Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/10/2014
Publication Date: 2/25/2015
Publication URL: http://handle.nal.usda.gov/10113/60768
Citation: Leite, F.L., Reinhardt, T.A., Bannantine, J.P., Stabel, J.R. 2015. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity. Veterinary Microbiology. 175(2-4):275-285.
Interpretive Summary: Johne's disease is a chronic, debilitating intestinal disorder in cattle characterized by diarrhea, reduced feed intake, weight loss and death. Cattle usually become infected as young calves by ingesting feces containing the causative bacteria. However, symptoms of disease do not usually present themselves until the animals reach 3 to 5 years of age or even older. During this time the animal is infected and may be shedding the organism in its feces without showing any clinical signs of disease. In addition to reduced production by these animals through reduced milk production, they also present a potential infective threat to the rest of the herd. New tools to diagnose and control Johne’s disease are essential to prevent further economic hardship within the livestock industry. The current study utilized newer laboratory methods to identify proteins that are present in the bacteria and some of these proteins were reactive with sera from infected cows. These results suggest that they may be useful as reagents for improved diagnostic tests or better vaccines. Incorporation of more refined tools will help to control and manage Johne’s disease in dairy herds.
Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) to identify individual proteins within the complexes. Identity of individual proteins within complexes was further confirmed by MS/MS upon excision of spots from 2D SDS-PAGE gels. Among the seven putative membrane complexes observed, major membrane protein (MAP2121c), a key MAP antigen involved in invasion of epithelial cells, was found to form a complex with cysteine desulfurase (MAP2120c). Other complexes found included those involved in energy metabolism (succinate dehydrogenase complex) as well as a complex formed by Cfp29, a characterized T cell antigen of M. tuberculosis. To determine antigenicity of proteins, Western blot was performed on replicate 2D SDS-PAGE gels with sera from noninfected control cows (n=9) and naturally infected cows in the subclinical (n = 10) and clinical (n=13) stages of infection. Clinical animals recognized MAP2121c in greater proportion than subclinical and control cows, whereas cysteine desulfurase recognition was not differentiated by infection status. To further characterize antigenicity, recombinant proteins were expressed for 10 of the proteins identified and evaluated in an interferon-gamma (IFN-gamma) release assay as well as immuno-blots. This study reveals the presence of protein complexes in the cell envelope of MAP, suggesting protein interactions in the envelope of this pathogen. Furthermore the identification of antigenic proteins with potential as diagnostic targets was characterized.