Characterization of Ethanol Extracted Cell Wall Components of <i>Mycobacterium avium</i> Subsp. <i>paratuberculosis</i>

Antigens extracted using ethanol (EtOH) and incorporated in the EtOH vortex ELISA (EVELISA) test have previously shown high specificity and sensitivity for detecting <i>Mycobacterium avium</i> subspecies <i>paratuberculosis</i> (<i>Map</i>) and <i>M. bovis</i> infections in cattle. The objective of this study is to define the components present in the EtOH extract. We show that this extract is composed of lipid, carbohydrate, and proteins on the surface of the bacilli, and that EtOH removes the outer layer structure of <i>Map</i> which comprise these elements. To identify proteins, polyclonal antibodies to the EtOH prep were produced and used to screen a <i>Map</i> genomic expression library. Seven overlapping clones were identified with a single open reading frame, MAP_0585, common to all. MAP_0585, which encodes a hypothetical protein, was recombinantly produced and used to demonstrate strong reactivity in sera from hyperimmunized rabbits, but this protein is not strongly immunogenic in cattle with Johne&#8217;s disease. A panel of monoclonal antibodies was used to determine the presence of additional proteins in the EtOH extract. These antibodies demonstrated that a well-known antigen, termed MPB83, is present in <i>M. bovis</i> EtOH extracts and a fatty acid desaturase (MAP_2698c) is present in <i>Map</i> EtOH extracts, while lipoarabinomannan was common to both. The lipid and carbohydrate components of the extract were analyzed using thin layer chromatography and lectin binding, respectively. Lectin biding and protease treatment of the EtOH extract suggest the antigenic component is carbohydrate and not protein. These results give further insight into this important antigen prep for detecting mycobacterial diseases of cattle.