The primary objectives of the following experiments were to: 1) determine the impact of a directed microbial (DFM) blend on digestibility and microbial populations on horses fed low (LQ) and high (HQ) quality hay, 2) evaluate the differences in diet digestibility, microbial populations, and blood metabolites due to DFM supplementation to the exercising horse, 3) assess the impact of short-chain fructooligosaccharide (scFOS) supplementation on digestibility and the fecal microbial ecosystem in senior (SR) vs. mature (MA) horses, and 4) determine the relationship between insulin dysregulation (ID) and morphometric neck measurements (MNM) in the non-obese stock-type horse. Results indicated that: 1) Supplementation with DFM tended to be beneficial in enhancing CP digestibility. Feeding CP beyond requirements may contribute to excess excretion of Escherichia coli (EC) in HQ which was supported by 16S rRNA analysis. Differences in the fecal microbial ecosystem were detected between LQ and HQ. The phylum Saccharibacteria was identified in both hay qualities even though it has been lightly reported in equine literature. 2) Supplementing DFM to moderately exercised horses tended to enhance DM and ADF digestibility but there was no evidence of a dietary modulation to the fecal bacteria isolated. 3) No differences were found between MA and SR when fed scFOSded ration balancer which indicates that scFOS may help mitigate decreases in digestibility experienced by SR. Senior horses had less diversity in their fecal bacterial population, which may help explain why SR are more prone to ailments such as colic and impaired immune function. 4) Obesity is an important genetic factor for ID however it should not be the only determining factor, as ID can occur in non-obese individuals. One individual out of 62 tested horses was identified as ID. Morphometric neck measurements (MNM) may help pinpoint horses that are at a greater risk for ID, but more research is needed to validate MNM in both obese and non-obese stock-type horses. There were correlations between glucose sampled before an oral sugar test (OST) and insulin post-OST but a fasting OST would still be recommended in assessing ID.

The prevalence of obesity is increasing globally. Obesity is characterized by increased fat mass and is a risk factor for type 2 diabetes (T2D). Obesity is associated with hyperglycaemia, hyperinsulinemia, insulin resistance and chronic inflammation. Currently, the most effective and durable treatment for obesity and its comorbidities is bariatric surgery. Bariatric surgery changes food intake, energy balance and the composition of gut microbiota. Bariatric surgery can lower blood glucose and put T2D into remission. It was unknown if bariatric surgery-induced changes in the gut microbiota was an independent yet sufficient factor to lower blood glucose. Fecal microbiota transplantation (FMT) was performed on conventional (specific-pathogen-free, SPF) and germ-free (GF) mice using fecal material obtained from patients before surgery and 12 months after bariatric surgery. We tested FMT into mice from the same patients before and after vertical sleeve gastrectomy (VSL) and biliopancreatic diversion with duodenal switch (BPD/DS). FMT did not alter body weight, fat mass, glucose tolerance or glucose transporter mRNA expression in all intestine segments in SPF mice. FMT lowered blood glucose during an oral glucose load in GF mice receiving bacteria after VSL and BPD/DS bariatric surgery. Post-BPD/DS surgery FMT decreased Glut1 transcript level in the ileum and increased Glut1 transcript level in the TA muscle of GF mice, but did not change GLUT1 protein levels. Post-BPD/DS surgery FMT also decreased goblet cell count, villus height and crypt depth in the ileum of GF mice. We conclude that changes in the gut microbiota caused by bariatric surgery is a standalone factor that can lower blood glucose and alter gut morphology. / Thesis / Master of Science (MSc) / Type 2 diabetes is a chronic disease that involves high blood sugar (i.e. glucose), which can damage many parts of the body leading to serious complications. Diabetes is a growing global problem and is the seventh leading cause of death. Obesity is one of the largest factors leading to type 2 diabetes. Bariatric surgery reduces obesity and is to date the most effective method to lower blood glucose and reverse type 2 diabetes. Bariatric surgery alters gut anatomy and the types of bacteria that inhabit the gut. Gut bacteria can change obesity and blood glucose levels, but it was not known if the bacterial community present after bariatric surgery was a factor that is sufficient to lower blood glucose. We found that transferring gut bacteria from humans after bariatric surgery into mice lowers the blood glucose and alters the gut barrier structure where food is absorbed. It is not yet clear how this happens, but these findings show that a change in gut microbes is a standalone factor that can alter host blood glucose. Finding the glucose lowering factor in bacteria may be a new treatment to combat type 2 diabetes.

