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The prophylactic treatment of neonatal broiler chicks with lymphokines derived from S. enteritidis-immurazed chickens (SE-ILK) was evaluated for its effect on the birds' resistance to an experimental infection S. enterica ser. gallinarum (SG). On the day of hatch, chicks were injected intraperitoneally with either SE-ILK, control non-immune lymphokines (NILK), or were left untreated. Thirty minutes later, all chicks were orally gavaged with either 10(4) colony forming units (CFU) or 10(6) CFU SG. The chicks were observed twice daily for 10 days for morbidity and mortality. Chicks that died during the experiment had their livers cultured for SG. The survivors were killed and their livers, spleens and caecal tonsils cultured for SG. The prophylactic treatment of chickens with SE-ILK induced significant protection against extraintestinal SG infection when compared to the NILK-treated or non-treated controls as evidenced by: (1) a significant reduction (P< 0.005) in the mortality of chicks challenged with either 10(4) and 10(6) CFU SG; (2) an increased average weight gains of chicks challenged with either 10(4) and 10(6) CFU SG; and (3) a significant (P< 0.001) reduction in the number of chicks with organs culture-positive for SG. The results suggest that the prophylactic administration of SE-ILK can confer non-specific protection to chicks against a pathogenic species of Salmonella resulting in reduced morbidity, mortality, and organ infectivity caused by SG infections of broiler chicks, while enhancing performance during the first 10 days of Ufe.

Heterophils play an indispensable role in the immune defense of the avian host. To accomplish this defense, heterophils use sophisticated mechanisms to both detect and destroy pathogenic microbes. Detection of pathogens through the toll-like receptors (TLR), FC and complement receptors, and other pathogen recognition receptors has been recently described for the avian heterophil. Upon detection of pathogens, the avian heterophil, through a network of intracellular signaling pathways and the release and response to cytokines and chemokines, responds using a repertoire of microbial killing mechanisms including production of an oxidative burst, cellular degranulation, and production of extracellular matrices of DNA and histones (HETs). In this review, the authors describe the recent advances in our understanding of the avian heterophil, its functions, receptors and signaling, identified antimicrobial products, cytokine and chemokine production, and some of the effects of genetic selection on heterophils and their functional characteristics.

Salmonella enterica serovar Enteritidis is one of the most prevalent Salmonella serovars in poultry and is often associated with human salmonellosis. S. Enteritidis is known to suppress nitric oxide (NO) production in infected chicken macrophage HD11 cells, while dead S. Enteritidis stimulates a high level of NO production, suggesting a bacterial inhibitory effect on NO production. Based on these observations, the present study was conducted to evaluate whether NO production in S. Enteritidis-infected HD11 cells can be used as a biomarker to identify molecules that kill intracellular Salmonella. Since Salmonella are known to manipulate the host cell kinase network to facilitate intracellular survival, we screened a group of pharmaceutical inhibitors of various kinases to test our hypothesis. A protein kinase A inhibitor, H-89, was found to reverse the suppression of NO production in S. Enteritidis-infected HD11 cells. Production of NO in S. Enteritidis-infected HD11 cells increased significantly following treatment with H-89 at or above 20 µM. Inversely, the number of viable intracellular Salmonella decreased significantly in cells treated with H-89 at or above 30 µM. Furthermore, the growth rate of S. Enteritidis in culture was significantly inhibited by H-89 at concentrations from 20 to 100 µM. Our results demonstrate that NO-based screening using S. Enteritidis-infected HD11 cells is a viable tool to identify chemicals with anti-intracellular Salmonella activity. Using this method, we have shown H-89 has bacteriostatic activity against Salmonella, independent of host cell protein kinase A or Akt1 activity.

Macrophages are important innate immune cells that play a central role in the first line defense against microbial infection. Although, chicken macrophages are equipped with effective antimicrobial mechanisms that can be readily activated in response to microbial infection, many Salmonella strains are able to survival inside the macrophage. A previous study has shown that S. Enteritidis was better equipped to survive in the chicken macrophages than S. Heidelberg, in which S. Enteritidis, but not S. Heidelberg, was able to suppress the host’s nitric oxide response. The interaction between chicken macrophages and Salmonella as well as intracellular survival of Salmonella in chicken macrophages remains poorly understood. Here, we used a species-specific immunometabolism kinome peptide array to identify pathways that are involved in chicken macrophages’ response to Salmonella infection. The kinome assay was performed on chicken HD-11 macrophages collected at 0.5, 2, and 6 hr post-infection with either S. Heidelberg or S. Enteritidis. We observed significant changes of phosphorylation (p ≤ 0.05) in a large number of peptides during the infection: 390, 449, and 575 peptides for S. Enteritidis and 185, 470, and 442 for S. Heidelberg at 0.5, 2, and 6 hr, respectively. Additionally, a relatively small number of peptides that showed infection-induced phosphorylation changes in opposite directions when comparing the two serovars: 142 peptides at 0.5 hr, 141 at 2 hr, and 82 at 6 hr. Analyzing phosphopeptides, we found that a number of pathways involved in immunity, signal transduction, cellular process, and metabolism were significantly altered, in some case differentially, during the infection with the two serovars.

We used an anti-inflammatory dose of dexamethasone (DEX) and Salmonella enteritidis (SE)-immune lymphokines (ILK) followed by oral SE challenge to chicks to determine the effects of these treatments on SE organ invasion and in vitro function of PMNs derived from peripheral blood. Endpoints included percent protection against SE organ invasion, numbers of peripheral blood PMNs, and in vitro PMN adherence, chemotaxis, and SE killing. SE organ invasion was significantly reduced in chicks treated with either ILK alone or DEX + ILK compared to controls. Chicks treated with either DEX alone or DEX + ILK responded with a significant increase in numbers of peripheral blood PMNs as compared to controls, while numbers of PMNs in the peripheral blood from chicks treated with ILK alone were not significantly increased. PMN adherence and percent SE killing by PMNs derived from chicks treated with either ILK alone or DEX + ILK were significantly increased compared to controls. Chemotaxis of PMNs derived from chicks treated with either ILK alone or DEX alone significantly increased 2-fold over control levels. Interestingly, chemotaxis of PMNs derived from chicks that received DEX + ILK was similar to controls. Generally, ILK abated the anti-inflammatory effects of DEX on PMNs in these assays, except for chemotaxis. We interpret these data to suggest that ILK may confer protection to chicks against the early phase of SE organ invasion by inducing an inflammatory response predominated by activated PMNs.

We previously reported that the prophylactic, intraperitoneal administration of supernatants from concanavalin A-stimulated T cells derived from Salmonella enteritidis (SE)-immune White Leghorn hens (i.e., SE-immune lymphokines or ILK), conferred protection to neonatal White Leghorn chicks against SE organ invasion. In the present study, we evaluated the effects of in ovo administration of ILK on hatchability, hatch weight, in vitro bactericidal activity of heterophils, and protection against SE organ invasion in neonatal White Leghorn chicks. On Day 18 of embryogenesis, injections were made into the amnion with either ILK or nonimmune ILK (NILK) or were not injected (untreated). On the day of hatch, whole blood was collected from 20 of the chicks per treatment group for heterophil isolation. All remaining chicks were orally challenged with 5 x 10(4) cfu SE. Twenty-four hours after SE challenge, organs (liver and spleen) from the chicks were cultured for SE. Hatchability of ILK- and NILK-treated chicks was not different from that of untreated chicks. Hatch weights of ILK-treated chicks were approximately 1 g less (P < .05) than that of NILK-treated or untreated chicks. In vitro bactericidal activity of peripheral blood heterophils derived from ILK-treated chicks was increased (P < .05) above activity from heterophils derived from NILK-treated or untreated chicks. Organ invasion with SE was markedly and significantly decreased in the ILK-treated chicks as compared with chicks treated with NILK or untreated chicks. These results suggest that in ovo administration of ILK confers protection to neonatal chicks against SE organ infectivity at hatch.

