Detection of respiratory viruses in shelter dogs maintained under varying environmental conditions

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

Detection

of

respiratory

viruses

in

shelter

dogs

maintained

under

varying

environmental

conditions

Francielle

Liz

Monteiro

_,

_Juliana

_Felipetto

_Cargnelutti

_,

_Mathias

_Martins

_,

Deniz

Anziliero

_,

_Magnólia

_Martins

_Erhardt

_,

_Rudi

_Weiblen

_,

_Eduardo

_Furtado

_Flores

a_{UniversidadeFederaldeSantaMaria(UFSM),DepartamentodeMedicinaVeterináriaPreventiva,SetordeVirologiaVeterinária,}

SantaMaria,RS,Brazil

b_{FaculdadeMeridional(IMED),DepartamentodeMedicinaVeterinária,PassoFundo,RS,Brazil}

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Articlehistory:

Received23April2015

Accepted20February2016

Availableonline19July2016

AssociateEditor:JoãoPessoaAraújo

Junior Keywords: CDV CPIV CAdV-2 CaHV-1

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ThreedogsheltersinRioGrandedoSulwereinvestigatedforassociationsbetweenthe

occurrenceofrespiratoryvirusesandshelterenvironmentalconditions.Nasalsecretions

randomlycollectedduringthecoldseasonweretestedviaPCR,andthisdatacollectionwas

followedbynucleotidesequencingoftheamplicons.Inshelter#1(poorsanitaryand

nutri-tionalconditions,highanimaldensityandconstantcontactbetweendogs),78%(58/74)of

thenasalsampleswerepositive,35%(26/74)ofwhichwereinsingleinfectionsand44%

(32/74)ofwhichwereincoinfections.Shelters#2 and#3 hadsatisfactorysanitaryand

nutritionalconditions,outdoorsexerciseareas(#2)andanimalclusteringbygroups(#3).

Inshelter#2,9%(3/35)ofthesampleswerepositiveforCanineparainfluenzavirus(CPIV),

and6%(2/35)werepositiveforCanidherpesvirus1(CaHV-1).Inshelter#3,9%(7/77)ofthe

sampleswerepositiveforCanineadenovirustype2(CAdV-2),and1%(1/77)werepositivefor

Caninedistempervirus(CDV).Theampliconsequences(CPIVandCDVnucleoproteingene;

CAdV-2E3gene;CaHV-1glycoproteinBgene)showed94–100%nucleotideidentitywith

Gen-Banksequences.OurresultsdemonstratethatCPIV,CAdV-2andCDVarecommonindog

sheltersandthattheirfrequenciesappeartoberelatedwithenvironmentalandnutritional

conditions.Theseresultsindicatetheneedforcontrol/preventionmeasures,including

vac-cinationandenvironmentalmanagement,tominimizetheseinfectionsandimprovedog

health.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Correspondingauthor.}

E-mail:eduardofurtadoflores@gmail.com(E.F.Flores).

