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Item The dynamics of drug resistance evolution and diagnosis in Mycobacterium tuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics/Genomics, Massey University, Manawatu, New Zealand(Massey University, 2024-07-30) Fong, Yang (Richard)Tuberculosis (TB) remains a critical global health challenge with over 10.4 million new cases annually, complicated by rising antimicrobial resistance (AMR) threatening to surpass cancer mortality by 2050. This PhD thesis establishes a systematic diagnostic framework addressing AMR challenges through progressive research from fundamental microbiome characterization to innovative diagnostic applications in resource-limited settings like Myanmar. The "Microbiome Dataset from the Upper Respiratory Tract of Patients Living with HIV, HIV/TB and TB from Myanmar" establishes the foundational understanding of microbial community structures in complex clinical presentations (n=309 isolates). This microbiome characterization reveals critical signatures that directly inform direct sequencing strategies for enhanced MTBC detection in polymicrobial environments, addressing a fundamental challenge in AMR detection. Next, the "Genomic Profiling of Mycobacterium tuberculosis Strains, Myanmar" validates and expands these microbiome-informed approaches through comprehensive whole genome sequencing surveillance, establishing genotype-phenotype correlations that achieve 97.8% concordance with phenotypic testing. This genomic profiling directly addresses AMR surveillance gaps by enabling rapid resistance prediction. Subsequently by "Unveiling Hr-TB in Myanmar: Comprehensive Genotypic and Phenotypic Insights for Improved TB Management" demonstrates targeted application of microbiome-informed diagnostic approaches to isoniazid mono-resistant TB, a clinically critical AMR variant frequently missed by conventional methods. The integrated microbiome-genomic approach enhances MTBC detection accuracy by 23% compared to standard methods, reducing diagnostic time from weeks to under one week. Future perspectives translate these discoveries into field-deployable MDA primer systems for point-of-care AMR detection using portable MinION sequencing technology. This systematic progression from microbiome foundation to diagnostic innovation establishes a replicable technological blueprint for next-generation TB AMR diagnostics, supporting Myanmar's National TB Control Program while providing a framework for global TB elimination efforts Keywords: Mycobacterium tuberculosis (MTB), Tuberculosis (TB), Antimicrobial Resistance (AMR), Isoniazid Mono-Resistant (Hr-TB), Drug-Resistant Tuberculosis (DR-TB), Whole-genome sequencing (WGS), Resistance associated Mutations, Epidemiology, Surveillance, Rapid Diagnosis, Microbiome, Yangon, Myanmar.Item Genetic diversity of microbes and its impacts on host switching : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2025-08-25) Dos Anjos Almeida, ValterLand use around forests, leading to habitat overlap, increases the risk of anthroponotic and zoonotic pathogen/microorganism transmission between humans, livestock, and wildlife. This scenario is exemplified by Buhoma, a village where most residents are primarily subsistence farmers who raise livestock and cultivate crops near the Bwindi Impenetrable National Park (BINP) in Southwestern Uganda, creating an ideal interface for understanding these dynamics. Given the significance of this geographic location and the underrepresentation of African populations in global microbiome research, this project aims to characterise the gut microbiome diversity and potential novel bacterial species, as well as microorganism sharing within a multi-host system in a rural area with documented interactions between humans, livestock, and wildlife. For microbial detection, DNA extracted from 553 faecal samples collected from humans, gorillas, and livestock was processed and sequenced to analyse microbial species communities in different hosts using shotgun high-throughput sequencing and bioinformatics tools. The analysis of 2,411 bacterial metagenome-assembled genomes (MAGs) confirmed the presence of numerous putative novel bacterial species in the gut microbiomes of the Ugandan hosts studied in this project. While many species were host-specific, others were detected across multiple host gut microbiomes from Buhoma and BINP, and in different host samples from other geographic locations (by comparing the MAGs generated in this thesis with other publicly available genomes). Among the bacterial MAGs assembled in this project, genera such as Prevotella and Treponema—considered markers of the gut microbiome in individuals with traditional agricultural lifestyles—and Campylobacter—which comprises species that are leading causes of bacterial gastroenteritis globally—were further investigated. These genera were found to contain numerous putative novel species, contributing to our knowledge of the human and animal gut microbiomes and enhancing our understanding of the species associated with them. The results of this study reveal a high diversity of previously unknown microbes recovered from human, gorilla, and livestock gut microbiomes collected simultaneously at the same wild rural interface. The Treponema genus includes species that inhabit a wide range of hosts and types of microbiotas. In the Ugandan host samples, 75 Treponema MAGs were reconstructed, enabling the identification of sixteen intestinal species, including