Microbiology & Immunology - Theses
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ItemAustralian venoms and care of the envenomed patient( 1979)Investigations described in this thesis were undertaken by the candidate between 1967 and 1979 in the Department of Immunology Research at the Commonwealth Serum Laboratories, Parkville. A number of chapters have been shortened following the publication of relevant papers. The summaries and conclusions are drawn from this published material and only new data presented in the actual chapters. By this combination, advantage is taken of efforts of the editors of journals to compress data and eliminate material of only peripheral interest. The department's involvement in venom research began in a small way with a revival of interest into aspects of the venom of the Sydney funnel-web spider (Atrax robustus). The arrival in the laboratory in 1967 of post mortem samples from a young man whose death followed a bite by a blue-ringed octopus (Hapalochlaena maculosa) added further impetus to the study of venoms. By 1970 it had become clear that although areas of immunological research such as antilymphocyte globulins were promising, the explosion of the population of immunological workers encouraged the writer to direct attention more towards venom research. The use of immunological techniques, particularly aimed at the detection and quantitation of venom, appeared to offer some very interesting avenues for investigation. The eventual development of these procedures had immediate clinical and forensic applications. For the first time specific venom components could be detected in the plasma and urine of human snake bite victims. Apart from forensic uses, clinical syndromes could be ascribed to specific snake venoms and the degree of envenomation at the time of blood sampling accurately determined. More recently determination of plasma levels of snake venom in monkeys has allowed some rationalisation of first aid measures.
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ItemGenetic manipulation of type D Pasteurella multocida for vaccine development( 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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ItemControlled production of tryptophan by genetically-manipulated strains of Escherichia coli( 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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ItemAntigenicity and immunological studies of sea wasp (Chironex fleckeri) and sea snake venoms( 1977)The studies encompassed in the major portion of this thesis record investigations of the antigenicity of sea wasp venom and their extension to the development of an active immunizing agent. They follow an extensive study of the venom's biological properties involved in its toxicity and the potential counteraction of these by antivenene. The latter studies have led to the introduction to clinical medicine of an antivenene effective not only against the venom of Australia's most venomous marine creature (Chironex fleckeri) but also against a closely related and visually similar venomous jellyfish (Chiropsalmus quadrigatus). Potentially a more important drug for the medical armamentarium,an immunizing agent in the form of a venom toxoid vaccine, has been researched, developed and evaluated in laboratory animals. When further safety testing has been carried out to satisfy the requirements of the New Drug Evaluation Committee, it will be possible to evaluate the vaccine in humans. If adequate antibody responses are obtained, it should be possible to offer protection against lethal sea wasp envenomation to those exposed to risk in tropical waters. Studies on venoms of another family of marine venomous creatures, the sea snakes, also form a part of this thesis. They were undertaken with a view to studying their relationship to the Australian tiger snake as expressed in neutralization of their venoms by a selection of antivenenes. The work reported in the first sea snake paper utilized the usually practised in vitro combination method, and the second sought to confirm or deny the poorly documented claim that in vitro combination was not an adequate guide to in vivo (clinical) usage by re-assessing the values using the in vivo protection method in laboratory animals. A substantial denial of the previously accepted view that in vitro combination might not be a good guide to in vivo experience, emphasized the need for statistical treatment of the results. It is hoped that such analyses will be widely accepted as the preferred method of evaluating results in future, as in other biological fields. (From Introduction)
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ItemStudies in the etiology of rheumatic fever( 1952)The aim of the present study has been prompted by an endeavour to find on the basis of the allergic theory, the sensitizing substance or substances which may be responsible for the rheumatic process. This search for the sensitizing antigen involved two processes. Firstly, the preparation of a variety of substances from sources, which on the basis of the allergic theory were believed to have etiological significance and secondly, the utilization of a suitable method to demonstrate that an antigen-antibody reaction does occur. The successful finding of the sensitizing agent would establish the validity of the allergic theory. The failure to find it, however, would be no proof against it, though it may be incompatible with some of the hypotheses that have been postulated in an attempt to specify the mechanism of the allergic process eg. the hypothesis of auto-antibodies. The complexity of the problem which is testified by the still obscure nature of the disease after 50 years of research necessitated a variety of approaches, at times highly empirical, in the present study. Some of these have been designed to clarify doubtful results of previous investigations. Others have never been tried before. Indeed, to find some original approach to the problem of rheumatic fever, that has escaped the notice of previous investigators is a task of its own. Not less arduous is the task of studying the literature which has accumulated on the subject during the last fifty years. Waksman (1949), in a paper dealing with the etiology of rheumatic fever, lists over 700 references, at the end of which, he refers to other authors for an “exhaustive review” of the subject. (From Introduction)
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ItemUrinary tract infection in patients with spinal cord injury( 1975)This study was made to ascertain the aetiology, origin and pathways of infection as well as the reason for recurrence of urinary tract infection in male patients with permanent indwelling catheter, at the Spinal Injuries Unit, Austin Hospital, Australia. There has been no detailed bacteriological report published since the Unit opened in 1956. Since urinary infections are frequent during the life of spinal paralytics, it is important in the management of such patients to determine whether it is relapse or re—infection which plays the major role of infection. The main body of this thesis presents the findings of a detailed search for wall—defective bacteria (L—forms) in an attempt to confirm or deny the hypothesis that persistent L—forms play a part in the repeated isolation of the same organism from a patient over a number of years. The aetiology of urinary infection occurring in new admissions was a reflection of the ward flora present at different times of survey, which in turn depended on the disinfection measures taken. Gram—negative, urea splitting organisms such as Klebsiella, Proteus, and Providence as well as the nosocomial bacteria Acinetobacter, Pseudomonas and Serratia were found to be more common in causing urinary infection than the traditional Escherichia coli and Streptococcus faecalis. The chlorhexidine solution used for bladder irrigation was shown to be the source of Acinetobacter infections acquired during 1971-2 in new admissions. From weekly examinations during 1971-2 of new admissions there was little evidence that the urinary infections came from the patients' own faecal or nasal flora even when Klebsiella, Proteus or Pseudomonas were regularly isolated from such material. (Open document to view complete abstract)
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ItemImmunobiology and molecular virology of fowl adenoviruses( 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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ItemThe development of attenuated Salmonella typhimurium as a vaccine vector( 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)