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Us as humans are colonised by many microbial communities (the human microbiome) that interact with and regulate the host's physiology, and have been linked with several diseases. The high number of interactions that intercurre between the microbiome and the host requires rigorous statistical approaches to link any condition of interest to microbiome data. Many publicly available microbiome datasets are available that allow to study such interactions. However, strong inconsistencies are found among the reported associations when looking at the same condition in different studies. On the road to consistent statistical microbiome analyses that rely on public data, lack of standardisation and availability are barriers to define reproducible and generalisable associations. The main aim of my PhD was the development of meta-analytical approaches to identify microbial signatures as general hallmarks of health versus disease, integrating diverse cohorts and conditions. During my PhD training, I first explored the associations between the oral microbiome and peri-implantitis, an oral disease of dental implants, in which I defined a microbial signature discriminating diseased from control samples. I further developed and applied discriminative models to multiple colorectal cancer (CRC) cohorts, showing that the microbial signature defined on CRC samples is shared across different populations. To be able to further generalised microbial signature with host's conditions through a meta-analysis approach, I collected and analysed 20,533 public metagenomes from 90 cohorts, that are available through the curatedMetagenomicData (cMD) version 3, an R package providing standardised taxonomic and functional profiles and manually curated metadata. The cMD3 resource was used to derive an easy-to-compute oral-to-gut introgression score that I found systematically associated in a large meta-analysis of twelve diseases and with ageing. Finally, I applied the meta-analysis approach to study diet interventions in mice, exploiting a novel approach able to profile the unexplored fraction of microbiomes, and showing associations driven by previously uncharacterised species. Overall, this thesis contributes to strengthening the links between human and animal microbiomes in normal and altered host conditions.

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.

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.

River ecosystems across the US and globally face numerous stressors that impact both ecological function and water quality. In 2015-16, municipalities along the Dan River in southern Virginia experienced repeated taste and odor (TandO) issues in their drinking water that originated from the river source water. Given that the source of TandO issues during these events were unknown, this research aimed to identify patterns in the distributions of river microorganisms that could help identify potential biological causes. Monthly water, sediment, and periphyton samples were collected for a full year from the Smith and Dan Rivers to quantify actinomycete, fungi, and chlorophyll a concentrations, which have historically been linked to TandO problems, and to characterize changes in microbial community structure. Although no significant TandO event occurred during the study period, the work produced unique and valuable data that describe patterns of microbial populations and communities in a river ecosystem. Results from the study show the abundances of actinomycetes, fungi, and chlorophyll a expressing seasonal and regional variation by habitat. From a broader ecological perspective, microbial communities sampled from water, sediment, and periphyton were each unique from each other regardless of river reach and season sampled. Overall, this research adds to our understanding of river ecology by detailing the microbial abundance and diversity in three river habitats, including periphyton, that can be used to predict sources of river TandO in future events, and offers new questions regarding how microbial diversity changes over space and time. / Master of Science / In 2015-16, cities along the Dan River in Virginia experienced multiple taste and odor (T&O) events that led to earthy and musty odors in drinking water. As part of a larger project looking at a range of possible chemical and biological sources of T&O, this research aimed to identify changes in abundance of river microorganisms that might indicate potential biological causes to T&O events. Monthly samples of water, sediment, and algal growth were collected for a year from 12 sites on the Smith and Dan Rivers. Samples were analyzed for abundances of three known T&O causing groups of organisms—actinomycetes, fungi, and photoautotrophs—and to characterize changes in total microbial communities as an indicator of ecological change occurring along the rivers. Although a significant T&O event did not occur during the study period, the research produced valuable descriptions of how important microorganisms change in a freshwater ecosystem. Actinomycetes elevated in the lower Dan River during fall, fungi elevated during the spring, and chlorophyll a was highest in the upper Smith River during winter, suggesting that photoautotrophic growth was more likely to be linked to previous T&O events. The diversity and makeup of the microbial communities in the rivers was primarily dependent on where they were growing (water, sediment, or periphyton) and secondarily on the season or the river reach. Combined, these results will help to identify causes of future T&O events in the Dan River and also provide new insights into ecological patterns of microorganisms in river ecosystems.

Systems biology is an interdisciplinary field that focuses on elucidating complex biological processes (systems) by investigating the interactions among its components through an iterative cycle composed of data generation, data analysis and mathematical modeling. Our contributions to systems biology revolve around the following two axes: - Data analysis: Two data analysis projects, which were initiated when I was a co-op at GlaxoSmithKline, are discussed in this thesis. First, next generation sequencing data generated for a phase I clinical trial is analyzed to determine the altered microbial community in human gut before and after antibiotic usage (Chapter 2). To our knowledge, there have not been similar comparative studies in humans on the impacts on the gut microbiome of an antibiotic when administered by different modes. Second, publicly available gene expression data is analyzed to investigate human immune response to tuberculosis (TB) infection (Chapter 3). The novel feature of this study is systematic drug repositioning for the prevention, control and treatment of TB using the Connectivity map. - Mathematical modeling: Polynomial dynamical systems, a state- and time- discrete logical modeling framework, is used to model two biological processes. First, a denitrification pathway in Pseudomonas aeruginosa is modeled to shed light on the reason of greenhouse gas nitrous oxide accumulation (Chapter 4). It is the first mathematical model of denitrification that can predict the effect of phosphate on the denitrification performance of this bacterium. Second, an iron homeostasis pathway linked to iron utilization, oxidative stress response and oncogenic pathways is constructed to investigate how normal breast cells become cancerous (Chapter 5). To date, our intracellular model is the only expanded core iron model that can capture a breast cancer phenotype by overexpression and knockout simulations. / Ph. D.

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.