It is important to develop the P.C.R. and the restriction fragments length polymorphism (R.F.L.P.) technologies to identify genome segments since they constitute quick, sensitive, and specific methodologies for the detection of Mycobacterium avium subsp. paratuberculosis (Mptbc), a microorganism that produces animal paratuberculosis and that is related to Crohn's disease in humans, Our Objectives were: 1. To isolate Mptbc strains from feces and organs and to apply a P.C.R. protocol to confirm the IS900 insertion sequence. 2. To establish, by R.F.L.P., the genetic pattern of the strains isolated and 3. To analyze cellular and extracellular proteins expressed in Mptbc, Feces and organs of deer were cultured in culture media (yolk-egg Herrold, with and without mycobactin, adding pyruvate and antibiotics) for the isolation of Mptbc. The strains isolated were amplified by P.C.R. and studied by R.F.L.P. and Immunoblotting (IB). Twelve Mptbc strains, 6 from feces and 6 from organs, were isolated. The strains isolated developed only in mycobactin Herrold medium, showing their characteristic dependence. The analysis by P.C.R. was positive starting from strains developed in the culture medium. When feces were inoculated with a Mptbc strain, the detection rate by R CA was of 100%, demonstrating the highest specificity for IS900 in a 1:1000 dilution of the problem sample, but it was negative starting from the raw sample (feces). The isolations revealed an identical "A" type genetic pattern of R.F.L.P.,R, with the probe of 217 base pairs (endonuclease BstEII and PstI). The immunoblotting detected protein antigens of 65 KDa (thermal shock), 42 ADa, 35 KDa, and 28 KDa, corresponding to Mptbc from an animal with clinical symptoms and characteristic histologic lesions in organs. 1. A single "A" genetic pattern was identified in the population studied. 2. The IS900 sequence specific of Mptbc was amplified. The result was not successful when feces without previous isolation were used. 3. Proteins excreted and contained in the bacterial soma of an animal were identified, but it is necessary to characterize Mptbc specific antigens better to increase the sensitivity of immunological tests.
