Investigação epidemiológica de estomatite vesicular por achados clínicos em bovinos e equinos no Estado do Maranhão / Stomatitis vesicular in cattle and horse in the Maranhão state

A Estomatite Vesicular (EV) é uma doença infecciosa que acomete equinos, bovinos, suínos, mamíferos silvestres e humanos. Por apresentar sinais clínicos semelhantes a outras doenças vesiculares, principalmente, febre aftosa, sua presença em determinadas regiões pode interferir no intercâmbio comercial internacional dos animais, seus produtos e subprodutos. Apesar de sua importância, a epidemiologia e a manutenção do vírus no ambiente não estão totalmente esclarecidas dificultando a aplicação de medidas de controle efetivas. A doença já foi diagnosticada em todas as regiões brasileiras. Bovinos com sialorréia, perda do epitélio lingual, lesões abertas com bordas amareladas nas gengivas, lábios, língua e mucosa oral e equinos com sialorréia e lesões abertas na mucosa oral e lábios foram observados e notificados ao Serviço Veterinário Oficial do Estado do Maranhão, Agência Estadual de Defesa Agropecuária do Maranhão (AGRD/MA). Amostras de soro de equinos e bovinos com sintomas de EV foram coletadas para investigação por ELISA e por neutralização viral, além do diagnóstico diferencial para Febre Aftosa (FA). Fragmentos epiteliais de bovinos com lesões na língua foram coletados para identificação molecular do agente. Todos os animais foram negativos para FA. Todos os bovinos e equinos foram reativos para EV nos testes sorológicos. A partir dos fragmentos epiteliais de bovinos enviados ao Instituto Biológico de São Paulo para PCR, foi possível caracterizar o agente como VesiculovirusIndiana III (Alagoas/VSAV).

Vesicular stomatitis (VS) is an infectious viral disease that affects bovines, equines, swine, wild animals and humans. As it is indistinguishable from other vesicular diseases, mainly Foot and Mouth Disease (FMD), it causes restrictions in commercial livestock trade at national and international levels and also significant economic losses. As the epidemiology and maintenance of VS virus in nature are not clearly understood it is difficult to take effective control measures. VS was diagnosed in some regions of Brazil, such as Minas Gerais, Santa Catarina, São Paulo and Alagoas. Cattle and horses with clinical symptoms of drooling, shedding of the lingual epithelium, presence of vesicles on the oral mucosa were observed and reported to the National Animal Health Office health of Maranhão State, Brazil. Samples of serum of these animals were collected and sent to Laboratório Nacional de Agropecuaria for ELISA and virus neutralization and differential diagnosis for Foot and Mouth Disease (FMD). The results of ELISA confirmed the VS. In the differential diagnosis, the results were negative for FMD. Samples of bovine epithelial tissue for VS by PCR confirmation of diagnosis were collected and sent to Biological Institute of São Paulo. Molecular results confirmed the VesiculovirusIndiana III (Alagoas/VSAV) infection.

Expressing of N gene encoding nucleocapsid protein of vesicular stomatitis virus and elementary application in ELISA / 生物工程学报

The gene encoding the nucleocapsid (N) protein of vesicular stomatitis virus (VSV-NJ) was subcloned from pMD-VN5, and inserted into pBAD/Thio TOPO vector. The recombinant plasmid was identified by restriction analysis and PCR. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted N gene. The results of SDS-PAGE and Western immunoblotting revealed that the N protein was expressed in Escherichia coli LGM194 in a high level and the recombinant fusion protein, which contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. It had a molecular mass of approximately 63.5 kD and immunologically reactive activity. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of vesicular stomatitis using 186 serum samples from experimentally infected goats and guinea-pigs with VSV-NJ and VSV-IN, and from field origin and reference serum samples. The sensitivity and specificity of the ELISA were compared with those of the standard microtiter serum neutralization (MTSN) tests. The ELISA and MTSN test results were highly correlated for detection of VSV antibodies. The ELISA was as sensitive as the SN assay in detecting positive serum to VSV. The correlation between SN titers and ELISA titers was statistically significant. These data suggest that the recombinant fusion N protein of VSV could be used as a recombinant test antigen for the serodiagnosis of vesicular stomatitis. The ELISA based on the reconmbinant nucleocapsid protein may offer the best combination of rapidity, sensitivity, simplicity, economy, and laboratory biosafety of any of the methods yet developed for VSV serodiagnosis. This study lay on foundation for the development of the diagnosis methods in serology for VSV.