Bautista-Gómezb, Raúl Fajardo Mu ˜noza, Israel Quijano Hernándezc

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

Veterinary

Microbiology

Identification

of

co-infection

by

rotavirus

and

parvovirus

in

dogs

with

gastroenteritis

in

Mexico

Ariadna

Flores

Ortega

a

_,

_José

_Simón

_{Martínez-Casta ˜neda}

a,∗

,

Linda

G. Bautista-Gómez

b

,

Raúl

Fajardo

Mu ˜noz

a

_,

_Israel

_Quijano

_Hernández

c

a_Centro_de_{Investigación}_y_Estudios_Avanzados_en_Salud_Animal,_Facultad_de_Medicina_Veterinaria_y_Zootecnia,_Universidad_Autónoma

delEstadodeMéxico,Toluca,EstadodeMéxico,México

b_Centro_{Universitario}_Amecameca,_Universidad_Autónoma_del_Estado_de_México,_Amecameca_de_Juárez,_México

c_Hospital_Veterinario_de_{Peque ˜nas}_Especies,_Facultad_de_Medicina_Veterinaria_y_Zootecnia,_Universidad_Autónoma_del_Estado_de_México,

Toluca,EstadodeMéxico,México

a

r

t

i

c

l

e

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o

Articlehistory:

Received26October2016

Accepted7March2017

Availableonline24June2017

AssociateEditor:MilianeSouza

Keywords:

Dog Mexico Parvovirus Rotavirus

a

b

s

t

r

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c

t

ThisisthefirstreportoncirculatingcaninerotavirusinMexico.Fiftysamplesfromdogswith gastroenteritiswereanalyzedusedpolymerasechainreactionandreversetranscription polymerasechainreactioninordertoidentifyparvovirusandrotavirus,respectively;7%of dogswereinfectedwithrotavirusexclusively,while14%wereco-infectedwithbothrotavirus andparvovirus;clinicalsignsinco-infecteddogsweremoresevere.

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

Infectiousgastroenteritisareoneofthemaincausesofdog

hospitalization,etiologicalagentsidentificationisachallenge forveterinarians,giventhatgastroenteritisetiologyiscaused

by diverse pathogenic agents, mainly co-infections among

virusorbacteria.1–3

Canineparvovirus(CPV-2)isamemberoftheParvoviridae

family,belongingtotheProtoparvovirusgenusandCarnivore

Protoparvovirustype1species.Diversereportsindicatethatis themostdiagnosedviralagentingastroenteritis.4,5_Over_the

pastyears,CPV-2hasdevelopednewantigenicvariants.In

∗ _{Corresponding}_author_at_:_Centro_de_{Investigación}_y_Estudios_Avanzados_en_Salud_Animal,_Facultad_de_Medicina_Veterinaria_y_Zootecnia, UniversidadAutónomadelEstadodeMéxico,CarreteradeCuotaToluca-Atlacomulco,Kilómetro15.5,CódigoPostal50200,Toluca,Estado deMéxico,Mexico.

E-mail:jsmartinezc@uaemex.mx(J.S.Martínez-Casta ˜neda).

1980CPV-2originalstrain wasreplacedbythevariant

des-ignatedtype2a(CPV-2a),in1984wasidentifiedCPV-2band

in2001,CPV-2cwasdetectedandreportedinItaly.Itis

pos-sible that CPV-2c isthe predominantvariant in numerous

countries.6

Currently,thereissomecontroversyoverclinical character-isticsofdiseaseassociatedwiththesethreevariants;however, someauthorssuggesttherearenoneclinicaldifferences.7

Clinical signs of canine parvovirosis include fever,

anorexia, lethargy, depression, vomiting, mucoid to

http://dx.doi.org/10.1016/j.bjm.2017.03.008

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brazilian journal of microbiology48(2017)769–773

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Fig.1–(A)Parvovirusamplicons.Thepictureshowsa2.0%agarosegelcontainingPCRproducts(275bp)fromdifferent samples.Lane1:DNAlengthmarker(generuler100–1000bpDNALadderplus,Fermentas);lanes2,4and8:CPV-positive samples.Lanes6and10:CPV-negativesamples.(B)Rotavirusamplicons(379bp).Lane1:DNAlengthmarker(generuler 100–1000bpDNALadderplus,Fermentas);lanes3,6,7and9:CRV-positivesamples;lanes2,4,5,8and10:CRV-negative samples.

hemorrhagic diarrhea and sometimes leukopenia;

how-ever, some reports show parvovirus in dogs with atypical

clinicalsigns,andtheauthorssuggestthatthisislikelydue

toCPV-2evolution.8_On_the_other_hand,_the_disease_can_vary

depending on the patient; actually,CPV-2c can infect both

pupsandadults.9

Afurtherfactorimplicatedinvariabilityofclinicalprofiles

inparvovirusisassociationwithothergastroentericviruses

such as canine distemper, canine coronavirus and canine

rotavirus.

