Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

Aquaculture is currently the fastest growing animal production sector. Because the aquaculture sector is growing at rapid rates, certain materials for feed, specifically marine protein sources, are becoming increasingly expensive and unsustainable. To counteract the reliance on fishmeal (FM) and other marine protein sources in the industry plant protein (PP), specifically soybean meal (SBM), has been investigated to replace FM as a protein source. Unfortunately, SBM when given in high quantities (greater than 30%) has been shown to negatively affect fish performance including retarded growth, intestinal inflammation, reduction of spawn quality, as well as dysbiosis in the gut microbiome, most likely due to presence of antinutritional factors such as saponins and tannins in SBM. The goal of this thesis was to investigate the effect of nutritional programming (NP) with SBM-based diet on gut microbiota in broodstock and progeny fish. Three feedings trials were conducted to test the efficacy of 3 approaches towards improving the use of PP in fish.The first trial (Chapter 2), tested the effect of NP on larval zebrafish (Danio rerio). NP is the theory of introducing an early nutritional stimulus to an animal that will ‘program’ the animal to better utilize the stimuli later in its adult life. The zebrafish were programmed in their larval stages, and the trial lasted for 65 days. There was a significant effect on growth performance for the programmed group (NP-PP) in terms of weight gains, as the NP-PP group grew better compared to the non-programmed group (NP-FM) and negative control (-control). There was no significant effect on the gut microbiome in terms of alpha or beta diversity, however, there were significant changes in the relative abundance (RA) of the gut microbiome throughout time in the NP-PP and the NP-FM groups. The findings of the study support that early NP of zebrafish with SBM improves growth performance on PP diet, but the gut microbiome does not seem to be a mechanism for NP.The second feeding trial (Chapter 3) focused on NP induced in the zebrafish broodstock with dietary SBM. For two weeks, the broodstock fish were fed with either a SBM diet or a FM diet so that gametogenesis occurred with either a FM or PP diet. This phase was called the ‘broodstock programming’ stage. The broodstock were then spawned, and the larval fish were separated into four different treatments: 1) SBM broodstock progeny, fed SBM for the entire trial (PPBS-PP) 2) SBM broodstock progeny fed FM the entire trial (PPBS-FM), 3) FM broodstock progeny fed FM the entire trial (+ control, FMBS-FM), and 4) FM broodstock progeny fed SBM the entire trial (- control, FMBS-PP). The PPBS-PP group achieved similar weight gains compared to all other treatments in terms of grams, but was numerically greater than the FMBS-PP treatment. There were no differences detected in gut microbiome alpha or beta diversity in any of the groups, however, there was significant change observed of certain bacterial phyla between the ‘programmed broodstock’, larval fish, and fish at the end of the trial, 48 days post hatch. Overall, this trial suggests that parental programming does not improve PP utilization in the progeny of zebrafish. It also appears that the gut microbiome is not a mechanism of parental programming. The third feeding trial (Chapter 4), was conducted on largemouth bass (Micropterus salmoides). This chapter had a similar experimental design as the first trial (Chapter 2), and larval largemouth bass were programmed with dietary SBM. This trial had an additional group added to it, which included a dietary saponin-programmed group. The study found that the NP with SBM diet or dietary saponin did not improve PP utilization and growth performance of largemouth bass in its pre-adult age. The study also found that the NP with SBM diet or dietary saponin did not have any effect on the largemouth bass gut microbiome, and there does not seem to be any gut microbiome modification associated with the NP in this fish species. Overall, NP can be used to improve dietary PP utilization but optimal timing and PP delivery method must be well assessed to ensure successful PP exposure and adaptation in different species. Nevertheless, the gut microbiome does not seem to be affected by NP and therefore is not considered the mechanism behind NP. Finally, studies on both zebrafish and largemouth bass presented major shifts in the gut microbiome as the fish aged. In addition, the core microbiomes of both species appeared to become more pronounced as the fish become adults. There seem to be an evolutionary tie between host and its gut microbiome. More studies, however, should further investigate this and the genetic effects on gut microbiota development and its heritability.