thirteen putative novel species. Three of the detected species were found in human samples from this study, as well as in publicly available Treponema genomes recovered from animals such as pigs, cattle, wild boars, roe deer, and goats. This detection underscores the potential for interspecies transmission and the establishment of these bacteria in different hosts. Overall, these findings highlight the complex interplay between humans, animals, and their gut microbiomes, emphasising the need for further research to elucidate the implications for public health. Prevotella species are prevalent and abundant components of microbial communities associated with mammals. This bacterial genus influences individual clinical and metabolic responses to dietary changes and overall health conditions. In humans, Prevotella species have been identified in various body sites such as the skin, oral cavity, vagina, and gastrointestinal tract. In the gut microbiome, they are commonly linked with diets abundant in plant-based foods and are particularly prevalent among agriculturalist populations, where they tend to dominate the microbiota. In the investigation of this genus, 37 MAGs were assembled, all belonging to yet-to-be-described species. Phylogenetic analysis revealed that they represented fifteen putative novel intestinal Prevotella species. The nimJ gene, which confers resistance to metronidazole (a nitroimidazole-class drug), was detected in one of the human intestinal Prevotella species. Given the widespread use of antibiotics like metronidazole for treating both humans and animals in regions of Uganda, including Buhoma, the presence of this gene provides evidence that Prevotella species can acquire resistance to commonly used nitroimidazole-class drugs in areas where their use is prevalent. Of the seven putative novel species identified in human gut microbiomes, two were also present in publicly available genomes in the gut microbiomes from pigs and rhesus macaques living in different geographic locations, providing evidence that the same Prevotella intestinal species can inhabit multiple hosts. Overall, these findings expand knowledge of intestinal Prevotella by introducing numerous previously unknown species, offering a species-level perspective for future studies on Prevotella’s impact on health. Campylobacter species are leading causes of bacterial gastroenteritis worldwide, yet their diversity and transmission dynamics at wildlife–livestock–human interfaces remain poorly characterised. I investigated Campylobacter diversity among sympatric mountain gorillas, livestock, and humans in Uganda, assembling 44 Campylobacter MAGs representing seven species—including five putative novel taxa. Three novel species were found in mountain gorillas, livestock harboured C. vicugnae (goats) and C. sp017646085 (cattle), while human gut microbiomes revealed Candidatus Campylobacter infans (not associated with disease) and C. sp900539255, which was significantly enriched in clinical samples (p=0.001) and displayed unique sulfur and nitrogen metabolic pathways. Antimicrobial resistance genes, including blaOXA-471_1, were detected in Ca. C. infans MAGs. Overall, our findings demonstrate high Campylobacter diversity at the human–animal interface in Buhoma and highlight the importance of expanding reference databases for accurate surveillance and effective public health interventions. In summary, this thesis introduces a substantial amount of new information on gut microbiome bacterial species in gorillas, livestock, and humans living at a wild-livestock-human interface. It also provides detailed insights into three bacterial genera present in the gut microbiomes of human populations, as well as other mammals such as gorillas, cattle, and goats.Item Escherichia coli genes that are required for the Ff phage lifecycle : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Manawatū, New Zealand(Massey University, 2025) den Hartog, Sam TerryFf phages have remarkably few genes yet are able to perform the complex task of infecting Escherichia coli via a process involving F pilus binding and retraction at the start of its life cycle, and releasing progeny from a host cell by a secretion-like process without killing it. The Ff phage-encoded assembly-secretion (egress) machinery operates across all layers of E. coli envelope – inner membrane, periplasm, peptidoglycan and outer membrane. Nevertheless, phage encodes only three proteins that build a functional assembly-secretion machine. In contrast, analogous trans-envelope systems in bacteria, that mediate pilus assembly, conjugation, DNA transfer and protein secretion require tens of proteins. To date, only a few host proteins required for Ff phages infection and DNA replication have been identified, of which only one was found to be involved in assembly, and only in one of three known Ff phages. Because the Ff phages are able to be assembled and secreted using a strategy similar to those that complex transenvelope secretion-assembly systems do yet only use 3 phage-encoded proteins for this, a hypothesis can be made that the Ff phage is using the host (E. coli)-encoded proteins to complete its lifecycle. The hypothesis that many host proteins are involved in the Ff lifecycle was tested by systematic screening of 212 E. coli single knockout mutants, which were mated to become F positive, in a Ff phage plaque and phagemid transduction assays. The screen found 66 E. coli proteins which were required for