Accurate immunodiagnosis of bovine paratuberculosis is among others hampered by the lack of specific antigens. One of the most frequently used antigen preparations is purified protein derivative, also known as tuberculin, produced from heat processed culture filtrates. This crude extract has limitations when used in diagnostic assays due to the presence of cross reactive antigens. The aim of the current study was to analyse the qualitative composition of tuberculins of the major mycobacterial pathogens and subsequently identify novel paratuberculosis specific antigens.

Using one dimensional gel electrophoresis followed by tandem mass spectrometry analysis of purified protein derivatives from Mycobacterium avium subspecies paratuberculosis(MAP), Mycobacterium avium subspecies avium(MAA) and Mycobacterium bovis we identified 156, 95 and 132 proteins respectively. Subsequently, comparative sequence analysis led to the selection of a MAP specific protein (MAP1718c). This protein as well as MAP3515c (4 AA difference with the homologous protein in MAA) and MAP1138c (LprG, the homologue of an interesting M. tuberculosis antigen) were expressed as recombinant proteins in E. coli for use in lymphocyte proliferation assays and serum antibody ELISA. While lymphocyte proliferation responses did not indicate substantial diagnostic potential of the antigens tested, the antibody titers measured by ELISA specific for MAP1138c, but not MAP1718c and MAP3515c, in serum from paratuberculosis infected cows (N=20) were significantly higher (p<0.05) than those in serum from control animals (N=20), despite the conserved nature of this protein.

In conclusion this study showed that a combination of proteomics and genomics starting from complex protein mixtures can reveal novel antigens supporting the development of more accurate diagnostics in mycobacterial diseases.