http://dx.doi.org/10.1016/j.bjm.2016.07.002

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC

Introduction

Canineinfectiousrespiratorydisease(CIRD)maybeassociated

withsinglevirusinfectionsorwithamultifactorialetiology

andareassignedtoinfectiousagentsthatreplicate

sequen-tiallyorinsynergy.1_{ThemainviralagentsinvolvedinCIRD}

includeCaninedistempervirus(CDV),Canineparainfluenzavirus

(CPIV),Canineadenovirustype2(CAdV-2)andCanidherpesvirus

1(CaHV-1).2

InBrazil,CDVinfectionisendemicindogpopulations,is

associatedwithrespiratoryand/ormultisystemicdisease,and

causesthousandsofdeathseachyear.3,4_{Duetoitsimpacton}

animalhealth,CDVisoneofthemostimportantinfectious

diseasesindogs.2,5_{SimilarlytoCDV,CAdV-2hasaworldwide}

distributionandisamajoragentofcanineinfectious

tracheo-bronchitis(CIT)or“kennelcough”,adiseasecharacterizedby

restrictedinfectionoftherespiratorysystem.6_CPIVhasawide

distributionin caninepopulations withan estimated

sero-prevalencerangingfrom30to70%.7_{CPIVinfectionisrelatedto}

highpopulationdensity;thevirusishighlytransmissibleand

presentswithrapiddisseminationbetweenanimals.2_CaHV-1

hasaworldwidedistributionandisassociatedwith

respira-toryandreproductivedisease.8_{LikeotherAlphaherpesviruses,}

CaHV-1establisheslatentinfectionsinnervegangliaandcan

periodicallyreactivatetheinfection.9_{Anestimated30–100%}

ofdomesticdogshaveantibodiestoCaHV-1.10

The transmission of respiratory viruses occurs through

directorindirectcontactbetweenanimals,primarilythrough

contaminatednasalsecretionsandaerosols.1_{CIRDmayaffect}

dogsofbothgendersandages;puppiesunder90daysoldare

moresusceptible,aswellasimmunosuppresseddogs,animals

withoutahistoryofvaccination;vaccinationfailuresor

mater-nalimmunitymayalsocontribute.11_{Thediseasepresentsa}

seasonalpatternwithahigherincidenceincoldmonths.12

ThediagnosisofCIRDislargelybasedontheepidemiology,

clinicalsignsandresponsetotherapy.However,anetiologic

diagnosisrequirestheidentificationoftheagentorits

prod-ucts(proteinsornucleicacids).4_{Vaccinationislargelyused}

topreventorcontrolrespiratoryinfectionsindogsandhelps

minimizeclinicaldisease;however,currentvaccinesarenot

alwayseffective.11

In Brazil, despite the wide distribution of these

infec-tionsandinformalreportsbyveterinarians,veryfewreports

describeviral respiratory disease in dogs.13–18 _{Additionally,}

thereislittleinformationregardingtheseinfectionsinlocal

environmentswithhighdensitiesandconstantanimal

move-ment suchas dog shelters. The identification of the more

commonrespiratoryvirusesindogsinvarious

epidemiologi-calconditionsisessentialfordevelopingefficientcontroland

preventionmeasures.

Thus,theobjectiveofthisstudywastoinvestigate

respira-toryviralinfectionsindogsinshelters.Forthis,threeshelters

locatedinRioGrandedoSulstate,Brazil,presentingdiverse

sanitaryandnutritionconditionswereincludedinanattempt

toassociatetheoccurrenceofviralinfectionswiththe

condi-tionsobserved.Thevirusesweredetectedinnasalsecretions

viapolymerasechainreaction(PCR)andfocusedonthemain

agents involved in this condition (CDV, CPIV, CAdV-2 and

CaHV-1).

Material

and

methods

DogsfromthreeshelterslocatedinRioGrandedoSulstate

(RS),Brazil,wereincludedinthisstudy.Twoshelters(#1and

#2)arelocatedinCachoeiradoSul(30◦0221Sand52◦53

38W),andoneshelter(#3)islocatedinPassoFundo(28◦15

46Sand52◦2424W).Thesamplecollectionwasperformed

in 2014 in monthsof low temperatures (July and August).

Fig. 1 illustratesthe environmental conditions observedin

theseshelters.Shelterselectionwasperformedtoincludea

varietyofshelterconditions,includingthosewithlow

tem-peratureseasons,varyingpopulationdensities(totakeinto

account the appearance and frequency ofcleaning of the

premises),andthenutritionalstatesofthedogs(takinginto

consideration the type offood and the frequencyof

feed-ingaccordingtohealthy/unhealthyanimalappearance).The

selectionoftheanimalswithineachshelterwasperformed

randomly.

Shelter#1hostsstraydogsandcatsofbothgenders,ofall

agesandprimarilycrossbredanimals.Theanimalpopulation

oftheshelterwas150dogsand30catsonthedateof

samp-ling. Theyoungdogs(sixmonthsuptotwoyearsold) and

adults(morethantwoyearsold)weremaintainedin

individ-ualcages/smallbarnsheldbyleashesinanopenspace,and

mostanimalshaddirectandindirectcontactwitheachother.

