Regional epithelial cell diversity in the small intestine of pigs.

Understanding regional distribution and specialization of small intestinal epithelial cells is crucial for developing methods to control appetite, stress, and nutrient uptake in swine. To establish a better understanding of specific epithelial cells found across different regions of the small intestine in pigs, we utilized single-cell RNA sequencing (scRNA-seq) to recover and analyze epithelial cells from duodenum, jejunum, and ileum. Cells identified included crypt cells, enterocytes, BEST4 enterocytes, goblet cells, and enteroendocrine (EE) cells. EE cells were divided into two subsets based on the level of expression of the EE lineage commitment gene, NEUROD1. NEUROD1hi EE cells had minimal expression of hormone-encoding genes and were dissimilar to EE cells in humans and mice, indicating a subset of EE cells unique to pigs. Recently discovered BEST4 enterocytes were detected in both crypts and villi throughout the small intestine via in situ staining, unlike in humans, where BEST4 enterocytes are found only in small intestinal villi. Proximal-to-distal gradients of expression were noted for hormone-encoding genes in EE cells and nutrient transport genes in enterocytes via scRNA-seq, demonstrating regional specialization. Regional gene expression in EE cells and enterocytes was validated via quantitative PCR (qPCR) analysis of RNA isolated from epithelial cells of different small intestinal locations. Though many genes had similar patterns of regional expression when assessed by qPCR of total epithelial cells, some regional expression was only detected via scRNA-seq, highlighting advantages of scRNA-seq to deconvolute cell type-specific regional gene expression when compared to analysis of bulk samples. Overall, results provide new information on regional localization and transcriptional profiles of epithelial cells in the pig small intestine.

Cells lining the intestinal tract (i.e., epithelial cells) provide a barrier to the outside environment but also play important specialized roles in nutrient absorption and secretion of mucus or hormones involved in controlling appetite and digestion. While similar cell types can be found throughout the small intestine, they have even more specialized function depending on region of the small intestine. Identification and characterization of intestinal epithelial cells are foundational to promoting pig intestinal health for optimal growth. Our research identified six types of epithelial cells across the small intestine of pigs. Enterocytes, an absorptive cell type, shared commonalities with human enterocytes, but a population of enteroendocrine cells, which secrete hormones, was unique to pigs. The location of certain epithelial cells in the intestine was identified and informed the relationship between various epithelial cell types. Overall, a clearer understanding of specific epithelial cells in the porcine intestine is provided, proving a critical foundation to further research aimed at maximizing pig intestinal health.

Chicken Intestinal Mycobiome: Initial Characterization and Its Response to Bacitracin Methylene Disalicylate.

The gastrointestinal (GI) tract harbors a diverse population of microorganisms. While much work has been focused on the characterization of the bacterial community, very little is known about the fungal community, or mycobiota, in different animal species and chickens in particular. Here, we characterized the biogeography of the mycobiota along the GI tract of day 28 broiler chicks and further examined its possible shift in response to bacitracin methylene disalicylate (BMD), a commonly used in-feed antibiotic, through Illumina sequencing of the internal transcribed spacer 2 (ITS2) region of fungal rRNA genes. Out of 124 samples sequenced, we identified a total of 468 unique fungal features that belong to four phyla and 125 genera in the GI tract. Ascomycota and Basidiomycota represented 90% to 99% of the intestinal mycobiota, with three genera, i.e., Microascus, Trichosporon, and Aspergillus, accounting for over 80% of the total fungal population in most GI segments. Furthermore, these fungal genera were dominated by Scopulariopsis brevicaulis (Scopulariopsis is the anamorph form of Microascus), Trichosporon asahii, and two Aspergillus species. We also revealed that the mycobiota are more diverse in the upper than lower GI tract. The cecal mycobiota transitioned from being S. brevicaulis dominant on day 14 to T. asahii dominant on day 28. Furthermore, 2-week feeding of 55 mg/kg BMD tended to reduce the cecal mycobiota α-diversity. Taken together, we provided a comprehensive biogeographic view and succession pattern of the chicken intestinal mycobiota and its influence by BMD. A better understanding of intestinal mycobiota may lead to the development of novel strategies to improve animal health and productivity.IMPORTANCE The intestinal microbiota is critical to host physiology, metabolism, and health. However, the fungal community has been often overlooked. Recent studies in humans have highlighted the importance of the mycobiota in obesity and disease, making it imperative that we increase our understanding of the fungal community. The significance of this study is that we revealed the spatial and temporal changes of the mycobiota in the GI tract of the chicken, a nonmammalian species. To our surprise, the chicken intestinal mycobiota is dominated by a limited number of fungal species, in contrast to the presence of hundreds of bacterial taxa in the bacteriome. Additionally, the chicken intestinal fungal community is more diverse in the upper than the lower GI tract, while the bacterial community shows an opposite pattern. Collectively, this study lays an important foundation for future work on the chicken intestinal mycobiome and its possible manipulation to enhance animal performance and disease resistance.

