Identification of Strain-Specific Sequences That Distinguish a Mycoplasma gallisepticumVaccine Strain from Field Isolates

ABSTRACTDespite attempts to control avian mycoplasmosis through management, vaccination, and surveillance, Mycoplasma gallisepticumcontinues to cause significant morbidity, mortality, and economic losses in poultry production. Live attenuated vaccines are commonly used in the poultry industry to control avian mycoplasmosis; unfortunately, some vaccines may revert to virulence and vaccine strains are generally difficult to distinguish from natural field isolates. In order to identify genome differences among vaccine revertants, vaccine strains, and field isolates, whole-genome sequencing of the M. gallisepticumvaccine strain ts-11 and several “ts-11-like” strains isolated from commercial flocks was performed using Illumina and 454 pyrosequencing and the sequenced genomes compared to the M. gallisepticumRlowreference genome. The collective contigs for each strain were annotated using the fully annotated Mycoplasmareference genome. The analysis revealed genetic differences among vlhAalleles, as well as among genes annotated as coding for a cell wall surface anchor protein (mg0377) and a hypothetical protein gene, mg0359, unique to M. gallisepticumts-11 vaccine strain. PCR protocols were designed to target 5 sequences unique to the M. gallisepticumts-11 strain: vlhA3.04a, vlhA3.04b, vlhA3.05, mg0377, and mg0359. All ts-11 isolates were positive for the five gene alleles tested by PCR; however, 5 to 36% of field isolates were also positive for at least one of the alleles tested. A combination of PCR tests for vlhA3.04a, vlhA3.05, and mg0359was able to distinguish the M. gallisepticumts-11 vaccine strain from field isolates. This method will further supplement current approaches to quickly distinguish M. gallisepticumvaccine strains from field isolates.