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Rapid diagnostic (LFD) and ELISA for detection of non-structural protein antibodies to FMD virus Foot-and-mouth disease (FMD) is a major livestock disease of transboundary importance, affecting cloven-hoofed animals including cattle, pigs, goats and sheep. It is estimated that the annual direct economic loss due to FMD in India is around Rs. 20,000 crores. The Department of Animal Husbandry Dairying and Fisheries, Government of India has initiated FMD control program (FMD-CP), which aims to progressively reduce the incidence of the disease through regular mass vaccination. In situations where a large proportion of the animal population is vaccinated, it is essential to differentiate infected from vaccinated animals (DIVA), to distinguish between antibodies produced by vaccination and those developed naturally as a result of viral infection. Therefore, development of rapid and precise diagnostics facilitating DIVA would be valuable in the sero-surveillance of FMD when active immunization is ongoing. Demonstration of antibodies to non-structural proteins (NSPs) as a result of virus infection forms the basis of using NSP tests as a DIVA tool. Presence of antibodies particularly to 3ABC is a reliable indicator of virus activity in vaccinated or naïve populations. OIE has prescribed ELISA using NSPs such as 3ABC protein, combined with blot assay as index screening system for demonstrating freedom from infection. A in-house recombinant ABC based rapid test and ELISA have been developed at IVRI Bangalore, using 3ABC antigen produced in insect cells (Fig. 1). Indian Veterinary Research Institute, Bangalore Campus has developed two diagnostic kits for FMD using insect cell expressed recombinant protein antigen, 3ABC. Both the tests are rapid, accurate and highly cost effective as compared to similar commercial kits. Both tests have been validated by testing different types of serum samples and blood samples (Rapid test) representing an endemic situation. Serum N P Serum Serum samples P N Blood Serum samples LFD strips showing negative (N) and positive (P) results using serum or blood samples. Rapid test: Rapid tests that don’t need sophisticated instruments and trained man power are value additions to the available tools under field conditions. Pen-side diagnostics in the form of immunochormatographic strip tests or Lateral Flow Device (LFD) have been increasingly adapted as diagnostic tools in human and veterinary medicine. Probably the most popular and well known examples of lateral flow tests are home pregnancy tests. The application of LFDs has extended to other clinical conditions including infectious diseases The lateral flow tests offer greater access, affordability and convenience with wider acceptability for rapid diagnosis. IVRI has developed a LFD (FMD NSP-Antibody Rapid Test) for detection of NSP antibodies to FMDV infection. The test, which uses recombinant protein antigen expressed in insect cells, is validated and its performance has been comparable with ELISA. The test is rapid, sensitive, specific for diagnosis of FMD. The biggest advantage with this LFD test is, it does not require cold chain for transport or storage of the kit, and results can be read with naked eyes without the need of specialized, costly instruments and trained personnel. The test can be performed in less than 10 minutes, at the farm level by the veterinarians and farmers. Field demonstration of the rapid test using blood samples. ELISA plate showing test samples along with positive (PC) and negative controls (NC). BG indicates background. Indirect ELISA: Enzyme labelled antibody based immunoassays provide highly sensitive systems, allowing detection and quantitation of antigen-specific antibodies in serum or plasma samples. Baculovirus expressed recombinant 3ABC antigen based ELISA developed by IVRI has performance highly comparable to similar commercial kits. Diagnostic sensitivity and specificity for the ELISA were found to be 98 and 97.4, respectively. The developed ELISA test compared well with two commercial kits showing high agreement between the assays for a set of known naïve and experimentally FMDV infected bovine sera. The ELSA kit developed is a highly cost effective substitute for similar commercial diagnostics, making it very useful in the sero-surveillance of FMD. Salient features of the test systems • LFD test can be performed by the farmers or field veterinarians. Test does not need cold chain maintenance thus are handy under field conditions. • First such diagnostic developed at IVRI and validated on large scale successfully • ELISA is a laboratory test, requiring trained personnel to perform and interpret the results. • Both the tests are highly specific and sensitive and enable differentiation of FMDV infected from vaccinated population. • Assays can be used to detect antibodies in different species of animals that are susceptible to FMD (cattle, buffaloes, sheep, goats and pigs and other ungulates including wild species). • The assays are highly cost effective compared to similar commercial kits marketed world wide