Amostras fecais de cães com gastrenterite, até 6 meses de idade, foram testadas para a presença do parvovírus canino (CPV-2) pela reação de hemaglutinação (HA) e confirmadas pela reação de inibição da hemaglutinação (HI). Quarenta das 79 amostras, recebidas no período de abril de 1995 a junho de 1997, foram consideradas positivas. Aproximadamente 70% destas amostras foram obtidas de animais entre 2 e 4 meses de idade, época em que o risco de contraírem a infecção pelo CPV-2 é alto apesar da vacinação. Nenhuma variação sazonal da infecção pelo parvovírus canino pôde ser observada, e um estudo retrospectivo realizado na PolVet - UFF mostrou que em um período de 6 anos (1991-97), casos de gastrenterite ocorreram durante todos os anos em Niterói, sem uma sazonalidade definida

Adão Onofre dos SANTOS

Alexandre de Pina COSTA

Ana Maria Vianna PINTO

Bianca Mendes MACIEL

Jussara Pereira do NASCIMENTO

Ledy do Horto dos Santos OLIVEIRA

Liliane Maria Valentim WILLI

Maria Cristina Nobre e CASTRO

Norma Vollmer LABARTHE

Rita de Cássia Nasser CUBEL GARCIA

Brazilian Journal of Veterinary Research and Animal Science

Rita de Cássia Nasser Cubel Garcia

Ana Maria Vianna Pinto

Alexandre de Pina Costa

Bianca Mendes Maciel

Ledy do Horto dos Santos Oliveira

Jussara Pereira do Nascimento

Adão Onofre dos Santos

Maria Cristina Nobre e Castro

Liliane Maria Valentim Willi

Norma Vollmer Labarthe

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Braz. J. veto Res. animo Sei.,São Paulo, V. 37, n.Z, p. 132-135,2000.CORRESPONDENCE TO:Rita Cubel GarciaDepartamento de Microbiologia eParasitologiaInstituto Biomédico da UniversidadeFederal FluminenseRua Prot. Hernani Meio, 10124210-130 - Niterói - RJe-mail: rcgarcia@centroin.com.brl-Departamento de Microbiologia eParasitologia do Instituto Biomédico daUFF, Niterói - RJ2-Departamento de Virologia do InstitutoOswaldo Cruz I FIOCRUZ - RJ3-Departamento de Clínica Médica dePequenos Animais da PoliclínicaVeterinária da UFF, Niterói - RJCanine parvovirus infection in puppies withgastroenteritis in Niterói, Rio de Janeiro, Brazilfrom 1995 to 1997 *Infecção pelo parvovírus canino em filhotes comgastrenterite em Niterói, Rio de Janeiro, Brasilde 1995 a 1997Rita de Cássia Nasser CUBEL GARCIA); Ana Maria Vianna PINTO);Alexandre de Pina COSTA); Bianca Mendes MACIEL);Ledy do Horto dos Santos OLIVEIRA); Jussara Pereira do NASCIMENT()2;Adão Onofre dos SANTOS); Maria Cristina Nobre e CASTR()1;Liliane Maria Valentim WILLP; Norma Vollmer LABARTHE3SUMMARYFecal samples from puppies with gastroenteritis less than 7 months old were examined for canine parvovirus infection(CPV-2) by hemagglutination (HA) and subsequent hemagglutination-inhibition (HI) tests. Forty ofthe 79 samples coUectedfrom April 1995 to June 1997 were found to be positive. About 70% of these samples were from 2 to 4 months old puppies,age in which they are at increased risk of developing CPV-2 infection, despite of vaccination. No seasonal distribution ofcanine parvovirus cases was found and it was supported by the results of a retrospective study performed at PoIVet-UFF,which showed that gastroenteritis cases occurred throughout the year, for a six-year period (1991-97) in Niterói, Rio de Janeiro.UNITERMS: Canine parvovirus; Gastroenteritis; Haemagglutination tests.INTRODUCTIONThe first cases of enteritis and myocarditis causedby canine parvovirus (CPV -2) were recognizedin the United States in 1978. Since then, CPV-2infection had spread rapidly throughout the world+'.Initial outbreaks of parvovirus in dogs werecharacterized by high morbidity and mortality in ali agegroups. In Brazil, the first cases of hemorrhagic enteritiswere first observed in Campinas and lately detected in SãoPaulo during 1979-19801•9• After that, CPV -2 infections havealso been diagnosed in Rio Grande do Su114•Parvovirus infection in dogs causes an acute diseasecharacterized by