Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates

Abstract

Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates

“…The developed assays aim to support routine diagnostics by determining the successful vaccination of the animals or confirming the M. gallisepticum-free status of a flock. Based on the diagnostic application of previously established MAMAs for the discrimination of live Mycoplasma vaccine strains from wild strains (38), submitting the DNA pool of samples from a small group of animals (at least 4 pools from 20 birds/house) to test the presence of the vaccine/pathogen is the most appropriate method to reflect the status of a flock. In order to achieve the most definite results of the discrimination of M. gallisepticum vaccine and wild-type strains, the combined use of all presented PCR tests is recommended.…”

Development, validation and field evaluation of a quantitative real-time PCR able to differentiate between field Mycoplasma synoviae and the MS-H-live vaccine strain

“…Furthermore, Kreizinger and others described a melt-curve and agarose gel based Mismatch Amplification Mutation Assays (Kreizinger et al, 2015). Although this assay was able to distinguish between the ts + MS-H vaccine strain, its tsre-isolates and wild-type M. synoviae isolates could be used directly on tracheal swab samples; genotyping in case of mixtures of strains (the presence of both field M. synoviae as well as MS-H vaccine strain) was unreliable as ambiguous melting curves were obtained.…”

“…Recently, polymorphisms in the obg gene of the MS-H-live vaccine strain have been described, which may represent important targets for the development of diagnostic differentiating PCR tests (Shahid et al, 2013a, b;Shahid et al, 2014;Kreizinger et al, 2015).…”

Development of mismatch amplification mutation assays for the differentiation of MS1 vaccine strain from wild-type Mycoplasma synoviae and MS-H vaccine strains

“…The ts - MS-H re-isolate was identified by the MS-H1 and MS-H2 MAMAs, based on the point mutations at nt367 (G) and nt629 (C) in the obg gene [12]. …”