Characterization of p30 membrane protein gene of Mycoplasma agalactiae isolates by polymerase chain reaction and restriction endonuclease enzyme assay

Seven hundred and eight (708) samples collected from the goats of Gujarat were screened for Mycoplasma agalactiae by culture and PCR using 16S rRNA based genus specific and species-specific primers. Amplification of the p30 membrane protein gene was carried out using specific primers and the resultant amplicons were subjected to restriction enzyme analysis. The isolates yielded 715 bp and 360 bp products with genus-specific and species-specific primers, respectively and were identified as M. agalactiae. Amplification of the p30 membrane protein gene yielded a 730 bp product. Restriction enzyme analysis of the 730 bp amplicon of p30 gene with RsaI and MboII yielded 2 fragments (654 bp and 74 bp) and 3 fragments (111 bp, 375 bp and 244 bp), respectively. Digestion with Sau3AI yielded two fragments (461 bp and 269 bp) while digestion with AluI resulted in 3 fragments (342 bp, 328 bp and 60 bp). The results of the present study revealed the presence of polymorphism at the respective positions of p30 membrane protein gene of M. agalactiae isolates examined by the restriction digestion. These polymorphisms can result into changes in pathogenesis and persistence inside the host and require further investigation of immunological outcome of these polymorphisms. Key words: Mycoplasma agalactiae, polymerase chain reaction (PCR), restriction enzyme, p30.