Actually,rotavirusesareclassifiedasdistinctmembersof thefamilyreoviridae,genusrotavirus,comprisingfivespecies (AtoE)andtwotentativespecies(FandG).Caninerotavirusis

adouble-strandedRNA,non-envelopedvirusthatpossesses

asegmentedgenomeandthatisapproximately60–75nmin

diameter;fewisolatesofrotavirushavebeenreportedindogs,

thesehavebeenclassifiedasserotypesG3andP5A,grouped

intogroupA,rotavirusofthisgroupcauseneonataldiarrhea

inhumanandmanyanimalspecies;ithasbeendemonstrated

thatdirectinterspecies transmissionbetweenheterologous

strains are key mechanisms ingenerating rotavirusstrain

diversityinnewhosts.Humaninfectionforrotavirusofcanine originhasbeenreported.10,11

Clinical signs ofthe disease includemoderate enteritis,

mainlyinpupsyoungerthantwoweeksold,12,13_and_it_also

causeslethargy,anorexia,fever,diarrheaandvomiting.

Gen-erally,patientsrecoverwithintwoweeks;however,thereare

reportsoffatalsevereenteritisindogsundertwoweeksold.14

The presence of antibodies against rotavirus has been

demonstrated in a high percentage of adult dogs (80%).15

Rotavirus infection does not have pathognomonic clinical

signsandmostdogscanbeasymptomatictoinfectionand

occasionallysignscanbeconfusedwithparvovirus,therefore, laboratorytestfordifferentialdiagnosesarenecessary.3,13_In

Mexico,itremainsunknownwhetherrotavirusiscirculating

amongstcaninepopulationsandifitplaysaprimaryroleas

etiologicagentingastroenteritis.

From March through June 2015, we conducted

non-probability sampling in dogs attending the small animal

veterinaryhospitaloftheAutonomousUniversityoftheState

ofMexico(UAEM,byitsacronyminSpanish).Fiftydogsof

dif-ferentages,breeds,non-vaccinateddogswithgastroenteritis

signswereselected.Onerectalswabwastakenfromeachdog

tobeusedinPCRtesting(parvovirusidentification),whilea

secondswabwastakenforRT-PCRtesting(rotavirus

identifi-cation).Swabswerestoredat−80◦Cuntilprocessing.

TheRotateqvaccine(Merck,USA),whichcontainsrotavirus

serotypesG1P[8],G2P[4],G3P[8]andG9P[8],wasusedas posi-tivecontrol,andinthecaseofparvovirustheEdo.Mex1isolate

wasused(previouslyidentifiedinourlaboratoryandwhich

correspondstoCPV2-cgenotype).

InordertoextractRNAfromrotavirus,eachswabwas

sus-pendedin200␮LofDiethylpyrocarbonate(DEPC1%)-treated

water and centrifuged 7min at 5000×g. Supernatant was

processedusingtheGeneJETViralDNAandRNAPurification

kit(Thermoscientific,USA),followingmanufacturer’s instruc-tions.

Rectalswabsusedforparvovirusdetectionweresuspended

in500␮Lofsterilewater,centrifuged1minat5000×g.200␮L

ofsupernatantwasprocessedforDNAextractionusingthe

QIAampDNAMiniKit(Qiagen,USA)followingthe

manufac-turer’sinstructions.

Inthecaseofrotavirus,two-stepRT-PCRwasperformed

foreachofthesamples,usingtheImProm-IITM_Reverse

Tran-scriptionSystemkit(Promega,USA).Inthefirststep,cDNA

synthesis was accomplished.3␮LofRNA ofeach

purifica-tionplus2␮L(0.5␮g/reaction)ofprimerdTwasusedineach reaction.Allreactionswereincubatedat70◦_C_for₁₅_min_and

subsequently at 4◦_C _for ₅_min. _Next, ₁_␮L _of _ImProm-IITM

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Fig.2–(A)The250nucleotidessequencedfromtheamplifiedfragmentsofcaninerotavirussamples;sequencesobtained werecomparedwithsequencesreportedinGenBank:GU565078.1,JX866792.1,andEU708927.1.(B)The250nucleotides sequencedfromtheamplifiedfragmentsofcanineparvovirussamples;sequencesobtainedwerecomparedwithreported sequencesinGenBank:KP694306.1,KP694305.1,andKP694304.1.

