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Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in North America
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Permanent Link: http://ufdc.ufl.edu/AA00010464/00001
 Material Information
Title: Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in North America
Series Title: Parasites & Vectors
Physical Description: Archival
Language: English
Creator: Beall, Melissa J.
Alleman, A. Rick
Breitschwerdt, Ed B.
Cohn, Leah A.
Couto, C. Guillermo
Dryden, Michael W.
Guptill, Lynn C.
Iazbik, Cristina
Kania, Stephen A.
Lathan, Patty
Little, Susan E.
Roy, Alma
Sayler, Katherine A
Stillman, Brett A.
Welles, Elizabeth G.
Wolfson, Wendy
Yabsley, Michael J.
Publisher: BioMed Central
Publication Date: 2012
 Subjects
Subjects / Keywords: Ehrlichia
E. canis
E. chaffeensis
E. ewingii
dog
tick
prevalence
 Notes
Abstract: Background: This study evaluated the exposure of dogs to three different Ehrlichia spp. in the south and central regions of the United States where vector-borne disease prevalence has been previously difficult to ascertain, particularly beyond the metropolitan areas. Methods: Dog blood samples (n = 8,662) were submitted from 14 veterinary colleges, 6 private veterinary practices and 4 diagnostic laboratories across this region. Samples were tested for E. canis, E. chaffeensis and E. ewingii specific antibodies using peptide microtiter ELISAs. Results: Overall, E. canis, E. chaffeensis and E. ewingii seroprevalence was 0.8%, 2.8%, and 5.1%, respectively. The highest E. canis seroprevalence (2.3%) was found in a region encompassing Arkansas, Louisiana, Oklahoma, Tennessee and Texas. E. chaffeensis seroreactivity was 6.6% in the central region (Arkansas, Kansas, Missouri, and Oklahoma) and 4.6% in the southeast region (Georgia, Maryland, North Carolina, South Carolina, Tennessee and Virginia). Seroreactivity to E. ewingii was also highest in the central region (14.6%) followed by the southeast region (5.9%). The geospatial pattern derived from E. chaffeensis and E. ewingii seropositive samples was similar to previous reports based on E. chaffeensis seroreactivity in white-tailed deer and the distribution of human monocytic ehrlichiosis (HME) cases reported by the CDC. Conclusions: The results of this study provide the first large scale regional documentation of exposure to E. canis, E. chaffeensis and E. ewingii in pet dogs, highlighting regional differences in seroprevalence and providing the basis for heightened awareness of these emerging vector-borne pathogens by veterinarians and public health agencies.
 Record Information
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution.
Resource Identifier: doi - 10.1186/1756-3305-5-29
System ID: AA00010464:00001

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RESEARCH OpenAccessSeroprevalenceof Ehrlichiacanis Ehrlichia chaffeensis and Ehrlichiaewingii indogsin NorthAmericaMelissaJBeall1*,ARickAlleman2,EdBBreitschwerdt3,LeahACohn4,CGuillermoCouto5,MichaelWDryden6, LynnCGuptill7,CristinaIazbik5,StephenAKania8,PattyLathan9,SusanELittle10,AlmaRoy11,KatherineASayler2, BrettAStillman1,ElizabethGWelles12,WendyWolfson11andMichaelJYabsley13AbstractBackground: Thisstudyevaluatedtheexposureofdogstothreedifferent Ehrlichia spp.inthesouthandcentral regionsoftheUnitedStateswherevector-bornediseaseprevalencehasbeenpreviouslydifficulttoascertain, particularlybeyondthemetropolitanareas. Methods: Dogbloodsamples(n=8,662)weresubmittedfrom14veterinarycolleges,6privateveterinary practicesand4diagnosticlaboratoriesacrossthisregion.Samplesweretestedfor E.canis E.chaffeensis and E.ewingii specificantibodiesusingpeptidemicrotiterELISAs. Results: Overall, E.canis E.chaffeensis and E.ewingii seroprevalencewas0.8%,2.8%,and5.1%,respectively.The highest E.canis seroprevalence(2.3%)wasfoundinaregionencompassingArkansas,Louisiana,Oklahoma, TennesseeandTexas. E.chaffeensis seroreactivitywas6.6%inthecentralregion(Arkansas,Kansas,Missouri,and Oklahoma)and4.6%inthesoutheastregion(Georgia,Maryland,NorthCarolina,SouthCarolina,Tennesseeand