We characterized the inflammatory cell influx in day-old chicks induced by the i.p. administration of live Salmonella enteritidis (SE) and lymphokines from concanavalin A-stimulated SE-immune T lymphocytes (ILK). An i.p. injection of ILK along with 5 × 103 cfu SE increased the survival rate of chicks 48 h later from 70% (ILK-treated controls) compared with 25% (saline-treated). The injection of both the ILK and live SE (but not formalin-killed SE) resulted in an increased influx of inflammatory heterophils into the peritoneum that peaked at 4 h after the injections with no increase in peritoneal macrophages. The heterophil accumulation was not influenced by polymyxin B, but was sensitive to heat treatment (100 C for 1 h) of the ILK, suggesting that lipopolysaccharide (LPS) did not contribute to the induced accumulation of heterophils. Treatment of the chicks with nordihydroguaiaretic acid or indomethacin did not abrogate the induced heterophil accumulation, suggesting that arachidonic acid metabolites were not involved in the SE/ILK-induced accumulation of peritoneal heterophils. The results from the current studies indicate that 1) ILK-mediated resistance to SE-induced mortality is mediated by a rapid influx of inflammatory heterophils to the site of infection; 2) live SE, during invasion, are vital for the site-directed migration of the heterophils; and 3) the mechanisms of induced heterophil accumulation are unknown but involve neither LPS nor arachidonic acid metabolites.

Poultry is a major reservoir for foodborne Salmonella serovars. Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg are the most prevalent serovars in U.S. poultry. Information concerning the interactions between different Salmonella species and host cells in poultry is lacking. In the present study, the above mentioned Salmonella serovars were examined for invasion, intracellular survival, and their ability to modulate oxidative burst and nitric oxide (NO) responses in chicken macrophage HD11 cells. All Salmonella serovars demonstrated similar capacity to invade HD11 cells. At 24 h post-infection, a 36-43% reduction of intracellular bacteria, in log(10)(CFU), was observed for Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg, whereas a significantly lower reduction (16%) was observed for Salmonella Enteritidis, indicating its higher resistance to the killing by HD11 cells. Production of NO was completely diminished in HD11 cells infected with Salmonella Typhimurium and Salmonella Enteritidis, but remained intact when infected with Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg. Phorbol myristate acetate-stimulated oxidative burst in HD11 cells was greatly impaired after infection by each of the five serovars. When newly hatched chickens were challenged orally, a high rate (86-98%) of systemic infection (Salmonella positive in liver/spleen) was observed in birds challenged with Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg, and Salmonella Kentucky, while only 14% of the birds were Salmonella Senftenberg positive. However, there was no direct correlation between systemic infection and in vitro differential intracellular survival and modulation of NO response among the tested serovars.

Previously conducted studies using two chicken lines (A and B) show that line A birds have increased resistance to a number of bacterial and protozoan challenges and that het- erophils isolated from line A birds are functionally more responsive. Furthermore, when stimulated withToll-like receptor (TLR) agonists, heterophils from line A expressed a totally different cytokine and chemokine mRNA expression pattern than heterophils from line B. A large-scale gene expression profile using an Agilent 44K microarray on heterophils isolated from line A and line B also revealed significantly differential expression in many immune-related genes following Salmonella enteritidis (SE) stimulation, which included genes involved in the TLR pathway. Therefore, we hypothesize the differences between the lines result from distinctive TLR pathway signaling cascades that mediate heterophil function and, thus, innate immune responsiveness to SE. Using quantitative RT-PCR on mRNA from heterophils isolated from control and SE-stimulated heterophils of each line, we profiled the expression of all chicken homologous genes identified in a reference TLR pathway. Several differentially expressed genes found were involved in the TLR-induced My88-dependent pathway, showing higher gene expression in line A than line B heterophils following SE stimulation. These genes included the TLR genes TLR4, TLR15, TLR21, MD-2, the adaptor proteins Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP ), Tumor necrosis factor-receptor associated factor 3 (TRAF3), the IkB kinases transforming growth factor-b-activating kinase 1 (TAK1), IKK+ and IKKa, the transcription factors NFkB2 and interferon regulatory factor 7, phosphatidylinositol-3 kinase (PI-3K ), and the mitogen- activated protein kinase p38. These results indicate that higher expression of TLR signaling activation of both MyD88-dependent and TRIF-dependent pathways are more beneficial to avian heterophil-mediated innate immunity and a complicated regulation of downstream adaptors is involved in stronger induction of a TLR-mediated innate response in the resis- tant line A.These findings identify new targets for genetic selection of chickens to increase resistance to bacterial infections.

Young poultry exhibit a transient colonization by some food-borne pathogens, including Salmonella, during the first week of life that stems from immature innate and acquired defense mechanisms. Consequently, modulation of the hosts' natural immune response is emerging as an important area of interest for food animal producers, including the poultry industry. Toll-like receptor (TLR) agonists have been shown to boost the innate immune response in young chickens and increase their resistance to colonization by Salmonella enterica serovar Enteritidis. The objective of the present study was to determine if pretreatment with loxoribine, a TLR7 agonist and immune modulator, protects young chicks from Salmonella Enteritidis organ invasion. Loxoribine (0-100 μg) was administered intra-abdominally to 1-d-old broiler chicks, and 4 h later, the birds were challenged orally with Salmonella Enteritidis. Twenty-four hours postchallenge, birds were euthanized and the liver and spleen aseptically removed and cultured for Salmonella Enteritidis. This was carried out on 3 separate occasions using 26 to 50 chicks per dose per experiment. Pretreatment of chicks with loxoribine (6.25-25 μg) significantly (P ≤ 0.05) reduced liver and spleen organ invasion by Salmonella Enteritidis. Higher doses (50-100 μg) of loxoribine had no effect. The results obtained in this study indicate that there is a potential application for using loxoribine to increase protection of young chicks when they are most susceptible to infections with Salmonella.

The ability of heterophils and monocytes from domestic chickens and turkeys to phagocytize and kill Salmonella enteritidis (SE) was investigated using both a standard plate count (chickens only) and direct microscopic examination by staining with acridine orange (both chickens and turkeys). Optimal phagocytosis of SE by both chicken and turkey phagocytes required opsonization of the bacteria by antibodies as determined by both assays. However, heterophils from both the turkeys and chickens phagocytized more SE than did monocytes from either type of fowl. Regardless of the presence or absence of antibodies, heterophils were more efficient bactericidal cells than were the monocytes since only heterophils killed the intracellular bacteria while the majority of nonopsonized SE survived in the monocytes. Phagocytosis of SE did not result in an increase in the amount of superoxide anion released by the heterophils suggesting that the ultimate fate of the bacteria in these cells was not related to an oxygen-dependent mechanism.

The gastrointestinal (GI) tract is the largest interface between an animal’s internal milieu and its exterior environment. As such, it forms a physical barrier between the two environments. However, the function of the GI tract in the well-being of an animal is more complex than this passive role. The GI tract not only regulates the selective entry of nutrients while keeping vigilant against pathogens, it is largely responsible for shaping the immune response. Through specialized receptors and other general mechanisms, the GI tract senses changes in its environment and actively responds to the changes. These responses allow the intestine to contribute to the defense against microbes as well as control and regulate the local immune response. In addition, the luminal microbial ecosystem is a highly complex community of primarily bacterial microbes that communicates extensively with itself and the host. The microbial community has major influences on the host, including effects on nutrient absorption, cancer, inflammation, host metabolism, barrier function, and gut function (neuromotor, immunological, vascular) among others. Regulation of the immune response is the basis for the use of probiotics and prebiotics reviewed in this chapter.

Yeast species such as Saccharomyces cerevisiae are known to be potent activators of the immune system. S. cerevisiae activates the innate immune system by engaging pattern recognition receptors such as toll like receptor 2 (TLR2) and dectin-1. In the current project, we express the immunogenic envelope protein E2 of bovine viral diarrhoea virus (BVDV) on the surface of S. cerevisiae. After successful expression, components of the innate and adaptive immune response induced by the recombinant S. cerevisiae in vitro were analysed to determine if expression in yeast enhances the immunogenicity of the viral protein. Recombinant S. cerevisiae stimulated production of the chemokine CXCL-8 in primary bovine macrophages, but did no stimulate production of reactive oxygen species (ROS) in the same cells. Additionally, bovine macrophages primed with S. cerevisiae expressing viral envelope proteins had a greater capacity for stimulating proliferation of CD4+ T-cells from BVDV-free animals compared to macrophages primed with envelope protein alone or S. cerevisiae without envelope protein expression. Heat inactivation of recombinant S. cerevisiae increased ROS production and capacity to stimulate CD4+ T-cells in macrophages but did not alter CXCL-8 release compared to the live counter-part. Additionally, heat-inactivation of recombinant S. cerevisiae induced less INFγ and IL-4 but equal amounts of IL-10 compared to live yeast T-cell cultures. Our studies demonstrate a use for S. cerevisiae as a vehicle for transporting BVDV vaccine antigen to antigen-presenting cell in order to elicit cell-mediated immunity even in naïve animals.

