Comparison of yeast-derived commercial feed additives on Salmonella Enteritidis survival and microbiota populations in rooster cecal in vitro incubations.

Yeast-derived products have become more of an interest in the poultry industry as of late because of their use in modulating the gastrointestinal tract (GIT) microbiome to both improve production parameters and prevent infection. This study aimed to evaluate the effects of various yeast-derived products on Salmonella enterica inoculation in un in vitro rooster cecal incubations and associated effects on the cecal microbiome. Cecal contents were obtained from 53-wk old White Leghorn H & N Nick Chick roosters (n = 3) fed a wheat-based, commercial-type basal diet. Cecal contents were diluted 1:3000 in anaerobic dilution solution (ADS) in an anaerobic chamber, with 20 mL aliquoted to each serum bottle. There were three controls (n = 3): basal diet only, diluted cecal contents only, and basal diet and diluted cecal contents; and five treatments containing the basal diet and diluted cecal contents (n = 3): Citristim® (ADM), ImmunoWall® (ICC), Maxi-Gen Plus® (CBS Bio Platforms), Hilyses® (ICC), and Original XPC® (Diamond V). All treatments were applied at a rate of 2.5 kg/tonne or less. All groups were inoculated with a nalidixic acid-resistant strain of Salmonella Enteritidis at 10^7 CFU/mL and incubated at 37 deg C. Samples were collected at 0, 24, and 48 h for S. Enteritidis enumeration and 16S rDNA microbial sequencing. Salmonella data were log-transformed and analyzed in a two-way ANOVA with means separated using Tukey's HSD (P≤0.05). Genomic DNA was extracted, and resulting libraries were prepared and sequenced using an Illumina MiSeq. Sequencing data were analyzed in QIIME2 (2021.4) with diversity metrics (alpha and beta), and an analysis of the composition of microbiomes (ANCOM) was performed. Main effects were considered significant at P≤0.05, with pairwise differences considered significant at Q≤0.05. There was an interaction of treatment and time on the enumeration of Salmonella where treatments of Citristim, Immunowall, Hilyses, and XPC reduced Salmonella by 1 log CFU/mL compared to the controls. At 48 h, each yeast product treatment reduced Salmonella by 3 log CFU/mL compared to the controls. There was no main effect of treatment on the alpha diversity metrics, richness, or evenness (P > 0.05). Treatment affected the beta diversity, abundance, and phylogenetic differences, but there were no pairwise differences (P>0.05, Q>0.05). Using ANCOM at the genus level, the taxa Synergistes, Alloprevotella, Sutterella, and Megasphaera abundance were significantly different (W = 154,147,145,140, respectively). These results demonstrate the potential of these yeast-derived products to reduce foodborne pathogens, such as Salmonella Enteriditis, in vitro, without negatively disrupting the cecal microbiome.

Conversion of Phenolic Acids in Canola Fermentation: Impact on Antimicrobial Activity against Salmonella enterica and Campylobacter jejuni.

Canola meal (CM) is commonly used in poultry feeds. CM has a high protein content but also contains high levels of antimicrobial phenolic acids. Lactic acid bacteria can alter CM phenolic composition during fermentation and influence its antimicrobial activity against pathogens. Fermented CM was analyzed for phenolic composition using tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). Sinapic acid and derivatives were the major phenolic acids in CM. Growth of lactobacilli in CM was attenuated when compared to cereal substrates. Glucosides and esters of sinapic acid were extensively hydrolyzed during fermentation with Lactiplantibacillus plantarum and Furfurilactobacillus milii. Lp. plantarum transformed hydroxycinnamic acids to dihydro, 4-vinyl, and 4-ethyl derivatives, Ff. milii reduced hydroxycinnamic acids to dihydroderivatives, but Limosilactobacillus reuteri did not convert hydroxycinnamic acids. The minimum inhibitory concentration of phenolic extracts was assessed with lactobacilli, Salmonella, and Campylobacter jejuni as indicator strains. Fermentation of CM with Lp. plantarum or Ff. milii increased the antimicrobial activity of phenolic extracts against Salmonella enterica and Campylobacter jejuni. Fermentation with Lm. reuteri TMW1.656 but not fermentation with Lm. reuteri TMW1.656ΔrtcN increased the antimicrobial activity of extracts owing to the production of reutericyclin. This study demonstrates that fermentation of CM with lactobacilli converts hydroxycinammic esters and may increase the antimicrobial activity of phenolic compounds in CM against pathogens.

