Bovine anaplasmosis in Turkey: First laboratory confirmed clinical cases caused by Anaplasma phagocytophilum

Bovine

anaplasmosis

in

Turkey:

First

laboratory

con

firmed

clinical

cases

caused

by

Anaplasma

phagocytophilum

Munir

Aktas

*

,

Sezayi

Özübek

DepartmentofParasitology,FacultyofVeterinaryMedicine,UniversityofFirat,23119Elazig,Turkey

ARTICLE INFO Articlehistory: Received13April2015

Receivedinrevisedform21May2015 Accepted25May2015

Keywords: Tick-bornefever PCR

Reverselineblot Cattle

ABSTRACT

Anaplasmaspeciesareobligateintracellularrickettsialpathogensthataffectthehealthofhumansand

otheranimals.ClinicalcasesofanaplasmosiscausedbyAnaplasmaphagocytophilumwereevaluated,and

thefrequencyofbovineAnaplasmaspecieswasdeterminedincattle.Bloodsamplesandthinblood

smearswerecollectedfrom10cattleexhibitingclinicalsignsoftick-bornefever.Inaddition,blood

sampleswerecollectedfrom123apparentlyhealthycattlefromthesamearea.DNAwasscreenedby

reverselineblotassayforthepresenceofthehypervariableV1regionofthe16SrRNAgeneofAnaplasma/

Ehrlichiaspecies.IntracytoplasmicinclusionbodiesofA.phagocytophilumwereobservedinneutrophils

of6sickanimals.Parasitemiarangedfrom0.2to1.6%inindividualslides.Reverselineblotshowed45.1%

(60/133)ofthesampledcattletobepositiveforoneormoreoffiveAnaplasmaspecies.Thefrequencyof

singleinfectionswas20.3%(27/133),whilemixedinfectionswerefoundin24.8%(33/133)ofsamples

with six different combinations of species and a maximum of four pathogens detected. A.

phagocytophilumwasthemostprevalent(41/133,30.8%)followedbyAnaplasmamarginale(25/133,

18.8%),Anaplasmacentrale(24/133,18%),Ehrlichiasp.strainOmatjenne(18/133,13.5%)andAnaplasma

bovis(1/133,0.7%).ThisisthefirstreportofA.bovisinacowfromTurkey.Thisisalsothefirstreportof

clinicalcasescausedbyA.phagocytophilumincattlefromthecountry.Therefore,A.phagocytophilum

shouldbetakenintoaccountasdifferentialdiagnosisincasesofhighfeverandanorexiainpastured

animals.

ã2015ElsevierB.V.Allrightsreserved.

1.Introduction

Bovineanaplasmosisisatick-bornerickettsialdiseasecaused byAnaplasma phagocytophilum,Anaplasma marginale, Anaplasma bovis,andAnaplasmacentrale(Dumleret al.,2001).A. phagocy-tophilumisthecausative agentofhumangranulocytic anaplas-mosisandoftick-bornefeverinawiderangeofhosts,including cattle(Woldehiwet,2010).Thepresenceofthepathogenisclosely related tothe occurrence of Ixodes ricinus, the mostabundant speciesoftickinEurope(Woldehiwet,2010).A.phagocytophilum causesfebrilediseaseincattlevaryingfromundetectableclinical signs to serious complications, including death (Pusterla and Braun,1997;Stuen etal.,2005).Thediseaseischaracterizedby high fever, cough, abortion, decreased milk production, and anorexia(Woldehiwet,2010).Incattle,theincubationperiodafter experimentalinoculationwasreportedtobe4–9dayswithfever lasting1–13days(Pusterlaetal.,1997).