Virginia).Seroreactivityto E.ewingii wasalsohighestinthecentralregion(14.6%)followedbythesoutheastregion (5.9%).Thegeospatialpatternderivedfrom E.chaffeensis and E.ewingii seropositivesampleswassimilartoprevious reportsbasedon E.chaffeensis seroreactivityinwhite-taileddeerandthedistributionofhumanmonocytic ehrlichiosis(HME)casesreportedbytheCDC. Conclusions: Theresultsofthisstudyprovidethefirstlargescaleregionaldocumentationofexposureto E.canis E.chaffeensis and E.ewingii inpetdogs,highlightingregionaldifferencesinseroprevalenceandprovidingthebasis forheightenedawarenessoftheseemergingvector-bornepathogensbyveterinariansandpublichealthagencies. Keywords: Ehrlichia E.canis E.chaffeensis E.ewingii ,dog,tick,prevalenceBackgroundDogsaresusceptibletoinfectionwithmultiple Ehrlichia spp.,including E.chaffeensis and E.ewingii,whichare predominantlytransmittedby Amblyommaamericanum (lonestartick),andto E.canis ,whoseprimaryvectoris consideredtobe Rhipicephalussanguineus (browndog tick)[1-4]. Amblyommaamericanum iscommonlyfound ondogsandpeopleinthesoutheasternandsouthcentral UnitedStates;indeed,humanmonocyticehrlichiosis causedby E.chaffeensis isthemostfrequentlydiagnosed tick-bornediseaseinthesouthernU.S.[5,6].Thenumber ofhumanmonocyticehrlichiosiscasesreportedannually hasalsorisensteadilyfrom200patientsintheyear2000 toover900patientsin2009[7,8].Infectionswith E.ewingii arealsowelldocumentedinbothdogsand humansinthesoutheasternandsouthcentralU.S. [4,9-12].Thegeographicrangeof A.americanum has expandeddramaticallyinrecentdecadestoincludemany areasoftheMidwestandNortheast,resultinginaconcomitantincreaseintheregionsatriskforautochthonous transmissionof E.chaffeensis and E.ewingii todogsand people[6,13].Investigativefieldsurveyshaveimplicated othertickspecies,including Dermacentorvariabilis *Correspondence:melissa-beall@idexx.com1IDEXXLaboratories,Inc.,Westbrook,ME,USA FulllistofauthorinformationisavailableattheendofthearticleBeall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 2012Bealletal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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(Americandogtick)and R.sanguineus ,whichhavebeen implicatedassecondaryvectorsof E.chaffeensis and/or E.ewingii [14-16],buttherelativeimportanceofthese ticksinmaintainingasustainedcycleofinfectioninnatureremainstobedetermined. Rhipicephalussanguineus ticks,thevectorsfor E.canis arealsocommoninthewarmerclimatesofthesouthern U.S.,dueinparttotheirtropicalorMediterraneanorigin andgeneralintoleranceofcoldtemperatures[17,18]. Browndogticksarethoughttohavebeenintroducedto theAmericas,mostlikelyontickinfesteddogsfrom Europe.Becausethistickcansurviveinsidedwellings whereverdogsarepresent, R.sanguineus isnowconsideredtobeendemicthroughouttheU.S.withhigherprevalenceinparticulargeographicregions[19-21].Brown dogticksareknownorstronglysuspectedtotransmita numberofotherpathogenstodogsinNorthAmerica, including Bartonellavinsonii subsp. berkhoffii Rickettsia rickettsii, Babesiacanis Hepatozooncanis ,and Anaplasmaplatys [17,22]. Diagnosisofanehrlichialinfectioncanbeperformed usingvisual,serologicormolecularmethods. Erhlichia spp.replicateinsideamembraneboundvacuole(i.e., morula)thatcansometimesbeobservedbylightmicroscopicexaminationofstainedbloodsmearsinsideeither monocytes(E.canis and E.chaffeensis) orgranulocytes ( E.ewingii ).Detectionofantibodiescanbeperformed byimmunofluorescentassay(IFA)orenzymelinked immunosorbantassays(ELISA)butcross-reactivity betweenantibodiesto Ehrlichia speciesispossible[23]. Polymerasechainreaction(PCR)isthemostcommon molecularmethodusedtodiagnosean Ehrlichia spp. infection,particularlyindogswithacuteillnesswhere theonsetofclinicalsignsmayprecedeameasurable antibodyresponse. Serologicsurveysofdogsforexposuretotick-borne pathogenshavebeenusedtoidentifyareaswhereboth peopleanddogsareatriskofacquiringinfection[24-28]. However,prospectivesurveysinvolvingdogshaveoften beenlimitedtoasmallgeographicregion,suchasaportionofasinglestate[29-31].Reportsoflargescaleretrospectiveanalysesmaybeskewedtoareaswithhigher humanpopulationdensityandthushigherpetdogdensity.However,theseregionsmaynotrepresentregions withthehighesttickexposure.Forexample,inarecent publicationdescribingexposuretocommontick-borne pathogensinnearlyonemilliondogsfromtheU.S.