Neonatal poultry exhibit a transient susceptibility to infectious diseases during the first week of life that stems from inefficient host defense mechanisms. Yet, the initial host immune response to pathogens is a critical determinant of disease resistance and susceptibility. With this context in mind, novel ways to stimulate or modulate the hosts’ natural immune response is emerging as an important area of interest for food animal producers including the poultry industry. Specifically, we have been investigating new modulation strategies tailored around the selective stimulation of the host's immune system, and particularly rapid acting innate immunity, as an alternative to direct targeting of microbial pathogens. One such approach that we have been investigating is the use of a group of cationic peptides produced by a Gram-positive soil bacterium, Brevibacillus texasporus (BT peptides). We have previously shown that, provided as a feed additive, BT peptides significantly induced a concentration-dependent protection against cecal colonization and extraintestinal colonization by Salmonella enterica serovar Enteritidis (SE). This protection is not the result of direct antibacterial activity of the BT peptides on the SE since the concentrations used were below the minimum inhibitory concentration for SE. We also found that BT are not absorbed in the intestine, but still induce a significant up-regulation in the functional efficiency of peripheral blood heterophils and monocytes. The mechanisms of this immune modulation are unknown. Here, using in vitro models for measuring: (1) leukocyte oxidative burst, (2) changes in leukocyte cytokine and chemokines gene expression profiles, and (3) phosphorylation of the mitogen activated protein kinases (MAPKs) in leukocytes, we evaluated the role of BT peptides as priming mediators for heterophil and monocyte responses at the level of cell function, gene transcription/expression, and cell phosphorylation following stimulation with inflammatory agonists. BT peptides primed both heterophils and monocytes for an increased oxidative burst and up-regulation in transcription of the pro-inflammatory cytokines IL-1β and IL-6 and inflammatory chemokines CXCLi1 and CXCLi2 induced by inflammatory agonists. In addition, BT peptides induced a rapid (10 min) phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2) and p38 kinase pathways in primary chicken heterophils. Taken together, we conclude that BT peptides, acting through MAPK pathways, enhance leukocyte functional and pro-inflammatory cytokine and chemokine gene transcription activities. These small cationic peptides may prove useful as immune modulators in neonatal poultry.

A class of innate receptors called the triggering receptors expressed on myeloid cells (TREM) has been discovered and shown to be involved in innate inflammatory responses. The TREM family has been found in the chicken genome and consists of one activating gene (TREM-A1) and two inhibitory genes (TREM-B1 and TREM-B2). However, to date, there have been no reports on the effects of activating the TREM molecules on the functional activity of the primary avian polymorphonuclear cell, the heterophil. To characterize the activation of avian heterophils, we evaluated the effect of receptor ligation on heterophil effector functions. A specific agonistic antibody (Ab) was generated against the peptide sequence of chicken TREM-A1 38-51aa (YNPRQQRWREKSWC). To study TREM-A1 mediated activation, purified peripheral blood heterophils were incubated with various concentrations of the anti-TREM-A1 Ab or control Ab against an irrelevant antigen. Activation via TREM-A1 induces a significant increase in phagocytosis of Salmonella enteritidis, a rapid degranulation, and a dramatic up-regulation in gene expression of the pro-inflammatory cytokine, IL-6, and the inflammatory chemokine, CXCLi2. However, we found no direct TREM-A1 stimulation of the heterophil oxidative burst. Like mammalian TREM, avian TREM-A1 ligation synergizes with the activation of Toll-like receptor-4 (TLR4) ligand, LPS. In addition, the synergistic activity of LPS and TREM-A1 resulted in a significantly (p⩽0.05) increased production of an oxidative burst. Taken together, these results suggest, unlike in mammalian neutrophils, TREM-A1 engagement activates a differential functional activation of avian heterophils, but like mammalian neutrophils, acts in synergy with TLR agonists. These results provide evidence of the function of TREM-A1 in heterophil biology and avian innate immunity.

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.

The intestinal microbial ecosystem is a dense and diverse population that can be utilized to reduce pathogenic bacterial populations that affect animal production efficiency and the safety of food products. Strategies to capture and utilize this complex natural resource have been developed that reduce the populations of foodborne pathogenic bacteria and eliminate pathogens that negatively impact animal production or food safety on the farm. Products used in animals to reduce pathogens in the food supply include probiotics, prebiotics and competitive exclusion cultures, as well as bacteriocins and bacteriophage (bacterial viruses). The individual efficacy of any of these compounds is due to specific microbial ecological factors within the gut of the food animal and its native microflora that alter the competitive pressures of the gut. This review explores the ecology behind the efficacy of these products against foodborne pathogens that inhabit food animals.

Two-week-old Leghorn chicks injected intravenously with 5-fluorouracil (5-FU) were found to have a three- to fivefold reduction (P < .05) in circulating polymorphonuclear leukocytes (PMN) 5 to 10 days later. The reduction in PMN was dose-dependent with a dosage of 200 mg/kg body weight of 5-FU, causing suppression of PMN to almost undetectable levels. Increasing the dosage of 5-FU to 400 mg/kg had no further effect on reducing the number of PMN in the circulation nor were overt clinical signs of toxicity observed. Single per os administration of 10(6) viable Salmonella enteritidis (SE) to individual groups of chicks treated or not treated with 5-FU at 2, 4, 6, 8, 10, or 12 days postinjection resulted in a two- to sixfold increase in the incidence of SE organ invasion (liver and spleen) beginning on Day 4 postinjection and continuing through Day 12 postinjection. The greatest increase in organ invasion occurred on Days 8 through 10 postinjection and was inversely proportional to the greatest reduction in the number of circulating PMN. Using 5-FU, a granulocytopenic chicken model was developed that can be used to study the defensive role of avian heterophils against infectious agents. The results from these preliminary experiments indicate that PMN play a significant role in reducing organ invasion by SE in Leghorn chicks.

Byrnes S. , Baton R. and Kogut M. 1993. In vitro interleukin-1 and tumor necrosis factor-alpha production by macrophages from chickens infected with either Eimeria maxima or Eimeria tenella . International Journal for Parasitology 23 : 639–645. We measured the in vitro production of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-) by macrophages from chickens during and immediately following an infection with one of two different species of Eimeria , E. maxima and E. tenella . Quantitatively, the amounts of IL-1 produced during each infection were nearly identical regardless of the oocyst dose of each parasite. TNF production followed a biphasic pattern of increased production with the first peak associated with the pathogenesis of disease and the second peak associated with the development of protective immunity. These experiments together with others we have reported imply cells taken from chickens infected with coccidia have a greater capacity to produce cytokines upon stimulation in vitro than cells from non-infected birds. The production of significantly greater amounts of TNF during the days 3–6 after inoculation correlates with the appearance of the most characteristic local and systemic pathophysiological changes in the host induced by the coccidia. The excessive release of TNF in response to a heavy coccidial infection may account for many of the pathological features observed with avian coccidiosis probably through the release of other mediators.

A procedure for the isolation and primary culture of epithelial cells from the mucosal region of the upper ileum in the chicken is described. When cells are isolated using this technique, normal in vivo-like morphology and viability are obtained and can be maintained in short-term as well as primary cultures exceeding 3 wk. This procedure for isolation of enterocytes is easily performed and provides access to primary mucosal epithelial cell cultures for pathologic and physiologic investigations of isolated chicken enterocyte function.

We assayed two classes of immunoregulatory cytokines, colony-stimulating factors (CSF) and interferon (IFN), during and immediately after a primary coccidial infection in chickens. Coccidial infection induces significant alterations in serum colony-stimulating activity (CSA) and these alterations immediately precede the characteristic biphasic leukocytosis. CSA rose sharply during the first 24 h post-inoculation (PI), but returned to control levels by 48 h PI. At this time, we detected an increase in peripheral blood leukocytes which peaked at 96 h PI. A second phase of CSA increase began 96 h PI and peaked at 120-144 h PI which again preceded the second phase of leukocytosis. We also examined the production of IFN during the first 20 days PI. Splenic T cells from Eimeria maxima-infected chickens produced significantly less IFN on day 5 PI compared to T cells from the coccidia-free controls. By days 10 and 15 PI, there was no significant difference in IFN production between the T cells of infected and non-infected chickens. However, by day 20 PI, IFN production by the T cells of the infected birds produced significantly more IFN than the control T cells. The results of our studies indicated the differential production of two different cytokines by chickens during and following a primary coccidial infection. Based on these experiments, CSF may be some of the first cytokines produced during an E. maxima-infection, while IFN may be one of the later cytokines produced.