A.marginaleisthemostwidelydistributedofthementioned speciesworldwide,andcausesdiseasecharacterizedby progres-sive anemia, weakness, fever, anorexia, decreased milk yield, jaundice, abortion, and sometimes death, while A. centrale infection results in mild anemia in most cases (Dumler et al., 2001;Kocanetal.,2010).Anaplasmabovisismorecloselyrelatedto A.phagocytophilumthantoA.marginaleorA.centrale(Dumleretal., 2001).CattleinfectionswithA.bovishavebeenreportedmainlyin AsiaandAfrica,andlittleisknownaboutitsepidemiology(Dumler et al., 2001). A. bovis infection can lead to anorexia, fever, incoordination, pale mucous membranes, and enlargement of lymphnodesincattle(Kaufmann,1996).

Tick-borne hemoprotozoan parasites in domestic ruminants andtickshavebeendocumentedinTurkey(Aktasetal.,2005,2007 Altayetal.,2007,2008),butthereisapaucityofinformationabout the presence of rickettsiae in the country. In recent molecular studies, A. phagocytophilum, A. marginale, A. centrale, and the Ehrlichiasp.strainOmatjennehavebeendetectedinsomeareasof Turkey(Gokceetal.,2008;Aktasetal.,2009,2010,2011,2012). However,todate,nolaboratoryconfirmedclinicalcaseofnatural infectionbyA.phagocytophilumhasbeenreportedinthecountry. We performed a PCR-based RLB survey on symptomatic and *Correspondingauthor.Tel.:+904242370000;fax:+904242388173.

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

http://dx.doi.org/10.1016/j.vetmic.2015.05.021

0378-1135/ã2015ElsevierB.V.Allrightsreserved.

ContentslistsavailableatScienceDirect

Veterinary

Microbiology

asymptomaticcattleintheBartınprovinceofthewesternBlack SearegionofTurkey.Theaimsofthestudyweretoevaluateclinical cases of anaplasmosis, and to determine the distribution and frequency of bovine Anaplasma spp. This is the first report of A.bovisincattlefromTurkey.Itisalsothefirstreportofclinical casesassociatedwithA.phagocytophilumincattlefromTurkey. 2.Materialsandmethods

2.1.Studyarea,bloodsamplingandmicroscopicexamination Thisstudy was conductedin Bart_ın province,a coastal area 25–300mabovesealevel,inthewestBlackSearegionofTurkey (41
530N,32
450E).Forestvegetationgenerallyconsistsof conifer-oustrees.Ithasahumidoceanicclimatewithmeanannualrainfall of1000mmandmeanannualtemperatureof13.4
C.Cattleinthe areaare maintainedunder small-scale family farming systems. Duringtheday,thecattlegrazeoncommunalpastures,generally 7–9h per day. In the evening the cows are milked and held overnight in enclosures. In May/June 2014, 4 heifers, 2 female calves,and4lactatingcows(crossbreedsofJersey,Simmental,and brown Swiss) with symptoms typical of tick-borne disease, includinghighfever,anorexia, cough,and decreasedmilkyield, weresubjected to a routine physicalexamination by a private veterinary clinician, and clinical signs were recorded. Blood samples were drawn from the jugular vein into anticoagulant (K3-EDTA) and stored at 4
C until DNA extraction. Thin blood smearswerepreparedfromtheEDTA-treatedsamples.Theblood samplesandsmearsweretransportedonicetotheDepartmentof Parasitology,FiratUniversity.Astheanimalswerenotcheckedfor thepresence of ixodid tick infestations, nodata regarding tick speciesonthecattlewereavailable.However,I.ricinuswas the mostprevalenttickspeciespreviouslyreportedfeedingoncattle nearthestudysite(Aktasetal.,2012).Thecattleweretreatedwith anintramuscularinjectionoflong-actingoxytetracyclineatadose of 20mg/kg body weight, with some animals also receiving supportive therapy (iron supplements, dextrose, vitamins) (Table1).