[25], only6.4%ofsamplescamefromthestatesofKansas, Oklahoma,Missouri,Arkansas,Louisiana,andMississippi,whereverydensepopulationsof A.americanum ticksarefound.Inaddition,themajorityofresults reportedwerefromurbanareaswithinthestates.To combatthebiasinherentinsuchsurveysandtobetter documenttheprevalenceofexposureto Ehrlichia spp.in thesouthandcentralU.S.,s amplescollect edfromdogs presentingprimarilytoregio nalveterinarycollegeswere testedfor E.canis E.chaffeensis and E.ewingii specific antibodies.Abetterunderstandingofthedistributionof exposuretothesethreeehrlichialagentsindogswillprovideinsightintoareaswherepeopleanddogsareat greatestriskofinfection.MethodsCanineSerumSamplesAtotalof8,662canineserumsampleswerecollectedfrom 14veterinarycolleges,4commercialdiagnosticlaboratory locationsand6privateveterinarypractices.Participating veterinarycollegesincludedAuburnUniversity,University ofFlorida,UniversityofGeor gia,UniversityofIllinois, KansasStateUniversity,LouisianaStateUniversity(LSU), MississippiStateUniversity,UniversityofMissouri,North CarolinaStateUniversity(NCSU),TheOhioStateUniversity,OklahomaStateUniversity(OKSU),PurdueUniversity,UniversityofTennessee,andTexasA&MUniversity (TAMU).Participatingcommercialdiagnosticlaboratories werelocatedinDallas,TX,Baltimore,MD,Totowa,NJ, andNorthGrafton,MA.Theprivatepracticeswere locatedinArkansas,Missour i,NorthCarolinaandTennessee.Themajorityofsamplescollectedfromveterinary collegesandfromalldiagnosticlaboratoriesconsistedof serumthatremainedafterperformanceofrequesteddiagnostictestsunrelatedtothisstudy.Thesesampleswere chosenrandomly,withoutregardtoclinicalsignsorsuspecteddiagnosis,fromamongallremainingserumsamplesfromdogs 6monthsofage.Serumsamples collectedfromdogsinvolvedinsheltermedicineprograms representedallsamplesfromTexasA&Mand183/307 serumsamplesfromLouisianaStateUniversity.Samples submittedfromNCSUincludedanequalnumber(250 each)ofrandomlychosensamplesandsamplespreviously submittedtotheNCSUVector-BorneDiseaseDiagnostic Laboratory(VBDDL)forserologicaltesting.Morethan halfoftheserumsamplescollectedfromOKSU(360/503) wereoriginallysubmittedforbrucellosistesting.Forsamplessubmittedfromprivateveterinarypractices,asmall additionalvolumeofblood(approximately2ml)wascollectedwithinformedownerc onsentspecificallyforthe purposesofthisstudyatthetimeofbloodcollection relatedtoroutineveterinarycare;sampleddogswerechosenfromthegeneralpopulationandnotselectedbasedon particularclinicalsigns.AlthoughmostsampleswerecollectedbetweenJanuary2009andOctober2010,250samplesfromtheNCSU-VBDDLwerecollectedduringthe first6monthsof2006.DatacollectionLimitedpatientinformationwasavailableforthesamples.However,thedateofsamplecollection,thedog sBeall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 Page2of11

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actualorapproximateage,andtheaddresszipcodeof thedog sowner,veterinaryhospitalorshelterwas recorded.Neitherbreednorgenderofdogswas recorded.SerologyAllserumsamplesweretestedby3microtiterplateELISAs,eachoneusingaspecies-specificpeptideforthe detectionofantibodiesreactiveto E.canis E.chaffeensis and E.ewingii .Syntheticpeptideswerederivedfromthe E.ewingii p28protein(EESP),the E.canis p16protein, andthe E.chaffeensis variable-lengthPCRtarget(VLPT) protein[32,33].Testingwasperformedaspreviously describedwithminormodifications[32].Sampleswere initiallyscreenedoneachofthe3microtiterplateELISAsusinganindirectassayprotocolandconfirmedusing thesamepeptideELISAbutwithapeptide-labeleddirect assay.Briefly,intheindirectassay,dilutedsamples (1:100)wereincubatedineachofthe3species-specific ELISApeptide-coatedwellsfor30minutes,thewells werewashed(300 L/wellwithPBSplusdetergent),and horseradishperoxidase-la beledanti-canineantibody (1:1000dilutioninadiluentcontainingnon-specificproteinanddetergent)wasaddedtoeachwellandincubated