Enteric microbiota influences enteric viral infections, and host response to these pathogens and vaccines. Using gnotobiotic (Gn) pigs transplanted with human gut microbiota (HGM), we studied the effects of HGM on the immune response to oral rotavirus vaccination and rotaviral disease. We also used HGM transplanted Gn pigs to determine the effects of HGM on human norovirus infection. Despite commercially available vaccines, human rotavirus is a leading acute gastroenteritis in children, especially those in developing countries. Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in all age groups worldwide, and no vaccines are commercially available. Further understanding of how enteric microbiota influences these viral diseases may identify therapeutic targets. In our rotavirus study, pigs were colonized with HGM from an infant with low fecal concentrations of enteropathy biomarkers and responded well to their first dose of oral rotavirus vaccine (healthy human gut microbiota "HHGM"); or pigs were colonized with HGM from an infant with high fecal concentrations of enteropathy biomarkers and a poor response to the first dose of oral rotavirus vaccine (unhealthy human gut microbiota "UHGM"). HHGM colonized pigs had stronger cell-mediated and mucosal immune response to oral rotavirus vaccine compared to UHGM pigs based on the number of rotavirus-specific IFN-γ producing T cells in the ileum, spleen, and blood, and trends towards higher rotavirus specific antibody titers in intestinal contents, respectively. Significant correlations between multiple Operational Taxonomic Units (OTUs) of bacteria and frequencies of IFN-γ producing T cells at the time of human rotavirus challenge existed, suggesting that certain members of the microbiota influenced the immune response to the vaccine. After the vaccinated pigs were challenged with human rotavirus, HHGM pigs had less severe and shorter duration of viral shedding and diarrhea compared to UHGM pigs, suggesting that HHGM facilitated development of stronger protective immunity. These results demonstrated that composition of the enteric microbiota influenced host immune response to oral vaccination. In the norovirus study, Gn pigs were colonized with HHGM to determine the effects of microbiota on HuNoV infection. Colonized pigs shed more virus for a longer duration than non-colonized pigs, and also had higher viral titers in the duodenum and distal ileum. Diarrhea was more severe 4-10 days post-infection and lasted longer in colonized compared to non-colonized pigs. Twenty-seven genes related to the immune system were highly upregulated in HuNoV infected, colonized pigs compared to non-colonized controls. These result showed that HHGM influenced infectivity of HuNoV in the Gn pig model and altered host gene expression related to the immune system. These studies showed that HHGM can improve the host immune response and efficacy of rotavirus vaccine, but it can also enhance infection and clinical disease in HuNoV infected Gn pigs. Depending on the virus, gut microbiota may be beneficial or detrimental to the host. Those developing future treatments aimed at altering microbiota to prevent or ameliorate one viral pathogen need to consider the potential for enhancing a different pathogen. These studies demonstrated the usefulness of HGM transplanted Gn pigs for evaluation of microbiota influence on infection and immunity of enteric viral pathogens. / Ph. D. / Gut microbiota influences intestinal viral infections, and host response to these pathogens and vaccines. Using gnotobiotic (Gn) pigs transplanted with human gut microbiota (HGM), we studied the effects of HGM on the immune response to oral rotavirus vaccination and rotaviral disease. We also used HGM transplanted Gn pigs to determine the effect of HGM on human norovirus infection. Despite commercially available vaccines, human rotavirus is a leading acute gastroenteritis in children, especially those in developing countries. Human norovirus (HuNoV) is a leading cause of vomiting and diarrhea in all age groups worldwide, and no vaccines are commercially available. Further understanding of how gut microbiota influences these viral diseases may identify therapeutic targets. In our rotavirus study, pigs were colonized with HGM from an infant without evidence of intestinal disease based on fecal analysis, and who responded well to the first dose of oral rotavirus vaccine (healthy human gut microbiota “HHGM”); or pigs were colonized with HGM from an infant with evidence of potential intestinal dysfunction and a