Campylobacter jejuni (C. jejuni) is one of the most common causes of human bacterial enteritis worldwide primarily due to contaminated poultry products. Previously, we found a significant difference in C. jejuni colonization in the ceca between two genetically distinct broiler lines (Line A (resistant) has less colony than line B (susceptible) on day 7 post inoculation). We hypothesize that different mechanisms between these two genetic lines may affect their ability to resist C. jejuni colonization in chickens. The molecular mechanisms of the local host response to C. jejuni colonization in chickens have not been well understood. In the present study, to profile the cecal gene expression in the response to C. jejuni colonization and to compare differences between two lines at the molecular level, RNA of ceca from two genetic lines of chickens (A and B) were applied to a chicken whole genome microarray for a pair-comparison between inoculated (I) and non-inoculated (N) chickens within each line and between lines. Our results demonstrated that metabolism process and insulin receptor signaling pathways are key contributors to the different response to C. jejuni colonization between lines A and B. With C. jejuni inoculation, lymphocyte activation and lymphoid organ development functions are important for line A host defenses, while cell differentiation, communication and signaling pathways are important for line B. Interestingly, circadian rhythm appears play a critical role in host response of the more resistant A line to C. jejuni colonization. A dramatic differential host response was observed between these two lines of chickens. The more susceptible line B chickens responded to C. jejuni inoculation with a dramatic up-regulation in lipid, glucose, and amino acid metabolism, which is undoubtedly for use in the response to the colonization with little or no change in immune host defenses. However, in more resistant line A birds the host defense responses were characterized by an up-regulation lymphocyte activation, probably by regulatory T cells and an increased expression of the NLR recognition receptor NALP1. To our knowledge, this is the first time each of these responses has been observed in the avian response to an intestinal bacterial pathogen.

Toll-like receptors (TLRs) are a critical component of the innate immune response of mammalian and avian species. While most mammalian TLRs have been well characterized, the chicken-specific TLR15 has not been extensively studied. We recently demonstrated that TLR15 is differentially expressed between Salmonella-susceptible-and-resistant chickens, indicating a potential role in the innate immune response to infection with Salmonella. The aim of the present study was to gain better insight into the nature of the ligand for TLR15 by characterizing gene expression patterns of TLR15 by heterophils in response to numerous bacterial-derived TLR agonists LPS, flagellin, CpG oligodeoxynucleotides, lipotechoic acid (LTA), peptidoglycan (PGN), and Pam3CSK4 (PAM), stimulation with live Salmonella enterica serovar Enteritidis (SE-used as a positive control), chicken isolates of Escherichia coli (EC) and Enterococcus gallinarum (EG), the equine-specific pathogen Rhodococcus equi, and stimulation with heat-killed, and formalin-killed SE, EC, and EG. TLR15 expression increased significantly in response to stimulation with live, heat-killed and formalin-killed SE, EC, and EG, but was unaffected by stimulation with known TLR agonists and R. equi. Overall, these observations demonstrate that the individual TLR agonists are not the ligand for TLR15, and that TLR15 recognizes a unique, non-secreted, heat-stabile component of both Gram-negative and Gram-positive bacteria commonly found in and/or capable of causing disease in chickens.

The BT/TAMUS 2032 (BT) cationic peptides are a group of related cationic peptides produced by a Gram-positive soil bacterium, Brevibacillus texasporus. Cationic amphiphilic peptides produced by host cells have been found to stimulate or prime the innate immune responses in mammals, but little information is available on the effects of bacterial-produced peptides on host immunity. We have previously shown that BT, provided as a feed additive for 4 days after hatch, significantly induced protection against extraintestinal colonization by Salmonella enterica serovar Enteritidis. We also found that feeding BT significantly upregulated the functional efficiency of heterophils, the avian equivalent to mammalian neutrophils. The objective of the present study was to further evaluate the effect of BT as a nonantibiotic, antibacterial compound and a stimulator of the innate immune response of young chickens. BT, provided as a feed additive at three different concentrations (12, 24, or 48 ppm) for 4 days after hatch, significantly increased protection against Salmonella enterica serovar Enteritidis cecal colonization in a concentration-dependent manner. We also confirmed our previous results that the functional activities of heterophils from chickens fed the BT rations were significantly upregulated. In addition, we also found that the functional activities of peripheral blood monocytes were significantly increased in a concentration-dependent manner when compared with monocytes isolated from chickens fed a control diet. This is the first report of bacterial cationic peptides providing protection against Salmonella cecal colonization. The significance of these data is that the orally delivered cationic peptides stimulate the innate response during the first week after hatch, normally a time of immunologic inefficiency and increased susceptibility to bacterial infections. We speculate that BT given as a feed additive during the first week after hatch could provide increased protection against a variety of bacterial pathogens because of the nonspecific nature of the innate response.

The scavenger receptors (SRs) comprise structurally and functionally divergent groups of cell surface and secreted proteins that play an important role in innate immune defenses. Searching translated chicken genomic databases revealed many proteins homologous to mammalian SRs. SR mediated immune functions (oxidative burst, degranulation, phagocytosis, nitric oxide (NO) production, and cytokine expression) were evaluated in chicken heterophils, peripheral blood mononuclear cells (PBMC), and a chicken macrophage cell line (HD11) using various SR class A and B ligands. Results showed that the SR-A ligands, fucoidan, poly(I) and poly(G), but not SR-B ligands, phosphatidylserine and LDL, stimulated dose-dependent NO production in HD11 cells. However, SR-A ligands failed to induce NO in chicken monocytes. Quantitative RT-PCR indicated that SR ligands differentially regulated the gene expression of cytokines and chemokine in HD11 cells with a strong up-regulation of the cytokines IL-1 beta and IL-6 and the chemokine MIP-1 beta, but had no effect on IL-4, IL-12, IFN-gamma, and IFN-beta. SR-B ligands did not alter expression of these genes. SR-A ligands had no stimulatory effect on functional response in heterophils. However, LDL, a SR-B ligand stimulated oxidative burst in both heterophils and PBMC. Additionally, results indicate that SRs are involved in bacterial binding in macrophages.

Cytokines are soluble, low molecular weight polypeptides and glycopeptides produced by a broad range of cell types of haematopoietic and nonhaematopoietic origin that have suppressive or enhansive effects on cellular proliferation, differentiation, activation, and motility. Like hormones, cytokines mediate cellular responses through autocrine, paracrine, and endocrine activities. Both inflammatory responses and specific immune responses to invasive microbes, which were evolved to protect the host from pathogens, are controlled by a complex network of cytokines. As regulators of the initiation and maintenance of host defenses, cytokines ultimately determine the type of response generated and the effector mechanisms generated to mediate resistance. As effector molecules, cytokines are produced transiently and locally to control the amplitude and duration of the response. Likewise, excessive or insufficient production of a cytokine or cytokines may contribute significantly to the pathophysiology of disease. Therefore, cytokines play pivotal but paradoxical roles in both in the regulation of inflammation and immunity. The use of exogenous cytokines against infectious agents in poultry medicine has centered on: (a) their use as adjuvants for vaccines, (b) their direct effects on inducing protection against infections and/or the undesired effects of immune responses elicited by pathogens, and (c) their ability to stimulate the ontogeny and activation of neonatal host defenses. This monograph will review what has been reported about the use of cytokines in poultry in these three main areas.

Previously, our laboratory reported an existing relationship between ontogenesis of heterophil activity and susceptibility to Salmonella enteritidis infection in poultry during the first week post-hatch. The prophylactic administration of S. enteritidis-immune lymphokines has been shown to enhance heterophil function in vitro and reduce S. enteritidis organ invasion in 1-day-old chicks. However, how long the heterophils remain activated is currently unknown. The objective of this research was to evaluate the duration of enhanced heterophil phagocytosis of S. enteritidis following the prophylactic administration of a single injection of S. enteritidis-immune lymphokines to neonatal chicks. Administration of S. enteritidis-immune lymphokines on the day of hatch resulted in a significant increase in heterophil phagocytic activity (P < 0.05) through day 5 post-hatch. No significant differences in phagocytic activities of heterophils from control and S. enteritidis-immune lymphokinetreated chicks was demonstrated from day 6 to 14 post-hatch. These data suggest that the administration of S. enteritidis-immune lymphokines on day of hatch potentiates heterophil phagocytic activity during a critical period of susceptibility to Salmonella infection in neonatal chicks. The immune lymphokine-induced enhancement of heterophil activity subsides by day 5 as the chick's natural defenses mature and are able to resist infections without exogenous augmentation.