for30minutes.Plateswerewashedasabove,3,3 ,5,5 teteramethylbenzidine(TMB)substratesolution(50 L/ well)wasadded,andopticaldensitywasdeterminedat 650nm;reactivesamplesweredenotedbyanyabsorbancevalue>2timesthenegativecontrolvalue.Reactive sampleswereconfirmedinduplicatebyadding50ulof theserumsampleand100 Lofaspecies-specificpeptidehorseradishperoxidaseconjugate(0.5to2.0 g/mL indiluent)tothemicrotiter-plateELISAcoatedwiththe species-specificpeptide.Thiswasallowedtoincubatefor 60min,microtiterwellswerethenwashed6times,incubatedwithTMBsubstrate(100 L/well)andreadat650 nmasdescribedabove. Resultsfor E.canis immunofluorescenceassays(IFA) wereavailablefor249samplesfromtheVBDDLat NCSU.Testingwasperformedfollowingpreviously describedstandardproce duresoftheVBDDLservice [34]. E.canis antigens(NCSUJakestrain)weregrown in vitro inDH82cellsbytheVBDDL.Seropositivesamples weredefinedashavingendpointtiters 64usingadilutionscaleof1:16-1:8192.AnalysisSeroprevalenceforeachpathogenwasdeterminedby countyandstateusingzipcodedataofthepatient,shelterorveterinarypracticesubmittingthesample.Atleast 50sampleswererequiredfromastateinordertocalculateseroprevalenceasdepictedinthemaps.Seroprevalenceresultsbycountywerecalculatedifatleast5 sampleswereobtainedfromacounty.Geographic regionsforcomparisonofseroprevalenceweredeterminedaccordingtotheexpectedtickpopulationpressure.Forseroprevalenceof E.canis ,whichistransmitted primarilyby R.sanguineus ,theregionwiththegreatest expectedtickpressurewasdefinedtoincludeAlabama, Arkansas,Florida,Georgia,Louisiana,Mississippi,North Carolina,Oklahoma,SouthCarolina,Tennesseeand Texasandthisregionwasco mparedtoallotherstates. Forseroprevalenceof E.ewingii and E.chaffeensis ,which aretransmittedprimarilyby A.americanum ,theregions expectedtohavethegreatest A.americanum pressure weredefinedascentral(Arkansas,Kansas,Missouri,and Oklahoma),orsoutheastern(Georgia,Maryland,North Carolina,SouthCarolina,TennesseeandVirginia),and thesetworegionswerecomparedtoallotherstates. Comparisonofseroprevalencebetweentwogeographic regions,aswellascomparisonsbetweengroupsofdogs, wasmadeusingtheChi-squaretest( P <0.05considered significant)whileaone-w ayANOVAwithsignificance assignedat P <0.05wasusedforcomparisonsof3or moreregions.RatesofHME permillioninthepopulationwerecalculatedbaseduponthereportedcasesper statefortheyears2008-2009dividedbytheU.S.census dataperstateforthesameyears.ComparisonofHME ratesto Ehrlichia spp.seroprevalencewasperformed usingalinearregressionforthosestateswithatleast50 caninesamplesandthecoefficientofdetermination(R2) wascalculated.AnalyseswereperformedusingGraphPad Prismv.5(GraphPadSoftware,LaJolla,CA).ResultsThe8,662canineserumsamplesoriginatedfromdogsin 41states,withmorethan600samplesfromeachofthe statesofTexas,Florida,Miss ouriandGeorgia(Table1). Manyuniversitiesenrolledinthestudycollectedatleast 300sampleswiththemostbeingsubmittedbytheUniversityofFlorida(Table2).Theaverageageofthedogs sampledwas6.6years(range0.5-23years).Theaverage ageofdogsresidinginshelterswasyounger(3.4years) thantheotherdogssampled(7.0years)aswastheageof dogstestedforbrucellosis(3.1years). Ofthe8,662samplestested,618(7.1%)weredeterminedtohaveantibodiestoatleastoneofthethree Ehrlichia species.Antibodiesto E.canis E.chaffeensis or E.ewingii weredetectedin71(0.8%),240(2.8%)and439 (5.1%)samples,respectively(Table1).In132samples, antibodiestomorethanone Ehrlichia specieswere detected.Themajorityoftheseco-exposeddogshad antibodiestoboth E.ewingii and E.chaffeensis (121/132; 92%).Sevendogshadantibodiesto E.ewingii and E.canis ,3dogshadantibodiesto E.canis and E.chaffeensis andonlyonedogwasfoundtohaveantibodiesto allthree Ehrlichia species.Dogswithantibodiesto E.chaffeensis weresignificantlymorelikelytohaveBeall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 Page3of11

