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    Genomics and eDNA provide a holistic understanding of microbial communities and zoonoses in Aotearoa's waters : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology at Massey University, Palmerston North, New Zealand
    (Massey University, 2024-03-18) Davis, Meredith Taylor
    In Aotearoa, water quality and freshwater ecosystem health is declining. Much of that decline has been blamed on livestock farming and is quantified by using invertebrate communities or Escherichia coli levels. However, the wider microbial community, particularly archaea and eukaryotes, have been overlooked in the current ecosystem health measures. Some of the more well-known impacts of microorganisms on waterways are sporadically measured (e.g., algae blooms, drinking water contamination by faeces, and shellfish toxins) but the drivers of their community structure, both individually and as a whole, in Aotearoa is poorly understood. This thesis is a start at rectifying this knowledge gap. The studies in this thesis were approached with a One Health perspective and provide a quantitative, holistic analysis of microbial communities by investigating three significant challenges facing Aotearoa’s freshwater ecosystems. Those challenges - the spread of waterborne disease, eutrophication, and microbial biogeochemical cycling - were investigated using microbiological cultures and environmental DNA, analysed with metagenomic and other molecular methods. I was able to determine that targeted testing for genetic loci associated with antimicrobial resistance and Shiga toxin-producing E. coli virulence was a useful in monitoring three Canterbury waterways for human health. Furthermore, enteropathogenic human and bovine strains of E. coli appeared unresponsive to in-stream nitrate-nitrogen concentrations of 0, 1, and 3 mg/L and native in-stream biota in microcosms. However, environmentally sourced E.coli imported as part of the in-stream biota survived longer in NO3-N concentrations of 1 and 3 mg /L than at 0 mg/L. Microorganism groups (e.g., archaea, bacteria, and microbial eukaryotes) responded to different environmental, spatio-temporal, and physico-chemical drivers depending on taxonomic level. As a group, lotic pressures and dispersal outweighed other drivers in community structuring. Archaeal communities were highly correlated with Austral season and the most abundant functional groups reflected a likely response to common agricultural pollutants found the Waiotahe catchment and in many rural rivers across Aotearoa (e.g., nitrogen pollution and livestock waste/effluents). The drivers commonly associated with bacterial survival (e.g., conductivity and temperature) were less important than dispersal and lotic pressures, particularly at lower taxonomic levels. Cultured E. coli concentrations from sediment and/or the water column were poorly indicators of Campylobacter, Enterobacter, and Enterococcus relative abundances. Additionally, neither Enterobacter nor Enterococcus relative abundances were correlated with E. coli/E. cloacae group concentrations or Campylobacter relative abundances. These findings have important implications for water quality monitoring and recreational human health risk assessments in Aotearoa. Currently, microbiological water testing is limited to bathing season (i.e., late spring to early autumn) and to culturing either Enterococcus in saline/brackish water or E. coli in freshwater. Effective water quality monitoring must include both water and benthic substrates to accurately portray the entire riverscape. Genetic loci associated with zoonotic human pathogens are present in some of Aotearoa’s waterways and they are likely a result of catchment land use, livestock farming, and effluent contamination. Additionally, genetic loci can be detected with collection methods similar to those employed for current water quality monitoring using Escherichia coli and some molecular methods are more specific (i.e., not proxies). Metagenomic methods allowed for the discernment of microbial communities and core biomes from genetic information extracted from environmental samples. Microbial communities were affected by many different in-stream conditions; however, dispersal and the pressures associated with lotic systems proved to be more important than adjacent land use, precipitation levels, or season. In contrast, archaeal communities were better explained by season. It is clear that water monitoring in Aotearoa needs an overhaul and to incorporate new technology in a thoughtful and ecologically informed manner. A review of the current methods and new technologies should be undertaken by a multi-disciplinary group of experts in the fields of microbiology, epidemiology, and freshwater and microbial ecology. Community buy in and the inclusion of Māori values and indigenous rights should be at the forefront of any proposed changes to freshwater restoration and conservation.