Small,mediumand largedogshad individualcages/houses

shelterswithinthesamearea(approximately1m2_).Puppies

were maintained incollective cagesindoorswithout direct

contactwithadultanimals.Shelter#1wasvisitedinthecold

seasonwhenthetemperaturesrangedof5–10◦C.Atthevisit,

several animals presented withnasal discharge, indicating

respiratoryinfection.Sanitaryandnutritionalconditionswere

inadequate,andthelocalityhadnotrecentlybeencleaned.

Theanimalsdidnotreceivegoodqualityfoodandwerenot

fedsufficientamounts.

Shelter#2hostsstraydogsofbothgendersindependentof

ageandprimarilycrossbredanimals.Onthedayofthevisit,

thenumberofanimalswas70.Duringtheday,theanimals

remained outdoors infenced areas or heldbyleashes and

weregroupedaccordingtogenderandage.Duringthenight,

theanimalswereallocatedindoors,incollectivecages,with

approximately10animals/cage.Thedogshadawideareain

whichtorunandexerciseduringthedayandhadcontactwith

eachother.Shelter#2wasvisitedwhentheoutdoor

tempera-tureswerebetween10and15◦C.Thesanitaryandnutritional

conditionswerefairtogood;theanimalswerefedonceper

day,andthecageswerecleanedthreetimesperweek.

Shelter#3hostsstraydogsandcatsofbothgenders,

vary-ing ages and primarilycrossbredanimals. Atthe visit, 180

dogsand20catswerepresentintheshelter.Thedogswere

allocated according to gender and age incollective fenced

barnsthathadatleastonedoghouseperanimal.The

ani-malshad constant directcontactwith other animalsfrom

thesamecage/barn.Theindividualareawasapproximately

3m2_{/animal.Theshelterwasvisitedwhentheambient}

tem-peraturesrangedfrom5to10◦C.Thesanitaryandnutritional

conditionsweregood,thecageswerecleanedonceperday,

theanimalshadclean wateradlibitumand werefedthree

Fig.1–Conditionsofthesheltersincludedinthisstudy.(A)Shelter#1.(B)Shelter#2.(C)Shelter#3.

Nasal swabs of 74, 35 and 77 dogs in shelters #1, #2

and#3,respectively,wererandomlycollected;approximately

50%ofthedogsineachshelterweresampled.Aftersample

collection, the swabs were maintained in RNAlater

solu-tion(LifeTechnologies,Carlsbad,CA,USA),andthesamples

remainedindryiceduringtransporttothelaboratorywhere

they were stored at−80◦_{C. All proceedings involving}

ani-mal manipulation were performed under the supervision

of a veterinarian and according to the recommendations

of the Brazilian Committee of Animal Experimentation

(ComitêBrasileirodeExperimentac¸ãoAnimal–COBEA,law

#6.638, May 8, 1979). This research was approved by the

institutionalEthics and Animal WelfareCommittee(UFSM,

ComitêdeÉticaeExperimentac¸ãoAnimal:approvalnumber

080/2014).

