Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Distribuição do vírus rábico no sistema nervoso central em ruminantes naturalmente infectados / Distribution of rabies vírus in the central nervous system in naturally infected ruminants

Com o objetivo de identificar a distribuição das lesões do vírus rábico no sistema nervoso central de casos espontâneos de raiva em ruminantes e comparar as técnicas de imunofluorescência direta (IFD), inoculação em camundongos (ICC) e presença de corpúsculos de Negri para o diagnóstico da doença foram analisados materiais proveniente de 48 casos de raiva, incluindo amostras de córtex frontal, temporal, parietal e occipital, hipocampo, tálamo, colículo rostral e caudal, cerebelo, ponte, medula oblonga, núcleo da base e porções da medula cervical, torácica e lombar. De 48 amostras analisadas, todas foram positivas na IFD e na ICC e em 30 (62,5 por cento) foram encontrados corpúsculo de Negri (CN). No entanto, houve diferenças importantes no resultados dos três testes nas diferentes regiões do SNC avaliadas. Nos cortes de córtex cerebral, em 38 bovinos, a presença de corpúsculos de inclusão foi baixa (11 por cento-37 por cento) assim como a positividade para IFD e ICC (60-80 por cento). Pelo contrário, todas as amostras de ponte, tálamo e medula testados foram positivas para IFD e ICC. Em outras regiões do tronco encefálico e também no cerebelo a positividade para ICC e IFD foi de 60 por cento a 96,7 por cento. No cerebelo foi encontrada a maior frequência (88,2 por cento) de corpúsculos de inclusão. Em oito ovinos as provas de ICC e IFD foram positivas em todos os cortes e foram observados corpúsculos de inclusão em três animais. Foram analisados somente dois casos de caprinos encontrando-se corpúsculos de inclusão em um e ambos foram positivos para IFD e ICC. Os resultados obtidos nesse trabalho sugerem que a conduta recomendada pelo Manual Técnico de Controle da Raiva dos Herbívoros (MTCRH) permite o diagnóstico de raiva associando o estudo histológico aos testes de IFD e ICC que incluem cerebelo, tálamo e tronco encefálico que apresentam alta positividade para as provas de IFD e ICC. No entanto, a melhor conduta é a de incluir metade do encéfalo cortado longitudinalmente e amostras de medula. Isto permite examinar por IFD e ICC uma ou mais regiões onde essas provas apresentam maior positividade e, posteriormente, se essas provas fossem negativas, retornar ao material original e examinar outras regiões. Por outro lado, a coleta de amostras dos locais recomendados pela MTCRH, assim com a coleta de metade do encéfalo, podem prejudicar o diagnóstico de outras doenças para o qual é necessário o estudo de todo o encéfalo após a fixação em formaldeído, para constatar a simetria e a distribuição das lesões. Nestes casos, com base nos resultados obtidos neste trabalho, pode ser recomendado para diagnóstico laboratorial de raiva o envio exclusivo de porções da medula cervical, dorsal e lombar, já que as três porções apresentaram 100 por cento de positividade nas provas de IFD e ICC. Além disso, o estudo histológico de todas as porções do cérebro incluídas neste trabalho permitirá complementar o diagnóstico.

With the aim to study the distribution of lesions the rabies virus in spontaneous cases of rabies in ruminants and to determine the efficiency of the direct fluorescent antibody test (DFA), mouse inoculation (MI) and presence of Negri bodies in the diagnosis of the disease, 48 cases of the rabies were examined. Samples of frontal, temporal, parietal and occipital cerebral cortex, hippocampus, thalamus, rostral and caudal colliculi, cerebellum, pons, medulla oblongata, basal nuclei and sections of the cervical, thoracic and lumbar spinal cord were examined. Of the 48 samples examined all were positive on DFA and MI, and in 30 (62.5 percent) Negri bodies were observed. However there were differences in the results of the three tests among different regions of the central nervous system. In the samples of the cerebral cortex in 38 cattle, the frequency of inclusion bodies was low (11-37 percent), and so was the positivity to DFA and MI (60-80 percent). In contrast, all samples of thalamus, pons and spinal cord were positive to DFA and MI. In other regions of the brain stem, positivity to these tests varied between 60 percent and 96.7 percent. On histologic examination, the major frequency of Negri bodies (88.2 percent) was observed in the cerebellum. In eight sheep the DFA and MI tests were positive in all sections of the CNS examined and Negri bodies were found in three sheep. Only two goats were examined; both were positive in DFA and MI tests and in one Negri bodies were found. These results suggest that the recommendations of the Brazilian Technical Manual for Rabies of Herbivores is adequate for rabies diagnosis, because their recommendations include the histologic study and the examination of cerebellum, and sections of the brainstem with high positivity to DFA and MI tests. However, a better recommendation is to send for DFA and MI half of the brain cut longitudinally and samples of the spinal cord, which will permit to examine one or two sections, and if those are negative to get back to the material and examine the rest of the sections. In contrast, to collect samples of the brain or half brain can be inappropriate for the diagnosis of other diseases of the CNS, for which the study of the whole fixed brain is necessary to recognize the symmetry or distribution of lesions. In these situations by the results obtained here, it can be recommended to send different sections of the spinal cord for DIF and MI tests and to fix the whole brain for gross and histologic examinations.