the phage lifecycle, most of which had impaired the infection step, which is dependent on the F-pilus as the primary receptor. Of the mutants that failed to be infected by Ff phage, there is some overlap with previously identified mutants that were defective in F pilus conjugation donor function, which indicates that there is an overlap between the F-pilus Ff receptor and conjugation function. Some mutants identified in this thesis were not functional as donors in conjugation, hence they added to the tally of E. coli chromosomal genes that cannot conjugate using the F pilus. Majority of the chromosomal mutants required for the Ff lifecycle found in this screen encode proteins that play a role in stress response pathways or control the envelope morphology. Many proteins encoded by mutated genes that did not fall into these two broad categories were also found. Due to the large number of mutants found that are implicated in the Ff phage infection and conjugation, from just a small systematic screen in this study, a further genetic screen is warranted to find the true extent of E. coli genes that are required for filamentous phage infection and conjugation. Phage infection and conjugation play major roles in horizontal gene transfer between bacteria. Thus finding E. coli genes that are vital for these processes is crucial to fully understand the mechanisms of inter-bacterial horizontal gene transfer. Having a complete list of genes involved in gene transfer mediated by filamentous phage infection and conjugation would give more targets to control the spread of antibiotic resistance during antibiotic treatment, or to develop other biotechnology applications.Item Biofilm formation of Vibrio parahaemolyticus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology at Massey University, Campus Manawatū, New Zealand(Massey University, 2024-05-20) Wang, DanVibrio parahaemolyticus in seafood can cause food poisoning. There is increasing concern with the increase in reports of illness globally believed to be due to climate change affecting sea temperatures. Biofilm formation of V. parahaemolyticus is an additional concern as biofilms are more resistant to cleaning and sanitation than planktonic cells. However, little is known about the biofilm formation of V. parahaemolyticus. Strain variation and the factors determining biofilm formation were investigated in this study with the aim to provide information that can be used to design more effective control strategies. This study identified two robust biofilm forming strains (PFR30J09 and PFR34B02) from nine V. parahaemolyticus seafood isolates. Comparative genome analysis unveiled 136 unique accessory genes in robust biofilm formers. Protein-protein-interaction analysis showed interactions between UDP-glucose metabolism (Gene ontology (GO): 0006011), cellulose biosynthesis (GO: 0030244), rhamnose metabolism (GO: 0019299) and O antigen biosynthesis (GO: 0009243). Cellulose contributed to robust biofilm formation. Cellulose biosynthesis was identified as being acquired from within the order Vibrionales. The cellulose synthase operons consisting of genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC were present in 15.94% (22/138) of V. parahaemolyticus. Strong biofilm-forming V. parahaemolyticus showed greater resistance to sanitizers of biofilm cells than the weaker biofilm forming cells. The effective concentrations of sodium hypochlorite for inactivating most V. parahaemolyticus biofilm cells were higher than the recommended concentration. Available chlorine of 1176 mg/L inactivated 1.74-2.28 log10 CFU/cm2 of biofilm on stainless steel surfaces and 4704 mg/L inactivated > 7.00 log10 CFU/cm2 of biofilm (to undetectable levels, < 10 CFU/cm2), except for biofilms formed by the strong biofilm formers. Peracetic acid (PAA) at 200 ppm (89.56 mg/L PAA, 471.64 mg/L hydrogen peroxide) inactivated > 5.00 log10 CFU/cm2 of biofilm from stainless steel surfaces (except for those the strong biofilm formers, see Figure 4.4). RNA sequencing (RNA-seq) identified 74 differentially expressed genes when comparing planktonic and biofilm cells of V. parahaemolyticus. These represented the rearrangement of nucleotide and energy metabolism in biofilm cells. Biosynthesis of secondary metabolites, purine and pyrimidine metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation were deemed essential in the young V. parahaemolyticus biofilms. Genes of purH, purF, pdhA are potential genetic targets for biofilm prevention and control of V. parahaemolyticus. Understanding V. parahaemolyticus biofilm formation will help to design strategies to overcome the limitations of chemical sanitizers, improving product safety and quality in the seafood industry.Item Phenotypic characterisation of members of the Lachnospiraceae family isolated from ruminants : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand(Massey University, 2023) Rajasekaran, KrsanaMicrobial fermentation in the rumen employs the metabolic capacity of microorganisms to degrade lignocellulose from the diets consumed by ruminant animals. Advances in genomic, metagenomic and culture independent methods for studying microbiomes have caused a lag in the functional characterisation of isolated cultures. Moreover, understanding the interactions between microbes during rumen fermentation may help in producing strategies to improve animal productivity and address environmental impact issues such as enteric methane emissions. In this study it is demonstrated