Comparative performance of different antigens on the lateral flow assay (LFA) platform for the rapid serodiagnosis of paratuberculosis

Abstract

Paratuberculosis is a globally prevalent disease, that adversely affects the economy of livestock farming. Control is largely based on early detection followed by 'Test and Cull' or 'Test and Segregate' Policy. Implementation of paratuberculosis control is a special challenge due to the non-availability of point of care diagnostics (PoCD). Therefore, the present study aimed to optimize and evaluate a lateral flow assay (LFA) for the rapid serodiagnosis of paratuberculosis in ruminant species, especially in the view of the resource-limited areas. Performance of three different antigenic preparations including native purified protoplasmic antigen (nPPA-LFA), commercial purified protoplasmic antigen (cPPA-LFA), and a cocktail of recombinant secretory proteins (RP-LFA) was evaluated as detection reagents for coating LFA strips. Comparative performance of the optimized LFA was also evaluated with gold standard tissue culture, fecal PCR (polymerase chain reaction), and plate ELISA. In addition, the onsite testing of animals belonging to different farms (endemic), species, and regions using optimized LFA was also done to highlight the on-farm testing approach. Findings revealed recombinant secretory proteins based LFA (RP-LFA) had a higher sensitivity of detection compared to other antigens. RP-LFA had a sensitivity of 77.7%, 75.44%, and 75.16% in comparison to gold standard tissue culture, fecal PCR, and plate ELISA, respectively. The specificity of RP-LFA was 100% with all reference tests. In comparison to plate ELISA, RP-LFA had a detection limit of 100% when the S/P ratio of the serum sample is ≥1.0 and 80% when the S/P ratio range of 0.8-1.0. Using RP-LFA, on-farm testing of 608 animals was done and 283 (46.5%) were found positive. Kappa analysis of present RP-LFA revealed 'good strength of agreement' with gold standard tissue culture, fecal PCR, and plate ELISA. Optimized RP-LFA had no cross-reactivity with bovine tuberculosis (bovine TB). The RP-LFA was found reproducible, user-friendly and test results can be interpreted within five minutes. In conclusion, the findings of the present study advocate the huge potential of LFA-based PoCD in the rapid diagnosis and control of paratuberculosis.

Keywords: Antibody detection; Lateral flow assay; Paratuberculosis; Point of care diagnosis; Recombinant proteins.