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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND IMMUNOLOGICAL CHARACTERISTICS OF JOHNE'S DISEASE Title: Immunogenicity of Proteome-determined Mycobacterium avium subsp. paratuberculosis-specific Proteins in Sheep with Paratuberculosis

Authors
item Hughes, Valerie - MOREDUN RESEARCH INST.
item Bannantine, John
item Denham, Susan - MOREDUN RESEARCH INST.
item Smith, Stuart - MOREDUN RESEARCH INST.
item Garcia-Sanchez, Alfredo - MOREDUN RESEARCH INST.
item Sales, Jill - THE KING'S BUILDING
item Paustian, Michael
item Mclean, Kevin - MOREDUN RESEARCH INST.
item Stevenson, Karen - MOREDUN RESEARCH INST.

Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 21, 2008
Publication Date: December 1, 2008
Citation: Hughes, V., Bannantine, J.P., Denham, S., Smith, S., Garcia-Sanchez, A., Sales, J., Paustian, M., Mclean, K., Stevenson, K. 2008. Immunogenicity of Proteome-determined Mycobacterium avium subsp. paratuberculosis-specific Proteins in Sheep with Paratuberculosis. Clinical and Vaccine Immunology. 15(12):1824-1833.

Interpretive Summary: In this study, we used a comparative proteomic approach to identify differences in protein profiles of Mycobacterium avium subspecies avium with Mycobacterium avium subspecies paratuberculosis. The paratuberculosis subspecies causes Johne’s disease whereas the avium subspecies is simply a ubiquitous environmental bacterium. The strong similarity in the proteins produced by these two bacteria cause a problem in the diagnosis of Johne’s disease. From this comparative analysis, we discovered 32 proteins from the Johne’s causing bacterium that were not produced by the environmental bacterium. These proteins are considered important potential targets for specific diagnostic tests. The remaining part of the study is dedicated to evaluating the serological reactivity of these 32 proteins. The major finding is that a subset of these proteins were shown to be reactive as well as specific. Thus, they are good candidates for building an improved diagnostic test for Johne’s disease.

Technical Abstract: Mycobacterium avium subspecies paratuberculosis causes paratuberculosis, a chronic granulomatous enteritis. Detecting animals with paratuberculosis infections is difficult because the currently available tools have low sensitivity and lack specificity being prone to spurious positive test results caused by exposure to environmental Mycobacterium avium complex organisms. To generate candidate antigens for incorporation into a specific test for paratuberculosis, subspecies-specific proteins were determined by proteomic comparison of M. avium subspecies paratuberculosis and M. avium subspecies avium. Analysis was aimed at revealing proteins only expressed (or predominant) in the protein profile of M. avium subspecies paratuberculosis. Two-dimensional gel electrophoresis resolved approximately 1000 protein spots from each subspecies. Proteome analysis identified protein spots whose expression profile appeared markedly increased in M. avium subsp. paratuberculosis and 32 were identified by analysis of their tryptic peptide profile by matrix assisted laser desorption –time of flight analysis. 30 of these proteins were cloned and their recombinant proteins expressed. Ovine paratuberculosis sera were used to assess their immunoreactivity by ELISA, western blot and dot blot analysis. Seventeen proteins were detected in at least one of the immunoassays and eleven proteins were detected by ELISA with an optical density in excess of the cutoff of 0.1 in four out of six sera tested. The immunoreactivity of these proteins indicates their potential as unique diagnostic antigens for the development of a specific serological detection of paratuberculosis.

Last Modified: 7/28/2014