Species Identification of Avian Mycoplasmas by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Analysis

Abstract

Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were used to detect and differentiate four pathogenic species (Mycoplasma gallisepticum, M. iowae, M. meleagridis, and M. synoviae) and ten nonpathogenic species of avian mycoplasma. A sequence of 1026 base pairs within the gene for 16S ribosomal RNA (16S rRNA) from avian mycoplasmas was successfully amplified by PCR with oligonucleotide primers (M16SPCR5' and M16SPCR3') common to all avian mycoplasmas tested. Restriction endonucleases (REs) with unique restriction sites, selected by computer-assisted analysis of known sequences of the amplified segment of avian mycoplasma, were then used to digest the PCR products. After electrophoresis of the resulting RE fragments, the RFLP patterns were compared. Combinations of up to six REs (HpaI, HhaI, HaeIII, HphI, FokI, and NlaIV) produced unique RFLP patterns by which the 14 species of avian mycoplasmas could be differentiated. The newly classified avian species M. imitans was also investigated by this method; M. imitans and M. gallisepticum gave identical RFLP patterns with the REs used in this study. The results obtained by the PCR and RFLP analysis were in agreement with current methods for species identification of avian mycoplasmas.