Characterization and Comparison of the Rumen Luminal and Epithelial Microbiome Profiles Using Metagenomic Sequencing Technique.

Bacterial dysbiosis as a result of nutritional, bacterial, viral, and parasitic gastrointestinal infections can adversely affect the metabolism, productivity, and overall health of cattle. The purpose of this project was to characterize the commensal microbiota present in two locations of the rumen concomitantly in vivo with the animals undergoing habitual husbandry, as it was hypothesized that there are major differences in the commensal microbiota present in the two locations of the adult bovine major forestomach. A surgically fitted rumen cannula was used to allow ruminal lumen contents and mucosal biopsies to be collected from six crossbred yearling steers. In order to assess as much environmental and individual steer microbiota variation as possible, each animal was randomly sampled three times over a 3 week period. 16S rRNA sequencing was performed to provide a detailed descriptive analysis from phylum to genus taxonomic level. Significant differences were observed between luminal and epimural bacterial populations in the bovine rumen. As expected, a core microbiome composed by Firmicutes and Bacteroidetes represented over 90% of the microbiome, however, further analysis showed distinct diversity and distribution of the microbiome between the two locations. Characterizing the gastrointestinal microbiome in vivo is imperative. The novelty and the contribution of this study to the literature is the use of live cattle which allowed real-time sample collections and analysis of the rumen microbiome providing an understanding of what is normal in the live animal.

Longitudinal Analysis of the Intestinal Microbiota in the Obese Mangalica Pig Reveals Alterations in Bacteria and Bacteriophage Populations Associated With Changes in Body Composition and Diet.

Due to its immunomodulatory potential, the intestinal microbiota has been implicated as a contributing factor in the development of the meta-inflammatory state that drives obesity-associated insulin resistance and type 2 diabetes. A better understanding of this link would facilitate the development of targeted treatments and therapies to treat the metabolic complications of obesity. To this end, we validated and utilized a novel swine model of obesity, the Mangalica pig, to characterize changes in the gut microbiota during the development of an obese phenotype, and in response to dietary differences. In the first study, we characterized the metabolic phenotype and gut microbiota in lean and obese adult Mangalica pigs. Obese or lean groups were created by allowing either ad libitum (obese) or restricted (lean) access to a standard diet for 54 weeks. Mature obese pigs were significantly heavier and exhibited 170% greater subcutaneous adipose tissue mass, with no differences in muscle mass compared to their lean counterparts. Obese pigs displayed impaired glucose tolerance and hyperinsulinemia following oral glucose challenge, indicating that a metabolic phenotype also manifested with changes in body composition. Consistent with observations in human obesity, the gut microbiota of obese pigs displayed altered bacterial composition. In the second study, we characterized the longitudinal changes in the gut microbiota in response to diet and aging in growing Mangalica pigs that were either limit fed a standard diet, allowed ad libitum access to a standard diet, or allowed ad libitum access to a high fat-supplemented diet over an 18-week period. As expected, weight gain was highest in pigs fed the high fat diet compared to ad libitum and limit fed groups. Furthermore, the ad libitum and high fat groups displayed significantly greater adiposity consistent with the development of obesity relative to the limit fed pigs. The intestinal microbiota was generally resilient to differences in dietary intake (limit fed vs ad libitum), though changes in the microbiota of pigs fed the high fat diet mirrored changes observed in mature obese pigs during the first study. This is consistent with the link observed between the microbiota and adiposity. In contrast to intestinal bacterial populations, bacteriophage populations within the gut microbiota responded rapidly to differences in diet, with significant compositional changes in bacteriophage genera observed between the dietary treatment groups as pigs aged. These studies are the first to describe the development of the intestinal microbiota in the Mangalica pig, and are the first to provide evidence that changes in body composition and dietary conditions are associated with changes in the microbiome of this novel porcine model of obesity.

Integrative Longitudinal Analysis of Metabolic Phenotype and Microbiota Changes During the Development of Obesity.

