Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/2/2003
Publication Date: N/A
Technical Abstract: Johne's disease, a chronic inflammatory ailment caused by infection with Mycobacterium paratuberculosis, is one of the most prevalent and costly diseases of dairy cattle worldwide. Accumulated evidence from several decades of research suggests that a critical factor for control and eradication of the disease from dairy herds includes the availability of a sensitive and specific test that will allow close monitoring of infection within farms. Unfortunately, existing microbiological, serological, or immunologic assays for the identification of infected animals are inadequate for this task. This is primarily because of the slow-growing nature of the organism, and the previous lack of information on M. paratuberculosis specific genes or proteins that would enable the development of specific and sensitive assays. Thus, the recent characterization of the complete genome sequence of M. paratuberculosis in our laboratory (funded by the USDA) has been a major step forward in meeting this critical need. Through a comparative genomic approach with closely related mycobacteria followed by extensive PCR analysis with several isolates, 21 M. paratuberculosis-specific genes have been identified. Nucleotide sequences representing each gene were amplified and cloned into an E. coli expression vector encoding the maltose binding protein (MBP) affinity tag. All 21 of the MBP fusion proteins were successfully purified under denaturing conditions. Purified recombinant M. paratuberculosis proteins were used in immunoblotting studies with sera from rabbits and mice immunized with whole cell preparations of M. paratuberculosis. These studies showed that five of the 21 gene products are produced by M. paratuberculosis and are antigenic. Immunoblot analysis with a panel of sera from nine healthy and ten clinical cattle shows the same five M. paratuberculosis proteins are also detected within the context of infection. Collectively, these data show we have identified novel sequences that are unique to the M. paratuberculosis genome and propose to utilize these unique sequences for the rational development of highly specific and sensitive PCR-based and antigen-based assays that will allow the detection of the bacterium in laboratory and field settings.