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Item Updating the genomic taxonomy and epidemiology of Campylobacter hyointestinalis(Springer Nature Limited, 2018-02-05) Wilkinson DA; O'Donnell AJ; Akhter RN; Fayaz A; Mack HJ; Rogers LE; Biggs PJ; French NP; Midwinter ACCampylobacter hyointestinalis is a member of an emerging group of zoonotic Campylobacter spp. that are increasingly identified in both gastric and non-gastric disease in humans. Here, we discovered C. hyointestinalis in three separate classes of New Zealand ruminant livestock; cattle, sheep and deer. To investigate the relevance of these findings we performed a systematic literature review on global C. hyointestinalis epidemiology and used comparative genomics to better understand and classify members of the species. We found that C. hyointestinalis subspecies hyointestinalis has an open pangenome, with accessory gene contents involved in many essential processes such as metabolism, virulence and defence. We observed that horizontal gene transfer is likely to have played an overwhelming role in species diversification, favouring a public-goods-like mechanism of gene ‘acquisition and resampling’ over a tree-of-life-like vertical inheritance model of evolution. As a result, simplistic gene-based inferences of taxonomy by similarity are likely to be misleading. Such genomic plasticity will also mean that local evolutionary histories likely influence key species characteristics, such as host-association and virulence. This may help explain geographical differences in reported C. hyointestinalis epidemiology and limits what characteristics may be generalised, requiring further genomic studies of C. hyointestinalis in areas where it causes disease.Item Extensive epigenetic modification with large-scale chromosomal and plasmid recombination characterise the Legionella longbeachae serogroup 1 genome(Springer Nature Limited, 2022-04-06) Slow S; Anderson T; Murdoch DR; Bloomfield S; Winter D; Biggs PJLegionella longbeachae is an environmental bacterium that is the most clinically significant Legionella species in New Zealand (NZ), causing around two-thirds of all notified cases of Legionnaires’ disease. Here we report the sequencing and analysis of the geo-temporal genetic diversity of 54 L. longbeachae serogroup 1 (sg1) clinical isolates, derived from cases from around NZ over a 22-year period, including one complete genome and its associated methylome. The 54 sg1 isolates belonged to two main clades that last shared a common ancestor between 95 BCE and 1694 CE. There was diversity at the genome-structural level, with large-scale arrangements occurring in some regions of the chromosome and evidence of extensive chromosomal and plasmid recombination. This includes the presence of plasmids derived from recombination and horizontal gene transfer between various Legionella species, indicating there has been both intra- and inter-species gene flow. However, because similar plasmids were found among isolates within each clade, plasmid recombination events may pre-empt the emergence of new L. longbeachae strains. Our complete NZ reference genome consisted of a 4.1 Mb chromosome and a 108 kb plasmid. The genome was highly methylated with two known epigenetic modifications, m4C and m6A, occurring in particular sequence motifs within the genome.Item Klebsiella pneumoniae in New Zealand sea lions : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Manawatū, New Zealand(Massey University, 2018) Pinpimai, KomkiewKlebsiella pneumoniae has been circulating in New Zealand sea lions since the outbreaks during the breeding seasons of 2001/02 and 2002/03 in Sandy Bay, on Enderby Island, Auckland Islands. A large number of pups have since died from K. pneumoniae every year during the breeding season. In order to prevent and control this infection, baseline data including bacterial phenotype and genotype, geographic distribution of the pathogen, and the immune response to the pathogen, have to be established. In this study, hypervirulent (HV) K. pneumoniae was isolated from different sources including New Zealand sea lion (NZSL) pups from different breeding sites, and characterised using a combination of biochemical, phenotypic tests, serological analysis and genotyping via whole genome sequencing. Isolates from pups, substrate samples from different breeding sites, a NZSL adult and birds, all had a close genetic relationship. The isolates have the same basic characteristics including a hypermucoviscous phenotype, serotype 2, and sequence type 86. This suggested clonality of this pathogen. The geographic distribution of the pathogen was found to be Enderby Island, Dundas Island, Campbell Island, and the Otago Peninsula (New Zealand mainland). The isolates analysed were all susceptible to commonly used antibiotics, with the exception of ampicillin. The HV isolates from pups were able to utilise a wide panel of carbon and nitrogen sources and had activity in a wide range of pH from 4.5 to 10, supporting the ability of this pathogen to survive in diverse environments. The findings in this thesis also suggest that the environment can be a reservoir for a short time period. For the long term, between breeding seasons, New Zealand sea lion adults and birds that live around the breeding site are potential reservoirs. The HV isolates from pups were resistant to some innate immune responses, including serum killing ability, oxidative killing ability and phagocytosis by neutrophils and monocytes. Overall, this study provided phenotypic and genotypic information on K. pneumoniae isolated from NZSL pups, as well as some information about innate immune responses to this pathogen, which can aid in the prevention and control of this infection.Item Diagnostic investigation into summer mortality events of farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Manawatū, New Zealand.