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Johne's disease (JD) is a contagious, chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The aim of this study was to identify antigenic proteins from the MAP cell envelope (i.e. cell wall and cytoplasmic membranes) by comparing MAP, M. avium subsp. hominissuis (MAH) and M. smegmatis (MS) cell envelope protein profiles using a proteomic approach. Composite two-dimensional (2D) difference gel electrophoresis images revealed 13 spots present only in the image of the MAP cell envelope proteins. Using serum from MAP-infected cattle, immunoblot analysis of 2D gels revealed that proteins in the 13 spots were antigenic. These proteins were identified by liquid chromatography tandem mass spectrometry as products of the following genes: sdhA, fadE25_2, mkl, citA, gapdh, fadE3_2, moxR1, mmp, purC, mdh, atpG, fbpB and desA2 as well as two proteins without gene names identified as transcriptional regulator (MAP0035) protein and hypothetical protein (MAP1233). Protein functions ranged from energy generation, cell wall biosynthesis, protein maturation, bacterial replication and invasion of epithelial cells, functions considered essential to MAP virulence and intracellular survival. Five MAP cell envelope proteins, i.e. SdhA, FadE25_2, FadE3_2, MAP0035 and DesA2 were recombinantly expressed, three of which, i.e. SdhA, FadE25_2 and DesA2, were of sufficient purity and yield to generate polyclonal antibodies. Immunoblot analysis revealed antibodies reacted specifically to the respective MAP cell envelope proteins with minimal cross-reactivity with MAH and MS cell envelope proteins. Identification and characterization of MAP-specific proteins and antibodies to those proteins may be useful in developing new diagnostic tests for JD diagnosis.

Identification of antigenic proteins from Mycobacterium avium subspecies paratuberculosis cell envelope by comparative proteomic analysis