Previously, the exact phylogenetic relationship between the giant panda (Ailuropoda melanoleuca) and the red panda (Ailurus fulgens) has been unclear, but with the advent of molecular sequencing technologies, these two animals are now known to be distantly related. Although taxonomically distinct, these two panda species have several things in common, primarily their almost exclusively bamboo diet, but also their similar physical adaptations to their diet and their sympatry in some locations. What was unknown was if their similarities also extended to their gastrointestinal tract (GIT) microbiota. Using next-generation 16S rRNA parallel sequencing technologies, we uncovered several factors that impact bacterial communities of the pandas and also found that the two panda species harbor distinct microbial communities. In general, the Firmicutes and the Proteobacteria dominate both pandas’ gut microbiomes, with lesser contributions by the Actinobacteria and the Bacteroidetes. However, for the red panda, sex, age, and season significantly alter GIT microbiota. For giant pandas, we found that bacterial communities differed significantly between their normal fecal samples and mucus excretions. Together, these data display the plasticity of their bacterial communities, with several factors shaping GIT microbiota. As both species are highly threatened, understanding the relationship between the panda and their gut microbiome has given insight into the overall health of these uniquely herbivorous carnivores.

The objective of this research was to use Bacillus subtilis-based probiotics alone or combined with various additional antibiotic alternatives (yeast-derived prebiotics and zinc supplementation) to maintain gut health and improve growth performance of antibioticree broilers. Three consecutive studies have been conducted to evaluate dietary effects on intestinal morphology, digestive organ development, microbiota, and growth performance of commercial broilers under different coccidia-challenge circumstances. In the first study, broilers were raised under commercial conditions, without coccidia-challenge (Chapter III). Broilers fed diets supplemented with antibiotics exhibited the highest body weight gain (BWG) from d 15 to 27. Broilers fed diets supplemented with B. subtilis or prebiotics + B. subtilis exhibited higher BWG from d 28 to 41 and higher BWG from d 0 to 41 as compared to birds fed control diets. In the second study, broilers were exposed to a clinical coccidia-challenge (Chapters IV and V). Interaction and main effects of the dietary supplement and coccidial vaccination were studied. Diets supplemented with Prebiotics + B. subtilis facilitated broilers to reach a similar feed conversion ratio (FCR) as to the antibiotic control group from d 0 to 56. Feeding diets supplemented with B. subtilis to coccidial vaccinated broilers reduced microbiota diversity by increasing the proportion of an antibiotic-resistant bacterium, Rikenella microfusus. In the third study, interaction and main effects of the subclinical coccidia-challenge and the dietary supplement were studied (Chapters VI and VII). The dietary anticoccidial supplementation increased feed intake and BWG and decreased FCR of broilers from d 15 to 28. However, dietary B. subtilis supplementation did not improve any growth performance of broilers. The combined use of zinc and B. subtilis lowered the high mortality of broilers fed diet supplemented with B. subtilis or zinc alone. Additionally, the dietary supplementation of zinc lowered the proportion of Clostridium in cecal contents of broilers. In conclusion, different nutrition strategies should be considered when broiler chickens are under different health circumstances. Bacillus subtilis-based probiotics have potential to replace the antibiotics, but not anticoccidial feed additives.

