Advances, challenges and tools in characterizing bacterial serine, threonine and tyrosine kinases and phosphorylation target sites.

INTRODUCTION: The cellular response to infection by bacterial pathogens involves a complex and highly regulated series of pathways that carry messages to various parts of the cell. These messages are transferred using post-translational modifications including phosphorylation by kinases. Understanding the host's signaling pathways is valuable in identifying potential treatment targets, but the bacterial signaling pathways and host-pathogen crosstalk are equally important to the development of therapeutics. Areas covered: This review summarizes some of the recent findings related to the bacterial phosphoproteome and especially serine/threonine/tyrosine sites, including methods and considerations for identifying novel phosphosites. We also consider the bioinformatics tools that have been developed to sift through the large volume of data in these studies and connect them to biologically relevant knowledge about pathways and function. Literature databases used include PubMed and Google Scholar from April 2018 to December 2018. Expert opinion: While the field has developed significantly in the past decade of research, high-quality experimental sequence data remains the limiting factor to future research into bacterial phosphoproteomics. As more proteomes are explored, it will be easier to tailor tools and techniques to prokaryotes. It will be necessary to consider the phosphoproteome in the broader biological context, through interdisciplinary collaborations.

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.

Induction of tissue- and stressor-specific kinomic responses in chickens exposed to hot and cold stresses.

Defining cellular responses at the level of global cellular kinase (kinome) activity is a powerful approach to deciphering complex biology and identifying biomarkers. Here we report on the development of a chicken-specific peptide array and its application to characterizing kinome responses within the breast (pectoralis major) and thigh (iliotibialis) muscles of poultry subject to temperature stress to mimic conditions experienced by birds during commercial transport. Breast and thigh muscles exhibited unique kinome profiles, highlighting the distinct nature of these tissues. Against these distinct backgrounds, tissue- and temperature-specific kinome responses were observed. In breast, both cold and hot stresses activated calcium-dependent metabolic adaptations. Also within breast, but specific to cold stress, was the activation of ErbB signaling as well as dynamic patterns of phosphorylation of AMPK, a key regulatory enzyme of metabolism. In thigh, cold stress induced responses suggestive of the occurrence of tissue damage, including activation of innate immune signaling pathways and tissue repair pathways (TGF-ß). In contrast, heat stress in thigh activated pathways associated with protein and fat metabolism through adipocytokine and mammalian target of rapamycin (mTOR) signaling. Defining the responses of these tissues to these stresses through conventional markers of pH, glycolytic potential, and meat quality offered a similar conclusion of the tissue- and stressor-specific responses, validating the kinome results. Collectively, the results of this study highlight the unique cellular responses of breast and thigh tissues to heat and cold stresses and may offer insight into the unique susceptibilities, as well as functional consequences, of these tissues to thermal stress.

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.

From mouth to macrophage: mechanisms of innate immune subversion by Mycobacterium avium subsp. paratuberculosis.

Johne's disease (JD) is a chronic enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). The high economic cost and potential zoonotic threat of JD have driven efforts to develop tools and approaches to effectively manage this disease within livestock herds. Efforts to control JD through traditional animal management practices are complicated by MAP's ability to cause long-term environmental contamination as well as difficulties associated with diagnosis of JD in the pre-clinical stages. As such, there is particular emphasis on the development of an effective vaccine. This is a daunting challenge, in large part due to MAP's ability to subvert protective host immune responses. Accordingly, there is a priority to understand MAP's interaction with the bovine host: this may inform rational targets and approaches for therapeutic intervention. Here we review the early host defenses encountered by MAP and the strategies employed by the pathogen to avert or subvert these responses, during the critical period between ingestion and the establishment of persistent infection in macrophages.

Chicken intestinal organoids: a novel method to measure the mode of action of feed additives.

There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or "mini-guts" has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses.

