The objective of this study was to assess oregano essential oil (OEO) as a pre-harvest intervention to reduce Salmonella enterica serovar Enteritidis (SE) colonization in broilers. Two identical floor-pen trials were conducted with a total of 200 Ross 708 straight-run chicks (0 to 43 days). Each trial included 2 pens (50 chickens/pen). All chickens received a common basal corn-soybean meal (C-SBM) diet from 0 to 4 days-of-age and followed a phase-feeding program consisting of starter, grower, and finisher diets to match nutritional requirements. At day 4, pens were assigned to either the basal corn-SBM diet without OEO (control) or the same diet supplemented with OEO (300 g/ton in the feed from 4 to 43 days and 6 oz/gal of stock solution from 28 to 43 days via the water source; Ecodiar®, Nutrinae LLC). At 7 days, chickens were challenged via oral gavage with SE (1 × 10⁵-10⁶ CFU/0.5 mL per chick). At day 43, one cecal pouch per bird was collected for SE enrichment to determine prevalence, and cecal content was analyzed for SE load using standard microbiological procedures. Data were analyzed using Student's t-test, with significance declared at P < 0.05. OEO supplementation reduced SE prevalence from 49% in the control to 34% (P = 0.03). Cecal SE counts were also reduced by approximately 1 log₁₀ with OEO compared with the control (2.7 × 10³ vs. 1.9 × 10² CFU/g; P = 0.02). Mechanistically, OEO supplementation significantly (P ≤ 0.05) reduced protein expression of key inflammatory cytokine (IL-21) and chemokine (MIP-1β) in the cecal tonsil compared to tissue from control-fed chickens. Therefore, OEO supplementation in the feed and water reduced SE prevalence and load in broilers, indicating greater resistance to colonization by reducing inflammation in important lymphoid tissue. This effect may potentially reduce flock-level transmission risk and supports OEO application as a pre-harvest strategy to reduce SE colonization in market-age broilers.

Removal of growth-promoting antibiotics (AGPs) is associated with the emergence of chronic, low-level gut inflammation induced by environmental stressors, especially diet components. However, there have been no reports demonstrating an increased susceptibility of birds to pathogenic bacteria such as Salmonella following the removal of AGPs from the animal feed. For the present studies, we hypothesize that a dietary-derived, low-grade chronic intestinal inflammation increases the susceptibility of young broiler chickens to intestinal colonization, extraintestinal infection, and the pathogenicity of Salmonella enterica serovar Enteritidis (SE). Two separate experiments were conducted separating a total of 250 birds/experiment into 2 feeding regimes: a normal basal corn-soy diet and a corn-soy + 30 % rice bran starting on the day of hatch through 28 days. Each diet contained 25 non-challenged control birds with the remaining 100 birds/diet divided into groups of 25 that were orally challenged with SE at either 1-, 7-, 14-, or 21-days post-hatch. At 28 days post-hatch, all birds were euthanized to evaluate SE intestinal colonization and organ invasion, and all birds and left-over feed were weighed to evaluate weight gain, feed intake, and feed conversion. The high NSP diet had a detrimental effect on the animals’ performance as evidenced by significant reduction in weight gain and feed intake and increased feed conversion. Birds fed the high NSP diet for at least 7 days were found to have significantly more detectable SE in both the ceca and in the liver compared to the control corn-soy fed birds. Lastly, there was a significant increase in SE recovery in birds fed the high NSP diet for at least 7 days. These results provide evidence that the diet-induced chronic gut inflammation created a more advantageous colonization environment for Salmonella. The consequence of low-grade chronic intestinal inflammation remains a major issue within the poultry industry as it is felt economically since it affects FCR and renders birds more susceptible to enteric pathogen colonization and invasion.