Thinbloodsmearswereairdried,fixedinabsolutemethanolfor 5min,andstainedwith10%Giemsastainfor30min.Sampleswere examined for intracytoplasmic Anaplasmaspp. inclusion bodies under oil immersion (1000). In each blood smear, 20 fields (minimumof5000redbloodcells)werescreenedforthepresence ofA.marginaleandA.centraleintheerythrocytes.Aminimumof 500neutrophilswerealsoscreenedineachsmear,andthenumber of cells containingAnaplasmabasophilic morulae was recorded

and the parasitemia was calculated as the percent of infected neutrophils (Stuen et al., 2011). Counts were made without knowledgeofthemolecularresults.

2.2.DNAisolation

Genomic DNA was extracted from 200

m

L of EDTA anti-coagulated blood with a QIAamp DNA Blood Minikit (Qiagen, Hilden, Germany) accordingto themanufacturer’sinstructions. DNAwaselutedin100

m

Lelutionbufferandstoredat80
_Cfor

later analysis. Negative control purifications using sterilized de-ionizedwaterwereperformedtomonitorcross-contamination. DNA concentrations (ng/

m

L) and purity (A260nm/A280nm) were

determinedbyspectrophotometry(NanoDrop1ND-2000UV/vis Spectrophotometer, Thermo Fisher Scientific Inc., Wilmington, Delaware,USA).

Inaddition,DNAsamplespreviouslyisolated(May/June2013) from123cattleinthesameprovince,withnoclinicalsymptoms, wereincluded.Allfurtheranalysiswasconductedonsamplesfrom bothsymptomaticandasymptomaticcattle.

2.3.PCRamplification

Toimproveonthequalityoftheamplification,a nestedPCR assay was performed using two universal primer pairs. The primers EC12A (50-TGATCCTGGCTCAGAACGAACG-30) and EC9 (50-TACCTTGTTACGACTT-30)wereusedforinitialamplificationof an approximately 1450bp fragment of the 16S rRNA gene in AnaplasmaandEhrlichiaspp.(Chenetal.,1994).ThePCRreactions wereperformedinPCRSprint(ThermoElectronCorporation,USA) aspreviouslydescribed(Aktasetal.,2010).Thenestedampli fica-tion performed using the RLB primers 16S8FE (50 -GGAATTCA-GAGTTGGATCMTGGYTCAG-30) and BGA1B-new (Biotin-50 -CGGGATCCCGAGTTTGCCGGGACTTYTTCT-30) produced 492– 498bpcorrespondingtothehypervariableV1regionofAnaplasma and Ehrlichiaspp.(Schoulsetal.,1999;Bekkeretal.,2002).For nestedPCRamplification,1

m

LoffirstroundPCRproductwasused asthetemplate.ThePCRwasperformedinatotalreactionvolume of25

m

Lcontaining2.5

m

Lof10PCRbuffer[100mMTris–HCl(pH 9), 500mM KCl,1% Triton X-100], 250

m

Mof each of the four deoxynucleotidetriphosphates,2UTaqDNApolymerase (Prom-ega,Madison,WI,USA),and10pmolofeachprimer.Atouchdown PCRprogramwasperformed:twocyclesof20sat94
C,30sat 67
C,and30sat72
C,followedbyasuccessionoftwo-cycleswith conditions identical to the previous cycles with the annealing temperature reduced by 2
C until it reached 57
C. A further

Table1

Summaryofclinicalandlaboratoryfindingsincattleexhibitingclinicalsignsofanaplasmosis. Animal

ID

Age Fever (
_C)

Mainclinicalsigns Laboratoryfindings Outcomea

Blood smear

Infectedleukocytes (%)

