|Robbe Austerman, Suelee|
|Huntley, Jason - IOWA STATE UNIV|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 12, 2004
Publication Date: February 12, 2004
Citation: Stabel, J.R., Bannantine, J.P., Robbe Austerman, S., Paustian, M., Huntley, J. 2004. Mycobacterium paratuberculosis: genome to disease control[abstract]. Meeting Abstract. Technical Abstract: There are four broad objectives within this research project that are united in the goal of control of this disease on the farm. The main objectives are: 1) Sequence the complete M. paratuberculosis genome; 2) Develop highly sensitive and specific diagnostic tests for detection of M. paratuberculosis through identification and characterization of unique bacterial genes and proteins and study of host immune responses during the different stages of disease, 3) Identify immunogens of M. paratuberculosis by random and directed expression library immunization, and 4) Identification of a diagnostic tool that can detect early infection in multiple species. Johne's disease has become a high priority disease in the US cattle industry. Herd prevalence of Johne's disease is estimated to be 22-40% as determined by a recent NAHMS survey of dairy cattle. There are no adequate estimates of herd prevalence in beef cattle or in small ruminants such as sheep or goats in the US. The economic impact of this disease on the dairy industry was estimated to be over $200 million per year in 1996 and is growing each year with the continued spread of this disease. In addition, M. paratuberculosis has been implicated as a causative factor in Crohn's disease, a chronic inflammatory bowel disease of human beings, which has served as a further impetus to control this disease in our national cattle industry. The dairy industry must be assured that they are providing a clean, safe product for consumers. The genome sequence project was initiated to identify new immunogens that will be useful in the development of diagnostic tools for the detection of M. paratuberculosis infection and as vaccine candidates to protect against infection and clinical disease. This project has the objective of analyzing the DNA of M. paratuberculosis for identification and analysis of unique genetic sequences that can be used to develop diagnostic tools for the detection of M. paratuberculosis infection. From this analysis, over 29 genes specific to M. paratuberculosis were identified. Each of theses 29 genes will be cloned and expressed in Escherichia coli. Recently, the focus has been narrowed to 13 unique genes that had not been previously described. These genes have been expressed and purified and antigenic analysis of these proteins is underway. We have incorporated one M. paratuberculosis-specific gene that is present in 6 copies within the genome into a nested PCR assay for the detection of M. paratuberculosis in fecal samples. This assay will be an alternative to the current PCR detection assays for M. paratuberculosis that utilize the IS900 gene sequence. Although the IS900 gene sequence is highly sensitive in PCR assays it is currently under patent restriction for use in diagnostic laboratories. We are in the process of evaluating these antigens in a whole blood interferon-g assay for the detection of M. paratuberculosis infection in cattle. We have also identified immunogens of M. paratuberculosis by random expression library immunization of mice. A M. paratuberculosis genomic DNA library was partitioned into 78 sublibraries containing an average of 1500 clones each. Approximately 2 µg of DNA from each sublibrary was delivered by gene gun to the abdomen of each mouse and then again at boosting 3 weeks later. Mice were challenged with live M. paratuberculosis and then necropsied after 3 months for evaluation of tissue colonization. Preliminary results suggest that 7 sublibraries thus far may be protective against M. paratuberculosis challenge by reduced recovery of viable M. paratuberculosis from spleen and mesenteric lymph nodes of immunized mice compared to infected controls. Sequencing of two of these protective libraries has been completed. Smaller gene pools from one of the sequenced libraries were randomly generated and used to immunize mice in further experiments. After challenge, tissue colonization will be assessed for each gene subgroup to characterize which gene(s) are generating protective responses in the host. We hope to extrapolate this information to evaluate the protective gene pools in a calf infection model in the near future.