Aquatic animals, such as fish and shellfish, provide important economic and nutritional benefits for human society. Due to overexploitation of natural fish sources through traditional wild-caught fisheries, aquaculture (generally described as fish farming or culturing) has grown into an economically important industry. A major focus area for the aquaculture field is related to sustainability by ensuring the health and welfare of the aquatic animals. Communities of microorganisms inhabiting the various niches of a given host comprise its microbiome and provide several key health benefits. The microbiome impacts nutrient acquisition, gut homeostasis, protection against pathogens, and immune system modulation. Therefore, much attention has been placed on studying how various culturing conditions and host factors impact the microbiomes of aquatic animals. Here, multiple studies were conducted to elucidate the impacts of various parameters on the microbiomes of rainbow trout, steelhead trout, and Nile tilapia, including dietary supplementation, administration of probiotics and animal age. Though there is a significant correlation between the diet fed to fish and their microbiome communities, small dietary changes such as the inclusion of a dried and lysed yeast product, acting as a protein source alternative to unsustainable fishmeal did not significantly alter the intestinal adherent microbiome of rainbow trout. Moreover, an optimal percentage of yeast replacement that did not negatively impact weight gain for the aquaculture-raised fish was identified, suggesting its efficacy for the industry. Similarly, the intestinal adherent microbiomes of steelhead trout were not significantly altered by diet supplementation with a Bacillus subtilis probiotic. The total microbiome of steelhead trout (mucosa combined with digesta) was instead significantly changed when they were only fed the probiotic additive at an early stage of intestinal development. This change in the microbiome of steelhead trout correlated with a significant increase in weight gain compared to fish only fed the probiotic during later stages of intestinal development. These findings also corroborate previous observations wherein the intestinal microbiome of fish varies during their developmental stages but then stabilizes over time. Determining the core set of bacteria present in fish microbiomes, independent of treatment variables, is another important factor when considering attempts to manipulate the microbiome. To that end, a literature review was conducted in which the phyla Firmicutes, Proteobacteria and, to a lesser extent, Actinobacteria, Bacteroides, and Tenericutes were identified as likely members of the rainbow trout core microbiome. Bacterial families identified as part of the core phyla included Lactobacilliaceae that are commonly used as probiotics and Mycoplasmataceae that lack cell walls. Preventing dysbiosis of the rainbow trout microbiomes will be crucial to ensuring the health of the fish hosts and increasing longevity and profitability of the aquaculture industry. Another important aquaculture-raised species is the Eastern oyster. This animal is critical for the ecological health of the Chesapeake Bay, and it is also an important source of revenue. A significant portion of the revenue flow is the harvest and sale of live oysters for consumption. Unfortunately, consumption of raw or undercooked oysters is the most common route of infection by the human pathogen Vibrio parahaemolyticus (VP) as oysters are a natural reservoir for VP. This bacterium is responsible for a debilitating acute gastroenteritis with potential to cause fatal septicemia. Despite efforts to mitigate infection by this CDC-reportable pathogen, cases continue to increase. The understudied host-microbe relationship between the Eastern oyster and VP has been implicated as a path to research for potential future therapeutics. A novel culturing system for oysters was created using fermentation jars within a BSL-2 ready biosafety cabinet. Using this system, the effect of harvest season was tested against the inoculation efficiency of VP. It was found that higher native Vibrio levels within the oysters were present during the summer compared to the winter. Moreover, addition of the bacteriostatic antibiotic chloramphenicol (Cm) enabled a higher inoculation efficiency by VP during both the summer and winter compared to oysters not exposed to the antibiotic. During the winter, exposure to Cm led to the highest inoculation efficiency (~100%). These findings confirm the importance of the existing microbial communities against exogenous inoculation. Therefore, a year-long study was conducted to investigate the microbiome of oysters during each season. This pan-microbiome study identified a significant impact of harvest season on the microbiome structure. An increased diversity, including higher levels of Cyanobacteriaceae, was observed during the summer. Whereas an increase in Arcobacteriaceae was observed during the winter. Bacteria that persisted throughout the year included Mycoplamataceae and Spirochaeteacae; these families may represent potential members of the Eastern oyster core microbiome. Further work is needed to study the localization patterns of VP within oysters. Such work includes further optimization of immunohistochemistry (IHC) and intracellular colonization assay methods under development here. Collectively, studies of the oyster-microbe interactions will help improve aquaculture methods and identify mitigation targets to reduce VP-related clinical infections. / Doctor of Philosophy / Fish and shellfish provide important economic and nutritional benefits for human society across the globe. Unfortunately, over-fishing of traditional sources of fish and shellfish has led to a reduced supply for world markets, even as the human population increases. Aquaculture, or fish farming, has been around for centuries, but its role in society has significantly increased in the past 50 years. It currently provides about half of fish and other aquatic products on the market today. To better maintain and increase the sustainability and profitability of this industry, more focus is being