Molecular detection and differentiation of mycoplasma species in swine lung tissues

Mycoplasma species are ubiquitous and cause both primary and secondary infections in swine. Most of these Mycoplasma species are fastidious and require stringent growth requirements. Molecular detection and differentiation of these Mycoplasma species directly from field samples have greater diagnostic value. A total of 57 porcine tissue samples collected from different regions of Tamil Nadu suspected of respiratory tract infections were screened with a PCR assay targeting the 16SrRNA gene of Mycoplasma and a multiplex PCR to identify and differentiate M. hyopneumonia, M. focculare, and M. hyorhinis infections. The PCR-based screening with 16SrRNA genus-specific primer confirmed Mycoplasma infection in four (7%) of the 57 samples screened with a specific amplicon of 717 bp. Multiplex PCR analysis of all 57 samples showed a similar positivity rate of 7% (n=4), demonstrating the presence of three 1129 bp and one 754 bp amplicons specific to M. hyorhinis and M. flocculare genome respectively in pneumonic lesions of swine from Tamil Nadu. The specificity of the M. hyorhinis genome was further confirmed by sequencing and BLAST analysis. A BLAST homology search revealed 99% sequence identity with European Mycoplasma isolates. Phylogenetic analysis in MEGA X, through the Maximum Likelihood tree (MLT) method revealed specific clustering along with M. hyorhinis species. The assembled partial contig sequence M. hyorhinis was deposited in the GenBank database under the accession number MW822751.