Molecular analysis of pathogenicity of Mycoplasma synoviae

Abstract

Mycoplasma synoviae is a major pathogen of poultry globally, causing chronic respiratory disease and arthritis. Vaccination is an effective means for the control of the disease. The MS-H vaccine is a live attenuated temperature sensitive strain developed through chemical mutagenesis of an Australian field strain, 86079/7NS. Rarely, the temperature-sensitive MS-H strain may revert to a non-temperature-sensitive phenotype. Due to reversion to non-temperature-sensitive phenotype, vaccine isolates may be difficult to distinguish from field strains. Therefore, there is an urgent need for a reliable and rapid assay that can differentiate the MS-H vaccine from field isolates to evaluate the efficacy of the vaccination programs. However, developing such assays depends on a better understanding of molecular basis behind attenuation of MS-H.

Comparison of the whole genome of MS-H with that of its parent strain 86079/7NS in previous studies has shown 32 mutations in the MS-H genome, one of which was a frameshift mutation early in an oligopeptide permease transporter gene oppF. In this study polyclonal antibodies raised against peptides upstream and downstream of the mutation in OppF revealed that only N-terminus of the OppF was expressed in MS-H while the full version was expressed in 86079/7NS. The potential of the recombinant full-length OppF, N- (OppF-N) and C termini (OppF-C) of OppF, upstream and downstream of the mutation site, was evaluated for the discrimination of antibody responses to MS-H versus field strains in indirect ELISA. Comparison of optical densities obtained from the ELISAs based on OppF, OppF-N and OppF-C revealed that the indirect ELISA based on OppF-C had the potential to differentiate between MS-H and field strain antibody responses.

The impact of the oppF mutation on the MS-H phenotype was investigated in vitro using a mutant of MS-H complemented with a wild-type copy of oppF gene in the MS-H genome. The results revealed that truncation of oppF impacts on growth characteristics of the MS-Hand provide insight into the molecular pathogenesis of M. synoviae and perhaps of broader mycoplasma species.

In addition, to characterise the function of OppF protein in MS-H, the metabolite profile of MS-H was compared with that of its field reisolate which only differed in its oppF gene. The liquid chromatography–mass spectrometry analysis revealed a significant decrease in the abundances of amino acids in MS-H compared to that in its field reisolate. This result was consistent with the role of OppF as a peptide transporter. Also, differences found in the level of other metabolites in MS-H reflected some compensation of the OppF function by pathways involving other metabolites.

Moreover, in this study genomes of five M. synoviae isolates from MS-H vaccinated chicken flocks were determined and analysed to confirm their relationship with MS-H and also examine the stability of oppF mutation and other genetic markers of the MS-H vaccine. A total of 25 single nucleotide substitution/insertion/deletion including that of oppF existed among the isolates examined. Four of these mutations were those that had been reported between MS-H and its parent strain 86079/7NS, while the other 21 were found only in the MS-H reisolates tested in this study. Therefore, 28 out of 32 mutations previously detected between MS-H and 86079/7NS remained consistent in 5 MS-H reisolates. These results confirmed that M. synoviae isolates in this study were true MS-H reisolates and that majority of the mutations found in the MS-H vaccine are stable after passage in vivo under field condition.

The studies described in this thesis have contributed to expansion of our understanding of putative virulence determinants in M. synoviae and laid foundations for the development of a DIVA (Differentiation of Infected and Vaccinated Animals) test to examine variants of the MS-H vaccine. Also, this thesis provided some information on the dynamics of MS-H population after passage in vivo.