Inflammatory bowel disease (IBD), characterised by chronic intestinal inflammation, is hypothesised to arise from the interplay between susceptibility genes, the immune system, environmental factors, and gut microbiota. Akkermansia muciniphila is a symbiotic bacterium that accounts for 1-5% of the human fecal microbiota. This microbe has been hailed as a next-generation probiotic, principally with regards to its plethora of beneficial host interactions, including the ability to influence mucin secretion and strengthen the intestinal barrier. Though a clear-cut role and mechanism by which A. muciniphila influences inflammatory conditions is unknown, evidence indicates this microbe is depleted in IBD, suggesting it may have protective effects that are lost in these conditions. Here, we investigate the role and mechanism of A. muciniphila in intestinal inflammation and its influence on intestinal barrier function by utilizing barrier-disrupting models of colitis, including dextran sulphate sodium (DSS) and Trichuris muris. Though only minor benefits were derived from this microbe in germ-free mice, in specific pathogen-free (SPF) mice, administration of pasteurized A. muciniphila in a DSS recovery model ameliorated inflammation severity and promoted recovery compared to controls. When gavaged prior to DSS administration, both live and pasteurized A. muciniphila failed to diminish inflammatory markers indicating minimal preventative effects. T. muris-infected SPF mice treated with live A. muciniphila showed increased levels of Th2 and anti-inflammatory cytokines, decreased worm burden, and enhanced levels of the mucin, Muc5ac, compared with those receiving control broth or pasteurized bacteria. Further, both live and pasteurized A. muciniphila ameliorated the severity of inflammation in mucin 2 deficient (Muc2-/-) mouse model of spontaneous colitis, indicating that these protective effects are Muc2-independent. These observations provide us not only with an enhanced understanding of the role A. muciniphila plays in the pathogenesis of intestinal inflammatory conditions but also may fuel novel avenues of treatment for those with IBD. / Thesis / Master of Science (MSc) / Akkermansia muciniphila is a bacterium that accounts for 1-5% of the human fecal microbiota and has been shown to stimulate intestinal mucus production and strengthen the gut barrier. Though several studies have linked inflammatory bowel disease (IBD) with decreased levels of A. muciniphila, the precise role of this microbe in gut inflammation is unknown. In this research, we investigate the role of A. muciniphila in gut barrier function and inflammation. Across several experimental models, we find that supplementation with live, and in some cases, pasteurized A. muciniphila, can help curb established inflammation and promote a more anti-inflammatory gut environment. We also identify that these changes are independent of this bacteria’s ability to influence mucin 2, the main building block of intestinal mucus. This study has the potential to both enhance our understanding of microbial influence in intestinal inflammation and may also lead to the development of future treatments for IBD.

No description available.

Interest in the gut bacterial community residing in the human intestine, otherwise known as the gut microbiota, has exploded in recent years. The gut microbiome has been linked to chronic diseases such as obesity, suggesting interventions that target the microbiome may be useful in treating obesity and its complications. Dietary pulses (e.g., common beans) are composed of nutrients and compounds that possess the potential to modulate the gut bacteria composition and function which can in turn improve appetite regulation and chronic inflammation in obesity. This review summarizes the current state of knowledge regarding the connection between the gut microbiome and obesity, appetite regulation, and systemic and adipose tissue inflammation. More specifically, it highlights the efficacy of interventions employing dietary common beans as a means to improve appetite regulation and inflammation in obesity in both rodent models and in humans. Collectively, results presented and discussed herein provide insight on the gaps in knowledge necessary for a comprehensive understanding of the potential of beans as a treatment for obesity while highlighting what further research is required to gain this understanding.

Cardiovascular disease (CVD) is a major cause of mortality and morbidity worldwide, often with heart failure as the terminal stage. Clinical studies have associated elevated levels of trimethylamine N-oxide (TMAO), a gut-derived metabolite, with adverse outcomes of CVD. As of today, TMAO's effects on cardiac structure and function are not well understood. In this study, both male and female TMAO-treated hearts showed functional deficits based on electrocardiography and echocardiography results. Immunohistochemistry results showed signs of hypertrophic cardiomyopathy in TMAO-treated male hearts while female TMAO-treated hearts showed signs of dilated cardiomyopathy. Neither TMAO group showed signs of fibrosis. Overproduction of reactive oxygen species was only observed in male TMAO-treated hearts. At the level of individual cardiomyocytes, significant delays in time to reach maximum contraction and dilation were only seen in TMAO-treated male hearts along with higher contractile force. Overall, TMAO-treated hearts show significant functional deficits with altered structure in a sex-specific way. Our study utilizes a variety of methods to comprehensively characterize features of TMAO-induced heart failure in both males and females which extends our current knowledge from human clinical associations. / Master of Science / Cardiovascular disease (CVD) is a major cause of mortality and morbidity worldwide, often with heart failure as the terminal stage. Clinical studies have associated elevated levels of trimethylamine N-oxide (TMAO), a compound derived from eggs, red meat and seafood, with adverse outcomes of CVD. As of today, TMAO's impact on the heart is not well understood. After supplementing mice with TMAO, we discovered deficiencies in heart function coupled with altered heart structure showing signs of hypertrophic cardiomyopathy in males and dilated cardiomyopathy in females. In-depth experiments suggest that TMAO-induced cell stress could be a potential underlying cause of previously mentioned changes but the specific mechanisms require further investigation. Overall, TMAO-treated hearts show significant functional deficits with altered structure in a sex-specific way. Our study utilizes a variety of methods to characterize features of TMAO-induced heart failure aiming to unravel relevant biological changes in both male and female mice which extends our knowledge from human clinical associations.

