Identification of enteric viruses circulating in a dog population with low vaccine coverage

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Veterinary

Microbiology

Identification

of

enteric

viruses

circulating

in

a

dog

population

with

low

vaccine

coverage

Christian

D.B.T.

Alves

_,

_Oscar

_F.O.

_Granados

_,

_Renata

_da

_F.

_Budaszewski

_,

André

F.

Streck

_,

_Matheus

_N.

_Weber

_,

_Samuel

_P.

_Cibulski

_,

_Luciane

_D.

_Pinto

_,

Nilo

Ikuta

_,

_Cláudio

_W.

_Canal

a_{UniversidadeFederaldoRioGrandedoSul(UFRGS),LaboratóriodeVirologia,FaculdadedeVeterinária,PortoAlegre,RS,Brazil} b_{UniversidadedeCaxiasdoSul(UCS),FaculdadedeMedicinaVeterinária,LaboratóriodeImunologia,CaxiasdoSul,RS,Brazil} c_{UniversidadeLuteranadoBrasil(ULBRA),LaboratóriodeDiagnósticoMolecular,Canoas,RS,Brazil}

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Articlehistory:

Received26August2017 Accepted14February2018 Availableonline15March2018 AssociateEditor:FernandoSpilki

Keywords: Distemper Parvovirus Diarrhea Dog Co-infection

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Althoughtheuseofvaccineshascontrolledentericdiseasesindogsinmanydeveloped countries,vaccinecoverageisstillunderoptimalsituationinBrazil.Thereisalarge popula-tionofnonimmunizeddogsandfewstudiesabouttheidentificationofthevirusesassociated withdiarrhea.Toaddressthissituation,stoolsamplesfrom325dogs wereanalyzedby polymerasechainreactionforthedetectionofcommonentericvirusessuchasCanine ade-novirus(CAdV),Caninecoronavirus(CCoV),Caninedistempervirus(CDV),Caninerotavirus(CRV) andCarnivorousprotoparvovirus1(canineparvovirus2;CPV-2).Atleastoneofthesespecies wasdetectedin56.6%(184/325)ofthesamples.Thevirusesdetectedmostfrequentlyin eitherdiarrheicornondiarrheicdogfeceswereCPV-2(54.3%ofthepositivesamples),CDV (45.1%)andCCoV(30.4%),followedbyCRV(8.2%)andCAdV(4.9%).Onlyoneagentwas detectedinthemajorityofthepositivesamples(63%),butco-infectionswerepresentin37% ofthepositivesamplesandmainlyincludedCDVandCPV-2.Thedatapresentedhereincan improvetheclinicalknowledgeinregionswithlowvaccinecoverageandhighlighttheneed toimprovethemethodsusedtocontroltheseinfectiousdiseasesindomesticdogs.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Gastrointestinal disorders are frequently reported in com-panionanimalclinicsasleadingtosevere dehydrationand deathinSouthAmerica.1,2_{Theycanhavebacterial,parasitic}

orviraletiologies.3_{Virusesassociatedwithentericillnesses}

∗ _{Correspondingauthor.}

E-mail:claudio.canal@ufrgs.br(C.W.Canal).

indogsareanimportantcauseofmortalityinnonprotected populations.2 _{Among these, canine parvovirus (CPV-2) and}

canine coronavirus (CCoV) are considered the most com-mon viral enteric pathogens in dogs worldwide.4–8 _Canine

distemper virus(CDV) isendemictoSouthAmerica andis frequentlyassociatedwithentericdisorders.8–11_Canine

ade-novirustype1(CAdV-1)iscommonlylinkedtohepatitisbut wasalsoassociatedtoseveregastroenteritisincluding vomi-tinganddiarrhea.12_{Thecaninerotavirus(CRV)isanunusual}

https://doi.org/10.1016/j.bjm.2018.02.006

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

entericpathogenindogsbutisimportantduetoitszoonotic potential.13,14

Thesearchformultiplepathogensinfecalsamplesfrom

dogs can mirror common exposure but can also show

interactionsbetweenpathogensdeterminingoraggravating disease.15,16_{Moreover,thereisalackofresearchsearchingfor}

multipleviralpathogensindogs,whichcoulduncoverthereal etiologyofcaninegastroenteritis.Therefore,thepresentstudy aimedtoverifythefrequencyofcanineentericviruses(CPV-2, CCoV,CDV,CAdV1andCRV)instoolsamplesfromdogs.