RLB

brt18 2 40.5 Depression,nasaldischarge,anorexia Positive 0.2 Aph+Am+Ac Recovery

brt45 2 41.5 Anorexia,coughing,diarrhea Positive 1.2 Aph Recovery

brt46 1 40.9 Recumbency,diarrhea,nasalandeyedischarge Positive 0.8 Aph+Am+Ac+Emo Recovery brt42 2 41 Weakness,anorexia,swellingofthehindlimbs Nodata – Aph+Am+Ac+Emo Recovery

brt3 6 39 Weakness,decreaseofmilkproduction,anorexia Negative – Aph Recovery

brt17 4 39.8 Weakness,decreaseofmilkproduction,anorexia Positive 1.6 Aph+Am+Ac+Emo Recovery

brt4 1 40.5 Weakness,coughing,anorexia Positive 0.4 Aph+Am+Ac+Emo Recovery

brt41 4 40 Weakness,decreaseofmilkproduction,coughing,anorexia Nodata – Aph+Am+Ac+Emo Recovery brt40 2 41.2 Anorexia,coughing,nasalandeyedischarge Positive 0.6 Aph+Am+Ac Recovery brt39 3 41.2 Decreaseofmilkproduction,coughing,anorexia,nasal

discharge

Negative – Aph+Am+Ac Recovery

Abbreviations:RLB–reverselineblotting;Aph–Anaplasmaphagocytophilum;Am–Anaplasmamarginale;Ac–Anaplasmacentrale;Emo–Ehrlichiasp.strainOmatjenne.

a

40cyclesof45sat94,57,and72
Cfollowed,withafinalextension at72
C for 10min.DNAfromA.marginale, A.phagocytophilum, A.centrale,andEhrlichiasp.strainOmatjennepositivebyRLBand DNA sequencing(GenBank accession nos. GU201518, FJ172530, GU223364,GU201519,respectively)obtainedinpreviousstudies (Aktasetal.,2010,2011)wereusedaspositvecontrolsinthePCR and for the subsequent RLB hybridization. In addition, the A/E-probeencodingtheAnaplasmaandEhrlichiaprobesequences, provided by a TBD-RLB Kit (Isogen Life Science, Maarssen, Netherland),wasalsousedaspositivecontrol.Apositivecontrol couldnotbeusedforA.bovis,becauseDNAfromthisagentwasnot available.Sterilizedde-ionizedwaterwasusedasnegativecontrol. Inordertominimizeriskofcontamination,DNAextractions,PCR preparation, PCR amplification, and agarose gelelectrophoresis wereperformedinseparaterooms.

2.4.Reverselineblotting(RLB)

The RLB membrane was prepared as described previously (Aydinetal.,2013).Briefly,to20

m

LofthePCRproduct,2SSPE/ 0.1%SDS was addedto a finalvolume of 150

m

L and held in a Thermal Cycler at 99
_{C for 10min and denatured for RLB}

hybridization. The biotinylated Anaplasma/Ehrlichia nested PCR products were hybridized with catch-all and species-specific oligonucleotideprobeslinkedtoanRLBmembrane(Table2).The primersandoligonucleotideprobeswereprovidedbytheMidland CertifiedReagentCompany(Texas,USA).

2.5.DNAsequencing

Onesampleobtainedfromasymptomaticanimal(brt45)tested positive with microscopy and RLB for A. phagocytophilum was selectedfor16SrRNAgenesequencing.The16SrRNAgenewas amplifiedusing20pmolofprimersEC12AandEC9toproducea PCRampliconofapproximately1450bp.ThePCRproductswere purifiedusingtheQIAquickPCRPurificationKit(Qiagen,Hilden, Germany) and submitted to a commercial laboratory (_Iontek, Turkey) for sequencing. The product was separated in an automated DNA genetic analyzer (ABI 310 Prism; PerkinElmer Corporation,Foster City, CA). Each constructwas sequencedat least three times. Sequencing results were evaluated with Chromas-Lite (www.technelysium.com.au). Sequence homology searchesweremadebyBLASTnanalysisofGenBank(www.ncbi. nlm.nih.gov), and multiple alignments were performed with ClustalW2 (www.ebi.ac.uk). Primers were deleted from DNA sequencespriortoBLASTcomparison.

