,Konya,Turkey}

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Articlehistory:

Received31October2014

Receivedinrevisedform11February2015 Accepted3March2015

Availableonline20March2015 Keywords: Hepatozoon PCR Sequence Canine Turkey

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Caninehepatozoonosisisatick-borneprotozoaldiseasecausedbyHepatozoonspp.Twospeciesof Hep-atozoonarecurrentlyknowntoinfectdogsasHepatozooncanisandH.americanum.AlthoughH.canis generallycausesachronicinfectionwithrelativelymildclinicalalterationscomparedtoH.americanum, infectionbyH.caniscanbelife-threatening.ThediseaseiswidespreadinUSA,Africa,Europe,South Amer-ica,andAsia.TodeterminethefrequencyofinfectionwithHepatozoonspp.instraydogsfromCentral AnatoliaRegionofTurkey,atotalof221bloodsamplescollectedoverathree-yearperiodwere evalu-atedbyusinggenusspecificPolymeraseChainReaction(PCR)designedtoamplifyafragmentof666bp locatedin18SrRNAgeneofHepatozoonspp.Eight(3.61%)bloodsampleswerepositiveforHepatozoon spp.Fortheclassificationofspecies,allpositivePCRproductswerepurifiedwithaPCRpurificationkitand sequenced.Sequencingresultsofeightrepresentativeampliconsindicatedthat6were98–99%identical tothesequenceofH.canisandtheother2sequenceswere95–97%identicaltothesequenceof Hepato-zoonspp.SoitwasnamedHepatozoonsp.MF.Aphylogenetictreewasconstructedfromthesequences ofthetick-borneagentsidentifiedpreviouslyandinthisstudyusingtheneighbor-joiningmethod.The nucleotidesequenceswerecomparedtotheH.canissequencesreportedinTurkeyusingthenucleotide BasicLocalAlignmentSearchTool(BLAST)program.Theresultsofthisstudyaresignificantintermsof thepresenceofanovelcanineHepatozoongenotype.

©2015ElsevierGmbH.Allrightsreserved.

Introduction

Hepatozoonspeciesareapicomplexanparasitesfromthefamily

Hepatozoidae.Therearemorethan300speciesinthegenus

Hepa-tozoonandabout46oftheminfectmammals(Smith,1996).Canine

hepatozoonosisis a tick-bornedisease caused bytheprotozoal

agentsHepatozooncanisandHepatozoonamericanumtransmitted

bythebrowndogtick,RhipicephalussanguineusandtheGulfCoast

tick,Amblyommamaculatumrespectively(Banethetal.,2003;Little

etal.,2009).H.canisisthemostcommonspeciesassociatedwith

caninehepatozoonosisinEurope,Asia,AfricaandLatinAmerica

(BanethandVincent-Johnson,2005;Karagencetal.,2006;Little etal.,2009;Aktas,2014;Kaewkongetal.,2014).H.canisaffectsthe

reticuloendothelialsystemorganssuchasspleen,lymphnodesand

夽 NucleotidesequencedatareportedinthispaperisavailableinGenBank,EMBL 21andDDBJdatabasesunderaccessionnumbersfromKF439864toKF439867.

∗ Correspondingauthor.Tel.:+903382262000/4310;fax:+903382262134. E-mailaddress:veterinermfa@gmail.com(M.F.Aydin).

bonemarrow,anditcausesthesymptomssuchaselevatedwhite

bloodcellcounts,stiffness,pain,weightloss,lethargy,anemiaand

fever;althoughgenerallychronicormilddisease,theinfectioncan

belife-threateninginsevereclinicalmanifestationsassociatedwith

highparasiteload(Baneth,2011).InlifecycleofcanineHepatozoon

spp.,dogsserveasintermediatehostthatmerogonyand

gameto-gonystagesofdevelopmenttakeplacein,andticksactasdefinitive

hostthatsexualdevelopmentandsporogonyphasesoccur(Baneth

etal.,2007).

