Les populations de bivalves exploités subissent régulièrement des épizooties qui affaiblissent voire déciment les stocks, et qui peuvent avoir des conséquences majeures pour l’aquaculture. Ces maladies, dues à des virus, bactéries, ou parasites, se développent particulièrement au printemps et en été. Ces périodes de l’année offrent également des conditions propices aux efflorescences de micro-algues toxiques, dont des dinoflagellés du genre Alexandrium. Ainsi, le risque de co-occurrence d’efflorescences d’Alexandrium sp. et de maladies infectieuses chez les bivalves est élevé. Or, ces micro-algues synthétisent et excrètent des neurotoxines et des composés cytotoxiques responsables d’altérations physiologiques chez les bivalves. L’objectif de cette thèse est d’évaluer les effets combinés d’une exposition à Alexandrium sp. et d’une infection par des agents pathogènes sur la physiologie des bivalves, à travers l’étude de différentes interactions tripartites bivalve – pathogène – Alexandrium sp. Les résultats de ce travail indiquent que différents profils de réponse existent en fonction des espèces impliquées dans ces interactions. Ainsi, une exposition à Alexandrium sp. peut augmenter le taux d’infection par des agents pathogènes chez des bivalves ou au contraire le diminuer. Les réponses hémocytaires associées peuvent traduire l’implication des défenses immunitaires dans ces modulations hôte-pathogène. De plus, l’exposition à des agents pathogènes peut interférer avec le processus d’accumulation de toxines algales dans les tissus des bivalves, illustrant la complexité de ces interactions. Ces résultats, associés à l’observation de lésions tissulaires chez les bivalves peuvent traduire l’altération des activités de nutrition (filtration, digestion…). Ce travail de thèse apporte une meilleure compréhension de l’implication des efflorescences toxiques dans le développement des maladies touchant les bivalves d’intérêt commercial, mais également de l’implication de l’environnement biotique des bivalves sur l’accumulation de phycotoxines réglementées. / Bivalve populations undergo regular epidemics that weaken or decimate exploited stocks and thus limit aquaculture. These diseases are caused mainly by viruses, bacteria or parasites, and occur primarily during spring and summer. This period of the year also provides favorable conditions for toxic dinoflagellate blooms, including species of the genus Alexandrium. Thus, the risk of Alexandrium sp. blooms and infectious diseases co-occurring in bivalves is high. However, these micro-algae synthesize and excrete toxins and cytotoxic compounds responsible for physiological changes in bivalves and could lead to an immuno-compromised status.The objective of this thesis is to evaluate the combined effects on bivalve physiology of exposure to the toxic dinoflagellate, Alexandrium sp., and infection by pathogens, through the study of different bivalve - pathogen - Alexandrium sp. tripartite interactions. The results of this work highlight the species-specific nature of these impacts.Thus, exposure to Alexandrium catenella reduces the herpesviruses infection in oyster Crassostrea gigas, whereas the dinoflagellate A. fundyense increases the susceptibility of C. virginica oyster to the parasite Perkinsus marinus, probably via immuno-suppression, as suggested by the partial inhibition of hemocyte responses. Additionally, the effect of a toxic algal bloom on oyster susceptibility to opportunistic diseases when exposed to a new microbial environment (simulating a transfer) was evaluated. Hemocyte responses to a changing microbial environment were suppressed by exposure to A. catenella, although no new bacterial infection was detected.Finally, exposure to pathogens or to a new microbial environment interferes with the processes by which oysters exposed to A. catenella accumulate algal toxins, illustrating the complexity of these interactions. These results provide a better understanding of the involvement of toxic algal blooms in the development of diseases affecting commercial bivalve species, but also of the involvement of the bivalve biotic environment in the accumulation of regulated toxins.