3.Results

3.1.Clinicalsignsanddirectevidenceofinfectiousagent

Themostprevalentclinicalsignsexhibitedwereanorexiaand hyperthermia (>39
C). Decreased milk yield was observed in lactating cows. Some animals experienced diarrhea and cough (Table1).Nohematologicalprofilesorbiochemicalparametersfor theanimals withsymptomsof tick-bornefever wereavailable; therefore, calculationof correlationwith clinicalsigns was not possible.Ofthe10Giemsa-stainedblood smearsobtainedfrom clinicalcases,eightwereevaluatedandassessedforthepresence ofAnaplasmainclusionbodies.NoA.marginaleorA.centralewere detected by microscopy, whereas intracytoplasmic inclusion bodies compatible with A. phagocytophilum were observed in granulocytic neutrophils of six animals (Fig. 1). Microscopic findingsrevealedparasitemiaof0.2–1.6%.Clinicalandmicroscopic findingswereconfirmedbyRLBpositive signalsforA. phagocy-tophilum. Allanimals received treatment and recovered within 5–8days.

3.2.Frequencyoftick-borneAnaplasmaspeciesincattle

FrequencyofAnaplasmaorEhrlichiaspeciesinsingleandmixed infectionsisshowninTable3.Ofthe133PCRproducts,60(45.1%) hybridized to the Anaplasma/Ehrlichia catch-all probe, and all exhibited positive reactions with their corresponding specific probes. The frequency of single infections was 20.3% (27/133), whilemixedinfectionswerefoundin33(24.8%)sampleswithsix speciescombinationsandamaximumoffourpathogensdetected insevenanimals,fiveofwhichweresymptomatic(Table3).Atotal of 30.8%of thestudiedanimalswerepositivefor A. phagocyto-philum,indicatingaconsiderablenumberofanimalsexposedto anaplasmosis.ThesecondmostcommonlyfoundpathogenwasA. marginaleat18.8%. Othertick-bornepathogens identifiedinthe blood samples included A. centrale (18%), Ehrlichia sp. strain Omatjenne(13.5%),andA.bovis(0.7%).

3.3.DNAsequencing

Anearlyfull-lengthsequence(1404bp)ofthe16SrRNAgeneof A. phagocytophilum was obtained and deposited in the EMBL/ GenBank databaseunderaccession numberKP745629. ABLAST searchrevealedanucleotideidentityof99.86%withthe16SrRNA geneofA.phagocytophilumWebsterstrain(Accessionno.U02521).

Table2

Anaplasmaspecificoligonucleotideprobesequencesusedinthisstudy.

