Evaluation of the immunogenicity of recombinant stress-associated proteins during Mycobacterium avium subsp. paratuberculosis infection: Implications for pathogenesis and diagnosis

The aim of this study was to assess the immunogenicity of recombinant stress-associated proteins of Mycobacterium avium subsp. paratuberculosis in sheep infected with the organism compared to control sheep. Five proteins – MAP2411, ClpP, Ppa, MAP0593c and GreA – which were identified previously in in vitro stress or dormancy responses of M. paratuberculosis to hypoxia, nutrient starvation and heat, were cloned, expressed and purified as His-tag recombinant proteins from the pET-15b vector in a BL21(DE3)pLysS strain of E. coli. The immunogenicity of MAP2411 did not differ between infected and control sheep. However, the serological reactivity of the other recombinant antigens, and combinations of them, varied according to the histological stage of paratuberculosis. Interestingly, the sera from some animals with paucibacillary lesions, which were not immunoreactive in a commercial paratuberculosis ELISA that was based on non-defined native antigens, recognised the recombinant antigens. We infer from their differential immunogenicity in infected and control sheep that four of the stress-associated proteins were expressed by M. paratuberculosis in vivo. These data provide fundamental information on host–mycobacterial interactions and have conceptual implications for the development of future diagnostic tests for early immune responses in animals infected with mycobacteria.