Melbourne Veterinary School - Theses
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ItemViruses of the other mammals: genomics and epidemiology of marsupial herpesviruses( 2018)Improving wildlife population health requires an understanding of the infectious agents within those populations. Historical accounts of herpesviruses in marsupials indicate that they can have a significant impact on animal health. This evidence is strongest for the macropodid alphaherpesviruses, but with improvements in molecular diagnostics the discovery of novel viruses has outpaced our understanding of their impact and significance. This thesis aimed to expand our knowledge of marsupial herpesviruses by examining relationships between marsupial herpesviruses and other herpesviruses, and by describing the clinical significance of infection. This thesis also aimed to improve diagnostic tools for detecting herpesvirus infection in marsupials. The core genomes of three marsupial herpesviruses were determined; macropodid alphaherpesvirus 1 (MaHV1, infecting wallabies), phascolarctid gammaherpesvirus 1 (PhaHV1, infecting koalas) and vombatid gammaherpesvirus 1 (VoHV1, infecting wombats). MaHV1 had a similar genome arrangement to other simplexviruses, but contained gene clusters that may be unique to the macropodid simplexviruses. PhaHV1 and VoHV1 had a shared gene arrangement and were likely to have speciated from a common ancestor. Over 30 new ORFs were identified within the genomes. Functional enzymatic characterisation was performed on two viral NTPDase homologs encoded within the two gammaherpesviruses. NTPDase activity was confirmed for the PhaHV1 homolog but not the VoHV1 homolog. Koalas are host to two divergent gammaherpesvirus species, PhaHV1 and -2. To understand the clinical significance of each individual virus a large molecular epidemiological study of 810 koalas from 7 separate geographic regions was conducted. Samples were tested using a rapid and differential PCR-HRM assay. Available signalment and clinical observation data was analysed in comparison to infection status through univariable and multivariable logistic regression analysis. Additional factors considered were location, year, body condition, fecundity in females, as well as the presence of other infectious agents (Chlamydia pecorum and koala retrovirus). PhaHV1 and -2 were present in 17% and 22% of koalas tested (state-wide), although some variation from the state average was observed in particular populations. Neither virus was associated with a particular sex. PhaHV1 detection was uniquely associated with the presence of koala retrovirus as well as increasing age. PhaHV2 detection did not change with age, which may indicate differences in how these two viruses are acquired over the life of the animal. Both viruses were positively associated with genital tract abnormalities, lowered fertility in females, emaciated body condition, urinary tract infection (wet bottom) and detection of C. pecorum, although the strength of these associations varied by sex and herpesvirus species. To further the development of herpesviruses serological tools, this thesis examined the ability of four commercially-available immunoglobulin-binding reagents to bind serum antibodies from 17 species within the Marsupialia and Monotremata. Serum samples were assessed for binding using immunoblots and ELISAs to three microbially-derived proteins; staphylococcal protein A, streptococcal protein G and peptostreptococcal protein L, and to an anti-kangaroo antibody. The inter- and intra-familial binding patterns of the reagents to serum immunoglobulins varied and evolutionary distance between animal species was not an accurate predictor of the ability of reagents to bind immunoglobulins.
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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.