placed on the health of the fish. The microbiome is the collection of communities of microorganisms, including bacteria, fungi, and archaea, that inhabit various environments including animal hosts. The majority of this dissertation focuses on the impact of factors like diet and age on the microbiomes of aquaculture-raised animals, especially fish. Dietary changes such as the addition of dried yeast-products had a significant impact on fish health but not on the microbiome communities. However, a common probiotic, Bacillus subtilis, did significantly increase not only the growth rate of trout but it also significantly altered the total intestinal microbiome found in the feces and the intestinal mucosal layer. Moreover, it was found that early exposure of the animals to the probiotic had enhanced benefits even though the microbiome appeared to stabilize over time as the fish developed. Maintaining or improving the microbiomes of fish, paying close attention to the microbes that exist as part of a core group of bacteria always present, is vital to ensuring fish health and understanding vertebrate host-microbe relationships. Thus, an analysis of the core microbiome of trout was performed. The final set of projects within this dissertation focused on the relationship between the Eastern oyster, a mollusk native to the Chesapeake Bay, and the bacterial human pathogen Vibrio parahaemolyticus (VP). VP is the leading cause of seafood-borne acute gastroenteritis worldwide, and efforts are needed to mitigate the increasing rate of human infections. Therefore, a simple system using fermentation jars within the laboratory biosafety cabinet was designed to enable safe culture of oysters that were exposed to VP under experimentally controlled conditions. Oysters harvested during the summer naturally harbored higher amounts of native Vibrio organisms in contrast to the winter oysters that harbored much lower levels. A separate microbiome analysis revealed large shifts in the oyster microbiome between summer and winter, although some microbes were continually present. The lower levels of existing Vibrio species detected in winter oysters may have allowed for the higher efficiency of inoculation of winter animals by VP. In fact, these winter animals had Vibrio microbiomes that were completely dominated by the inoculated strain which will enable future work to observe the pattern by which VP localizes, or colonizes, the oysters. Ultimately, these efforts may lead to the development of future disease mitigation strategies against VP.

Animals are ecological landscapes that host communities of microbes often referred to as microbiomes. These microbes can be transferred between individuals when they come into contact, such as during mating. Microbes that reside in or on any aspect of a host that becomes exposed to the reproductive tract or gametes comprise the reproductive microbiome. These microbes within the reproductive microbiome are important to overall host biology because they can influence host reproductive function, and thus play a role in shaping host ecology, evolution, and fitness. Though previous work has revealed much about the impact of beneficial and pathogenic microbes within the reproductive tract, much is left to be learned from describing the dynamic nature of the reproductive microbiome, and ultimately, how it affects host fitness. For my dissertation, I asked questions regarding how and why reproductive microbiome diversity varies among individuals. For instance, does reproductive microbiome diversity vary with respect to the number of mates or mating activity? Does reproductive microbiome diversity vary with host age or breeding stage? Are there fitness consequences associated with differences in reproductive microbiome diversity? To explore these questions, I studied tree swallows (Tachycineta bicolor), a socially monogamous bird in which both females and males engage in extra-pair mating activity. I focused on the cloacal microbiome as it is the site of contact during mating, and thus where microbes can be sexually transferred between individuals. I found that social partners did not have more similar cloacal microbiome diversity compared to other individuals in the same population, and that cloacal microbiome diversity was similar between sexes (Chapter II). By combining an observational approach with a hormone implant manipulation, I found that neither the number of sires per brood nor the increased mating activity of females significantly influenced cloacal microbiome richness or community structure. However, female age and breeding stage did significantly correlate with cloacal microbiome richness and community structure (Chapter III). Based on these findings, I hypothesized that the effect of mating activity on variation in the cloacal microbiome may only be detectable over a female's lifetime, and not within a single breeding season. In addition, I found evidence for a relationship between lay date and cloacal microbiome structure, after controlling for age. And I found that older females lay earlier in the season compared to younger, first-time breeding females (Chapter IV). These results provide support for a relationship between lay date and the cloacal microbiome and highlight the importance of age to this relationship. Lastly, I discussed future steps that can be taken to extend the framework established by my dissertation research, and thereby gain further insight into the factors shaping the reproductive microbiome (Chapter V). / Doctor of Philosophy / Animals host communities of microbes often referred to as microbiomes, and these microbes can be transferred between individuals when they come into contact, such as during mating. Microbes that reside in or on any part of a host that becomes exposed to the reproductive tract make up the reproductive microbiome. These microbes within the reproductive microbiome are important to an animal's biology because they can influence reproductive function, such as fertilization and pregnancy success. Though previous work has revealed much about the impact of beneficial and pathogenic microbes within the reproductive tract, much is left to be learned from describing