Veterinary Science - Theses
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ItemAdjuvant-mediated manipulation of immune memory resilience( 2017)Vaccine-based protection relies on rapid and augmented immune recall following pathogen encounter. With the evidence that some pathogens are capable of manipulating host immune responses, the quality of the vaccine-induced immune responses plays an essential role in protection. In this context, vaccines should be focused on developing immune recall responses resistant to manipulation by such pathogens, a characteristic termed “immune memory resilience”. The overall aim of this study was to induce resilient immune memory in the face of adjuvant-mediated manipulation, taking advantage of locally induced immune responses in a sheep lymphatic cannulation model. Since the immune responses could be studied in real-time as they occur at the local lymph node and in the absence of generalized recirculation we compared induction of resilient immune memory using two adjuvants (alum and Quil A). Alum was used as a Th2 biased immune response inducing adjuvant; while Quil A was used to induce Th1 biased immune responses. The antibody isotype concentrations (IgG2 for Th1 and IgG1 for Th2) were monitored. Immune memory was not resilient when induced with a single vaccine of one adjuvant and subjected to manipulation by the alternate adjuvant. Interestingly, however, two vaccinations with the same adjuvant dramatically increased the level of resilience (i.e. quality) of the immune memory, in vivo, upon opposite adjuvant challenge (chapter 3). The absolute dilemma of using large animal models (specifically sheep) in immunological research involves lack of an immunological toolkit. With the availability of sheep genome-sequencing data, RT-qPCR has become the most reliable and cost-effective way to analyse immune responses in vivo. Thus, identification of the most consistently transcribed reference genes for sheep efferent lymphatic cellular fraction was carried out in the current study (chapter 4). Using appropriately standardized reference genes for normalization, the induction of resilient immune memory in vivo following two vaccinations with the same adjuvant and challenge with the alternate adjuvant was studied (chapter 5). Differentiating Th1 and Th2 subset biased immune responses following each vaccination was focused in terms of master regulator transcription factor expression (T-bet for Th1 and GATA3 for Th2) and signature cytokine gene expression (IFN-γ for Th1 and IL-4 for Th2) in vivo. Resilient immune memory induction with two vaccines of the same adjuvant was closely regulated at master regulator transcription factor and signature cytokine gene expression. Th1 or Th2 lineage-specific signature cytokine producing cells also showed Th1 or Th2 lineage bias following each vaccination in vivo. Therefore the current study substantially enhanced the understanding of resilient immune memory induction in the face of adjuvant-mediated manipulation while establishing the foundation to use sheep lymphatic cannulation model in vaccine studies in vivo.
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ItemImmunological and virological investigations arising from the 2007 Australian equine influenza outbreak( 2016)During Australia’s equine influenza (EI) outbreak, horses were vaccinated in Victoria prophylactically using a recombinant canarypox- vectored vaccine. Humoral and cell–mediated immune responses were monitored following an accelerated primary course reduced to 14 days. To demonstrate proof of freedom from EI, nasal swabs were taken from diseased and normal horses. Quantitative PCR was performed on samples from 559 horses, all negative for EI. Shedding of equine herpesviruses -1,-2 and -4 was correlated with the horse’s clinical status.
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ItemAssessment of Helicobacter pylori antioxidant enzymes as therapeutic targets( 2012)Helicobacter pylori is a significant gastric pathogen in humans, with gastritis and gastric neoplasia frequently resulting from chronic colonisation. The organism is reliant on its antioxidant enzyme system for survival, and this thesis proposes that components of this system – namely, the antioxidant enzymes superoxide dismutase (HpSOD), catalase (HpKatA), and thiolperoxidase (HpTpx) - present a viable therapeutic target in H. pylori infection. Using a pET Escherichia coli expression system, recombinant forms of the three enzymes from H. pylori were created and then trialled as vaccine antigens administered with an appropriate adjuvant via systemic and mucosal routes. Moreover, in order to assess for synergistic action, the enzymes were assessed both individually and in combination. These trials demonstrated that superoxide dismutase and thiolperoxidase are protective antigens, but no additional protection was observed with the combination vaccine. The essential requirement for the antioxidant system in H. pylori further led to speculation that inhibition of enzyme function may be an alternative mode of therapy. Monoclonal antibodies directed against the three enzymes were generated via hybridomas derived from hyperimmunised mice. Antibodies were characterised by class and isotype, binding properties and ability to inhibit enzyme function, in order to determine the most promising candidates for therapeutic trials. After demonstrating the presence of systemic antibodies at the gastrointestinal mucosal surface in a mouse trial, the candidate antibodies were administered via intraperitoneal injection to mice infected with H. pylori. Although the antibody therapy did not impair colonisation, there was a significant increase in a number of pro-inflammatory cytokines within gastric tissue following administration of the anti-HpSOD antibody. Investigating further, it was found that incubation of macrophages in the presence of recombinant HpSOD attenuated the cytokine response to lipopolysaccharide. These findings suggested that HpSOD may possess previously undescribed immunosuppressive properties, potentially facilitating long-term colonization. Possible pathway mechanisms are discussed, as well as supportive findings from work on other chronic pathogens.