poor response to the first dose of oral rotavirus vaccine (unhealthy human gut microbiota “UHGM”). HHGM colonized pigs had a stronger immune response to the oral rotavirus vaccine compared to UHGM pigs. Significant correlations between multiple Operational Taxonomic Units (OTUs) of bacteria and frequencies of rotavirus-specific immune cells at the time of human rotavirus challenge existed, suggesting that certain members of the microbiota influenced the immune response to the vaccine. After the vaccinated pigs were challenged with human rotavirus, HHGM pigs had less severe and shorter duration of viral shedding and diarrhea compared to UHGM pigs, suggesting that HHGM enhanced vaccine efficacy. These results demonstrated that composition of the gut microbiota influenced host immune response to oral vaccination. In the norovirus study, GN pigs were colonized with HHGM to determine the effects of microbiota on HuNoV infection. Colonized pigs shed more virus for a longer duration than non-colonized pigs, and also had higher viral titers in sections of small intestine. Diarrhea was more severe 4-10 days after infection and lasted longer in colonized compared to non-colonized pigs. Twenty-seven genes related to the immune system were highly upregulated in HuNoV infected, colonized pigs compared to controls. These result showed that HHGM influenced infectivity of HuNoV in the Gn pig model and altered host gene expression related to the immune system. These studies showed how HHGM improved the host immune response and efficacy of rotavirus vaccine, but conversely enhanced infection and clinical disease in HuNoV infected pigs. Depending on the virus, gut microbiota may be beneficial or detrimental to the host. Those developing future treatments aimed at altering microbiota to prevent or ameliorate one viral pathogen need to consider the potential for enhancing a different pathogen. These studies showed the usefulness of HGM transplanted Gn pigs for evaluation of microbiota influence on infection and immunity of intestinal viruses.

The commensal intestinal microbiota contributes substantially to intestinal development in the early neonatal period by mechanisms that are not yet elucidated but could contribute to novel strategies to improve intestinal health. A series of gnotobiotic experiments using isolator-reared caesarian section-derived piglets inoculated at 1 d of age with selected bacteria and euthanized at 14 or 15 days of age were performed to investigate intestinal morphology, inflammation and digestive function. In Experiment 1, piglets were maintained germfree (GF), mono-associated with Escherichia coli (EC), mono-associated with Lactobacillus fermentum (LF) or conventionalized with sow feces (CV). Increased (P<0.05) gene expression of Fas ligand (FasL) and tumor necrosis factor (TNF?) in EC and CV as compared to LF and GF pigs coincided with increased apoptotic and proliferative activity. Toll-like receptors (TLR) 2, 4 and 9 were differentially regulated (P<0.05) by colonizing species. In Experiment 2 using the same animals as Exp. 1, increased turnover of brush border enzymes was indicated by reduced (P<0.05) specific activity of aminopeptidase N (APN) and lactase (LPH) and increased expression of APN in CV and EC as compared to GF and LF pigs. Reduced enzyme activity to gene expression ratio corresponded with an in vitro assay of microbial inactivation of APN. In Experiment 3, probiotic Lactobacillus sp., L3777, and Bifidobacteria sp., B5445, did not induce expression of inflammatory cytokines in mono-association but di-association with E. coli increased (P<0.05) inflammatory and anti-inflammatory mediators and resulted in a high rate of sepsis (50%) relative to E. coli mono-association. Induced expression of inflammatory cytokines by commensal bacteria through TLR and other means, appear to play a substantial role in microbially-induced enterocyte turnover. Enterocyte immaturity did not account for reduced enzyme activity associated with inflammation as increased expression of APN in response to microbial colonization was observed, suggesting a host response pathway enabling effective competition with the intestinal microbiota for available peptide nutrients. Probiotic bacteria were relatively benign in mono-association but may have facilitated increased translocation of <i>E. coli</i> in di-association. Gnotobiotic animal models are essential to demonstrate outcomes of host response characterized by communication among numerous cell types, although are of significant technical difficulty.