Materials

and

methods

Ethicsandsamplecollection

Fecalsampleswerecollectedfromtherectalampullaeof325 dogsatveterinaryclinicsduring2008and2014andstoredat −20◦_{C.Theanimalssampledwerefromeightofthefederal}

StatesofBrazil (RioGrandedoSul,SantaCatarina,Paraná, Rio de Janeiro, São Paulo, Mato Grosso do Sul, Rondônia andAcre)(Fig.1).Themajorityoftheanalyzeddog popula-tionwasunvaccinated(282/325),24animalswerevaccinated butwithincompletevaccineprotocolsand19received com-pleteprotocols(Table1).Dogsdefinedasvaccinatedreceived thecompletevaccinationprotocolusingthreedosesof poly-valentvaccine, whilethe onesdefined as vaccinated with incompleteprotocols receivedoneortwodoses.Polyvalent vaccinesusedindogsinBrazilareconstitutedbyCPV-2,CDV, CCoV,CAdV2,canineparainfluenzavirusandLeptospiraspp. withminordifferencesaccordingthedifferentmanufacturers. Thesanitarystatuswasdefinedbytheclinicduringsample collectionwhere82/325wereapparentlyhealthy,77/325 pre-sentedentericdisease-associatedsignsand166/325hadthis informationnotdetermined.TableS1describes associated-informationaboutall dogsamplesanalyzedinthe present study.

TheprojectwasregisteredwiththeEthicsCommitteeon the Use ofAnimals (CEUA) ofUniversidade Federal doRio GrandedoSulunderprotocolnumber#24984.

DNA/RNAisolationandreversetranscription(RT)

Fecalsamplesweredilutedto20%(w/v)inphosphate-buffered saline(PBS,pH7.4)andstoredat−80◦_{Cforfurtheranalysis.}

DNAwasisolatedfromthesupernatantusing guanidine-isothiocyanateandasilica-based17_{commercialkit(NewGene}

Preamp,SimbiosBiotecnologia,Cachoeirinha,RS,Brazil). Total RNAwas extractedusing TRIzol® LSReagent (Life Technologies®, Carlsbad,CA,USA). ThecDNA was synthe-sized with SuperScript®III Reverse Transcriptase Kit (Life Technologies®,Carlsbad,CA,USA)usingreverseprimersfor eachtarget9,18–21_{inatotalvolumeof20␮L.}

PCR

PCRandRT-PCRwereperformedusingpreviouslypublished protocols.TheCPV-2PCRprotocolaimedtoamplify583bpof theVP2gene,18_{whiletheCAdVassaytargeted509bpofthe}

E3codingregion.19_{ForCDVdetection,RT-PCRwasperformed}

toamplify afragmentof479bpofthe Ngene, followedby nestedPCRthataimstoamplifya286bpfragment.9_TheCCoV

protocolamplified410bpoftheMgene,20_{andtheCRVprotocol}

targeted1062bpofthe VP7gene.21 _{ThePCRproductswere}

electrophoresedin2%agarosegels,visualizedunderUVlight andcomparedwitha100bpmolecularweightladder(Ludwig Biotecnologia,Cotia,SP,Brazil).

PCRandRT-PCRpositivesamplesweresubmittedforDNA sequencing(ACTGeneAnálisesMolecularesLtda.,Alvorada, RS, Brazil) in order to confirm the identities of amplified viruses. PCRamplificationproductswere purifiedusingthe NucleoSpinExtractIIKit(Macherey-Nagel,Düren,Germany). After,bothstrandsweresequencedwithanABIPRISM3100 GeneticAnalyzer(AppliedBiosystems,FosterCity,CA,USA)

City City City City Sáo paulo Niterói Bagé Cachoeirinha Canoas Caxias do sul Glorinha Gravataí Lavras do sul Novo hamburgo Passo fundo Pelotas Porto alegre Rio pardo Santana do livramento São francisco de paula Santa maria Taquara Viamão TOTAL TOTAL TOTAL City City City City CPV-2 CPV-2 CDV CDV CCoV CCoV CAdV CAdV CRV CRV CPV-2 CDV CCoV CAdV CRV

Total tested CPV-2 CDV CCoV CAdV CRV Total tested

CPV-2 CPV-2 CDV CDV CCoV CCoV CAdV CAdV CRV CRV Total tested Total tested Total tested Total tested