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Bacteria, especially symbiotic species in the gastrointestinal tract, have lived with human for long time and are involved in many aspects of host physiology. There is growing evidence that microbiota-gut-brain axis plays an important role in modulating the response to stress in both human and animals. Alterations in the gut microbiota can change the central nervous system function through effects on the endocrine, immune and nervous systems. Recent studies suggest that probiotic treatment may help to maintain resistance against the detrimental effects of stress though the microbiota-gut-brain axis. However, how potentially beneficial bacteria interact with specific immune and neural components, to mediate beneficial effects on behavior remain unclear. Using chronic social defeat stress, a model often used in post-traumatic stress disorder research, we found that administration of Lactobacillus rhamnosus JB-1 beginning 48 hours following chronic social defeat led to persistence of fear memory and social deficits. These effects were associated with changes in gene expression related to emotion and memory in the hippocampus. This was in contrast to previous studies showing that probiotic intervention during social defeat prevents stress induced deficits in social behavior. This indicates that timing of L. rhamnosus treatment in relation to stress exposure has important implications for effects of the bacteria on behavior. In relation to the mechanism of action of L. rhamnosus on behavior, we demonstrate through depletion and adoptive transfer experiments that CD4+CD25+ T cells in mice treated with JB-1 were necessary and sufficient for JB-1 induced anxiolytic and antidepressant-like effects. Evidence also suggested that Ly6Chi monocytes may be a downstream target inhibited by Tregs involved in the behavioral effects of the bacteria. We observed that JB-1 could also reduce the number of activated microglia in the hippocampus, and attenuate hypothalamic-pituitary-adrenal axis reactivity with the integrity of vagus nerve. Crucially we demonstrated that JB-1 induced promotion of peripheral Tregs, reduction in microglia activation in the hippocampus, and attenuation of HPA axis reactivity, were all inhibited following vagotomy indicating that vagus nerve integrity is required to maintain immune and endocrine linkages from gut microbes to the brain. These studies demonstrate prerequisites for beneficial probiotic effects on stress related behaviours including a specific time window in relation to stress exposure, the activation of regulatory immune cells, and undisrupted vagal nerve signalling. These findings highlight the inter-systemic communication of the microbiota-gut-brain axis in the stress response, and might help to unveil more therapeutic opportunities in relation to stress-related mood disorders. / Thesis / Doctor of Philosophy (PhD) / Excessive exposure or dysregulated responses to stress in human and animals induces behavioral changes and the development of mood disorders. The Microbiota-gut-brain axis plays an important role in maintenance of homeostasis. However, crosstalk between the different components of microbiota-gut-brain axis, and how specific microbes can modulate these interactions, remains unclear. Thus, we sought to understand the mechanism of inter-systemic communication linking a specific gut microbe to changes in stress response and behavior. We observed immunoregulation by regulatory T cells were essential in Lactobacillus rhamnosus JB-1 induced anxiolytic and antidepressant-like effects. We also found the integrity of vagus nerve was necessary for JB-1 induced promotion of regulatory T cells and decrease in microglial activation and attenuation of hypothalamic-pituitary-adrenal axis that are associated with the anxiolytic effects of the bacteria. We also identified that the temporal relationship between exposures to stress and the bacteria is important as ingestion of JB-1 directly after chronic social defeat lead to persistence of fear memory and social deficits. This work will help us to understand mechanisms underlying the microbiota-gut-brain axis, which may allow for the development of novel microbe based therapeutic intervention against mood disorders.