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ItemInvestigations into the molecular basis of pathogenicity of the Australian infectious bronchitis viruses( 2012)Infectious bronchitis virus (IBV) is a highly infectious pathogen of poultry and has been detected in commercial flocks worldwide. Due to the labile nature of its large single-stranded RNA genome, numerous strains with widely varying properties have been detected and characterised so far. Australian IBV strains have been shown, using molecular techniques, to be highly divergent from international strains. The work described in this thesis aimed to identify and characterise a number of Australian IBV strains and to relate their pathogenicity to the genomic structure of the 3' end of their genome. Initially, high resolution melt (HRM) curve analysis was used to detect novel strains of IBV in field submissions. One such strain, N1/08, was found to have emerged as a result of recombination between subgroup 2 and subgroup 3 strains. An in vivo study determined that the pathogenicity of N1/08 was more similar to subgroup 2 strains. HRM curve analysis also detected a variant strain in the commercial preparation of the IBV vaccine, VicS. Two subpopulations were isolated from the VicS vaccine, namely VicS-v and VicS-del. It was determined that VicS-v and VicS-del grew similarly in embryonated chicken eggs, but that the pathogenicity of VicS-del was significantly lower than that of VicS-v in vivo. Remarkably, an equal ratio of VicS-v and VicS-del appeared more pathogenic in vivo than VicS-v alone. This suggested that there may be a synergistic relationship between VicS-v and VicS-del in vivo. Genetic characterisation of the VicS-del structural protein gene region found a number of differences compared to VicS-v, including a 40 bp deletion in the 3' UTR, providing an explanation for the distinct HRM curve pattern of the VicS vaccine. In order to investigate whether differences in the nucleotide sequence of the structural protein genes were reflected in differential transcription of the novel strains studied, the transcriptomes of N1/08, VicS-v and VicS-del and other reference Australian IBVs were examined. No difference in quantity of RNA transcripts was observed, however differences were observed in the number and lengths of transcripts between strains, including the lack of a gene 3 transcript for subgroup 2 strains. This investigation also found that the substantial gene rearrangements and mutations present in subgroup 2 strains may have abolished transcription of gene 3, which encodes a structural envelope protein.
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ItemCharacterization of the eg95 gene family in Echinococcus granulosus G6 genotype: implications for the efficacy of the EG95 vaccine against genotypes other than G1( 2011)Cystic echinococcosis (CE) in humans and livestock animals is caused by infection with the cestode parasite Echinococcus granulosus. The parasite has a worldwide distribution, causing human morbidity and mortality as well as economic losses. E. granulosus presents significant intraspecific variability and several different genotypes have been described varying with respect to host preference and other biological characteristics. The G1 genotype is the variant most commonly associated with human infections worldwide, with genotype G6 also causing a significant proportion of human CE cases. A recombinant vaccine, designated EG95, has been developed for use in animals to interrupt the cycle of transmission of E. granulosus, with a view to decreasing the transmission of the parasite to humans. The vaccine is based on an antigen expressed in the oncosphere of E. granulosus. EG95 is encoded by members of a family of closely related genes in the G1 genotype. Potential variability in the protein target of the EG95 vaccine in different genotypes of E. granulosus could lead to parasites of genotypes other than G1 being insusceptible to the vaccine. Currently there is no reliable information about the likely efficacy of the EG95 vaccine against genotypes other than G1. The principal aim of the research described in this thesis was to provide information about the variability of the eg95-related gene(s) and their predicted protein products in the G6 genotype of E. granulosus, and other genotypes, also providing data that could be used for assessment of the antigenicity of the EG95-related proteins in each genotype. Using genomic DNA cloning techniques, seven members of an eg95 –related gene family were characterised from the G6 genotype of E. granulosus. Three proteins are predicted to be encoded by these genes, with two genes considered to be pseudogenes. Investigations were undertaken to determine the ability of two of theEG95-related proteins, encoded by genes in the G6 genotype, to react with specific antibodies in the sera of sheep vaccinated with EG95 and shown to be protected against a challenge infection with the G1 genotype. Proteins encoded by genes in the G6 genotype displayed only limited antigenic cross-reactivity with the current EG95 vaccine antigen, suggesting that the current vaccine may not protect animals against an infection with parasites of the G6 genotype. Data presented in this thesis provides the information that would enable a G6 genotype-specific vaccine to be developed against E. granulosus, should this be considered a desirable addition to the available tools for control of CE transmission.