Differential Impact of Subtherapeutic Antibiotics and Ionophores on Intestinal Microbiota of Broilers.

Antimicrobial growth promoters (AGPs) are commonly used in the livestock industry at subtherapeutic levels to improve production efficiency, which is achieved mainly through modulation of the intestinal microbiota. However, how different classes of AGPs, particularly ionophores, regulate the gut microbiota remains unclear. In this study, male Cobb broiler chickens were supplemented for 14 days with or without one of five commonly used AGPs including three classical antibiotics (bacitracin methylene disalicylate, tylosin, and virginiamycin) and two ionophores (monensin and salinomycin) that differ in antimicrobial spectrum and mechanisms. Deep sequencing of the V3-V4 region of the bacterial 16S rRNA gene revealed that two ionophores drastically reduced a number of rare bacteria resulting in a significant decrease in richness and a concomitant increase in evenness of the cecal microbiota, whereas three antibiotics had no obvious impact. Although each AGP modulated the gut microbiota differently, the closer the antibacterial spectrum of AGPs, the more similarly the microbiota was regulated. Importantly, all AGPs had a strong tendency to enrich butyrate- and lactic acid-producing bacteria, while reducing bile salt hydrolase-producing bacteria, suggestive of enhanced metabolism and utilization of dietary carbohydrates and lipids and improved energy harvest, which may collectively be responsible for the growth-promoting effect of AGPs.

High Throughput Screening for Natural Host Defense Peptide-Inducing Compounds as Novel Alternatives to Antibiotics.

A rise in antimicrobial resistance demands novel alternatives to antimicrobials for disease control and prevention. As an important component of innate immunity, host defense peptides (HDPs) are capable of killing a broad spectrum of pathogens and modulating a range of host immune responses. Enhancing the synthesis of endogenous HDPs has emerged as a novel host-directed antimicrobial therapeutic strategy. To facilitate the identification of natural products with a strong capacity to induce HDP synthesis, a stable macrophage cell line expressing a luciferase reporter gene driven by a 2-Kb avian ß-defensin 9 (AvBD9) gene promoter was constructed through lentiviral transduction and puromycin selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 584 natural products. A total of 21 compounds with a minimum Z-score of 2.0 were identified. Secondary screening in chicken HTC macrophages and jejunal explants further validated most compounds with a potent HDP-inducing activity in a dose-dependent manner. A follow-up oral administration of a lead natural compound, wortmannin, confirmed its capacity to enhance the AvBD9 gene expression in the duodenum of chickens. Besides AvBD9, most other chicken HDP genes were also induced by wortmannin. Additionally, butyrate was also found to synergize with wortmannin and several other newly-identified compounds in AvBD9 induction in HTC cells. Furthermore, wortmannin acted synergistically with butyrate in augmenting the antibacterial activity of chicken monocytes. Therefore, these natural HDP-inducing products may have the potential to be developed individually or in combinations as novel antibiotic alternatives for disease control and prevention in poultry and possibly other animal species including humans.

Butyrate: A Double-Edged Sword for Health?

Butyrate, a four-carbon short-chain fatty acid, is produced through microbial fermentation of dietary fibers in the lower intestinal tract. Endogenous butyrate production, delivery, and absorption by colonocytes have been well documented. Butyrate exerts its functions by acting as a histone deacetylase (HDAC) inhibitor or signaling through several G protein-coupled receptors (GPCRs). Recently, butyrate has received particular attention for its beneficial effects on intestinal homeostasis and energy metabolism. With anti-inflammatory properties, butyrate enhances intestinal barrier function and mucosal immunity. However, the role of butyrate in obesity remains controversial. Growing evidence has highlighted the impact of butyrate on the gut-brain axis. In this review, we summarize the present knowledge on the properties of butyrate, especially its potential effects and mechanisms involved in intestinal health and obesity.