severe vomiting and diarrhea. Disease ismost commonly seen in puppies from 6 weeks to 6'monthsof age. Most adult dogs have beco me immune throughvaccination ar natural infection'.Infected dogs can shed more than 109 tissue culture* Supported by CNPq, Pfizer Saúde Animal.132infectious dose (TCIDso) of CPV -2 per gram of feces.Consequently feces constitute the major source of infectionfor susceptible animals and they also represent the mostuseful material for diagnosis-".Several methods have been used to detect CPV -2 infecal samples. Electron microscopy by negative staining, virusisolation in cell cultures and enzyme immunoassay-'ê"-".But, for rapid diagnosis, the most frequently used is thehemagglutination (HA) assay followed by hemagglutination-inhibition (HI) test with a CPV -2-specific antiserumv-":".A preliminary study conducted in São Paulo byMehnert et ai. 11, in order to identify the viral agents thatcause hemorrhagic gastroenteritis in dogs, showed thatcanine parvovirus was responsible for the most of the cases.The purpose of the present study is to determine theoccurrence of parvovirus infection in puppies suffering fromgastroenteritis in Niterói, Rio de Janeiro, during a two-year period.CUBEL-GARCTA, R.C.N.; VIANA-PINTO, A.M.; COSTA, A.P.; MACIEL, B.M.; OLIVEIRA, L.H.S.; NASCIMENTO, l.P.; SANTOS, A.O.;NOBRE E CASTRO, M.C.; WILLI, L.M.Y.; LABARTHE, N.Y. Canine parvovirus infection in puppies with gastroenteritis in Niteroi, Rio deJaneiro, Brazil from 1995 to 1997. Braz. J. veto Res, animo Seio,São Paulo, v. 37, n. 2, p. 132-135, 2000.MAlERIALANDMElHODThe seventy-nine fecal samples in this study collectedfrom April 1995 to June 1997 from puppies less than 7months of age with gastroenteritis were obtained fromdifferent sources. Fifty-one were from Policlínica Veterinária,Universidade Federal Fluminense (Polvet), 20 samples werefrom a private owned Animal Hospital, Veterinária dasMeninas (Vet Men), both located in Niterói city. Another 8samples subrnitted for diagnosis were from dogs seen bypractitioners at Rio de Janeiro city.Approximately 10% of fecal material suspensionswere prepared in Hank's balanced salt solution. After vortexand centrifugation the supematants were treated with 1/10volume of chloroform as described previously".For the HA test, serial 2-fold dilutions of the 10%fecal suspensions were made in BABS buffer pH 9.0 (I.5MNaCl, 0.5M H3B03, I.OM NaOH, 0.2% BSA). Then, an equalvolume (50 !J.I)ofO.5% porcine orrhesus monkey erythrocytesdiluted in VAD buffer pH 6.0 (O.15M NaCl, 0.3M Na2HP04,O.l5M NaH2P04) were added and incubated at 4°C. Testswere read after the erythrocyte controls had settled completely.Samples with HA titers ~ 8 were considered negative.Samples with HA titers ~ 16 were confirmed as positive inthe HI test"-".The specific anti-CPV -2 serum used in HI test wasobtained after immunizing adult guinea pigs withParvoguard" vaccine (Solvay Saúde Animal Ltda.)". Initially,serum sample was heated at 56°C for 30 rninutes, treatedwith 25% Kaolin and then adsorbed with 50% erythrocytesuspension. Serial 2-fold dilutions of serum in BABS weremade in rnicroplates, and an equal volume (25 !J.I)of 10%fecal suspensions (diluted to contain 8 UHA/50 !J.I)was addedto each well. After incubation, 50 ul of 0.5% erythrocytesuspension diluted in VAD buffer pH 6.0 was added. HIendpoints were determined after an ovemight incubationperiod at 4°C. Samples were considered positive when theHA reaction was inhibited by the antiserum. Each test seriesincluded appropriate erythrocyte, antigen (vaccine) andserum controls".From a total of 79 samples subrnitted for diagnosis,39 were from mongrel puppies, 7 from Doberman pinschers,and 4 from Rottweilers. Another 20 samples were distributedamong the following breeds: German shepherd (7), Fila (4),Boxer (3), Husky (3), Cocker spaniel (2), Dachshund (2),Poodle (2), Yorkshire (2), Basenji (1), Beagle (1), Setter (1)and Terrier (1).RESULTSFecal samples from 42 male and 37 female puppiessuffering from diarrhea and vorniting were tested for thepresence of CPV -2 by HAJHI tests, and 40 (23 males and 17females) of these 79 samples were positive. As shown inTab. 