Targeted Molecular Typing of Pathogenic Avian Mycoplasmas

Intraspecies identification (DNA "fingerprinting") of pathogenic avian mycoplasmas is a powerful tool for epidemiological studies and monitoring strain identity. However the only widely method available for Mycoplasma gallisepticum (MG) and M. synoviae (MS)wasrandom amplified polymorphic DNA (RAPD). This project aimed to develop alternative and supplementary typing methods that will overcome the major constraints of RAPD, such as the need for isolation of the organism in pure culture and the lack of reproducibility intrinsic in the method. Our strategy focussed on recognition of molecular markers enabling identification of MG and MS vaccine strains and, by extension, pathogenic potential of field isolates. Our first aim was to develop PCR-based systems which will allow amplification of specific targeted genes directly from clinical material. For this purpose we evaluated the degree of intraspecies heterogeneity in genes encoding variable surface antigens uniquely found in MG all of which are putative pathogenicity factors. Phylogenic analysis of targeted sequences of selected genes (pvpA, gapA, mgc2, and lp) was employed to determine the relationship among MG strains.. This method, designated gene targeted sequencing (GTS), was successfully employed to identify strains and to establish epidemiologically-linked strain clusters. Diagnostic PCR tests were designed and validated for each of the target genes, allowing amplification of specific nucleotide sequences from clinical samples. An mgc2-PCR-RFLP test was designed for rapid differential diagnosis of MG vaccine strains in Israel. Addressing other project goals, we used transposon mutagenesis and in vivo and in vitro models for pathogenicity to correlated specific changes in target genes with biological properties that may impact the course of infection. An innovative method for specific detection and typing of MS strains was based on the hemagglutinin-encoding gene vlhA, uniquely found in this species. In parallel, we evaluated the application of amplified fragment length polymorphism (AFLP) in avian mycoplasmas. AFLP is a highly discriminatory method that scans the entire genome using infrequent restriction site PCR. As a first step the method was found to be highly correlated with other DNA typing methods for MG species and strain differentiation. The method is highly reproducible and relatively rapid, although it is necessary to isolate the strain to be tested. Both AFLP and GTS are readily to amenable to computer-assisted analysis of similarity and construction of a data-base resource. The availability of improved and diverse tools will help realize the full potential of molecular typing of avian mycoplasmas as an integral and essential part of mycoplasma control programs.