Le microbiome bactérien constitue l’ensemble des bactéries retrouvées au sein d’un espace. Le rôle du microbiome pulmonaire dans l’asthme est mal défini, mais il est maintenant établi que celui de patients asthmatiques diffère de celui d’individus sains. Cependant, l’influence des conditions environnementales et de la médication sur le microbiome pulmonaire est méconnue et leurs effets difficiles à contrôler chez l’humain. De plus, la stabilité du microbiome dans le temps demeure controversée. L’hypothèse de l’étude est que les microbiomes pulmonaire, nasal et oral de chevaux non-médicamentés varient avec l’environnement et que le statut asthmatique affecte principalement le microbiome pulmonaire. Six chevaux atteints d’asthme équin sévère et 6 chevaux sains ont été gardés dans 3 environnements distincts (exposition antigénique faible, modérée et élevée). Dans chaque environnement, la fonction pulmonaire a été évaluée et des lavages bronchoalvéolaires (LBA), oraux et nasals ont été prélevés. La région V4 du gène de l’ARN ribosomal 16S a été séquencée (Illumina MiSeq 4) et analysée à l’aide du logiciel Mothur et du progiciel Vegan dans R. Les communautés bactériennes pulmonaires, orales et nasales sont fortement regroupées par conditions environnementales et l’effet de l’environnement est plus marqué chez les chevaux sains. Le microbiome pulmonaire des chevaux asthmatiques diffère de celui des chevaux sains au niveau taxonomique de la famille, avec une tendance à une surreprésentation des Pasteurellaceae, contrairement aux microbiomes nasal et oral qui ne sont pas différents entre les deux groupes. Les familles bactériennes Neisseriaceae, Lachnospiraceae et Bacteroidaceae, dont certaines espèces sont potentiellement pathogènes, ont été seulement retrouvées dans les LBAs de chevaux asthmatiques. Cette étude montre que les microbiomes bactériens pulmonaires des chevaux sains et asthmatiques ne recevant pas de médication sont différents et varient selon le niveau d’exposition antigénique. Cette différence étant présente principalement lorsque les chevaux asthmatiques ont une inflammation pulmonaire suggère que le microbiome pulmonaire altéré dans l’asthme n’est pas inhérent, mais coïncide avec l’inflammation pulmonaire. Le rôle du microbiome dans la perpétuation de l’inflammation reste à investiguer. / Bacterial microbiome is defined as the whole bacterial population found within a space. The role of the pulmonary microbiome in asthma is poorly defined, but it is now well established that the one of asthmatic patients differs from that of healthy individuals. However, the influence of environmental conditions and medication on pulmonary microbiome is poorly known and effects difficult to control in humans. Moreover, microbiome stability over time remains controversial. The hypothesis of this study is that the pulmonary, nasal and oral microbiomes of unmedicated horses vary with the environment and that asthmatic status does affect the pulmonary microbiome. Six horses with severe equine asthma and 6 healthy horses were kept in 3 distinct environments (low, moderate and high antigen exposure). In each environment, pulmonary function has been evaluated and bronchoalveolar lavages (BALs), nasal and oral washes were collected. The V4 region of the 16S rRNA gene was sequenced (Illumina MiSeq 4) and analyzed using the Mothur software and the Vegan package in R. Pulmonary, oral and nasal bacterial communities are strongly grouped by environmental conditions and the effect of the environment is more pronounced in healthy horses. The pulmonary microbiome of asthmatic horses differs from that of healthy horses at the family level of taxonomic designation, with a tendency towards an overrepresentation of Pasteurellaceae, unlike nasal and oral microbiomes which are not different between the two groups. The bacterial families Neisseriaceae, Lachnospiraceae and Bacteroidaceae with pathogenic potential were only found in the BALs of asthmatic horses. This study shows that the lung bacterial microbiomes of healthy and asthmatic horses receiving no medication are different and vary accordingly to the antigenic exposure level. This difference is present mainly when asthmatic horses have a strong pulmonary inflammation which suggest that the altered pulmonary microbiome is not inherent but coincident with pulmonary inflammation. Its role in the perpetuation of inflammation remains to be investigated.