No description available.

Background: Approximately 40% of global human immunodeficiency virus-1 (HIV) transmission occurs in the female genital tract (FGT). Epithelial cells lining the FGT comprise the first barrier to HIV-1 entry. The functions of these cells are influenced by female sex hormones and the mucosal microbiota. Studies have suggested that hormonal environment and a dysbiosis of the FGT microbiota may lead to inflammation in the genital mucosa and enhance HIV acquisition. A Lactobacillus dominant microenvironment in the FGT is considered to have protective functions against sexually transmitted pathogens, however the interaction between sex hormones and lactobacilli and their effect on epithelial cell functions remains to be determined. Methods of Study: For these studies, primary genital epithelial cells (GECs) were isolated from hysterectomy tissues obtained following patient consent. GEC cultures were grown to confluence on cell culture inserts in the presence or absence of the female sex hormones estrogen (E2), progesterone (P4), or medroxyprogesterone acetate (MPA). Polarized monolayers were exposed to two probiotic strains of Lactobacillus: L. reuteri (RC-14) or L. rhamnosus (GR-1), or the most common strain of bacteria found in the FGT, L. crispatus in the presence or absence of HIV-1. Cell viability, barrier integrity, and innate inflammatory factors were among the primary measures performed. Results: In our system, cell viability was unaltered in the presence of Lactobacillus species and/or female sex hormones. All three strains of bacteria (L. crispatus and probiotic lactobacilli GR-1 and RC-14) significantly increased GEC barrier integrity, as measured by transepithelial electrical resistance (TER). Both GR-1 and RC-14 significantly reduced GEC barrier permeability as measured by a dextran dye leakage assay, whereas L. crispatus did not. Conversely, hormones did not alter barrier integrity nor barrier permeability. However, hormones did alter secretion of cytokines and chemokins by GECs. GECs grown in the presence of estrogen decreased TNF-α, IL-1α, IL-1β and IL-8 secretion in comparison to no hormone treatment, while GECs grown in the presence of MPA significantly decreased MIP-1α and TNF-α secretion. In the presence of HIV both GR-1 and RC-14 were able to confer an increase in barrier integrity similar to that observed with GR-1 and RC-14 treatment alone. Addionally, GECs grown in the presence of E2 and MPA displayed a less inflammatory (TNF-α, IL-1α, and IL-1β) environment when exposed to HIV compared to no hormone and P4. Interstingly, the decrease in inflammation was not observed when measuring chemokines such as IL-8 and RANTES. Furthermore, probiotic bacteria were able to significantly reduce HIV mediated increases in TNF-α when grown in the presence of no hormone, P4, and MPA. A similar trend was observed for GECs grown in the presence of E2 however, given that E2 reduced the TNF-α response mediated by HIV, results were not significant. Overall, probiotic lactobacilii GR-1 and RC-14 enhanced GEC barrier functions while E2 and MPA appeared to exert an anti-inflammatory effect on epithelial cell innate responses in both the presence and absence of HIV. Conclusions: In our system, probiotic lactobacilli enhanced GEC barrier functions and estrogen appeared to exert an anti-inflammatory effect on epithelial innate responses. Enhanced barrier function and decreased inflammation correlate with decreased in HIV acquisition and replication. These studies provide an insight into how factors in the genital microenvironment can affect HIV acquisition in the FGT, and will subsequently assist in the development of prophylactic strategies to reduce HIV transmission. / Thesis / Master of Science (MSc) / Approximately 40% of global HIV transmission occurs in the female genital tract. Although women make up more than 50% of infected individuals worldwide, the details regarding how HIV infection starts in the female genital tract remains poorly understood. The cells that line the genital tract are the first barrier against HIV entry. These cells are influenced by common factors within the genital tract microenvironment such as female sex hormones and natural bacterial populations. Previous studies have suggested that certain hormonal contraceptives or a build-up of pathogenic bacteria within the genital tract, leads to an inflammatory microenvironment and may enhance HIV acquisition. Comparatively, ‘good bacteria’ within the microenvironment have been shown to have protective effects against sexually transmitted infections. For this study, we were interested in understanding how different hormones (estrogen, progesterone and progesterone based hormonal contraceptives) and ‘good bacteria’ (specifically probiotic strains of lactobacilli), affect the cells that line the genital tract and local inflammation in the presence and absence of HIV. Therefore, we obtained cells that line the genital tract (epithelial cells) from women undergoing hysterectomies. The cells were grown in the presence or absence of hormones, exposed to ‘good bacteria’ and then challenged with HIV. In our system, probiotic lactobacilli enhanced genital epithelial cell barrier functions and estrogen appeared to exert an anti-inflammatory effect on epithelial cells. Furthermore, when genital epithelial cells were pre-treated with lactobacilli and exposed to HIV, lactobacilli treatment was able to protect against HIV mediated barrier disruption. Lactobacilli treated genital epithelial cells also reduced inflammatory markers in the presence HIV. Enhanced barrier function and decreased inflammation correlate with decrease in HIV infection and replication. This study provides insight into how factors in the genital microenvironment can affect HIV infection in the female genital tract and suggests potential prophylactic strategies to reduce HIV infection.