The role of T lymphocytes on the development of turkey coccidia in chickens was investigated to further document the involvement of immune mechanisms in the reactions of non-specific hosts to infections with species of Eimeria. All chickens treated with the T cell-specific immunosuppressive drug, cyclosporin A (CsA), 1, 2 or 3 days before or for 3 consecutive days immediately before inoculation with sporulated oocysts of Eimeria from the turkey, shed oocysts in their feces 7–12 days after inoculation. Those chickens treated with CsA after inoculation produced no oocysts although in one experiment a small number of oocysts were discharged by birds injected with CsA 1 day after inoculation. High serum levels of CsA were maintained in the CsA-treated chickens for at least 24 h post-inoculation with the oocysts. Immunosuppressive activity of the drug was determined by measuring lymphocyte blastogenesis in response to concanavalin A. These results demonstrate that the suppression of T lymphocyte activity in a non-specific host early during an infection with a heterologous species of Eimeria permits the complete intracellular development of the parasite. Our findings are suggestive of a role for T lymphocytes in preventing the development of Eimeria in non-specific hosts possibly via a lymphokine-mediated mechanism.

Background Salmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis and usually results from the consumption of contaminated poultry products. The mechanism of SE resistance in chickens remains largely unknown. Previously, heterophils isolated from broilers with different genetic backgrounds (SE-resistant [line A] and -susceptible [line B]) have been shown to be important in defending against SE infections. To dissect the interplay between heterophils and SE infection, we utilized large-scale gene expression profiling. Results The results showed more differentially expressed genes were found between different lines than between infection (SE-treated) and non-infection (control) samples within line. However, the numbers of expressed immune-related genes between these two comparisons were dramatically different. More genes related to immune function were down-regulated in line B than line A. The analysis of the immune-related genes indicated that SE infection induced a stronger, up-regulated gene expression of line heterophils A than line B, and these genes include several components in the Toll-like receptor (TLR) signaling pathway, and genes involved in T-helper cell activation. Conclusion We found: (1) A divergent expression pattern of immune-related genes between lines of different genetic backgrounds. The higher expression of immune-related genes might be more beneficial to enhance host immunity in the resistant line; (2) a similar TLR regulatory network might exist in both lines, where a possible MyD88-independent pathway may participate in the regulation of host innate immunity; (3) the genes exclusively differentially expressed in line A or line B with SE infection provided strong candidates for further investigating SE resistance and susceptibility. These findings have laid the foundation for future studies of TLR pathway regulation and cellular modulation of SE infection in chickens.

Salmonella enterica serovar Enteriditis (SE) causes a majority of foodborne illness in the U.S. A more productive avian innate immune response could reduce bacterial colonization and the incidence of infection in humans. However, quantification and comparison of the toll-like receptors (TLR), a component of the innate immune system that recognize bacterial pathogens, and their response to SE colonization across the avian gastrointestinal (GI) tract has not been reported. Therefore, we assessed these changes using real-time qRT-PCR to measure expression of TLR 1LA, 2A, 2B, 3, 4, 5, 7, 15, and 21 in the duodenum, jejunum, ileum, cecal tonsil, ceca, and large intestine of uninfected and SE-infected 2-day-old broiler chickens. Samples were collected soon after hatch to approximate natural SE exposure and to measure initial changes in the immune response to infection. All TLRs had measurable expression within the duodenum, jejunum, ileum, cecal tonsil, ceca, and large intestine. The general expression pattern, with the exception of TLR 21, showed distal GI segments had higher TLR mRNA expression than proximal segments. Infected chickens had increased expression of TLR 1LA, 2A, 4, and 15 in distal GI segments and upregulation of TLR 2B, 3, and 15 in proximal segments, including the duodenum. Interestingly, SE-infection caused downregulation of TLR 5, with no change in TLR 7 or 21. Overall, we provide a comprehensive report of mRNA expression profiles for the TLR family of innate immune receptors in the GI tract of 2-day-old broilers and their differential response to SE colonization.

Campylobacter jejuni is one of the most common causes of acute enteritis worldwide. Chickens are believed to be the main reservoir of C. jejuni. The role that host genetics play in resistance/susceptibility to C. jejuni colonization in broilers is still not clear. Day-old broilers from 2 parental lines (A and B) and their F(1) reciprocal crosses (C and D) were challenged orally with 10(5) cfu of C. jejuni to address the role of genetics in determining resistance/susceptibility to C. jejuni colonization in broilers. Cloacal swabs were collected on 6, 10, and 13 d postinoculation (dpi), and cecal contents cultured for C. jejuni on 7 and 14 dpi. The number of C. jejuni colonies in the cloacal swabs and cecal contents of each bird were recorded at each time point. Significantly fewer bacteria were found in the cecal contents from line A than B (P < 0.05) and cross D (A male x B female) when compared with cross C (A female x B male) at both 7 and 14 dpi. There was a significant correlation between C. jejuni counts in cloacal swabs and those in cecal contents. The results indicated that a paternal effect might be one of the important genetic factors influencing resistance to C. jejuni colonization in broilers.

Pattern recognition receptors (PRRs) are essential for recognition of conserved molecular constituents found on infectious microbes. Toll-like receptors (TLRs) are a critical component of the PRR repertoire and are coupled to downstream production of cytokines, chemokines, and antimicrobial peptides by TLR adaptor proteins. Our laboratory previously demonstrated a role for TLR function in the differential innate response of two lines of chickens to bacterial infections. The aim of the present study was to elucidate the role of TLRs in the differential innate responsiveness by measuring differences between lines A (resistant) and B (susceptible) in heterophil mRNA expression of selected TLRs (TLRs 4, 5, and 15) and TLR adaptor proteins (MyD88, TRIF, and TIRAP) in response to stimulation with Salmonella enterica serovar Enteritidis (SE). Although heterophils from both lines had significantly increased expression of TLR 15 mRNA in response to stimulation with SE, heterophils from chickens resistant to infection with SE had significantly greater levels of TLR 15 mRNA expression prior to and following stimulation with SE than heterophils from chickens susceptible to infection with SE. No significant differences were noted between lines in nonstimulated levels of TIRAP, but upon SE stimulation, line A birds had higher levels of expression than B birds. No significant differences were found in heterophils between lines for mRNA expression of TLRs 4 and 5 nor MyD88 and TRIF. These data indicate that differences in the gene expression of TLR 15 by heterophils likely accounts for some of the observed differences between the lines in their susceptibility to infection.

Functional and molecular techniques have both been employed to measure the production of cytokines following influenza infection. Historically, the use of functional or antibody-based techniques was employed in mammalian immunology. In avian immunology, only a few commercial antibodies are available to measure avian cytokines. However, the determination of the genomic sequence of Gallus gallus species has made it possible to measure cytokine induction without monoclonal antibody- or functional-based tests, but rather based on molecular techniques. Although these tests do not measure functionally expressed cytokines, the lack of reagents to identify and quantify avian cytokines makes them critical to extend any measure of cytokine response. Measurement of cytokine induction, based on the design of primers and probes for RT-PCR or real-time RT-PCR for the cytokine mRNA, has become one of the more recent technologies reported to measure avian cytokines. It is important to note that small nucleotide polymorphisms between different lines of birds may result in substandard results when using published primer and probe sequences. This requires empirical testing to ensure adequate results.

Previous studies using F1 reciprocal crosses and two parental lines of broilers show the sire is instrumental in determining the in vitro leukocyte function and cytokine/chemokine profile. Since the innate immune response is the primary means young chickens have to protect themselves, we hypothesize utilizing a novel genomics approach to select sires based on an elevated pro-inflammatory cytokine and chemokine profile. By identifying sires with increased pro-inflammatory cytokine (interleukin [IL]-1β and IL-6) and chemokine (CXCLi2 and CCLi2) mRNA expression levels, we expect the progeny will also have elevated profiles. We characterized the pro-inflammatory cytokine and chemokine profile of 119 sires using quantitative real-time RT-PCR (qRT-PCR) and identified two populations with inherently high and low mRNA expression levels of IL-1β, IL-6, CXCLi2, and CCLi2. Select high and low sires were then used to produce progeny for the second phase of the trial. Blood samples were collected from 214 progeny and the cytokine and chemokine mRNA expression levels determined. Progeny from high sires had significantly ( P ≤ 0.02) higher cytokine (IL-1β and IL-6) and chemokine (CXCLi2 and CCLi2) mRNA expression levels compared to progeny from low sires. We have identified a broiler population of sires with higher and lower than average pro-inflammatory cytokine/chemokine mRNA expression levels and used them to produce progeny with similar profiles.

