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.

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.

Noroviruses (NoVs) are a cause of nonbacterial acute gastroenteritis affecting all ages. NoV infections result in over 200,000 pediatric deaths in developing countries annually. Vaccine development has been hindered by the lack of cell culture systems and small animal models; thus, vaccine development has relied upon recombinant VP1 capsid proteins, such as virus-like particles (VLPs) and P particles. P particles are a novel vaccine candidate derived from expression of the VP1 protruding (P) domain, while VLPs require expression of the full-length VP1. My studies utilize a gnotobiotic (Gn) pig model of human NoV infection and diarrhea to evaluate the protective efficacy and T cell responses induced by P particles and to compare them with prior NoV infection (NoVPO) and VLPs. Gn pigs received 100 µg of P particles (LoPP) or VLPs, 250 µg P particles (HiPP), or adjuvants only intranasally at post-inoculation day (PID) 0, 10, and 21. Monophosphoryl lipid A and chitosan were used as mucosal adjuvants. At PID 28, a subset of pigs were orally challenged with 10 median infectious doses (ID50) NoV. NoVPO, LoPP, HiPP, and VLPs provided partial protection from diarrhea (83%, 47%, 60%, and 60% protection rates, respectively). Only NoVPO and HiPP provided protection from shedding (49% and 60% protection rates, respectively) and also reduced the number of CD25- regulatory T cells (Tregs) in duodenum following challenge. NoV primary infection induced an overall pro-Treg and low, transient Th1 response. LoPP induced stronger overall T cell responses compared to VLPs, including activated CD4+ T cells and duodenal CD8+IFN-γ+ T cells, suggesting that P particles are more immunogenic than VLPs. I also evaluated the effects of simvastatin, a cholesterol-reducing drug that increases NoV infectivity, on P particle vaccine efficacy. Simvastatin abolished P particle-induced protection and significantly increased diarrhea severity. Simvastatin reduced total numbers of duodenal mononuclear cells, IFN-γ+ T cells pre-challenge, and Tregs post-challenge, indicating that simvastatin impairs development of immune system and immune responses. Findings from these studies elucidate potential mechanisms behind P particle-induced immunity and reveal the negative effects of simvastatin on NoV-induced protective immunity. The knowledge will facilitate the development of effective NoV vaccines. / Ph. D.

My dissertation research focused on studying mechanisms of immunomodulation by probiotic lactobacilli on innate and T cell immune responses induced by rotavirus infection and vaccines in a gnotobiotic pig model of human rotavirus (HRV) infection and vaccination. We first studied the effects of probiotics on antigen-presenting cells (APCs) through TLR activation. We found that a mixture of Lactobacilli acidophilus strain NCFM (LA) and L. reuteri (ATCC# 23272) induced strong TLR2-expressing APC responses and virulent HRV induced a TLR3 response. Probiotics and HRV had an additive effect on TLR2- and TLR9-expressing APC responses, consistent with the adjuvant effect of lactobacilli. Dose effects of LA on T cell immune responses were investigated. We found that low dose LA significantly enhanced frequencies of HRV-specific IFN-γ producing CD4⁺ and CD8+ T cells whereas high dose LA reduced frequencies of HRV-specific IFN-γ producing CD4+ T cells. Low dose LA reduced frequencies of induced regulatory (iTreg) cells and TGF-β expression in the iTreg cells whereas high dose LA increased frequencies of iTreg cells and IL-10 expression in the iTreg cells. The dose effects of LA were independent of HRV infection/vaccination. In addition, we demonstrated that TCR-γδ T cells play an important role in modulating immune responses to rotavirus infections. All three γδ T cell subsets showed evidence of activation after HRV infection by increasing TLR2, TLR3, TLR9 expression and IFN-γ production during the acute phase of infection. There was an additive effect between lactobacilli and HRV in inducing total γδ T cell expansion in ileum and in recruiting the cells from blood. HRV infection induced a significant expansion of the CD2+CD8+ γδ T cell subset in the ileum. This subset mainly exerts regulatory functions as evident by expressing FoxP3, secreting TGF-β and IL-10 or increasing production of the anti-inflammatory cytokines by CD4+ and/or CD8+ αβ T cells in the co-cultures. CD2+CD8- and CD2-CD8- γδ T cell subsets have mainly pro-inflammatory and anti-viral functions as evident by secreting IFN-γ or promoting CD4+ αβ T cell proliferation and IFN-γ production. This knowledge will facilitate the development of more effective vaccination and therapeutic strategies to protect children and young animals against rotavirus gastroenteritis. / Ph. D.

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