Evaluation of IS900 PCR for Detection and IS1311 PCR via Restriction Enzyme Analysis for Strain Typing of Mycobacterium avium subspecies paratuberculosis in Ruminant Fecal Samples.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic granulamatous infectious enteritis known as Johne's disease in ruminants and is characterized by progressive diarrhea, weight loss, and reduced productivity. MAP shedding in feces makes fecal samples valuable for diagnostic screening. Based on clinical symptoms, 220 fecal samples from cattle and buffaloes with a history of chronic diarrhea were collected. Each sample was stained with Ziehl- Neelsen staining technique to detect acid fast positive bacteria. The DNA was collected from highly acid fast bacterial samples and was subjected to isolation of Mycobacterium avium subsp. paratuberculosis (MAP). Out of 71 acid fast bacterial samples; 22 were highly acid fast positive. Only 2 samples were found to be positive in isolation for MAP. DNA from these isolates were then extracted and subjected to IS900 PCR hence, confirming the presence of MAP in the samples. The positive samples were also subjected to IS1311 PCR and were analyzed by Restriction enzyme analysis (REA). The PCR-REA confirmed the presence of 'Cattle type' of strain of the two isolates. Sequence analysis further demonstrated a high degree of similarity between the two isolates and strong phylogenetic identity with previously reported global MAP sequences, with the highest similarity (99.3%) to a Chinese MAP isolate (MW546854_China). [ABSTRACT FROM AUTHOR]