Functional and molecular techniques have both been employed to measure the production of cytokines following influenza infection. Historically, the use of functional or antibody-based techniques was employed in mammalian immunology. In avian immunology, only a few commercial antibodies are available to measure avian cytokines. However, the determination of the genomic sequence of Gallus gallus species has made it possible to measure cytokine induction without monoclonal antibody- or functional-based tests, but rather based on molecular techniques. Although these tests do not measure functionally expressed cytokines, the lack of reagents to identify and quantify avian cytokines makes them critical to extend any measure of cytokine response. Measurement of cytokine induction, based on the design of primers and probes for RT-PCR or real-time RT-PCR for the cytokine mRNA, has become one of the more recent technologies reported to measure avian cytokines. It is important to note that small nucleotide polymorphisms between different lines of birds may result in substandard results when using published primer and probe sequences. This requires empirical testing to ensure adequate results.

The toll-like receptors (TLRs) recognize microbial pathogens and pathogen-associated molecular patterns and trigger inflammatory immune responses to control the infection. Here, we examined functional innate immune responses to Salmonella enteritidis (SE, live or formalin-killed) and various TLR agonists including lipoteichoic acid (LTA) and peptidoglycan (PGN) from Staphylococcus aureus and synthetic lipoprotein Pam3CSK4 (PAM), poly I:C (synthetic double-stranded RNA analog), lipopolysaccharide (LPS) from S. enteritidis, flagellin (FGN) from S. typhimurium, loxoribine (LOX) and R837 (synthetic anti-viral compounds), and CpG oligodeoxydinucleotide (CpG ODN)by measuring antimicrobial activities including oxidative burst and degranulation in heterophils and nitric oxide production in peripheral blood monocytes. Our results demonstrate differential nitric oxide responses to TLR agonists in turkey monocytes. LTA and CpG ODN were the most potent stimuli for nitric oxide induction followed by PAM, poly I:C, and LPS, whereas FGN, PGN, LOX, R837, and control ODN stimulated little or no nitric oxide production. Live SE stimulated significantly less NO production than formalin-killed SE (FKSE). Although FKSE induced significant degranulation and oxidative burst, most TLR agonists stimulate little oxidative burst and degranulation responses in turkey heterophils.

Previous work has shown that flagellin (FGN) is a potent stimulator in vitro of phagocytic cell functions of chickens. The purpose of this study was to define the effects of FGN on the inflammatory response to Salmonella enteritidis (SE) in chickens. Intra-abdominal (IA) FGN administration caused significant increases in peripheral blood leukocytes (PBL) compared with SE-injected controls at 4 and 8 h postinjection (Por=0.05). The heterophil (PMN) was the predominant cell responsible for the increased numbers of leukocytes in the peripheral blood. In the abdominal cavity, leukocyte infiltrates were significantly greater in FGN-injected (8 h) and SE/FGN-injected (4 and 24 h) birds than in the SE-injected control birds. Again, the predominant leukocyte infiltrating the abdominal cavity was the PMN. Bovine serum albumin (BSA)-injected protein control birds showed no increases in PBL or in abdominal cell leukocyte infiltrates over saline-injected controls. In IA challenge studies, FGN reduced SE-associated mortality (26%) compared with mortality of 51% in the SE group (Por=0.05). BSA-injected/SE-challenged chicks had mortality similar to that of the SE group. The data suggest that FGN is a potent stimulator of a heterophil-mediated innate immune response in vivo, protecting against bacterial infections in chickens.

Toll-like receptors (TLRs) recognize microbial components and initiate the innate immune responses that control microbial infections. We have investigated the innate immune response of chicken monocytes to ligands of TLR3 and TLR9, poly I:C (an analog of viral double-stranded RNA) and CpG-ODN (a CpG-motif containing oligodeoxydinucleotide) by measuring the induction of nitric oxide (NO) synthesis in chicken monocytes. Our results show that poly I:C and CpG-ODN synergized the induction of NO. When stimulated separately, CpG-ODN induced significant NO production in the chicken monocytes; whereas, poly I:C stimulated very little NO production. In combination, CpG-ODN and poly I:C induced significantly higher level of NO in chicken monocytes than either agonist alone. The addition of poly I:C prior to or simultaneously with CpG-ODN was required for the synergy. No synergistic effects on NO production were observed when monocytes were stimulated with combinations of CpG-ODN or poly I:C with other TLR agonists. Unlike chicken monocytes, cells of a chicken macrophage cell line, HD11, were readily stimulated to produce NO by both CpG-ODN and poly I:C with no synergism on NO induction when HD11 cells were stimulated by a combination of CpG-ODN and poly I:C. Using a pharmacological inhibitor, we also demonstrated that double-stranded RNA-dependent protein kinase (PKR) is indispensable for stimulation of NO production by CpG-ODN alone or in combination with poly I:C in both chicken peripheral blood monocytes and HD11 macrophage cells. Our results show that a combination of bacterial DNA and dsRNA induces an enhanced inflammatory immune response that has both antiviral and antibacterial activity in primary chicken monocytes.

We evaluated the innate immune response to various synthetic CpG-containing oligodeoxynucleotides (CpG ODNs) by measuring nitric oxide production in the peripheral blood monocytes from turkey poults. The results indicate that the presence of the CpG dinucleotide in ODNs was a prerequisite for activation of turkey monocytes and induction of nitric oxide (NO) synthesis. CpG motifs and sequence structure of the ODNs were also found to influence stimulatory activity greatly. The most potent CpG ODN to induce NO synthesis in turkey monocytes was human-specific CpG ODN M362, followed by CpG ODN 2006 (human), CpG ODN#17 (chicken) and CpG ODN 1826 (mouse). The optimal CpG motif for NO induction was GTCGTT. Phosphorothioate modification of CpG ODNs also significantly increased stimulatory activity. Compared with chicken monocytes, turkey monocytes appeared to be less sensitive to CpG motif variation, whereas chicken monocytes were found to respond more strictly to human-specific CpG ODNs or ODNs that contain GTCGTT motifs.

The TLR agonists, flagellin (FLG) and lipopolysaccharide (LPS) stimulate functional activation and cytokine gene expression via the extracellular signal regulated kinase 1/2 (ERK1/2) MAP kinase cascade. However, the upstream mechanisms of these signaling events remain unknown. In mammals, the small GTP-binding protein Ras mediates ERK1/2 activation through activation of downstream effectors Raf-1-MEK1/2-ERK1/2 in response to a variety of stimuli. It is not clear whether this classic Ras cascade plays a role in TLR signaling in avian cells. In the present study, we investigated the role of Ras in FLG- and LPS-mediated signaling in ERK activation in chicken heterophils. Treatment of heterophils with LPS caused a rapid (within 5 min) activation of Ras-GTP. The role of Ras activation in LPS-induced stimulation of ERK1/2 was corroborated when the specific Ras inhibitor, FTI-277, inhibited ERK1/2 activation. The classic Ras-mediated pathway of ERK1/2 activation by LPS was confirmed when the specific Raf-1 inhibitor, GW 5074, and the MEK1/2 inhibitor, U0126, both reduced ERK activation by 51–60%. Of more interest was that treatment of the heterophils with FLG did not activate Ras-GTP. Likewise, neither FTI-277 nor GW 5074 had any effect on FLG-mediated activation of ERK1/2. Another small GTPase, Rap1, has been shown to play a role in mammalian neutrophil function. Using a Rap1-GTP pull-down assay, we found that FLG stimulation, but not LPS, of avian heterophils induced a rapid and transient Rap1 activation. Rap1 has been shown to activate the ERK1/2 via a different Raf family member B-Raf whose downstream effector is MEK1/2. We show here that FLG stimulation of heterophils induces the phosphorylation of Rap1. The FLG induction of the Rap1 → B-Raf → MEK1/2 → ERK1/2 cascade was confirmed by the reduction of ERK1/2 activation by the specific Rap1 inhibitor (GGTI-298) and U0126. The results demonstrate that for the first time that the small GTPase Ras family is involved in TLR signaling of avian heterophils with the TLR agonists LPS (Ras) and FLG (Rap1) inducing differential signaling cascades to activate the downstream ERK MAP kinase.