RNAandDNAextractionfromnasalswabswereperformed

usinganRTPDNA/RNAvirusextractionkit(Invitek,Hayward,

CA,USA)accordingtothemanufacturer’sinstructions.After

RNAextraction,complementaryDNA(cDNA)wassynthesized

usingan enzymeSuperScript IIIReverse Transcriptase Kit

(Life Technologies, Carlsbad, CA, USA). The PCR reactions

wereinitiallystandardizedtooptimizetheconcentrationof

each reagent. Viruses obtained from two commercial

vac-cineswereusedascontrolsforCDV, CPIVand CAdV-2.For

CaHV-1,nucleicacidextractedfromtheliverofapuppy

natu-rallyinfectedwithCaHV-1wasusedasacontrol.17_Ultrapure

waterwas usedas anegative controlin all reactions. The

primers used in all reactions are described in Table 1. All

reactionswereperformedusingatotalvolumeof25␮Lwith

2␮Lof totalDNA (100–200ng) according tothe PCR

condi-tionsdescribedforeachvirus.PrimerstoCPIVwereobtained

usingtheCloneManager7program(http://www.scied.com),

andthesequencesareshowninTable1.PCRreactiontoCPIV

was performedina25␮Lvolumeusing100–200ngof

tem-plate DNA,12.5␮Mofeachprimer,2.5mMMgCl2,10mMof

dNTPs,1×reactionbufferand0.75unitsofTaqpolymerase

(Invitrogen,Carlsbad,CA,USA).ThePCRconditionwasone

stepat95◦Cfor7min,followedby35cyclesofdenaturation

(95◦C,45s),primerannealing(50◦C,1min),extension(72◦C,

45s),and afinalextensionof7minat72◦C.ThePCR

prod-uctswereresolvedin1.5%agarosegelstainedwithGelRed®

(Biotium,Hayward,CA,USA)andvisualized underUVlight

afterelectrophoresis(60V,40min).

For nucleotide sequencing, 90␮L of each PCR

prod-uct was purified using a PureLink® _{Quick Gel}

Extrac-tion and PCR purification Combo Kit (Life

Technolo-gies, Carlsbad, CA, USA) according to the manufacturer’s

instructions. Positive samples were randomly selected and

sequenced in quadruplicates in an automatic sequencer

ABI-PRISM 3100 Genetic Analyzer (Applied Biosystems,

Foster City, CA, USA). The obtained sequences were

ana-lyzed using the Staden Package for consensus sequences

achievement.22_{Thematrixofsampleidentitywithsequences}

deposited in GenBank was performed using the BioEdit

Table1–PrimersusedforthedetectionofcaninerespiratoryvirusesviaPCRindogsamplesfromsheltersintheRio

GrandedoSulstate.

Virus Amplifiedgene Primers(5–3) Ampliconlength Reference CDV Nucleoprotein TTCTGAGGCAGATGAGTTCTTC

CTTGGATGCTATTTCTGACACT

829pb 19

CPIV Nucleoprotein GAGGCTCGACGAATAATC

GTTCGGCTTGAGTTAGACC

532pb Thisreference

CAdV-2 E3 CGCGCTGAACATTACTACCTTGTC

CCTAGAGCACTTCGTGTCCGCTT

1030pb 20

CaHV-1 GlycoproteinB CCTAAACCTACTTCGGATGA

GGCTTTAAATGAACTTCTCTGG

450pb 21

Table2–InfectionsandcoinfectionsofrespiratoryvirusesindogsheltersinRioGrandedoSulstate.

Shelter +/total(%) Singleinfection–n(%) Coinfection–n(%) CPIV CAdV-2 CaHV-1 CDV CDV

CAdV-2 CPIV CAdV-2 CPIV CDV CPIV CAdV-2CDV #1 58/74(78) 22(30) 4(5) nd nd 2(3) 17(23) 3(4) 10(14) #2 5/35(14) 3(9) nd 2(6) nd nd nd nd nd #3 7/77(9) nd 6(8) nd 1(1) nd nd nd nd nd,notdetected.

Sequence Alignment Editor Software suite, version 7.0.5.3

(http://www.mbio.ncsu.edu/bioedit/bioedit.html).

Results

and

discussion

Thepresent study investigated the presenceofrespiratory

virusesindogsofthreesheltersinRioGrandedoSulstate,

Brazil,throughvirus detection innasal secretionsvia PCR.

Consideringthepreviousserologicalstudiesoncanine

respi-ratory viruses in Brazil,14,15 _{the primary difference of the}

presentstudywasthedirectdemonstrationandidentification

ofthevirusesinvolvedinCIRD.

