Molecular Detection of Mycobacterium avium paratuberculosis by Large Sequence Polymorphisms (LSPs)

Abstract

Background: Mycobacterium avium subspecies paratuberculosis is a significant pathogen causing Johne’s disease. Rational evaluation of the prevalence of this organism in animals has been restricted by the lack of accurate diagnostic techniques. DNA microarray-based and computational study of Mycobacterium avium subsp. paratuberculosis strain K10 discovered 17 distinct large sequence polymorphisms present. Of these 17 LSPPs, 7 were with a specificity of greater than 98% and in some cases, sensitivity of up to 95% suggesting that the LSPs are ideal for diagnosing M. avium subsp. paratuberculosis. Methods: A total of 218 fecal samples were collected from cattle (n=180) and buffaloes (n=38) for detection of M. avium subsp. paratuberculosis (MAP) using Large Sequence Polymorphisms (LSPs) and IS900 PCR. The fecal samples were subjected to Ziehl-Neelsen staining, isolation and PCR. A total of 90 fecal samples were found ZN staining positive and were subjected to isolation of MAP, growths were seen in two fecal samples after 16 weeks of incubation. Result: Out of 218 fecal samples tested, 7 samples were positive by both LSP PCR viz; LSPP2 (MAP0284), LSPP4 (MAP0865), LSPP11(MAP2154) LSPP12 (MAP2182c), LSPP12 (MAP2188c), LSPP15 (MAP3774), LSPP16 (MAP3815) and IS900 sequence with an amplicon size of 600, 597, 375, 430, 719, 621, 611 and 229 bp. DNA was also extracted from the isolates (n=2) and were confirmed by PCR targeting Large Sequence Polymorphism (LSPs) and IS900. Detection of MAP in all the 7 fecal samples both by IS900 and by 7 LSPPs indicates the detection potential of selected 7 LSPPs (LSPP2, LSPP4, LSPP11, LSPP12 (MAP2182c), LSPP12 (MAP2188c), LSPP15, LSPP16) in fecal samples of cattle and buffaloes suspected for Johne’s disease.