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 heterophils isolated from line A birds are functionally more responsive. Furthermore, when stimulated with Toll-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 IκB kinases transforming growth factor-β-activating kinase 1 (TAK1), IKKε and IKKα, 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 resistant line A. These findings identify new targets for genetic selection of chickens to increase resistance to bacterial infections.

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 and Campylobacter are the two leading causes of bacterial-induced foodborne illness in the US. Food production animals including cattle, swine, and chickens are transmission sources for both pathogens. The number of Salmonella outbreaks attributed to poultry has decreased. However, the same cannot be said for Campylobacter where 50–70% of human cases result from poultry products. The poultry industry selects heavily on performance traits which adversely affects immune competence. Despite increasing demand for poultry, regulations and public outcry resulted in the ban of antibiotic growth promoters, pressuring the industry to find alternatives to manage flock health. One approach is to incorporate a program that naturally enhances/modulates the bird’s immune response. Immunomodulation of the immune system can be achieved using a targeted dietary supplementation and/or feed additive to alter immune function. Science-based modulation of the immune system targets ways to reduce inflammation, boost a weakened response, manage gut health, and provide an alternative approach to prevent disease and control foodborne pathogens when conventional methods are not efficacious or not available. The role of immunomodulation is just one aspect of an integrated, coordinated approach to produce healthy birds that are also safe and wholesome products for consumers.

Economic pressure on the modern poultry industry has directed the selection process towards fast-growing broilers that have a reduced feed conversion ratio. Selection based heavily on growth characteristics could adversely affect immune competence leaving chickens more susceptible to disease. Since the innate immune response directs the acquired immune response, efforts to select poultry with an efficient innate immune response would be beneficial. Our laboratories have been evaluating the innate immune system of two parental broiler lines to assess their capacity to protect against multiple infections. We have shown increased in vitro heterophil function corresponds with increased in vivo resistance to Gram-positive and Gram-negative bacterial infections. Additionally, there are increased mRNA expression levels of pro-inflammatory cytokines/chemokines in heterophils isolated from resistant lines compared to susceptible lines. Collectively, all data indicate there are measurable differences in innate responsiveness under genetic control. Recently, a small-scale selection trial was begun. We identified sires within a broiler population with higher and/or lower-than-average pro-inflammatory cytokine/chemokine mRNA expression levels and subsequently utilized small numbers of high-expressing and low-expressing sires to produce progeny with increased or decreased, respectively, pro-inflammatory cytokine/chemokine profiles. This novel approach should allow us to improve breeding stock by improving the overall immunological responsiveness. This will produce a line of chickens with an effective pro-inflammatory innate immune response that should improve resistance against diverse pathogens, improve responses to vaccines, and increase livability. Ongoing work from this project is providing fundamental information for the development of poultry lines that will be inherently resistant to colonization by pathogenic and food-poisoning microorganisms. Utilization of pathogen-resistant birds by the poultry production industry would significantly enhance the microbiological safety of poultry products reaching the consumer.

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.