<p>Carbohydrases have been used to improve fiber utilization in monogastric animals. However, their effects on animal performance and nutrient digestibility have been inconsistent in pigs. The efficacy of carbohydrases has been suggested to depend on enzyme activity and fiber composition, but recent evidence suggests other factors like environmental conditions may play significant role. The effect of heat stress (HS) on the efficacy of a multienzyme carbohydrase blend in growing pigs was investigated. It was hypothesized that HS alters the efficacy of carbohydrases. Ninety-six growing pigs (gilts:barrows; 1:1) (initial BW of 20.15±0.18 kg) were randomly assigned to 6 treatments, with 8 replicates of 2 pigs replicate pen in a 3×2 factorial arrangement: three levels of carbohydrase (0, 1X or 2X) and two room temperature conditions (20^oC constant or cyclical 28^oC night time and 35^oC daytime). The 1X is the recommended commercial dose of the enzyme (50g/tonne) and provides 1250 visco-units endo-β-1,4-xylanase, 4600 units α-L-arabinofuranosidase and 860 visco-units endo-1,3(4)-β-glucanase per kg of feed. The 2X dose was 100g/tonne. Pigs were fed ad libitum for 28 days and 1 pig per pen was sacrificed at d28. Heat stress increased the respiratory rate and skin temperature (P<0.001). There was no enzyme × temperature effect on response criteria. Enzyme treatment quadratically increased BW d28 (P=0.025), ADG (P=0.022) and average daily feed intake (ADFI) (P=0.032) with 1X being the highest. Heat stress reduced the BW at d14 (P=0.002) and d28 (P=0.006), average daily gain (ADG) (P=0.005) and ADFI (P<0.001). However, there was a trend of increased gain: feed ratio (G/F) (P=0.093) in the HS pigs compared to the thermoneutral (TN). Heat stress increased apparent jejunal digestibility (AJD) of energy (P=0.039) and apparent ileal digestibility (AID) of calcium (P=0.007). ADFI was positively correlated to ADG (r=0.57; P<0.001) but negatively correlated to G/F (r=-0.42; P=0.003). Enzyme supplementation increased in vitro viscosity at 3, 4 and 5 hours (P value). Heat stress reduced serum glucose concentration on d1 (P=0.0002) but increased serum non esterified fatty acid (NEFA) concentration (P=0.002). A similar trend to NEFA was observed in triacylglycride (TAG). In the jejunum, carbohydrases had trend for increased villi height (P=0.07) while HS reduced villi height (P=0.02). Heat stress increased the jejunal mRNA abundance of IL1β in the jejunum (P<0.001). There was a trend for a reduction in ileal MUC2 (P=0.092), occludin (P=0.091) due to HS, with the trend increasing in PEPT1 (P=0.064). There was no effect of HS on alpha diversity of fecal microbiome, but sampling day affected beta diversity. There was an increase in the abundance of pathogenic bacteria (like Clostridium) in the HS group. Heat stress did not alter the efficacy of carbohydrase but both carbohydrase and HS modulate pig performance independently.</p>

Depression and anxiety disorders affect upwards of one in six individuals at some point in their life making them the most prevalent mental illnesses today. Recent evidence has suggested a possible correlation between the human gut microbiota and the development of depressive and anxiety-like symptoms through a signaling pathway termed the microbiota-gut-brain axis. In both animals and individuals suffering from depression and anxiety-like symptoms, alterations in their gut microbial composition seem to compromise the function of this pathway. In addition to this microbiota-gut-brain axis, other microbiota-derived molecules have been linked to symptoms of depression and anxiety. Given this emerging role of the gut microbiome and gut–brain axis, it is crucial to understand the factors shaping our gut microbiome in order to determine potential therapeutic strategies to treat depression and anxiety. Following a concise review of the human microbiome, depression/anxiety, and the gut-brain axis, I will examine the gut microbiota role as a regulator of depression and anxiety. In addition, other biological markers associated with both the gut microbiome and these disorders will be reviewed. Lastly, I will evaluate the gut microbiome as a prospective therapeutic target for mental illnesses such as depression and anxiety.

The human gut microbiome is important for health and development, and understanding its functioning and dynamics are of great medical importance. The microbiome food web is largely characterized by chains of byproduct cross-feeding (where metabolites of one organism are used as nutrients for another), yet a recent study have shown that the average length of the chains are considerably shorter than what metabolic capabilities of present species allow for. Here, I use evolving populations of digital organisms to investigate if recurring perturbations are a potential constraint of byproduct cross-feeding chains. I evolved digital populations in an environment unconstrained by energy loss between trophic levels and then exposed them to a period of recurring perturbations, where a fraction of the population was removed at 100 random points in time. Perturbations caused a substantial decrease in cross-feeding chain length, with increased frequency as perturbation intensity increased. In some communities, effects persisted after the perturbation period had ended. Tracking evolution of resource use during and after the perturbation period revealed that organisms descending from long-chained ancestors often evolved a shorter chain, suggesting that they adapted to perturbations by losing the ability to consume low-level resources. The evolutionary loss of resource consumption could explain the persisting effects on cross-feeding chains. Though my study suggests that perturbations can limit the length of byproduct cross-feeding chains, further studies are necessary toconclude if effects remain in environments with a more realistic energy transfer between trophic levels.

The composition and metabolic activity of the microbiome affect many aspects of health, and there is current interest in dietary constituents that may affect this system. The purpose of this study was to evaluate the effects of a mix of probiotics, a mix of prebiotics and a bioactive protein fraction on the microbiome, when fed to mice individually and in combination. Mice were fed the total western diet (TWD) supplemented with prebiotics, probiotics, and Tri-Factor (bioactive proteins) individually and in combination for four weeks. Subsequently, effects on the composition of gut microbiome, gut short chain fatty acids (SCFAs) concentration, gut inflammation and integrity of the mucosal barrier were analyzed. Ruminococcus gnavus was increased in mice gut microbiome after feeding prebiotics. Bifidobacterium longum was increased after feeding probiotics. Probiotic was associated with higher level of Clostridium neonatale. The treatments affected beta-diversity with exception of Tri-Factor, but not alpha diversity of microbiome. All treatments were associated with lower plasma zonulin, compared to the control group, indicating an effect on gut permeability. There were no treatment effects on cecal or fecal SCFAs, and the treatments did not affect gut inflammation as measured by fecal calprotectin.

