Chicken intestinal mycobiome: Biogeography, succession, and response to in-feed antibiotics

In-feed antibiotics increase animal performance primarily through modulation of the intestinal microbiota. However, the exact mechanism of action is not understood. Furthermore, a majority of research on intestinal microbiota has focused solely on the intestinal bacterial population with very little being known about smaller populations such as fungi. Deep sequencing of the internal transcribed spacer 2 (ITS2) region of fungal rRNA genes was utilized in two studies to characterize the biogeography of the chicken intestinal fungal community, the mycobiome, along the gastrointestinal tract of broiler chickens on days 28 and 42 and its possible shift in response to bacitracin methylene disalicylate (BMD), a commonly used in-feed antibiotic. Intestinal luminal contents were also collected from the duodenum, jejunum, ileum, and cecum at seven different time points throughout an entire production cycle to determine the succession of the mycobiome. The phyla Ascomycota and Basidiomycota were found to be predominant regardless of age or GI location. Genera commonly associated with feed ingredients or soil such as Microascus, Gibberella, Trichosporon, and Aspergillus were the most abundant. However, their abundance varied greatly between studies indicating a strong environmental influence. A clear succession of the cecal mycobiome was observed, in which specific fungal consortia moved down the intestinal tract as birds aged indicating that this population is transient in nature. Dietary supplementation of BMD at a subtherapeutic level of 55 mg/kg resulted in a decrease in the cecal fungal diversity. To further elucidate effect of antibiotics on cecal bacterial populations, broiler chicks were supplemented with or without one of five commonly used antibiotics for 14 days followed by deep sequencing of the V3-V4 region of the bacterial 16S rRNA gene. Although each antibiotic modulated the gut microbiota differently, the closer the antibacterial spectrum, the more similarly the microbiota is regulated with treatments classified as ionophores having the greatest effect. Importantly, all antibiotics had a strong tendency to enrich butyrate- and lactic acid-producing bacteria, while reducing bile salt hydrolase-producing bacteria, suggesting in-feed antibiotics improve animal growth performance through enhanced metabolism and utilization of dietary carbohydrates and lipids and improved energy harvest

Tissue expression of claudin-1 and claudin-2 tight junction proteins in chickens

Understanding gut homeostasis is an active area of research in livestock animals, as it is key to improving animal health and efficiency. Tight junction proteins are responsible for preventing the transport of foreign materials and microorganisms between cells in epithelial and endothelial tissue layers while also regulating the passage of water and ions. Their location and function within these protective barrier tissues suggest that tight junction proteins play a significant role in the tissue integrity and innate immunity of the host. The purpose of this study is to examine the expression patterns of mRNAs for different tight junction proteins in various tissues of broiler chickens. Tissue samples were collected from four 30-day old, healthy broiler chickens raised under standard care. Total RNA was isolated, reverse transcribed, and quantitative polymerase chain reaction (qPCR) was performed to measure the mRNA expressions of two major tight junction proteins, namely claudin-1 and claudin-2, in the skin, brain, lung, and various segments of the digestive tract. Fold differences among tissue types was calculated using the Ct method normalized to the expression of a house-keeping gene, GAPDH. Our results indicated that claudin-1 mRNA was abundant in the skin, lung, spleen, and pancreas, with approximately 250-, 300-, 200-, and 150-fold higher than the brain, respectively. Meanwhile, claudin-2 mRNA was highly expressed in the duodenum, spleen, and pancreas, showing approximately 900-, 650-, and 700-fold higher than the brain, respectively. Understanding the tissue expression patterns of major tight junction proteins represent an important first step in identifying the strategies to modulate their expression in the intestinal tract, thereby improving gut health, immunity, and production efficiency of chickens

