Submitted to: Book Chapter
Publication Type: Book / chapter
Publication Acceptance Date: 12/10/2005
Publication Date: 4/28/2006
Citation: Bannantine, J.P., Paustian, M. 2006. Identification of diagnostic proteins in Mycobacterium avium subspecies paratuberculosis by whole genome analysis approach. In: O'Connor, L., editor. Methods im Molecular Biology: Diagnostic Bacteriology Protocols. 2nd edition. Totowa, NJ: Humana Press. p. 185-196. Interpretive Summary: This communication represents an invited book chapter written specifically to describe in great detail the methods used by our laboratory to identify protein antigens from a whole genome analysis of M. paratuberculosis. The emphasis is on the methodology used more so than on what results were obtained using the methods. Basically, we analyzed the M. paratuberculosis genome sequence for genes that are present uniquely in M. paratuberculosis. This was accomplished by comparing this genome to other closely related mycobacterial genomes that have been sequenced. We then performed DNA amplification studies such as PCR to survey additional mycobacteria that are not represented by a sequenced genome. Once all the unique M. paratuberculosis gene were discovered, we began to produce proteins from these genes or coding sequences. These proteins could then be analyzed to see if they are immunogenic or illict antibodies.
Technical Abstract: Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is an economically significant veterinary pathogen that causes Johne’s disease in cattle and sheep. There is a critical need for improved diagnostic tests to detect M. paratuberculosis infection in these animals. As with many other animal diseases, efforts need to be concentrated on the development of simple, rapid, noninvasive tests that can be performed by veterinarians or animal producers without expensive laboratory equipment. With the genome sequence of M. paratuberculosis now complete, we have taken a different strategy to identify novel proteins that are present uniquely in M. paratuberculosis and are antigenic in the context of infected cattle. Through a whole genome comparison of M. paratuberculosis with other sequenced mycobacterial genomes, we identified a collection of over 90 genes that are present uniquely in M. paratuberculosis. This list has been further trimmed to 39 following PCR amplification of unique genes using genomic DNA template from several mycobacterial species and isolates. A selection of the remaining genes has been cloned and expressed in E. coli and purified by affinity chromatography. Successfully purified proteins were analyzed using sera from rabbits immunized with M. paratuberculosis. Furthermore, to identify antigens in the context of disease, sera from cattle with Johne’s disease as well as healthy control cattle are used in immunoassays. Using this methodology, the first protein antigens specific to M. paratuberculosis were identified.