How did antibiotic growth promoters increase growth and feed efficiency in poultry?

It has been hypothesized that reducing the bioenergetic costs of gut inflammation as an explanation for the effect of antibiotic growth promoters (AGPs) on animal efficiency, framing some observations but not explaining the increase in growth rate or the prevention of infectious diseases. The host's ability to adapt to alterations in environmental conditions and to maintain health involves managing all physiological interactions that regulate homeostasis. Thus, metabolic pathways are vital in regulating physiological health as the energetic demands of the host guides most biological functions. Mitochondria are not only the metabolic heart of the cell because of their role in energy metabolism and oxidative phosphorylation, but also a central hub of signal transduction pathways that receive messages about the health and nutritional states of cells and tissues. In response, mitochondria direct cellular and tissue physiological alterations throughout the host. The endosymbiotic theory suggests that mitochondria evolved from prokaryotes, emphasizing the idea that these organelles can be affected by some antibiotics. Indeed, therapeutic levels of several antibiotics can be toxic to mitochondria, but subtherapeutic levels may improve mitochondrial function and defense mechanisms by inducing an adaptive response of the cell, resulting in mitokine production which coordinates an array of adaptive responses of the host to the stressor(s). This adaptive stress response is also observed in several bacteria species, suggesting that this protective mechanism has been preserved during evolution. Concordantly, gut microbiome modulation by subinhibitory concentration of AGPs could be the result of direct stimulation rather than inhibition of determined microbial species. In eukaryotes, these adaptive responses of the mitochondria to internal and external environmental conditions, can promote growth rate of the organism as an evolutionary strategy to overcome potential negative conditions. We hypothesize that direct and indirect subtherapeutic AGP regulation of mitochondria functional output can regulate homeostatic control mechanisms in a manner similar to those involved with disease tolerance.

Phenotype Alterations in the Cecal Ecosystem Involved in the Asymptomatic Intestinal Persistence of Paratyphoid Salmonella in Chickens.

The gastrointestinal ecosystem involves interactions between the host, gut microbiota, and external environment. To colonize the gut of poultry, Salmonella must surmount barriers levied by the intestine including mucosal innate immune responses and microbiota-mediated niche restrictions. Accordingly, comprehending Salmonella intestinal colonization in poultry requires an understanding of how the pathogen interacts with the intestinal ecosystem. In chickens, the paratyphoid Salmonella have evolved the capacity to survive the initial immune response and persist in the avian ceca for months without triggering clinical signs. The persistence of a Salmonella infection in the avian host involves both host defenses and tolerogenic defense strategies. The initial phase of the Salmonella-gut ecosystem interaction is characteristically an innate pro-inflammatory response that controls bacterial invasion. The second phase is initiated by an expansion of the T regulatory cell population in the cecum of Salmonella-infected chickens accompanied by well-defined shifts in the enteric neuro-immunometabolic pathways that changes the local phenotype from pro-inflammatory to an anti-inflammatory environment. Thus, paratyphoid Salmonella in chickens have evolved a unique survival strategy that minimizes the inflammatory response (disease resistance) during the initial infection and then induces an immunometabolic reprogramming in the cecum that alters the host defense to disease tolerance that provides an environment conducive to drive asymptomatic carriage of the bacterial pathogen.

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.

Applying different morphometric intestinal mucosa methods and the correlation with broilers performance under Eimeria challenge.