Salmonella is one of the leading bacterial foodborne pathogens and is commonly found in poultry. The objective of this study was to determine if feeding an oregano-based antibiotic alternative could reduce the burden of Salmonella in broilers. Day-of-hatch straight-run broiler chicks were provided a basal corn-soymeal diet. After four days on the basal diet, birds were assigned to a dietary treatment group (n = 50 chicks/group/experiment). The groups were control with no oregano feed additive or a diet supplemented with an oregano oil product (300 g/ton). At 7-days-of-age, chicks were challenged via oral gavage (0.5 mL) with Salmonella enterica serovar Enteritidis (1 × 104-5 colony forming units [cfu]/chick). Fourteen-days post-challenge (dpc), 50 birds from each group were euthanized. At necropsy, the liver and one cecal pouch were removed and enriched for S. Enteritidis and cecal content was analyzed for S. Enteritidis enumeration. The study was conducted twice. Data were analyzed to determine differences in S. Enteritidis recovered between the groups with P < 0.05 significant. No differences were observed in the percent positive liver samples in the controls compared to those on the oregano diet. There was a reduction (P = 0.003) in the positive ceca between controls (61 %) compared to chickens on the oregano diet (40 %). The S. Enteritidis recovered from cecal content was also reduced (P < 0.001) in chickens on the oregano diet (3.2 × 103 cfu/g) compared to controls (3.5 × 105 cfu/g). Cytokine (IL-1β, IL-18) and chemokine (CXCL8) mRNA expression was also 1.9-2.2-fold higher (P ≤ 0.04) in the cecal tonsil of chickens on the oregano diet compared to controls indicating immunomodulatory affect. Collectively, these data demonstrate addition of an oregano-based feed additive to a broiler diet reduces S. Enteritidis colonization and invasion in the ceca and may be an important pre-harvest intervention strategy to reduce the burden of this highly important foodborne pathogen.

The poultry cecum is considered the "gold standard" for detecting Salmonella in poultry because it has the largest concentration of Salmonella in the bird. The current study investigated alternative anatomical sampling locations for detecting Salmonella in preharvest turkeys. In this study, 36-day-old turkeys were challenged with a cocktail of Salmonella Heidelberg, S. Reading, and S. Infantis by oral gavage (N = 78). The birds were sampled on Day 0-, 6-, 9- and 12-days post-challenge. Across all serotypes, total Salmonella recovery from the different anatomical locations was significantly higher in samples taken from the cloaca, crop, vent feathers, and foot pad samples when compared to the cecal samples (P < 0.05). Similarly, Salmonella results for individual serotypes demonstrated a higher recovery of S. Reading and S. Infantis within the cloaca, and vent feathers samples compared to the ceca. Salmonella Heidelberg recovery was significantly higher in the joints, and bone marrow when compared to the cecal samples. Study results demonstrate that Salmonella can spread systemically in turkeys without demonstrated morbidity and can be recovered at other anatomical locations at higher rates than found in ceca. In addition, Salmonella serotypes translocated differently within the bird and can be recovered at different anatomical locations.

Campylobacter spp., and especially C. jejuni, are leading causes of bacterial-derived foodborne illness in humans. Poultry are known reservoirs, and in many instances, Campylobacter are thought to be commensal organisms harmlessly residing in the gastrointestinal tract of poultry; therefore, human illness could result from mishandling or consuming raw or undercooked chicken products contaminated with Campylobacter. The objective of this study was to prime the chickens with a low dose of C. jejuni at a young age followed by a higher dose later and monitor changes to key inflammatory and regulatory cytokines and chemokines. Broiler chicks (2-days-of-age) were administered 1 × 103 colony forming units (cfu)/mL C. jejuni, (0.5 mL, orally) followed by a second dosage of 1 × 107 cfu/mL (1 mL, orally) at 28-d-of-age. At 35-d-of-age, the chickens were necropsied and the cecal tonsil was collected. The study was conducted twice. Cytokine (IL1β, IL6, IL10, IFNγ, TGFβ4) and chemokine (CXCL8) mRNA expression was measured in the cecal tonsil. Expression of IL1β, IL10, TGFβ4, and CXCL8 mRNA were significantly higher (2.6 - 5.8-fold; P ≤ 0.04) in the cecal tonsils of the chickens subjected to the two doses of C. jejuni. The C. jejuni challenges had no effect on the mRNA expression levels of IL6 and IFNγ (P > 0.05). These data indicate repeated exposure to C. jejuni stimulates a diverse and robust cytokine response in important lymphoid tissue indicating the host recognizes the C. jejuni as pathogenic and mounts a response yet a persistent infection is still established.

