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Development, improvement and application of existing diagnostic technologies for FMD - SE1123

Description
Foot-and-mouth disease (FMD) remains economically the most important disease that affects susceptible farm livestock. The threat of an introduction into the UK is ever present as the disease remains endemic in many countries in the Middle East, Africa and Asia. FMD is highly contagious and has the potential for rapid spread, as was graphically illustrated during the epidemic which occurred in the UK in 2001 when there were 2030 designated cases of disease. Consequently, rapid and accurate diagnosis of FMD is required for the implementation of effective control measures in the event of an outbreak. Clinical disease caused by both swine vesicular disease and vesicular stomatitis is indestinguishable from that of FMD and this necessitates the availability of test procedures to differentiate these vesicular viruses.

Diagnosis of disease is principally dependent on the detection of virus, antigen or genome. However, in circumstances where either vaccination is not employed or where there has been no evidence of previous infection, tests for the detection of virus antibody can also be used. In addition, serology is also needed for import/export testing to demonstrate freedom from infection and in disease surveillance studies, while antibody quantification is important in assessing the efficacy of vaccination campaigns. Diagnostic test procedures need to be highly sensitive, specific and swift and must be continuously assessed and refined to ensure their optimal performance in the face of evolution of pathogens and to incorporate technical improvements in related fields of virology.

The recognition and serotyping of antigen and infectious virus is currently undertaken in the laboratory by an indirect sandwich ELISA and virus isolation in sensitive cell cultures while the detection of viral genome is done by molecular assays and principally by RT-PCR procedures. The development of the latter molecular assays for the diagnosis of FMD and other vesicular diseases is the subject of a separate Defra project, SE1121. The existing, routinely employed antigen ELISA employs type-specific polyclonal antisera as both capture and detecting antibodies. An improvement in assay specificity has already been demonstrated in principle by using a recombinant integrin protein, which has been demonstrated to be pan-reactive to FMD viruses (FMDVs) of all serotypes, as the capture ligand in combination with type-specific monoclonal antibodies (mabs) as the detectors. FMDV mabs will continue to be examined for their spectrum of reaction against FMDVs of all serotypes and subtypes and those exhibiting the desired characteristics of virus type-specificity but broad intra-typic reactivity will be selected to establish an improved antigen ELISA. Lateral flow devices incorporating mabs and/or other ligands will continue to be evaluated for pen-side vesicular virus antigen detection and as a means of support to veterinary clinical judgement of disease outbreaks.

Primary calf thyroid cells and a pig kidney cell line (IB-RS-2) are currently used for vesicular virus isolation studies. Although these cells are highly sensitive, the former suffer from the continual need to source thyroid glands to produce fresh cell culture suspensions, which is difficult, expensive and labour intensive, while the latter are permanently infected with classical swine fever virus. Alternative candidate cell cultures will be sourced and new cell lines will be produced by integrin transfection or immortalised by oncogene transfection or by using the enzyme telomerase. All the cell lines that are either acquired or generated will be screened for their susceptibility to FMD and other vesicular virus replication. The outcome may either identify existing, alternative cell lines for use or establish new cell lines of suitable sensitivity for vesicular virus diagnosis.

Validation of the solid phase competition ELISA (SPCE) for the detection of FMDV antibodies at a single serum dilution is currently being extended to all seven FMDV serotypes. Improvements in standardisation and simplification of the SPCE may arise from novel approaches using FMDV type-specific or pan-reactive mabs instead of polyclonal guinea pig antibodies as the competing reagent, pan-reactive recombinant integrin as the capture ligand instead of type-specific polyclonal rabbit antibodies and recombinant antigen instead of inactivated cell culture grown antigens of each FMDV serotype.
Objective
Objective 1 : Establish an indirect sandwich ELISA for routine FMDV antigen detection using integrin ávâ6 recombinant protein as the capture ligand and FMD mabs as detectors

Objective 2 : Further evaluate prototype lateral flow devices for FMD, SVD and VS diagnosis

Objective 3 : Develop novel immunoassays for FMD serology using polyclonal and monoclonal antibodies; recombinant integrin ávâ6; and recombinant antigens

Objective 4 : Source suitable candidate cell lines for vesicular virus diagnosis and generate new cell lines by integrin transfection or alternative immortalisation techniques and screen for their sensitivity to vesicular virus replication
Project Documents
• Final Report : Development, improvement and application of existing diagnostic technologies for FMD   (339k)
Time-Scale and Cost
From: 2006

To: 2009

Cost: £355,942
Contractor / Funded Organisations
Institute for Animal Health (BBSRC)
Keywords
Animal Diseases              
Animal Health              
Biotechnology              
Diagnosis              
Foot and Mouth              
GM Non-Food              
Plants and Animals              
Fields of Study
Animal Health