The upper respiratory tract (URT), including the nasal and oral cavities, is a reservoir for pathogenic and commensal microbial species, collectively known as the microbiota. Microbial colonization of the URT occurs right after birth, and URT microbial composition has been linked to development of respiratory infections, allergy, and asthma, though few direct mechanisms have been uncovered. Thus, I set out to establish animal models for characterizing the URT microbiota, and its role in infections. I found that nasal washes, a predominant method for measuring URT bacterial colonization, were insufficient for completely extracting the URT microbiota. The age and source of mice greatly affected the composition of the microbiota, which could be transferred to germ-free mice via cohousing. I also established that mice colonized with the Altered Schaedler’s Flora in the gut microbiota have no cultivable URT microbiota. To test the function of the URT microbiota, I colonized mice with Streptococcus pneumoniae, the leading cause of bacterial pneumonia worldwide. I show that the presence of a nasal microbiota increases permissiveness to pneumococcal infection in murine models. Addition of a single URT isolate, Actinomyces naeslundii, increased pneumococcal adherence to human respiratory epithelial cells in vitro and increased pneumococcal dissemination in vivo in a sialidase-dependent manner. The microbiota affects expression of several host genes throughout the respiratory tract involved in pneumococcal pathogenesis. Together, this work establishes new models for assessing the URT microbiota, and highlights the contribution of the URT microbiota to pneumococcal pathogenesis and identifies druggable targets to prevent and treat infections. / Dissertation / Doctor of Philosophy (PhD) / Bacteria living in the gut have been shown to benefit our health, but the role of bacteria living in our respiratory tract is relatively unknown. I describe the methods for characterizing the bacteria in the nose of a mouse as a model of the human nose. I found that pockets of the mouse nose are colonized by different bacteria. I also characterized a mouse model that had bacteria in the gut without nasal bacteria. I used this mouse model to understand infections with Streptococcus pneumoniae, the worldwide leading cause of bacterial pneumonia. The mice without nasal bacteria were protected from infections, which was due to a nasal bacteria helping S. pneumoniae escape from the nasal tissue. This work established new models for understanding how bacteria affect respiratory health, and identified new targets for protecting against infections.

This study investigated the microbial colonization and maternal influences on the neonatal calf gut microbiome. Microbiome samples were collected from dams (n = 6) and calves (n = 6) using sterile flocked swabs. The vaginal, oral, and fecal bacterial communities were examined from the dam and the fecal community of calves was examined from birth to 60 d of age. Microbial communities varied by anatomical location and age of the calf. Metagenomic analysis 16s ribosomal DNA revealed ten phyla associated with microbiomes of the dam and the same ten phyla associated with calf feces at various time points: Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, Verrucomicrobia, Spirochaetes, Tenericutes, Fibrobacteres, and Lentisphaerae. Overall, the calf meconium and fecal microbiome is influenced by a combination of the maternal vagina, oral, and fecal microbiomes. Further studies will be needed to identify the transference mechanisms of maternal microbes to offspring and the associated host-microbial interactions. / Master of Science in Life Sciences / This study investigated the microbial colonization and maternal influences on the neonatal calf gut microbiome. Microbiome samples were collected from dams (n = 6) and calves (n = 6) using sterile flocked swabs. The vaginal, oral, and fecal bacterial communities were examined from the dam and the fecal community of calves was examined from birth to 60 d of age. Microbial communities varied by anatomical location and age of the calf. Metagenomic analysis 16s ribosomal DNA revealed ten phyla associated with microbiomes of the dam and the same ten phyla associated with calf feces at various time points: Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, Verrucomicrobia, Spirochaetes, Tenericutes, Fibrobacteres, and Lentisphaerae. Overall, the calf meconium and fecal microbiome is influenced by a combination of the maternal vagina, oral, and fecal microbiomes. Further studies will be needed to identify the transference mechanisms of maternal microbes to offspring and the associated host-microbial interactions.