Padronização de um teste ELISA indireto para diagnóstico da cinomose canina / Standardization of an indirect ELISA test for canine diagnosis diagnosis

A cinomose é uma enfermidade causada pelo vírus Canine Distemper Virus (CDV). Essa doença afeta principalmente cães, mas também acomete outras espécies domésticas e selvagens. A imunidade do animal está relacionada ao grau que a esse patógeno vai atingir o organismo do indivíduo. Ela afeta a respiração do animal, pode causar vômito, diarreia, convulsões, podendo levar o animal à óbito. O objetivo do presente trabalho foi padronizar um teste ELISA indireto com antígeno de superfície para o diagnostico cinomose utilizando amostras de soro canino. Para padronização da técnica, fez-se necessário o estudo da diluição do antígeno para identificar a melhor concentração para sensibilização da placa. O teste foi aplicado primeiramente com diferentes diluições do antígeno para detecção do melhor desempenho do antígeno. Feito isso, foi testado em um banco de soro de 45 animais comprovadamente positivos no teste ELISA comercial e em soro de 45 animais comprovadamente negativos no teste ELISA comercial, posteriormente foi calculado o ponto de corte, especificidade e sensibilidade do teste. O teste ELISA indireto se mostrou com excelência como um teste de diagnóstico para a cinomose canina, obtendo-se ponto de corte de densidade óptica de 0,229, sensibilidade de 95,5% e especificidade de 84,4%.

Distemper is a disease or the disease by the CDV virus, Distemper Virus. This disease mainly affects dogs, but also affects other domestic and wild species. The animal's immunity is related to the degree to which it will reach the individual's organism. It affects the animal's breathing, can cause vomiting, diarrhea, convulsions, and can lead to death. The aim of the present work test was to standardize an indirect ELISA for distemper diagnosis in experiments using a surface antigen. For the study of technical identification, it was necessary to specify the antigen for the best concentration of plaque sensitization. The test was initially applied with different dilutions of the antigen to detect the best performance of the antigen. This was tested in a serum bank of 45 animals proven positive in the commercial ELISA test and in the serum of 45 animals proven negative in the commercial ELISA test, later it was tested on the cut-off point, specificity and sensitivity of the test. The indirect ELISA test proved to be excellent as a diagnostic test for canine distemper, with an optical density cut-off of 0.229, sensitivity of 95.5% and specificity of 84.4% being obtained.

First description of sarcoptic mange in a wild coati (Nasua narica), in Ecuador, and cooccurrence of canine distemper virus / Primeira descrição da sarna sarcóptica em um coati selvagem (Nasua narica), no Equador, e coocorrência do vírus distemper canino

Abstract We present a case of Sarcoptes and canine distemper virus (CDV) infection in a white-nosed coati (Nasua narica) that was trapped in the dry tropical forest of Cerro Blanco reserve, located in the coastal region of Ecuador. Sarcoptic mange is a highly contagious and zoonotic disease with worldwide distribution that causes epidemics. Mange is produced by Sarcoptes mites that causes severe epidermal damage. Secondary infections and physiological constrictions without treatment can lead to death of the host. In addition, cooccurrence of canine distemper virus was detected via iiRT-PCR from serum samples. Physical analyses showed that 90% of the skin was affected by severe alopecia due to the sarcoptic mange infection. The presence of mites and histopathological analyses confirmed the diagnosis of infection. This coati was taken to a veterinary clinic and was fed every day, but it died after four days. This is the first report of sarcoptic mange and the first report of CDV in white-nosed coatis in South America. Further studies are needed in this region, to seek out other suspected cases, given the high capacity for disease transmission. Preventive actions to avoid epidemic and zoonotic episodes are needed.

Resumo Apresentamos um caso de Sarcoptes e infecção pelo vírus da cinomose canina (CDV) em um quati-do-nariz-branco (Nasua narica) que ficou preso na floresta tropical seca da reserva de Cerro Blanco, localizada na região costeira do Equador. A sarna sarcóptica é uma doença altamente contagiosa e zoonótica de distribuição mundial que causa epidemias. A sarna é produzida por ácaro do gênero Sarcoptes que causa graves danos epidérmicos. Infecções secundárias e constrições fisiológicas sem tratamento podem levar à morte do organismo. Além disso, a coocorrência do vírus da cinomose canina foi detectada, via iiRT-PCR, a partir de amostras de soro. As análises físicas mostraram que 90% da pele estava afetada por alopecia severa devido à infecção pelo ácaro da sarna sarcóptica. A presença de ácaros e análises histopatológicas confirmaram o diagnóstico de infecção. Esse quati foi levado a uma clínica veterinária e foi alimentado todos os dias, mas morreu após quatro dias. Esse é o primeiro relato de sarna sarcóptica e o primeiro relato de CDV em quatis-de-nariz-branco na América do Sul. São necessários mais estudos nessa região, para buscar outros casos suspeitos, dada a alta capacidade de transmissão da doença. Ações preventivas para evitar episódios epidêmicos e zoonóticos, são necessárias.

