Development of mismatch amplification mutation assay for the rapid differentiation of Mycoplasma gallisepticum K vaccine strain from field isolates.

Mycoplasma gallisepticum causes respiratory diseases and reproduction disorders in turkeys and chickens. The infection has considerable economic impact due to reduced meat and egg production. Because elimination programmes are not feasible in a large number of poultry farms, vaccination remains the only effective measure of disease control. Differentiating vaccine strains from field isolates is necessary in the control of vaccination programmes and diagnostics. The aim of this study was to develop a polymerase chain reaction based mismatch amplification mutation assay (MAMA) for the discrimination of K vaccine strain (K 5831, Vaxxinova Japan K.K.). After determining the whole genome sequence of the K strain, primers were designed to detect seven different vaccine-specific single nucleotide polymorphisms. After evaluating preliminary results, the MAMA-K-fruA test detecting a single guanine-adenine substitution within the fruA gene (G88A) was found to be the most applicable assay to distinguish the K vaccine strain from field isolates. The detected K strain-specific single nucleotide polymorphism showed genetic stability after serial passage in vitro, but this stability test should still be evaluated in vivo as well, investigating a large number of K strain re-isolates. The MAMA-K-fruA assay was tested on a total of 280 culture and field samples. The designed assay had 102 and 103 template copy number/µl sensitivity in melt-curve analysis based and agarose-gel based assays, respectively, and showed no cross reaction with other avian Mycoplasma species. The new MAMA provides a time- and cost-effective molecular tool for the control of vaccination programmes and for diagnostics.

Genotyping Mycoplasma gallisepticum by multilocus sequence typing.

Mycoplasma gallisepticum causes chronic respiratory disease and reproductive disorders in many bird species, resulting in considerable economic losses to the poultry industry. Maintenance of M. gallisepticum-free flocks is the most adequate method to control infection. To this end, monitoring systems and vaccination programs with live vaccine strains are applied worldwide. There is strong demand for efficient epidemiological investigation tools to distinguish M. gallisepticum strains in order to control disease. Up to now, multilocus sequence typing (MLST) has been regarded as gold standard for genotyping bacteria due to its good reproducibility and high discriminatory power. The aim of this study was to develop an MLST assay which can determine phylogenetic distances between M. gallisepticum strains. After analysing more than 30 housekeeping genes, six loci (atpG, dnaA, fusA, rpoB, ruvB, uvrA) were selected for the MLST assay due to their genomic location and high diversity. Examination of 130 M. gallisepticum strains with this MLST method yielded 57 unique sequence types (STs) with a 0.96 Simpson's index of diversity. Considering the large number of STs and high diversity index, this MLST method was found to be appropriate to discriminate M. gallisepticum strains. In addition, the developed method was shown to be suitable for epidemiological investigations, as it confirmed linkage between related strains from outbreaks in different farms. Besides, MLST also suggested high impact of extensive international trade on the spread of different M. gallisepticum strains. Furthermore this method can be used for differentiation among vaccine and field strains.