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Application of RT-PCR for the diagnosis of FMD and other vesicular diseases 2 - SE1124

Control of foot-and-mouth (FMD) outbreaks is dependent upon early recognition of infected animals, which requires familiarity with clinical signs of the disease and the ability to accurately and rapidly detect FMD virus (FMDV) in clinical samples using laboratory tests. Of the established diagnostic approaches, virus isolation (VI) in cell culture is considered to be the “gold standard”. This method can be highly sensitive (depending upon the cell culture system used), although it is slow taking between 1-4 days to generate a result. Other assays such as antigen-capture ELISA (Ag-ELISA) are more rapid, but they have lower analytical sensitivity and are inappropriate for use with certain sample types. It is now recognised that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role for the rapid and sensitive detection of FMDV in a wide range of clinical sample types. During previous Defra-funded projects (SE1119 and SE1121), automated real-time RT-PCR (rRT-PCR) assays for the detection of FMDV and other viruses (SVDV and VESV) causing vesicular diseases of livestock have been developed. As part of the validation of these assays, studies to compare the performance of the rRT-PCR with VI and Ag-ELISA have been undertaken and have shown that rRT-PCR is an extremely sensitive and rapid procedure for routine laboratory diagnosis of FMD. These methods have now been adopted by the OIE (included into the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals) and are in routine use in the National Reference Laboratory at IAH-Pirbright for the diagnosis of suspect cases of FMD in the UK. During the recent FMD outbreak (in August 2007), rRT-PCR was used as a front-line diagnostic tool to support the field investigation and control of the disease. In addition to confirming the presence of FMDV on two premises, for the first time we have used this assay to identify cattle with FMDV viraemia prior to the presentation of clinical signs. During this recent outbreak, the majority of samples received from suspect cases were blood samples (mixture of EDTA and serum tubes). Learning from these recent experiences, during this new project we aim to develop optimised and improved protocols using new high through-put robotic equipment to process blood samples that might be received during an outbreak.

Laboratory-based rRT-PCR methods can generate a result within a few hours of receipt of a sample. However, the time taken to transport suspect clinical material to a central laboratory (such as Pirbright) can be lengthy, and this delay can preclude laboratory confirmation. Notably, the implementation of the control strategy adopted by the UK government in the 2001 epizootic: to slaughter the animals on infected premises within 24 hours, meant that in the majority of cases, diagnosis based on clinical signs was not substantiated by laboratory investigation. Retrospective analyses have indicated that reliance upon clinical diagnosis alone resulted in over-reporting of FMD, since the presence of FMDV on 23% of farms designated as infected premises could not be corroborated by rRT-PCR, VI or Ag-ELISA. These factors have given rise to the idea that improvements to the reliability of diagnosis may be achieved by using rapid and sensitive tests that can be used in situ or close to a suspect premises, without transferring the samples to a central laboratory. The use of rapid diagnostic assays was recommended in two major reports arising from the 2001 outbreak (Royal Society Report on Infectious Diseases of Livestock, 2002 & Foot and Mouth Disease 2001: Lessons to be Learned Enquiry, 2002). During SE1121, we evaluated a portable PCR instrument (Bioseeq™; Smiths Detection, Watford) for the detection of FMDV, to assess whether this equipment can potentially be used for field diagnosis of FMD. The results from this preliminary study were encouraging, indicating that it is possible to develop a sensitive rRT-PCR assay for FMDV on the Bioseeq™ platform which can generate a result within 60 minutes. Building upon these results, future work on the Bioseeq™ will address the development of a simple-to-use and robust template extraction protocol, further optimisation of the speed of the assay, and evaluation of the methodology in the field. The recent development of portable equipment for PCR has made molecular diagnosis of FMD in the field an achievable goal. However, this approach relies on precision thermocycling requiring instrumentation which can be fragile, prohibitively expensive and that will require decontamination when transferred from one site to another. As an alternative to PCR, we have also explored the use of isothermal (single temperature) amplification methods for the detection of FMDV. During SE1121, we evaluated a nucleic acid sequence based amplification (NASBA) assay for FMDV. We have also previously developed a loop-mediated amplification (LAMP) assay for FMDV. Since the specific amplification step for both of these assay formats occurs at a constant temperature, there is less reliance upon expensive equipment. Furthermore, the products of the RT-LAMP assay are generated in abundance so that positive LAMP reactions can be visualised with the naked eye. We plan to perform further validation of this technology and (funding permitting from an external collaborator) will design, fabricate and test a disposable device suitable for the field detection of FMDV.

The objectives of this new project are to continue the development and validation of molecular assays suitable for the routine diagnosis of FMD. These methods will underpin the future diagnostic activities of the National Reference Laboratory for FMD and will be included into the contigency plan for the response in the UK to FMD.
Project Documents
• EVID4 - Final project report : SE1124 Final Report evid 4 - Final   (763k)
Time-Scale and Cost
From: 2008

To: 2012

Cost: £884,092
Contractor / Funded Organisations
Institute for Animal Health (BBSRC)
Animal Diseases              
Animal Health              
Foot and Mouth              
Plants and Animals              
Fields of Study
Animal Health