Oligonucleotideprobe Sequence(5’–0_{3) Reference}

Anaplasma/Ehrlichia TTATCGCTATTAGATGAGCC Schoulsetal.,1999

Ehrlichia/Anaplasmacatch-all GGGGGAAAGATTTATCGCTA Bekkeret al.,2002

Anaplasmamarginale GACCGTATACGCAGCTTG Bekkeretal.,2002

Anaplasmacentrale TCGAACGGACCATACGC Bekkeretal.,2002

Anaplasmabovis GTAGCTTGCTATGAGAACA Bekkeretal.,2002

Ehtlichiasp.strainOmatjenne CGGGTTTTTATCATAGCTTGC Bekkeretal.,2002

Anaplasmaphagocytophilum1 TTGCTATAAAGAATAATTAGTGG Schoulset al.,1999

Anaplasmaphagocytophilum3 TTGCTATGAAGAATAATTAGTGG Schoulsetal.,1999

Anaplasmaphagocytophilum5 TTGCTATAAAGAATAGTTAGTGG Schoulsetal.,1999

Anaplasmaphagocytophilum7 TTGCTATAGAGAATAGTTAGTGG Schoulsetal.,1999

AnaplasmaphagocytophilumA-HGE GCTATAAAGAATAGTTAGTGG Schoulsetal.,1999

4.Discussion

Theclinicalfindingsoffever,anorexia,anddecreasedmilkyield fromthediseasedanimalspointedtoatick-bornedisease.These clinicalsignswereconsistentwithpreviousreportsthat anaplas-mosisin sheepand cattlenormallyproducedmild tomoderate symptoms that are seldom fatal unless complicated by other infections (Pusterla and Braun,1997; Stuen, 2007; Laloy et al., 2009;Guyotetal.,2011).IntracytoplasmicinclusionbodiesofA. phagocytophilum were observed in leukocytes of the diseased cattle (Fig. 1). These clinical and microscopic findings were con_firmedbyPCR-basedRLBhybridizationandDNAsequencing. Although tick-borne fever caused by A. phagocytophilum is widespread in Europe (Hofmann-Lehmann et al., 2004; Laloy etal.,2009;Guyotetal.,2011;Cecietal.,2014),thediseasehasnot beenconsideredamajorpathogeninTurkey.Here,forthefirst time,wedescribedclinicaldiseaseincattle.Apossiblereasonthat thediseasehasnotbeenpreviouslydiagnosedmaybethelackof awarenessandexperienceofveterinarypractitioners.Sinceglobal saleand transportofcattle,especially fromEurope,is common practice,importationofcattle infectedwithA.phagocytophilum mayplayarole.

Diagnosisofbovineanaplasmosisisusuallybasedonclinical findingsanddetectionofinclusionbodiesinGiemsa-stainedthin bloodsmears.However,morulaewerenotobserved inthetwo clinicalcasesthatwereRLBpositiveforA.phagocytophilum.This finding is supported by a previous report of Anaplasma DNA detectedasearlyasfivedaysbeforeinclusionswereobservedby bloodsmearmicroscopy(Stuenetal.,2011).Wefoundparasitemia

of0.2–1.6%inanimals,consistentwiththeviewthatthepercentof infected leukocytes varies markedly (Pusterla et al., 1997). In addition,arecentstudyfoundtheinfectionratetobesignificantly differentondays3,6,7,and8inexperimentallyinfectedsheep (Stuenetal.,2011).Therefore,moleculardiagnostictoolsrepresent amoresensitivemethod,andthetimeframefordetectionofA. phagocytophilum is longerwhen usingmolecularmethods than withthinbloodsmears(Pusterlaetal.,1997;Niederetal.,2012). The results of this survey indicated the presence of A. phagocytophilum, A.marginale, A.centrale,A.bovis,and Ehrlichia sp.strainOmatjennecirculatinginthesampledcattleinthewest Black Sea region of Turkey. Most of these pathogens were anticipated, since theyhave beenpreviously reported in cattle andticksinareasofTurkey(Gokceetal.,2008;Aktasetal.,2010, 2011;Aktas,2014).However,thisisthefirsttimethatA.bovishas beendetectedincattlefromTurkey,andthisgivesgreaterinsight intodistributionandphylogeneticdiversityofbovine anaplasmo-sis.HighprevalenceofAnaplasmainfections(45.1%)wasobserved here compared to the findings of studies conducted in other provinces of Turkey (Gokce et al., 2008; Aktas et al., 2011). Seasonal,geographic,andclimaticdifferencesmighthave contrib-uted.Amongthedetectedpathogens,A.phagocytophilumwasthe mostfrequentlyfound,presentin30.8%ofthesamples(41/133). TheabundanceofI.ricinus,avectorofA.phagocytophilum,might explainthehigherpositiverateofA.phagocytophiluminthearea. A.marginaleandA.centralewerefoundin18.8%and18%ofthe cattle examined, respectively, thus confirming the presence of thesespeciesinthestudyarea,asreportedelsewhereinTurkey (Aktas et al., 2011, 2012).Although A. bovis has beenreported Fig.1.Bloodsmearsshowinginclusionbodies(morulaenexttothenucleus;arrows)ofAnaplasmaphagocytophiluminleukocytesfromnaturallyinfectedcattle. Table3