Mix(finalconcentration0.5mMeachdNTP),4.6␮LMgCl2(final

concentration1.5mM),4␮LImProm-IITM_5X_Reaction_Buffer,

0.5␮LRecombinantRNasinRibonucleaseInhibitor(Promega,

USA)(2500Units) and 3.9␮Lnuclease-free watertoreach a

finalvolumeof15␮L.

PCR was performed using the primers VP6F

(5′_{-GACGGVGCRACTACATGGT-3}′₎ _and _VP6R ₍₅′

-GTCCAATTCATNCCTGGTGG-3′₎ _designed _to _amplify _a

379bp fragment of VP6 gene (GenBank accession

num-berKJ940164.1);theseprimersarelocatedinthenucleotides

750–769and920–939respectively.Thereactionwasperformed

using1.5␮Lofbothprimersata20␮molconcentrationplus

3␮LofcDNA,5␮LofGoTaq®GreenMasterMix(Promega,USA)

reactionbuffer(pH8.5)containing400␮Mofeachnucleotide

dATP, dGTP, dCTP, dTTP, 2.5␮L of MgCl2 and 0.5␮Lof taq

polymerase. The reaction included 11␮L of nuclease-free

watertoafinalvolumeof25␮L.

Allreactionswereperformedunderthe following

condi-tions:1cycleat94◦_C₅_min,_followed_by₃₅_cycles_at₉₄◦_C₁_min, 61◦_C₁_min,₇₂◦_C_for₂_min_and_a_final_extension_at₇₂◦_C₅_min.

Ampliconswere loadedina2.0%agarosegel(stained with

0.5mg/mLofethidiumbromide)andvisualizedbymeansofa

UVBio-ImagingSystemsMiniBisProtransilluminator(Fig.1).

Inthecaseofparvovirus,100ngofDNAfromeach

sam-ple was used for PCR. Previously, we designed a pair of

primers:ParvoInt2FB(5′_{-TCAAGCAGATGGTGATCCAAG-3}′₎_and

ParvoInt2CR(5′_{-GGTACATTATTTAATGCAGTTA-3}′₎_to_amplify_a

275bpfragmentoftheVP2genefragment.Theseprimersare

locatedbetweennucleotides1107–1130and1360–1382ofthe

genesequence(GenBankaccessionnumberFJ0051962c).

Reac-tionscontained2␮Lofbothprimersata20␮Mconcentration,

plus12.5␮LofGoTaq®GreenMasterMix(Promega,USA)

con-tainingtheenzymeDNApolymerase,reactionbuffer(pH8.5),

400␮MofeachnucleotidedATP,dGTP,dCTP,dTTPand3mM

MgCl2.28.5␮Lofnuclease-freewaterwasaddedtoobtaina

finalvolumeof50␮L.

ThefollowingconditionswereusedinallPCRreactions:

onecycleat94◦_C_for₅_min,₃₅_cycles_at₉₄◦_C_for₃₀_s,₅₂◦_C_for 1min,72◦_C_for₁_min_and_a_final_extension_at₇₂◦_C_for₅_min.

Theelectrophoresisofampliconswasachievedina2%agarose

gel(stainedwith0.5mg/mLethidiumbromide)andvisualized

ina UVBio-ImagingSystemsMiniBis Protransilluminator

(Fig.1).

Inordertoverifyprimerspecificity,ampliconsfrom

posi-tivecontrolsweresequenced.Sequencingwasaccomplished

throughthefollowingprocedure:first,PCRproductswere

puri-fied from agarose gel using the Wizard® SV Gel and PCR

Clean-UpSystem(Promega,USA).Next,30ngofpurifiedPCR

product was sequencedby means ofthe Dye® Terminator

v.3.1 Sequencing Kit(Applied Biosystems, USA).Sequences

wereprocessedina3100sequenceanalyzer(Applied

Biosys-tems, Foster City, CA, USA) and reaction sequences were

analyzedinaPRISM® 3130xl ABI analyzer(Applied

Biosys-tems, USA). Due to inconsistencies on the extremes of

theobtainedsequences,thesewereeditedconsideringonly

250-nucleotide-long final for the alignment, DNA BLAST

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) was utilized for

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brazilian journal of microbiology48(2017)769–773

Table1–Clinicalsignsandhematologicfindingsindogs infectedwithcanineparvovirus(CPV-2),dogsinfected withcaninerotavirus(CRV)andco-infected

(parvovirus–rotavirus)dogswithgastroenteritis.

