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 signalling. 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 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 [AviPlus®P]) 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 d14 and challenged with a cocktail of Clostridium perfringens strains (107) on d17-19. On d20-21 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 (848.6g) than controls (796.2g). 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 three 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 immunometabolic responses Salmonella Enteritidis and Salmonella Heidelberg induce in chicken macrophages

Arsenault, RyanSalmonella is a burden to the agriculture and health sectors as a result of the high number of illnesses, food contamination, and recalls. Salmonella Enteritidis (S. Enteritidis) is one of the most prevalent serotypes isolated from poultry. Salmonella Heidelberg (S. Heidelberg), which is becoming more prevalent than S. Enteritidis, is one of the five most isolated serotypes. Many animals including poultry are carriers of Salmonella but do not show any symptoms. Thus, it is more difficult for producers to avoid the processing and the distribution of contaminated products especially due to the restriction of antibiotic use in food animals. Salmonella invades host cells and exploits host mechanisms for its own benefits. For example, Salmonellae are capable of surviving in macrophages whose role is to kill pathogenic bacteria. Understanding the mechanism by which Salmonella infects and creates a suitable niche in hosts will reveal a potential target for the treatment and prevention of Salmonella contamination without the use of antibiotics. 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. Kinome peptide array data and available literature showed significant changes in the phosphorylation states of mTOR and AMPK peptides in chickens during Salmonella infections. Therefore, focusing on the AMPK-mTOR signaling cascade, we demonstrated that S. Enteritidis and S. Heidelberg infections induced differential kinase activities in metabolic and immune related peptides of HD11 chicken macrophages. Metabolic flux assays measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) demonstrated that, i) S. Enteritidis at 30 minutes post infection increased glucose metabolism ii) S. Heidelberg at 30 minutes post infection decreased glucose metabolism iii) Both Salmonella infections induce increased oxygen metabolism. Gentamicin protection assays performed at 30 minutes and 2 hours post infection revealed that S. Enteritidis bacteria are more invasive than S. Heidelberg. Furthermore, flow cytometry results showed increased apoptotic/dead cell population in S. Enteritidis infections compared to S. Heidelberg. These results show different immunometabolic responses of HD11 macrophages to S. Enteritidis and S. Heidelberg infections.University of Delaware, Department of Biological SciencesM.S

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 Comparison of the Immunometabolic Effect of Antibiotics and Plant Extracts in a Chicken Macrophage-like Cell Line during a <i>Salmonella</i> 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