Diagnóstico de cinomose canina por meio de teste imunocromatográfico e sua correlação com achados clínicos e hematológicos no semiárido da Paraíba / Diagnosis of canine distemper by immunochromatographic test and its correlation with clinical and hematological findings in the semiarid region of Paraíba

Objetivou-se através deste trabalho, determinar a prevalência de cinomose canina no semiárido da Paraíba, através de testes rápidos imunocromatográficos, correlacionando-a com os principais achados clínicos e hematológicos. Foram analisadas 67 fichas de animais em que foram realizados testes rápidos para pesquisa de antígeno em amostras nasais e oculares no período de janeiro a dezembro de 2019. Observou-se que 47% (32/67) dos cães analisados foram positivos para cinomose canina. As variáveis que apresentaram diferença estatística significativa (p<0,05) para a infecção foram animais sem raça definida 60% (21/35), animais não vacinados 70% (29/42), e período seco do ano, sendo o mês de agosto (40%; 13/32), com maior ocorrência. Os principais sistemas afetados foram o respiratório 61% (17/28), oftalmológico 70% (22/31), nervoso 69% (13/19), dermatológico 45% (9/20), e gastrintestinal 42% (6/14). As principais alterações hematológicas foram anemia 66% (23/32), leucopenia 76% (19/25) e linfopenia 48% (15/31). Concluiu-se que foi elevada a ocorrência de cinomose canina em animais com suspeita clínica no Semiárido Paraibano, e animais sem raça definida, não vacinados, no período seco do ano foram mais diagnosticados com a enfermidade.

The objective of this study was to determine the prevalence of distemper canine distemper vírus (CDV) infection in the semi-arid region of Paraíba, using rapid immunochromatographic tests, correlating it with the main clinical and hematological findings. 67 records of animals were analyzed in which rapid tests were performed for antigen research in nasal and ocular from January to December 2019. It was observed that 47% (32/67) of compulsory dogs were positive for canine distemper. The variables that defined difference difference (p <0.05) for infection were mixed breed animals 60% (21/35), unvaccinated animals 70% (29/42), and dry period of the year, being the August (40%; 13/32), with greater occurrence. The main affected systems were the respiratory 61% (17/28), ophthalmological 70% (22/31), nervous 69% (13/19), dermatological 45% (9/20), and gastrointestinal 42% (6/14 )) The main changes were hematological, anemia 66% (23/32), leukopenia 76% (19/25) and lymphopenia 48% (15/31). It was concluded that the occurrence of canine distemper in animals with clinical suspicion in the Semiarid Paraibano was high, and non-vaccinated mixed-breed animals in the dry period of the year were more diagnosed with the disease.

Construction and identification of an infectious clone for CDV-3 strain of canine distemper virus / 生物工程学报

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.

Características epizootiológicas da infecção natural pelo vírus da cinomose canina em Belo Horizonte / Natural canine distemper virus infection epizootiology characteristics in Belo Horizonte

O perfil epizootiológico da cinomose canina em Belo Horizonte é desatualizado e não alberga algumas características relevantes. Uma análise recente da distribuição do vírus em relação às características do hospedeiro e do meio ambiente associada aos principais sinais clínicos e achados laboratoriais são importantes para se adotarem medidas estratégicas para o controle da enfermidade. Objetivou-se, assim, determinar as características epizootiológicas da infecção pelo vírus da cinomose canina associada à variedade de sinais clínico-neurológicos e laboratoriais em Belo Horizonte, auxiliando no diagnóstico precoce da infecção e na diminuição das taxas de morbidade e mortalidade da doença. A avaliação do perfil epizootiológico de 90 cães revelou que a doença é mais frequente em animais adultos (um a seis anos de idade) e que não receberam vacinas conforme recomendado pelos protocolos. Os sinais clínicos extraneurais e neurais foram variados, com predomínio para manifestações gastrentérica e respiratória, mioclonia e déficit motor, respectivamente. O exame do fluido cérebro-espinhal demonstrou predomínio de proteinorraquia associada à pleocitose linfocítica. O teste de imunocromatografia para pesquisa de antígeno com amostras do fluido cerebroespinhal foi eficaz para identificar a doença em pacientes com sinais neurológicos, diferentemente das amostras do swab conjuntival, que não devem ser utilizadas.(AU)