The commensal intestinal microbiota contributes substantially to intestinal development in the early neonatal period by mechanisms that are not yet elucidated but could contribute to novel strategies to improve intestinal health. A series of gnotobiotic experiments using isolator-reared caesarian section-derived piglets inoculated at 1 d of age with selected bacteria and euthanized at 14 or 15 days of age were performed to investigate intestinal morphology, inflammation and digestive function. In Experiment 1, piglets were maintained germfree (GF), mono-associated with Escherichia coli (EC), mono-associated with Lactobacillus fermentum (LF) or conventionalized with sow feces (CV). Increased (P<0.05) gene expression of Fas ligand (FasL) and tumor necrosis factor (TNF?) in EC and CV as compared to LF and GF pigs coincided with increased apoptotic and proliferative activity. Toll-like receptors (TLR) 2, 4 and 9 were differentially regulated (P<0.05) by colonizing species. In Experiment 2 using the same animals as Exp. 1, increased turnover of brush border enzymes was indicated by reduced (P<0.05) specific activity of aminopeptidase N (APN) and lactase (LPH) and increased expression of APN in CV and EC as compared to GF and LF pigs. Reduced enzyme activity to gene expression ratio corresponded with an in vitro assay of microbial inactivation of APN. In Experiment 3, probiotic Lactobacillus sp., L3777, and Bifidobacteria sp., B5445, did not induce expression of inflammatory cytokines in mono-association but di-association with E. coli increased (P<0.05) inflammatory and anti-inflammatory mediators and resulted in a high rate of sepsis (50%) relative to E. coli mono-association. Induced expression of inflammatory cytokines by commensal bacteria through TLR and other means, appear to play a substantial role in microbially-induced enterocyte turnover. Enterocyte immaturity did not account for reduced enzyme activity associated with inflammation as increased expression of APN in response to microbial colonization was observed, suggesting a host response pathway enabling effective competition with the intestinal microbiota for available peptide nutrients. Probiotic bacteria were relatively benign in mono-association but may have facilitated increased translocation of <i>E. coli</i> in di-association. Gnotobiotic animal models are essential to demonstrate outcomes of host response characterized by communication among numerous cell types, although are of significant technical difficulty.

Clostridioides difficile (C. difficile) and human rotavirus (HRV) are leading causes of bacterial and viral gastroenteritis worldwide. Treatment and vaccination options for both pathogens have significant limitations. C. difficile infections are treated with antibiotics, which is paradoxical as C. difficile itself is associated with antibiotic usage. In the United States, two live oral attenuated vaccines (Rotarix and RotaTeq) are licensed for protection against HRV. Since receiving approval from the World Health Organization (WHO), Rotarix and RotaTeq have been widely implemented into global national childhood immunization schedules, with one report finding 59 countries using Rotarix and 25 using RotaTeq. However, these vaccines have much lower efficacy rates in low- and middle-income countries. Because of these caveats, there is an urgent need to generate novel prophylaxes and treatments for C. difficile and HRV. In order to address this need, animal models that replicate the nuances of each infection are imperative. We have developed gnotobiotic (Gn) pig models for each pathogen. Gn pigs infected with spores of the hypervirulent UK1 strain of C. difficile develop classical signs of infection, including watery diarrhea and weight loss. Gross necropsy reveals colonic distention and discoloration, and histopathological evaluation shows volcano lesions, pseudo membrane formation, and epithelial cell erosion. Gn pigs infected with a G4P[6] strain of HRV also display pathogen specific signs of infection, including diarrhea, fecal rotavirus shedding, and damaged intestinal villi. A dose response study of the G4P[6] strain revealed diarrhea and virus shedding occurred at all tested doses, however the most severe diarrhea and virus shedding, measured by cumulative diarrhea score, area under the curve (AUC) of diarrhea, peak virus titer, and AUC of virus shedding, were all detected in the highest dose group. Based on the presentation of clinical signs of infection, 105 fluorescent focus units was selected as the optimal challenge dose for future studies. These models enable us to test candidate therapeutics, but also elucidate unique replicative features of the pathogens. For example, we found that HRV can replicate in the salivary glands and nasal cavity of Gn pigs in addition to the small intestine. HRV infection primed immune responses in the ileum, tonsils, and facial lymph nodes; infection also induced high levels of systemic and mucosal rotavirus specific antibody responses. Moving forward, we hope to expand upon this replication study to identify what cell types within the glands are infected as well as look at local cellular immune responses to HRV infection. Additional future directions include determining the protective efficacy of next generation HRV vaccines and evaluating effectiveness of an engineered probiotic yeast in reducing severity of C. difficile infection and disease. The Gn pig models of C. difficile and G4P[6] HRV are clinically relevant, and they will continue to serve as useful tools to better our understanding of pathogenesis, infection, and prevention of these