CPV-2 CDVCCoVCAdV CRV Total tested

CPV-2 CDV CCoV CAdV CRV Total tested

Ji-paraná Rio branco Cuiabá Cambé Concórdia Florianópolis Londrina Maringá Tamarana TOTAL TOTAL TOTAL TOTAL TOTAL 1 1 0 0 0 0 0 3 3 3 21 21 1 1 17 3 0 0 0 0 0 0 0 0 300 600 900 1.200 km 0 0 0 1 1 1 3 3 1 5 7 12 0 0 0 0 2 2 4 6 0 2 21 22 1 8 10 3 3 3 39 46 1 89 8 0 12 7 7 0 0 0 0 0 0 22 22 3 0 0 0 0 0 0 0 0 0 4 4 2 2 RS SC PR SP RJ RO MT AC 0 0 0 29 29 0 0 0 0 0 0 0 0 4 4 0 2 0 1 1 1 11 14 5 0 0 0 9 2 18 0 2 0 0 1 2 51 47 41 1 1 15 1 8 0 0 0 0 0 0 0 0 0 7 3 3 4 0 14 0 0 0 0 0 0 0 0 0 0 0 0 2 2 0 0 0 1 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 1 0 6 0 0 0 0 0 0 3 1 1 4 4 4 4 1 1 1 1 1 3 4 4 34 2 17 1 3 48 1 11 4 10 12 6 9 162

Fig.1–MapofBrazil.ThemapindicatestheBrazilianfederativestateoforiginofeachsampledescribingthecitiescollected

andnumberofpositivesamplesforeachevaluatedvirus.RS:RioGrandedoSul;SC:SantaCatarina;PR:Paraná;SP:São

Table1–Detectionofentericvirusesindogswithdifferenthealthandimmunizationstatus.

Virus Healthstatus Vaccinationstatus

Entericdisease Healthy Vaccinated Incomplete Non-vaccinated

CPV-2 36(46.8%) 10(12.2%) 5(26.3%) 13(54.2) 82(29.1%) CDV 32(41.6%) 18(22%) 0(0%) 7(29.2%) 76(27%) CCoV 12(15.6%) 20(25.4%) 0(0%) 12(50%) 44(15.6%) CRV 2(2.6%) 1(1.2%) 1(5.3%) 0(0%) 14(5%) CAdV 0(0%) 1(1.2%) 0(0%) 0(0%) 9(3.2%) Total 77 82 19 24 282

usingaBigDyeTerminatorv.3.1cyclesequencingkit(Applied Biosystems).

Thecommercialvaccine Recombitek® (Boehringer

Ingel-heim,IngelheimamRhein,Germany)wasusedasapositive

controlfortheCPV-2andCDVassays,whileVanguardPlus®

(Pfizer,Wayne,PA, USA)wasusedforCCoVand CAdVand

RotaTeq(MerckSharp&DohmeCorp.,Kenilworth,NJ,USA) wasusedfortheCRVprotocol.

Results

Genomic amplification productsofat leastone virus were

detectedin56.6%(184/325)ofthesamples.CPV-2wasthemost commonlydetectedampliconin54.3%(100/184)ofthe posi-tivesamples,followedbyCDV (45.1%,83/184),CCoV(30.4%, 56/184), CRV (8.2%, 15/184) and CAdV (4.9%, 9/184). Fig. 1

describestheresultsforeachanalyzedvirusperBrazilian loca-tion.

Regardingthesanitarystatus,CPV-2wasthemostfrequent virusindogsdisplayingsignsofentericdisease,followedby CDV,CCoVandCRV,withnodetectionofCAdV-positive

ani-mals(Table 1). CCoV was the mostfrequentin apparently

healthydogs,followedbyCDV,CDV,CPV-2,CRVandCAdV.No significantdifferencewasobservedbetweengroups.

Thevaccinationstatuswasalsoevaluatedinthepresent study.Invaccinateddogs,onlyCPV-2andCRVweredetected

(Table1).Indogsthatreceivedtheincompletevaccine

proto-col,CPV-2wasthemostfrequent,followedbyCCoV,CDVand nodetectionofCRVandCAdV-positiveanimals.In unvacci-nateddogs,CPV-2wasalsothemostfrequentdetectedvirus, followedbyCDV,CCoV,CRVandCAdV.Nosignificant differ-encewasobservedbetweengroups.