Week-Old Chicks with High Bacteroides Abundance Have Increased Short-Chain Fatty Acids and Reduced Markers of Gut Inflammation.

As important commensals in the chicken intestine, Bacteroides are essential complex carbohydrate degraders, and short-chain fatty acid (SCFA) producers that are highly adapted to the distal gut. Previous studies have shown large variation in Bacteroides abundance in young chickens. However, limited information is available regarding how this variation affects the gut microbiome and host immunity. To investigate how elevated or depleted Bacteroides levels affect gut microbial functional capacity and impact host response, we sampled 7-day-old broiler chickens from 14 commercial production flocks. Week-old broiler chickens were screened and birds with low Bacteroides (LB) and high Bacteroides (HB) abundance were identified via 16S rRNA gene amplicon sequencing and quantitative PCR (qPCR) assays. Cecal microbial functionality and SCFA concentration of chickens with distinct cecal Bacteroides abundance were profiled by shotgun metagenomic sequencing and gas chromatography, respectively. The intestinal immune responses of LB and HB chickens were assessed via reverse transcription qPCR. Results showed that the gut microbiota of the LB group had increased abundance of lactic acid bacteria pyruvate fermentation pathway, whereas complex polysaccharide degradation and SCFA production pathways were enriched in the HB group (P < 0.05), which was supported by increased SCFA concentrations in the ceca of HB chickens (P < 0.05). HB chickens also showed decreased expression of interleukin-1ß and increased expression of interleukin-10 and tight-junction protein claudin-1 (P < 0.05). Overall, the results indicated that elevated Bacteroides may benefit the 7-day broiler gut and that further work should be done to confirm the causal role of Bacteroides in the observed positive outcomes. IMPORTANCE To date, limited information is available comparing distinct Bacteroides compositions in the chicken gut microbial communities, particularly in the context of microbial functional capacities and host responses. This study showed that possessing a microbiome with elevated Bacteroides in early life may confer beneficial effects to the chicken host, particularly in improving SCFA production and gut health. This study is among the first metagenomic studies focusing on the early life chicken gut microbiota structure, microbial functionality, and host immune responses. We believe that it will offer insights to future studies on broiler gut microbial population and their effects on host health.

Minimum phosphorus requirements for laying hen feed formulations.

The objective of this contribution was to summarize from scientific literature the optimal concentration of nonphytate phosphorus (NPP) in feed for laying hens. The considered studies were one meta-analysis from 2012 and original studies published since then. Dietary treatments in the studies included variation in supplementation with mineral P sources and phytase. The studies investigated different periods of production and varied in duration but data were insufficient to analyze such factors in a systematic way. No study showed a positive effect on performance and eggshell when the NPP concentration was increased above 2.2 g NPP/kg of feed without the use of phytase. At such level, no consistent impairment of various bone quality traits were found but only few studies on bone quality traits were published. Overall, the data suggested that not more than 2.2 g NPP/kg of feed is needed for laying hens in different stages of production. This value can be reduced when phytase is added to the feed. Such reduction may differ depending on factors such as phytate content of the feed and phytase dosage. However, data are insufficient for calculating precise values of reduction. While phytate degradation in laying hens was markedly increased by phytase supplementation in several studies, effects of phytase supplementation on performance and bone traits in laying hens were less conclusive probably because the hens were supplied more than their NPP requirement. Transition to a system based on digestible P for laying hens similar to broiler chickens may support more precise P nutrition and more sustainable egg production in the future.

Early-life ß-glucan exposure enhances disease resilience of broiler chickens to a natural Clostridium perfringens infection.

Necrotic enteritis (NE) is an economically important disease in poultry. Colonization by the opportunistic pathogen C. perfringens occurs early after hatch and induces host immune tolerance, which allows it to persist as part of the bird's commensal microflora. ß-glucan, a yeast cell wall component, is well characterized for its immunomodulatory capacity, and is a strong driver of innate immune memory. In this study, we assessed the effectiveness of ß-glucan to reduce severity of NE, when co-administered with heat-killed C. perfringens via intra-abdominal route at day 1 of age. We found that this early-life exposure in the presence of ß-glucan did not reduce intestinal C. perfringens loads or lesion severity during a subsequent NE outbreak. However, it improved ileal morphology, prevented liver and spleen weight decline, and preserved feed efficiency in challenged birds. Molecular analyses revealed metabolic changes consistent with innate immune memory. Together, our results suggest that ß-glucan can reduce the negative impacts of NE by influencing the context in which C. perfringens is first encountered.