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The emerging evidence on the interconnectedness between the gut microbiome and host metabolism has led to a paradigm shift in the study of metabolic diseases such as obesity and type 2 diabetes with implications on both underlying pathophysiology and potential treatment. Mounting preclinical and clinical evidence of gut microbiota shifts, increased intestinal permeability in metabolic disease, and the critical positioning of the intestinal barrier at the interface between environment and internal milieu have led to the rekindling of the “leaky gut” concept. Although increased circulation of surrogate markers and directly measurable intestinal permeability have been linked to increased systemic inflammation in metabolic disease, mechanistic models behind this phenomenon are underdeveloped. Given repeated observations of microorganisms in several tissues with congruent phylogenetic findings, we review current evidence on these unanticipated niches, focusing specifically on the interaction between gut permeability and intestinal as well as extra-intestinal bacteria and their joint contributions to systemic inflammation and metabolism. We further address limitations of current studies and suggest strategies drawing on standard techniques for permeability measurement, recent advancements in microbial culture independent techniques and computational methodologies to robustly develop these concepts, which may be of considerable value for the development of prevention and treatment strategies.

The bacteria in host-associated microbial communities influence host health through various mechanisms, such as immune stimulation or the release of metabolites. However, viruses that target bacteria, called bacteriophages (phages), may also shape the animal microbiome. Most phage lifecycles can be classified as either lytic or temperate. Lytic phages infect and directly kill bacterial hosts and can directly regulate bacterial population size. Temperate phages, in contrast, have the potential to undergo either a lytic cycle or integrate into the bacterial genome as a prophage. As a prophage, the phage may alter bacterial host phenotypes by carrying novel genes associated with auxiliary metabolic functions, virulence-enhancing toxins, or resistance to other phage infections. Lytic phages may also carry certain auxiliary metabolic genes, which are instead used to takeover bacterial host functions to better accommodate the lytic lifecycle. In either case, the ability to alter bacterial phenotypes may have important ramifications on host-associated communities. This dissertation focused on the genetic contributions that phages, and particularly prophages, provide to the bacterial members of two separate host-associated communities: the honey bee (Apis mellifera) gut microbiome and the amphibian skin microbiome. My second chapter surveyed publicly available whole genome sequences of common honey bee gut bacterial species for prophages. It revealed that prophage distribution varied by bacterial host, and that the most common auxiliary metabolic genes were associated with carbohydrate metabolism. In chapter three, this bioinformatic pipeline was applied to the amphibian skin microbiome. Prophages were identified in whole genome bacterial sequences of bacteria isolated from the skin of American bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), Spring peepers (Pseudacris crucifer) and American toads (Anaxyrus americanus). Prophages were additionally identified in publicly available genomes of non-amphibian isolates of Janthinobacterium lividum, a bacteria found both on amphibian skin and broadly in the environment. In addition to a diverse set of predicted prophages across amphibian bacterial isolates, several Janthinobacterium lividum prophages from both amphibian and environmental isolates appear to encode a chitinase-like gene undergoing strong purifying selection within the bacterial host. While identifying the specific function of this gene would require in vitro isolation and testing, its high homology to chitinase and endolysins suggest it may be involved in the breakdown of either fungal or bacterial cellular wall components. Finally, my fourth chapter revisits the honey bee gut system by investigating the role of geographic distance in bacteriophage community similarity. A total of 12 apiaries across a transect of the United States, from Virginia to Washington, were sampled and honey bee viromes were sequenced, focusing on the lytic and actively lysing temperate community of phages. Although each apiary possessed many unique bacteriophages, apiaries that were closer together did have more similar communities. Each bacteriophage community also carried auxiliary carbohydrate genes, especially those associated with sucrose