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antibodiesto E.ewingii thanwereeitherdogsseronegativefor E.chaffeensis ( P <0.0001)ordogsseropositive for E.canis ( P =0.0051). Atleastoneseroreactivesamplewasidentifiedfrom eachof25states,andseroreactivityforatleast2ofthe3 Ehrlichia specieswasfoundinsamplesfrom22states (Figure1).Theregionexpectedtohavethegreatest R. sanguineus tickpressure(Alabama,Arkansas,Florida, Georgia,Louisiana,Mississippi,NorthCarolina,Oklahoma,SouthCarolina,TennesseeandTexas)didnot Table1Distributionofcaninesamplesbystateand Ehrlichia spp.seroprevalencerelativetoreportedcasesofHME.State TotalNumberSamplesTotal Ehrlichia (%) E.canis (%) E.ewingii (%) E.chaffeensis (%)HMEcases/M* Alaska 2 0 0 0 0 N Alabama 337 15(4.5)1(0.3)12(3.6) 5(1.5) 1.8 Arkansas 84 37(44.0)3(3.6)31(36.9)18(21.4) 21.7 Arizona 1 0 0 0 0 0 California 5 0 0 0 0 0.04 Colorado 5 0 0 0 0 N Connecticut 97 0 0 0 0 0.3 Delaware 27 0 0 0 0 23.3 Florida 733 27(3.7)4(0.5)19(2.6) 9(1.2) 0.6 Georgia 662 51(7.7)1(0.2)36(5.4) 23(3.5) 1.9 Hawaii 2 0 0 0 0 N Iowa 14 0 0 0 0 N Illinois 489 17(3.5) 08(1.6) 9(1.8) 2.4 Indiana 553 4(0.7) 0 4(0.7) 3(0.5) 0.3 Kansas 457 35(7.7)4(0.9)31(6.8) 5(1.1) 1.1 Kentucky 16 3(18.8)1(6.3)3(18.8) 0 2.9 Louisiana 274 6(2.2)4(1.5)2(0.7) 0 0 Massachusetts 241 2(0.8) 0 2(0.8) 2(0.8) 2.3 Maryland 254 16(6.3)2(0.8)9(3.5) 9(3.5) 8.3 Maine 13 0 0 0 0 0.8 Michigan 9 0 0 0 0 0.5 Minnesota 7 0 0 0 0 2.1 Missouri 663 190(29)5(0.8)151(22.8)85(12.8) 28.4 Mississippi 151 9(6.0) 0 9(6.0) 2(1.3) 1.0 Montana 1 0 0 0 0 N NorthCarolina 403 41(10.2)3(0.7)21(5.2) 23(5.7) 4.7 Nebraska 62 1 1(1.6)0 0 1.4 NewHampshire 28 0 0 0 0 4.2 NewJersey 257 10(3.9)1(0.4)7(2.7) 5(1.9) 9.0 NewYork 188 3(1.6) 0 3(1.6) 1(0.5) 3.7 Ohio 428 5(1.2)4(0.9)1(0.2) 0 1.0 Oklahoma 514 47(9.1)9(1.8)37(7.2) 5(1.0) 33.2 Pennsylvania 96 1(1.0) 01(1.0) 0 0.7 RhodeIsland 24 0 0 0 0 9.0 SouthCarolina 34 7(20.6)3(8.8)2(5.9) 3(8.8) 0.3 SouthDakota 2 0 0 0 0 0 Tennessee 181 18(9.9)5(2.8)14(7.7) 5(2.8) 10.9 Texas 893 23(2.6)18(2.0)5(0.6) 1(0.1) 0.7 Virginia 385 48(12.5)2(0.5)31(8.1) 25(6.5) 8.4 Wisconsin 9 0 0 0 0 4.0 WestVirginia 30 1 0 0 1(3.3) 0.3 Unknown 31 1 0 0 1 Total 8662 618(7.1)71(0.8)439(5.1)240(2.8)*AverageincidenceofHMEcasespermillionasreportedfor2008and2009bypassivesurveillance;MMWRJune25,2010(2008)andMay13,2011(2009)(Nnotreported)Beall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 Page4of11

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Table2Numberofcaninesamplessubmittedbyinstitutionandaveragepatientage.Location Numberofsamplestested Averagepatientage(yrs) AuburnUniversity 538 7.2 UniversityofFlorida 645 8.3 UniversityofGeorgia 514 8.0 UniversityofIllinois 425 8.0 KansasStateUniversity 521 7.4 LouisianaStateUniversity 307 3.7 MississippiStateUniversity 182 7.0 UniversityofMissouri 614 8.0 NorthCarolinaStateUniversity 500 7.1 OklahomaStateUniversity 503 3.1 TheOhioStateUniversity 501 NA PurdueUniversity 609 8.2 TexasA&MUniversity 381 3.1 UniversityofTennessee 108 6.2 PrivateVeterinaryClinics(6) 314 6.1 CommercialLaboratories(4) 2000 7.0(NA=notavailable) Figure1 Seroprevalencebystateofallthree Ehrlichia spp., E.canis E.chaffeensis and E.ewingii ,indogs Beall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 Page5of11

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havesignificantlymore E.canis seropositivesamples (1.2%)comparedtoallotherstatescombined(0.5%; P = 0.07)(Figure2).However,thecentralportion(Arkansas, Louisiana,Oklahoma,Tennessee,Texas)ofthisregion hadsignificantlymoresamplesseroreactivefor E.canis thantheeasternportion(Alabama,Florida,Georgia,Mississippi,NorthCarolina,SouthCarolina)ofthisregion (2.0%vs.0.5%respectively; P =0.0121).Coincidentwith A.americanum distribution,seroreactivityto E.chaffeensis (Figures3,4)was6.6%inthecentralregion(Arkansas,Kansas,Missouri,andOklahoma)and4.6%inthe southeastregion(Georgia,Maryland,NorthCarolina, SouthCarolina,TennesseeandVirginia).Seroprevalence of E.chaffeensis inbothofthese A.americanum indigenousregionswassignificantlyhigherthantheseroprevalencefromallotherstatescombined(0.7%; P <0.0001). However,seroreactivityto E.ewingii (14.6%)wassignificantlyhigherinthecentralregioncomparedwiththe seroprevalenceinthesoutheasternregion(5.9%)and whencomparedtoallotherstatescombined(1.4%; P < 0.0001)(Figures3,4). Todetermineiftheseroprevalenceof