Previously we demonstrated that increased in-vitro heterophil function translates to increased in-vivo resistance to Salmonella enteritidis infections in broilers (line A > B). Heterophils produce cytokines and modulate acute protection against Salmonella in neonatal poultry. We hypothesized that heterophils from S. enteritidis-resistant chickens produce an up-regulated pro-inflammatory cytokine/chemokine response compared with S. enteritidis-susceptible chickens. In this study, heterophils were isolated 1, 14, and 28 days post-hatch, treated with RPMI or phagocytic agonists, and the cytokine/chemokine mRNA expression assessed using quantitative real-time reverse transcriptase-polymerase chain reaction. At all time-points, heterophils from S. enteritidis-resistant chickens (line A) had higher levels of pro-inflammatory cytokine/chemokine mRNA expression upon stimulation compared with heterophils from S. enteritidis-susceptible chickens (line B). Furthermore, heterophils from line A chickens had decreased mRNA expression of transforming growth factor-beta4, an anti-inflammatory cytokine, compared with line B. These data indicate a relationship between cytokine/chemokine mRNA expression by heterophils and determining overall immune competence. Therefore, heterophil functional efficiency, accompanied by evaluating cytokine/chemokines produced by heterophils, may be useful biomarkers for breeders to consider when developing new immunocompetent lines.

Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern (PAMP) found in the cell wall of gram-negative bacteria and, in mammals, is recognized by the Toll-like receptor 4 (TLR4) in conjunction with the serum protein, lipopolysaccharide-binding protein (LBP), and the CD14 co-receptor. We have found that chicken heterophils constitutively express multiple TLRs including TLR4. Interestingly, ultrapure LPS from Salmonella minnesota directly induced the functional activation of heterophils without the presence of LBP. However, the role of LBP and CD14 in the recognition of LPS and the induction of innate immunity, including cellfunctional activation and the transcription of cytokine and chemokine genes in chicken heterophils, is not known. As previously seen, in the absence of chicken serum, heterophil exposure to ultrapure LPS from Salmonella minnesota stimulated an increased degranulation response. However, the presence of 5% chicken serum, presumed to be a source of LBP, increased heterophil degranulation by 84%. In addition, the presence of either soluble recombinant human LBP (rhLBP, 68%) or CD14 (39%) also induced the up-regulation of the heterophil degranulation response. Incubation of heterophils with either chicken serum or rhLBP also significantly induced the up-regulation of pro-inflammatory cytokine (IL-1beta, IL-6, and IL-18) and chemokine (CCLi4, CXCLi1, CXCLi2, and the CXC receptor 1) mRNA expression. Moreover, polyclonal antibodies directed against rat CD14 and human TLR4, but not antibodies against human TLR2, blocked LPS-mediated degranulation and up-regulation of the pro-inflammatory cytokine and chemokine mRNA expression. These data clearly demonstrate that LBP and CD14/TLR4 engagement is directly involved in LPS-mediated functional activation and innate immune gene expression in chicken heterophils.

Heterophils isolated from distinct broilers (lines A and B) differ in function and cytokine gene expression profiles. Nothing is known about Toll-like receptor (TLR) expression nor functional activation and cytokine/chemokine gene expression of line A and B heterophils when stimulated with TLR agonists. We found that line A and B heterophils express the same range of TLRs. All the bacterial TLR agonists, peptidoglycan, the synthetic lipoprotein Pam3CSK4, ultra-pure lipopolysaccharide, and flagellin all induced significantly greater functional activation of heterophils from line A compared to B. Only stimulation with the guanosine analog, loxoribine, (LOX) induced a significantly greater functional response in B over A. Additionally, all heterophils from line A stimulated with the bacterial TLR agonists had dramatic upregulation of pro-inflammatory cytokine and chemokine mRNA expression, whereas heterophils from line B had little or no upregulation of these genes. However, stimulation of all heterophils from line B with the bacterial TLR agonists and LOX induced a significant upregulation of IFN-alpha, with little transcription of this cytokine gene in line A heterophils. These findings suggest that the difference in heterophil functional efficiency between these parent lines is due to recognition of pathogens and activation of signaling pathways that induce innate cytokine and chemokine responses.

Neutrophil oxidative burst/degranulation activities were measured using CpG in vitro stimulation. Neutrophils isolated from pigs 6 weeks of age were incubated with treatment for 1 hr at 39°C. Neutrophils had increased degranulation activity when treated with CpG when compared to the activity of cells treated with non CpG. Neutrophils treated with CpG exhibited an increase in oxidative burst. CpG resulted in levels of activity comparable to those observed with PMA stimulation. Two of the swine CpG (p2006 and pD19) were chosen for administration to swine to observe the effects on S. choleraesuis (SC) infection. Pigs were administered 1mg of CpG and subsequently challenged with SC. SC numbers in cecal contents in one CpG group were reduced by two to three logs when compared to control numbers and other CpG groups. In addition, reductions in SC positive rectal swabs, liver, spleen, jejunal, ileal, and colon colonization were observed. Introduction The mammalian neutrophil is a first line of defense against invading pathogens and represents an important part of the innate immune defenses. The immunostimulatory activities of certain DNA antigens expressed by bacteria and other pathogens have been found to be linked to the recognition of these antigens by host receptors in the Toll-like receptor (TLR) family of receptors (Hemmi et al, 2000). Bacterial DNA has been found to be a potent immune stimulator and this activity has been linked to the presence of a high frequency of unmethylated CpG dinucleotides. CpG oligodeoxynucleotides (CpG-ODN) have been found to interact with TLR9 (Hemmi et al, 2000; Bauer et al, 2001). This interaction leads to a cascade of intracellular signaling resulting in gene expression and the production of immunomodulatory molecules including cytokines, helping to formulate aspects of both the innate and the adaptive host responses to invading organisms (Freytag, Clements 2005). CpG-ODN could be used as adjuvants for vaccines to increase their effectiveness as well as be used on their own as immune stimulators in domestic food animals and in human medicine (Freytag, Clements 2005; Verfaillie et al, 2005). Salmonellae are known to colonize the gut of domestic swine and pose a risk for food born disease in humans. In addition, multiple serotypes are known to cause disease in swine including Salmonella choleraesuis, S. Typhimurium, and S. typhisuis, along with many others. Disease can vary from enterocolitis, septicemia, pneumonia to meningitis/encephalitis and mortality. In humans, food born disease from contaminated food products, including pork products, remains a continuing public health concern. The purpose of the present investigation was to compare the effects of CpG-ODN known to stimulate immune responses in humans, pigs, and mice on neutrophils from swine in vitro and to determine if the oral administration of CpG-ODN in weaned pigs would reduce gut colonization and fecal excretion of S. choleraesuis in vivo. Materials and Methods Neutrophils were isolated from pigs (approx. 10kg body weight) using density gradient centrifugation. CpG-ODN The following CpG-ODN and non-CpG containing control (n2041) (Biosource International) were used for in vitro and in vivo (marked with “x”) assays: pD19 G*G*T*G*C*A*T*C*G*A*T*G*C*A*G*G*G*G*G*G x p2006 T*C*A*T*C*G*A*T*T*T*A*T*C*G*A*T*T*A*T*C*G*A*T x n2041 C*T*G*G*T*C*T*T*T*C*T*G*G*T*T*T*T*T*T*T*C*T*G*G x CpG#17 G*T*C*G*T*T*G*T*C*G*T*T*G*T*C*G*T*T Degranulation assay Neutrophils (8x10 cells/ml) were incubated with respective stimulant for 1 hour at 39°C. Tubes were then transferred to an ice bath for 10 minutes. Tubes were centrifuged at 250x g for 10min. at 4°C. Supernatants were then removed and stored at 4C. Each sample supernatant (25μl) or appropriate standard was added to each well in a costar flat bottom elisa plate #3915, non-treated, opaque and incubated with 50μl of freshly prepared substrate for 4h at 41°C in the dark. Stop solution was then added (200μl). Liberated 4-methylumbelliferone was P O S T E R P R E S E N T A T IO N S 272 SafePork 2005 measured fluorimetrically (excitation: 355nm, emission: 460nm) with a GENios Plus Fluorescence Microplate Reader (Tecan US Inc.). Values were extrapolated from the standard curve. Oxidative burst assay Production of ROS by swine neutrophils during oxidative burst was measured by the oxidation of DCFH-DA to fluorescent DCF. Neutrophils (1 ml at 8x10 cells/ml) were incubated with stimulants and DCFH-DA (10μg/ml) for 1 h at room temperature. Aliquots (150μl) were then placed into black 96-well plates and the fluorescence was measured using a GENios Plus Fluorescence Microplate Reader (Tecan US Inc.) at 485 nm excitation and 530 emission wavelengths. The fluorescent units were recorded (RFU). In vivo S. choleraesuis study Twelve pigs, average weight 26kg, were randomly assigned to 1 of 4 groups. Pigs were orally administered the respective treatment or control (1mg/pig) in 2ml volumes. Twenty four hours later, pigs were challenged by oral gavage with 109 colony-forming units (cfu) of S. choleraesuis. Rectal swabs were obtained daily from each pig and pigs in all groups were euthanized on day 7. At necropsy, tissues and contents from organs and the gut were cultured for the presence of S. choleraesuis using standard isolation techniques. Results Results from in vivo culture of daily rectal swabs and tissue cultured for the presence of S. choleraesuis are presented below. < CFU determinations in cecal contents were as follows: < 2041 – 3.4 x 10 CFU/g content < p2006 – 3.5 x 10 CFU/g content < pD19 – 10 CFU/g content < SC control – 4.4 x 103 CFU/g content Total # of positive samples/group in ICLN, Liver, spleen, jejunum, ileum, cecum, and colon combined: < 2041 – 17/21 < p2006 – 15/21 < pD19 – 8/21 < SC control – 16/21 Total Number of daily rectal swabs positive for S. choleraesuis over 7 days: < 2041 – 15/21 < p2006 – 17/21 < pD19 – 7/21 < SC control – 14/21 Results of the in vitro degranulation and oxidative burst assays are presented in Figures 1 and 2. Discussion The use of CpG sequences as adjuvants or as direct immunostimulators presents a novel tool for the reduction of pathogens in food animals as well as people. The induction of the host’s endogenous immune mechanisms to fight infection and/or disease is also one way in which food animal producers could reduce the use of antibiotics as prophylactic or growth promoter measures, although a balance between growth potential of livestock and the energy costs of host immune functions must be considered. In the present study, CpG DNA (pD19) was found to reduce Salmonella colonization in the gut and organs of swine. In addition, neutrophils isolated from the peripheral blood of pigs showed increased functional characteristics in the presence of CpG DNA (p2006, CpG 17, pD19) sequences than those not exposed to these sequences. Studies in chickens have also shown the effectiveness of CpG DNA in protecting against Salmonella infections and in stimulating the innate immune functions of the avian host (He et al, 2005). Although on their own, CpG 0 5 1 0 1 5 2 0 2 5 3 0 3 5 Neutrophil Degranulation % ch an ge fr om ne ga tiv e co nt ro l 2041 p2006 pD19 CpG#17 PMA

