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

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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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Amoebae, Pathogenic free-living amoebae, Naegleria fowleri, Thermal pools, Chlorine, Acanthamoeba culbertsoni, Soil protozoa, Micro-organisms