Gut Microbiota Is a Major Contributor to Adiposity in Pigs

Different breeds of pigs vary greatly in their propensity for adiposity. Gut microbiota is known to play an important role in modulating host physiology including fat metabolism. However, the relative contribution of gut microbiota to lipogenic characteristics of pigs remains elusive. In this study, we transplanted fecal microbiota of adult Jinhua and Landrace pigs, two breeds of pigs with distinct lipogenic phenotypes, to antibiotic-treated mice. Our results indicated that, 4 weeks after fecal transplantation, the mice receiving Jinhua pigs’ “obese” microbiota (JM) exhibited a different intestinal bacterial community structure from those receiving Landrace pigs’ “lean” microbiota (LM). Notably, an elevated ratio of Firmicutes to Bacteroidetes and a significant diminishment of Akkermansia were observed in JM mice relative to LM mice. Importantly, mouse recipients resembled their respective porcine donors in many of the lipogenic characteristics. Similar to Jinhua pig donors, JM mice had elevated lipid and triglyceride levels and the lipoprotein lipase activity in the liver. Enhanced expression of multiple key lipogenic genes and reduced angiopoietin-like 4 (Angptl4) mRNA expression were also observed in JM mice, relative to those in LM mice. These results collectively suggested that gut microbiota of Jinhua pigs is more capable of enhancing lipogenesis than that of Landrace pigs. Transferability of the lipogenic phenotype across species further indicated that gut microbiota plays a major role in contributing to adiposity in pigs. Manipulation of intestinal microbiota will, therefore, have a profound impact on altering host metabolism and adipogenesis, with an important implication in the treatment of human overweight and obesity

Butyrate, Forskolin, and Lactose Synergistically Enhance Disease Resistance by Inducing the Expression of the Genes Involved in Innate Host Defense and Barrier Function

The rising concern of antimicrobial resistance highlights a need for effective alternatives to antibiotics for livestock production. Butyrate, forskolin, and lactose are three natural products known to induce the synthesis of host defense peptides (HDP), which are a critical component of innate immunity. In this study, the synergy among butyrate, forskolin, and lactose in enhancing innate host defense, barrier function, and resistance to necrotic enteritis and coccidiosis was investigated. Our results indicated that the three compounds synergistically augmented the expressions of multiple HDP and barrier function genes in chicken HD11 macrophages. The compounds also showed an obvious synergy in promoting HDP gene expressions in chicken jejunal explants. Dietary supplementation of a combination of 1 g/kg sodium butyrate, 10 mg/kg forskolin-containing plant extract, and 10 g/kg lactose dramatically improved the survival of chickens from 39% to 94% (p < 0.001) in a co-infection model of necrotic enteritis. Furthermore, the three compounds largely reversed growth suppression, significantly alleviated intestinal lesions, and reduced colonization of Clostridium perfringens or Eimeria maxima in chickens with necrotic enteritis and coccidiosis (p < 0.01). Collectively, dietary supplementation of butyrate, forskolin, and lactose is a promising antibiotic alternative approach to disease control and prevention for poultry and possibly other livestock species

Effect of Salmonella Typhimurium Colonization on Microbiota Maturation and Blood Leukocyte Populations in Broiler Chickens

Reducing Salmonella in commercial chickens is vital to decreasing human salmonellosis infections resulting from contact with contaminated poultry and poultry products. As the intestinal microbiota plays an important role in preventing pathogen colonization, we sought to understand the relationship between Salmonella infection and the cecal microbiota and the host immune system. Day-of-hatch broiler chicks were assigned to three treatments: control, artificial (SA), and natural (SN) Salmonella infection. At seven days of age, control and SA birds were inoculated with PBS or Salmonella Typhimurium, respectively. Five SA birds were transferred to SN cages to facilitate natural infection. Cecal content and blood samples were collected at 0, 8, 14, and 21 days of age for microbiota and leukocyte analysis, respectively. A significant change in microbiota composition was observed in both groups as noted by a decrease in Lactobacillus and Escherichia and an increase in Bacteroides. Leukocyte analysis revealed a decrease in the percentage of circulating monocytes at 7 days post-infection while a decrease in thrombocyte and an increase in heterophil percentages were seen at 14 days post-infection. Taken together, these results demonstrate the ability of Salmonella to modulate the intestinal microbiota to facilitate colonization. Additionally, results indicated an early role of monocytes and thrombocytes during colonization, followed by heterophils

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 β-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

1,25-Dihydroxyvitamin-D3 Induces Avian β-Defensin Gene Expression in Chickens.

