qRT-PCR to detect FMD virus

Summary

Of all exotic diseases, foot and mouth disease (FMD) presents the greatest threat to Australia's livestock industries.  An outbreak of FMD would have a severe impact through loss of both productivity and exports. This virus can be difficult to detect in sheep and there has been significant evidence of dispersal of FMD virus (FMDV) through the movement of undetected, infected sheep. Consequently, there is a need for a laboratory based method for rapid testing to support large scale surveillance. Recent research has shown that FMDV can be detected in oral fluids/oral swabs for lengthy periods using virus isolation/culture indicating that there is merit in using oral swabs for FMDV surveillance because of the ease of sample collection and capacity to detect virus for longer than from blood. Virus isolation is, however, labour intensive, expensive and relatively slow and amplifies infectious virus, requiring expensive containment laboratory facilities. In contrast, quantitative 'real time' reverse transcription polymerase chain reaction (qRT-PCR) technology is very sensitive and allows rapid detection of viral nucleic acid (RNA) without amplifying infectious virus.

A combination of qRT-PCR assays to detect FMDV RNA and collection of oral swabs would provide an efficient option for large scale FMDV surveillance – either during an outbreak or during 'proof of freedom' testing. Since such an approach would be of considerable value in Australia, the McGarvie Smith Institute and NSW Department of Primary Industries, in partnership with MDC, provided funds to undertake such a study. A large collection of plasma and oral swab samples from experimentally infected sheep was available at the Central Veterinary Institute, Lelystad, The Netherlands and were offered for use in this research. These were invaluable because the time of infection and stages of infection were accurately defined and virus isolation, a 'gold standard against which to evaluate the high throughput qRT-PCR assay, had been completed on each sample. The management of the German Federal Reference Laboratories at the Friedrich Loeffler Institute (FLI), Greifswald agreed to host Australian scientists to undertake this study.

Objectives

The objectives of this project were to:Confirm the capacity of qRT-PCR to detect FMDV in oral swabs from sheep;Define the onset and duration of detection of FMDV in oral fluid and plasma;Estimate the level of pooling of samples that might be achieved without compromising the capacity to detect FMDV in a flock.