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Systemic Lupus Erythematosus (SLE) stands as a multifaceted autoimmune disorder, characterized by a spectrum of clinical manifestations and the generation of autoantibodies against self-antigens. Our focus was on the pivotal role of B cells in the development of SLE. The study also underscored the significant contribution of regulatory B (Breg) cells in the context of SLE, suggesting their potential as key regulators of the disease process. Our results provided a deeper understanding of the intricate interplay between B cells and SLE, offering insights that were valuable for both scientific research and future designs of therapeutic approaches. Cutting-edge single-cell RNA sequencing was employed to analyze the differences in splenic Breg subsets and their molecular profiles across different stages of lupus development in mice. Transcriptome-based changes in Bregs during active disease were confirmed through phenotypic analysis. These findings provided crucial insights into the dynamic role of B cells in the pathogenesis of SLE. In addition, we delved into the intricate connection between SLE and the gut microbiota. A literature review offered a comprehensive analysis of current research, with a particular emphasis on potential interactions between bacterial flagellin and Toll-Like Receptor 5 (TLR5) on immune cells. These interactions garnered substantial attention due to their potential implications in the pathogenesis of SLE. We synthesized existing research, providing valuable insights into the complex interplay between SLE and the microbiota and suggesting promising avenues for further investigation and potential therapeutic interventions. In the final study, we explored lupus-like disease in mice with global Tlr5 deletion, initially expecting disease attenuation. Surprisingly, the results revealed an exacerbation of lupus-like symptoms, particularly in female mice lacking Tlr5. Future research will seek to uncover the mechanisms by which Tlr5 deletion modulates interactions between the host and the gut microbiota, ultimately contributing to the exacerbation of lupus-like disease. / Doctor of Philosophy / Systemic Lupus Erythematosus (SLE) is characterized by a range of health issues and the body attacking itself. In this exploration, we journey through the intricate landscape of SLE, uncovering key players and unexpected twists. In this dissertation, we journeyed through the intricate landscape of SLE, uncovering key players and unexpected twists. In this dissertation, we closely examined these immune cells, revealing how different types of B cells contributed to SLE's development. We also introduced the enigmatic regulatory B (Breg) cells, which acted as potential peacekeepers in this autoimmune reaction. Our results illuminated the complex relationship between B cells and SLE, offering insights that benefited both researchers and those seeking new treatments. We employed cutting-edge technology, single-cell RNA sequencing, to scrutinize the genetic fingerprints of B cells in mice with SLE. The results unveiled changes in Breg cells during active disease, providing critical clues about how B cells impacted SLE progression. In addition, this dissertation took us into the microscopic world of our gut inhabitants, the microbiota. We dived into a treasure trove of research, focusing on how interactions between bacterial flagellin and various microbiota elements affected immune cells through a special receptor called Toll-Like Receptor 5 (TLR5). These interactions, like hidden clues, had piqued scientists' interest for their potential role in SLE development. We synthesized existing research, offering valuable insights into the complex interplay between SLE and our microbiota. The discussion also suggested promising paths for future research and potential therapies. In the final study, we encountered a plot twist. We anticipated that deleting the Tlr5 gene would improve lupus-like disease in mice. To our surprise, the opposite happened. Lupus-like symptoms worsened, especially in female mice lacking Tlr5. Clinical signs included enlarged spleens and lymph nodes, increased immune cell activity, and kidney inflammation. But Tlr5 deletion didn't change the mice's metabolism or the leaky gut. Instead, it reshaped their gut's microbial residents. Future research aimed to uncover how Tlr5 deletion altered the interactions between the host and gut microbes, ultimately making lupus-like disease more severe. In a nutshell, this journey through SLE's complex world provided a deeper understanding of its intricacies. We met the B cells, explored the microbiota, and encountered surprises along the way. These discoveries were vital pieces of the puzzle, bringing us closer to unlocking the secrets of SLE and, perhaps, finding new ways to manage and treat this challenging autoimmune disorder.

Microbiota-released extracellular vesicles (MEVs) have emerged as key players in intercellular signaling in host-microbiome communications. However, their role in gut-brain axis signaling has been poorly investigated. Here, we performed deep multi-omics profiling of MEVs generated ex-vivo and from stool samples to gain insight into their role in gut-brain-axis signaling. Metabolomics unveiled a wide array of metabolites embedded in MEVs, including many neurotransmitter-related compounds such as arachidonyl-dopamine (NADA), gabapentin, glutamate, and N-acylethanolamines. To test the biodistribution of MEVs from the gut to other parts of the body, Caco-2, RIN-14B, and hCMEC/D3 cells showed the capacity to internalize labeled MEVs through an endocytic mechanism. Additionally, MEVs exhibited dose-dependent paracellular transport through Caco-2 intestinal cells and hCMEC/D3 brain endothelial cells. Overall, our results revealed the capabilities of MEVs to cross the intestinal and blood-brain barriers to delivering their cargo to distant parts of the body.