A microencapsulated feed additive containing organic acids and botanicals has a distinct effect on proliferative and metabolic related signaling in the jejunum and ileum of broiler chickens.

Well designed and formulated natural feed additives have the potential to provide many of the growth promoting and disease mitigating characteristics of in-feed antibiotics, particularly feed additives that elicit their effects on targeted areas of the gut. Here, we describe the mechanism of action of a microencapsulated feed additive containing organic acids and botanicals (AviPlus®P) on the jejunum and ileum of 15-day-old broiler-type chickens. Day-of-hatch chicks were provided ad libitum access to feed containing either 0 or 500 g/MT of the feed additive for the duration of the study. Fifteen days post-hatch, birds were humanely euthanized and necropsied. Jejunum and ileum tissue samples were collected and either flash frozen or stored in RNA-later as appropriate for downstream applications. Chicken-specific kinome peptide array analysis was conducted on the jejunum and ileum tissues, comparing the tissues from the treated birds to those from their respective controls. Detailed analysis of peptides representing individual kinase target sites revealed that in the ileum there was a broad increase in the signal transduction pathways centering on activation of HIF-1α, AMPK, mTOR, PI3K-Akt and NFκB. These signaling responses were largely decreased in the jejunum relative to control birds. Gene expression analysis agrees with the kinome data showing strong immune gene expression in the ileum and reduced expression in the jejunum. The microencapsulated blend of organic acids and botanicals elicit a more anti-inflammatory phenotype and reduced signaling in the jejunum while resulting in enhanced immunometabolic responses in the ileum.

M2 Polarization and Inhibition of Host Cell Glycolysis Contributes Intracellular Survival of Salmonella Strains in Chicken Macrophage HD-11 Cells.

Salmonella enterica is a group of facultative, gram-negative bacteria. Recently, new evidence indicated that Salmonella could reprogram the host metabolism to increase energy or metabolites available for intracellular replication. In this study, using a chicken-specific kinomic immunometabolism peptide array analysis, we found that infection by S. Enteritidis induced significant phosphorylation changes in many key proteins of the glycolytic pathway in chicken macrophage HD-11 cells, indicating a shift in glycolysis caused by Salmonella infection. Nitric oxide production and changes of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) represented by extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively, were measured in chicken macrophages infected with three Salmonella strains (S. Enteritidis, S. Heidelberg, and S. Senftenberg). The infection reduced glycolysis and enhanced OXPHOS in chicken macrophages as indicated by changes of ECAR and OCR. Salmonella strains differentially affected macrophage polarization and glycolysis. Among three strains tested, S. Enteritidis was most effective in downregulating glycolysis and promoting M2 polarization as measured by ECAR, ORC, and NO production; while S. Senftenberg did not alter glycolysis and may promote M1 polarization. Our results suggested that downregulation of host cell glycolysis and increase of M2 polarization of macrophages may contribute to increased intracellular survival of S. Enteritidis.

Impact of a Blend of Microencapsulated Organic Acids and Botanicals on the Microbiome of Commercial Broiler Breeders under Clinical Necrotic Enteritis.

Previously, the supplementation of a microencapsulated blend of organic acids and botanicals improved the health and performance of broiler breeders under non-challenged conditions. This study aimed to determine if the microencapsulated blend impacted dysbiosis and necrotic enteritis (NE) in broiler breeders. Day-of-hatch chicks were assigned to non-challenge and challenge groups, provided a basal diet supplemented with 0 or 500 g/MT of the blend, and subjected to a laboratory model for NE. On d 20-21, jejunum/ileum content were collected for microbiome sequencing (n = 10; V4 region of 16S rRNA gene). The experiment was repeated (n = 3), and data were analyzed in QIIME2 and R. Alpha and beta diversity, core microbiome, and compositional differences were determined (significance at p ≤ 0.05; Q ≤ 0.05). There was no difference between richness and evenness of those fed diets containing 0 and 500 g/MT microencapsulated blend, but differences were seen between the non-challenged and challenged groups. Beta diversity of the 0 and 500 g/MT non-challenged groups differed, but no differences existed between the NE-challenged groups. The core microbiome of those fed 500 g/MT similarly consisted of Lactobacillus and Clostridiaceae. Furthermore, challenged birds fed diets containing 500 g/MT had a higher abundance of significantly different phyla, namely, Actinobacteriota, Bacteroidota, and Verrucomicrobiota, than the 0 g/MT challenged group. Dietary supplementation of a microencapsulated blend shifted the microbiome by supporting beneficial and core taxa.

Broiler chickens with 1950s genetics display a stable immune profile as measured by Kinome, mRNA expression, and metabolism when stimulated early in life with CpG.

Significant changes in growth potential and feed conversion have been bred into the modern broiler chicken for well over 60 yr. These metabolic changes have had significant effects on the immune performance as well. To better understand these genetic differences in immunometabolism we studied the immune response of the modern broiler and the Athens Canadian Random Bred (ACRB) heritage broiler strain. We injected newly hatched modern broiler and ACRB chicks intraabdominally with CpG oligonucleotide, an immunostimulatory synthetic oligonucleotide. We conducted species-specific kinome array analysis and gene expression analysis on jejunum and cecal tonsil tissue. We also performed metabolic analysis of blood cells. In the modern birds, there is an initial inflammatory response to the injection at d 3 post-hatch with activation of PI3K-Akt, JAK-STAT, and NF-κB signaling, and IL-1ß and IL-6 mRNA expression. By d 15 post-hatch this response changed to deactivation and downregulation of these immune responses in modern but not heritage broilers. Metabolic analysis showed an increase in glycolysis in peripheral blood mononuclear cells from modern birds given CpG, but no difference in ACRB. These results show that the ACRB birds may have a less inflammatory and more stable immune profile in response to immune stimulation than the modern broilers, possibly resulting in a more disease resistant phenotype overall.