Singleinfectionswereobservedinthemajorityofthe pos-itive samples (63.0%, 116/184), where CPV-2 was the most frequentlydetected(44.0%,51/116),followedbyCDV(28.4%, 33/116), CCoV (19.8%, 23/116), CRV (4.3%, 5/116) and CAdV (3.4%,4/116)(Table2).Co-infections(samplespositivefortwo ormoreviruses)wereobservedin37.0%(68/184)ofthepositive samples.Amongthem,dualinfectionswerethemost com-mon(86.8%,59/68),althoughtriple(10.3%,7/68)andquadruple (2.9%,2/68)infectionswere alsofound.Regarding the sani-tary status,co-infectionswere observedin31.2% (24/77)of thedogspresentingenteric-associated clinicalsignsand in 15.9%(13/82)ofapparentlyhealthydogs(Table3).Whenthe vaccinationstatuswasevaluated(Table3),co-infectionswere observedin5.3%(1/59) ofthevaccinated dogs,in45.8% of dogsthatreceivedincomplete protocolsofvaccinationand

in19.9%oftheunvaccinateddogs.Nosignificantdifference wasobservedbetweengroups.

Themostfrequentvirusesinvolvedinco-infectionswere CDVandCPV-2,witheachonedetectedin73.5%(50/68)and 72.1%(49/68)ofthesamplespositiveformorethanonevirus, respectively, followed by CCoV (48.5%, 33/68), CRV (14.7%, 10/68)and CAdV(7.4%,5/68). ThecombinationofCDV and CPV-2 wasthe mostfrequentlyobserved(52.9%,36/68 CDV +CPV-2positivesamples),althoughother viruseswerealso detectedinsixofthesesamples(Table1).

PCRandRT-PCRpositivesamplesweresubmittedforDNA sequencingand confirmedamplificationofthe wildstrains andnocontaminationwithpositivecontrols(datanotshown).

Discussion

Inthepresentstudy,weverifiedthefrequencyoffivecanine viralenteropathogensinadogpopulationwithlow vaccina-tioncoveragewhereCPV-2wasthemostfrequentlydetected virus(Table2).Thedatapresentedhereinagreewithprevious studieswhereCPV-2isreportedasthemostcommoncauseof severediarrheainpuppies.7,15,16,22_{However,thefrequencyof}

CPV-2foundinourstudy(54.3%)washigherwhencompared withtherangesreportedinotherstudies(16–48.7%).7,15,16,22

TheCPV-2prevalencevariesbetweenstudies,dependingon the inclusion criteriafor participation.However,since vac-cination againstcanine pathogens other than rabiesisnot mandatoryinBrazil,the vaccinecoveragefortheseviruses mustbedrasticallylower,whichcontributestothehighrate ofdetectionobservedinourstudy.

CCoV isgenerallythe second mostcommon viralagent detectedindiarrheicdogs,7,15,16,22 _{but inthepresentstudy,}

CDVhadahigherfrequencyofdetection(Table2).However, thefrequencyofdetectionobservedforCCoVwasstillhigher thanthatreportedotherstudies.7,15,16,22 _{SomeCCoVstrains}

can cause severe diarrhea and intestinal damage indistin-guishablefromthosecausedbyCPV-2.6

CDV isendemictoBrazil8,23 _{butnottotheregions}

sam-pled in previous studies.7,16,22 _{This could explainthe high}

ratesofCDVinfectiondetectedinthepresentstudy.Moreover, CDVcausesamultisystemicdiseasewithimmunodepression thatcanfavorinfectionbyotherpathogensincluding other diarrhea-associatedviruses.24

CRV and CAdV were also identified in 8.2 and 4.9% of thedogstested,respectively.Bothviralagentsarelinkedto diarrheaandvomiting13_{andshouldnotbeexcludedinthe}

Table2–Singleinfectionandco-infectionratesindogswithpositivePCRdetectionofcommondiarrhea-associatedviral agents. CDV CPV-2 CCoV CRV CAdV Singleinfection 30/113(26.5%) 51/113(45.1%) 23/113(20.3%) 5/113(4.4%) 4/113(3.5%) CDV+CPV-2 30/67(44.7%) 30/67(44.7%) CPV-2+CCoV 10/67(14.9%) 10/67(14.9%) CDV+CCoV 9/67(13.4%) 9/67(13.4%) CCoV+CRV 4/67(5.9%) 4/67(5.9%) CDV+CAdV 2/67(2.9%) 2/67(2.9%) CPV-2+CRV 1/67(1.5%) 1/67(1.5%) CPV-2+CAdV 1/67(1.5%) 1/67(1.5%) CCoV+CAdV 1/67(1.5%) 1/67(1.5%) CRV+CAdV 0 0 CDV+CPV-2+CCoV 3/67(4.4%) 3/67(4.4%) 3/67(4.4%) CDV+CCoV+CRV 2/67(2.9%) 2/67(2.9%) 2/67(2.9%) CDV+CPV-2+CAdV CDV+CPV-2+CRV 1/67(1.5%) 1/67(1.5%) 1/67(1.5%) CDV+CCoV+CAdV CPV-2+CCoV+CAdV CPV-2+CCoV+CRV 1/67(1.5%) 1/67(1.5%) 1/67(1.5%) CCoV+CRV+CAdV CDV+CPV-2+CCoV+CRV 1/67(1.5%) 1/67(1.5%) 1/67(1.5%) 1/67(1.5%) CDV+CPV-2+CCoV+CAdV 1/67(1.5%) 1/67(1.5%) 1/67(1.5%) 1/67(1.5%) Total 83/184 100/184 23/184 15/184 9/184

Table3–Singleinfectionandco-infectionratesindogswithdifferenthealthandimmunizationstatus.