(Massey University, 2020) Brosnahan, CaraSalmon farming is the second highest value aquaculture species in New Zealand and produces approximately 88% of the global market of farmed Chinook salmon, Oncorhynchus tshawytscha (Tucker, 2014). New Zealand salmon are free of many significant diseases affecting salmonids globally (Diggles, 2016). Therefore, disease is one of the greatest threats to this New Zealand aquaculture species. Biosecurity, early detection, and characterisation of new or emerging diseases is vital for management and sustainability of the aquaculture industry. Elevated mortalities termed ‘summer mortalities’ with no cause identified have occurred in certain farmed Chinook salmon populations in the Marlborough Sounds since 2012. This study identified two potential bacterial pathogens involved in summer mortalities; New Zealand rickettsia-like organism (NZ-RLO) and Tenacibaculum maritimum. Distribution of NZ-RLO and T. maritimum within farmed Chinook salmon populations, phylogenetic analysis of these pathogens and the pathogenicity of two strains of NZ-RLO were assessed to provide an understanding of the role of NZ-RLO and T. maritimum in summer mortalities. Additionally, new diagnostic tests were developed to efficiently detect these pathogens. Identification of NZ-RLO in the summer mortalities was the first detection in New Zealand. Tenacibaculum maritimum had been reported in New Zealand previously, however it had not been associated with mortalities. This study confirmed three strains of NZ-RLO with restricted geographical distribution. Two strains of NZ-RLO were found exclusively in areas where fish experienced summer mortalities and were associated with clinical signs of disease, indicating certain strains of NZ-RLO were likely primary pathogens. Widespread distribution of T. maritimum was detected within farmed salmon and no association was found with T. maritimum and clinical signs of disease in areas experiencing summer mortalities, indicating T. maritimum was unlikely to be a primary pathogen. This study proves that laboratory exposure of salmon to two strains of NZ-RLO caused disease and mortalities however, the differences between the two strains suggest NZ-RLO2 may be more pathogenic. This study suggests NZ-RLOs are likely to be involved in summer mortalities as primary pathogens however, the interaction between the pathogens and environment is likely to have amplified the levels of mortalities during these events.Item Temperature- and host-dependent transcriptional responses in the entomopathogenic bacterium, Yersinia entomophaga MH96 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany Campus, New Zealand(Massey University, 2020) Paulson, Amber RoseYersinia entomophaga MH96 is a virulent pathogenic bacterium that is infective towards a broad range of insects and is under development as a biopesticide. MH96 produces insecticidal toxin complex called Yen-TC that is secreted at temperatures of 25 °C and below and has been shown to be the primary virulence factor (VF) during per os challenge against the New Zealand grass grub, Costelytra giveni and other agricultural pests (Hurst et al., 2011a, 2019). New insights into the pathobiology of MH96 during insect infection were gained from the in vivo transcriptome, including identification of a core secreted weaponry of co-expressed/co-secreted VFs, including Yen-TC and other exoenzymes; however, many other diverse types of VFs, including toxins, effectors, fimbriae, secretion systems, efflux pumps, iron acquisition, stress response and metabolic adaptation were also identified as highly expressed under in vivo conditions. A small DNA-binding protein, Yen6, was shown to be under thermoregulation at the transcriptional level and host-dependent-regulation at the post-transcriptional level and contributed to virulence during intrahemocoelic infection of Galleria mellonella at 37 °C. The in vivo transcriptome of Δyen6 and in vitro DNA-binding specificity analysis provided evidence that Yen6 is a novel LytTR-containing regulator that activates a ribose uptake/metabolism gene cluster, rbsD-xylG-rbsC-xylF-rbsK-ccpA, and represses a fructose uptake/metabolism gene cluster, IIA-fruK-IIB and a gene for RNA-binding protein yhbY during infection at 37 °C. Another small DNA-binding protein, Yen7, was also implicated as a potential temperature-dependent activator of Yen-TC component genes and over-expression of yen7 resulted in restored secretion by MH96 at 37 °C; however, deletion of yen7 did not abrogate Yen-TC production. Experimental investigations into potential regulatory linkages between Yen6 and yen7 were undertaken, and evidence to date does not support Yen6 as transcriptional repressor of yen7. A 17.5 Kb unstable element within the genome of MH96 with linkages to Yen-TC and toxin secretion, motility and cell shape was identified. Overall the findings presented in this thesis represent the most detailed investigation of MH96 pathogenesis to date, reinforcing MH96 as one of the most highly entomopathogenic bacteria known to humankind; yet suggesting