Melbourne Veterinary School - Theses
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ItemExploration of the basis of virulence attenuation and development of recombinant derivatives in a Mycoplasma synoviae live vaccine( 2017)Mycoplasma synoviae is an important avian pathogen that is responsible for significant economic losses in the poultry industry worldwide. It is able to infect chickens, turkeys and other avian species, causing subclinical respiratory tract infections, synovitis, and egg abnormalities. A live attenuated temperature-sentitive (ts+) vaccine strain, MS-H, is widely used in many countries to prevent infection with M. synoviae. Due to its ts+ phenotype, MS-H does not grow at the core body temperature of birds, but colonises the upper respiratory tract and establishes a solid protection against wild-type M. synoviae. In rare cases, the ts+ MS-H strain may revert to a non-temperature-sensitive (ts−) phenotype, but this is not associated with a significant increase in the pathogenicity in MS-H re-isolates, indicating that factors other than temperature sensitivity may be involved in the attenuation of the vaccine. To better characterise the molecular basis of the vaccine attenuation, MS-H and its parent 86079/7NS were subjected to whole genome sequencing. Comparative analysis of the genome organisations of the MS-H, 86079/7NS and other published M. synoviae strains revealed a number of features unique to MS-H that can be used as novel genetic markers in routine diagnostic tests. In M. synoviae strains MS-H and 86079/7NS, a 50-kb genomic inversion was observed for the first time amongst M. synoviae strains. Two newly identified horizontally transferred genes that encode a deoxyribose-phosphate aldolase and an ATP-dependent DNA helicase were noted for MS-H and 86079/7NS. Genomic comparison of MS-H and its parent strain 86079/7NS revealed 33 single nucleotide polymorphisms (SNPs) (excluding the highly variable vlhA gene), including 11 nonsynonymous SNPs that may havIe functional consequences. A frameshift mutation causing a premature termination of translation was found in oppF-1, a gene that encodes an ATP-binding protein that is a component of an oligopeptide transporter. This frameshift is likely to have inactivated the OppF protein function, suggesting that this mutation may play a significant role in the attenuation of MS-H. A nested PCR combined with high-resolution melting (HRM) curve analysis, based on the frameshift mutation within oppF-1 gene, was developed. This assay could reliably differentiate MS-H from all field strains tested in this study and was efficient for both cultures and swabs from clinical cases. In addition, this study investigated the potential for use of the MS-H vaccine as a delivery vector for foreign antigens using infectious bronchitis virus (IBV) as a model, aiming at the development of recombinant poultry vaccine candidates, especially for those pathogens affecting the upper respiratory system. To achieve this, expression vectors were constructed based on an oriC plasmid (pMAS-LoriC), which is capable of freely replicating in M. synoviae. Gibson assembly was used as a versatile strategy for constructing the vectors. The variable lipoprotein haemagglutinin gene (vlhA) promoter was used to drive expression of partial S1 and N IBV genes in MS-H. The expression of IBV N or S1 mRNA in MS-H recombinants was detected by quantitative real-time PCRs (qPCRs). Western blotting confirmed the expression of IBV partial S1 and N proteins using polyclonal antibodies specific for S1 and IBV respectively. This is the first report of successful expression of a non-native gene in M. synoviae. Thus the studies reported in this thesis have contributed to (1) development of our understanding of putative virulence factors in M. synoviae, (2) development of an assay to discriminate MS-H from field strains, and (3) laid the foundation for the development of novel recombinant vaccines to control other upper respiratory pathogens.