The epizootiology profile of canine distemper in Belo Horizonte is outdated and does not harbor some important characteristics. A recent analysis of the virus distribution in relation to host and environmental characteristics associated with the main clinical signs and laboratory findings are important for adopting strategic measures to control the disease. The aim of this study was to determine the epizootiology characteristics of canine distemper virus infection associated with a variety of clinical and neurologic signs and laboratory findings in Belo Horizonte, helping to detect early infection and reduce morbidity and mortality rates. The evaluation of the epizootiology profile of 90 dogs revealed that the disease is more frequent in adult animals (1-6 years of age) and did not receive vaccines as recommended by the protocols. Extra neural and neural clinical signs were varied, with predominance for gastrointestinal and respiratory manifestations and myoclonus and motor deficit, respectively. Examination of the cerebrospinal fluid of 16 dogs showed a predominance of increase protein associated with lymphocytic pleocytosis. The immunochromatography test for antigen screening with samples of cerebrospinal fluid in 76 animals with neurological signs was effective in identifying the disease, unlike conjunctival swab samples, which should not be used.(AU)

Neuron-specific enolase as biomarker for possible neuronal damage in dogs with distemper vírus / Enolase neuronal específica como um possível biomarcador para lesão neuronal em cães com cinomose

Neuron-specific enolase (NSE) is a biomarker of neuronal cell lysis, which demonstrates stability in extracellular fluids such as blood and cerebrospinal fluid. To the authors knowledge there is no research information comparing the use of NSE in dogs with and without encephalitis, putting in evidence the importance of that biomarker to detect neuronal damage in dogs. The objective was to compare the serum NSE levels in dogs with and without encephalitis, and to determine the serum NSE levels in normal dogs. Thirty eight dogs were evaluated, 19 dogs with encephalitis (EG Group) and 19 dogs without encephalitis (CG Group). The criteria for inclusion in the EG Group were presence of neurological signs in more than one part of the CNS (multifocal syndrome) and positive molecular diagnosis for canine distemper virus; for the CG Group were an age between 1 to 7 years and be clinically normal; NSE were measured in serum using an ELISA assay, and the results were compared. In the EG Group the NSE values were higher with significant difference (P=0.0053) when compared with the CG Group. NSE is a biomarker that can be measured in serum samples of dogs to monitor neuronal lesions in encephalitis.(AU)

Enolase neuronal específica (NSE) é um biomarcador de lise de neurônios, que demonstra estabilidade em fluidos extracelulares como sangue e líquido cerebrospinal. Para o conhecimento dos autores, não há informações de pesquisa que comparem o uso de NSE em cães com e sem encefalite, evidenciando a importância desse biomarcador para detectar danos neuronais em cães. O objetivo foi comparar os níveis séricos de NSE em cães com e sem encefalites, e determinar os níveis séricos de NSE em cães saudáveis. Trinta e oito cães foram avaliados, 19 cães com encefalites (Grupo EG) e 19 cães sem encefalite (Grupo CG). O critério para inclusão no Grupo EG foi presença de sinais neurológicos em mais de uma estrutura do SNC (síndrome multifocal) e positividade no diagnóstico molecular para o vírus da cinomose canina; para o Grupo CG foi idade entre 1 e 7 anos e ser clinicamente normal; NSE foram mensuradas em amostras séricas usando o método de ELISA, e os resultados comparados. No Grupo EG os valores de NSE foram altos com diferença significativa (P=0.0053) quando comparado com o Grupo CG. NSE é um biomarcador que pode ser mensurado em amostras séricas de cães para monitorar lesões neuronais em encefalites.(AU)

Antigenic and immunogenic properties of the canine distemper virus nucleocapsid protein expressed in Escherichia coli employing codon optimized synthetic gene / Propriedades antigênicas e imunogênicas da proteína do nucleocapsídeo do virus da cinomose canina expressa em Escherichia coli empregando gene sintético com codons otimizados