the dynamic nature of the reproductive microbiome as a whole and how it affects an animal's reproductive success. For my dissertation, I sought to understand how and why reproductive microbiome diversity varies among individuals, especially in relation to mating. To explore these questions, I studied tree swallows (Tachycineta bicolor), a species of bird in which females and males will pair up to breed and rear young, yet both may mate with individuals other than their partner. I focused on the microbiome within the cloaca of birds as it is where contact is made during mating, and thus where microbes can be sexually transferred between individuals. I found that social partners did not have more similar cloacal microbiome diversity compared to other individuals in the same population, and that cloacal microbiome diversity was similar between females and males (Chapter II). Since tree swallows frequently mate with multiple partners and it is possible for bacteria to be acquired though each mating attempt, I proposed that it is important to consider the number of mates per individual when assessing the diversity of the cloacal microbiome. I then performed observational and experimental studies where I assessed the number of mates per female and manipulated female sexual activity with hormone implants. I found that neither the number of sires per brood nor the increased mating activity of females influenced cloacal microbiome diversity, however, female age and breeding stage were significantly associated with cloacal microbiome diversity (Chapter III). Based on these findings, I hypothesized that the effect of mating activity on variation in the cloacal microbiome may only be detectable over a female's lifetime, and not within a single breeding season. In addition, I found evidence for a relationship between lay date and cloacal microbiome structure, after controlling for age. And I found that older females lay earlier in the season compared to younger, first-time breeding females (Chapter IV). Taken together, these results provide support for a relationship between lay date and the cloacal microbiome and highlight the importance of age to this relationship. Lastly, I discussed future steps that can be taken to extend the framework established by my dissertation research and gain further insight into factors shaping the reproductive microbiome (Chapter V).

Ruminant livestock maintain an important role in meeting the nutrient requirements of the global population through their unique ability to convert plant fiber into human-edible meat and milk products. Volatile fatty acids (VFA) produced by rumen microbial fermentation of feed substrates represent around 70% of the ruminant animal's metabolic energy supply. Rumen fermentation profiles may directly impact productivity because the types of VFA produced are utilized at differing efficiencies by the animal. Improving our understanding of factors that control these fermentative outcomes would therefore aid in optimizing the productive efficiency of ruminant livestock. Improvements in animal efficiency are now more important than ever as the livestock industry must adapt to continue meeting the nutritional needs of a growing global population in the context of increased resource restrictions and requirements to lower the environmental impact of production. The relationship between diet and VFA ultimately supplied to the animal is complex and poorly understood due to the influence of numerous nutritional, biochemical, and microbial variables. The central aim of this body of work was therefore to explore and characterize how fermentation dynamics, rumen environmental characteristics, and the rumen microbiome behave in response to variations in the supply of fermentative substrate. The objective of our first experiment was to describe a novel in vitro laboratory technique to rank livestock feeds based on their starch degradability. This experiment also compared the starch degradation rates estimated by the in vitro method to the rates estimated by a traditional in situ method using sheep. A relationship between the degradation rates determined by these two procedures was observed, but only when feed nutrient content was accounted for. While this in vitro approach may not be able to reflect actual ruminal starch degradation rates, it holds potential as a useful laboratory technique for assessing relative differences in starch degradability between various feeds. Our second experiment aimed to measure changes in VFA dynamics, rumen environmental characteristics, and rumen epithelial gene expression levels in response to dietary sources of fiber and protein designed to differ in their rumen availabilities. Conducted in sheep, this study utilized beet pulp and timothy hay as the more and less available fiber source treatments, respectively, and soybean meal and heat-treated soybean meal as the more and less available protein source treatments, respectively. Results indicated that rumen environmental parameters and epithelial gene expression levels were not significantly altered by treatment. However, numerous shifts in response to both protein and fiber treatments were observed in fermentation dynamics, especially in interconversions of VFA. The objective of the third investigation was to assess whether the rumen microbiome can serve as an accurate predictor of beef and dairy cattle performance measurements and compare its predictive ability to that of diet explanatory variables. The available literature was assembled into a meta-analysis and models predicting dry matter intake, feed efficiency, average daily gain, and milk yield were derived using microbial and diet explanatory variables. Comparison of model quality revealed that the microbiome-based predictions may have comparable accuracy to diet-based predictions and that microbial variables may be used in combination with diet to improve predictions. In our fourth experiment, the objective was to investigate rumen microbial responses to the fiber and protein diet treatments detailed in Experiment 2. Responses of interest included relative abundances of bacterial populations at three taxonomic levels (phylum, family, and genus) in