RESUMEN
A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.
Asunto(s)
Enterovirus Humano B/aislamiento & purificación , Virus de la Fiebre Aftosa/aislamiento & purificación , Análisis por Micromatrices/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virus del Exantema Vesicular del Cerdo/aislamiento & purificación , Animales , Bovinos , Enterovirus Humano B/clasificación , Enterovirus Humano B/genética , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/clasificación , Virus de la Fiebre Aftosa/genética , Sensibilidad y Especificidad , Ovinos , Porcinos , Enfermedad Vesicular Porcina/diagnóstico , Enfermedad Vesicular Porcina/virología , Exantema Vesicular del Cerdo/diagnóstico , Exantema Vesicular del Cerdo/virología , Virus del Exantema Vesicular del Cerdo/clasificación , Virus del Exantema Vesicular del Cerdo/genéticaRESUMEN
In this study, we wished to assess the prevalence and determine the sources of three zoonotic bacterial pathogens (Salmonella, Campylobacter, and Escherichia coli O157:H7) in the Salmon River watershed in southwestern British Columbia. Surface water, sewage, and animal faecal samples were collected from the watershed. Selective bacterial culture and PCR techniques were used to isolate these three pathogens and indicator bacteria from these samples and characterize them. Campylobacter was the most prevalent pathogen in all samples, followed by Salmonella, and E. coli O157:H7. E. coli O157:H7 and Salmonella isolation rates from water, as well as faecal coliform densities correlated positively with precipitation, while Campylobacter isolation rates correlated negatively with precipitation. Analysis of DNA extracted from water samples for the presence of Bacteroides host-species markers, and comparisons of C. jejuni flaA-RFLP types and Salmonella serovars from faecal and water samples provided evidence that human sewage and specific domestic and wild animal species were sources of these pathogens; however, in most cases the source could not be determined or more than one source was possible. The frequent isolation of these zoonotic pathogens in the Salmon River highlights the risks to human health associated with intentional and unintentional consumption of untreated surface waters.