Altered Toll-like receptor 9 signaling in Mycobacterium avium subsp. paratuberculosis-infected bovine monocytes reveals potential therapeutic targets.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle. The complex, multifaceted interaction of M. avium subsp. paratuberculosis with its host includes dampening the ability of infected cells to respond to stimuli that promote M. avium subsp. paratuberculosis clearance. By disrupting host defenses, M. avium subsp. paratuberculosis creates an intracellular environment that favors the establishment and maintenance of infection. Toll-like receptors (TLRs) are important sensors that initiate innate immune responses to microbial challenge and are also immunotherapeutic targets. For example, TLR9 contributes to host defense against M. avium subsp. paratuberculosis, and its agonists (CpG oligodeoxynucleotides [ODNs]) are under investigation for treatment of Johne's disease and other infections. Here we demonstrate that M. avium subsp. paratuberculosis infection changes the responsiveness of bovine monocytes to TLR9 stimulation. M. avium subsp. paratuberculosis inhibits classical TLR9-mediated responses despite a 10-fold increase in TLR9 expression and maintained uptake of CpG ODNs. Other TLR9-mediated responses, such as oxidative burst, which occur through noncanonical signaling, remain functional. Kinome analysis verifies that classic TLR9 signaling is blocked by M. avium subsp. paratuberculosis infection and that signaling instead proceeds through a Pyk2-mediated mechanism. Pyk2-mediated signaling does not hinder infection, as CpG ODNs fail to promote M. avium subsp. paratuberculosis clearance. Indeed, Pyk2 signaling appears to be an important aspect of M. avium subsp. paratuberculosis infection, as Pyk2 inhibitors significantly reduce the number of intracellular M. avium subsp. paratuberculosis bacteria. The actions of M. avium subsp. paratuberculosis on TLR9 signaling may represent a strategy to generate a host environment which is better suited for infection, revealing potential new targets for therapeutic intervention.

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.

A Comparison of the Immunometabolic Effect of Antibiotics and Plant Extracts in a Chicken Macrophage-like Cell Line during a Salmonella Enteritidis Challenge.

Immunometabolic modulation of macrophages can play an important role in the innate immune response of chickens triggered with a multiplicity of insults. In this study, the immunometabolic role of two antibiotics (oxytetracycline and gentamicin) and four plant extracts (thyme essential oil, grape seed extract, garlic oil, and capsicum oleoresin) were investigated on a chicken macrophage-like cell line (HD11) during a Salmonella Enteritidis infection. To study the effect of these substances, kinome peptide array analysis, Seahorse metabolic assay, and gene expression techniques were employed. Oxytetracycline, to which the bacterial strain was resistant, thyme essential oil, and capsicum oleoresin did not show any noteworthy immunometabolic effect. Garlic oil affected glycolysis, but this change was not detected by the kinome analysis. Gentamicin and grape seed extract showed the best immunometabolic profile among treatments, being able to both help the host with the activation of immune response pathways and with maintaining a less inflammatory status from a metabolic point of view.

A microencapsulated feed additive containing organic acids and botanicals has a distinct effect on proliferative and metabolic related signaling in the jejunum and ileum of broiler chickens.

Well designed and formulated natural feed additives have the potential to provide many of the growth promoting and disease mitigating characteristics of in-feed antibiotics, particularly feed additives that elicit their effects on targeted areas of the gut. Here, we describe the mechanism of action of a microencapsulated feed additive containing organic acids and botanicals (AviPlus®P) on the jejunum and ileum of 15-day-old broiler-type chickens. Day-of-hatch chicks were provided ad libitum access to feed containing either 0 or 500 g/MT of the feed additive for the duration of the study. Fifteen days post-hatch, birds were humanely euthanized and necropsied. Jejunum and ileum tissue samples were collected and either flash frozen or stored in RNA-later as appropriate for downstream applications. Chicken-specific kinome peptide array analysis was conducted on the jejunum and ileum tissues, comparing the tissues from the treated birds to those from their respective controls. Detailed analysis of peptides representing individual kinase target sites revealed that in the ileum there was a broad increase in the signal transduction pathways centering on activation of HIF-1α, AMPK, mTOR, PI3K-Akt and NFκB. These signaling responses were largely decreased in the jejunum relative to control birds. Gene expression analysis agrees with the kinome data showing strong immune gene expression in the ileum and reduced expression in the jejunum. The microencapsulated blend of organic acids and botanicals elicit a more anti-inflammatory phenotype and reduced signaling in the jejunum while resulting in enhanced immunometabolic responses in the ileum.

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.