addition to estimations of community richness and diversity. Numerous population shifts were observed in response to fiber treatment. Prominent fibrolytic population abundances as well as richness and diversity estimations were found to be greater with timothy hay treatment and lower with beet pulp whereas pectin degraders increased in abundance on beet pulp. Microbial responses associated with protein treatment were not as numerous but appeared to reflect taxa with roles in protein metabolism. These four investigations revealed that significant changes can occur in VFA fermentation and rumen microbial populations when sources of nutrient substrates provided in a ruminant animal's diet are altered and that a new approach may be useful in investigating degradation of another important substrate for fermentation (starch) in a laboratory setting. Our findings also determined that animal performance can be predicted to a certain extent by rumen microbial characteristics. Collectively, these investigations offer an improved understanding of factors that influence the process of converting feed to energy sources in the ruminant animal. / Doctor of Philosophy / Ruminant animals, such as beef cattle, dairy cattle, and sheep, play a major role in delivering essential nutrients to the human population through their provision of meat and dairy products. The current growth projections of the global population, in addition to increased concerns surrounding greenhouse gas emissions and restrictions on resources such as land and water make it important for us to consider ways of optimizing the productivity of these animals. A unique feature of ruminants is their ability to conduct microbial fermentation of large amounts of plant matter in their rumens to produce energetically valuable compounds called volatile fatty acids (VFA), which are the primary source of energy that the animals use for growth, reproduction, and milk production. One promising way of improving animal productivity is to increase the amount of energy from the diet that becomes available to fuel the animal's body processes; however, the process of converting feed to VFA is complicated and currently not well understood. The overall aim of this body of work was therefore to explore various nutritional, ruminal, and microbial factors that are known to impact fermentation in order to 1) increase our understanding of how these factors interconnect and 2) put us in a better position to manipulate these factors for optimal animal performance. The goal of our first experiment was to devise and use a novel laboratory technique to rank livestock feeds based on the degradability of their starch content, which is an important substrate for VFA fermentation. Our observations indicate that this technique may be a useful tool to help us determine relative differences between feeds based on their starch degradabilities in a laboratory setting. Our second experiment investigated the effects of feeding varying sources of fiber (beet pulp and timothy hay) and protein (heat-treated and untreated soybean meals) to sheep in terms of their VFA fermentation, rumen conditions, and the expression of certain key genes in the epithelial tissue of the rumen wall. While rumen environmental characteristics and epithelial gene expression remained largely unchanged, numerous key aspects of VFA fermentation, predominantly carbon exchanges between different VFA, were altered in response to nutrient source. The third investigation described in this work examined the ability of the microbial populations responsible for rumen fermentation to explain variation in beef and dairy cow productivity compared with the ability of diet characteristics to explain this variation. Using statistical methods to analyze the reports currently available in scientific literature, our findings indicate that the rumen microbiome and diet may exert independent effects on productivity levels and that the microbiome may be used to enhance diet-based predictions of animal performance. Finally, we explored variations in the sheep rumen microbiome in response to the diet treatments utilized in Experiment 2. We observed minimal impact of protein source on the microbiome, but numerous microbial responses were evident when fiber source was varied. These responses included decreases of fiber-degrading bacterial populations and increases in pectin-degrading populations when beet pulp was fed compared to timothy hay. Taken together, these experiments help to provide us with a more comprehensive picture of the numerous factors involved in the process of converting feed to a usable form of energy for ruminant livestock.

Little is known about the microbiome composition associated with peri-implantitis in developing countries. A recent study found a high prevalence of peri-implantitis in a group of Moroccan patients. We hypothesized that a distinct microbiome may be associated with this disease in Moroccan subjects, and the aim of this study was to investigate the composition of the microbiome in peri-implantitis sites and sites without peri-implantitis. The study material consisted of 35 dental patients with dental implants: 22 of these had peri-implantitis, and 13 were without peri-implantitis. Among these subjects, dental plaque samples were collected from 50 peri-implant sites as follows: in the peri-implantitis subjects, 22 samples were from peri-implantitis sites (peri-implantitis patient diseased sites) and 15 samples from sites without peri-implantitis (peri-implantitis patient control sites); and 13 samples from implants from subjects without peri-implantitis (non-peri-implantitis patient control sites). The samples were sequenced for the V1-V3 region of the 16S rRNA gene, and the resultant sequences were classified at the species level using a previously described Blastn-based algorithm. Downstream analysis of the data was performed with Phyloseq, Microbiome, Vegan and MaAsLin packages in R, using a false discovery rate (FDR) cutoff of 0.25. Fifty-six species and 30 genera were identified per sample on average. No significant differences were found between the groups in terms of species richness and alpha diversity. However, beta