E.chaffeensis and E.ewingii indogsmightcorrelatewiththenumberof HMEcasesreportedper1,000,000inthehumanpopulation,alinearregressionwasperformedonthedata.If samplesfromallstateswereincluded,thecoefficientof determination(R2)wasonly0.47and0.37for E.ewingii and E.chaffeensis ,respectively.However,whenthesamplesfromdogswithsignificantlyloweraverageage(those fromLSU,OKSU,TAMU)wereomittedfromtheanalysis,thecoefficientofdeterminationincreasedto0.72and 0.73for E.ewingii and E.chaffeensis ,respectively(Figure 5).TherewasasignificantcorrelationbetweenHMEcases reportedpermillionpeopleandseroprevalenceof E.ewingii and E.chaffeensis indogs( P <0.0001)butnotwithseroprevalenceof E.canis indogs( P =0.704). E.canis IFAresultswerecomparedwiththatofthe Ehrlichia species-specificELISAsfor249samplesobtained fromtheNCSU-VBDDL. E.canis IFAidentified5seropositivesampleswithtitersofgreaterthanorequalto1:64. The Ehrlichia speciesELISAsidentifiedthesame5samplesasseropositivewith3seroreactivefor E.canis ,one seroreactivefor E.chaffeensis ,andoneseropositivefor both E.chaffeensis and E.ewingii .ThepeptideELISAs detected20additional Ehrlichia seroreactivesamples withinthegroupthathadbeennegativeby E.canis IFA. Figure2 Seroprevalencebystateof E.canis indogs Beall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 Page6of11

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Ofthese20,only2wereseropositivefor E.canis andthe remainderhaddetectableantibodiesto E.chaffeensis E. ewingii ,orboth(Table3).Inthissampleset,the E.canis IFAdidnotdetectthe E.ewingii -onlyseroreactivesamples (n=7)identifiedbythespecies-specificpeptideELISA.DiscussionThisstudyevaluatedtheexposureofdogstothree Ehrlichia species,withaparticularfocusondogsfromthe southandcentralregionsoftheU.S.,usingpeptide-based species-specificELISAassays.Basedonthisstudy,dogs Figure3 Seroprevalencebystateof E.chaffeensis (green)and E.ewingii (purple)indogs Beall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 Page7of11

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weremostcommonlyexposedto E.ewingii withantibodiesidentifiedin5.1%ofallsamplestested.Antibodiesto E.chaffeensis wereidentifiedin2.8%ofdogstested.This predominanceof E.ewingii infectionindogshasbeen reportedpreviouslyinstudiesfromMissouri,Arkansas, andOklahoma[4,10];thepresentstudyconfirmsand extendsthatfindingoveramuchwidergeographicarea. Antibodiesto E.ewingii and E.chaffeensis wereidentified indogsfrom23and20ofthe41statesconsidered,respectively,suggestingawidespreaddistributionoftheseagents intheeasternU.S.Thispatternparallelsthedistribution andintensityof A.americanum ticksinnature[35]. Further,itisconsistentwithdataderivedbylessspecific serologicassaysusingserafromdeeranddogs[25,35,36]. Figure4 Seroprevalencebycountyof E.chaffeensis (green)and E.ewingii (purple)indogsforthemid-Atlantic,southeasternand centralregionsoftheU.S ..Ablackdotindicatesthatatleastoneseroreactivesamplewasidentifiedinthatcounty. Beall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 Page8of11

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Thegeographicdistributionof E.chaffeensis and E.ewingii seroreactivedogsinthepresentstudyisalso largelyconsistentwithreportsofHMEor E.ewingii infectionsinpeople(seeTable1).However,thecorrelationwithHMEwasnotidentifiedunlesstheinstitutions withabiasedselectionofyoungdogsinthepopulation (shelterandkennel/breederdogs)wereexcludedfrom theanalysissuggestingt hatcanineexposuremaybe influencedbythetimespentinanendemicenvironment. Areviewofindividualstatesrevealedsomeinstances wherethe E.chaffeensis and E.ewingii seroprevalencein dogsexceededthereportednumberofHMEcasesin people.Forinstance, E.chaffeensis and E.ewingii were identifiedinapproximatelythesameproportionofdogs testedfromKansas(1.1%;6.8%)andMississippi(1.3%; 6.0%)asfromTennessee(2.8%;7.7%)butnocasesof HMEwerereportedfromKansasorMississippiin2008 whereas64HMEcaseswerereportedinpeoplefrom Tennesseeinthatyear[37].Resultsfrom2009showan increaseinthenumberofHMEcasesinKansasand Mississippi,witheachreporting6clinicalcases,while73 