H.canisinfectionsindogshavebeendescribedinworldwide,

includingtheUSA,SouthAmerica,Africa,EuropeandAsia(Vojta

etal.,2009),onlyasmallnumberofcasesandepidemiological

sur-veyshavebeenreportedinTurkeytodate(Tüzdil,1933;Voyvada

etal.,2004;Karagencetal.,2006;Aktasetal.,2013).Turkeyis

locatedbetween the36–42◦ northernparallels and the26–45◦

easternmeridiansformingalandbridgebetweenEuropeandAsia

withherlandareaof770,760km2_{consistingofsevengeographical}

regionsandshowingdifferentecologicalandclimateconditions.

Althoughtherehavebeenanumberofstudiesoftheprevalence

of tick-borneprotozoanparasites (Theileriaand Babesia) in tick

http://dx.doi.org/10.1016/j.ttbdis.2015.03.004

Fig.1.Turkeymapshowingthestudyarea.

vectorsanddomesticanimalsinTurkey(Altayetal.,2008;Aydin

etal.,2012,2013,2015).Thereispaucityoncaninetick-borne

dis-easesthatrepresentswholecountry.Thisstudywasplannedto

determinetheprevalenceof Hepatozoon spp.infections indogs

fromtheCentralAnatoliaRegionofTurkeybyusingPCR.

Materialsandmethods

Studyareaandcollectionoffieldsamples

Theinvestigationwascarried outon221apparently

asymp-tomatic dogs from different locations throughout Konya and

KaramanprovinceslocatedinCentralAnatoliaRegionofTurkey

(Fig.1)during2010–2013.

Allanimalswereexaminedforgeneralclinicalinvestigations

suchasrectal temperature,visualobservationof mucous

mem-branes,abnormalitiesinsizeofsubcutaneouslymphnodes.The

informationabouttheage,sexandbreedofanimalswasrecorded

(datanotshown).Afterclinicalinspections,bloodsampleswere

takenfromthevenacephalicaantebrachiiintosteriletubeswith

K3EDTA-anticoagulant.

DNAextraction

Onehundred twenty-fivemicroliters ofblood was addedto

250␮loflysisbuffer(0.32Msucrose,0.01MTris,0.005MMgCl2,1%

TritonX-100,pH7.5).Themixturewascentrifugedat11.600×gfor

1min.Thepelletwaswashedthreetimeswith250_␮llysisbuffer

bycentrifugation.Thesupernatantwasdiscarded,andthefinal

pel-letwasresuspendedin100␮lofPCRbuffer(50mMKCl,10mM

Tris–HCl(pH8),0.1%TritonX-100,pH8.3).ProteinaseK(50␮g/ml)

wasaddedtothepelletsuspension,andthemixturewasincubated

at56◦Cfor1h.Finally,thesampleswereheatedat100◦Cfor10min

(Aydinetal.,2013).GenomicDNAswerekeptat−20◦_Cuntiluse.

Polymerasechainreactionandagorosejelelectrophoresis

Amplificationof the666bpfragmentof 18S ssrRNAgene of

Hepatozoon spp. was performed using PCR protocol using the

forwardHepF5-ATACATGAGCAAAATCTCAAC-3 and thereverse

HepR 5-CTTATTATTCCATGCTGCAG-3 primers (Inokuma et al.,

2002).ThePCRwasperformedinatouchdown thermocyclerin

atotal reactionvolumeof25_␮lcontainingPCRbuffer[750mM

Tris–HCl (pH 8.8), 200mM (NH4)2SO4, 0.1% Tween 20], 5mM

MgCl2,125␮M deoxynucleotide triphosphates,1.25U TaqDNA

polymerase(Promega,Madison, WI,USA),primers(20pmol/␮l)

andtemplateDNA.TouchdownPCRwasperformedwith50cycles

of94◦Cfor20s,67–59◦Cfor45s,and72◦Cfor45s,followedbya

finalextensionat72◦Cfor10min.Theannealingtemperaturewas

decreasedeverysecondcyclewith2◦Ctoa“touchdown”

temper-atureof57◦C.

