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.

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.

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.

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.

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.

The Differential Phosphorylation-Dependent Signaling and Glucose Immunometabolic Responses Induced during Infection by Salmonella Enteritidis and Salmonella Heidelberg in Chicken Macrophage-like cells.

Salmonella is a burden to the poultry, health, and food safety industries, resulting in illnesses, food contamination, and recalls. Salmonella enterica subspecies enterica Enteritidis (S. Enteritidis) is one of the most prevalent serotypes isolated from poultry. Salmonella enterica subspecies enterica Heidelberg (S. Heidelberg), which is becoming as prevalent as S. Enteritidis, is one of the five most isolated serotypes. Although S. Enteritidis and S. Heidelberg are almost genetically identical, they both are capable of inducing different immune and metabolic responses in host cells to successfully establish an infection. Therefore, using the kinome peptide array, we demonstrated that S. Enteritidis and S. Heidelberg infections induced differential phosphorylation of peptides on Rho proteins, caspases, toll-like receptors, and other proteins involved in metabolic- and immune-related signaling of HD11 chicken macrophages. Metabolic flux assays measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) demonstrated that S. Enteritidis at 30 min postinfection (p.i.) increased glucose metabolism, while S. Heidelberg at 30 min p.i. decreased glucose metabolism. S. Enteritidis is more invasive than S. Heidelberg. These results show different immunometabolic responses of HD11 macrophages to S. Enteritidis and S. Heidelberg infections.