werereportedfromTennessee[7].Diseasereporting oftenvariesfromyeartoyear,andunderreportingof HMEisthoughttocommonlyoccur[5].Alternatively, infectionwithother Ehrlichia spp.mayberesponsiblefor thehighnumberofHMEpatientsreportedfromregions withlowcanineseroprevalenceasnovel Ehrlichia spp. continuetobedescribed[38-40]. Interestingly,121(1.4%)dogsinthepresentstudyhad antibodiestoboth E.ewingii and E.chaffeensis ,suggestingsomedegreeofsimultane ousorsequentialinfection withthesetwoagents,bothofwhicharetransmittedprimarilyby A.americanum ticks.Co-infectionindogswith multiple Ehrlichia spp.hasbeenreportedfromMissouri andNorthCarolina,baseduponmolecularmethods [10,34].Humanco-infectionwith E.chaffeensis and E.ewingii hasnotbeenreported,butthefindingsfrom thepresentstudy,thesharedvectortick,andtheinability todistinguishinfectionsw iththesetwoagentsbyless specificserologictechniques,suchasIFA,suggestscoinfectionshouldbeconsideredinpeople,particularly whenmoreseverediseaseisevident.Experimentalinfectionwithmultiplerickettsialagentsindogshasbeen showntoleadtomoreseverediseasethaninfectionwith asinglepathogen[41]. E.canis isawellrecognizedtick-bornepathogenofdogs knowntocausemonocyticehrlichiosis.Inthisstudyexposureto E.canis washighestintheregionofTexas,Louisiana,ArkansasandOklahoma,whichisconsistentwiththe densityoftheprimarytickvector, R.sanguineus .However, theoverallseroprevalenceof E.canis wasmuchlower comparedtotheseroprevalenceof E.chaffeensis and E.ewingii ,whichmaybeconsistentwiththeincreasein A.americanum numbersandexpandinggeographicdistributionofthistickspecies[5].Additionally,previousserosurveyswhichhaveutilizedmethodslike E.canis IFAor theSNAP3DxorSNAP4Dxtestkitstomeasure E.canis seroprevalencemayhavedetectedehrlichialantibodiesotherthanthosespecificfor E.canis resultinginan overestimateof E.canis exposure[23,42].Thisdiffers fromthepresentstudywhichutilizedapeptidethatisspecificfor E.canis anddoesnotdetectantibodiesto E.chaffeensis or E.ewingii [32].Finally,theadventofmore efficaciousandlong-actingacaracidescouldbehavingan impacton E.canis exposureratesindogsduetothe resultingsuppressionofthenaturalmaintenancecyclefor E.canis .Boththegreaterhostspecificityof R.sanguineus ticksrelativeto A.americanum andthelackofwildlife reservoirfor E.canis favorasuppressionofthenatural maintenancecyclefor E.canis relativeto E.chaffeensis and E.ewingii .Nosuchsuppressionof A.americanum maintenancecyclesindeerorotherwildlifeinhabiting naturalenvironmentswouldbeexpectedtobeongoing currently. Figure5 Correlationbetweentheaveragenumberofreported casesofHME/millionpeoplebystateandseroprevalencefor E.chaffeensis (grey)and E.ewingii (black)indogsbystate AnalysisexcludedHMEratesandcanineseroprevalencefromthose stateswheretheveterinaryinstitutionshadabiasedcollectionof samplesfromyoungdogswithinthepopulation. Table3Comparisonofserologicresultsusing E.canis IFA andthethree Ehrlichia speciesELISAsEhrlichia speciesbyELISA: E.canis IFA/Ehrlichia speciesELISAs Pos/NegPos/PosNeg/PosNeg/Neg E.canis 032 E.ewingii 007 E.chaffeensis 016 E.ewingii + E.chaffeensis 015 Total0520224Atotalof249serumsamplesfromtheVBDDLatNCSUhadpreviouslybeen testedby E.canis IFA.Titersof1:64andgreaterwereconsideredpositive.Beall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 Page9of11

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Inadditiontotheresultsofthisstudy,resultsfrompreviousexperimentalinfectionstudieswith E.chaffeensis and E.ewingii [32]aswellasregionalfieldstudies[4] havedemonstratedthesensitivityandspecificityofthese peptidereagentsforantibodydetectionininfectedcanine samples.Tofurtherevaluatetheperformance,alimited numberofsampleswereusedtocomparetheresultsof thethree Ehrlichia ELISAswith E.canis IFAasperformedbytheNCSUVBDDL.Whencomparedto E.canis IFA,20/249samples(8%)werereactiveonthe species-specificELISAsthatdidnotdemonstratereactivitybyIFAtesting.Resultsofapreviousstudydemonstratedthatupto8%ofsampleswithreactivityto Ehrlichia speciespeptidereagentsonSNAP3Dx(p30/ p30-1)maytestnegative(titers<1:64)on E.canis IFA [23].Theidentificationofsampleshavingantibodiesto