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IS900 RFLP Analysis of Mycobacterium avium subsp. Paratuberculosis of Iranian Isolates and Analyze Using Bioinformatics Tools

Aims and objectives: Mycobacterium avium subspecies paratuberculosis was first diagnosed and reported in imported cattle from England during 1962-1965. Further studies have shown that paratuberculosis is scattered throughout Iranian ruminants population. In spite of heavy economic loses; little genetic information is available. So, present study was carried out in order to obtain a better understanding of the MAP genetic diversity in Iranian isolates. Methods: During a 7-month period, 36 isolates of MAP was collected from milk and feces obtained from sheep, goat and cattle originating 10 provinces plus a further eleven archived MAP isolates and two MAP vaccine strains (MAP 316F & III &V) and two D4 reference strains (Weybridge and Turkish) were included and digested by restriction endonucleases PstI. Digested DNA was hybridised with a standard IS900 probe. Genomic fingerprints were scanned by camera and analysed using the software Gel pro analyzer & Bionumerix software package version 6.5 (Applied Maths Belgium). Dendrogram and Minimum Spanning Tree was draw using arithmetic averages (UPGMA) for Iranian isolates and vaccine strains. Results: ten RFLP (Pst I) types were detected and designated as B (bovine, ovine and caprine), and G (bovine, and caprine) and eight new type (three of them singletons and five cluster) in accordance with the study by pavlik et al 1999. The largest cluster (G strain) contained 45.8% of Iranian isolates (n = 22) then type II (B strain), represented by 11 isolates, recovered from all the three animals in six provinces. A single type was identified only in cattle from three provinces. The identical RFLP type was shown by MAP vaccine strains were different from the Iranian isolates. Two D4 reference strains had restriction pattern but did not hybridize to IS900 sequence. Dendrogram result showed B profiles close to G profile and MAP vaccine strains profiles (>98). The results of Minimum Spanning Tree also showed that all of the clusters and orphan were derived directly or indirectly from B profile with multiple hosts throughout the country. This pattern has been seen from throughout the world and multiple hosts. Conclusions: RFLP analysis showed that the Iranian isolates have perhaps originated from B specific profile but it still need to more future works using genotyping methods.