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    Identification and characterisation of novel virulence factors from Dothideomycete pathogens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2023) McCarthy, Hannah
    The Dothideomycetes class of fungi contains some of the most important plant pathogens, including Dothistroma septosporum and Fulvia fulva. Dothistroma needle blight, caused by D. septosporum, is a devasting disease of pines that has been increasing in severity and incidence worldwide. Tomato leaf mould, caused by F. fulva, has recently risen in importance after overcoming resistance breeding efforts. There is now an urgent need to identify novel genes from these pathogens that encode virulence factors, such as effector proteins. These proteins contribute to pathogen virulence, so the study of virulence factors not only enables a deeper understanding of how pathogens and their hosts interact at the molecular level, but also provides information that may lead to the development of new methods for disease control. As such, the aim of this thesis was to identify and characterise new candidate virulence factors from D. septosporum and F. fulva. Candidate virulence factor CfEcp11-1 from F. fulva, previously identified to trigger plant defences in wild tomato cultivars, was a particular focus in Chapter 2. Two new homologues of CfEcp11-1 were identified from Fusarium oxysporum and suggested to be part of the same Leptosphaeria AviRulence and Suppressing (LARS) effector family. Recognition of CfEcp11-1 by the tomato receptor was also examined through the design of chimeric and mutant protein sequences. CRISPR/Cas9 was used for the first time in F. fulva to disrupt CfEcp11-1 and generate single- and double-copy mutants; to the best of our knowledge this was the first report of multiple gene copy disruption by CRISPR/Cas9 in a fungal pathogen. In Chapter 3, candidate virulence factors from D. septosporum and F. fulva were identified through a prediction pipeline, which selected proteins with predicted roles as effectors that manipulate plant defences, or as transcription factors which regulate the expression of other genes. Because D. septosporum and F. fulva are hemibiotrophic pathogens, they transition from colonising living plant tissue to killing and feeding on dead tissue, a transition termed the necrotrophic switch. It is currently unknown what mechanisms govern this important disease process, so a key focus was on candidate virulence factors with a possible role in the necrotrophic switch. In Chapter 4, some of these candidates were further characterised, and new candidates identified, from proteomic analysis of the culture filtrates of D. septosporum and F. fulva grown in different conditions. Existing transcriptomic data were used to assess which of these proteins were likely to be functional in planta. Among those identified were several characterised effectors, such as Cf/DsEcp2-1, Cf/DsEcp20-1, Cf/DsEcp20-3, and CfAvr4E, which were secreted in culture despite having known functions in planta. Novel candidate virulence factors from D. septosporum and F. fulva were also identified in this analysis, including a Nis1 domain-containing protein from D. septosporum with a possible role in the necrotrophic switch. This analysis illustrates that in culture proteomic analysis can be a useful tool for the identification of candidate effector proteins. In Chapter 5, two candidate virulence factor genes of D. septosporum with predicted roles in the necrotrophic switch were disrupted through CRISPR/Cas9 gene editing; this was the first use of this method for gene disruption in D. septosporum. The disruption mutants were tested for virulence on the P. radiata host. One of the mutants was disrupted in DsCE3, which was suggested to be a virulence factor, with a possible role in the necrotrophic switch. Overall, the results presented in this thesis have provided new research methodologies as well as valuable knowledge about the molecular tools these two pathogens use to invade their hosts. Whilst further work is required, these developments will ultimately aid future disease control strategies in pine and tomato.
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    The susceptibility of pathogenic free-living amebae to chemotherapeutic agents : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1979) Donald, Jennifer Jane
    The treatment of infections caused by pathogenic free-living amebae (PFLA) has, until only recently, been far from successful. The continued screening of chemotherapeutic agents against amebae of the genera Naegleria and Acanthamoeba is therefore of the utmost importance. Seven chemotherapeutic agents, amphotericin B, rifampicin, tetracycline, polymyxin B sulphate, 5-fluorocytosine, miconazole and R41,400 were screened for activity against a non-pathogenic and pathogenic species of Naegleria and a non-pathogenic and pathogenic species of Acanthamoeba in axenic culture. For the Naegleria spp. amphotericin B, miconazole and R41,400 were found to be active. Acanthamoebae spp. were found to be susceptible only to 5-fluorocytosine and R41,400. The possible use of combinations of drugs against the amebae was also investigated in axenic culture. For Naegleria fowleri (MsT) amphotericin B with either tetracycline or rifampicin showed a synergistic effect. Polymyxin B sulphate and 5-fluorocytosine showed synergistic activity against Acanthamoeba culbertsoni (A-1) but when polymyxin B was combined with tetracycline or rifampicin no significant additive effect was seen. After axenic culture testing the susceptibility of the pathogenic species, N. fowleri (MsT) and A. culbertsoni (A-1), to the agents which showed activity, was investigated in a Vero cell culture system. For N. fowleri (MsT) the results of axenic testing were confirmed with amphotericin B, miconazole and R41,400 protecting the monolayer from the destructive effects of the amebae. 5-Fluorocytosine inhibited the formation of cytopathic effect (CPE) when the cell cultures were inoculated with A. culbertsoni (A-1) but viable amebae were still present. R41,400 had no effect on A. culbertsoni (A-1) at concentrations at or above those which were cytotoxic to the Vero cells. The use of combinations of drugs was also investigated in Vero cell culture. Amphotericin B and rifampicin showed an antagonistic rather than a synergistic effect when used against N. fowleri (MsT) in cell culture but amphotericin B and tetracycline showed synergistic activity. For A. culbertsoni (A-1) the synergistic activity of polymyxin B and 5-fluorocytosine was confirmed. The lack of an additive effect between polymyxin B and either tetracycline or rifampicin was also shown in cell culture. The new imidazole derivative R41,400, which showed promise against N. fowleri (MsT) in in vitro tests was then tested in the in vivo situation. Mice experimentally infected with N. fowleri (MsT) were treated once or twice daily intraperitoneally with different doses of R41,400. At the higher dosage levels tested the drug appeared to have a deleterious effect, the average time for death being less than that for the controls.
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    The safety of ready-to-eat meals under different consumer handling conditions : a thesis presented in partial fulfilment of the requirements for the degree of Master in Food Technology at Massey University, Manawatū, New Zealand
    (Massey University, 2016) Jiang, Fan
    Microbial count is an important index to measure the safety status of a food. This trial aimed to determine the safety of eight meals (four meats and four vegetarians) by using the agar plate counting method to measure the populations of total bacteria and specific pathogenic microorganisms during four day’ abusing. The results showed that chicken & lemon sauce, pork & cranberry loaf and lasagne veg can be considered as acceptable after a series of handling steps including heating and holding in different environments. BBQ beef, quiche golden and pie rice & vegetable were all marginal for the microbial load before heating, but afterwards all of them were acceptable. Casserole chickpea and hot pot sausage were in marginal for the microbial load by the end of trial.