Neurochemicals, such as the catecholamines, serve as critical regulators controlling the immune response in the pathogenesis of inflammation. Overproduction of one of these neurochemicals, namely norepinephrine, has been previously shown to occur concomitant to the development of diet-induced gut inflammation. As a pro-inflammatory neuroimmune modulatory chemical, norepinephrine can dysregulate the immune response to inflammation. In order to ameliorate the increased production of norepinephrine, we have utilized a microbial endocrinology-based approach that utilizes an Enterococcus faecium probiotic to produce the anti-inflammatory neurochemical dopamine. As shown in the present study, the feed incorporated E. faecium probiotic converts the precursor L-dopa to dopamine with high efficiency to produce significant amounts of dopamine within the gastrointestinal tract. In replicate broiler feeding trials utilizing a high non-starch polysaccharide (NSP) inflammation-inducing diet in combination or not with L-dopa alone or in combination with the dopamine-producing E. faecium probiotic, the NSP diet induced a large increase in norepinephrine concomitant to the development of inflammation that was abrogated in the groups fed the L-dopa precursor in combination with the dopamine-producing E. faecium. Less, though still significant, amelioration of the norepinephrine increase was achieved in the group only fed the L-dopa precursor. The present report represents the first use of a dopamine-producing probiotic to mechanistically influence the production of another neurochemical that is intimately involved in the pathophysiology of gut inflammation. As such, this study demonstrates that the use of a Microbial Endocrinology-designed probiotic can serve as a means by which to prevent and/or control the development of gut inflammation in poultry.

Maintenance of intestinal health is critical to successful poultry production and one of the goals of the poultry production industry. For decades the poultry industry has relied upon the inclusion of antibiotic growth promoters (AGP) to achieve this goal and improve growth performance. With the removal of AGPs, the emergence of chronic, low-level gut inflammation has come to the forefront of concern in the poultry industry with the diet being the primary source of inflammatory triggers. We have developed a dietary model of low-grade, chronic intestinal inflammation in broilers that employs feeding a high nonstarch polysaccharides (NSP) diet composed of 30% rice bran to study the effects of this inflammation on bird performance and physiology. For the present studies, we hypothesize that the low-grade chronic inflammation causes neurons in the intestinal enteric nervous system to secrete neurochemicals that activate immune cells that drive the inflammation and negatively affect bird performance. To test our hypothesis, 1-day-old broiler chickens were weighed and divided into 2 dietary regimes: a control corn-soybean diet and a group fed a high NSP diet (30% rice bran). At 7-, 14-, 21-, and 28-d posthatch (PH), birds were weighed, fecal material collected, and 5 birds were sacrificed and sections of duodenal and cecal tissues excised, and duodenal and cecal contents collected for ultra-high performance liquid chromatography analyses (UHPLC). UHPLC revealed 1000s-fold increase in the concentration of norepinephrine (NOR) in birds fed the high NSP diet compared to the control fed birds. Further, the fecal concentrations of NOR were also found to be significantly elevated in the birds on the NSP diet throughout all time points. There were no differences in weight gain nor feed conversion from 1 to 14 d PH, but birds fed the high NSP diet had significantly reduced weight gain and feed conversion from 14 to 28 d PH. The results revealed that a dietary-induced low-grade chronic inflammatory response increased NOR production in the gut which negatively affected bird performance. This study suggests that neuroimmune pathways may serve as a mechanistic target for the development of new interventions to decrease the incidence of chronic inflammation and thereby benefit performance.

