In silico screened Mycobacterium avium subsp. paratuberculosis (MAP) recombinant proteins upregulated under stress conditions are immunogenic in sheep

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johneʹs disease (JD) in ruminants. MAP is known to enter a dormant phase outside the host, typically on soil. In vitro experiments have reported regulation of certain MAP genes when exposed to stressors similar to what is thought to produce dormancy. It is believed that in vivo regulation of dormancy genes and associated proteins by MAP may play a role in evading the host defence mechanisms and induce the host immune response against these dormancy-related proteins. Five proteins encoded by dormancy-related genes that were previously found to be upregulated under stress conditions and predicted through in silico analysis to possess immune epitopes (three hypothetical proteins and two proteins involved in fatty acid metabolism) were selected. Recombinant proteins were produced, purified and evaluated by indirect enzyme-linked immunosorbent assay (ELISA) for immunogenicity using a panel of sera obtained from sheep unexposed and exposed to MAP. The antibody levels of the exposed group were significantly higher than the unexposed group (P < 0.001). Individually, the five proteins were found to discriminate between sera from sheep exposed to MAP compared to unexposed sheep. At 91% diagnostic specificity, the diagnostic sensitivity of the recombinant antigen ELISA ranged from 24% to 42% and AUCROC from 0.7015 to 0.8405.