how cultured strains are required to accurately describe the functional traits of rumen bacteria. Members of the Lachnospiraceae family are one of the most abundant bacterial groups in the rumen, however, many of its isolated members are yet to be fully characterised or properly classified. In this study, the genomes of 45 Lachnospiraceae strains sequenced in the Hungate 1000 project were functionally annotated using the web-based annotation tool, Protologger. These predictions were then compared with phenotypic traits from the corresponding strains, uncovered using microscopy, carbon utilisation testing, and by analyses of short-chain fatty acid production, and headspace hydrogen. The results indicate how the genome can assist in the culturing and studying of rumen microorganisms but should not be solely relied on for the elucidation of functional traits. Phenotypic characterisations of the 45 Lachnospiraceae strains revealed a preference for the resultant soluble components of cellulose degradation rather than hemicellulose. Starch and pectin were more readily fermented in comparison to cellulose and xylan. End product analysis revealed that the studied strains produce acetate, butyrate and propionate, products known to contribute to host health and nutrition. Ethanol, formate, lactate and less commonly succinate were produced as fermentation products demonstrating the potential of the strains to participate in interspecies metabolite transfers. A subset of the strains including members of the genera Lachnospira, Eubacterium and Oribacterium as well as unclassified Lachnospiraceae bacterium strains were shown to produce methanol from pectin degradation. End products of fibrolytic fermentation by the 45 Lachnospiraceae strains can potentially act as substrates for methanogenic archaea. The results of this study help to improve the knowledge surrounding the poorly studied Lachnospiraceae family and increases the overall utility of the Hungate 1000 culture collection. Additionally, the comparison between genotypic predictions and the phenotyping, accentuates the importance of culture-based studies, providing an incentive to continue cultivating representative strains from the rumen environment to clarify how various microorganisms are contributing to rumen fermentation.Item 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 TaylorIn 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.Item A rapid and accessible method to profile and identify antibiotic resistance genes in bacteria using multiplex amplicon panels and Oxford Nanopore Technology : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Albany, New Zealand(Massey University, 2023) McGrath, NickThe spread of antibiotic resistance is severely burdening agriculture and healthcare industries. Bacteria have readily gained resistance to nearly all commercially available antibiotics through the use and overuse of antibiotics in both industries. Current methods to detect resistance genes in bacteria are either outdated and slow or too expensive and inaccessible. A method that is rapid and accessible is necessary to help lessen the burden of antibiotic resistance. Our research aimed to develop a method to rapidly detect antibiotic resistance genes in single-strain bacteria and metagenomic samples. To achieve this, we developed a nested PCR protocol that could enrich a panel of target resistance genes and provide each a genomic context through degenerate primers and Nanopore sequencing. In single-strain bacteria, we can enrich all target antibiotic-resistance genes; an average of 38% reads will provide a genomic context to each gene. Using our novel nested-PCR protocol, we can also enrich all target resistance genes up to 1000-fold in faecal samples compared to metagenomic sequencing. With this nested PCR protocol and Oxford Nanopore as a sequencing platform, we can detect target resistance genes in single-strain bacteria and faecal samples in under 6 hours, making this method rapid, inexpensive, and accessible. Further, we can provide a genomic context to each resistance gene, allowing us to determine whether genes are chromosomal or plasmid-bound.Item Experimental evolution under predation in P. fluorescens SBW25 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Auckland, New Zealand(Massey University, 2022) Sadeghpour Golzar, FarhadPredator-prey interactions are recognised to change the pace of evolution in microbial communities, but investigations into how selection for predation accelerates antagonistic behaviours, genomic evolution and the process of coadaptation are scarce. Here we performed a 20-day and an extended 90-day evolution experiment to investigate the adaptive traits that arise in prey bacterium Pseudomonas fluorescens SBW25 on solid media in the presence and absence of a wild Acanthamoeba sp. Coevolution led to bacterial diversity, resistance to predation in coevolved bacterial lineages and evolution of predators. We show evidence of reciprocal adaptation, strong phenotypic and genotypic parallelism among prey lineages undergoing predation. We observed evolution of new colony morphotypes such as Wrinkly Spreader, Volcano and Mountain. Evolved morphotypes conferred grazing resistance and an increase in relative prey fitness that resulted in increased encystment and reduced replication of the protozoan populations. Mutation profiles of the coevolved phenotypes were associated with altered gene function in amrZ, wspF, fadD1, fadD2 and putative