Despite common occurrence and importance of canine distemper disease the majority of tests currently available for diagnosis are hampered by either low sensitivity or specificity. In this study it was evaluated antigenic and immunogenic characteristics of a conserved region of nucleocapsid protein of canine distemper virus (rCDV NP) expressed in Escherichia coli employing a codon optimized synthetic gene. The expression of rCDVNP in Star strain (mean 300μg/mL, purified) was confirmed by SDS-PAGE and Western blot analysis by using His-Tag monoclonal antibodies. Western blot and ELISA, employing positive and negative control dog sera, demonstrated the rCDVNP antigenicity. The rCDVNP was inoculated in hens and immunoglobulin Y (IgY) was purified from the egg yolk. The mean yield of IgY was 28.55mg/mL. IgY reacted with the recombinant protein as demonstrated by Western blot and ELISA assays. In summary, our findings demonstrated that rCDVNP is antigenic since CDV positive dog sera recognized the protein in vitro. Additionally, the rCDVNP proved to be immunogenic in hens being possible to isolate a high concentration of specific IgY antibodies from the egg yolk. Taken together, these results indicate that the rCDVNP along with the specific IgY could be useful tools for development of the canine distemper immunodiagnostic assays.(AU)

Apesar da ocorrência comum e importância da cinomose canina, a maioria dos testes atualmente disponíveis para diagnóstico são prejudicados pela baixa sensibilidade ou especificidade. Neste estudo foram avaliadas características antigênicas e imunogênicas de uma região conservada da proteína do nucleocapsídeo do virus da cinomose canina (rCDV NP) expressa em Escherichia coli empregando um gene sintético e codons otimizados. A expressão na cepa Star (média de 300μg/mL, purificada) foi confirmada por SDS-PAGE e Western blot utilizando anticorpos monoclonais anti-His-Tag. A antigenicidade da rCDVNP foi demonstrada por western blot e ELISA empregando soros de cães positivos e negativos. A rCDVNP foi inoculada em galinhas e imunoglobulina Y (gY) foi obtida e purificada a partir da gema. A produção média de IgY foi 28.55mg/mL. Anticorpos IgY reagiram com a proteína recombinante, quando analisados por Western blot e ELISA. Em resumo, nossos achados demonstram que a rCDVNP produzida é antigênica, uma vez que os anticorpos de soro de cães positivos para CDV reconheceram a proteína in vitro. Além disso, a rCDVNP foi imunogênica em galinhas, sendo possível isolar anticorpos IgY específicos a partir da gema do ovo em altas concentrações. Tomados em conjunto, estes resultados indicam que a rCDVNP juntamente com a IgY específica podem ser ferramentas úteis para elaborar ensaios de imunodiagnóstico de cinomose canina.(AU)

Detection of viral infections in wild Korean raccoon dogs (Nyctereutes procyonoides koreensis)

Wild raccoon dogs (Nyctereutes procyonoides koreensis) may play a role transmitting several pathogens to humans and pet animals. Information concerning the incidence of rabies, canine distemper virus (CDV), canine parvovirus (CPV), canine adenovirus type 2 (CAdV-2), canine parainfluenza virus type 5 (CPIV-5), and canine herpesvirus (CHV) is needed in wild raccoon dogs. In total, 62 brain samples of raccoon dogs were examined for rabies virus (RABV) and CDV, and 49 lung samples were screened for CDV, CAdV-2, CPIV-5, and CHV. No RABV, CAdV-2, CPIV-5, or CHV was identified, but nine CDV antigens (8.1%, 9/111) were detected. Moreover, 174 serum samples from wild raccoon dogs were screened for antibodies against the five major viral pathogens. The overall serosurveillance against CDV, CPV, CAdV-2, CPIV-5, and CHV in wild raccoon dogs was 60.3%, 52.9%, 59.8%, 23.6%, and 10.3%, respectively. Comparisons of the sero-surveillance of the five pathogens showed that raccoon dogs of Gyeonggi province have slightly higher sero-positive rates against CDV, CPV, and CHV than those of Gangwon province. These results indicate high incidences of CDV, CPV, and CAdV-2 in wild raccoon dogs of two Korean provinces and a latent risk of pathogen transmission to companion and domestic animals.

Genetic characterization of the haemagglutinin gene in canine distemper virus strains from naturally infected dogs in Brazil / Caracterização genética do gene da hemaglutinina em vírus da cinomose canina de cães naturalmente infectados no Brasil

Canine distemper is one of the major infectious diseases in dogs and wild animals, resulting in high morbidity and mortality. The H gene has the greatest genetic variability among the genes encoded by the canine distemper virus (CDV) genome, and it has been used to characterise field samples, allowing the identification of specific lineages. Variation in the H gene can allow the virus to evade recognition by vaccine-induced antibodies, resulting in vaccine failure. The purpose of this study was to characterise H gene in CDV strains from naturally infected dogs in the state of São Paulo. The phylogenetic analysis revealed that Brazilian CDV strains were genetically related to the circulating CDV strains in Uruguay, Argentina, and Europe. We found no evidence of South America 2 and 3 CDV lineages circulating in Brazilian dogs. The degree of genetic divergence between wild Brazilian CDV strains and vaccine strains may suggest the possibility of vaccine failures and consequently the occurrence of canine distemper outbreaks.(AU)