PCRproductwasvisualizedusingGelDoc(Bio-Rad,Hercules,

CA,USA) in a 1.5%agarose gel stainedwith ethidiumbromide

afterelectrophoresis(100V,60min).PCRproductswerepurified

usingtheQiaquickpurificationkit(Qiagen,Hilden,Germany)and

sequencing was conducted by a commercial company (Iontek,

Istanbul,Turkey).Sequencesweredeterminedinbothdirections

(usingtheforwardandreverseprimers).

Phylogeneticanalysis

The sequences of the partial 18S rRNA gene of Hepatozoon

identified in this studyhave beendeposited in GenBankunder

the Accession Nos.: KF439864–KF439867. Each construct was

sequencedatleastthreetimesandsubjectedtoBLASTsimilarity

searches.Aphylogenetictreewasconstructedfromthesequences

of the 18SrRNA genes of sometick-borne agents infect

carni-voresusingtheneighbor-joiningmethodbytheMAFFTMultiple

Sequence Alignment Software Version 7 (Katoh and Standley,

2013).Thenucleotidesequencesusedinthisstudyareavailable

inGenBankunderthefollowingaccessionnumbers:DQ111764for

Babesiacanisrossi;AM183216forBabesiacanisvogeli;HM212628

for Theileria annae; JQ867388 for Hepatozoon felis; DQ439540,

DQ060329,KC584777andFJ497019forH.canis.

Results

Outof221fieldsamples,8(3.61%)werefoundpositiveinterms

ofHepatozoonspp.bytouchdownPCR.

Allofthepositivesampleswereconfirmedbysequence

compar-isons.TwoHepatozoonsequenceswereidentified.Distributionand

frequencyofcanineHepatozoonspeciesdetectedbyPCRinCentral

AnatoliainTurkeywassummarizedinTable1.

The partial sequences of the 18S rRNA genes for H. canis

and Hepatozoon sp. MF were deposited in the EMBL/GenBank

databasesunderaccessionnumbersfromKF439864–KF439867.H.

canissequencesshared99%identitywiththepreviouslyreported

sequencesforthe18SrRNAgeneofH.canis,alsotheothersequence

Fig.2.Neighbor-joininganalysisofthe18SrRNAgeneofthecanineBabesia,TheileriaandHepatozoonwasdeterminedinthisstudyandthosepresentintheGenBank database.Numbersabovethebranchdemonstratebootstrapsupportfrom1000replications.ThetreewasconstructedbyusingtheMAFFTMultipleSequenceAlignment SoftwareVersion7.Theorigin(Country)andGenBankaccessionnumbersareindicatedinparentheses.Sequencesdescribedinthisstudyareindicatedinbold.Scalebar representsnucleotidesubstitutionsperposition.

Fig.3.Nucleotidesequencesofpartial18SrRNAforHepatozoonspp.isolatesdeterminedinthisstudy(KF439864–KF439867)werealignedwiththepublished(DQ060328, DQ060327,DQ060325,JQ867390,JQ867389,KF034777andKF034776)18SrRNAsequencesoftheH.canisisolatesinTurkeyasareference.Adotindicatesanucleotidethat isconservedrelativetothepublishedsequence.

Table1

DistributionandfrequencyofcanineHepatozoonspp.detectedbyPCR(n=221). Province Noof examineddogs Hepatozoon canis Hepatozoon sp.MF Total Karaman 50 – 1(2%) 1(2%) Konya 171 6(3.50%) 1(0.58%) 7(4.09%) Total 221 6(2.71%) 2(0.9%) 8(3.61%)

Nucleotidesequencesofpartial18SrRNAgeneforHepatozoon spp.determinedinthisstudy(KF439864–KF439867)werealigned withthepublished(DQ060328,DQ060327,DQ060325,JQ867390, JQ867389,KF034777andKF034776)18SrRNAgenesequencesof theH.canisinTurkeyasareference(Fig.3).

Basedonthe18SrRNAgenesequencealignments,98%identity

wasfoundamong2isolatesofH.canisdeterminedinthisstudy

while3%,2%and1%geneticdifferencewasdeterminedwiththe

isolatesofH.canisfromManisa(DQ060325),Bodrum(DQ060327)

andAydin(DQ060328)provincesofTurkeyrespectively.Alsono

geneticdifferencewasdeterminedforH.canisbetweenKonya

iso-lates(KF439866,KF439867)andtheisolates(JQ867389,JQ867390)

obtainedfromDiyarbakirprovinceofTurkey.The2Hepatozoonsp.