The intestinal wall has on its surface, protrusions called villi that are responsible for the absorption of nutrients. Commonly, these structures have their dimensions measured to related more area surface with better absorption. However, the measurement of these villi neglects the inflammation and the presence of immature cells that increase the surface area but affect negatively the absorption and compromise the animal performance. The measurements of villi/crypt are traditional tools in animal research; however, they may overlook alterations that impact the mucosal functionality. This study aimed to compare the morphometry of the intestinal villi/crypt with the I See Inside (ISI) scoring methodology, exploring their correlation with zootechnical performance. Therefore, broilers were grouped as nonchallenged (NC) and challenged with Eimeria (CH) and jejunum samples were collected at 22 d for histological analysis. The same villi were submitted to the ISI methodology, which is based on the scoring of 8 parameters related to the inflammatory process, and the measurements of villus height (VH), villus width (VW), crypt depth (CD), crypt width (CW), VH:CD ratio and villi absorptive surface (VAS). The CH group presented higher ISI total score, VW, CD, CW and lower VH, VH:CD, and VAS in comparison to the NC group. While the villi/crypt morphometry did not exhibit correlations with performance, the presence of Eimeria oocysts and the ISI total score was positively correlated (P < 0.05) with the feed conversion ratio (FCR), demonstrating a statistical interaction between high ISI scores and worse performance. In conclusion, a larger villus is not related to better intestinal functionality when this enlargement is unleashed by the immune processes occurring inside. The scoring system that evaluates the type of alteration observed has a direct impact on the animal's zootechnical performance which is not observed with the single metric surface evaluation.

Leukocyte Response to Campylobacter Intra-Abdominal Infection in One Day Old Leghorn Chickens.

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.

Addition of a protected complex of biofactors and antioxidants to breeder hen diets confers transgenerational protection against Salmonella enterica serovar Enteritidis in progeny chicks.

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-1ß 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.

Morphological Assessment and Biomarkers of Low-Grade, Chronic Intestinal Inflammation in Production Animals.

The complex interaction between the intestinal mucosa, the gut microbiota, and the diet balances the host physiological homeostasis and is fundamental for the maximal genetic potential of production animals. However, factors such as chemical and physical characteristics of the diet and/or environmental stressors can continuously affect this balance, potentially inducing a state of chronic low-grade inflammation in the gut, where inflammatory parameters are present and demanding energy, but not in enough intensity to provoke clinical manifestations. It's vital to expand the understanding of inflammation dynamics and of how they compromise the function activity and microscopic morphology of the intestinal mucosa. These morphometric alterations are associated with the release of structural and functional cellular components into the feces and the blood stream creating measurable biomarkers to track this condition. Moreover, the identification of novel, immunometabolic biomarkers can provide dynamic and predictors of low-grade chronic inflammation, but also provide indicators of successful nutritional or feed additive intervention strategies. The objective of this paper is to review the mechanisms of low-grade inflammation, its effects on animal production and sustainability, and the biomarkers that could provide early diagnosis of this process and support studies of useful interventional strategies.

Chicken-Specific Kinome Analysis of Early Host Immune Signaling Pathways in the Cecum of Newly Hatched Chickens Infected With Salmonella enterica Serovar 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.

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.

Role of diet-microbiota interactions in precision nutrition of the chicken: facts, gaps, and new concepts.

In the intestine, host-derived factors are genetically hardwired and difficult to modulate. However, the intestinal microbiome is more plastic and can be readily modulated by dietary factors. Further, it is becoming more apparent that the microbiome can potentially impact poultry physiology by participating in digestion, the absorption of nutrients, shaping of the mucosal immune response, energy homeostasis, and the synthesis or modulation of several potential bioactive metabolites. These activities are dependent on the quantity and quality of the microbiota alongside its metabolic potential, which are dictated in large part by diet. Thus, diet-induced microbiota alterations may be harnessed to induce changes in host physiology, including disease development and progression. In this regard, the gut microbiome is malleable and renders the gut microbiome a candidate 'organ' for the possibility of precision nutrition to induce precision microbiomics-the use of the gut microbiome as a biomarker to predict responsiveness to specific dietary constituents to generate precision diets and interventions for optimal poultry performance and health. However, it is vital to identify the causal relationships and mechanisms by which dietary components and additives affect the gut microbiome which then ultimately influence avian physiology. Further, an improved understanding of the spatial and functional relationships between the different sections of the avian gut and their regional microbiota will provide a better understanding of the role of the diet in regulating the intestinal microbiome.

From crypts to enteroids: establishment and characterization of avian intestinal organoids.

