Microbiology & Immunology - Theses

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    Genetic manipulation of type D Pasteurella multocida for vaccine development
    Wright, Catherine Louise ( 1997-10)
    Progressive Atrophic Rhinitis (PAR) is a serious complex disease of young swine characterized by sneezing, atrophy of the nasal turbinates, shortening and twisting of the snout and reduction in weight gain. Although the aetiology of the disease is complex, infection with the bacterium toxigenic Pateurella multocida, is considered essential. A dermonecrotic toxin (DNT) produced by toxigenic strains of type D P. multocida is central to the resorption of the nasal bone structures characteristic of the infection. The P. multocida DNT gene toxA has been previously cloned, sequenced and genetically manipulated in order to develop a vaccine for PAR. These earlier studies demonstrated that DNT-specific antibodies produced in pigs by vaccination with the purified genetically inactivated DNT derivative (toxoid) resulted in the protection of the animals against experimentally induced PAR. An alternative approach to using a subunit vaccine for PAR is to express the toxoided gene from P. multocida either from the chromosome or a plasmid thus providing a live vaccine that could present to the porcine immune system a full spectrum of bacterial antigens in addition to the DNT. (For complete abstract open document)
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    Controlled production of tryptophan by genetically-manipulated strains of Escherichia coli
    Cowan, Peter J. ( 1992)
    The tryptophan productivity of the genetically-manipulated strain JP4153 was increased 2.5-fold by introducing pMU78, a medium copy-number plasmid carrying a feedback-resistant trp operon. JP4153(pMU78) produced 23.5 g/l of tryptophan at a rate of 0.7 g/l/h when grown at 37 degrees C in a defined glucose and ammonium salts medium in a bench-scale fermentor. During prolonged cultivation in the presence of antibiotic, the recombinant strain generated faster-growing, production-defective variants, which harboure mutated derivatives of pMU78. Insertion sequences were responsible for the two predominant types of mutation. The plasmid element ISI02 mediated deletions extending into the promoter-proximal region of the plasmid-borne trp operon. ISI0-Right, a chromosomal element, inserted into the promoter/trpE region of the plasmid. Three methods were employed to increase the structural stability of JP4153(pMU78) during the course of the production process. First, the growth of seed cultures was carried out at 30 degrees C, the permissive temperature for the trpS378 mutation carried by the host strain. Second, the seed culture medium was modified by the addition of yeast extract, which appeared to reduce the selective disadvantage conferred by the plasmid. Third, ISI02was deleted from pMU78 to create pMU88. (For complete abstract open document)
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    Immunobiology and molecular virology of fowl adenoviruses
    Erny, Katrina M. ( 1990)
    Three fowl adenoviruses (FAVs) representing 3 serotypes were examined both in vitro and in vivo. The choice of FAVs for this study was influenced by their suitability for future development as recombinant viral vectors to deliver vaccines to commercial poultry flocks. In vivo studies of these viruses demonstrated the wide range of immunogenicity and pathogenicity to be found amongst FAVs. Pathogenicity did not correlate with serotype, however, a direct relationship was evident between pathogenicity and stimulation of a systemic antibody response. It was also shown that this systemic antibody was not required for protection against re-infection with homologous FAVs, thereby implicating local and cell mediated immunity in resistance to FAV infection. A causal relationship was demonstrated between a group of highly virulent FAVs and the poultry disease, inclusion body hepatitis. This is one of very few reports establishing a direct relationship between FAV and disease. It is envisaged that different FAV recombinant vectors might be administered as an aerosol either simultaneously or consecutively at different stages of chicken development. The feasibility of these vaccination strategies was demonstrated and concluded that simultaneous infection with different FAV serotypes does not compromise the individual immune responses to each virus. Consecutive vaccination demonstrated the importance of using serologically distinct FAV serotypes. The in vivo cross protection between serotypes 4 and 10, along with the demonstration of strong homologies between these serotypes at the genomic level supported the proposal that these 2 classical serotypes should be combined. The in vitro examination of FAV showed that the replication kinetics of non-oncogenic FAV are very similar to those of the highly oncogenic FAV, CELO virus. For each of the 3 FAVs studied, a restriction enzyme map was constructed. These physical maps of the 45-46 Kb FAV genomes were particularly useful for identifying specific regions of the genome transcribed late in infection. For one of these FAVs, a transcription map was constructed for the late phase of replication between 10 and 24 hpi. This transcription map demonstrated similarities between FAV and human adenovirus (HAV) not previously reported. These studies provide a basis upon which to conduct future research toward the development of recombinant FAV viral vectors. The immunological studies predict that such vectors would be very flexible for the delivery of foreign genes to chickens at various ages to stimulate either local or systemic immune responses as required. The restriction enzyme and transcription maps will be invaluable to research directed at manipulation of specific regions of the FAV genome and identification of essential and non-essential genes.
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    The development of attenuated Salmonella typhimurium as a vaccine vector
    Dunstan, Sarah Jane ( 1997)
    Investigation of attenuated Salmonella typhimurium vaccine vectors in the murine model is an important step in the development of new and effective multivalent S. typhi vaccines. This thesis examined a number of parameters involved in the effective delivery of heterologous antigens from live attenuated S. typhimurium. S. Typhimurium was engineered to express the model heterologous antigen C fragment and the affects of (i) the type of attenuating mutation the S. Typhimurium harbours, (ii) the type of promoter used to direct expression of the heterologous antigen, and (iii) the type of plasmid used to encode the heterologous antigen, were examined. In addition, the immunobiological consequences of encoding murine interleukin-6 (mIL-6) in S. Typhimurium were investigated. Information obtained from optimisation studies was used to construct a novel S. Typhimurium/rotavirus vaccine, which was evaluated in the murine model. (From Summary)