A cinomose canina é uma das principais doenças infecciosas em cães e animais selvagens, resultando em alta morbidade e mortalidade. O gene H tem uma das maiores variabilidades genéticas entre os genes codificados pelo vírus da cinomose canina (CDV), e tem sido utilizado para caracterizar as estirpes de CDV, permitindo a identificação de linhagens específicas. A variação no gene H pode permitir que o vírus evite o reconhecimento por anticorpos induzidos pela vacina, resultando em falha vacinal. O objetivo deste estudo foi caracterizar o gene H em estirpes de CDV de cães infectados naturalmente no estado de São Paulo. A análise filogenética revelou que as estirpes de CDV brasileiras estão geneticamente relacionadas as estirpes circulantes no Uruguai, na Argentina e na Europa. Não foi encontrada nenhuma evidência da circulação no estado de São Paulo das linhagens América do Sul 2 e 3. O grau de divergência genética entre linhagens selvagens de CDV brasileiras e as estirpes vacinais podem sugerir a possibilidade de falhas vacinais e consequentemente a ocorrência de surtos de cinomose canina.(AU)

Establishment of canine kidney cell line for canine distemper virus replication / 동물의과학연구지

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin-Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.

Detection of canine respiratory coronavirus from dogs with respiratory disease / 동물의과학연구지

Canine respiratory coronavirus (CRCoV) is commonly associated with canine kennel cough worldwide. Clinically infected dogs present coughing, sneezing, and nasal discharge. Severe infections may progress to pneumonia. Through serological surveys, CRCoV has been identified as a worldwide pathogen found in the respiratory tracts of dogs suffering from mild or severe respiratory disease. In this study, three dogs were obtained from a dog kennel. Over the previous 5 days, the dogs showed coughing, sneezing, and nasal discharge. To detect the etiologic pathogen, we performed multiplex RT-PCR (mRT-PCR) to amplify the genes encoding canine influenza virus matrix protein, canine distemper virus nucleocapsid protein, and CRCoV spike protein. Dot blotting was achieved with a CRCoV-specific probe. Nasal-secreting CRCoV was detected by the 442 bp CRCoV-positive PCR reaction in the nasal swabbing samples from dogs. Further, CRCoV-positive reactions by dot blot hybridization were detected in the nasal swabbing samples from dogs. In conclusion, we detected CRCoV in kenneled dogs with respiratory disease in Korea. Multiplex RT-PCR was able to detect successfully CRCoV infection in dogs. We suggest that mRT-PCR would be useful and effective for monitoring CRCoV infection in various kinds of dogs.

Efeitos colaterais do uso da ribavirina, prednisona e DMSO em cães naturalmente infectados pelo vírus da cinomose / The side effects of ribavirin, prednisone and DMSO in dogs naturally infected by canine distemper virus

O estudo tem o objetivo de identificar efeitos indesejáveis da ribavirina, prednisona e DMSO em cães naturalmente infectados com o vírus da cinomose. Foram utilizados 60 cães apresentando quadro neurológico da cinomose com evolução de 10 dias. Os animais foram internados e receberam tratamento de suporte; foram avaliados diariamente e realizados hemograma, dosagem bioquímica e exame de urina tipo I. Os grupos 1 e 2 foram tratados com ribavirina e sua associação com DMSO; os grupos 3 e 4 com DMSO e prednisona e o grupos 5 com ribavirina e prednisona e o grupo 6 com ribavirina, prednisona e DMSO. Os animais foram anestesiados para a colheita de líquor, medula óssea e sangue, antes do tratamento para diagnóstico através da RT-PCR. As amostras negativas foram analisadas pela técnica de hn-PCR. Todos os animais apresentaram resultado positivo em pelo menos uma das duas reações. O efeito adverso da ribavirina e a sua associação com a prednisona foi a anemia hemolítica, que foi confirmada pela observação de bilirrubina na urina apenas dos cães tratados com ribavirina.