diversity analysis by PERMANOVA (Adonis) identified a statistically significant difference (FDR=0.024) between the peri-implantitis patient diseased sites and non-peri-implantitis patient control sites. Compared to non-peri-implantitis patient control sites, diseased but not control sites in patients with peri-implantitis showed significantly higher levels of Peptostreptococcus stomatitis and Mogibacterium spp. However, both diseased and control sites in patients with peri-implantitis had higher abundance of Olsenella uli, Atopobium spp. and Actinomyces spp. compared to non-peri-implantitis patient control sites. No differences at FDR ≤ 0.25 were found between diseased and control sites in patients with peri-implantitis, but Porphyromonas endodontalis tended to be elevated in diseased sites while Veillonella parvula tended to increase in control sites. These findings suggest a distinct dysbiotic microbiome is associated with peri-implantitis sites in Moroccan patients. / Oral Biology

The variability in calf management can change the physiological state of the calf as they are weaned or attain puberty. It is up to the producer to ensure that the calves develop properly to meet their expected needs on the farm. While there are guidelines from the NRC in place, there is a substantial range in the amount of protein and fat that a calf can be fed. This physiological state can be reflected in the proteins produced in tissues, the expression of gene regulatory pathways, or even the microbes present in the gut. The purpose of this study was to examine how an increase in dietary energy in milk replacer of pre-weaned Holstein heifers impacts the microbial profile of the rumen as well as the transcriptome in tissues related to growth and metabolism. Our hypothesis was that pre-weaned Holstein heifers on milk replacer diets with lower dietary energy will have a different rumen microbiome composition and a different transcriptome in growth related tissues. Holstein heifer calves (n = 36) were assigned randomly to 1 of 2 milk replacer diets: restricted (R; 20.9% CP, 19.8% Fat; n = 18) or enhanced (E; 28.9% CP, 26.2% Fat; n = 18). Calves were euthanized and rumen fluid was collected at pre-weaning (8 wks; n = 6) or post-weaning (10 wks; n = 6). Liver (L), adipose (A), and longissimus dorsi (LD) tissues were collected at pre-weaning (8 wks; n = 12). Average daily gain (ADG) and gain-to-feed ratio (G:F) were calculated for each calf. Analysis of ADG and G:F was performed using a PROC GLM in SAS with diet as the main effect; E calves had increased ADG and G:F compared to R calves. For rumen samples, libraries were constructed from extracted DNA and DNASeq was conducted using a paired-end analysis at 100 bp using Illumina HiSeq 2500. Operational taxonomic unit (OTU) clustering analysis was conducted using the 16s rRNA Greengenes reference. A PERMANOVA analysis was conducted in R to determine OTU populations for age and treatment. There was no difference in microbiome composition between pre-weaning and post-weaning calves (P = 0.761). Microbiome composition differed between E and R calves (P < 0.001). Bacteroidetes and Firmicutes represented the most abundant phyla for both E and R calves. Enhanced calves had 49.4% (5141 reads) Bacteriodetes and 36.4% (3789 reads) Firmicutes; whereas, R calves had 31.6% (2491 reads) Bacteriodetes and 41.1% (3236 reads) Firmicutes. For L, A, and LD samples, libraries were constructed from extracted RNA for RNA-Seq analyses. RNA-Seq analysis was performed using CLC Genomics Workbench and the Robinson and Smith Exact Test was used to identify differentially expressed genes between diets. There were 238 differentially expressed genes in A, 227 in LD, and 40 in L. Of the differentially expressed genes, 10 appeared in at least 2 tissues. PANTHER was used to identify functional categories of differentially expressed genes. The majority of genes were associated with metabolic processes (A = 112, 26.7%; L = 16, 32.0%; LD = 81, 34.0%) or cellular processes (A = 93, 22.1%; L = 13, 26.0%; LD = 73, 30.7%). In E calves, upregulated genes included those regulating NADH dehydrogenation (LD = 17, A = 5; i.e. ND1, ND4), gluconeogenesis (LD = 2, A = 6; i.e. ALDOB, PCK2), and cell proliferation (LD = 2, A = 3; i.e. GADD45A, CDKN1A). There was a difference in both the transcriptome and rumen microbiome of calves fed differing levels of dietary energy. The calves on the R diet had a rumen microbial composition more similar to a younger calf, while the composition of E calves was more similar to a mature calf. The change in regulation of genes involved in the cell cycle and ATP synthesis in response to dietary energy could explain the change in ADG between diets. Because the R calves appeared to have stunted development of their microbiomes and an expression profile similar to oxidative stress, it is possible that the R diet did not meet the nutritional requirements of that calves. / Master of Science / Changes in the way a calf is raised from birth can affect the biological processes that occur when they change from liquid to solid feed or reach reproductive maturity. While there are guidelines in place in how much a calf should be fed, there is still a large range in the amount of protein and fat in the liquid feed. The change in nutrition levels changes the biological processes occurring in the calf, which are reflect by changes in expression of genes in different parts of the calf as well the levels of microbes in the gut. The purpose of this study was to examine how the change in protein and fat in the liquid feed of female calves affects the microbes in the first section of the stomach, the rumen, as well as the genes expressed in parts of the calf associated with growth. Our hypothesis was that female calves fed liquid diets with lower protein and fat will have different rumen microbes and a different level of gene expression in growth related tissues. Female calves (n = 36) were randomly assigned 1 of 2 diets at birth: restricted (R; 20.9% Crude Protein, 19.8% Fat; n = 18) or enhanced (E; 28.9% Crude Protein, 26.2% Fat; n = 18). Calves were euthanized and rumen contents were collected at removal of the liquid feed (8 wks; n = 6) or 2 wks after calves were switched to an all dry feed diet (10 wks; n = 6). Liver (L), adipose (A), and longissimus dorsi (LD) tissues were collected at removal of the liquid feed (8 wks; n = 12). Bacterial DNA was extracted from the rumen samples and RNA was extracted from L, A, and LD samples. DNA and RNA were sequenced at the University of Missouri DNA Core Lab. Microbiome composition differed between E and R calves (P < 0.001). Enhanced calves had 49.4% Bacteriodetes and 36.4% Firmicutes; whereas, R calves had 31.6% Bacteriodetes and 41.1% Firmicutes. There were 238 differentially expressed genes in A, 227 in LD, and 40 in L. Of the differentially expressed genes, 10 appeared in at least 2 tissues. In E calves, upregulated genes included those regulating NADH dehydrogenation (LD = 17, A = 5; i.e. ND1, ND4), gluconeogenesis (LD = 2, A = 6; i.e. ALDOB, PCK2), and cell growth (LD = 2, A = 3; i.e. GADD45A, CDKN1A). There was a difference in both the gene expression and rumen microbiome of calves fed differing levels of protein and fat. The calves on the R diet had a rumen microbial composition more similar to a younger calf, while the composition of E calves was more similar to a mature calf. Because the R calves appeared to have stunted development of their microbiomes and an expression profile similar to oxidative stress, it is possible that the R diet did not meet the nutritional requirements of that calves.

The building plumbing microbiome has important implications, especially in terms of its role as a reservoir and conduit for the spread of opportunistic pathogens (OPs), such as Legionella pneumophila. This dissertation applied next-generation DNA sequencing tools to survey the composition of building plumbing microbiomes and assessed hypothetical factors shaping them. A challenge to identifying key factors shaping building plumbing microbiomes is untangling the relative contributions of influent water quality, provided by drinking water utilities, and those of building-level features, such as pipe materials. To this end, standardized pipe rigs were deployed at the treatment plants and in distal portions of the water distribution system at five water utilities across the eastern U.S. Source water and treatment practices appeared to be the overarching factors shaping the microbial taxonomic composition at the tap, with five key water chemistry parameters identified (total chlorine, pH, P, SO42- and Mg2+). Hot water plumbing is of particular interest because OPs tend to proliferate in warm water environments and can be inhaled in aerosols when showering. Two identical lab-scale recirculating hot water rigs were operated in parallel to examine the combined effects of water heater temperature set point, pipe orientation, and water use frequency on the hot water plumbing microbiome. Our results revealed distinct microbial taxonomic compositions between the biofilm and water phases. Importantly, above a threshold of 51 °C, water heater temperature, pipe orientation, and water use frequency together incurred a prominent shift in microbiome composition and L. pneumophila occurrence. While heat shock is a popular means of remediating L. pneumophila contamination in plumbing, its broader effects on the microbiome are unknown. Here, heat shock was applied to acclimated lab-scale hot water rigs. Comparison of pre- versus post- heat shock samples indicated little to no change in either the microbial composition or L. pneumophila levels at the tap, where both water heater temperature and water use frequency had the most dominant effect. Overall, this dissertation contributes to advancing guidance regarding where to most effectively target controls for OPs and also advances research towards identifying the features of a 'healthy' built environment microbiome. / PHD / Drinking water is often misconceived to be “sterile,” whereas in reality the water distribution and plumbing systems that convey the water to the consumer represent a robust microbial habitat. While it is not possible, or even desirable, to kill all of the microbes present in drinking water, the Safe Drinking Water Act in the U.S. enforces measures to purify and disinfect water at the treatment plant and keep bacterial numbers low in water mains and up to the consumer property line. However, current regulatory frameworks are designed to protect against fecal- (e.g., raw sewage and manure) derived pathogens, whereas recently opportunistic pathogens (OPs), including Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa have come to the forefront as the leading source of tap-water related illness in the U.S. and other developed countries. In contrast to traditional fecal pathogens, building plumbing systems are a natural habitat for OPs, where they can readily proliferate. Currently there are no provisions within the Safe Drinking Water Act or other regulations to protect consumers specifically from OPs. There are also no “silver bullet” remedial measures that consistently and reliably defend against OPs colonizing building building plumbing, particularly when aiming to protect against multiple types of OPs. A major challenge in preventing and remediating OP proliferation in building plumbing is that they tend to be protected from disinfectants, such as chlorine, inside amoeba hosts and within the slimy layer that forms on the surface of pipe walls called “biofilm”. With the recent advent over the past decade of next-generation DNA sequencing, there are new reasons to take interest in the microbial composition of tap water. In particular, next-generation DNA sequencing has provided new insight into the composition of the human microbiome, e.g., the microbes that naturally inhabit our skin, gut, and lungs, and has revealed striking relationships with human health (e.g., obesity, diabetes, asthma, autism, allergies). The question naturally arises with respect to the factors shaping the human microbiome, with role of the “built environment” being of fundamental interest. The built environment; including homes, offices, schools, hospitals, and vehicles, is where most humans in developed countries spend > 90% of their time. Tap water is likely an important feature