A blend of microencapsulated organic acids and botanicals reduces necrotic enteritis via specific signaling pathways in broilers.

Necrotic enteritis (NE) is a devastating disease that has seen a resurgence of cases following the removal of antibiotics from feed resulting in financial loss and significant animal health concerns across the poultry industry. The objective was to evaluate the efficacy of a microencapsulated blend of organic (25% citric and 16.7% sorbic) acids and botanicals (1.7% thymol and 1% vanillin [AviPlusP]) to reduce clinical NE and determine the signaling pathways associated with any changes. Day-of-hatch by-product broiler breeder chicks were randomly assigned to a control (0) or supplemented (500 g/MT) diet (n = 23-26) and evaluated in a NE challenge model (n = 3). Birds were administered 2X cocci vaccine on d 14 and challenged with a cocktail of Clostridium perfringens strains (107) on d 17 to 19. On d 20 to 21 birds were weighed, euthanized, and scored for NE lesions. Jejunal tissue was collected for kinome analysis using an immuno-metabolism peptide array (n = 5; 15/treatment) to compare tissue from supplement-fed birds to controls. Mortality and weight were analyzed using Student's t test and lesion scores analyzed using F-test two-sample for variances (P < 0.05). The kinome data was analyzed using PIIKA2 peptide array analysis software and fold-change between control and treated groups determined. Mortality in the supplemented group was 47.4% and 70.7% in controls (P = 0.004). Lesions scores were lower (P = 0.006) in supplemented birds (2.47) compared to controls (3.3). Supplement-fed birds tended (P = 0.19) to be heavier (848.6 g) than controls (796.2 g). Kinome analysis showed T cell receptor, TNF and NF-kB signaling pathways contributed to the improvements seen in the supplement-fed birds. The following peptides were significant (P < 0.05) in all 3 pathways: CHUK, MAP3K14, MAP3K7, and NFKB1 indicating their importance. Additionally, there were changes to IL6, IL10, and IFN- γ mRNA expression in tissue between control- and supplement-fed chickens. In conclusion, the addition of a microencapsulated blend of organic acids and botanicals to a broiler diet reduced the clinical signs of NE that was mediated by specific immune-related pathways.

Feeding of yeast cell wall extracts during a necrotic enteritis challenge enhances cell growth, survival and immune signaling in the jejunum of broiler chickens.

Necrotic enteritis (NE) is one of the most common and costly diseases in the modern broiler industry, having an estimated economic impact of $6 billion dollars annually. Increasing incidents of NE have resulted from restrictions on the use of antibiotic feed additives throughout the broiler industry. As such, finding effective antibiotic alternatives has become a priority. In this study, an experimental model of NE was used, comprising a commercial infectious bursal disease virus vaccine and Clostridium perfringens (C. perfringens) inoculation. Yeast cells wall (YCW) components, ß-glucan (BG), and mannoproteins (MPTs) were evaluated for their effects on disease development. Chicken-specific immunometabolic kinome peptide arrays were used to measure differential phosphorylation between control (uninfected), challenged (infected), and challenged and treated birds in duodenal, jejunal, and ileal tissues. Treatment groups included crude YCW preparation, BG, MPT, or BG+MPT as feed additives. Data analysis revealed kinome profiles cluster predominantly by tissue, with duodenum showing the greatest relative signaling and jejunum showing the greatest response to treatment. BG, MPT, and BG+MPT cluster together, separate from controls and challenge birds in each tissue. Changes in signaling resulting from the treatments were observed in cell growth and survival responses as well as immune responses. None of the treatments of disease challenge returned the profiles to control-like. This is attributable to immune modulation and metabolic effects of the treatments generating distinct profiles from control. Importantly, all the treatments are distinct from the challenge group despite being challenged themselves. Only BG+MPT treatment had a significant effect on bird weight gain compared with the NE challenge group, and this treatment had the greatest impact on gut tissue signaling in all segments. The signaling changes elicited by BG+MPT during an NE challenge were increased cell growth and survival signaling, reducing cell death, apoptosis and innate inflammatory responses, and generating compensatory signaling to reduce disease severity.

Administration of a Postbiotic Causes Immunomodulatory Responses in Broiler Gut and Reduces Disease Pathogenesis Following Challenge.

With the reemergence of poultry diseases such as necrotic enteritis following the restriction of in-feed antibiotics, the search for antibiotic alternatives has become critically important. Postbiotics are non-viable bacterial products or metabolic byproducts from probiotic microorganisms that have positive effects on the host or microbiota. These are a promising alternative to antibiotics. Here, we describe the mechanism of action of a postbiotic in the context of a Clostridium perfringens (C. perfringens) challenge model. By using performance measurements and a peptide array kinome analysis, we describe the kinotypes and signal transduction changes elicited by the postbiotic with and without C. perfringens challenge. The postbiotic improves lesion scores, C. perfringens counts and mortality compared to challenge groups without the postbiotic, and it improves weight gain in the most severely challenged birds. The postbiotic predominantly affects the innate immune response and appears immunomodulatory. In the context of infection, it reduces the proinflammatory responses and generates a homeostatic-like response. This postbiotic is a viable alternative to antibiotics to improve poultry health in the context of C. perfringens pathogen challenge.