The present study aims at the identification of undesirable effects of ribavirin, predinisone and DMSO in dogs naturally infected by canine distemper virus. The research analyzed 60 dogs with clinical neurological signs and 10 days of evolution. The animals were hospitalized for the appropriate support treatment; were daily observed, and complete blood cells count, biochemical analysis, and urine exam type I were conducted. Groups 1 and 2 were treated with ribavirin and its combination with DMSO; Groups 3 and 4 treated with prednisone and DMSO, Group 5 treated with ribavirin and prednisone, while Group 6 with ribavirin, prednisone and DMSO. Before the treatment, animals were anesthetized for the cerebrospinal fluid, bone marrow and blood samples collection for the diagnosis based on RT-PCR. The negative samples were analyzed using the hn-PCR technique. All the animals presented positive results in at least one of the 2 tests. The adverse result of ribavirin and its association with prednisone was characterized by haemolytic anemia, confirmed by the evaluation of bilirrubin occurrence only in the urine of dogs treated with ribavirin.

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

Canine distemper virus infection in a lesser grison (Galictis cuja): first report and virus phylogeny / Infecção pelo vírus da cinomose canina em um furão (Galictis cuja): primeiro relato e filogenia viral

Infectious diseases in wild animals have been increasing as a result of their habitat alterations and closer contact with domestic animals. Canine distemper virus (CDV) has been reported in several species of wild carnivores, presenting a threat to wildlife conservation. We described the first case of canine distemper virus infection in lesser grison (Galictis cuja). A free-ranging individual, with no visible clinical sigs, presented sudden death after one day in captivity. Molecular diagnosis for CDV infection was performed using whole blood collected by postmortem intracardiac puncture, which resulted positive. The virus phylogeny indicated that domestic dogs were the probable source of infection.

Doenças infecciosas em animais selvagens têm aumentado devido às alterações em seu habitat e ao maior contato com animais domésticos. A cinomose já foi descrita em diversas espécies de carnívoros selvagens, representando uma ameaça à conservação da vida selvagem. Nesse estudo é descrito o primeiro caso de infecção pelo vírus da cinomose em um furão (Galictis cuja). Um indivíduo de vida livre, sem sinais clínicos aparentes, apresentou morte súbita após um dia em cativeiro. Foi realizado o diagnóstico molecular para detecção do vírus da cinomose canina, sendo o resultado positivo. A filogenia do vírus indicou que cães domésticos foram a provável fonte de infecção.

Serologic Survey of Rabies Virus, Canine Distemper Virus and Parvovirus in Wild Raccoon Dogs (Nyctereutes procyonoides koreensis) in Korea

Oral rabies vaccination (ORV) program for the wild animals in rabies risk regions of Korea has been conducted since 2000. Evaluation of ORV program under field condition and information concerning the incidence of exposure to canine distemper and canine parvovirus (CPV) are needed in wild raccoon dogs (Nyctereutes procyonoides koreensis). Ninety four sera of wild raccoon dogs were screened for antibodies against rabies, canine distemper virus (CDV) and CPV in Korea. The overall prevalence of antibodies against rabies virus (RABV), CDV and CPV in wild raccoon dogs was 35.1%, 89.4% and 24.5%, respectively. Comparisons of sero-prevalences of RABV, CDV and CPV were assayed in two regions (Gyeonggi-do and Gangwon-do). The Gyeonggi-do (36.4%) showed higher sero-positive rate against CPV than Gangwon-do (20.8%). In contrast, Gangwon-do (41.7% and 97.2%) showed higher sero-positive rates against RABV and CDV than Gyeonggi-do (13.6% and 63.6%). These results indicate that there was severe circulation of CDV and CPV among wild raccoon dogs in the two regions of Korea. Furthermore, raccoon dogs showing a protective antibody titer (0.5 IU/ml) were 15.9%, suggesting that new rabies control program such as trap-vaccination-release (TVR) should be launched urgently in rabies risk regions.

Detecção molecular e análise filogenética do gene H de amostras do vírus da cinomose canina em circulação no município de Campinas, São Paulo / Molecular detection and phylogenetic analysis of the gene H from canine distemper virus isolates circulating at the municipality of Campinas, São Paulo

O vírus da cinomose canina (CDV), um Morbillivirus da família Paramyxoviridae, é o agente etiológico de doença neurológica e sistêmica em cães. O diagnóstico laboratorial da infecção requer o isolamento viral ou detecção do material genético do vírus em secreções ou tecidos de cães com suspeita clínica da doença. A diversidade genética entre os isolados de CDV pode ser aferida pelo sequenciamento efilogenia molecular do gene que codifica a hemaglutinina viral (gene H), havendo atualmente um especial interesse em comparar as amostras circulantes a campo com o genogrupo América-1, que abrange as cepas presentes nas vacinas disponíveis no mercado. No presente estudo, foi realizada a detecção molecular do gene H de CDV a partir de amostras biológicas colhidas ante- e post- -mortem de 15 cães com sinais clínicos sugestivos de cinomose na região metropolitana de Campinas, São Paulo. Dez dos 15 cães analisados tiveram ao menos um órgão positivo na detecção molecular e os amplicons obtidos foram submetidos ao sequenciamento nucleotídico seguido de análise filogenética molecular. De forma semelhante ao que já foi reportado para estudo analisando a diversidade do gene H em outros países, a reconstrução filogenética obtida para as amostras de casos de cinomose da região de Campinas demonstrou as mesmas foram agrupadas junto a amostras norte-americanas, europeias e japonesas recentes, em um grupo genético distinto do grupo de amostras clássicas de CDV, nomeado America-1, o qual engloba as estirpes vacinais Snyder Hill, Onderstepoort e Lederle.

