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    Pathogenic free-living amebae-occurrence in New Zealand thermal regions, together with investigations into their disinfection, immunity and virulence : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, New Zealand
    (Massey University, 1978) Cursons, Raymond Thomas Michael
    Pathogenic free-living amebae (PFLA), of which Naegleria fowleri was the predominant pathogenic species, were isolated from 6 out of 10 pools sampled from the Hamilton, Rotorua and Gisborne Health Districts. The majority of these PFLA contaminated-pools occurred in the Matamata-Taupo region, and this localized geographical distribution appeared to be influenced, in part, by the particular physical and chemical properties of the pool. 'High-risk' pools, which exhibited a high incidence of isolations of PFLA, were shown to be natural pools, i.e. soil enclosures, as opposed to concrete constructed pools. PFLA were also isolated from the soil, and it was thought that soil acted as a reservoir of PFLA. No seasonal distribution in the occurrence of PFLA in thermal pools was noticed. A comparative study on the disinfecting potential of chlorine, chlorine dioxide, ozone and deciquam 222 for PFLA showed that all 4 disinfectants possessed amebicidal properties, but only at higher levels than those normally used for disinfecting bacteria. Of the 4 disinfectants, deciquam 222 proved to be the most effective amebicide, followed by chlorine, chlorine dioxide and ozone. An immunological survey of normal human sera for the presence of antibodies to either pathogenic or non-pathogenic Naegleria and Acanthamoeba spp. established that human sera had a titre ranging between 1/5 - 1/20 for Naegleria spp. and between 1/5 - 1/80 for Acanthamoebae. No discrimination in titres was observed between blood groups or sexes and fluorescein-labelled class-specific immunoglobulins showed that these antibodies belonged mainly to both the Ig M and Ig G classes. It was also shown that fresh adult human sera, as opposed to cord or specific hyperimmune-rabbit sera, contained a heat-labile neutralizing factor which inhibited the formation of cytopathic effects (CPE) in Vero cell culture by Acanthamoeba culbertsoni, but not by N. fowleri. Homologous, as well as heterologous antigens of Naegleria spp. were however, shown to cross-react with both the in vitro, macrophage inhibition factor assay, and in vivo, delayed hypersensitivity, correlates of cell-mediated immunity. Finally, this study also demonstrated that both pathogenic and non-pathogenic species of Naegleria and Acanthamoeba secreted both an extracellular phospholipase 2 and lysophospholipase into their axenic cultures. The relative production of phospholipase 2 correlated with the formation of CPE in Vero cell culture by either amebae, or by cell-free filtrates from axenic cultures of amebae. The relative level of production of this enzyme appeared to influence the virulence and hence pathogenic-potential of these micro-organisms.
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    Bacterial attachment to meat surfaces : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2003) Narendran, Valarmathi
    The aim of this study was to optimise the hygienic efficiency of slaughter and dressing operations. Three strategic approaches, namely reducing and removing or killing the bacteria attached to meat surfaces, were considered. The second option of removal was selected for development, as current technology inevitably results in bacterial contamination, while killing bacteria on meat surfaces requires drastic treatments that may adversely affect quality parameters. The initial attachment mechanism between bacteria and the carcass surface (reversible attachment) was studied using the collagen film model system. Bacterial attachment to the collagen model was compared with attachment to cut beef muscle and uncut beef muscle using viable count procedure. Scanning electron microscopy and direct microscopic count procedure using an epifluorescence microscope was also developed using both collagen films mounted on microscope slides and collagen coated microscope slides. The collagen film viable count system was the method selected to model bacterial attachment to meat because of ease and consistency of quantification. There was no positive correlation between attachment and many bacterial cell surface factors such as charge, hydrophobicity, protein and polysaccharide surface molecules. Different eluents were used to identify the principal component interfering with single attachment mechanisms on electrostatic interaction and hydrophobic interaction chromatographic columns and on collagen film. Three components interfering with the isolated attachment mechanisms were identified. They were Tween, sodium chloride (NaCl) and mannose. Further column studies indicated that cell surface proteins play a more important role in cell surface negative charge and hydrophobicity than do surface polysaccharides. A wash solution was formulated using the components Tween, NaCl and mannose to reverse what were believed to be the major attachment mechanisms. Further trials with Tween, NaCl and mannose and increasing their concentrations and the application of increased vigorous rinsing also proved ineffective for washing the cells from meat surfaces. This result also supports the hypothesis that bacterial attachment to meat surface is very complex and multifactorial. Elution studies using 10 % Tri sodium orthophosphate pH 12.0 killed the cells rather than removing them and further work will be directed towards the killing.