MFisolatesshowed99%identitytoeachotherand3–5%genetic

dif-ferenceweredeterminedwiththeotherHepatozoonspp.isolates

fromtheworldintermsof18SrRNAgenesequences.

Discussion

Caninehepatozoonosisisamajorhealthproblemfordog

pop-ulationworldwide(Vincent-Johnsonetal.,1997;Holland,2001;

Baneth,2011;Chomel,2011).Thediseaseisfrequentlydiagnosed

bymicroscopicdetectionofgametocytes withintheneutrophils

instainedbloodsmears(EliasandHomans,1988;Ibrahimetal.,

1989).Serodiagnostictoolssuchasindirectfluorescentantibody

test and enzyme linked immunosorbent assay have been used

to detect the species-specific antibodies (Gonen et al., 2004;

Mylonakisetal.,2005;Karagencetal.,2006).PCRinblood and

buffycoathasbeenshownthebestdiagnosticassayfordetecting

H.canisindogs(Karagencetal.,2006;Otrantoetal.,2011;Aktas

etal.,2013).

In Turkey, the first detection of canine hepatozoonosis was

describedinadogin1933(Tüzdil, 1933).Voyvadaetal.(2004)

explainedthedetailedclinicaldescriptionofH.canisinfection

iden-tifiedby microscopy. In anepidemiological studyconducted in

AegeancoastofTurkey,Karagencetal.(2006)demonstratedthat

theprevalenceofhepatozoonosisamong349dogswas10.6%by

bloodsmearevaluationand25.8%bybloodPCR.Inanother

molec-ularsurveyconductedintheSouth-eastAnatoliaregion,15.87%of

63dogswerefoundpositiveforH.canisbyPCR;alsoH.canisandH.

felisinfectionwasdetectedintickR.sanguineus(Aktasetal.,2013).

Nostudyaboutcaninehepatozoonosishasbeenreportedinthe

centralpartofTurkeysofar.Thecurrentstudyconfirmedthe

pres-enceofH.canisandHepatozoonsp.MFbyusingthespecificprimer

set(HepFandHepR),whichamplifies666bpfragmentofthe18S

rRNAgenes.Ofthe221dogsexamined,8(3.61%)werefoundto

beinfectedwithHepatozoonspp.Bymeansofthisstudy,thefirst

detailedmolecularcharacterizationofH.caniswasmadeinCentral

AnatoliaRegionofTurkey.Anewgenotype,Hepatozoonsp.MFwas

discoveredindogs.H.canisandHepatozoonsp.MFisolates

identi-fiedinthisstudyshowed98–99%and95–97%similaritywiththe

Hepatozoonspp.isolatesidentifiedworldwiderespectively.When

comparedwiththeresultsofotherstudiesconductedinTurkey,the

prevalenceofcaninehepatozoonosiswaslowerinthisregionthan

thatofAegeancoastandsouth-easternregions(Karagencetal.,

2006;Aktasetal.,2013).Higherinfectionrateswererecordedto

beas25.8%byKaragencetal.(2006)inTurkey,20.3%bySasakietal.

(2008)inNigeria,57.8%byOtrantoetal.(2011)inItaly,11.4%by

Jittapalapongetal.(2006)inThailand,11.8%byVojtaetal.(2009)

in Croatiaand 79.2% bydeMiranda et al.(2014) inBrazil.The

lowerinfectionrateofH.canisinthisstudymightbeattributed

tothemainvectors,butthestudiesrelatedwiththeidentification

ordistributionofthearthropodvectorsofH.canishavenotbeen

organizedintheregionyet.

Inconclusion,twoHepatozoonspp.(H.canisandHepatozoonsp.

MF)wasdetectedindogsinthisstudy.Thesurveyrevealedthe

presenceofanovelcanineHepatozoongenotype.Thedataobtained

inthisstudydemonstratesthefirstgeneticallycharacterizationof

Hepatozoonspp.inTurkey.