Canine distemper virus (CDV), a Morbillivirus of the family Paramyxoviridae, is the etiological agent of neurological and systemic disease in dogs. The laboratory diagnosis of infection requires viral isolation or detection of genetic material of the virus in secretions or tissues of dogs with clinical suspicion of the disease. The genetic diversity among isolates of CDV can be assessed by sequencing and phylogenetic analysis of the gene that encodes the viral hemagglutinin (H gene), and there is currently a special interest in comparing the strains currently circulating in the field with the genogroup America-1, which comprises strains present in vaccines available in the market. In this study, the molecular detection of CDV gene H was performed from biological samples harvested ante-and post-mortem from 15 dogs with clinical signs suggestive of canine distemper in the metropolitan region of Campinas, São Paulo. Ten of the 15 dogs examined had at least one positive organ under molecular detection and the obtained amplicons were sequenced and further analyzed by molecular phylogenetic analysis. Similarly to what has already been reported on previous studies regarding the diversity of the gene H in other countries, the phylogenetic reconstruction obtained for the samples of cases of distemper from Campinas region showed they were grouped with the North American, European and Japanese newly described samples, a genetic group distinguished from classical samples of CDV, named America-1, which encompasses the vaccine strains Snyder Hill, Onderstepoort and Lederle.

Atypical necrotizing encephalitis associated with systemic canine distemper virus infection in pups

This report describes the naturally occurring atypical neuropathological manifestation of systemic canine distemper virus (CDV) infection in two 16-day-old Pit Bull pups. CDV-induced changes affected the gray and white matter of the forebrain while sparing the hindbrain. Histologically, there was necrosis with destruction of the nervous parenchyma due to an influx of inflammatory and reactive cells associated with eosinophilic intranuclear inclusion bodies within glial cells. Positive immunoreactivity against CDV antigens was predominantly observed within astrocytes and neurons. RT-PCR was used to amplify CDV-specific amplicons from brain fragments. These findings suggest the participation of CDV in the etiopathogenesis of these lesions.

Detecção do vírus da cinomose canina em cães assintomáticos e não vacinados / Canine distemper virus detection in asymptomatic and non vaccinated dogs

A quantitative real time polymerase chain reaction (PCR) revealed canine distemper virus presence in peripheral blood samples from asymptomatic and non vaccinated dogs. Samples from eleven domestic dogs with no signs of canine distemper and not vaccinated at the month of collection were used. Canine distemper virus vaccine samples in VERO cells were used as positive controls. RNA was isolated with Trizol®, and treated with a TURBO DNA-free kit. Primers were designed for canine distemper virus nucleocapsid protein coding region fragment amplification (84 bp). Canine b-actin (93 bp) was utilized as the endogenous control for normalization. Quantitative results of real time PCR generated by ABI Prism 7000 SDS Software showed that 54.5 percent of dogs with asymptomatic canine distemper were positive for canine distemper virus. Dissociation curves confirmed the specificity of the real time PCR fragments. This technique could detect even a few copies of viral RNA and identificate subclinically infected dogs providing accurate diagnosis of this disease at an early stage.

A reação em cadeia da polimerase (PCR) em tempo real revelou a presença do vírus da cinomose canina em amostra de sangue de cães assintomáticos e não vacinados. Amostra de onze cães domésticos sem nenhum sinal clínico de cinomose e que não foram vacinados no mês da coleta de sangue foram utilizados para análise. Amostra vacinal do vírus da cinomose canina em células VERO foi utilizada como controle positivo. O RNA total foi isolado utilizando-se Trizol®, e tratadas com o Kit TURBO DNA-free. Os iniciadores foram desenhados para amplificar a região do nucleocapsídeo viral com 319pb e 84pb para a PCR convencional e PCR em tempo real, respectivamente. O fragmento alvo da b-actina canina com 93pb foi utilizado como controle endógeno e normalizador. Resultados quantitativos da PCR em tempo real gerados pelo programa ABI Prism 7000 SDS demonstraram que 54,5 por cento dos cães assintomáticos foram positivos para o vírus da cinomose canina. As curvas de dissociação confirmaram a especificidade dos fragmentos da PCR em tempo real. A detecção precoce do RNA viral é importante para a identificação de cães subclinicamente infectados e limitar a difusão da doença.