degradation, and antimicrobial resistance genes. Combined, the results of these three studies suggest that bacteriophages, and particularly prophages, may be contributing to the genetic diversity of the bacterial community through nuanced relationships with their bacterial hosts. / Doctor of Philosophy / The microbial communities of animals, called "microbiomes", play important roles in the health of animals. The bacteria in these microbiomes can help strengthen the immune system, provide resistance to dangerous pathogens, and break down nutrients. However, bacteria are not alone in the microbiome; viruses are also present. Surprisingly, the vast majority of the world's viruses, even those living inside animals, infect bacteria. These viruses, called "bacteriophages" or "phages", can impact the bacterial communities in a microbiome. Phages can be grouped in to two broad categories based on lifecycle. Lytic phages kill the bacterial host directly after infection. Temperate phages, on the other hand, can either immediately kill the host like lytic phages or alternatively, become a part of the bacterial genome and live as prophages. Phages with both lifecycles can sometimes carry genes that, although not essential to the phage, may change the traits of the bacteria during infection. For example, some phages carry toxin genes, which bacteria use to cause disease in animals. Other phages might carry genes that provide antibiotic resistance or alter the metabolism of the infected bacteria. If a phage gene benefits the infected bacteria, the bacteria may begin interacting with its environment in a new way or may even become more abundant. Alternatively, phages that directly kill infected bacteria may have a negative effect on bacterial population sizes. To begin unraveling how phages influence bacterial species in microbiomes, I investigated two different animal systems: the Western honey bee (Apis mellifera) gut microbiome and the amphibian skin microbiome. I first identified prophages of several common bacterial species that reside in the honey bee gut (Chapter 2). Prophages were more common in certain bacterial species than others, and some possessed genes associated with the breakdown of sugars or pollen, suggesting they help honey bees process their food. Using similar techniques, I then identified prophages in bacteria isolated from the skin microbiomes of several amphibian species common in the eastern United States (American bullfrogs, Eastern newts, Spring peepers, and American toads) (Chapter 3). Most notably, the bacteria Janthinobacterium lividum may benefit from prophages that carry genes for potentially antifungal chitinase enzymes that destroy the fungal cell wall. Finally, I returned to the honey bee gut microbiome system by investigating how honey bee bacteriophage communities change over large geographic distances (Chapter 4). This study, which examined honey bees from 12 apiaries sampled from the east to west coast of the United States, looks primarily at lytic phage and temperate phage that are not integrated as prophage, but are instead seeking a bacterial host to infect. I found that nearby apiaries tended to have more similar communities of bacteriophages, compared to apiaries far away. Additionally, most bacteriophage communities carry genes associated with the breakdown of sugars like sucrose. Overall, these three studies show that phages, and especially prophages, contribute to the genetic landscape of the microbiome by broadly providing bacterial hosts with access to a diverse set of genes.

Microbiome plays an important role in human health. The analysis of association between microbiome and clinical outcome has become an active direction in biostatistics research. Testing the microbiome effect on clinical phenotypes directly using operational taxonomic unit abundance data is a challenging problem due to the high dimensionality, non-normality and phylogenetic structure of the data. Most of the studies only focus on describing the change of microbe population that occur in patients who have the specific clinical condition. Instead, a statistical strategy utilizing distance-based or similarity-based non-parametric testing, in which a distance or similarity measure is defined between any two microbiome samples, is developed to assess association between microbiome composition and outcomes of interest. Despite the improvements, this test is still not easily interpretable and not able to adjust for potential covariates. A novel approach, kernel-based semi-parametric regression framework, is applied in evaluating the association while controlling the covariates. The framework utilizes a kernel function which is a measure of similarity between samples' microbiome compositions and characterizes the relationship between the microbiome and the outcome