Clinical findings/typeof infection

%with CPV (n=27)

%with CRV (n=7)

%with CPV–CRV (n=11)

Abdominalpain 66.6 28.5 81.8

Anorexia 33.3 0 72.7

Lethargy 81.4 14.2 100

Vomiting 44.4 14.2 54.5

Fever(>39.5◦_C) _14.8 ₀ _36.3

Hemorrhagic diarrhea

70.3 14.2 90.9

Mucoiddiarrhea 29.6 85.7 9.0

Leukocytosis 0 28.5 18.1

Leukopenia 40.7 0 54.5

Panleukopenia 14.8 0 36.3

Clinicalsigns,physicalexamination,andlaboratorytestresultsin dogswithviralenteritis.Referencerangesfortotalleukocytecount: 6.0–17.0×109/L,panleukopeniareferstogenerallowcountsofall leukocytecount.

availableinthe GenBank,specifically withGU565078.1and JX866792.1 for rotavirus and KP682524.1 for parvovirus; sequencealignmentsanalyseswereperformedwiththeuseof ClustalWmethod,utilizingBioEditSequenceAlignmentEditor

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

Thesequencesobtainedwerematched100%withthe

Gen-Banksequences(Fig.2),whichindicatesthatprimersusedfor

PCRandRT-PCRinthisstudy,arespecificforgenome

ampli-ficationofbothviruses.Therefore,theanalysisofallsamples

wasaccomplishedusingbothPCRandRT-PCRprotocols.Out

of50dogs,82%(45/50)were positiveforatleastoneofthe

twoviruses, specifically,60% (27/45) were positive onlyfor

parvovirus,8%(7/45)werepositiveonlyforrotavirusand14%

(11/45)wereco-infected.Fivesamples,whichwerenot

posi-tiveforeitherparvovirusorrotavirus,werepositiveforcanine

distemperorSalmonellaspp.(datanotshown).

Asageneralobservation,theclinicalsignsofrotaviruswere

milderthatthoseofparvovirus;dogsinfectedwithrotavirus

exclusively, displayed abdominal pain, lethargy, vomiting,

mucoiddiarrhea andinsomecasesleukocytosis. However,

intheco-infecteddogsseveregastroentericsymptomswere

observedinadditiontoleukopenia, panleukopenia,mucoid

orhemorrhagicdiarrhea,vomitingandlethargy.These

symp-toms were evenmore severe that those observed in dogs

infectedwithonlyparvovirus(Table1).

Datafrom thepresentstudywere comparedtoprevious

studies,andourresultsshowarelativelyhighpercentageof

dogswithco-infection(14%)inrelationtofindingsinother

countries,forinstance2.4%hasbeenreportedinU.S.A., as

inferred from reverse passive hemagglutination (RPHA), in

Japan,1.2%wasestimatedfromviralisolationand2%using

electronmicroscopy,whileinItalytherewereno

identifica-tionsusingPAGE.1,2,4_These_low_percentages_might_be_related

tothetechniquesemployedinthesestudies,inthissense,in

thepresentstudyRT-PCRwasusedwhichhasdemonstrateda

highersensitivity.16_It_is_also_possible_that_contrasting_results

areinfluencedbythecountrywherestudieswereconducted,

asitiswelldocumentedthatdifferentrotavirusgenotypesare

associatedwithlow-incomeareas.Furthermore,other

stud-ieshavereportedthepresenceofrotavirusonlyinpupsless

thantwomonthsofageornewborns,11_however,_in_this_study

rotavirusinfectionwasdetectedindogsolderthan6months.

Thisreportisthefirststudydemonstratingthepresence

of circulating rotavirus in dogs from the State of Mexico.

Althoughrotavirusisoflittleimportanceforveterinariansas ithaslowmortalityrate,inthepresentstudyitwasobserved toplayanimportantroleinseverityofviralgastroenteritis,

especiallywhenassociatedtocanineparvovirus.Ontheother

hand,althoughparvovirusisconsideredthemostimportant

pathogenincaninegastroenteritis,rotavirusisofspecial inter-esttopublichealthduetoitszoonoticpotential.Insupportof this,thereexistreportsofinter-speciestransmissionofcanine rotavirusoftheG3grouptochildreninItalyandTaiwan.10

Althoughthisisthefirstreportofrotavirusincaninesof

the StateofMexico,futuremolecularepidemiology studies

shouldbeconductedtoidentifythecirculatinggenotypesin

dogpopulationsthroughoutMexico,asthiswillincreaseour

understandingontheimportanceofrotavirusindogs.

Financial

support

TheauthorswouldliketothanktotheFundaciónEducación

Superior-Empresa,A.C.forthefinancialsupport.

Conflicts

of

interest

Theauthorsdeclarednopotentialconflictsofinterestwith

respecttotheresearch,authorship,and/orpublicationofthis article.

Acknowledgement

AriadnaFloresthanktoConsejoNacionaldeCienciay

Tec-nologíaforthescholarshipawardedforpostgraduatedstudies

inUniversidadAutónomadelEstadodeMéxico,Programade

MaestriayDoctoradoenCienciasAgropecuariasyRecursos

Naturales.

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