possible relationship of megaesophagus and canine distemper in two German shepherd dogs

Megaesophagus [esophageal dilatation] is a neuromuscular disease. In this disorder, the esophagus is abnormally stretched and air is collected with food in it. Two male dogs aged 4 and 6-year-old, German shepherd breeds and weighing 18.4 and 23.2 kg were presented to the Veterinary Hospital of Shahid Chamran University with complaints of regurgitation, dehydration, anorexia, depression, dysphagia, myoclonus, hypersalivation, gurgling sound during swallowing and fatigue in the past month. They were not vaccinated against distemper and other viral diseases. Physical examination revealed a relatively thin body condition. A plain radiograph of the chest revealed advanced megaesophagus with aspiration pneumonia. Rapid antigenic test [immunochromatography assay] was positive for canine distemper. CBC was abnormal [leucopenia, lymphopenia and neutropenia]. Supportive treatments were administered for them. We advised the dogs be placed in a vertical position for 15 min after the meal. Only one dog was sent back home in good health four weeks later and the disease did not relapse for more than six months. The second dog had a symptom of megaesophagus and did not respond to supportive treatments completely. A possible relationship between megaesophagus and canine distemper infection has been reported in the present survey

Alterações citológicas do sangue periférico e da medula óssea de cães com cinomose / Cytological alterations of the bone marrow and peripheral blood of dogs with canine distemper

Avaliaram-se o mielograma, o hemograma e a ocorrência de apoptose no sangue periférico e na medula óssea de cães com cinomose de ocorrência natural. Foram utilizados 15 cães distribuídos em dois grupos: (a) controle - seis animais clinicamente saudáveis com RT-PCR negativa para o vírus da cinomose canina (CC); (b) infectado - nove animais com manifestações clínicas de CC e RT-PCR positiva. Dos cães com CC, oito (88,9 por cento) apresentaram anemia discreta a moderada (hematócrito: 30,6 por cento), normocítica (VCM: 67,9fL) e normocrômica (CHCM: 34,1g/dL). Todos os animais apresentaram contagens médias normais de leucócitos totais (11600 células/µL) e neutrófilos segmentados (8802 células/µL). Linfopenia foi observada em cinco animais (55,6 por cento) e desvio nuclear dos neutrófilos para a esquerda em oito (88,9 por cento). As contagens médias de linfócitos e neutrófilos bastonetes foram, respectivamente, 1054 e 1508células/µL. No mielograma, todos os animais apresentaram celularidade e relação M:E dentro dos limites de referência. O hemograma e a medula óssea dos cães-controle não apresentaram alteração e não havia células em apoptose no esfregaço sanguíneo desses animais. Nos cães com CC, a média do índice apoptótico foi 0,73 por cento no esfregaço sanguíneo e 1,87 por cento na medula óssea. A apoptose, portanto, pode estar envolvida na patogênese das alterações hematológicas observadas na CC.

The myelogram, the hemogram, and the occurrence of apoptosis in peripheral blood and bone marrow in dogs with canine distemper (CD) of natural occurrence were studied. Fifteen dogs were distributed into two groups: (a) control - six clinically healthy animals with RT-PCR negative for canine distemper virus (CDV); and (b) infected - nine animals showing clinical CD manifestations and RT-PCR positive. The majority of dogs with CD (88.9 percent) presented discrete to moderate (hematocrit: 30.6 percent), normocytic (MCH: 67.9fL) and normochromic (MCHC: 34.1g/dL) anemia. All animals showed total leukocytes counting (11,600 cells/µL) and segmented neutrophils (8,802 cells/µL) within the limits of reference. Lymphopenia and left shift neutrophils were observed in 55.6 percent and 88.9 percent of the dogs, respectively. Additionally, the average counts of lymphocytes and neutrophils were 1,054 and 1,508cells/µL, respectively. The myelogram of all animals presented cellularity and M:E relation within the limits of reference. Haemogram and bone marrow of the control dogs had no alteration. Moreover, no apoptotic cells were detected in the smear of the peripheral blood of control animals. On the other side, dogs with CD presented a higher apoptotic index (AI), both in the peripheral blood (AI: 0.73 percent) and in the bone marrow (AI: 1.87 percent). Therefore, apoptosis may contribute to hematological changes observed in CD.