Detection of Johne's Disease in Imported Sheep and Isolation of Sheep Strain of Mycobacterium avium subsp. paratuberculosis

Abstract

An imported sheep from New Zealand was diagnosed with Johne's disease from a real-time PCR test of fecal samples and was culled during the import quarantine. The DNA (0.03 to 0.163 pg/2.5 μl) of Mycobacterium avium subsp. paratuberculosis (MAP) was detected in the feces and a maximum of 14.3 pg/2.5 μl of DNA was detected in tissue specimens (ileum to cecum, mesenteric lymph nodes). In the bacterial cultivation of tissue samples using MGIT liquid medium, the growth of acid-fast bacteria was confirmed after 6 weeks of incubation and 200 to 600 pg/2.5 μl of MAP DNA was detected in the medium. DNA Typing by RFLP analysis of IS1311 PCR products revealed that the isolates were ovine-type but not bovine-type. The primary culture of the bacteria was only successful using MGIT liquid medium. However, a subculture of the isolate was possible from the liquid medium to Herrold's solid medium, and the colony growth was enhanced in the presence of 10％ CO₂. Chronic granulomatous enteritis was observed histopathologically and such lesions were consistent with the isolated areas of the bacteria. Real-time PCR and bacterial isolation using MGIT liquid medium are highly sensitive and efficient diagnostic methods for detecting ovine Johne's disease.