Obesity has increased at an alarming rate over the past two decades in the United States. In addition to increased body mass, obesity is often accompanied by comorbidities such as Type II Diabetes Mellitus and metabolic dysfunction-associated fatty liver disease, with serious impacts on public health. Our understanding of the role the intestinal microbiota in obesity has rapidly advanced in recent years, especially with respect to the bacterial constituents. However, we know little of when changes in these microbial populations occur as obesity develops. Further, we know little about how other domains of the microbiota, namely bacteriophage populations, are affected during the progression of obesity. Our goal in this study was to monitor changes in the intestinal microbiome and metabolic phenotype following western diet feeding. We accomplished this by collecting metabolic data and fecal samples for shotgun metagenomic sequencing in a mouse model of diet-induced obesity. We found that after two weeks of consuming a western diet (WD), the animals weighed significantly more and were less metabolically stable than their chow fed counterparts. The western diet induced rapid changes in the intestinal microbiome with the most pronounced dissimilarity at 12 weeks. Our study highlights the dynamic nature of microbiota composition following WD feeding and puts these events in the context of the metabolic status of the mammalian host.

In vivo Microbiome Profiling of the Luminal and Mucosal Surface of the Duodenum Using a Cannulated Yearling Bovine Model.

The gut microbiome provides important metabolic functions for the host animal. Bacterial dysbiosis as a result of bacterial, viral, and parasitic gastrointestinal infections can adversely affect the metabolism, productivity, and overall health. The objective of this study is to characterize the commensal microbiome present in the lumen and the mucosal surface of the duodenum of cattle, as we hypothesize that due to metabolic processes and or host proprieties, there are differences in the natural microbiota present in the mucosal surface and luminal contents of the bovine duodenum. Duodenal lumen contents and mucosal biopsies were collected from six dairy crossbred yearling steers. A flexible video-endoscope was used to harvest biopsy samples via a T shaped intestinal cannula. In order to assess as much environmental and individual steer microbiota variation as possible, each animal was sampled three times over a 6 week period. The DNA was extracted from the samples and submitted for16S rRNA gene Ion Torrent PGM bacterial sequencing. A detailed descriptive analysis from phylum to genus taxonomic level was reported. Differences in the microbiome population between two different sites within the duodenum were successfully characterized. A great and significant microbiota diversity was found between the luminal and mucosal biopsy At the phylum taxonomic level, Firmicutes, and Bacteroidetes composed over 80% of the microbiome. Further analysis at lower taxonomic levels, class, family, and genus, showed distinct diversity and distribution of the microbiome. Characterizing the gastrointestinal microbiome in vivo is imperative. The novelty of this study is the use of live cattle undergoing customary husbandry allowing real-time analysis of the duodenum microbiome contributing to the literature with respect to the bovine duodenum microbiome.

Maternal Dietary Protein Intake Influences Milk and Offspring Gut Microbial Diversity in a Rat (Rattus norvegicus) Model.

Historically, investigators have assumed microorganisms identified in mother's milk to be contaminants, but recent data suggest that milk microbiota may contribute to beneficial maternal effects. Microorganisms that colonize the gastrointestinal tracts of newborn mammals are derived, at least in part, from the maternal microbial population. Milk-derived microbiota is an important source of this microbial inocula and we hypothesized that the maternal diet contributes to variation in this microbial community. To evaluate the relationship between a mother's diet and milk microbiome, we fed female rats a low- or high-protein diet and mated all individuals. Milk and cecal contents were collected from dams at peak lactation (14-day post-partum), and the bacterial composition of each community was assessed by 16S rRNA gene amplicon sequencing. Our findings revealed higher dietary protein intake decreased fecal microbial diversity but increased milk microbial and pup cecum diversity. Further, the higher dietary protein intake resulted in a greater abundance of potentially health-promoting bacteria, such as Lactobacillus spp. These data suggest that dietary protein levels contribute to significant shifts in the composition of maternal milk microbiota and that the functional consequences of these changes in microbial inocula might be biologically important and should be further explored.