1, the majority of the 79 samples were collected frompuppies within two, three or four days of the onset of thesymptoms. At least 50% of the samp1es tested within thisperiod were positive. Fifty-seven (72%) of the 79 samp1eswere from animals ranging from 2 to 4 months of age, and33 samples (58%) were positive (Tab. 2).The annual distribution of canine parvovirus cases fromApril 1995 to June 1997 was examined, From 20 samplesreceived in 1995, 18 (90%) were positive. Among 39 samplestested in '1996, CPV-2 was confmned for 17 (44%) while in1997 five (25%) of the 20 samples were positive.The distribution of the number of gastroenteritis casesin relation to the total number of dogs attending at PolVetfrom January 1991 to December 1997 is shown in Fig. l.Gastroenteritis could be diagnosed in about 10% of the dogsTable 1Distribution of confirmed CPV-2 cases in relation to the day ofdisease in Niterói from 1995 to 1997.* Not available for three animals.Table 2Distribution of confmned CPV-2 cases in relation to the age ofthe puppies in Niterói from 1995 to 1997.Age (months) 3 4 5 6 Total2N° of positivasamples / N° ofsamples tested3/6 10/18 12/20 11/19 2/8 2/8 40/79Table 3CPV-2 positive cases detected in vaccinated puppies in Niteróifrom 1995 to 1997.CaseA N° of days between( g~) vaccination and onsetmon s of the disease2nd doseBreed1S1 dose1 Mongrel 2 42 Siberian husky 3 83 Mongrel 2 244 Rottweiler 3 375 Boxer 3 496 Doberman pinscher 4 617 Yorkshire 5 838 Doberman pinscher 5 919 Cocker spaniel 4 NK*10 Rottweiler 4 4711 Fila 3 4812 Fila 3 50* Not known.17722133CUBEL-GARCIA, R.C.N.; VIANA-PINTO, A.M.; COSTA, A.P.; MACIEL, B.M.; OLIVEIRA, L.H.S.; NASCIMENTO, J.P.; SANTOS, A.O.;NOBRE E CASTRO, M.C.; WILLI, L.M.Y.; LABARTHE, N.Y. Canine parvovirus infection in puppies with gastrocnteritis in Niteroi, Rio deJaneiro, Brazil Irom 1995 to 1997. Braz. J. veto Res. animo Sci., São Paulo, v. 37, n. 2, p. 132-135, 2000.450400350 (24±9)~30000.g 250'õ 200Z 150100(24±9) (27±4)(33±10)(26± 7}(23±5)(22±7)(23±9) (21±6)(15±5)(I8±6)50o Jan Feb Mar Apr May Jun Jul Aug Sep oa Nov DeeMonths*~ Total nO of dogs • Total nO of GE gases DN° of GE cases in puppiesFigure 1Distribution of lhe total number of gastroenteritis (GE) cases andthe total number of dogs attending at PoIVet-UFF in lhe period of1991 to 1997. * Average month1y values of GE cases from 1991-1997. Number in parenlhesis means X ± (J of lhe GE cases in puppies.throughout the year and at least 70% of these cases occurredin puppies less than 7 months of age.From the 40 CPV -2 positive puppies, 27 had not beenvaccinated and for another one this data was not known.Among 12 positive puppies that had been vaccinated, 8 hadreceived only the first dose and in two of them (cases 1 and 2)enteritis symptoms began 4 and 8 days afier vaccination (Tab.3). A further 4 puppies were given two doses of vaccine, andthree of them received the second dose within 7 days beforethe onset of the disease (cases 9, 10, li). One of these was aRottweiler that died.DISCUSSIONCanine parvovirus emerged as a new pathogen ofdogs in the late 1970s. Initially seen as epidemic disease inall dogs, parvoviral enteritis now may be controlled byvaccination. Disease is commonly seen in I to 6 monthsold puppies due to vaccination failures because theinterference of maternal antibodies':",In the present study we were able to demonstrate, byHAJHI tests, canine parvovirus infection in 44% of puppieswith gastroenteritis during a two-year period. This highproportion was probably due to the age range of the puppiesstudied (2 - 4 months). At this age vaccination may not becompleted", and the majority