Evaluation of the immunogenicity of Mycobacterium avium subsp. paratuberculosis (MAP) stress-associated recombinant proteins

Abstract

The aim of this study was to assess the humoral immune responses in MAP-infected and uninfected sheep against 27 MAP stress-associated recombinant proteins that were regulated in in vitro models of physiological stress. These include evaluation of 5 proteins, which were previously reported by Gumber et al. (2009b), using serum samples from sheep with a wide range of disease stages. For purification of recombinant his-tagged proteins expressed as an insoluble protein, on-column refolding purification was applied as well as one-step denaturing purification. All purifications together resulted in a total of 48 recombinant antigen preparations. In antibody ELISA tests, 23 of these, representing 18 MAP proteins, showed significant differences in responses between infected and uninfected sheep. Recombinant antigen preparations MAP2281c, MAP3555 (refolded form), and MAP0711c (refolded form) when incorporated in an ELISA, had similar sensitivity to a commercial antibody ELISA test at the cutpoint of 90% specificity, and showed relatively high values in receiver operating characteristic (ROC) curve analysis. However, as some of the sera from uninfected sheep also reacted to recombinant antigens, further development of the assays is necessary prior to practical application. Compared to the commercial antibody ELISA, MAP0593c, MAP2281c, MAP2411, MAP3555, and MAP3200 detected more infected sheep with a lower grade of lesion, suggesting that these proteins identified in the in vitro models of stress were also expressed in vivo in MAP-infected sheep at an early stage of infection. This is consistent with the hypothesis of latency or dormancy in subclinical mycobacterial infection.