Previously we showed that Mycobacterium paratuberculosis culture filtrates (CFs) contain more
antigens that react with sera from infected cattle than do cellular extracts of the organism. The goal of the present study was to identify
proteins of potential diagnostic value among these CF
proteins.
Proteins of potential interest were first separated by 2-DE. Roughly 240 CF
protein spots were detected on CBB-stained
gels using Phoretix 2D software. Of these, 83% reacted with serum from M.
paratuberculosis-infected cattle in immunoblots. When bovine serum was absorbed with M. phlei
antigens, however, only 37 of these antigenic
protein spots were reactive. Twenty-four of these spots were selected for identification based on their immunoblot staining intensity and differences in pI and mass. A total of 14
proteins were ultimately identified by MS and BLAST searches as ModD, PepA, ArgJ, CobT,
Antigen 85C, and nine hypothetical
proteins. N-terminal
peptide analysis of PepA,
Antigen 85C, ModD, MAP1693c, MAP2168c, and MAP1022c showed that each
protein has 27-39
amino acids that may function as a
signal sequence suggesting they are secreted through a Sec-dependent pathway. These 14
proteins from M.
paratuberculosis CF are strong candidates for use as
antigens for improved serodiagnostic tests for bovine
paratuberculosis.