of these puppies had not beenvaccinated.As shown by our results, 12 of the positive puppieshad received one or two doses of vaccine. Using the HA assayit is not possible to discriminate between field and vaccinevirus, and it is known that both vaccine and wild CPV -2 canbe detected in fecal samples by this test from 3 to 9 days afieroral infectiorr=. In 7 of these vaccinated puppies, the findingof CPV -2 does not rea1lymean vaccine virus shedding, becausethe interval between the date of vaccination and days of diseasewas longer than 15 days. In 5 positive puppies the HA resultcould be due to the detection of vaccine virus because the fecaIsamples were collected from 1 to 8 days afier vaccination.Certain breeds like Rottweiler and Doberman pinscherare reported to be more susceptible to the development ofparvovirus disease". This could not be confirmed in our studybecause of the low number of samples received fromRottweilers and Doberman pinschers.In a study performed in Canada, the incidence of CPV-2 infection peaked in the months of July, August andSeptember, during the warm season'". No seasonal variationof CPV -2 infection in Niterói was apparent but the lownumber of samples received during a two-year period meansthis is not definitive. The data from PoIVet-UFF during 1991to 1997 show that gastroenteritis cases occur throughout theyear without a clearly defined seasonality and we detectedparvovirus infection throughout the study period.Our results confrnn that CPV -2 infection in puppiesis a significant cause of morbidity in the city of Niterói, Riade Janeiro.ACKNOWLEDGEMENTSThe authors wish to thank Drs. Cristiane OliveiraMilward and Mariluci Gomes Espariz (Veterinária dasMeninas) who generously provided fecal samples for testing.The authors also wish to thank Dr. David Brown (VirusReference Division, CPHL, London) for critical reading ofthe manuscript.RESUMOAmostras fecais de cães com gastrenterite, até 6 meses de idade, foram testadas para a presença do parvovírus canino (CPV-2) pela reação de hemaglutinação (HA) e confirmadas pela reação de inibição da hemaglutinação (HI). Quarenta das 79amostras, recebidas no período de abril de 1995 a junho de 1997, foram consideradas positivas. Aproximadamente 70%destas amostras foram obtidas de animais entre 2 e 4 meses de idade, época em que o risco de contraírem a infecção peloCPV -2 é alto apesar da vacinação. Nenhuma variação sazonal da infecção pelo parvovírus canino pôde ser observada, e umestudo retrospectivo realizado na PolVet - UFF mostrou que em um período de 6 anos (1991-97), casos de gastrenteriteocorreram durante todos os anos em Niterói, sem uma sazonalidade definida.134UNITERMOS: Parvovírus canino; Gastrenterite; Reações de hemaglutinação.CUBEL-GARCIA, R.C.N.; VIANA-PINTO, A.M.; COSTA, A.P.; MACTEL, B.M.; OLIVEIRA, L.H.S.; NASCIMENTO, J.P.; SANTOS, A.O.;NOBRE E CASTRO, M.C.; WILLI, L.M.Y.; LABARTHE, N.Y. Canine parvovirus infection in puppies with gastroenteritis in Niteroi, Rio deJaneiro, Brazil from 1995 to 1997. Braz, J. veto Res. animo Sci., São Paulo, v. 37, n. 2, p. 132-135, 2000.REFERENCES1- ANGELO, M.J.O.; HAGIWARA, M.K.; JULY, J.R.; CARVALHO,R.P.S.; BACCARO, M.R. Isolamento de parvovírus canino noBrasil. Revista da Faculdade de Veterinária e Zootecnia daUniversidade de São Paulo, v.25, n.l, p.123-34, 1988.2- APPEL, M.; PARRISH, C.R. Canine parvovirus type 2. In: APPEL,M.S. Virus infections of carnivores. Amsterdam: Elsevier, 1987.p.69-92.3- APPEL, M.J.G.; SCOTT, nw. CARMICHAEL, L.E. Isolation andimmunization studies of a canine parvo-like vírus from dogs withhaemorrhagic enteritis. Veterinary Record, v.I 05, n.8, p.156-9, 1979.4- CARMICHAEL, L.E.; JOUBERT, J.C.; POLLOCK, R.Y.H.Hemagglutination by canine parvovirus: serologic studies anddiagnostic applications. American Journal of VeterinaryResearch, vAI, n.5, p.784-91, 1980.5- CARMICHAEL, L.E.; JOUBERT, J.C.; POLLOCK, R.Y.H. Amodified live canine parvovirus strain with novel plaquecharacteristics. I. Viral attcnuation and dog response. CornellVeterinary, v.71, nA, pA08-21. 