Our results showed the occurrence of the main canine

respiratoryvirusesintheseshelterswithvaryingfrequencies

andcombinationsofsingleandmixedinfections.Inshelter#1,

78%ofthe74sampleswerepositiveforatleastonevirus;CPIV

wasthemostfrequentagent(71%ofthesamples).CPIVwas

detectedinsingle(30%)orinmixedinfectionsandwas

asso-ciatedwithCAdV-2(23%),CDV(4%),orboth(14%).CDVand

CAdV-2werefoundinahighpercentageofanimals,especially

incoinfections(Table2).Inshelters#2and#3,unlikeshelter

#1,asmallpercentageofsampleswerepositiveforthevirus

andonlyinsingleinfections.Inshelter#2,CPIVwasdetected

in9%ofthesamplesandCaHV-1wasdetectedin6%.In

shel-ter#3,9%ofthe sampleswere positiveforCAdV-2and 1%

forCDV(Table2).Thevaryingsanitaryandnutritional

condi-tionsandthedogcrowding/densityoftherespectiveshelters

mayexplaintheimportantdifferencesintheratesofpositive

animals.

Shelter#1hadprecariousnutritionalandsanitary

condi-tions,poorinfrastructureandpoorfoodquality(Fig.1A).In

shelter#2,the animalshad awideoutdoors areainwhich

toplayandexercise;however,thedogsofvaryingageshad

directcontact(Fig.1B).Fig.1Cshowsshelter#3with

individ-ualdoghousesandcageswithalowpopulationdensityand

goodsanitaryconditions(approximatelysixdogs/cage).

Fac-torsassociatedwithanimalovercrowding,suchasexcessive

noise, poor air quality and diet, in addition to bad

ken-nelcleaning,maycausestress,whichmayinturnpromote

theestablishmentanddisseminationofviralinfections.23–25

Thus,thepoorsanitaryandnutritionalconditionsofshelter

#1mayhavefavoredthehighrateofrespiratoryviruses.

Inthis shelter, CDV, CPIVand CAdV-2 were detected in

single or mixed infections, corresponding to 78% of the

positivedogs.CPIVwas detectedin71% ofthe samples,of

which30% weresingleinfections and 41%were associated

with CAdV-2 and/or CDV. CPIV is considered the primary

virusinvolvedinrespiratorydiseaseindogs,2,7,26–29 _andhas

beenmostfrequentlyreportedinconditionsofhighanimal

density.2 _{CPIVinfectionproducespathology inthetracheal}

epithelium15_{andfavorssecondaryrespiratoryinfectionsby}

otherpathogenssuchasCAdV-2.6

Inshelter#1,CDVwasdetectedonlyassociatedwith

CAdV-2and/orCPIV,correspondingto21%ofthepositivesamples.

CDVreplicationoccursinepithelialcellsandmacrophagesof

theupperrespiratorysystem,pharynxandtonsils,followed

bylymphnodeinfectionandsystemicdisseminationthatcan

evolvetomultisystemdiseaseandimmunosuppression.30_For

thisreason,bacterialsecondaryinfectionsareoftendetected

in dogswith distemperinaddition tocoinfectionbyother

viruses,suchasCAdV-2andCaHV-1.5,31,32

CAdV-2 detection in 45% of the samples from

shel-ter #1 may have been influenced by the high CPIV and

CDV infections in the kennel because CPIV promotes

pri-marylesionsinthetrachealepithelium15_{and CDVinduces}

immunosuppression.30_{Additionally,adenovirusesarehighly}

resistant inenvironmentalconditionsandremainviablein

theenvironmentforanextendedduration,therebyfavoring

disseminationamonganimals.6_{Notably,ahighprevalenceof}

CAdVindogpopulationshasbeenreportedinshelterswithout

ahistoryofvaccination.33,34

AninvestigationofrespiratoryvirusesindogsinGermany

analyzed 58 samplesof shelter animals with and without

respiratorysignsanddetected22.4%(13/58)tobepositivefor

CPIVandonepositiveforCAdV-2andCPIV.29_{Asimilarstudy}

performedinGermanyexamined68nasalswabsofdomestic

dogs28_{; inthisstudy, 7.4%(5/68) ofthe sampleswere}

posi-tiveforCPIV,2.9%(2/68)forCAdV-2and1.5%(1/68)forCDV.

