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.

The biological effects of microencapsulated organic acids and botanicals induces tissue-specific and dose-dependent changes to the Gallus gallus microbiota.

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 500 g/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 300 g/MT and 67% in the 500 g/MT group. Staphylococcaceae were 2% in NTC and 2 and 0% in 300 and 500 g/MT groups. Enterobacteriaceae decreased in the 500 g/MT (31%) and increased in the 300 g/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 500 g/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 500 g/MT compared to NTC and 300 g/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.

Dietary l-arginine supplementation influences growth performance and B-cell secretion of immunoglobulin in broiler chickens.

Our previous study has shown that high levels of l-arginine (ARG) have reduced serum and mucosal antibody concentrations. In order to provide a better understanding in the application of ARG supplementation in the poultry industry, the study was conducted to investigate the effect of high levels of ARG on performance and B-cell secretion of immunoglobulin M (IgM) and IgG development in broiler chickens. A total of 192 1-day-old male Arbor Acres Plus broilers were randomly allocated into 4 groups (8 replicates per group, 6 birds per replicate) fed diets containing one of four ARG concentrations (analysed): 9.8, 14.7, 19.1 and 23.4 g/kg respectively. Growth performance was measured based on body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR). Increasing ARG quadratically increased (p < 0.05) BWG and FI with reaching plateau at 14.7 g/kg, while linearly decreased (p < 0.05) FCR, indicating that maximal performance required ARG no more than 14.7 g/kg in diets. Serum IgG and IgM concentrations were linearly reduced (p < 0.05) with increasing ARG. Chickens fed 19.1 g/kg or 23.4 g/kg ARG had lower (p < 0.05) serum IgG or IgM than chickens fed 9.8 g/kg ARG. As for the mRNA expression of bursal IgG and IgM, they were significantly downregulated with increasing ARG (p < 0.05). Chickens on ARG (>19.1 g/kg) had a lower (p < 0.05) IgG and IgM mRNA expression than chickens fed 9.8 g/kg. Activator of transcription 3 (STAT3) mRNA expression was linearly reduced with increasing ARG (p < 0.05), the transcriptional repressor B-cell lymphoma 6 (BCL6) mRNA expression was quadratically (p < 0.05) responded, and these cytokines had the lowest expression at 19.1 g/kg. ARG supplementation (>14.7 g/kg) did not significantly improve the growth performance, while it may have a potential negative regulatory effect on B-cell-mediated humoral immunity in chickens associated with suppression of the STAT3 expression associated with the JAK/STAT3 pathway.

AMPK and mTOR: sensors and regulators of immunometabolic changes during Salmonella infection in the chicken.

Non-typhoidal Salmonella enterica induce an early pro-inflammatory response in chickens, but the response is short-lived, asymptomatic of clinical disease, results in a persistent colonization of the gastrointestinal (GI) tract, and can transmit infections to naïve hosts via fecal shedding of bacteria. The underlying mechanisms that facilitate this persistent colonization of the ceca of chickens by Salmonella are unknown. We have begun to concentrate on the convergence of metabolism and immune function as playing a major role in regulating the host responsiveness to infection. It is now recognized that the immune system monitors the metabolic state of tissues and responds by modulating metabolic function. The aim in this review is to summarize the literature that has defined a series of genotypic and phenotypic alterations in the regulatory host immune-metabolic signaling pathways in the local cecal microenvironment during the first 4 d following infection with Salmonella enterica serovar Enteritidis. Using chicken-specific kinomic immune-metabolism peptide arrays and quantitative real-time-PCR of cecal tissue during the early (4 to 48 h) and late stages (4 to 17 d) of a Salmonella infection in young broiler chickens, the local immunometabolic microenvironment has been ascertained. Distinct immune and metabolic pathways are altered between 2 to 4 d post-infection that dramatically changed the local immunometabolic environment. Thus, the tissue immunometabolic phenotype of the cecum plays a major role in the ability of the bacterium to establish a persistent cecal colonization. In general, our findings show that AMPK and mTOR are key players linking specific extracellular milieu and intracellular metabolism. Phenotypically, the early response (4 to 48 h) to Salmonella infection is pro-inflammatory, fueled by glycolysis and mTOR-mediated protein synthesis, whereas by the later phase (4 to 5 d), the local environment has undergone an immune-metabolic reprogramming to an anti-inflammatory state driven by AMPK-directed oxidative phosphorylation. Therefore, metabolism appears to provide a potential critical control point that can impact infection. Further understanding of metabolic control of immunity during infection should provide crucial information of the development of novel therapeutics based on metabolic modulators that enhance protection or inhibit infection.

