Isolation of Mycobacterium avium subsp. paratuberculosis and confirmation of cases by Nested-PCR

Paratuberculosis or Johne's disease is a chronic granulomatous disease of the small intestine that is caused by Mycobacterium avium subspecies paratuberculosis (MAP).The aim of this study was to determine the status of this disease in suspected animals and finally confirmation of isolates identity by molecular tests. For this purpose, 142 suspicious samples were cultured on plain and mycobactin J-supplemented Herrold's egg-yolk slants from Isfahan, Alborz, Tehran, North Khorasan, Khuzestan, Zanjan, Fars, Qazvin, Kerman and Golestan provinces. Forty seven isolates were obtained out of 142 suspicious samples. All specimens that were grown on mycobactin J-supplemented Herrold's egg-yolk slants were detected by Ziehl-Neelsen staining as acid Fast bacilia, and in 83 samples of the total, as well as mycobacterial strains, a 543 bp fragment was amplified using PCR-16s rRNA. Nested PCR was done on isolates and positive and negative controls. At the first stage, a 398 bp fragment was obtained by P90 and P91 primers, and in the next step, a 298 bp fragment was obtained using AV1 and AV2 primers, which indicates the presence of Mycobacterium paratuberculosis in the samples. Based on this study, Nested-PCR is suggested as a suitable rapid diagnostic method for disease cases.