Host defense peptides (HDPs) play a critical role in innate immunity. Specific modulation of endogenous HDP synthesis by dietary compounds has been regarded as a novel approach to boost immunity and disease resistance in animal production. 1,25-dihydroxy vitamin D3 (1,25D3) is well known as a powerful HDP inducer in humans, but limited information about the effect of 1,25D3 on HDPs in poultry is available. Here, we sought to examine whether 1,25D3 could stimulate avian β-defensin (AvBD) expression in chickens. We used chicken embryo intestinal epithelial cells (CEIEPCs) and peripheral blood mononuclear cells (PBMCs) to study the effect of 1,25D3 on the expression of AvBDs. We observed that 1,25D3 is able to up-regulate the expression of several AvBDs in CEIEPCs and PBMCs, whereas it increased the amounts of AvBD4 mRNA in CEIEPCs only in the presence of lipopolysaccharide (LPS). On the other hand, LPS treatment not only inhibited the expression of CYP24A1 but also altered the expression pattern of VDR in CEIEPCs. Furthermore, AvBDs were not directly regulated by 1,25D3, as cycloheximide completely blocked 1,25D3-induced expression of AvBDs. Our observations suggest that 1,25D3 is capable of inducing AvBD gene expression and is a potential antibiotic alternative through augmentation of host innate immunity as well as disease control in chickens

Cold stress initiates catecholaminergic and serotonergic responses in the chicken gut that are associated with functional shifts in the microbiome

ABSTRACT: Climate change is one of the most significant challenges facing the sustainability of global poultry production. Stress resulting from extreme temperature swings, including cold snaps, is a major concern for food production birds. Despite being well-documented in mammals, the effect of environmental stress on enteric neurophysiology and concomitant impact on host-microbiome interactions remains poorly understood in birds. As early life stressors may imprint long-term adaptive changes in the host, the present study sought to determine whether cold temperature stress, a prominent form of early life stress in chickens, elicits changes in enteric stress-related neurochemical concentrations that coincide with compositional and functional changes in the microbiome that persist into the later life of the bird. Chicks were, or were not, subjected to cold ambient temperature stress during the first week post-hatch and then remained at normal temperature for the remainder of the study. 16S rRNA gene and shallow shotgun metagenomic analyses demonstrated taxonomic and functional divergence between the cecal microbiomes of control and cold stressed chickens that persisted for weeks following cessation of the stressor. Enteric concentrations of serotonin, norepinephrine, and other monoamine neurochemicals were elevated (P < 0.05) in both cecal tissue and luminal content of cold stressed chickens. Significant (P < 0.05) associations were identified between cecal neurochemical concentrations and microbial taxa, suggesting host enteric neurochemical responses to environmental stress may shape the cecal microbiome. These findings demonstrate for the first time that early life exposure to environmental temperature stress can change the developmental trajectory of both the chicken cecal microbiome and host neuroendocrine enteric physiology. As many neurochemicals serve as interkingdom signaling molecules, the relationships identified here could be exploited to control the impact of climate change-driven stress on avian enteric host-microbe interactions

Effects of 1,25D₃ on the expression of AvBDs with or without the presence of LPS.

<p>CEIEPCs were treated with 20 ng/mL of 1,25D and 800 μg/mL for 12h. The relative gene expression was measured by qPCR and normalized to <i>GAPDH</i>. Each bar represents mean ± SD of the results from 2–3 independent experiments performed in triplicate. The bars without the same letter indicate differences significant at <i>P</i> < 0.05.</p