Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Proceedings
Publication Acceptance Date: 9/6/2004
Publication Date: 10/22/2004
Citation: Rodriguez, L.L., Jimenez, C., Herrero, M., Cornish, T.E., Wilson, W.C., Letchworth, G.J., Pauszek, S.J., George, M., Smoliga, G.R. 2004. Validation of a real-time rt-pcr for vesicular stomatitis virus. American Association of Veterinary Laboratory Diagnosticians. P. 40
Technical Abstract: Vesicular Stomatitis Virus (VSV) causes vesicular lesions in horses, cattle, and swine. The disease in cattle and swine clinically mimics foot-and-mouth disease. Control strategies for the two diseases differ radically and thus mandate rapid and accurate differentiation. The objective of this study was to validate a real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the diagnosis of VSV. Vesicular lesion samples were obtained from cattle, horses, and swine throughout Central America and shipped to the Laboratorio de Diagnostico de Enfermedades Vesiculares (LADIVES) in Panama. Samples from Costa Rica were split and half retained in Costa Rica. A portion of each sample was prepared for virus isolation by maceration in cell culture medium and filtration through a .22 micron spin filter before being inoculated on VERO cells. Cultures that did not have cytopathic effects were passed after three days. Cytopathogenic agents were tested by ELISA with antibodies against VSV-Indiana and New Jersey and the N and P genes were sequenced. A second portion of each lesion sample was prepared for RT-PCR by RNA extraction using a commercial extraction kit and tested by RT-PCR using a mixture of primers and probes. Approximately half of the samples yielded a cytopathogenic agent, most of which were identified as VSV-New Jersey by reaction to serotype specific antibodies, reaction with VSV-NJ primers and probes, and genomic sequence. Most samples from which VSV was isolated also reacted in the RT-PCR. However VSV was not isolated from many RT-PCR positive samples. We conclude that RT-PCR is more sensitive than cell culture for the identification of VSV from field samples, but additional primers and probes will be required to identify all Central American VSVs. An important secondary result of this research was the collection of hundreds of new VSV isolates. This collection encompassed a large portion of VSV cases occurring over a large and diverse geographic region during an entire year. The date of sampling and precise longitude, latitude, elevation, and ecological region are known from each sample. This benchmark collection constitutes a foundation from which many additional studies can arise.