Intestinal organoids (IO), known as "mini-guts", derived from intestinal crypts, are self-organizing three-dimensional (3D) multicellular ex vivo models that recapitulate intestine epithelial structure and function and have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions, and intestinal disease in mammals. However, studies on avian IO are limited and the development of long-term cultures of IO model for poultry research is lacking. Therefore, the objectives of this study were to generate crypt-derived organoids from chicken intestines and to optimize conditions for cell growth and enrichments, passages, and cryopreservation. Crypts were collected from the small intestines of birds at embryonic d-19 and ceca from layer and broiler chickens with ages ranging from d 1 to 20 wk, embedded in a basement membrane matrix, and cultured with organoid growth media (OGM) prepared in house. The crypt-derived organoids were successfully grown and propagated to form 3D spheres like structures that were cultured for up to 3 wk. Organoids were formed on d one, budding appeared on d 3, and robust budding was observed on d 7 and beyond. For cryopreservation, dissociated organoids were resuspended in a freezing medium. The characteristics of IO upon extended passages and freeze-thaw cycles were analyzed using reverse transcription (RT)-PCR, immunoblotting, and live cell imaging. Immunoblotting and RT-PCR using E-cadherin (the marker for epithelial cells), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, the marker for stem cells), chromogranin A (the marker for enteroendocrine cells), lysozyme (the marker for Paneth cells), and mucin (the biomarker for goblet cells) confirmed that IO were composed of heterogeneous cell populations, including epithelial cells, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both valproic acid and CHIR99021, a glycogen synthase kinase 3ß inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this is the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens. This model will facilitate elucidation of the mechanisms impacting host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry.

Potential Replacements for Antibiotic Growth Promoters in Poultry: Interactions at the Gut Level and Their Impact on Host Immunity.

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.

Reserpine improves Enterobacteriaceae resistance in chicken intestine via neuro-immunometabolic signaling and MEK1/2 activation.

Salmonella enterica persist in the chicken gut by suppressing inflammatory responses via expansion of intestinal regulatory T cells (Tregs). In humans, T cell activation is controlled by neurochemical signaling in Tregs; however, whether similar neuroimmunological signaling occurs in chickens is currently unknown. In this study, we explore the role of the neuroimmunological axis in intestinal Salmonella resistance using the drug reserpine, which disrupts intracellular storage of catecholamines like norepinephrine. Following reserpine treatment, norepinephrine release was increased in both ceca explant media and Tregs. Similarly, Salmonella killing was greater in reserpine-treated explants, and oral reserpine treatment reduced the level of intestinal Salmonella Typhimurium and other Enterobacteriaceae in vivo. These antimicrobial responses were linked to an increase in antimicrobial peptide and IL-2 gene expression as well as a decrease in CTLA-4 gene expression. Globally, reserpine treatment led to phosphorylative changes in epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), and the mitogen-associated protein kinase 2(MEK2). Exogenous norepinephrine treatment alone increased Salmonella resistance, and reserpine-induced antimicrobial responses were blocked using beta-adrenergic receptor inhibitors, suggesting norepinephrine signaling is crucial in this mechanism. Furthermore, EGF treatment reversed reserpine-induced antimicrobial responses, whereas mTOR inhibition increased antimicrobial activities, confirming the roles of metabolic signaling in these responses. Finally, MEK1/2 inhibition suppressed reserpine, norepinephrine, and mTOR-induced antimicrobial responses. Overall, this study demonstrates a central role for MEK1/2 activity in reserpine induced neuro-immunometabolic signaling and subsequent antimicrobial responses in the chicken intestine, providing a means of reducing bacterial colonization in chickens to improve food safety.

Protected Organic Acid and Essential Oils for Broilers Raised Under Field Conditions: Intestinal Health Biomarkers and Cecal Microbiota.