White Leghorn chickens from a common founder population have been divergently selected for high (HAS) or low (LAS) antibody responses to sheep red blood cells (SRBC) for 49 generations resulting in two diverse lines for this trait. Much has been studied in these two lines; however, the impact of these selection pressures on cytokine and chemokine expression is not fully understood. The purpose of this study is to determine if selection for antibody response to SRBC impacts cytokine and chemokine expression in peripheral blood leukocytes (PBL) and spleen from HAS and LAS chickens. Total RNA was isolated from PBL and spleen after which mRNA expression of cytokines (IL4, IL6, IL10, TGF-β4) and chemokines (CXCL8, CCL4) were determined by quantitative real-time RT-PCR (qRT-PCR). The data were analyzed using Student's t test comparing HAS and LAS (P<0.05) and are reported as corrected 40-CT. PBL and spleen samples were analyzed separately. With respect to PBL, expression of IL6 was higher (P<0.05) in PBL isolated from LAS chickens compared to those from the HAS line whereas there were no differences (P>0.05) in IL4, IL10, CXCL8, CCL4, or TGF-β4. The cytokine and chemokine mRNA expression profiles were different in the spleen between the two lines. IL4 and CXCL8 expression were higher (P<0.05) in spleen samples from HAS chickens than LAS. The expression of IL6, IL10, CCL4, or TGF-β4 in the spleens did not differ (P>0.05) between the lines. The data indicate that selection for specific antibody responses to SRBC impacts the cytokine and chemokine expression profile in PBL and spleens but in different ways in HAS and LAS. These studies provide insight into the influence that selection pressures for antibody responses have on different immune response components, specifically cytokines and chemokines typically involved in the innate response.

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.

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.

Addition of vitamins and antioxidants has been long associated with increased immunity and are commonly used in the poultry industry; however, less is known regarding their use in broiler breeder hens. The objective of this study was to determine if feeding a complex of protected biofactors and antioxidants composed of vitamins and fermentation extracts to broiler breeder hens conferred resistance against Salmonella enterica serovar Enteritidis (S. Enteritidis) in the progeny chicks. Three-day-old chicks from control- and supplement-fed hens were challenged with S. Enteritidis and necropsied 4- and 11-days postchallenge (dpc) to determine if there were differences in invasion and colonization. Serum and jejunum were evaluated for various cytokine and chemokine production. Fewer (P = 0.002) chicks from supplement-fed hens had detectable S. Enteritidis in the ceca (32.6%) compared to chicks from control-fed hens (64%). By 11 dpc, significantly (P < 0.001) fewer chicks from supplement-fed hens were positive for S. Enteritidis (liver [36%]; ceca [16%]) compared to chicks from the control hens (liver [76%]; ceca [76%]). The recoverable S. Enteritidis in the cecal content was also lower (P = 0.01) at 11 dpc. In additional to the differences in invasion and colonization, cytokine and chemokine production were distinct between the 2 groups of chicks. Chicks from supplement-fed hens had increased production of IL-16, IL-6, MIP-3α, and RANTES in the jejunum while IL-16 and MIP-1β were higher in the serum of chicks from the control-fed hens. By 11 dpc, production of IFN-γ was decreased in the jejunum of chicks from supplement-fed hens. Collectively, these data demonstrate adding a protected complex of biofactors and antioxidants to the diet of broiler breeder hens offers a measure of transgenerational protection to the progeny against S. Enteritidis infection and reduces colonization that is mediated, in part, by a robust and distinct cytokine and chemokine response locally at the intestine and systemically in the blood.