Mycobacterium avium subsp. paratuberculosis inhibits gamma interferon-induced signaling in bovine monocytes: insights into the cellular mechanisms of Johne's disease.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle and may have implications for human health. Establishment of chronic infection by M. avium subsp. paratuberculosis depends on its subversion of host immune responses. This includes blocking the ability of infected macrophages to be activated by gamma interferon (IFN-γ) for clearance of this intracellular pathogen. To define the mechanism by which M. avium subsp. paratuberculosis subverts this critical host cell function, patterns of signal transduction to IFN-γ stimulation of uninfected and M. avium subsp. paratuberculosis-infected bovine monocytes were determined through bovine-specific peptide arrays for kinome analysis. Pathway analysis of the kinome data indicated activation of the JAK-STAT pathway, a hallmark of IFN-γ signaling, in uninfected monocytes. In contrast, IFN-γ stimulation of M. avium subsp. paratuberculosis-infected monocytes failed to induce patterns of peptide phosphorylation consistent with JAK-STAT activation. The inability of IFN-γ to induce differential phosphorylation of peptides corresponding to early JAK-STAT intermediates in infected monocytes indicates that M. avium subsp. paratuberculosis blocks responsiveness at, or near, the IFN-γ receptor. Consistent with this hypothesis, increased expression of negative regulators of the IFN-γ receptors SOCS1 and SOCS3 as well as decreased expression of IFN-γ receptor chains 1 and 2 is observed in M. avium subsp. paratuberculosis-infected monocytes. These patterns of expression are functionally consistent with the kinome data and offer a mechanistic explanation for this critical M. avium subsp. paratuberculosis behavior. Understanding this mechanism may contribute to the rational design of more effective vaccines and/or therapeutics for Johne's disease.

The Study of Microbe-Host Two-Way Communication.

Back-and-forth intercommunication in host-pathogen interactions has long been recognized to play an important role in commensalism and microbial pathogenesis. For centuries, we have studied these microbes in our surroundings, yet many questions about the evolutionary cross-talk between host and microbe remain unanswered. With the recent surge in research interest in the commensal microbiome, basic immunological questions have returned to the fore, such as, how are vast numbers of microbes capable of coexisting within animals and humans while also maintaining a healthy functional immune system? How is the evasion and subversion of the immune system achieved by some microbes but not others? The intricate and important-to-remember two-way interaction and coevolution of host and microbe is the communication network we must tap into as researchers to answer these questions.

Kinome Analysis of Cattle Peripheral Lymph Nodes to Elucidate Differential Response to Salmonella spp.

Salmonella spp., contained within the peripheral lymph nodes (PLNs) of cattle, represents a significant source of contamination of ground beef. Herein is the first report where species-specific kinome peptide arrays designed for bovine biology were used to further the understanding of Salmonella spp. within these PLNs. For the purpose of this research, multiple comparisons of sub-iliac lymph nodes were made to include nodes from feedlot cattle that were infected with Salmonella spp. to those that were non-infected; seasonal differences in feedlot cattle harvested in either August or January; cull dairy cows compared to feedlot cattle; and PLNs from cattle experimentally inoculated with Salmonella spp. versus naturally infected animals. The first comparison of Salmonella-positive and -negative PLNs found that considering the kinotypes for these animals, the major distinguishing difference was not the presence or absence of Salmonella spp. in the PLNs but the concentration. Further, the majority of pathways activated were directly related to immune responses including innate immunity, thus Salmonella spp. within the PLNs activates the immune system in that node. Results from the comparison of feedlot cattle and cull dairy cows suggests that a Salmonella spp.-negative animal, regardless of type, has a more consistent kinome profile than that of a Salmonella spp.-positive animal and that the differences between feedlot and cull dairy cattle are only pronounced when the PLNs are Salmonella spp. positive. PLNs collected in the winter showed a much more consistent kinome profile, regardless of Salmonella status, suggesting that in the winter these cattle are similar, and this is not affected by the presence of Salmonella spp., whereas significant variability among kinotypes was observed for PLNs collected in the summer. The most distinct clustering of kinotypes observed in this study was related to how the animal was infected with Salmonella spp. There were significant differences in the phosphorylation state of the immune response peptides between experimentally and naturally infected animals, suggesting that the immune system is activated in a significantly different manner when comparing these routes of infection. Increasing our understanding of Salmonella spp. within cattle, and specifically within the PLNs, will ultimately help design effective pre-harvest intervention strategies as well as appropriate experimentation to validate those technologies.

Broiler chickens with 1950s genetics display a stable immune profile as measured by Kinome, mRNA expression, and metabolism when stimulated early in life with CpG.