Conflictofinterest

Theauthorsdeclarethattheysharenoconflictofinterest.

Acknowledgments

TheauthorsthankSüleymanGökmenandAkifKiris¸ci(DVM)for

theirkindhelpduringsamplecollectionandSezayiÖzübek(DVM,

PhDStudent)fortheassistanceonPCRoptimization.

References

Aktas,M.,2014.Asurveyofixodidtickspeciesandmolecularidentificationof tick-bornepathogens.Vet.Parasitol.200(3–4),276–283.

Aktas,M.,Özübek,S.,SayınIpek,D.N.,2013.MolecularinvestigationsofHepatozoon speciesindogsanddevelopmentalstagesofRhipicephalussanguineus.Parasitol. Res.112,2381–2385.

Altay,K.,Aydin,M.F.,Dumanli,N.,Aktas,M.,2008.MoleculardetectionofTheileria andBabesiainfectionsincattle.Vet.Parasitol.158(4),295–301.

Aydin,M.F.,Aktas,M.,Dumanli,N.,2012.TickInfestationsonsheepandgoatsinthe BlackSeaRegionofTürkiye.KafkasUniv.Vet.Fak.Derg.18(Suppl.A),A17–A22.

Aydin,M.F.,Aktas,M.,Dumanli,N.,2013.MolecularidentificationofTheileriaand BabesiainsheepandgoatsintheBlackSeaRegioninTurkey.Parasitol.Res.112 (8),2817–2824.

Aydin,M.F.,Aktas,M.,Dumanli,N.,2015.MolecularidentificationofTheileriaand BabesiaintickscollectedfromsheepandgoatsintheBlackSearegionofTurkey. Parasitol.Res.114(1),65–69.

Baneth,G.,2011.Perspectivesoncanineandfelinehepatozoonosis.Vet.Parasitol. 181,3–11.

Baneth,G.,Mathew,J.S.,Shkap,V.,Macintire,D.K.,Barta,J.R.,Ewing,S.A.,2003.

Caninehepatozoonosis:twodiseasesyndromescausedbyseparateHepatozoon spp.TrendsParasitol.19,27–31.

Baneth,G.,Samish,M.,Shkap,V.,2007.LifecycleofHepatozooncanis(Apicomplexa: Adeleorina:Hepatozoidae)inthetickRhipicephalussanguineusanddomestic dog(Canisfamiliaris).J.Parasitol.93(2),283–299.

Baneth,G.,Vincent-Johnson,N.,2005.Arthropod-borneinfectiousdiseasesofthe dogandcat.In:Shaw,S.E.,Day,M.J.(Eds.),Hepatozoonosis.,pp.78–88,UK, London.

Chomel,B.,2011.Tick-borneinfectionsindogs–anemerginginfectiousthreat.Vet. Parasitol.179,294–301.

deMiranda,R.L.,O’Dwyer,L.H.,deCastro,J.R.,Metzger,B.,Rubini,A.S.,Mundim,A.V., Eyal,O.,Talmi-Frank,D.,Cury,M.C.,Baneth,G.,2014.Prevalenceand molecu-larcharacterizationofHepatozooncanisindogsfromurbanandruralareasin SoutheastBrazil.Res.Vet.Sci.97(2),325–328.

Elias,E.,Homans,P.A.,1988.Hepatozooncanisinfectionindogs:clinicaland haema-tologicalfindingstreatment.J.SmallAnim.Pract.29,55–62.

Gonen,L.,Strauss-Ayali,D.,Shkap,V.,Vincent-Johnson,N.,Macintire,D.K.,Baneth, G.,2004.Anenzyme-linkedimmunosorbentassayforantibodiestoHepatozoon canis.Vet.Parasitol.122(2),131–139.

Holland,M.,2001.EmergingdiseasesinnorthernEurope.J.SmallAnim.Pract.42, 205–206.

Ibrahim,N.D., Rahamathulla,P.M.,Njoku,C.O., 1989.Neutrophil myeloperoxi-dasedeficiencyassociatedwithcaninehepatozoonosis.Int.J.Parasitol.19(8), 915–918.

