Submitted to: BMC Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/21/2005
Publication Date: 1/21/2005
Citation: Huntley, J., Stabel, J.R., Bannantine, J.P. 2005. Immunoreactivity of the mycobacterium avium subsp. paratuberculosis 19-kda lipoprotein. BMC Microbiology. 5(1):1-8. Interpretive Summary: We now have the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP) completed. The next step is functional genomics, which involves identifying functional of genes or analyzing them for diagnostic or vaccine potential. In this manuscript, we cloned a MAP gene sequence that has been shown to be an excellent stimulator of the immune response for M. tuberculosis infections. We evaluated this gene in three immunological assays and found that it is not as potential a stimulator of the immune response in cattle as compared to the TB counterpart in humans.
Technical Abstract: Background The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne’s disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed. Results MAP0261c is conserved in mycobacteria, showing a 95% amino acid identity in M. avium subspecies avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c was cloned, expressed, and purified as a fusion protein with the maltose-binding protein (MBP-19kDa) in Escherichia coli. IFN-g production was measured from 21 naturally infected and 9 control cattle after peripheral blood mononuclear cells (PBMCs) were stimulated with a whole cell lysate (WCL) of M. avium subsp. paratuberculosis or the recombinant MBP-19kDa. Overall, the mean response to MBP-19kDa was not as strong as the mean response to the WCL. By comparison, cells from control, non-infected cattle did not produce IFN-g after stimulation with either WCL or MBP-19kDa. To assess the humoral immune response to the 19-kDa protein, sera from cattle with clinical Johne’s disease were used in immunoblot analysis. Reactivity to MBP-19kDa protein, but not MBP alone, was observed in 9 of 14 infected cattle. Antibodies to the 19-kDa protein were not observed in 8 of 9 control cows. Conclusions Collectively, these results demonstrate that while the 19-kDa protein from M. avium subsp. paratuberculosis stimulates a humoral immune response and weak IFN-g production in infected cattle, the elicited responses are not strong enough to be used in a sensitive diagnostic assay.