of interest. This kernel-based regression model, however, cannot be applied in longitudinal studies since it could not model the correlation between the repeated measurements. We proposed microbiome association exact tests (MAETs) in linear mixed model can deal with longitudinal microbiome data. MAETs can test not only the effect of overall microbiome but also the effect from specific cluster of the OTUs while controlling for others by introducing more random effects in the model. The current methods for multiple variance component testing are based on either asymptotic distribution or parametric bootstrap which require large sample size or high computational cost. The exact (R)LRT tests, an computational efficient and powerful testing methodology, was derived by Crainiceanu. Since the exact (R)LRT can only be used in testing one variance component, we proposed an approach that combines the recent development of exact (R)LRT and a strategy for simplifying linear mixed model with multiple variance components to a single case. The Monte Carlo simulation studies present correctly controlled type I error and provided superior power in testing association between microbiome and outcomes in longitudinal studies. Finally, the MAETs were applied to longitudinal pulmonary microbiome datasets to demonstrate that microbiome composition is associated with lung function and immunological outcomes. We also successfully found two interesting genera Prevotella and Veillonella which are associated with forced vital capacity.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The aim of this study was to explore proteomics and microbiome profiling in pigs fed high fibre diets. In the first phase, maize cobs were ensiled using whey, molasses and exogenous enzymes in the laboratory and effects on ensiling characteristics and fibre levels were evaluated. In the second phase South African Windsnyer-type Indigenous pigs (SAWIP) and Large White x Landrace crosses (LW x LR) were fed diets containing ensiled maize cobs and evaluated on; diet preferences, nutrient digestibility and colonic fermentation; growth performance, carcass traits and blood metabolite profiles; the faecal microbiome; and serum and liver proteomic profiles. Ensiling maize cobs with molasses, whey and exogenous enzymes did not improve fermentation characteristics but exogenous enzymes reduced fibre fractions and energy content of maize cob silages. Dets containing two levels of maize cobs ensiled without any additive; a low (LMC) and high (HMC) maize cob inclusion levels and a control diet which did not have any maize cobs (CON) were formulated. The SAWIP preferred the CON diet more than (P < 0.05) diets with maize cobs while the LW x LR had no feed preferences. There was no correlation between preference and diet digestibility in both breeds. The SAWIP digested nutrients better (P < 0.05) than the LW x LR in the high fibre diets. There were no differences in the diversity of the core composition of gut bacterial communities between the breeds and diets. There were differences in the ratios of Bacteroidia to Clostridia between the SAWIP and LW x LR. Verrucomicrobiae was present in SAWIP and LW x LR on HMC diet and not on the CON diet. There was a breed x diet interaction (P < 0.05) for Oscillospira. Analysis of the microbiome revealed breed differences and no dietary differences. There were differences in serum and liver proteins and in serum metabolite levels. Two specific proteins identified were Guanidinoacetate N-methyltransferase-like isoform 1 associated with creatine biosynthetic and Catalase, which is involved in cholesterol metabolic processes. At the grower stage, the SAWIP consumed more feed per metabolic body weight than the LW x LR while at the finisher stage LW x LR consumed more feed per metabolic body weight (P < 0.05) than the SAWIP. The breed of pig influenced most of the growth performance and carcass parameters more than the diet did. The SAWIP demonstrated an adaptation to high fibre diets by consuming more feed than the LW x LR per metabolic body weight at the grower stage. The inclusion of ensiled maize cobs in diets did not negatively affect selected commercial pork cuts. Analysis of faecal microbiomes revealed differences that may explain the enhanced ability of the SAWIP to digest fibrous diets better than the LW x LR breed.Proteomics can identify biomarkers that evaluate the performance of pigs consuming high fibre diets. A proof of principle to assess serum and liver protein profiles of pigs fed a a high fibre diet using a sodium dodecyl sulphate polyacrylamide gel electrophoresis matrix-assisted laser desorption ionization mass spectrometry (SDS-PAGE /MALDI MS) workflow was established. Key words: ensiling, exogenous enzymes, palatability, fermentation, fibre, metageome, biomarkers.