E.chaffeensis and/or E.ewingii byELISAbutnotconfirmedpositiveby E.canis IFAmayreflectthevariability ofcross-reactiveantibodiesbetweenthese Ehrlichia species.DifferencesinassayformatbetweenELISAandIFA, suchastestingalessdiluteserumsampleandtheuseof highlyconcentratedimmunodominantepitopesonthe ELISA,mayaccountforsomeoftheobserveddifferences. Morestudiesareneededtodeterminethedegreewith whichcross-reactingantibodiesindogsexposedto E.chaffeensis and E.ewingii willreactwith E.canis IFA, theprimary Ehrlichia speciesusedasantigeninIFAtestingbyveterinarydiagnosticlaboratories.ConclusionsInthisstudy,wedocumentedthatdogsinthecentraland southcentralU.S.aremorecommonlyexposedto E.ewingii thanotherehrlichialspecies,andaremorecommonly exposedto E.chaffeensis thanto E.canis .Inthecaseof E.chaffeensis and E.ewingii ,thelonestarticktransmits theseinfectiousagentstobothdogsandpeople.Thedog hasbeendescribedasasentinelforvector-borneinfectionslikeLymeDiseaseandRockyMountainSpotted Fever[29,43-45].Theresultsofthisstudyprovidepreliminaryevidencethatdogscanbetestedusing Ehrlichia species-specificpeptidesandserveasaregionalorlocal sentineltogaugethepotentialriskforhumaninfection withthesetick-transmittedpathogens.Acknowledgements TheauthorswouldliketoacknowledgeJillSaucier,PauletteShields,Jennifer Sinsabaugh,andBrendonThatcherfortheirtechnicalassistancewiththe ELISAtesting;JulieBradleyandBarbHegartyfortheirtechnicalassistance withIFAtesting;PeterVonKameckeandJessicaLachtaraforassistancewith dataanalysisandmapping;Drs.WalterHoffmannandKarenSnowdenfor submittingsamplesfromtheUniversityofIllinoisandTexasA&MUniversity, respectively;Drs.BlaineAndrews,ScottLittle,EllenRatcliff,JimRussell,Mark Thompson,andKameronWorleyforsubmittingcaninesamplesfromprivate practice. ThisstudywasfundedbyIDEXXLaboratories,Inc. Authordetails1IDEXXLaboratories,Inc.,Westbrook,ME,USA.2UniversityofFlorida,College ofVeterinaryMedicine,Gainesville,FL,USA.3NorthCarolinaStateUniversity, CollegeofVeterinaryMedicine,Raleigh,NC,USA.4UniversityofMissouri, CollegeofVeterinaryMedicine,Columbia,MO,USA.5TheOhioState University,CollegeofVeterinaryMedicine,Columbus,OH,USA.6KansasState University,CollegeofVeterinaryMedicine,Manhattan,KS,USA.7Purdue University,CollegeofVeterinaryMedicine,WestLafayette,IN,USA.8UniversityofTennessee,CollegeofVeterinaryMedicine,Knoxville,TN,USA.9MississippiStateUniversity,CollegeofVeterinaryMedicine,MississippiState, MS,USA.10OklahomaStateUniversity,CollegeofVeterinaryMedicine, Stillwater,OK,USA.11LouisianaStateUniversity,CollegeofVeterinary Medicine,BatonRouge,LA,USA.12AuburnUniversity,CollegeofVeterinary Medicine,Auburn,AL,USA.13UniversityofGeorgia,CollegeofVeterinary Medicine,Athens,GA,USA. Authors contributions MJB,SELandLACwereprimarilyresponsibleforthefirstdraftofthe manuscript.Allauthorscriticallyreviewedandapprovedthefinal manuscript. Competinginterests MJB,andBSareemployeesofIDEXXLaboratories,Inc.EBB,LAC,MAD,SEL, andMJYhavereceivedfundingfromIDEXXLaboratories,Inc.foreducation, researchorconsulting. Received:26September2011Accepted:8February2012 Published:8February2012 References1.AnzianiOS,EwingSA,BarkerRW: Experimentaltransmissionofa granulocyticformofthetribe Ehrlichieae by Dermacentorvariabilis and Amblyommaamericanum todogs. AmJVetRes 1990, 51 :929-931. 2.EwingSA,DawsonJE,KocanAA,BarkerRW,WarnerCK,PancieraRJ,FoxJC, KocanKM,BlouinEF: Experimentaltransmissionof Ehrlichiachaffeensis ( Rickettsiales:Ehrlichieae )amongwhite-taileddeerby Amblyomma americanum ( Acari:Ixodidae ). JMedEntomol 1995, 32 :368-374. 3.GrovesMG,DennisGL,AmyxHL,HuxsollDL: Transmissionof Ehrlichia canis todogsbyticks( Rhipicephalussanguineus ). 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EmergInfectDis 2011, 17 :1710-1712.doi:10.1186/1756-3305-5-29 Citethisarticleas: Beall etal .: Seroprevalenceof Ehrlichiacanis Ehrlichia chaffeensis and Ehrlichiaewingii indogsinNorthAmerica. Parasites& Vectors 2012 5 :29. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Beall etal Parasites&Vectors 2012, 5 :29 http://www.parasitesandvectors.com/content/5/1/29 Page11of11