Wild-type and mutant AvrA- Salmonella induce broadly similar immune pathways in the chicken ceca with key differences in signaling intermediates and inflammation.

Salmonella enterica serovar Typhimurium (ST) is a serious infectious disease throughout the world, and a major reservoir for Salmonella is chicken. Chicken infected with Salmonella do not develop clinical disease, this may be the result of important host interactions with key virulence proteins. To study this, we inoculated chicken with mutant Salmonella Typhimurium that lacked the virulence protein AvrA (AvrA(-)). AvrA is referred to as an avirulence factor, as it moderates the host immune response. The lack of the AvrA virulence gene in ST resulted in reduced weight gain, enhanced persistence and greater extraintestinal organ invasion in chickens, as compared to wild-type (WT) ST. Kinome analysis was performed on inoculated cecal tissue. The majority of the signal transduction pathways induced by AvrA(-) and WT ST were similar; however, we observed alterations in innate immune system signaling. In addition, a leukocyte migration pathway was altered by AvrA(-) ST that may allow greater gut barrier permeability and invasion by the mutant. Cytokine expression did not appear significantly altered at 7 d post-inoculation; at 14 d post-inoculation, there was an observed increase in the expression of anti-inflammatory IL-10 in the WT inoculated ceca. This study is the first to describe mutant AvrA(-) ST infection of chicken and provides further insight into the Salmonella responses observed in chicken relative to other species such as humans and cattle.

Chicken-Specific Kinome Array Reveals that Salmonella enterica Serovar Enteritidis Modulates Host Immune Signaling Pathways in the Cecum to Establish a Persistence Infection.

Non-typhoidal Salmonella enterica induces an early, short-lived pro-inflammatory response in chickens that is asymptomatic of clinical disease and results in a persistent colonization of the gastrointestinal (GI) tract that transmits infections to naïve hosts via fecal shedding of bacteria. The underlying mechanisms that control this persistent colonization of the ceca of chickens by Salmonella are only beginning to be elucidated. We hypothesize that alteration of host signaling pathways mediate the induction of a tolerance response. Using chicken-specific kinomic immune peptide arrays and quantitative RT-PCR of infected cecal tissue, we have previously evaluated the development of disease tolerance in chickens infected with Salmonella enterica serovar Enteritidis (S. Enteritidis) in a persistent infection model (4-14 days post infection). Here, we have further outlined the induction of an tolerance defense strategy in the cecum of chickens infected with S. Enteritidis beginning around four days post-primary infection. The response is characterized by alterations in the activation of T cell signaling mediated by the dephosphorylation of phospholipase c-γ1 (PLCG1) that inhibits NF-κB signaling and activates nuclear factor of activated T-cells (NFAT) signaling and blockage of interferon-γ (IFN-γ) production through the disruption of the JAK-STAT signaling pathway (dephosphorylation of JAK2, JAK3, and STAT4). Further, we measured a significant down-regulation reduction in IFN-γ mRNA expression. These studies, combined with our previous findings, describe global phenotypic changes in the avian cecum of Salmonella Enteritidis-infected chickens that decreases the host responsiveness resulting in the establishment of persistent colonization. The identified tissue protein kinases also represent potential targets for future antimicrobial compounds for decreasing Salmonella loads in the intestines of food animals before going to market.

Electron-Beam-Inactivated Vaccine Against Salmonella Enteritidis Colonization in Molting Hens.

Electron-beam (eBeam) irradiation technology has a variety of applications in modern society. The underlying hypothesis was that eBeam-inactivated Salmonella enterica serovar Enteritidis (SE) cells can serve as a vaccine to control SE colonization and shedding in poultry birds. An eBeam dose of 2.5 kGy (kilograys) was used to inactivate a high-titer (10(8) colony-forming units [CFU]) preparation of SE cells. Microscopic studies revealed that the irradiation did not damage the bacterial cell membranes. The vaccine efficacy was evaluated by administering the eBeam-killed SE cells intramuscularly (1 x 10(6) CFU/bird) into 50-wk-old single comb white leghorn hens. On day 14 postvaccination, the hens were challenged orally with live SE cells (1 x 10(9) CFU) and SE colonization of liver, spleen, ceca, and ovaries determined on day 23. Blood samples were collected on days 0, 14, and 23 postvaccination and the sera were analyzed to quantify SE-specific IgG titers. The vaccinated chickens exhibited significantly (P < 0.0001) higher SE-specific IgG antibody responses and reduced SE ceca colonization (1.46 ± 0.39 logi10 CFU/g) compared to nonvaccinated birds (5.32 ± 0.32 log10 CFU/g). They also exhibited significantly lower SE colonization of the ovaries (1/30), spleen (3/30), liver (4/30), and ceca (7/30) compared to nonvaccinated birds. These results provide empirical evidence that eBeam-based SE vaccines are immunogenic and are capable of protecting chickens against SE colonization. The advantages of eBeam-based vaccine technology are that it is nonthermal, avoids the use of formalin, and can be used to generate inactivated vaccines rapidly to address strain-specific infections in farms or flocks.