Previous studies showed differences in in vitro heterophil function between parental (A>B) broilers and F1 reciprocal crosses (D>C). Our objectives were to (1) determine if in vitro variations translate to differences in resistance to Salmonella enteritidis (SE) and (2) quantitate cytokine mRNA in heterophils from SE-infected chicks. One-day-old chicks were challenged and organs were cultured for SE. Chicks with efficient heterophils (A and D) were less susceptible to SE compared to chicks with inefficient heterophils (B and C). Heterophils were isolated from SE-infected chicks and cytokine mRNA expression was evaluated using quantitative real-time RT–PCR. Pro-inflammatory cytokine mRNA was up-regulated in heterophils from SE-resistant chicks compared to susceptible chicks. This is the first report to quantitate cytokine mRNA in heterophils from SE-infected chicks. These data show a relationship between in vitro heterophil function, increased pro-inflammatory cytokine mRNA expression, and increased resistance to SE in 1-day-old chicks.

Unmethylated CpG oligodinucleotides (CpG-ODN) flanked by specific bases found in bacterial DNA are known to stimulate innate immune responses. In this study, synthetic CpG-ODNs were evaluated for their in vitro stimulation of leukocyte and in vivo protection against Salmonella enteritidis (SE) in neonatal chickens. Our studies showed that CpG-ODN stimulated bactericidal activities, releasing granules (degranulation) and generating reactive oxygen species (oxidative burst), in chicken heterophils and up regulated nitric oxide production in chicken peripheral blood monocytes. When day-old chickens were given (i.p.) synthetic CpG-ODNs followed by oral challenge of SE, a significant reduction (p < 0.05) of organ invasion by SE was observed in chickens pretreated with CpG-ODN containing the immunostimulatory GTCGTT motif. This CpG-OND also significantly reduced mortality of chickens with acute peritoneal infection of SE. Our study provides evidence that immunostimulatory CpG-ODN stimulated innate immune activities and enhanced the resistance to infectious pathogens in neonatal chickens.

Synthetic oligodeoxynucleotide containing unmethylated CpG motif (CpG-ODN) is immune stimulatory to chicken heterophils. Recognition of CpG-ODN by chicken heterophils leads to the mobilization and release of granules. This CpG-ODN-induced heterophil degranulation was chicken serum (CS)-dependent. Heat-denaturation and membrane filtration of CS revealed that the active serum cofactor(s) was likely a protein in nature with a molecule mass within 50,000 to 100,000. This serum cofactor(s) was heat-resistant at 56 degrees C for 1h. The involvement of a cell surface receptor in recognition of CpG-ODN was also demonstrated by (1) trypsin treatment of the heterophils abrogated the degranulation response and (2) CpG-ODN-induced heterophil degranulation was sensitive to the inhibition of Clathrin-dependent endocytosis. In addition, among various microbial agonists, including CpG-ODN, lipopolysaccharide, lipoteichoic acid, phorbol myristate acetate, and formalin-killed Salmonella enteritidis, CpG-ODN was the only agonist that displayed serum-dependent induction of degranulation in chicken heterophils. This is the first report that shows serum-dependent activation of leukocytes by CpG-ODN.

Heterophils, the principal avian polymorphonuclear leukocytes (PMNs) equivalent to the mammalian neutrophil, function as professional phagocytes against bacterial infections, mediate acute inflammation, and respond to cytokine stimulation to aid in regulation of innate host defenses. Interleukin-2 (IL-2) has been found to exercise an array of biological effects on other cell types besides T lymphocytes, including NK cells, B cells, monocytes, and neutrophils. In the present experiments, using real-time quantitative RT-PCR, we evaluated the role of rChIL-2 as a priming mediator controlling heterophil responses at the level of gene transcription by examining the expression of mRNA for pro-inflammatory (IL-1, IL-6, IL-8) and Th1 (IL-18 and IFN-) cytokine genes following stimulation with phagocytosis agonists; i.e., opsonized and nonopsonized Salmonella enteritidis. Peripheral blood heterophils were isolated and incubated with rChIL-2 from transfected COS cells. rChIL-2 selectively primed the heterophils for an increase in transcription of the pro-inflammatory cytokine IL-8 and of theTh1 cytokine IL-18 induced by all three phagocytic agonists. Although rChIL-2 priming modulated the expression of specific cytokine mRNA in heterophils stimulated by different phagocytic agonists, the rChIL-2 by itself did not directly induce gene expression of either the pro-inflammatory or Th1 cytokines. We propose that rChIL-2 could be priming heterophils solely to function as more efficient innate effector cells to limit bacterial growth through the selective increase of IL-8 and IL-18 gene expression. Published by Elsevier Ltd.

Toll-like receptors (TLRs) play an important role in the innate immune response of avian heterophils. We previously used the pharmacological inhibitors genistein, verapamil, chelerythrine, and pertussis toxin to investigate the upstream signaling events involved in TLR2-mediated oxidative burst in chicken heterophils. Only chelerythrine, a protein kinase C inhibitor, was found to significantly inhibit oxidative burst stimulated by the TLR2 agonist lipoteichoic acid (LTA). In the present study, we used selective pharmacological inhibitors to investigate the roles of phosphatidylinositol-3'-kinase (PI3-K), phospholipase C (PLC), calcium-dependent protein kinase C (PKC), extra-cellular signal regulated kinase (ERK), and nuclear translocation factor kappa B (NF-kappaB) on TLR2-mediated oxidative burst. U-73122 (a PLC inhibitor), wortmannin (a PI3-K inhibitor), PD 98059 (an ERK inhibitor), Gö 6976 (a PKC inhibitor) and Bay 11-7082 (a NF-kappaB inhibitor) significantly decreased LTA-stimulated oxidative burst in heterophils by 77%, 30%, 36%, 78%, and 61%, respectively. Activated TLR2 utilizes PI3-K, PLC, PKC, ERK, and NF-kappaB as signaling factors that mediate the oxidative burst of chicken heterophils.

Functionally, the innate immune system of immature chickens is inefficient during the first week posthatch. This immunological inefficiency enables pathogens such as Salmonella enterica serovar Enteritidis (SE) to invade and colonize the visceral organs of immature chickens. The objective of this study was to evaluate the effect of purified beta-glucan as an immunomodulator of the innate immune response. beta-glucan, as a feed additive, significantly provided protection against SE organ invasion in young chickens (P