Inokuma,H.,Okuda,M.,Ohno,K.,Shimoda,K.,Onishi,T.,2002.Analysisofthe 18SrRNAgenesequenceofaHepatozoondetectedintwoJapanesedogs.Vet. Parasitol.106,265–271.

Jittapalapong,S.,Rungphisutthipongse,O.,Maruyama,S.,Schaefer,J.J.,Stich,R.W., 2006.DetectionofHepatozooncanisinstraydogsandcatsinBangkokThailand. Ann.N.Y.Acad.Sci.1081,479–488.

Kaewkong,W.,Intapan,P.M.,Sanpool,O.,Janwan,P.,Thanchomnang,T., Kongk-lieng,A.,Tantrawatpan,C.,Boonmars,T.,Lulitanond,V.,Taweethavonsawat,P., Chungpivat,S.,Maleewong,W.,2014.Highthroughputpyrosequencing tech-nologyformoleculardifferentialdetectionofBabesiavogeli,Hepatozooncanis,

EhrlichiacanisandAnaplasmaplatysincaninebloodsamples.TicksTickBorne Dis.5(4),381–385.

Karagenc,T.I.,Pasa,S.,Kirli,G.,Hosgor,M.,Bilgic,H.B.,Ozon,Y.H.,Atasoy,A.,Eren, H.,2006.Aparasitological,molecularandserologicalsurveyofHepatozoon canisinfectionindogsaroundtheAegeancoastofTurkey.Vet.Parasitol.135, 113–119.

Katoh,K.,Standley,D.M.,2013.MAFFTmultiplesequencealignmentsoftware ver-sion7:improvementsinperformanceandusability.Mol.Biol.Evol.30(4), 772–780.

Little,S.E.,Allen,K.E.,Johnson,E.M.,Panciera,R.J.,Reichard,M.V.,Ewing,S.A.,2009.

NewdevelopmentsincaninehepatozoonosisinNorthAmerica:areview. Para-sitesVectors26(2),1–4.

Mylonakis,M.E.,Leontides,L.,Gonen,L.,Billinis,C.,Koutinas,A.F.,Baneth,G.,2005.

Anti-Hepatozooncanisserumantibodiesandgamontsinnaturally-occurring caninemonocyticehrlichiosis.Vet.Parasitol.129(3–4),229–233.

Otranto,D.,Dantas-Torres,F.,Weigl,S.,Latrofa,M.S.,Stanneck,D.,deCaprariis,D., Capelli,G.,Baneth,G.,2011.DiagnosisofHepatozooncanisinyoungdogsby cytologyandPCR.ParasitesVectors4,55.

Sasaki,M., Omobowale,O.,Ohta, K., Tozuka, M.,Matsuu, A.,Hirata, H., Not-tidge, H.O., Ikadai, H., Oyamada, T., 2008. A PCR-based epidemiological survey of Hepatozoon canis in dogs in Nigeria. J. Vet. Med. Sci. 70 (7), 743–745.

Smith,T.G.,1996.ThegenusHepatozoon(Apicomplexa:Adeleina).J.Parasitol.82, 565–585.

Tüzdil,A.N.,1933.BizdeilkdefagörülenbirHepatozooncanisvakası.TürkBaytarlar CemiyetiMecmuası13,35.

Vincent-Johnson,N.A.,Macintire,D.K.,Lindsay,D.L.,Lenz,S.D.,Baneth,G.,Shkap, V.,Blagburn,B.L.,1997.AnewHepatozoonspeciesfromdogs:descriptionof thecausativeagentofcaninehepatozoonosisinNorthAmerica.J.Parasitol.83, 1165–1172.

Vojta,L.,Mrljak,V.,Curkovic,S.,Zivicnjak,T.,Marinculic,A.,Beck,R.,2009. Molec-ularepizootiologyofcaninehepatozoonosis inCroatia.Int.J.Parasitol.39, 1129–1136.

Voyvada,H.,Pasa,S.,Unver,A.,2004.FirstclinicalcaseofpureHepatozooncanisof adoginTurkey.J.SmallAnim.Pract.45,613–617.