Obesity is a growing problem that impacts both adults and children. Obesity is linked to the development of unfavorable health outcomes like excess fat accumulation in the liver, a problem known as non-alcoholic fatty liver disease (NAFLD). Brown adipose tissue (BAT), a thermogenic body fat that can be turned on by cold, produces heat by consuming circulating lipids and glucose in a futile cycle. Less active cold-stimulated BAT is linked to obesity and type 2 diabetes in adults but whether this relationship exists in children is unknown. In rodents, increases in BAT activity are associated with reductions in NAFLD, effects that may be mediated through changes in the gut microbiome and reductions in peripheral serotonin. Whether the gut microbiome and serotonin play a role in regulating BAT activity in adults and children is not known. In this thesis, we have utilized magnetic resonance imaging (MRI) proton density fat fraction (PDFF) to assess BAT in the supraclavicular (SCV) region after three hours and one hour of whole-body cold exposure in adults and children, respectively. In 60 adults (aged 18-57 years), we examined whether there is a relationship between cold-stimulated BAT activity and liver fat (assessed via MRI) and whether gut microbiota plays a role in connecting these two tissues. In children, we investigated, BAT activity after whole-body cold exposure in 26 boys (aged 8-10 years). We also explored if BAT activity was different between boys with and without overweight/obesity. Finally, in young boys, we measured the levels of serotonin in platelet-poor plasma and its metabolic end product 5-hydroxyindole acetic acid (5-HIAA) in the urine. We explored if these measures of circulating serotonin were related to cold-stimulated BAT activity and if they were different between boys with and without overweight/obesity. The findings from this research indicate that higher cold-stimulated BAT activity is associated with lower liver fat in adults, but that this relationship is unlikely mediated through changes in the gut microbiota. Additionally, boys with overweight/obesity have lower cold-stimulated BAT activity and lower 5-HIAA in their urine compared to those with normal weight. Moreover, circulatory serotonin is negatively related to total adiposity. However, circulating serotonin is not related to cold-stimulated BAT activity in this cohort. These findings are important as they indicate for the first time that increasing BAT activity in adults and children could potentially be a new avenue for the treatment of NAFLD and obesity. / Thesis / Doctor of Philosophy (PhD) / Obesity is a risk factor for the accumulation of extra liver fat, a problem known as non-alcoholic fatty liver disease (NAFLD). Brown adipose tissue (BAT) is a kind of body fat that rather than storing calories like white fat burns calories when switched on by cold. Studies in adults have shown that people with obesity and type 2 diabetes have less active BAT suggesting switching it on may be helpful to promote weight loss and lower glucose. However, whether this relationship exists in children is not known. In rodents, increased BAT activity has also been linked to reductions in NAFLD, effects that might involve a hormone called serotonin, or changes in the gut microbiome but whether this is important in children and adults is also not understood. In this thesis, we utilized magnetic resonance imaging (MRI) to examine BAT activity after whole-body cold exposure in adults (3 hours) and children (1 hour). In 60 adults (aged 18-57 years), we report that higher cold-stimulated BAT activity is linked to NAFLD, but gut microbiota does not seem to play a role in this relationship. In 26 boys (aged 8-10 years), BAT is less responsive to cold in boys with overweight/obesity compared to boys with normal weight. Additionally, serotonin is lower in boys with overweight/obesity compared to boys with normal weight. These findings suggest that increasing BAT activity in adults and children could potentially be a new avenue for the treatment of NAFLD and obesity.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13536号 / 論医博第2276号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 妹尾 浩, 教授 中川 一路 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introducción: El incremento de pacientes con diabetes ha forjado la búsqueda de nuevas investigaciones y de nuevos enfoques terapéuticos preventivos de esta enfermedad. En los últimos años viene cobrando gran importancia el estudio de la asociación entre la diabetes tipo 2 y los microorganismos que habitan en el tracto gastrointestinal, responsables de modificar y contribuir positiva o negativamente con el metabolismo del huésped. Metodología: En el estudio participaron 26 pacientes con diabetes mellitus tipo 2 atendidos en el servicio de Endocrinología de un hospital nivel 4, durante el periodo de agosto de 2016 a febrero de 2017. De cada paciente se colectaron muestras de heces, datos clínicos y la frecuencia de consumo de alimentos. Se identificaron 13 bacterias de la microbiota intestinal en muestras fecales de pacientes diabéticos tipo 2 metabólicamente controlados frente a no controlados mediante la técnica de reacción en cadena de la polimerasa (PCR). Resultados: Se identificó al menos un género de bacterias colónicas en pacientes diabéticos controlados y no controlados en 5 (71,4%) y 11 (57,9%) de los casos respectivamente. Los géneros de bacterias más frecuentes encontrados en los pacientes controlados fueron Proteobacteria, Firmicutes y Prevotella; mientras que en los pacientes no controlados se encontró con mayor frecuencia Prevotella, Firmicutes y Clostridium. Conclusiones: La comunidad bacteriana que reside en el colon es única en cada individuo y varía constantemente. Se observó la presencia de 11 bacterias en total. No se encontró diferencia estadística en la comparación de ambos grupos. / Introduction: The increasing incidence of patients with the diagnosis of Diabetes Mellitus type 2 (DM2) has shifted the focus of new research on preventative therapeutic approaches. Recent evidence suggests an important association between the prognosis of patients with DM2 and their gastrointestinal bacterial microbiota linked to modifications that may positively or negatively change the host’s metabolism. Methodology: The study included 26 patients diagnosed with Diabetes Mellitus Type 2 in the Endocrinology service of a tertiary referral hospital, between August 2016 and February 2017. Stool samples were collected from each patient as well as their food consumption frequency record and relevant clinical data. The fecal bacterial microbiota was analyzed by polymerase chain reaction (PCR) to identify 13 different genera of bacterias from type 2 diabetic patients metabolically controlled versus uncontrolled type 2 diabetic patients. Results: At least one genus of colonic bacteria was identified in controlled and non-controlled diabetic patients in 5 (71.4%) and 11 (57.9%) respectively. The most frequent bacterial genera found in the controlled patients were Proteobacteria, Firmicutes and Prevotella; while in the non-controlled patients were Prevotella, Firmicutes and Clostridium Conclusions: The gut microbiota of a host is unique to each patient and varies constantly. It was observed the presence of 11 bacteria in total. No statistical difference was found in the comparison of both groups. / Tesis

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In this work we use the fruit fly Drosophila melanogaster as a model to identify bacterial genes that help bacteria to persist in their animal hosts. Early work on this model system established that dietary replenishment drives the composition of the D. melanogaster gut microbiota, and subsequent research has shown that some bacterial strains can colonize the fly for much longer than the flow of bulk diet through the gut. In this work we reveal that bacterial genes influence bacterial persistence by studying the correlation between bacterial genotype and persistence in the D. melanogaster gut microbiota. We performed an initial assay with 7 bacterial strains to establish that different bacterial strains persist differently independent of ingestion in the fly. We then repeated the assay with 41 different strains of bacteria in order to perform a metagenome wide association (MGWA) to find distinct bacterial genes that are significantly correlated with persistence. Based on the MGWA, we tested if 44 mutants from 6 gene categories affect bacterial persistence in the flies. We identified that transposon insertions in four flagellar genes (fliF, flgH, fliI, and flgE), one urea carboxylase gene, one phosphatidyl inositol gene, one bacterial secretion gene, and one antimicrobial peptide (AMP) resistance gene each significantly lowered colonization forming units (CFUs) that resulted from plating the gut content in Drosophila melanogaster. Follow-up experiments with the flagellar gene mutants revealed that each significant flagellar mutant was non-motile compared with the wild type. Taken together, these results reveal that there are bacterial genes that are involved in mechanisms, like bacterial motility, that help bacteria to persist in the fly gut.

No description available.