Using a previously characterized and described abdominal model to define the avian immune response to Salmonella intra-abdominal challenge in chickens, we have adapted this technique for the study of chickens' immune response to a Campylobacter intra-abdominal challenge. The intra-abdominal Campylobacter infection model facilitates the characterization of peripheral blood leukocyte dynamics and abdominal cell infiltrates. Day-of-hatch Leghorn chickens were injected intra-abdominally (IA) with Campylobacter jejuni [(CJ)1 × 108 colony-forming units (CFUs)]. Changes in peripheral blood leukocyte numbers and abdominal cell infiltrates were monitored at 0, 4, 8, and 24 h post-injection. Peripheral blood leukocyte numbers were also determined for 2 h post-injection. For mortality studies, birds were injected intra-abdominally with 1 × 108 CFUs CJ and mortalities were recorded for 72 h post-injection. In the peripheral blood of CJ-injected chicks, total white blood cell (WBC) numbers began increasing by 2 h post-injection, peaking at 4 h post-injection with the predominant cell type being polymorphonuclear leukocytes (heterophils). Total WBCs declined after 8 h and this decline continued at 24 h, with total WBC numbers approaching control values. The injection of CJ into the abdominal cavity caused a rapid rise in abdominal cell infiltrates with the predominant infiltrating leukocytes being heterophils. Peak abdominal heterophil infiltrates were observed at 8 h post-injection, declining only slightly by 24 h post-injection. Mortality in the CJ challenge groups reached 37%. Mortality in the Salmonella enteritidis positive control groups were greater than 50%. The data suggest that Campylobacter infection does stimulate the innate immune response in chickens when administered IA, however, the immune response and infection is not characterized with the high levels of mortality observed with a Salmonella infection. These data provide a basis for a more definitive characterization of chickens' immune response to Campylobacter and a model to evaluate intervention strategies to prevent the infection and colonization of poultry.

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.

Poultry is a major source of human foodborne illness caused by broad host range Salmonella serovars (paratyphoid), and developing cost-effective, pre-harvest interventions to reduce these pathogens would be valuable to the industry and consumer. Host responses to infectious agents are often regulated through phosphorylation. However, proteomic mechanisms of Salmonella acute infection biology and host responses to the bacteria have been limited concentrating predominately on the genomic responses of the host to infection. Our recent development of chicken-specific peptide arrays for kinome analysis of host phosphorylation-based cellular signaling responses provided us with the opportunity to develop a more detailed understanding of the early (4-24 h post-infection) host-pathogen interactions during the initial colonization of the cecum by Salmonella. Using the chicken-specific kinomic immune peptide array, biological pathway analysis showed infection with S. Enteritidis increased signaling related to the innate immune response, relative to the non-infected control ceca. Notably, the acute innate immune signaling pathways were characterized by increased peptide phosphorylation (activation) of the Toll-like receptor and NOD-like receptor signaling pathways, the activation of the chemokine signaling pathway, and the activation of the apoptosis signaling pathways. In addition, Salmonella infection induced a dramatic alteration in the phosphorylation events of the JAK-STAT signaling pathway. Lastly, there is also significant activation of the T cell receptor signaling pathway demonstrating the initiation of the acquired immune response to Salmonella infection. Based on the individual phosphorylation events altered by the early Salmonella infection of the cecum, certain conclusions can be drawn: (1) Salmonella was recognized by both TLR and NOD receptors that initiated the innate immune response; (2) activation of the PPRs induced the production of chemokines CXCLi2 (IL-8) and cytokines IL-2, IL-6, IFN-α, and IFN-γ; (3) Salmonella infection targeted the JAK-STAT pathway as a means of evading the host response by targeting the dephosphorylation of JAK1 and TYK2 and STAT1,2,3,4, and 6; (4) apoptosis appears to be a host defense mechanism where the infection with Salmonella induced both the intrinsic and extrinsic apoptotic pathways; and (5) the T cell receptor signaling pathway activates the AP-1 and NF-κB transcription factor cascades, but not NFAT.

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 immunometabolism 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 signifi-cant (P < 0.05) in all 3 pathways: CHUK, MAP3K14, MAP3K7, and NFKB1 indicating their importance. Additionally, there were changes to IL6, IL10, and IFN-g mRNA expression in tissue between control-and supplement-fed chickens. In conclusion, the addition of a micro encapsulated blend of organic acids and botanicals to a broiler diet reduced the clinical signs of NE that was mediated by specific immune-related pathways.