Significant changes in growth potential and feed conversion have been bred into the modern broiler chicken for well over 60 yr. These metabolic changes have had significant effects on the immune performance as well. To better understand these genetic differences in immunometabolism we studied the immune response of the modern broiler and the Athens Canadian Random Bred (ACRB) heritage broiler strain. We injected newly hatched modern broiler and ACRB chicks intraabdominally with CpG oligonucleotide, an immunostimulatory synthetic oligonucleotide. We conducted species-specific kinome array analysis and gene expression analysis on jejunum and cecal tonsil tissue. We also performed metabolic analysis of blood cells. In the modern birds, there is an initial inflammatory response to the injection at d 3 post-hatch with activation of PI3K-Akt, JAK-STAT, and NF-κB signaling, and IL-1ß and IL-6 mRNA expression. By d 15 post-hatch this response changed to deactivation and downregulation of these immune responses in modern but not heritage broilers. Metabolic analysis showed an increase in glycolysis in peripheral blood mononuclear cells from modern birds given CpG, but no difference in ACRB. These results show that the ACRB birds may have a less inflammatory and more stable immune profile in response to immune stimulation than the modern broilers, possibly resulting in a more disease resistant phenotype overall.

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

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

Informal nutrition symposium: leveraging the microbiome (and the metabolome) for poultry production.

Knowledge of gut microbiology of poultry has advanced from a limited ability to culture relatively few microbial species, to attempting to understand the complex interactions between the bird and its microbiome. The Informal Nutrition Symposium 2021 was intended to help poultry scientists to make sense of the implications of the vast amounts of information being generated by researchers. This paper represents a compilation of the talks given at the symposium by leading international researchers in this field. The symposium began with an overview of the historical developments in the field of intestinal microbiology and microbiome research in poultry. Next, the systemic effects of the microbiome on health in the context of the interplay between the intestinal microbiota and the immune system were presented. Because the microbiome and the host communicate and influence each other, the novel field of kinomics (the study of protein phosphorylation) as used in the study of the poultry microbiome was discussed. Protein phosphorylation is a rapid response to the complex of signals among the microbiome, intestinal lumen metabolites, and the host. Then, a description of why an understanding of the role of microbial endocrinology in poultry production can lead to new understanding of the mechanisms by which the gut microbiota and the host can interact in defined mechanisms that ultimately determine health, pathogenesis of infectious disease, and behavior was given. Finally, a view forward was presented underscoring the importance of understanding mechanisms in microbiomes in other organ systems and other species. Additionally, the importance of the development of new -omics platforms and data management tools to more completely understand host microbiomes was stressed.

A blend of microencapsulated organic acids and botanicals reduces necrotic enteritis via specific signaling pathways in broilers.

Necrotic enteritis (NE) is a devastating disease that has seen a resurgence of cases following the removal of antibiotics from feed resulting in financial loss and significant animal health concerns across the poultry industry. The objective was to evaluate the efficacy of a microencapsulated blend of organic (25% citric and 16.7% sorbic) acids and botanicals (1.7% thymol and 1% vanillin [AviPlusP]) to reduce clinical NE and determine the signaling pathways associated with any changes. Day-of-hatch by-product broiler breeder chicks were randomly assigned to a control (0) or supplemented (500 g/MT) diet (n = 23-26) and evaluated in a NE challenge model (n = 3). Birds were administered 2X cocci vaccine on d 14 and challenged with a cocktail of Clostridium perfringens strains (107) on d 17 to 19. On d 20 to 21 birds were weighed, euthanized, and scored for NE lesions. Jejunal tissue was collected for kinome analysis using an immuno-metabolism peptide array (n = 5; 15/treatment) to compare tissue from supplement-fed birds to controls. Mortality and weight were analyzed using Student's t test and lesion scores analyzed using F-test two-sample for variances (P < 0.05). The kinome data was analyzed using PIIKA2 peptide array analysis software and fold-change between control and treated groups determined. Mortality in the supplemented group was 47.4% and 70.7% in controls (P = 0.004). Lesions scores were lower (P = 0.006) in supplemented birds (2.47) compared to controls (3.3). Supplement-fed birds tended (P = 0.19) to be heavier (848.6 g) than controls (796.2 g). Kinome analysis showed T cell receptor, TNF and NF-kB signaling pathways contributed to the improvements seen in the supplement-fed birds. The following peptides were significant (P < 0.05) in all 3 pathways: CHUK, MAP3K14, MAP3K7, and NFKB1 indicating their importance. Additionally, there were changes to IL6, IL10, and IFN- γ mRNA expression in tissue between control- and supplement-fed chickens. In conclusion, the addition of a microencapsulated blend of organic acids and botanicals to a broiler diet reduced the clinical signs of NE that was mediated by specific immune-related pathways.