Despite varyingfrequencies,thesestudiesreportedCPIVto

bethemostfrequentrespiratoryvirus indogs,followedby

CAdV-2andCDV.

There are few reports of direct diagnosis of respiratory

virusesindogs;however,someserologicalstudieshavebeen

performedinBrazil.14,15_{InsouthernBrazil,aserological}

inves-tigation of817domesticdogswithoutavaccinationhistory

showedthat43%oftheanimalswereseropositivetoCAdVand

27.3%toCDV.14_{Asimilarstudywasconductedinapopulation}

of173dogsinshelters,alsofromtheRSstate,inwhich

anti-bodiestoCPIVandCDVweredetectedin51.4%and4.1–9.3%of

thesamples,respectively.15_{Thesestudiesshowedthat}

respi-ratoryvirusescirculateamongdomesticandshelterdogsin

southernBrazilinvaryingcombinationsandprevalencesthat

likelyreflectenvironmentalandepidemiologicaldifferences

betweenregionsanddogpopulations.

Inshelters#2and#3,therespiratoryvirusesweredetected

orCaHV-1(shelter#2)and9%byCDVorCAdV-2(shelter#3).

CaHV-1wasdetectedinsamplescollectedonlyfromthedogs

ofshelter#2,correspondingto6%ofthecollectedsamples.

AlthoughCaHV-1 may causerespiratory disease,the

infec-tionhasalsobeenassociatedwithother clinicaloutcomes,

includingreproductivedisease.35,36_{Duetotheabilityof}

CaHV-1toremainlatentinthehost,itsdiagnosisindogpopulations

shouldpreferentiallybeperformedviaserologicaltesting.37–40

Inthissense,wedetectedpositiveserologyforCaHV-1in7

outof8dogsinshelter#1(notshown).Atwo-year

longitu-dinalinvestigationinashelterintheUnitedStatesinvolving

211necropsieddogsshowedCaHV-1involvementin12.8%and

9.6%oftracheaandlungsamples,respectively,reinforcingthe

involvementofCaHV-1inrespiratorydiseaseindogs.2

Theidentityofthesequencedmatrixoftheshelter

sam-pleswithsequencesdepositedinGenBankrevealed96to99%

identity(KU341102,KU341103,KU341104andKU341105)with

the N gene (AJ009656.1, JF965338.1, KC590511.1, AY386315,

JF965338.1,KF856711.1,KP738610.1andJN381190.1)ofCDV,98

to99%identity(KU341106, KU341107,KU341108,KU341109)

with the N gene (EF543648.1, EF546391.1 and AY581307.1)

ofCPIV, 97–100% identity (KU315333, KU315334, KU315335,

KU315336 and KU315337) with the E3 gene (KF676978.1,

JX416842.1, JX416842.1, U77082.1 and GQ915311.1) of

CAdV-2, and 99–100% identity (KU315338 and KU315339) with

thegBgene(KJ946357.1,KJ676506.1,JX908000.1,HQ846625.1,

AF361073.1andAY582737.1)ofCaHV-1.

Thus,the resultsobtainedinthis research showedthat

respiratoryvirusinfections(CPIV,CDV,CAdV-2andCaHV-1)

are common in dogs housed in public shelters. The

fre-quencyanddisseminationoftheseviralinfectionsappearto

berelatedtoahighpopulationdensityandpoorsanitaryand

nutritionalconditions.Theseresultsalsoindicatetheneedfor

control/preventionmeasures,suchasvaccinationandgood

environmental conditions, to minimize these infections in

shelterdogs.CPIVinfectionappearstoplayanespecially

pre-dominantroleinwinterrespiratoryinfectionsindogshelters

andwarrantsfurtherpreventivemeasures.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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Coelhobycollaborationduringsamplecollections.F.L.

Mon-teiro,J.F.Cargnelutti,M.Martins,E.F.FloresandR.Weiblenare

researchfellowsfromCNPq.

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