MH96 has possibly maintained at least one core thermoregulatory mechanism more typical of an opportunistic pathogen.Item Pathogens associated with acute infectious canine tracheobronchitis in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Masters in Veterinary Studies in Virology at Massey University, Turitea, Palmerston North, New Zealand(Massey University, 2018) Sowman, Harriett RoseInfectious canine tracheobronchitis (ICT) or canine infectious respiratory disease, commonly known as kennel cough, is an acute, highly contagious respiratory disease that affects the larynx, trachea, bronchi, and occasionally the parenchyma of the lower respiratory tract. Several pathogens have been implicated in ICT including viruses, bacteria and mycoplamsa. Little is known about the prevalence of canine respiratory pathogens in New Zealand. Hence, the aim of this study was to identify potential respiratory pathogens from dogs that are affected by ICT in New Zealand, and compare agents found in diseased dogs to those found in healthy dogs. In house (IH) qPCR assays were developed for the detection of canine adenovirus type 2 (CAdV-2), canine herpesvirus (CHV) and canine parainfluenza (CPIV). A total of 96 dogs were sampled, including 47 healthy and 49 diseased dogs, which comprised three different groups of dogs: greyhounds, pet dogs, and working farm dogs. A questionnaire was included for each dog sampled. The samples collected were then subjected to the following tests: virus isolation, haemagglutination assay for CPIV, IH qPCR for CAdV-2 and CHV, as well as IDEXX RealPCR respiratory disease panel, and bovine respiratory coronavirus ELISA to detect antibody to canine respiratory coronavirus (CRCoV). Based on IDEXX qPCR, CPIV (7.3%), Bordetella bronchiseptica (7.3%) and Mycoplasma cynos (17.0%) were the most common agents detected in samples from diseased dogs, whereas CAdV-2 (10.6%) was the most common pathogen amongst healthy dogs. Based on IH qPCR, CAdV-2 infection was very common among all dogs sampled, with 34/47 (72%) positive diseased dogs and 37/47 (78.6%) positive healthy dogs. A total of 47/92 (51%) of dogs were positive for CRCoV antibodies, including 32/46 (69.6%) of diseased dogs and 14/46 (30.4%) of healthy dogs. In addition, acute serum samples from diseased dogs were significantly more likely to be positive for CRCoV antibodies compared to sera from healthy dogs (RR 5.22, CI 1.972, 14.115, p=0.0003). The results of this study suggest that CRCoV, M.cynos and potentially CPIV may have a role in ICT in New Zealand, however further investigation is required to support these findings. In addition, if one excluded dogs positive for CAdV-2 (as there was no difference in levels of detection of this virus between healthy and diseased dogs), then only 13/47 (27.6%) of diseased dogs were positive for at least one agent via IDEXX and IH qPCR. This suggests that other aetiological agents, not examined in this study, may have contributed to respiratory disease in sampled dogs. Techniques such as next generation sequencing may help to identify these pathogens.Item Glucose induced germ tube formation in Candida albicans : a thesis presented to Massey University in fulfilment of the requirements for a Masters of Science degree in Microbiology(Massey University, 2004) Sciascia, MiriamaCandida albicans is an opportunistic fungal pathogen that can cause a wide range of superficial and systemic infections. One of the many factors that have been implicated in C. albicans success as a pathogen is its ability to reversibly switch between a yeast form and a hyphal form (dimorphism). The dimorphic switch is triggered by a wide variety of stimuli which include temperature alone, pH alone, and serum. Serum is a potent inducer of germ tube formation and remains the medium of choice for rapid identification of C. albicans from other non-albicans Candida species. Recently it was shown that, in serum, glucose is the primary inducer of germ tubes in C. albicans strain A72 (Hudson and Farley, unpublished). In this study the ability of glucose, dialysed serum and serum filtrate to induce germ tube formation in a randomly chosen panel of clinical isolates of C. albicans was studied, and the role ot two putative glucose receptors and a putative glucose transporter in the transduction of the glucose signal was investigated. Dialysed serum (molecular weight, > 10 kDa) was less effective (P > 0.05,Students t- test) at inducing germ tube formation than serum. The addition of exogenous glucose alone to dialysed serum restored its ability to induce germ tube formation levels to those seen in serum in seven of the nine clinical isolates tested. Serum filtrate (molecular weight, > 10 kDa) induced germ tubes to levels indistinguishable trom those seen in serum (P > 0.05, Students t-test) in all but one of the clinical isolates tested. Buffered glucose was also able to induce germ tubes in all the clinical isolates tested and the percentage germ tube formation was not statistically significantly different from that obtained with serum in ten out of sixteen clinical isolates tested. The addition of urea to these assays had no statistically significant effect on the induction of germ tube formation. It was proposed that the induction of germ tube formation by glucose was mediated by a surface receptor and therefore the C. albicans genome was examined for genes encoding putative glucose receptors. Identified as possible receptors were orf19.1944 and