Introduction: Inflammatory pouch complications, including pouchitis, chronic pouchitis (CP) and a Crohn’s disease-like phenotype (CDL) of the pouch following ileal pouch-anal anastomosis (IPAA), are relatively common, and arise via unknown mechanisms. The phenotypic similarities between pouch inflammation and inflammatory bowel disease (IBD) suggest there may be common pathways involved in both disorders. The aim of this thesis is to investigate the serological, genetic and microbial factors contributing to the development of pouch inflammation in a large, well characterized patient cohort. Methods: Subjects with IPAA were recruited, and clinical and demographic information was obtained through medical chart review and patient questionnaire, allowing patients to be grouped based on post-surgical phenotype. Blood and tissue was collected for genetic, serological and microbial analyses. Anti-microbial antibodies were detected using enzyme-linked immunosorbent assay (ELISA), genotyping was carried out using the Illumina Goldengate custom SNP assay and Sequenome iPLEX platform, and tissue-associated microbial communities were assessed using 454 pyrosequencing. Results: Among our cohort, smoking was associated with CDL (P=0.003) and Ashkenazi Jewish heritage with CP (P<0.008). NOD2insC (rs2066847) (P=7.4x10-5), anti-CBir1 (P<0.0001) and ASCA (IgG) (P=0.03) were significantly associated with inflammatory pouch outcomes. Additional SNPs in NOX3, DAGLB, and NCF4 were also marginally associated with pouch outcome. A multi-variable risk model combining clinical, serologic and genetic markers was constructed and could differentiate between chronic pouch inflammatory phenotypes and no pouchitis. Genus level microbial analysis demonstrated that several organisms (Bacteroides, Parabacteroides, Blautia and Moryella) were detected less frequently among the inflammatory outcome groups (P<0.05). These associations remained significant even following adjustment for antibiotic use, smoking, country of birth and gender. Conclusions: CD-associated anti-microbial antibodies and genetic markers are associated with chronic inflammatory pouch phenotypes. Additionally, changes in the composition of the pouch associated microbiome are associated with inflammation. These observations suggest that similar mechanisms may be involved in non-surgical IBD and pouchitis.

Effects of a short 40-d dry period with only a close-up diet (SHORT) and a conventional 60-d dry period with a 39-d far-off and a 21-d close-up diet (CONV) on milk production, feed intake, blood and rumen parameters, liver gene expression and rumen microbiota profile were compared in 11 second-parity and 15 third and later parity cows. Milk production was recorded daily during the first 16 wks of lactation. Differential liver gene expression was assessed by affymetrix microarray analysis and DNA extracted from rumen samples was subjected to Illumina sequencing for exploring the microbiome profile. The SHORT treatment reduced milk yield and DMI after calving in third and later parity cows, but not in second-parity cows when compared to the CONV treatment. Cows on the SHORT treatment had higher concentrations of NEFA in blood plasma and tended to have higher liver TAG immediately after calving. These effects tended to be greater in third- and later parity cows compared to second-parity cows. Expression patterns of genes involved in β-oxidation at the first week of lactation compared to those at three weeks before calving showed lower hepatic β-oxidation capacity in cows on the SHORT treatment compared to those on the CONV treatment. During this period, the expression of DGAT, a key gene in the triglyceride synthesis, increased in SHORT-treatment cows while it remained unchanged in CONV-treatment cows. The expression patterns of genes involved in gluconeogenesis showed a higher capacity at first week after calving in cows on the SHORT compared to those on the CONV treatment. Our study also showed that the SHORT treatment increased the relative abundance of Firmicutes and reduced the relative abundance of Bacteroidetes compared to the CONV treatment and reduced the shifting of rumen microbiota from before to after calving. Results also demonstrated that the rumen microbiota was more stable in the SHORT treatment during the transition period. Based on these results, a 40-d dry period management with only a close-up diet might be beneficial for second parity cows. However, this treatment may be detrimental for older cows as excessive energy intake and fat deposition during the dry period in these animals result in lower milk production and higher mobilization of NEFA and accumulation of fat in the liver. / February 2016