Effect of Salmonella infection on cecal tonsil regulatory T cell properties in chickens.

Two studies were conducted to study regulatory T cell [Treg (CD4⁺CD25⁺)] properties during the establishment of a persistent intestinal infection in broiler chickens. Four-day-old broiler chicks were orally gavaged with 5 × 106 CFU/mL Salmonella enteritidis (S. enteritidis) or sterile PBS (control). Samples were collected at 4, 7, 10, and 14 d postinfection. There was a significant (P < 0.05) increase in the number of CD4⁺CD25⁺ cells by d 4 postinfection that increased steadily throughout the course of the 14-d infection, whereas the number of CD4⁺CD25⁺ cells in the noninfected controls remained steady throughout the study. CD4⁺CD25⁺ cells from cecal tonsils of S. enteritidis-infected birds had a higher (P < 0.05) IL-10 mRNA content than CD4⁺CD25⁺ cells from the noninfected controls at all time-points studied. The amount of IL-2 mRNA content in the cecal tonsil CD4⁺CD25⁻ cells from the infected birds did not differ (P > 0.05) when compared to that of noninfected control birds. At a lower effector/responder cell ratio of 0.25:1, CD4⁺CD25⁺ cells from cecal tonsils of Salmonella-infected birds suppressed T cell proliferation at d 7 and 14 post-S. enteritidis infection, while CD4⁺CD25⁺ cells from noninfected control groups did not suppress T cell proliferation. In the second studu, 1-day-old chickens were orally gavaged with PBS (control) or 1.25 × 108 CFU/bird S. enteritidis. At 7 and 21 d post-Salmonella infection, CD25⁺ cells collected from cecal tonsils of S. enteritidis-infected birds and restimulated in vitro with Salmonella antigen had higher (P < 0.05) IL-10 mRNA content compared to those in the control group. Spleen CD4⁺CD25⁺, CD4⁺, and CD8⁺ cell percentage did not differ (P > 0.05) between the Salmonella-infected and control birds. In conclusion, a persistent intestinal S. enteritidis infection increased the Treg percentage, suppressive properties, and IL-10 mRNA amounts in the cecal tonsils of broiler birds.

Selection for pro-inflammatory mediators yields chickens with increased resistance against Salmonella enterica serovar Enteritidis.

Salmonella is a leading cause of foodborne illness and can be transmitted through consumption of contaminated poultry; therefore, increasing a flock's natural resistance to Salmonella could improve food safety. Previously, we characterized the heterophil-mediated innate immune response of 2 parental broiler lines and F1 reciprocal crosses and showed that increased heterophil function and expression of pro-inflammatory mediators corresponds with increased resistance against diverse pathogens. A preliminary selection trial showed that individual sires had varying inherent levels of pro-inflammatory mediators and selection based on a high or low phenotype was passed onto progeny. Based on these results, we hypothesized selection of broilers for higher levels of the pro-inflammatory mediators IL-6, CXCLi2, and CCLi2 would produce progeny with increased resistance against Salmonella Enteritidis. Peripheral blood leukocytes were isolated from 75 commercial broiler sires, screened, and 10 naturally high and low expressing sires were selected and mated to randomly selected dams to produce the first generation of "high" and "low" progeny. The mRNA expression of CXCLi2 and CCLi2 were significantly (P ≤ 0.02) higher in the high progeny and were more resistant to liver and spleen organ invasion by Salmonella Enteritidis compared with low progeny. Production of the second generation yielded progeny that had differences (P ≤ 0.03) in all 3 mediators and further improved resistance against Salmonella Enteritidis. Feed conversion ratio and percent breast meat yield were calculated and were equal, whereas the high birds weighed slightly, but significantly, less than the low birds. These data clearly demonstrate that selection based on a higher phenotype of key pro-inflammatory mediators is a novel means to produce broilers that are naturally more resistant to Salmonella, one of the most important foodborne pathogens affecting the poultry industry.