1981.6- CUBEL, R.C.N.; PINTO, AM.Y.; OLIY.EIRA, L.H.S.; MACIEL, B.M.;COSTA, A.P.; LABARTHE, N.Y.; CASTRO, M.C.N.; SANTOS,A.O.; NASCIMENTO, lP. An enzyme immunoassay fordetectionof canine parvovirus in fecal samples. 11/: ENCONTRONACIONAL DE VIROLOGIA. 8., São Lourenço, 1996. Anais.São Lourenço: Sociedade Brasileira de Virologia, 1996. pA2.7- DURIGON, E.L.; ANGELO, M.J.O.; JEREZ, J.A.; TANAKA. H.;HAGIWARA. M.K. Comparação entre as reações dehemaglutinação (HA), isolamento do vírus em culturas celulares(CC). imunoeletrosmoforese (TEOF) e imunomicroscopiaeletrônica (IME) , para o diagnóstico etiológico da parvovirosecanina. Revista de Microbiologia, v.18, n.3, p.205-1 O, 1987.8- GLlCKMAN, L.T.; DOMANSKI, L.M.; PATRONCK, G.J.;VISINTAINER, F. Breed-related risk factors for canineparvovirus enteritis. Journal of American Veterinary MedicalAssoeiation, v.187, n.6, p.589-94, 1985.9- HAGIWARA, M.K.; JULY, J.R.; BACCARO, M.R.; ANGELO,M.J.O. Enterite hemorrágica em cães associada à infecção porum parvovírus. Arquivos do Instituto de Biologia, vA7, n.1I2,pA7-9, 1980.10- HOUSTON, D.M.; RIBBLE, C.S.; HEAD, L.L. Risk factorsassociated with parvovirus enteritis in dogs: 283 cases (1982-1991). Journal of American Veterinary Medical Association,v.208, nA, p.542-6, 1996.11- MEHNERT, D.U.; MONEZI, T.A.; PRADO, M.A.; HÁRSI, C.M.;CAVALIERO, M.J.; QUEIROZ, A.P.S.; MÜLLER, N.G.;ANGELO, M.J.O. Canine gastroenteritis in Brazil: Preliminaryresults of a viral etiological study. In: ENCONTRO NACIONALDE VIROLOGIA, 8., São Lourenço, 1996. Anais. São Lourenço:Sociedade Brasileira de Virologia, 1996. pA3.12- MOCHIZUKI, M.; HIDA, S.; HSUAN, s.w. SATO, H. Fecalexaminations for diagnosis of canine parvovirus infection. JapaneseJournal ofVeterinary Seience, vA6, nA, p.587-92, 198413- MOCHIZUKI, M.; SAN GABRIEL, M.C.; NAKATANI, H.;YOSHIDA, M. Comparison of polymerase chain reaction withvirus isolation and haemaggluunation assays for thc detectionof canine parvoviruses in faecal spccimcns. Research VeterinarySeience, v.55, n.l, p.60-3, 1993.14- MOOJEN, Y.; GONÇALVES, I.P.D.; PIZZOL, M.D.; OLIVEIRA.R.T.; SCHERER, H.Á.; RAVAZZOLO, A.P.; SIMANKE, A.T.;HOTZEL, I. Parvovirose canina: Diagnóstico laboratorialrealizado no período de 1980 a 1989, na Faculdade de Veterináriada UFRGS, Porto Alegre. Arquivos da Faculdade deVeterinária da UFRGS, v.20. n.único, p.209-23, 1992.15- POLLOCK, R.Y.H.; COYNE, M.J. Gastroenterology: CanineParvovirus. Veterinary Clinics of North America : Smallanimal practice, v.23. n.3, p.555-69, 1990.16- SANTOS, P.; PINTO, A.M.Y.; CUBEL GARCIA, R.C.N.:LABARTHE, N.Y.; OLIVEIRA, L.H.S. Padronização dereagentes e métodos utilizados na técnica de hcmaglutinaçãopara o diagnóstico laboratorial da parvovirose canina. RevistaBrasileira de Ciêneia Veterinária, vA, n.3, p.III-5, 1997.17- SENDA, M.; HIRAYAMA, N.; YAMAMOTO, H.; KURATA, K.An improved hemagglutination test for study of canineparvovirus. Veterinary Microbiology, v.12, n.l, p.I-6, 1986.18- TERAMOTO, Y.A.; MILDBRAND, M.M.; CARLSON, J.;COLLINS, J.K.; WINSTION, S. Comparison of enzyme-linkedimmunosorbent assay, DNA hybridization, hemagglutination, andelectron microscopy for detection of canine parvovirus infections.Journal of Clinical Microbiology, v.20, n.3, p.373-8, 1984.Received: 15/12/1998Accepted: 03/09/1999135

Infecção pelo Parvovírus Canino em filhotes com gastrenterite em Niterói, Rio de Janeiro, Brasil de 1995 a 1997

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Canine Parvovirus infection in puppies with gastroenteritis in Niterói, Rio de Janeiro, Brazil from 1995 to 1997

Canine Parvovirus infection in puppies with gastroenteritis in Niterói, Rio de Janeiro, Brazil from 1995 to 1997

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