Shanmugasundaram Karuppusamy¹, Lucy Muthairia², David Kelton³, Brandon Plattner⁴, Antonio Facciuolo2 and Gordon M. Kirby¹

Departments of Biomedical Sciences1, Molecular and Cellular Biology2, Population Medicine3, Pathobiology4, University of Guelph, Guelph, ON, Canada

Mycobacterium avium subsp. Paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants called Johne’s disease, a major production-limiting infection worldwide. Calves exposed to MAP in feces and milk develop a silent infection lasting several years without showing clinical signs. Cattle with subclinical infection intermittently shed MAP in feces and milk causing further spread of the disease. Diagnosis at early stages of Johne’s disease is difficult because current tests such as fecal culture, PCR and serological tests lack sensitivity and specificity, are time-consuming and expensive. There is a need for approaches to early diagnosis and prevention of clinical infection and the associated consequences i.e. profound weight loss, diarrhea and reduction in milk production. The purpose of this research is to develop new approaches to early diagnosis and prevention of Johne’s Disease using antibodies that target unique MAP cell wall and membrane epitopes. Using a proteomic approach involving 2 dimensional difference gel electrophoresis (DIGE), cell wall and membrane proteins of MAP, M. smegmatis (MS) and M. avium subsp. hominissuis (MAH) were compared. Ten unique MAP-specific cell wall proteins were found and shown to be immunogenic in Western blots using serum from Johne’s-infected cattle. Proteins were isolated from gel plugs and identified by LC-MS/MS analysis as Succinate dehydrogenase iron-sulfur subunit (SdhA), Acyl-CoA dehydrogenase E25 and E3 (FadE25 and FadE3), Acyl-ACP desaturase (DesA2), Ribonucleotide-transport ATP-binding protein ABC transporter (Mkl) and an uncharacterized protein; these MAP proteins are associated with energy and lipid metabolism and virulence. Rats were immunized with purified recombinantly expressed proteins for generation of polyclonal antibodies. Affinity purified antibodies to SdhA , FadE25 and DesA2 are specific to MAP proteins in western blot analysis and do not react with cell wall extracts from MS or MAH. Antibodies to FadE3, Mkl and an uncharacterized protein are currently being analyzed. Anti-serum is also being used in immunocytochemistry studies to determine specificity in smears of intact mycobacteria and tissue sections of intestines with associated lymphoid tissue from Johne’s-infected cows. Validation of these MAP-specific antigens could ultimately lead to the development of improved diagnostic tests and vaccine targets for Johne’s disease.