shaping the microbiome of the built environment, serving as a conduit for microbes into tiny droplets called aerosols, which can be inhaled into the lungs or otherwise inoculate the skin during showering or be transferred onto food during food preparation. Thus, there is interest in mapping out the microbiome of tap water and the factors that shape it, not only because of its potential to harbor OPs, but because of its potential general effect on built environment and human microbiomes. Long-term research could lead towards identifying which microbes serve a beneficial, or “probiotic,” role in preventing pathogen growth and benefiting human health. The purpose behind the body of research described in this dissertation was to apply newly available next-generation DNA sequencing tools towards mapping out the microbial composition characteristic of tap water, with emphasis on implications for preventing proliferation of OPs. Of particular interest was the relative role of what water utilities and building operators can do to protect public health. To this end, the DNA sequencing approach was applied to carefully controlled and replicated field- and laboratory-scale plumbing rigs to gain insight into the relative roles and interactions of the water quality provided by drinking water utilities and practical building-level engineering controls. Specific factors investigated included: stagnation (i.e., the tendency of water to sit unused in pipes in 8 hour cycles), pipe material (e.g., metallic versus plastic), pipe configuration (i.e., up or down flow to induce convective mixing vs stratification, respectively), water heater temperature set point (i.e., balancing hotter temperatures needed to kill pathogens versus lower temperatures desirable to save energy or prevent scalding), and heat-shock treatment (i.e., temporarily elevating the water heater temperature and flushing the system to kill off pathogens). There were several general findings that can be highlighted based on this research. First, based on comparison of standardized plumbing rigs installed at five water utilities in the U.S., the nature of the water provided by the local water utility was the overarching factor shaping the microbiome composition at the tap, moreso than pipe material or stagnation. Second, there exists an ideal threshold water heater temperature setting (51 °C based on the conditions of this study) above which there is a concordant shift in microbiome composition and decrease in L. pneumophila occurrence. Third, consistent water heater temperature setting above this threshold has a stronger long-term influence on the microbiome composition and L. pneumophila control than temporarily elevating the temperature for heat-shock treatment. Finally, biofilm and bulk water microbial compositions are extremely diverse in composition (e.g., thousands of species of microbes in each) and functional markers, and distinct from one anaother in terms of their characteristics under different operational conditions. In sum, this study takes a step towards better understanding building plumbing microbiome and identifies several promising engineering and control factors that can ultimately inform intentional engineering of the building plumbing microbiome, particularly with respect to protecting public health against OPs and potentially other microbiome-related ailments in the future.

Ecosystems comprise large groups of highly interdependent organisms. Cnidarians, such as sea anemones and corals, are keystone species in many marine ecosystems, especially coral reefs. Each individual cnidarian also constitutes an ecosystem unto itself, a "holo- biont", consisting of the host animal and accompanying microbial symbionts. To interro- gate cnidarian holobionts, I used computational approaches to analyze the transcriptomes of three cnidarians and build mechanistic models of their microbial symbionts. In par- ticular, I analyzed and annotated the transcriptomes of the cauliflower coral Pocillopora damicornis, the lined sea anemone Edwardsiella lineata, and the starlet sea anemone Ne- matostella vectensis, providing information about the molecular functions expressed by these organisms, and allowing development of a corresponding set of public databases: PocilloporaBase, EdBase, and an updated version of StellaBase, that facilitate access to the corresponding datasets. Additionally, I developed a method to infer the phylogenetic antiquity of transcripts. This method also allowed me to identify transcripts from other organisms (e.g., microbes) belonging to the anemone or coral holobiont. In parallel – in order better to understand the microbial symbionts that share envi- ronments with cnidarian hosts, I also developed new computer-simulation approaches for modeling metabolic interactions between different microbial species. These approaches are based on genome-scale stoichiometric reconstructions of metabolic networks and on Flux Balance Analysis (FBA). In addition to contributing to the development and testing of a new FBA-based platform for modeling communities in structured environments (Compu- tation Of Microbial Ecosystems in Time and Space, or COMETS), I used this platform for specific in silico experiments on microbial symbiosis. In particular, I computed all pairwise interactions between 582 different prokaryotic models, and identified global patterns of pu- tative positive (cross-feeding) vs. negative (food competition) interactions in this matrix of species pairs. I found that about 7% of the pairs yielded a greater biomass when grown together than when grown separately as monocultures. Despite existing challenges, such as the limitations of gap-filling steps in model construction and the need for a better knowl- edge of nutrient composition in natural environments, this approach could in the future help forecast shifts in the coral holobiont under likely scenarios of marine environmen- tal changes. In general, this work demonstrates how the integration of high-throughput sequencing technology and mechanistic systems-biology simulations, can provide unique tools to analyze interactions between microbes, and to mitigate or reverse adverse changes in marine ecosystems.