NumberofcattleinfectedwithsingleormultipleAnaplasmasp.detectedbytheRLB(n=133)

Natureofinfection A.phagocytophilum A.marginale A.centrale E.sp.Omatjenne A.bovis No.positivesamples(%)

Singleinfection + – – – – 24(18%) – + – – – 1(0.7%) – – – + – 2(1.5%) Subtotal 27(20.3%) Mixedinfection + – – – + 1(0.7%) + + + – – 1(0.7%) – + + + – 1(0.7%) + + + + – 7(5.3%) + – – + – 8(6%) – + + – – 15(11.3%) Subtotal 33(24.8%) Total 41(30.8%) 25(18.8%) 24(18%) 18(13.5%) 1(0.7%) 60(45.1%)

mainlyin Asianand Africancountries(Ooshiroetal.,2008;Liu etal.,2012), recentstudieshaveindicated thatthepathogenis presentincattleincontinentalEurope(Georgesetal.,2001;Ceci etal.,2014).A.bovisisusuallyassociatedwithsubclinicalinfection incattle,butsymptomsincludingfever,anorexia,anddepression can be present (Wanduragala and Ristic, 1993). In the present study, RLB results revealed co-infection of A. bovis and A. phagocytophiluminasampleobtainedfromanapparentlyhealthy cow.Thus,itisdifficulttoevaluatethepathogenesisofA.bovisin thisstudy.Moreepidemiologicalstudiesarerequiredtodetermine thepathogenicity,vectors,andreservoiranimalsoftheagent.

ItiswellknownthatRLBisusefulindetectingmixedinfections. Our results revealed that 33 animals (24.8%) presented co-infectionswithspeciesofAnaplasma/Ehrlichiainsixcombinations (Table3).Thefrequentdetectionofco-infections,alsoreportedby other(Georgesetal.,2001;dela Fuenteet al.,2005;Cecietal., 2014),isaninterestingfindingintick-bornediseasesandrequires further studies to better understand relationships between pathogensandthehostimmunesystem.

Inthisstudy, 13.5%ofsampleswerepositiveforEhrlichiaspecies (Ehrlichiasp.strainOmatjenne).Thepathogenwasinitiallyisolated fromsheepintheOmatjenne regionofNamibia(Allsoppetal., 1997).RecentstudiesdetectedthisEhrlichiaspeciesincattle(Aktas etal.,2011;Mtshalietal.,2013)andinAfricanbuffalo(Eygelaar et al., 2015). Although there is still little known about the pathogenicityofEhrlichiasp.strainOmatjenneinruminants,the resultssuggestthatcattlemaybeanaturalhostofthepathogen andcouldplayanimportantroleinitsepidemiology.

ThepresenceandfrequencyofbovineAnaplasmaspeciesinthe westBlackSearegionofTurkeyweredeterminedusingtheRLB hybridization assay. Four Anaplasma (A. phagocytophilum, A. marginale, A. centrale, A. bovis) and one Ehrlichia (Ehrlichia sp. strain Omatjenne species) infecting cattle were identified. A. phagocytophilumcausesclinicaldiseaseinsusceptiblecattleand considerableeconomiclosstothefarmsintheregion.Localanimal health officers and veterinarians should bemade awareof the presenceofthepathogen,and includeit intheirdiagnosesand treatmentstrategies.Thispathogenalsoinfectshumans,causing granulocytic anaplasmosis. This study suggests that human anaplasmosisshouldbeincludedinthedifferentialdiagnosisin thisregionincasesofinfluenza-likeillnessfollowingatickbite. ThisisalsothefirstreportofthepresenceofA.bovisincattlein Turkey.

Conflictofinterest

Theauthorsdeclarenoconflictsofinterest. Acknowledgements

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