Cloning and expression of cultural filtrate proteins from novel and native strains of Mycobacterium avium subspecies paratuberculosis and their application in ELISA based sero-diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is endemic in livestock leading to low per animal productivity. MAP as survives pasteurization, poses a public health problem because of high exposure to animals and humans. There is an urgent need for newer diagnostic tests with high specificity and sensitivity as the current ones suffer from lower sensitivity and specificity. In present study, six Mycobacterium avium subspecies paratuberculosis (MAP)-specific culture filtrate proteins (CFPs) were produced and evaluated for sero-diagnosis of MAP infection in goat and cattle herds in India. Genes encoding for six MAP-CFPs were amplified and cloned into easy cloning vector pJET1.2/pTZ57R followed by sub-cloning into expression vector pET28a (+)/pET22b (+) containing C-terminal Histidine. Recombinant CFPs (r-CFPs) expressions were optimized in Escherichia coli (Rosetta cells) and purified using Ni-NTA affinity chromatography. In SDS-PAGE, MAP CFPs viz., 1693c, 2168c, ModD, 85C, Pep AN and Pep AC showed 22, 24, 55, 38, 20 and 25 kDa molecular masses, respectively. Identity of these r-CFPs was further confirmed by immuno-blotting. We developed six different ELISAs using the six individual r-CFPs and one additional ELISA i.e. cocktail ELISA (c-ELISA) was prepared using cocktail of all 6 r-CFPs. The performance of all seven ELISAs were further evaluated against whole cell protoplasmic based indigenous ELISA (i-ELISA). c-ELISA showed almost similar sensitivity as shown by i-ELISA. However, individual r-CFP based ELISA could not reach up to the sensitivity of cocktail of six r-CFPs. None of the r-CFP showed any false positive (as compare to i-ELISA) thereby specificity was 100%. Results of ELISA tests based on cocktail of r-CFPs, ModD and 85C were quite similar to i-ELISA from goat sera whereas in cattle serum c-ELISA was comparable with i-ELISA. Our study showed a comparable specificity of c-ELISA for the diagnosis of JD and it may have applicability in region where disease is endemic. Future validation of c-ELISA against gold standard or confirmatory tests would give a better insight on its diagnostic potential over i-ELISA.