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The Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF 1 (oligopeptide permease transporter) which results in expression of a truncated OppF 1 . The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF 1 gene in the MS-H genome. Wild-type oppF 1 was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF 1 -tetM or with a similar vector lacking oppF 1 coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF 1 -tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF 1 detected bands of expected size for full-length OppF 1 in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF 1 -tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF 1 -tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF 1 -tetM (passage 27) showed that the chromosomal copy of the mutated oppF 1 had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF 1 impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species. RESEARCH HIGHLIGHTS The full-length OppF 1 was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF 1 impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF 1 in MS-H was replaced with wild-type oppF 1 .

Mycoplasma synoviae (MS) is an economically important avian pathogen worldwide, causing subclinical respiratory tract infection and infectious synovitis in chickens and turkeys. A temperature-sensitive (ts + ) live attenuated vaccine MS-H, derived from the Australian field strain 86079/7NS, is now widely used in many countries to control the disease induced by MS. Differentiation of MS-H vaccine from field strains is crucial for monitoring vaccination programs in commercial poultry. Comparison of genomic sequences of MS-H and its parent strain revealed an adenine deletion at nucleotide position 468 of the MS-H oppF-1 gene. This mutation was shown to be unique to MS-H in further comparative analyses of oppF-1 genes of MS-H re-isolates and field strains from Australia and other countries. Based on this single nucleotide, a combination of nested PCR and high-resolution melting (HRM) curve analysis was used to evaluate its potential for use in differentiation of MS-H from field strains. The mean genotype confidence percentages of 99.27 and 48.20 for MS-H and field strains, respectively, demonstrated the high discriminative power of the newly developed assay (oppF PCR-HRM). A set of 13 tracheal swab samples collected from MS-H vaccinated specific pathogen free birds and commercial chicken flocks infected with MS were tested using the oppF PCR-HRM test and results were totally consistent with those obtained using vlhA genotyping. The nested-PCR HRM method established in this study proved to be a rapid, simple and cost effective tool for discriminating the MS-H vaccine strain from Australian and international strains in pure cultures and on tracheal swabs., (Copyright © 2017 Elsevier B.V. All rights reserved.)