A Poultry Subclinical Necrotic Enteritis Disease Model Based on Natural Clostridium perfringens Uptake.

Necrotic enteritis (NE) in poultry is an opportunistic infection caused by Clostridium perfringens. Well-known as a multifactorial disease, NE development is under the influence of a wide range of environmental risk factors that promote the proliferation of pathogenic C. perfringens at the expense of nonpathogenic strains. Current in vivo NE challenge models typically incorporate pre-exposure to disease risk factors, in combination with exogenous C. perfringens inoculation. Our goal was to enhance current models using a natural uptake of C. perfringens from the barn environment to produce a subclinical infection. We incorporated access to litter, coccidial exposure (either 10× or 15× of the manufacturer-recommended Coccivac B52 Eimeria vaccine challenge; provided unspecified doses of E. acervulina, E. mivati, E. tenella, and two strains of E. maxima), feed composition, and feed withdrawal stress, and achieved the commonly observed NE infection peak at 3 weeks post-hatch. NE severity was evaluated based on gut lesion pathology, clinical signs, and mortality rate. Under cage-reared conditions, 15× coccidial vaccine-challenged birds showed overall NE lesion prevalence that was 8-fold higher than 10× coccidial vaccine-challenged birds. NE-associated mortality was observed only in a floor-reared flock after a 15× coccidial vaccine challenge.

The Use of Disinfectant in Barn Cleaning Alters Microbial Composition and Increases Carriage of Campylobacter jejuni in Broiler Chickens.

To maintain food safety and flock health in broiler chicken production, biosecurity approaches to keep chicken barns free of pathogens are important. Canadian broiler chicken producers must deep clean their barns with chemical disinfectants at least once annually (full disinfection [FD]) and may wash with water (water wash [WW]) throughout the year. However, many producers use FD after each flock, assuming a greater efficacy of more stringent cleaning protocols, although little information is known regarding how these two cleaning practices affect pathogen population and gut microbiota. In the present study, a crossover experiment over four production cycles was conducted in seven commercial chicken barns to compare WW and FD. We evaluated the effects of barn cleaning methods on commercial broiler performance, cecal microbiota composition, Campylobacter and Salmonella occurrence, and Campylobacter jejuni and Clostridium perfringens abundance, as well as on short-chain fatty acid (SCFA) concentrations in the month-old broiler gut. The 30-day body weight and mortality rate were not affected by the barn cleaning methods. The WW resulted in a modest but significant effect on the structure of broiler cecal microbiota (weighted-UniFrac; adonis P = 0.05, and unweighted-UniFrac; adonis P = 0.01), with notable reductions in C. jejuni occurrence and abundance. In addition, the WW group had increased cecal acetate, butyrate, and total SCFA concentrations, which were negatively correlated with C. jejuni abundance. Our results suggest that WW may result in enhanced activity of the gut microbiota and reduced zoonotic transmission of C. jejuni in broiler production relative to FD in the absence of a disease challenge. IMPORTANCE We compared the effects of barn FD and WW methods on gut microbial community structures and pathogen prevalence of broiler chickens in a nonchallenging commercial production setting. The results revealed that barn cleaning methods had little impact on the 30-day body weight and mortality rate of broiler chickens. In addition, the FD treatment had a subtle but significant effect on the broiler cecal microbiota with increased abundances of Campylobacter and decreased SCFA concentrations, which would support the adoption of WW as a standard practice. Thus, compared to FD, WW can be beneficial to broiler chicken production by inhibiting zoonotic pathogen colonization in the chicken gut with reduced cost and labor of cleaning.

Intestinal nutrition: role of vitamins and biofactors and gaps of knowledge.