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.

Linking norepinephrine production and performance to diet-induced low-grade, chronic inflammation in the intestine of broilers.

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.

Selection for high and low antibody responses to sheep red blood cells influences cytokine and chemokine expression in chicken peripheral blood leukocytes and splenic tissue.

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 2 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-ß4) 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-ß4. 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-ß4 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.

Salmonella enterica Typhimurium infection causes metabolic changes in chicken muscle involving AMPK, fatty acid and insulin/mTOR signaling.

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) infection of chickens that are more than a few days old results in asymptomatic cecal colonization with persistent shedding of bacteria. We hypothesized that while the bacterium colonizes and persists locally in the cecum it has systemic effects, including changes to metabolic pathways of skeletal muscle, influencing the physiology of the avian host. Using species-specific peptide arrays to perform kinome analysis on metabolic signaling pathways in skeletal muscle of Salmonella Typhimurium infected chickens, we have observed key metabolic changes that affected fatty acid and glucose metabolism through the 5'-adenosine monophosphate-activated protein kinase (AMPK) and the insulin/mammalian target of rapamycin (mTOR) signaling pathway. Over a three week time course of infection, we observed changes in the phosphorylation state of the AMPK protein, and proteins up and down the pathway. In addition, changes to a large subset of the protein intermediates of the insulin/mTOR pathway in the skeletal muscle were altered by infection. These changes occur in pathways with direct effects on fatty acid and glucose metabolism. This is the first report of significant cellular metabolic changes occurring systemically in chicken due to a Salmonella infection. These results have implications not only for animal production and health but also for the understanding of how Salmonella infection in the intestine can have widespread, systemic effects on the metabolism of chickens without disease-like symptoms.

Enhanced innate immune responses in a brood parasitic cowbird species: Degranulation and oxidative burst.

We examined the relative effectiveness of two innate immune responses in two species of New World blackbirds (Passeriformes, Icteridae) that differ in resistance to West Nile virus (WNV). We measured degranulation and oxidative burst, two fundamental components of phagocytosis, and we predicted that the functional effectiveness of these innate immune responses would correspond to the species' relative resistance to WNV. The brown-headed cowbird (Molothrus ater), an obligate brood parasite, had previously shown greater resistance to infection with WNV, lower viremia and faster recovery when infected, and lower subsequent antibody titers than the red-winged blackbird (Agelaius phoeniceus), a close relative that is not a brood parasite. We found that cowbird leukocytes were significantly more functionally efficient than those of the blackbird leukocytes and 50% more effective at killing the challenge bacteria. These results suggest that further examination of innate immunity in the cowbird may provide insight into adaptations that underlie its greater resistance to WNV. These results support an eco-immunological interpretation that species like the cowbird, which inhabit ecological niches with heightened exposure to parasites, experience evolutionary selection for more effective immune responses.

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.

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.

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.

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.

Systemic response to Campylobacter jejuni infection by profiling gene transcription in the spleens of two genetic lines of chickens.

Campylobacter jejuni (C. jejuni) is a leading cause of human bacterial enteritis worldwide with poultry products being a major source of C. jejuni contamination. The chicken is the natural reservoir of C. jejuni where bacteria colonize the digestive tract of poultry, but rarely cause symptoms of disease. To understand the systemic molecular response mechanisms to C. jejuni infection in chickens, total splenic RNA was isolated and applied to a whole genome chicken microarray for comparison between infected (I) and non-infected (N) chickens within and between genetic lines A and B. There were more total splenic host genes responding to the infection in resistant line A than in susceptible line B. Specifically, genes for lymphocyte activation, differentiation and humoral response, and Ig light and heavy chain were upregulated in the resistant line. In the susceptible line, genes for regulation of erythrocyte differentiation, hemopoiesis, and RNA biosynthetic process were all downregulated. An interaction analysis between genetic lines and treatment demonstrated distinct defense mechanisms between lines: the resistant line promoted apoptosis and cytochrome c release from mitochondria, whereas the susceptible line responded with a downregulation of both functions. This was the first time that such systemic defensive mechanisms against C. jejuni infection have been reported. The results of this study revealed novel molecular mechanisms of the systemic host responses to C. jejuni infection in chickens that warrant further investigation.