Identifying genes in Mycobacterium avium subspecies paratuberculosis that are essential for survival in dairy calves

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic wasting disease common in dairy cattle. Currently, there are no effective vaccines to prevent infection and fecal bacterial shedding. To determine potential targets for a live- attenuated vaccine strain 12 Holstein-Friesian dairy calves were inoculated with 5 x 10^10 CFUs on two consecutive days with a mutant library from MAP strain A1-157. MAP was cultured 2 and 4 mo post inoculation from intestinal tissues and lymph nodes. Quantitative PCR was used to assess changes in proportions of select gene mutants between intestinal tissues and the inoculum; this method failed to confirm previously described essential genes. After applying filtering parameters, targeted next generation sequencing discovered 42 genes absent from all tissues which are involved in DNA transcription, pathogenesis, metabolism and biosynthesis, and oxidation-reduction reactions. These results provide many potential genes for attenuation of MAP for the creation of candidate vaccine strains.