The objective of the present study was to evaluate the effect of protected organic acids (OA) and essential oils (EO) [P(OA + EO)] on the intestinal health of broiler chickens raised under field conditions. The study was conducted on four commercial farms. Each farm consisted of four barns, two barns under a control diet and two tested barns supplemented with P(OA + EO), totaling 16 barns [8 control and 8 under P(OA + EO)]. The control group was supplemented with antibiotic growth promoters [AGP; Bacitracin Methylene Disalicylate (50 g/ton) during starter, grower and finisher 1, and flavomycin (2 g/ton) during finisher 2]. The tested group was supplemented with 636, 636, 454, and 454 g/ton of P(OA + EO) during starter, grower, finisher 1 and 2, respectively. Eighty birds were necropsied (40/treatment; 20/farm; and 5/barn) to collect blood, jejunal tissue, and cecal contents. The data were submitted to analysis of variance (ANOVA) (P < 0.05) or Kruskal-Wallis' test and the frequency of antimicrobial resistant (AMR) genes was analyzed by Chi-Square test (P < 0.05). It was observed that the supplementation of P(OA + EO) reduced (P < 0.05) the histopathology scores, such as the infiltration of inflammatory cells in the epithelium and lamina propria and tended (P = 0.09) to reduce the serum concentration of calprotectin (CALP). The supplementation of P(OA + EO) reduced the serum concentration of IL-12 (P = 0.0001), IL-16 (P = 0.001), and Pentraxin-3 (P = 0.04). Additionally, P(OA + EO) maintained a cecal microbiota similar to birds receiving AGP. The substitution of AGP by P(OA + EO) reduced (P < 0.05) the frequency of four AMR genes, related to gentamicin (three genes), and aminoglycoside (one gene). Overall, the inclusion of P(OA + EO), and removal of AGP, in the diets of commercially raised broiler chickens beneficially changed the phenotype of the jejunum as shown by the lowered ISI scores which characterizes an improved intestinal health. Furthermore, P(OA + EO) significantly reduced the serum concentration of several inflammatory biomarkers, while maintaining the diversity and composition of the cecal microbiota similar to AGP fed chickens and reducing the prevalence of AMR genes.

Effects of Salmonella enterica ser. Enteritidis and Heidelberg on host CD4+CD25+ regulatory T cell suppressive immune responses in chickens.

Poultry infected with Salmonella mount an immune response initially, however the immune responses eventually disappear leading the bird to be a carrier of Salmonella. The hypothesis of this study is that Salmonella infection induces T regulatory cell numbers and cytokine production and suppress host T cells locally in the gut to escape the host immune responses. An experiment was conducted to comparatively analyze the effect of S. enterica ser. Enteritidis (S. Enteritidis) and S. enterica ser. Heidelberg (S. Heidelberg) infection on CD4+CD25+ T regulatory cell properties in chickens. A total of 144 broiler chicks were randomly distributed into three experimental groups of non-infected control, S. Enteritidis infected and S. Heidelberg infected groups. Chickens were orally inoculated with PBS (control) or 5x106 CFU/mL of either S. Enteritidis or S. Heidelberg at 3 d of age. Each group was replicated in six pens with eight chickens per pen. Chickens infected with S. Enteritidis had 6.2, 5.4, and 3.8 log10 CFU/g, and chickens infected with S. Heidelberg had 7.1, 4.8, and 4.1 log10 CFU/g Salmonella in the cecal contents at 4, 11, and 32 dpi, respectively. Both S. Enteritidis and S. Heidelberg were recovered from the liver and spleen 4 dpi. At 4, 11, and 32 dpi, chickens infected with S. Enteritidis and S. Heidelberg had increased CD4+CD25+ cell numbers as well as IL-10 mRNA transcription of CD4+CD25+ cells compared to that in the control group. CD4+CD25+ cells from S. Enteritidis- and S. Heidelberg-infected chickens and restimulated with 1 µg antigen in vitro, had higher (P < 0.05) IL-10 mRNA transcription than the CD4+CD25+ cells from the non-infected controls Though at 4dpi, chickens infected with S. Enteritidis and S. Heidelberg had a significant (P < 0.05) increase in CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, the CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, were comparable to that in the control group at 11 and 32dpi identifying that the host inflammatory response against Salmonella disappears at 11 dpi. It can be concluded that S. Enteritidis and S. Heidelberg infection at 3 d of age induces a persistent infection through inducing CD4+CD25+ cells and altering the IL-10 mRNA transcription of CD4+CD25+ cell numbers and cytokine production in chickens between 3 to 32 dpi allowing chickens to become asymptomatic carriers of Salmonella after 18 dpi.