The role of the microbiota in the health of the host is complex and multifactorial. The microbiota both consumes nutrients in competition with the host, but also creates nutrients that can be used by other microbes, but also the host. However, the quantitative impact of the microbiota on nutrient supply and demand is not well understood in poultry. The gastrointestinal tract is one of the largest points of contact with the external environment, and the intestinal microbiome is the largest and most complex of any system. Although the intestinal microbiota has first access to consumed nutrients, including vitamins, and is potentially a major contributor to production of various vitamins, the quantification of these impacts remains very poorly understood in poultry. Based on the human literature, it is clear that vitamin deficiencies can have systemic effects on the regulation of many physiological systems, beyond the immediate, direct nutrient functions of the vitamins. The impact of excessive supplementation of vitamins on the microbiota is not well understood in any species. In the context of poultry nutrition, in which substantial dietary excesses of most vitamins are provided, this represents a knowledge gap. Given the paucity of studies investigating the vitamin requirements of modern, high-producing poultry, the limited understanding of vitamin nutrition (supply and utilization) by the microbiome, and the potential impacts on the microbiome of the move away from dietary growth-promoting antibiotic use, more research in this area is required. The microbiota also contributes a vast array of other metabolites involved in intramicrobiota communication, symbiosis and competition that can also have an impact on the host. Myo-inositol and butyrate are briefly discussed as examples of biofactors produced by the microbiota as mediators of intestinal health.

Plasma metabolomic profiling reveals potential onset of lay biomarkers in broiler breeders.

Changes in the metabolic fingerprint of plasma during the onset of lay in broiler breeders were investigated. We used metabolomics to identify biomarkers of sexual maturity and to provide a comprehensive understanding of breeder metabolome during the pullet to hen transition period. A total of 36 pullets were used, in which 30 pullets were randomly assigned to one of 10 unique growth trajectories and 6 birds were assigned to an unrestricted group. The growth trajectories were designed using a 3-phase Gompertz growth model with 10 levels of BW gain in the prepubertal and pubertal growth phases ranging from the breeder-recommended target BW to 22.5% higher, in 2.5% increments. The BW trajectories were applied to each individual bird using a precision feeding (PF) system, which collected BW and feed intake data for each individual bird. The birds were classified based on age at first egg (AFE), and 12 pullets were chosen from the lower and upper AFE extremes (early and late onset of lay) at 18, 20, 22, 24, and 26 wk of age to run repeated blood plasma metabolomic assays. The metabolomic profile data were collected using a direct-injection liquid chromatography-tandem mass spectrometry and steroid assays. Univariate analysis identified 87 differential metabolites between the early- and late-onset of lay groups at 24 wk of age and 104 differential metabolites between the pullet and hen groups. Further investigation of differential metabolites showed 15 potential biomarkers for pullet to hen transition by analyzing the receiver operating characteristic (ROC) curve, mainly consisting of carnitine and choline metabolites. Differential metabolites during the pullet to hen transition were mainly associated with lipid, energy, and amino acid metabolism pathways, which gave clues to the physiological and metabolic shifts resulting from sexual maturation. At 24 wk of age, the main pathways involved in differentiation of the early- and late-onset of lay groups were related to lipid and amino acid metabolism. These metabolites could be involved in biosynthesis of egg yolk precursors in the liver.

Architecture of broiler breeder energy partitioning models.

A robust model that estimates the ME intake over broiler breeder lifetime is essential for formulating diets with optimum nutrient levels. The experiment was conducted as a randomized controlled trial with 40 Ross 708 broiler breeder pullets reared on 1 of 10 target growth trajectories, which were designed with 2 levels of cumulative BW gain in prepubertal growth phase and 5 levels of timing of growth around puberty. This study investigated the effect of growth pattern on energy efficiency of birds and tested the effects of dividing data into daily, 4-d, weekly, 2-wk, and 3-wk periods and the inclusion of random terms associated with individual maintenance ME and ADG requirements, and age on ME partitioning model fit and predictive performance. Model [I] was: MEId = a × BWb + c × ADGp + d × ADGn + e × EM + ε, where MEId was daily ME intake (kcal/d); BW in kg; ADGp was positive ADG; ADGn was negative ADG (g/d); EM was egg mass (g/d); ε was the model residual. Models [II to IV] were nonlinear mixed models based on the model [I] with inclusion of a random term for individual maintenance requirement, age, and ADG, respectively. Model [II] - 3 wk was chosen as the most parsimonious based on lower autocorrelation bias, closer fit of the estimates to the actual data (lower model MSE and closer R2 to 1), and greater predictive performance among the models. Estimated ME partitioned to maintenance in model [II] - 3 wk was 100.47 ± 7.43 kcal/kg0.56, and the ME requirement for ADGp, ADGn, and EM were 3.49 ± 0.37; 3.16 ± 3.91; and 2.96 ± 0.13 kcal/g, respectively. Standard treatment had lower residual heat production (RHP; -0.68 kcal/kg BW0.56) than high early growth treatment (0.79 kcal/kg BW0.56), indicating greater efficiency in utilizing the ME consumed. Including random term associated with individual maintenance ME in a 3-wk chunk size provided a robust, biologically sound life-time energy partitioning model for breeders.