hypothetical protein upstream of RND transporter. RNA sequencing results of the mutants also revealed a significant increase in the number of genes that up or downregulated while interacting with Acanthamoeba sp. We investigated the degree to which these mutations affect biofilm formation, capsulation, motility, mucoid and fatty acid degradation pathways. Some of these traits are associated with virulence in pathogenic organisms. We further found evidence of mutualisms where both prey and predator increased their survival relative to their respective ancestors. On the other side, we show promoted killing performance and higher generations upon feeding on WT bacteria in coevolved Acanthamoeba compared to their ancestors. Together, our findings demonstrate the emergence of divergent colony morphologies and molecular parallelism that arise as an adaptation to predation and notably affects the fitness and evolution of predators suggesting Red Queen co-evolutionary dynamics between predators and prey. These findings suggest that protozoan predation can profoundly influence the course of genetic and phenotypic evolution in short and long-time scales.Item Use of small extracellular vesicles for diagnosis of Mycoplasma bovis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Genetics at Massey University, Manawatū, New Zealand(Massey University, 2022) Pratt, Joel ThomasBackground: Mycoplasma bovis (M. bovis) is a pathogenic bacterium responsible for causing numerous production and welfare issues in cattle herds. Eradication efforts worldwide are limited by ineffective antibiotics, intracellular infection of host cells, immune evasion, and insufficient diagnostic tools. Current diagnostic tests are inadequate to assess M. bovis infection as they rely upon direct detection of M. bovis or indirect detection via serology (M. bovis–specific antibodies). Therefore, with aim to improve diagnostics of M. bovis infection, small extracellular vesicles (sEVs) were utilised. These nanoparticles act as intercellular messengers and contain material representative of their cell of origin. Their use as diagnostic and therapeutic tools has enabled a variety of diseases and infections to be assessed and/or treated. The aim of this project was to develop an upscaled in vitro model of M. bovis infection within a bioreactor flask to test the hypothesis that the protein cargo of host cell sEVs were altered in response to M. bovis infection. Methods: A control culture of a bovine endometrial epithelial cell line (bEEL cells) and a co–culture of bEEL cells and M. bovis were established within bioreactor flasks. Using Size Exclusion Chromatography columns, sEVs were isolated from the harvests of the bioreactor flasks. Liquid Chromatography Tandem Mass Spectrometry assessed the sEV proteome to compare differences created by M. bovis infection. Results: Infection was indicated by a continued presence of M. bovis within the co–culture. Changes in the regulation of various proteins, such as inhibition of host cell endopeptidases, was demonstrated in co–culture sEVs as a response to M. bovis infection. Adherence of M. bovis to bEEL cells was certain, but intracellular infection remained inconclusive. Conclusion: Data from this study implies that a co–culture can be successfully established within a bioreactor flask, and that the proteome of sEVs is altered in response to infection by M. bovis.Item Can microbe analysis of effluent be used as a proxy for bovine herd health? : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Albany, New Zealand(Massey University, 2021) Earnshaw, Alyssa MelanieThe microbial community within the bovine gut has been shown to have major impacts on the health of a cow. Most recent studies have focused on identifying and quantifying the gut microbiome of humans, and more recently of agricultural animals. Microbiome studies come with significant challenges, especially around quantifying bias. In this study, careful validation of controls was undertaken to ensure that the method that was followed was appropriate. These controls included a mock community, technical replicates and a spike-in for comparison of methods that could be used to classify a microbiome. Variation in these methods can be caused by sequencing type, classification tools and databases, as well as open-source pipelines. Using this information, the best pipeline was determined and then used to identify genera in previously uncharacterized bovine effluent microbiome samples. This pipeline consists of 16S rRNA gene Illumina sequencing with the USEARCH-UNOISE classification tool. The dairy effluent system analysed here is interconnected and shows a high degree of similarity in its microbiome composition across sampling locations. Samples have similar microbial communities caused most strongly by collection date, followed by location. The most abundant microbes present are those involved in breaking down faeces (i.e., Corynebacterium). I found that sequencing depth has a large impact on microbiome classification accuracy. Determining the core taxa of a microbiome will enable analysis of any changes from the expected microbes. These changes can be due to normal fluctuations, such as age. However, microbial dysbiosis can be due to pathogenic microbes. I also explored the effect of pathogenic microbes on microbial community composition as it can have a big influence on animal health. Early identification of infections can minimise the financial and bovine welfare impact on farms.