pathogens. / Doctor of Philosophy / Clostridioides difficile (C. difficile) and human rotavirus (HRV) both cause gastrointestinal related symptoms when they infect humans. Treatments available for C. difficile and HRV all have significant drawbacks. This represents a gap in knowledge which we aimed to fill by developing germ-free (gnotobiotic [Gn]) pig models of C. difficile and HRV infection and disease. Animal models that mimic the outcomes of disease seen in humans are essential for evaluating protectiveness of new therapeutics. The more similar the disease presentation, the more likely the treatment results will be translational to humans. We began with C. difficile; pigs were orally fed C. difficile and monitored for a week post infection for development of signs of infection. Inoculated pigs lost weight and developed diarrhea. Bacterial cells and toxins were isolated from fecal samples collected on various days post infection. Multiple changes were observed in infected pigs’ large intestinal tissues, including severe bleeding, tissue distension, and fluid buildup. Infected pigs also had significant upregulation of pro-inflammatory cytokines, indicating activation of the immune response. We performed a similar procedure for the establishment of the HRV model. Gn pigs were orally challenged with differing doses of G4P[6] HRV and followed for several days post infection. Consistent with HRV infection in children, the pigs developed watery diarrhea that lasted for multiple days. Small intestinal tissues collected at necropsy had several signs of damage, including blunted villi, fluid buildup, and immune cell invasion. These lesions were also consistent with HRV infection in humans. Taken all together, these results indicated successful establishment of both C. difficile and HRV models. While the primary goal of generating these models was to evaluate new treatments, a secondary goal was to use them to better our understanding of pathogen replication dynamics. For example, the small intestine was thought to be the primary site of HRV infection. Using a pig model of HRV, we expanded on this knowledge to show that HRV can replicate in the nose and salivary glands as well. Additionally, we found HRV infection to induce immune responses near the sites of infection, including the intestine, the tonsils, and the facial lymph nodes. Overall, these studies demonstrate the utility of germ-free pig models and are an important first step in generating more effective treatments for bacterial and viral infections.

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants. Plant growth-promoting rhizobacteria may enhance plant growth either by direct or indirect mechanisms. The direct mechanisms of action include nitrogen fixation,production of phytohormones and lowering of ethylene concentrations. The objective of this study was to determine whether Pseudomonas putida strain 6-8 isolated from the rhizosphere of legume crops grown in Saskatchewan fields was able to promote the growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms. Initial studies determined the effect of strain 6-8 and other known phytohormoneproducing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and growth chamber conditions. Variations in the results were observed, as there were significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly increased root dry weight, shoot dry weight and root surface area in pots in growth chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20- 18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in growth pouches and in pots in growth chambers when compared to other strains and the control. The ability of the PGPR strains to produce auxin and cytokinin phytohomones in pure culture and in the canola rhizosphere was tested using the enzyme linked immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and the concentration of the indoles produced increased with increasing concentrations of the precursor tryptophan. There were no significant differences among PGPR strains in production of indole-3-acetic acid (IAA) when assayed using ELISA. The concentrations of IAA secreted by PGPR strains were extremely low (0.19 µg/ml 9.80 µg/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was detected in supernatants obtained from canola growth pouches inoculated with PGPR strains, but there were no significant differences in the concentrations of IAA secreted among PGPR strains. Significantly higher concentrations of IPA and ZR were observed in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated control. Strain 6-8 produced siderophores, solubilized inorganic phosphate and used 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole nitrogen source. These traits are considered to be alternative mechanisms for direct plant growth promotion. A qualitative and quantitative study of root colonization by strain 6-8 was conducted by tagging the strain with green fluorescent protein in conjunction with confocal laser scanning microscopy and by conventional plating. The populations of strain 6-8 were higher on canola roots than on lentil roots by conventional plating. Similar results were also observed in confocal laser scanning microscopy (CLSM) studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil. Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct growth promoting characteristics. The ability of strain 6-8 to promote the growth of canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies may be related to these direct growth promoting characteristics. Strain 6-8 may have potential for development as a plant growth-promoting rhizobacterial inoculant.

