Isolation and Genotyping of Mycobacterium avium subsp paratuberculosis cattle strains from the contaminated Cow herds in province of Razavi Khorasan using

Paratuberculosis or Johne's disease is a disease of irreparable damage to the livestock industry and animal products imported to our country. Researchers around the world alert if necessary actions to control this disease is not done, one of the most dangerous financial losses to the national economy that the disease will be in there. Given the similar clinical and molecular diagnosis of isolation of the bacteria from patients with Crohn's disease in many people, Mycobacterium paratuberculosis in Crohn's disease in humans is estimated to be involved, The scientists of this bacterium as a major potential threat to public health and safety are considered. In this study, the presence of Mycobacterium avium subsp Paratuberculosis was investigated in cow fecal, manure and milk samples. Samples were gathered of dairy farms of cities in Razavi khorasan province, Iran. There were 3 fecal samples(rectal and manure samples) and raw milk samples including the bulk milk transporters, milk samples of cows with paratuberculosis clinical signs, and the farm bulk milk tanks. All these farms were not tested for presence of MAP but in clinical point of view were affected by John's disease. The samples were cultured on Herrold's egg yolk medium with and without mycobactin J. The samples, in which the bacteria could merely grow on Herrold's media containing mycobactin J, were considered to be positive. Additional tests such as colony morphology, acid fast staining and Nested PCR–IS%33 assay were performed to confirm of isolated bacteria. The positive and negative cases in each group with other groups were evaluated both observationally and statistically. The number of colonies was variable from to 3 colonies. The highest numbers of colonies were observed in the fecal samples taken from cows showing clinical signs of Johne's disease and the lowest numbers of colonies were observed in the milk samples taken from the farm bulk milk tanks. In addition, a number of fecal samples were tested by direct Nested PCR for presence of MAP. A variety of genotyping techniques has been used for typing of Mycobacterium Paratuberculosis , but RFLP, is a technique used at the highest international level for genotyping of Mycobacterium, including Mycobacterium Paratuberculosis. Thus, the present study, patterns of RFLP-IS%33, arising from the application of two restriction enzymes BstEII and PstI were compared with the reference models available. As expected, almost strains isolated from Cow samples (Fecal, manure and Milk) belonging to C and B types (type B-C). Type C is predominant type isolated from cattle in most European countries and patients with Crohn's disease in humans