TDK conference 2019

Mycoplasma gallisepticum is a facultative pathogen agent with global distribution which causes respiratory diseases and reproduction disorders in turkeys and chickens. The infection reduces feed intake, weight gain, egg production, and hatchability as well. M. gallisepticum has a strong economic impact, thus the infection must be controlled. The most viable solution is eradication followed by keeping the stocks free of the pathogen, however, this objective is unfeasible in many farms. Vaccines can be used for prevention and antimicrobial agents for treatment.

In the past decades, several vaccine strains have been developed of which vaccine strains F, ts-11, 6/85 and the most recently developed K strain are proved to be efficient. Differentiating the individual vaccine strains from each other and from the wild-type strains is necessary in the control of vaccination programs and diagnostics. Out of various discriminating methods, molecular biological techniques are used recently. PCR (polymerase chain reaction) based MAMA (mismatch amplification mutation assay) methods are available for the rapid and reliable identification of the F, ts-11 and 6/85 vaccine strains.

The aim of this study was to develop an efficient MAMA method for the determination of K strain (K 5831, Vaxxinova BV., the Netherlands). After whole genome sequencing, gene sequences of the K strain and other online available M. gallisepticum strains were investigated. Vaccine-specific mutations were identified by computer analysis. Four different SNPs (single nucleotide polymorphisms) were found to be appropriate for further examination. Tests were performed on 274 samples including DNA of clinical samples and field isolates originating from different countries all over the world.

Results confirmed that the SNP in the fruA gene of the K strain is the most efficient one triggering guanine-adenine substitution in the 88th nucleotide, inducing asparagine acid - asparagine amino acid change in the protein encoded. In this assay, the vaccine-specific primer contains an additional 14 base pairs long GC-clamp. This difference in size can be easily detected by conventional (agarose-MAMA) or real-time (melt-MAMA) PCR, as the smallest difference in melting temperatures of the K vaccine strain and wild-type strains was 4,91°C.

The designed assay has sufficient specificity and sensitivity to be applicable as a routine diagnostic method, which is a great step towards efficient control of vaccination programmes and diagnostics.