The chicken gastrointestinal tract (GIT) has a complex, biodiverse microbial community of ~ 9 million bacterial genes plus archaea and fungi that links the host diet to its health. This microbial population contributes to host physiology through metabolite signaling while also providing local and systemic nutrients to multiple organ systems. In a homeostatic state, the host-microbial interaction is symbiotic; however, physiological issues are associated with dysregulated microbiota. Manipulating the microbiota is a therapeutic option, and the concept of adding beneficial bacteria to the intestine has led to probiotic and prebiotic development. The gut microbiome is readily changeable by diet, antibiotics, pathogenic infections, and host- and environmental-dependent events. The intestine performs key roles of nutrient absorption, tolerance of beneficial microbiota, yet responding to undesirable microbes or microbial products and preventing translocation to sterile body compartments. During homeostasis, the immune system is actively preventing or modulating the response to known or innocuous antigens. Manipulating the microbiota through nutrition, modulating host immunity, preventing pathogen colonization, or improving intestinal barrier function has led to novel methods to prevent disease, but also resulted in improved body weight, feed conversion, and carcass yield in poultry. This review highlights the importance of adding different feed additives to the diets of poultry in order to manipulate and enhance health and productivity of flocks.

Clostridium perfringens (Cp) is a Gram-positive anaerobe that is one of the causative agents of necrotic enteritis (NE) in chickens, which leads to high mortality. Owing to the ban of administering antibiotics in feed to chickens, there has been an increase in the number of NE outbreaks all over the world, and the estimated loss is approximately 6 billion U.S. dollars. The best alternative method to control NE without antibiotics could be vaccination. In this study, we exposed three different strains of Cp to electron beam (eBeam) irradiation to inactivate them and then used them as a killed vaccine to control the colonization of Cp in broiler chickens. The vaccine was delivered to 18-day old embryos in ovo and the chickens were challenged with the respective vaccine strain at two different time points (early and late) to test the protective efficacy of the vaccine. The results indicate that an effective eBeam dose of 10 kGy inactivated all three strains of Cp, did not affect the cell membrane or epitopes, induced significant levels of IgY in the vaccinated birds, and further reduced the colonization of Cp strains significantly (p < 0.0001) in late challenge (JGS4064: 4 out of 10; JGS1473: 0 out of 10; JGS4104: 3 out of 10). Further studies are necessary to enhance the efficacy of the vaccine and to understand the mechanism of vaccine protection.

Background: Microencapsulated organic acids and botanicals have the potential to develop into important tools for the poultry industry. A blend of organic acids and botanicals (AviPlus®P) has previously shown to reduce Salmonella and Campylobacter in chickens; however, changes to the microbiota of the jejunum and ileum have not been evaluated. Microbiota diversity is linked to, but not correlated with, the efficacy of natural products; therefore, understanding the effects on the microbiota is necessary for evaluating their potential as an antibiotic alternative. Results: Ileal and jejunal segments from control and supplement-fed chickens (300 and 500g/metric ton [MT]) were subjected to alpha diversity analysis including Shannon’s diversity and Pielou’s Evenness. In both analytics, the diversity in the ileum was significantly decreased compared to the jejunum irrespective of treatment. Similarly, beta diversity metrics including Bray-Curtis dissimilarity index and Weighted Unifrac Distance Matrix, were significant (Q<0.05) for both tissue and treatments comparisons. Alpha and beta diversity analytics indicated compartmentalization effects between the ileum and jejunum. Additionally, analysis of communities in the microbiota (ANCOM) analysis showed Lactobacilliaceae predominated the total operational taxonomic units (OTU), with a stepwise increase from 53% in the no treatment control (NTC) to 56% in the 300g/MT and 67% in the 500g/MT group. Staphylococcaceae were 2% in NTC and 2 and 0% in 300 and 500g/MT groups. Enterobacteriaceae decreased in the 500g/MT (31%) and increased in the 300g/MT (37%) compared to the NTC (35%). Aerococcaceae was 0% for both doses and 7% in NTC. Ruminococcaceae were 0% in NTC and 2% and 1% in the 300 and 500g/MT. These changes in the microbial consortia were statistically (Q<0.05) associated with treatment groups in the jejunum that were not observed in the ileum. Least discriminant analysis effect size (LEfSE) indicated different changes directly corresponding to treatment. Enterobacteriaceae demonstrated a stepwise decrease (from NTC onward) while Clostridiaceae, were significantly increased in the 500g/MT compared to NTC and 300g/MT (P<0.05).Conclusion: The bioactive site for the microencapsulated blend of organic acids and botanicals was the jejunum, and dietary inclusion enhanced the GIT microbiota and may be a viable antibiotic alternative for the poultry industry.