orf19.5962. Orf19.3668, a putative glucose transporter, was also examined because its expression had been reported to increase during serum induced germ tube formation. Strains carrying homozygous deletions of each ORF were made and the phenotypes of the mutants investigated. None of the ORFs were found to be involved in glucose or serum mediated germ tube formation. However, orf19.1944 was shown to play a role in germ tube formation under embedded conditions.Item A novel model developed for quantitative microbial risk assessment in the pork food chain : a dissertation presented in partial fullfilment [sic] of the requirements for the degree of Doctor of Philosophy at Massey University, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand(Massey University, 2007) Titus, Simone MeganFood-borne diseases contribute substantially to morbidity and mortality rates worldwide. The deleterious impact of these diseases on human health, concurrent with the associated socioeconomic cost has led to an increased demand for the production of safe food globally. Consequently, agencies such as the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) have resolved to address this issue. In this vein, scientific, risk-based approaches which facilitate estimation of the probability of disease occurrence, the magnitude of the disease and efficacious control measures have been recommended for use internationally. Many pathogens have been implicated as aetiological agents of food-borne disease. The WHO has identified non-typhoidal Salmonella, Escherichia coli and thermophilic Campylobacter as zoonotic food-borne pathogens of greatest importance. These pathogens can be transmitted to humans through pork consumption. This thesis therefore proposes a suite of novel, mechanistic, semi-stochastic, quantitative, modular process risk models describing the propagation of these three pathogens from the live pig at the abattoir, to pork chops sold at retail. The model is developed for use in risk-based, quantitative microbial exposure assessments in New Zealand and can be employed to explore different intervention strategies targeted at mitigating contamination levels of these pathogens on pork chops. The models comprise multiple, coupled, differential and difference equations. These equations explicitly describe bacterial growth, inactivation, removal, cross-contamination and food partitioning occurring in continuous and discrete time in abattoirs and at retail. Distributions of pathogen numbers on the surface of carcasses, and prevalence levels are output by the models at different stages of abattoir processing and pork chop production. Both dressed pork carcases exiting abattoirs in New Zealand and pork chops at retail are predicted to contain low surface contamination levels of the pathogens under consideration, while a small percentage is estimated to be highly contaminated. Median contamination levels on dressed pork exiting the abattoir are predicted to be less than one cfu/cm2. Generally, there are large reductions in surface bacterial numbers for all three organisms from the time the live pig enters the abattoir, to sale of the pork chop at retail. The introduction of a second singeing procedure immediately postevisceration in the abattoir is predicted by our models, to be an effective mitigation strativ egy, with estimated reductions in median pathogen levels of 100%. This control measure is considered to be more effective than coverage of the anal region of the pig during evisceration. This latter mitigation strategy was predicted to result in 10% – 44% reduction of median pathogen contamination levels. At retail, pork chops are also estimated to contain low numbers of these pathogens. Therefore handling of the raw pork chop soon after purchase from retail outlets may be associated with a low risk of contracting salmonellosis, colibacillosis and campylobacteriosis. This risk can be further reduced by placing pork chops in a blast chiller for 12 hours prior to display. When this mitigation strategy was modelled the outputs indicated a 15% – 61% reduction in the maximum pathogen levels on pork chops, 44 – 100% reduction in the 10th – 90th range and 14% – 50% reduction in pathogen prevalence levels. Detailed investigation revealed the limitations of a specific modelling approach. We determined that the population-based modelling approach is not an appropriate alternative to the individual-based modelling approach when there is a large disparity in contamination levels between processed carcasses. Therefore the former technique should not be used in the presence of large heterogeneity with respect to the number of bacteria on the food unit of interest, or when bacterial populations input into the model are described with large variances. This thesis demonstrates the application of a suite of novel risk models in the pork food chain. We propose use in quantitative microbial exposure assessments. The applicability of these models is not only limited to the pork chain or to the above mentioned pathogens, but by modification of parameters, the entire model, or portions thereof can be extrapolated to other animal species undergoing similar abattoir procedures with pathogens of analogous epidemiological patterns. Finally the information provided by the models can be instrumental in assisting risk managers in their decision-making and policy development undertakings and provide guidance to effectively and strategically funnel limited resources.