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Mycobacterium avium and Mycobacterium avium subspecies paratuberculosis are two very closely related mycobacteria that are pathogenic for ruminants. M.a.paratuberculosis causes chronic granulomatous enteritis (paratuberculosis or Johne’s disease) in ruminants and has been implicated in Crohn’s disease of humans. IS901+ M.avium causes avian tuberculosis in birds and susceptible ruminants. Diagnosis of both diseases is difficult, particularly in the early stages and is hampered by the lack of diagnostic reagents specific for these organisms. Prevalence and epidemiological studies of these diseases have not been possible as a result of inefficient diagnosis and a requirement for strain-specific markers. The two organisms are genetically very similar sharing greater than 95% DNA homology although they are biologically distinct in terms of their growth characteristics, media supplements for in vitro growth, host range and pathogenicity.
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The aim of this project is to employ the recent technological advances within the field of comparative functional genomics to identify genes and gene products responsible for these phenotypic variations to aid identification of loci involved in pathogenicity and generate specific markers for diagnosis and epidemiological studies. This will greatly improve our understanding of these two economically important diseases and facilitate control.
The project has involved a large amount of development work to optimise and standardise the experimental and analytical procedures required to undertake the comparative proteome analyses. Procedures have been developed to extract organisms directly from infected tissue, grow organisms in vitro to a defined growth phase, prepare protein extracts, estimate protein concentrations, standardise electrophoresis and develop methods for silver staining gels that do not interfere with mass spectroscopy. Research to date employing 2D-gel electrophoresis and Matrix Assisted Laser Desorption/Ionization Mass Spectrometry) MALDI-MS) has identified a number of proteins that may be suitable for the specific diagnosis of M.a.paratuberculosis and epidemiological studies. In addition, proteins have been identified that are exclusively expressed or upregulated in vivo. These proteins are putative virulence factors that could represent potential targets for drug therapy or novel vaccines and will be investigated further. <P>