Statusofinfection Healthstatus Vaccinationstatus

Entericdisease Healthy Vaccinated Incomplete Non-vaccinated Negativea _{22(28.6%) 45(54.9%) 14(73.7%) 5(20.9%) 119(42.2%)} Singleinfectionb _{31(40.3%) 24(29.3%) 4(21.1%) 8(33.3%) 104(36.9%)} Dualinfectionc _{21(22.3%) 13(15.9%) 1(5.3%) 9(37.5%) 49(17.4%)} Tripleinfectiond _{3(3.9%) 0(0%) 0(0%) 2(8.3%) 5(1.8%)} Quadrupleinfectione _{0(0%) 0(0%) 0(0%) 0(0%) 2(0.7%)} Quintupleinfectionf _{0(0%) 0(0%) 0(0%) 0(0%) 0(0%)} Total 77 82 19 24 282

a _{Samplesnegativeforallthetestedviruses.} b _{Positiveforatleastoneofthetestedviruses.} c _{Positiveforatleasttwoofthetestedviruses.} d _{Positiveforatleastthreeofthetestedviruses.} e _{Positiveforatleastfourofthetestedviruses.} f _{Positiveforallthetestedviruses.}

pathogen,anditsfrequencyindogsneedstobedetermined tobetterassesstheriskofinfectioninhumans.

Thesamplesanalyzedinthepresentstudywereobtained byconveniencefromveterinaryclinicswhichcouldbias infor-mationregardingsanity.Nevertheless,CPV-2remainedasthe mostfrequentviralagentinthedifferentdoggroups(Table1). Theonly exception was indogs apparently healthy where CCoVandCDVweremorefrequentthanCPV-2butwithno statisticalsignificance.

Morethanone-third ofthe dogstestedforCPV-2,CCoV, CDV,CRVandCAdVwerepositiveforco-infections(Table2). Nosignificantdifferenceinco-infectionrateswereobserved between the groups regarding clinical signs and vaccina-tion status (Table 3). The occurrence of co-infection can increase the pathogenicityof the disease sincethese viral agentscanactasimmunosuppressants.12,24,25_Thereisalack

of studies searching for multiple viral pathogens in dogs,

which could uncover the real etiology and interactions in the clinics. Moreover, a single search for the more com-mon pathogens can reveal the real epidemiology of less common viral enteropathogens. Canine diarrhea can have other etiologiessuchasbacteriaand parasitesthatare fre-quentlyreported.15,16_{DespiteCPV-2beingthemostfrequently}

detected diarrhea-associated viral pathogen inthe present study,ahighdegreeofco-infectionswasobserved.These co-infection rates reinforcethatthe search formorecommon individualpathogenscoulduncovertherealepidemiologyof lesscommonviralenteropathogens.

Inthepresentstudy,stoolsamplesfromdogswere evalu-atedforthepresenceofviralpathogens,andCPV-2wasfound tobethemostcommon.Incontrastwithpreviousstudies,CDV wasthesecondmostcommonviralagentdetected,probably sinceitisendemictoBrazilandnottotheregionssampled inotherstudies.Moreover,thehighfrequencyofco-infected

dogsthatwasobservedcanincreasethepathogenicityofthe disease.Thedatapresentedhereincanimprovetheclinical knowledgeinregionswith lowvaccine coverageand high-lighttheneedtoimprovethemethodsofcontrollinginfectious diseasesindomesticdogs.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WearethankfultoSimbiosBiotecnologiaLtda.forkindly sup-plyingtheDNAextractionkits,andwethanktheveterinarians whocollected thesamples.Financialsupportwasprovided bythe Conselho Nacionalde Desenvolvimento Científico e Tecnológico(CNPq/Brazil),Coordenac¸ãodeAperfeic¸oamento de Pessoalde Nível (CAPES/Brazil), Fundac¸ão de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and Propesq/UFRGS.

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2018.02.006.

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