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants. Plant growth-promoting rhizobacteria may enhance plant growth either by direct or indirect mechanisms. The direct mechanisms of action include nitrogen fixation,production of phytohormones and lowering of ethylene concentrations. The objective of this study was to determine whether Pseudomonas putida strain 6-8 isolated from the rhizosphere of legume crops grown in Saskatchewan fields was able to promote the growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms. Initial studies determined the effect of strain 6-8 and other known phytohormoneproducing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and growth chamber conditions. Variations in the results were observed, as there were significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly increased root dry weight, shoot dry weight and root surface area in pots in growth chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20- 18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in growth pouches and in pots in growth chambers when compared to other strains and the control. The ability of the PGPR strains to produce auxin and cytokinin phytohomones in pure culture and in the canola rhizosphere was tested using the enzyme linked immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and the concentration of the indoles produced increased with increasing concentrations of the precursor tryptophan. There were no significant differences among PGPR strains in production of indole-3-acetic acid (IAA) when assayed using ELISA. The concentrations of IAA secreted by PGPR strains were extremely low (0.19 µg/ml 9.80 µg/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was detected in supernatants obtained from canola growth pouches inoculated with PGPR strains, but there were no significant differences in the concentrations of IAA secreted among PGPR strains. Significantly higher concentrations of IPA and ZR were observed in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated control. Strain 6-8 produced siderophores, solubilized inorganic phosphate and used 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole nitrogen source. These traits are considered to be alternative mechanisms for direct plant growth promotion. A qualitative and quantitative study of root colonization by strain 6-8 was conducted by tagging the strain with green fluorescent protein in conjunction with confocal laser scanning microscopy and by conventional plating. The populations of strain 6-8 were higher on canola roots than on lentil roots by conventional plating. Similar results were also observed in confocal laser scanning microscopy (CLSM) studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil. Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct growth promoting characteristics. The ability of strain 6-8 to promote the growth of canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies may be related to these direct growth promoting characteristics. Strain 6-8 may have potential for development as a plant growth-promoting rhizobacterial inoculant.

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It is well known that the elderly often manifest chronic low-grade inflammation. This phenomenon, called “inflamm-aging,” is postulated to contribute to increased susceptibility towards infectious diseases and an overall increase in frailty. We have proposed examining the gut microbiome as a potential mediator of these changes. Gut microbial communities influence the host immune system; often dictating an individual’s health status. Thus, harmful gut microbiome changes, termed dysbiosis, are associated with poor health in the elderly. We first sought to understand the key immunological, physiological and microbiome changes occurring with age (Chapter 3). Our data reveals immune impairments in aged mice, with increased intestinal permeability, systemic inflammation and alterations in the functions of myeloid cell populations. However, our aged germ-free (GF) mice are protected from these outcomes, indicating that the old microbiome may play a strong role in these age-associated impairments. To study this further, we have colonized young and old GF mice with the “young” or “old” microbiota in order to determine whether the relationship between microbial dysbiosis with age and health status is correlative or causative (Chapter 4). Interestingly, young GF mice colonized with old microbiota have significantly increased permeability, systemic inflammation and an influx of Ly6Chigh monocytes when compared to those colonized with the young microbiota. By using transgenic mice (TNF-/- mice), or by reducing systemic TNF levels via therapeutics, we were able to reduce some aspects of microbial dysbiosis and age-associated inflammation (Chapter 5). Our data suggests that harmful changes to the gut microbiome composition with age initiate a cycle of negative events that ultimately result in increased inflammatory myeloid cell recruitment, increased intestinal permeability and an overall increase in systemic inflammation in old mice. By identifying these key changes, we can work towards developing effective therapeutics that promotes healthy aging and protection against infectious diseases. / Thesis / Master of Science (MSc) / Élie Metchnikoff first coined the term “dysbiosis” when he described the imbalance in microbial populations that could result anywhere in the body. Since then, numerous studies have examined the role of the intestinal microbiota in defense against pathogens. Metchnikoff also suggested that the gut composition and function is altered with age and this can in turn; increase the host’s susceptibility towards infectious diseases. My research aims to characterize the role of microbial dysbiosis on the immune defects with age. To do so, I will be utilizing a unique set of mice, called gnotobiotic mice. These mice are housed under specific germ-free conditions and contain no microbiome. Thus, they provide us with the ideal model to study the effects of the microbiome on immune function. The findings from these studies will help in the development of preventative and therapeutic alternatives to provide the elderly with more years of healthy, independent living.

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