Application of high-resolution melting-curve analysis on pvpA gene for detection and classification of Mycoplasma gallisepticum strains

Mycoplasma gallisepticum (MG) is an avian species pathogen which causes heavy economic losses in the poultry industry. The purpose of this study was to determine genomic diversity of 14 MG field strains from chicken, Chuker partridge and peacock collected during 2009-2012 in Iran by polymerase chain reaction and partial sequencing of the pvpA gene. A High-Resolution Melting (HRM) technique was also developed and applied to differentiate between field and vaccine strains. Sequencing of the pvpA gene revealed a 51 nucleotide deletion, within DR-1 and DR-2, among MG strains from chicken and partridge whilst 63 nucleotides were deleted in MG strain from peacock. One nucleotide substitution was also observed among chicken MG strains. Phylogenetic analysis of the sequences clustered all of the Iranian MG strains into two clades or phylogeny groups; the strains from chicken and partridge in one group (group 1) and the strain from peacock into another group (group 4). HRM analysis has also produced comparable outcome to those of sequencing; four distinct melting curves which correspond to the three MG strains from chicken, Chukar partridge and peacock and ts-11 vaccine strain. Overall, findings of this study point towards a single source of infection for the chicken and partridge MG strains and likelihood of the strains being native and endemic in Iran. Peacock considered as an exotic species in Iran, hence the genetic distance for the pvpA gene. MG can be transmitted easily among different avian species and this distinct peacock strain may pose a threat to poultry industry. Our findings also show that molecular variation among pvpA gene of MG strains could be revealed using the relatively rapid and affordable FIRM technique.