Informal nutrition symposium: leveraging the microbiome (and the metabolome) for poultry production.

Knowledge of gut microbiology of poultry has advanced from a limited ability to culture relatively few microbial species, to attempting to understand the complex interactions between the bird and its microbiome. The Informal Nutrition Symposium 2021 was intended to help poultry scientists to make sense of the implications of the vast amounts of information being generated by researchers. This paper represents a compilation of the talks given at the symposium by leading international researchers in this field. The symposium began with an overview of the historical developments in the field of intestinal microbiology and microbiome research in poultry. Next, the systemic effects of the microbiome on health in the context of the interplay between the intestinal microbiota and the immune system were presented. Because the microbiome and the host communicate and influence each other, the novel field of kinomics (the study of protein phosphorylation) as used in the study of the poultry microbiome was discussed. Protein phosphorylation is a rapid response to the complex of signals among the microbiome, intestinal lumen metabolites, and the host. Then, a description of why an understanding of the role of microbial endocrinology in poultry production can lead to new understanding of the mechanisms by which the gut microbiota and the host can interact in defined mechanisms that ultimately determine health, pathogenesis of infectious disease, and behavior was given. Finally, a view forward was presented underscoring the importance of understanding mechanisms in microbiomes in other organ systems and other species. Additionally, the importance of the development of new -omics platforms and data management tools to more completely understand host microbiomes was stressed.

Effects of phytase supplementation on eggshell and bone quality, and phosphorus and calcium digestibility in laying hens from 25 to 37 wk of age.

Effects of dietary available phosphorus (aP) and Ca levels and an Escherichia coli 6-phytase supplementation were studied in Lohmann LSL-Lite hens from 25 to 37 wk of age. Eighty-four hens were used in a completely randomized design with 7 treatments. The treatments were a positive control (PC) diet with 0.45% aP, 3.70% Ca, and 0.16% Na from 25 to 28 wk and 0.38% aP, 3.73% Ca, and 0.15% Na from 29 to 37 wk; a negative control (NC) diet, similar to the PC diet, with 0.22% aP, 3.00% Ca, and 0.13% Na from 25 to 28 wk and 0.19% aP, 3.02% Ca, and 0.13% Na from 29 to 37 wk; the NC diets supplemented with phytase at 150 (NC + 150), 300 (NC + 300), 600 (NC + 600), or 1,200 (NC + 1,200) phytase unit (FTU)/kg; and the PC diet supplemented with phytase at 1,200 (PC + 1,200) FTU/kg. Hen performance, eggshell, and bone quality were measured on a 4-wk basis. Bone breaking strength and ash and apparent ileal digestibility (AID) of P and Ca were determined at 37 wk. One- and 2-way ANOVA were conducted, and Tukey's range test was used to compare multiple means where P ≤ 0.05. No differences in hen performance, eggshell quality, bone breaking strength, bone ash, and P digestibility were observed between the PC and the NC treatments. The NC hens had lower cortical (P < 0.001) and trabecular + medullary bone mineral density (P = 0.004) and total bone mineral content (P < 0.001) than the PC hens. The PC + 1,200 increased cortical bone mineral density (P < 0.001). The reductions of aP and Ca in the NC diet were not deficient for performance but had a minor impact on bone mineralization. The NC + 600 and NC + 1,200 increased AID of P (P = 0.024), and all phytase treatments except the NC + 150 increased AID of Ca (P = 0.010) compared with the NC diet.