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Home » Archive » 2015

TDK conference 2015

Development of a Molecular Biological Method for Differentiation of Wild-type and Vaccine Strains of Mycoplasma synoviae
Csepreghy Anna - year 4
HAS-CAR, Institute for Veterinary Medical Research
Supervisors: Gyuranecz Miklós DVM, Kreizinger Zsuzsa DVM

Abstract:

Mycoplasma synoviae is a worldwide spread bacterium, which can cause infectious synovitis, airsacculitis and eggshell apex abnormality in chickens and turkeys. The thermosensitive (ts+) MS-H vaccine strain is often used in the control of M. synoviae infections. The ability to differentiate the vaccine strain from wild-type isolates is essential for the evaluation of the effect of control programs. In certain cases, distinguishing the ts+ and ts- strains can be necessary, because the bacterium can lose its thermosensitive phenotype during in vivo passage. Fast and accurate identification of the M. synoviae strains is not possible with previously developed assays; moreover, some of the assays need special laboratory equipment.

In the current study we presented novel genotyping assays targeting two known single nucleotide polymorphisms (SNPs) on the obg gene which clearly differentiate the ts+ MS-H vaccine strain, ts- MS-H re-isolate and wild-type M. synoviae strains.

We developed two mismatch amplification mutation assays (MAMA) to identify SNPs. The assays do not need the cultured bacteria to be effective. Two SNP-specific primers are competing in the melt-MAMA, which is based on the analyses of the difference in the melting points of primers after a real-time polymerase chain reaction (PCR). We extended the sequence of one competing primer with a 14-15 base pair long GC-clamp, so that the melting points separate clearly. The other system is an agarose gel-based MAMA, which can be performed with conventional PCR, and we can differentiate the PCR products by size during gel electrophoresis. We adjusted the methods with the M. synoviae reference strain (NCTC 10124), the ts+ vaccine strain (Vaxsafe MS-H), and two synthetic control sequence (containing all the specific SNPs). We also tested the methods on clinical samples.

Our assays are specific and sufficiently sensitive to be used in practice. The presented methods are preferable to previous assays because they are rapid, and they can be performed directly on clinical samples. They are cost effective as they can be performed on basic laboratory equipment, and they do not require expensive reagents.

Application of these methods will help to differentiate the ts+ MS-H vaccine strain, ts- MS-H re-isolate and wild-type M. synoviae strains, which allows to track the M. synoviae infections, thus facilitating control programs.



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