During the first week after hatch, young chicks are vulnerable to pathogens as the immune system is not fully developed. The objectives of this study were to determine if supplementing the starter diet with a microencapsulated feed additive containing citric and sorbic acids, thymol, and vanillin affects in vitro functional activity of peripheral blood leukocytes (PBLs). Day-old chicks (n = 800) were assigned to either a control diet (0 g/metric ton [MT]) or a diet supplemented with 500 g/MT of the microencapsulated additive. At 4 D of age, peripheral blood was collected (100 birds per treatment), and heterophils and monocytes isolated (n = 4). Heterophils were assayed for the ability to undergo degranulation and production of an oxidative burst response while nitric oxide production was measured in monocytes. Select cytokine and chemokine mRNA expression levels were also determined. Statistical analysis was performed using Student t test comparing the supplemented diet to the control (P ≤ 0.05). Heterophils isolated from chicks fed the microencapsulated citric and sorbic acids, thymol, and vanillin had higher (P ≤ 0.05) levels of degranulation and oxidative burst responses than those isolated from chicks on the control diet. Heterophils from the supplemented chicks also had greater (P ≤ 0.05) expression of IL10, IL1β, and CXCL8 mRNA than those from control-fed chicks. Similarly, nitric oxide production was significantly (P ≤ 0.05) higher in monocytes isolated from birds fed the supplement. The cytokine and chemokine profile in monocytes from the supplement-fed chicks showed a significant (P ≤ 0.05) drop in IL10 mRNA expression while IL1β, IL4, and CXCL8 were unchanged. In conclusion, 4 D of supplementation with a microencapsulated blend made up of citric and sorbic acids, thymol, and vanillin enhanced the in vitro PBL functions of degranulation, oxidative burst, and nitric oxide production compared with the control diet. Collectively, the data suggest feeding broiler chicks a diet supplemented with a microencapsulated blend of citric and sorbic acids, thymol, and vanillin may prime key immune cells making them more functionally efficient and acts as an immune-modulator to boost the inefficient and undeveloped immune system of young chicks.

The use of natural products as feed additives in the poultry industry is increasing; however, most studies focus on performance and growth with little regard for determining mechanism. Our laboratory designed a chicken (Gallus gallus)-specific immunometabolic kinome peptide array. Using this tool to examine the active enzymes responsible for phosphorylation events (kinases) provides important information on host and cellular functions. The objective of this project was to determine if feeding a microencapsulated product comprised of a blend of organic acids and botanicals (AviPlus®P) impacts the intestinal kinome of broiler chickens (Gallus gallus). Day-of-hatch chicks were provided 0 or 500g/MT of the additive and jejunal and ileal segments collected for kinome analysis to determine the mode-of-action of the additive. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed by uploading the statistically significant peptides to the Search Tool for the Retrieval of Interacting Genes database. As a whole, GO and KEGG analysis showed similar activities in the ileum and jejunum. However, there were a small number of KEGG pathways that were only activated in either the ileum or jejunum, but not both. Analysis of the adipocytokine and PI3K-AKT signaling pathways showed differences between ileal and jejunal activity that were controlled, in part, by AKT3. Additionally, cytokine/chemokine evaluation showed the ileum had higher IL1β, IL6, IL10, TNFα, IFNγ, CXCL8, and CCL4 mRNA expression levels (P<0.05). As a whole, the data showed the addition of microencapsulated organic acids and botanicals to a broiler diet activated many of the same signaling pathways in the ileum and jejunum; however, distinctions were observed. Taken together, the findings of this study begin to define the mode-of-action that microencapsulated organic acids and botanicals have on two important intestinal segments responsible for nutrient digestion and absorption in chickens.