Targeted gene manipulation in a complex microbial community is an enabling technology for precise microbiome editing. This thesis introduces a new microbial therapeutic system dubbed Bacterial CRISPR-Transposase Reduction of Virulence In Situ (BACTRINS). BACTRINS is an in-situ microbiome engineering platform designed for efficient and precise genomic insertion of a desired payload and simultaneous knockout of target genes. When applied against a Shiga toxin-producing pathogen in the gut, this system delivers a CRISPR-associated transposase by bacterial conjugation for site-specific inactivation of the Shiga toxin gene and integration of a nanobody therapeutic payload to disrupt pathogen attachment. A single dose of this therapy resulted in high efficiency Shiga gene inactivation and improved survival in a murine infection model of Shiga-producing pathogen. This work establishes a new type of live bacterial therapeutic capable of reducing gut infections by transforming toxigenic pathogens into commensal protectors.

Alzheimer’s Disease is a devastating disease and a major medical challenge of the 21st century without preventative treatment available and absence of a cure. Due to the profound clinical impact on patients with the disease, and emotional and financial impact on their loved ones, there has been extensive research into therapeutic agents. Unfortunately, many of the studies are abandoned or fail due to a lack of complete understanding of the complex pathophysiology of Alzheimer’s Disease, and ineffective drug targets. While the etiology of disease remains controversial, the accumulation of both amyloid beta protein and neurofibrillary tangles has been the most supported theory for the past thirty years. Also in recent years, the gut microbiome has gained much attention for its unexplored potential and role in health and disease, and the Mediterranean Diet has been deemed the “healthiest” diet for the sixth consecutive year by health experts. This study reviews the current understood pathophysiology of AD and standard diagnostic criteria. This thesis aims to provide an overview of the neuroprotective effects of the Mediterranean Diet, the alterations of the gut microbiome that are associated with the Mediterranean Diet, and the alterations of the gut microbiome associated with Alzheimer’s Disease. Based on the analysis of the large number of studies presented, this thesis concludes that the Mediterranean Diet optimally modifies the ratio of Firmicutes:Bacteroidetes in the gut of people with Alzheimer’s Disease and reduces pathological and symptomatic progression of disease.

Life-history strategies are adaptations in behavior, physiology, and anatomy that influence survival and reproductive success. Variation in life-history strategies is often determined by adaptations to environmental conditions and trade-offs with sexually-selected signals. One of the aspects controlling life-history trade-offs is the endocrine system. Testosterone is a hormone that mediates several key aspects of male reproduction, yet little is known about the causes and consequences of variation in testosterone. Using rufous-collared sparrows (Zonotrichia capensis), a Neotropical songbird with a wide distribution, I explored geographical patterns of variation in testosterone levels and infection by haemosporidians, a type of blood parasite. I found that testosterone did not vary with elevation, nor predict haemosporidian infection, but males in breeding condition were more likely to be infected (Chapter I). High levels of testosterone have been associated with an increased number of sexual contacts and can suppress the immune response, thus it may increase the risk of sexually transmitted infections. By studying the communities of bacteria that reside in the cloaca of birds, I found that they were different depending on testosterone levels, and that high-testosterone males had higher relative abundance of Chlamydiae, a class of intracellular pathogens (Chapter II). During the breeding season there is an increase in physical contacts among individuals, testosterone levels increase in males, and there are additional energetic demands, all of which can increase exposure to bacteria or facilitate infection. I compared the cloacal microbiome of the same individuals between breeding and non-breeding seasons, and found that in males, but not in females, bacterial richness and phylogenetic diversity increased when birds were in reproductive condition. This suggested that the cloacal microbiome in birds is dynamic and responsive to breeding condition and sex of the host (Chapter III). Lastly, I synthesized the most relevant findings and suggested directions for future work (Chapter IV). I conclude that variation in testosterone is not always associated with immune suppression, and that the links among reproductive physiology, behavior, and the microbiome can provide insight into the evolution of life-history strategies. / Ph. D.