Long-chain glucomannan supplementation modulates immune responsiveness, as well as intestinal microbiota, and impacts infection of broiler chickens with Salmonella enterica serotype Enteritidis.

The zoonotic pathogen Salmonella enterica serotype Enteritidis (SE) causes severe disease in young chickens. Restriction on antibiotic use requires alternative SE control strategies such as nutritional solutions to improve the resistance of chickens. In this study, chickens were fed long-chain glucomannan (GM) or standard diet and challenged with SE at seven days of age. During 21 days post-infection (dpi), we determined numbers and responsiveness of natural killer (NK) and T cells in ileum and spleen, and SE-specific antibody titers in serum. Microbiota compositions in ileum and caeca were determined, as well as correlations of these with numbers and function of immune cells. Some of the samples in the control group had numerically higher CFUs than the GM-treated group. In addition, the relative abundance of SE based on DNA assessment was significantly lower at 21 dpi upon GM supplementation. At 3 dpi, numbers of intraepithelial NK cells were significantly higher, while activation of intraepithelial NK cells (7 dpi), numbers of intraepithelial cytotoxic CD8+ T cells (14 dpi) and SE-specific antibodies (14 dpi) were numerically higher. Furthermore, relative abundance of the commensal lactic acid bacteria (LAB) significantly increased with GM supplementation post-infection. Higher relative abundance of streptococci was associated with reduced SE in ileal and caecal contents at 21 dpi. Relative abundance of streptococci negatively correlated with SE counts and positively correlated with NK cell activation and SE-specific antibodies, which suggests involvement of the commensal LAB in NK cell responsiveness. These results indicate that GM supplementation modulates the immune system, intestinal microbiota and impacts SE infection of young chickens.

Transgenerational effects of innate immune activation in broiler breeders on growth performance and immune responsiveness.

The impact of transgenerational effects on growth performance and immunity has not yet been studied extensively within the poultry husbandry sector. An important factor is the impact of the hens on the physical well-being and fitness to the environment of the offspring. This study is the first to investigate the effect of stimulating the maternal innate immune system with lipopolysaccharides (LPS) or ß-glucan on growth performance and immune responses in the next generation. Transgenerational effects and consequences of these maternal treatments were further examined using a necrotic enteritis (NE) challenge model in the offspring. We show that offspring of LPS-treated broiler breeders have a higher feed efficiency from 14 to 21 days of age, that is, the period just after the NE challenge. Moreover, more broiler chickens with intestinal lesions after the NE challenge were found in the offspring of the LPS-treated broiler breeders. Both the LPS and ß-glucan maternal treatments resulted in transgenerational effects on blood-derived monocytes by showing a tendency of decreased IL1ß mRNA levels after ex vivo LPS stimulation. These data are a first indication that broiler breeder hens can affect immune responsiveness and feeding efficiency of their offspring in a transgenerational manner.

Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens.

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

Early Life Inoculation With Adult-Derived Microbiota Accelerates Maturation of Intestinal Microbiota and Enhances NK Cell Activation in Broiler Chickens.

Studies in mammals, including chickens, have shown that the development of the immune system is affected by interactions with intestinal microbiota. Early life microbial colonization may affect the development of innate and adaptive immunity and may contribute to lasting effects on health and resilience of broiler chickens. We inoculated broiler chickens with adult-derived-microbiota (AM) to investigate their effects on intestinal microbiota composition and natural killer (NK) cells, amongst other immune cells. We hypothesized that AM inoculation directly upon hatch (day 0) would induce an alteration in microbiota composition shortly after hatch, and subsequently affect (subsets of) intestinal NK cells and their activation. Microbiota composition of caecal and ileal content of chickens of 1, 3, 7, 14, 21, and 35 days of age was assessed by sequencing of 16S ribosomal RNA gene amplicons. In parallel, subsets and activation of intestinal NK cells were analyzed by flow cytometry. In caecal content of 1- and 3-day-old AM chickens, a higher alpha-diversity (Faith's phylogenetic diversity) was observed compared to control chickens, whereas ileal microbiota were unaffected. Regarding beta-diversity, caecal microbiota profiles could be clustered into three distinct community types. Cluster A represented caecal microbiota of 1-day-old AM chickens and 1- and 3-day-old control chickens. Cluster B included microbiota of seven of eight 3- and 7-day-old AM and 7-day-old control chickens, and cluster C comprised microbiota of all chickens of 14-days and older, independent of inoculation. In 3-day-old AM chickens an increase in the percentages of intestinal IL-2Rα+NK cells and activated NK cells was observed compared to control chickens of the same age. In addition, an increase in relative numbers of intestinal cytotoxic CD8αα+T cells was observed in 14- and 21-day-old AM chickens. Taken together, these results indicate that early exposure to AM shapes and accelerates the maturation of caecal microbiota, which is paralleled by an increase in IL-2Rα+NK cells and enhanced NK cell activation. The observed association between early life development of intestinal microbiota and immune system indicates possibilities to apply microbiota-targeted strategies that can accelerate maturation of intestinal microbiota and strengthen the immune system, thereby improving the health and resilience of broiler chickens.

Take care of the environment: housing conditions affect the interplay of nutritional interventions and intestinal microbiota in broiler chickens.

BACKGROUND: The intestinal microbiota is shaped by many interactions between microorganisms, host, diet, and the environment. Exposure to microorganisms present in the environment, and exchange of microorganisms between hosts sharing the same environment, can influence intestinal microbiota of individuals, but how this affects microbiota studies is poorly understood. We investigated the effects of experimental housing circumstances on intestinal microbiota composition in broiler chickens, and how these effects may influence the capacity to determine diet related effects in a nutrition experiment. A cross-sectional experiment was conducted simultaneously in a feed research facility with mesh panels between pens (Housing condition 1, H1), in an extensively cleaned stable with floor pens with solid wooden panels (H2), and in isolators (H3). In H1 and H2 different distances between pens were created to assess gut microbiota exchange between pens. Feed with and without a blend of medium-chain fatty acids (MCFA) was used to create differences in cecal microbiota between pens or isolators within the same housing condition. Male one-day-old Ross broiler chickens (n = 370) were randomly distributed across H1, H2, and H3. After 35 days cecal microbiota composition was assessed by 16S ribosomal RNA gene amplicon sequencing. Metabolic functioning of cecal content was assessed based on high-performance liquid chromatography. RESULTS: Microbial alpha diversity was not affected in broilers fed +MCFA in H1 but was increased in H2 and H3. Based on weighted UniFrac distances, the nutritional intervention explained 10%, whereas housing condition explained 28% of cecal microbiota variation between all broilers. The effect size of the nutritional intervention varied within housing conditions between 11, 27, and 13% for H1, H2, and H3. Furthermore, performance and metabolic output were significantly different between housing conditions. The distance between pens within H1 and H2 did not influence the percentage of shared genera or operational taxonomic units (OTUs). CONCLUSIONS: The cecal microbiota of broilers was modifiable by a nutritional intervention, but the housing condition affected microbiota composition and functionality stronger than the diet intervention. Consequently, for interpretation of intestinal microbiota studies in poultry it is essential to be aware of the potentially large impact of housing conditions on the obtained results.