In-Ovo Glutamine Administration Enhances Intestinal Development and Functions in Broiler Chickens: Insights from Enteroid Models.

BACKGROUND: Early life events play significant roles in tissue development and animal health in their later life. Early nutrition, through in-ovo delivery, has shown beneficial effects on improving intestinal health in broiler chickens. However, the underlying mechanism is not fully investigated. A recently developed enteroid culture technique allows investigations on intestinal epithelial functions that are close to physiologic conditions. OBJECTIVES: In this study, we evaluated the short- and long-term effects of in-ovo administration of glutamine (Gln) on intestinal epithelial development and functions by using intestinal enteroid culture and tissue electrophysiologic analysis. METHODS: A hundred eggs of commercial Cobb500 broilers were in-ovo injected with 0.2 mL of either phosphate-buffered saline (PBS) or 3% Gln at embryonic day 18 (E18). Chicks were killed on the day of hatch, and at 3- and 14-d posthatch. Enteroids were generated from the small intestine. After 4 d of culture, enteroids were harvested for 5-ethynyl-2'-deoxyuridine proliferation, fluorescein isothiocyanate-4 kDa dextran permeability, and glucose absorption assays. At day 3 (d3) and day 14 (d14), intestinal barrier and nutrient transport functions were measured by the Ussing chamber. The gene expression of epithelial cell markers, nutrient transporters, and tight-junction proteins were analyzed in both intestinal tissues and enteroids. RESULTS: In comparison with the PBS control group, in-ovo Gln increased intestinal villus morphology, epithelial cell proliferation, and differentiation, and altered epithelial cell population toward increased number of enteroendocrine and goblet cells while decreasing Paneth cells. Enteroids gene expression of nutrient transporters (B0AT1, SGLT1, and EAAT3), tight junction (ZO2), glucose absorption, and barrier functions were enhanced on the day of hatch. Long-term increases of intestinal di-peptide and alanine transport were observed at day 14 posthatch. CONCLUSIONS: Together our results suggested that the in-ovo injection of Gln stimulated intestinal epithelium proliferation and programmed the epithelial cell differentiation toward absorptive cells.

Establishment and characterization of an SV40 immortalized chicken intestinal epithelial cell line.

Primary chicken intestinal epithelial cells or 3D enteroids are a powerful tool to study the different biological mechanisms that occur in the chicken intestine. Unfortunately, they are not ideal for large-scale screening or long-term studies due to their short lifespan. Moreover, they require expensive culture media, coatings, or the usage of live embryos for each isolation. The aim of this study was to establish and characterize an immortalized chicken intestinal epithelial cell line to help the study of host-pathogen interactions in poultry. This cell line was established by transducing into primary chicken enterocytes the SV40 large-T antigen through a lentiviral vector. The transduced cells grew without changes up to 40 passages maintaining, after a differentiation phase of 48 h with epidermal growth factor, the biological properties of mature enterocytes such as alkaline phosphatase activity and tight junction formation. Immortalized enterocytes were able to generate a cytokine response during an inflammatory challenge, and showed to be susceptible to Eimeria tenella sporozoites invasion and generate a proper immune response to parasitic and lipopolysaccharide (Escherichia coli) stimulation. This immortalized cell line could be a cost-effective and easy-to-maintain model for all the public health, food safety, or research and pharmaceutical laboratories that study host-pathogen interactions, foodborne pathogens, and food or feed science in vitro.

Evaluating protective effects of botanicals under inflammation and oxidative stress in chicken apical-out enteroids.

Botanicals (BOTs) are well known for their anti-inflammatory and antioxidant activities. They have been widely used as feed additives to reduce inflammation and improve intestinal functions in agricultural animals. However, the effects of BOTs on chicken intestinal epithelial functions are not fully understood. The 3D apical-out chicken enteroids recapitulate the intestinal tissue, and allow convenient access to the luminal surface, thus serving as a suitable model for investigating gut functions. The aim of this study was to identify the roles of BOTs in protecting the intestinal epithelium in chicken enteroids under challenging conditions. Apical-out enteroids were isolated from the small intestines of 18 days-old chicken embryos. Lipopolysaccharide (LPS, 10 µg/mL) and menadione (400 µM) challenges were performed in the media with or without BOTs. Paracellular Fluorescein isothiocyanate-dextran 4kD (FD4) permeability, inflammatory cytokine gene expression, and reactive oxygen species (ROS) generation were analyzed post-BOTs and challenges treatments. Statistical analysis was performed using one-way ANOVA and post hoc multiple comparisons among treatments. The results showed that the LPS challenge for 24 h induced a 50% increase in FD4 permeability compared with nontreated control; thymol, thyme essential oil, and phenol-rich extract significantly (P < 0.02) reduced FD4 permeability by 25%, 41%, and 48% respectively, in comparison with LPS treatment. Moreover, the gene expression of inflammatory cytokines was upregulated, tight junction proteins and defensins were downregulated (P < 0.05) after 6 h of LPS treatment, while these BOTs treatments significantly restored the LPS-induced gene expression alterations (P < 0.05). Menadione oxidative challenge for 1 h significantly increased the ROS level compared with unchallenged control. Enteroids treated with thymol and thyme essential oils showed 30% reduced ROS levels, while the phenol-rich extract reduced them by 60%, in comparison with the challenged group (P < 0.0001). These data confirmed the role of BOTs in supporting the barrier function and reducing the disruptive effects of inflammation and oxidation in the chicken intestine.

Investigating the effects of essential oils and pure botanical compounds against Eimeria tenella in vitro.

Essential oils (EO) and natural bioactive compounds are well-known antibacterial and anti-inflammatory factors; however, little is known about their anticoccidial activity and mode of action. EO deriving from basil (BEO), garlic (GAR), oregano (OEO), thyme (TEO), and their main bioactive compounds were investigated for their anticoccidial proprieties and compared to salinomycin (SAL) and amprolium (AMP) in vitro. The invasion of Eimeria tenella sporozoites was studied on 2 cell models: Madin-Darby Bovine Kidney (MDBK) cells and primary chicken epithelial cells (cIEC). Invasion efficiency was evaluated at 2 and 24 h postinfection (hpi) with counts of extracellular sporozoites and by detection of intracellular E. tenella DNA by PCR. Results show that at both timepoints, the EO were most effective in preventing the invasion of E. tenella with an average reduction of invasion at 24 hpi by 36% in cIEC and 55% in MDBK. The study also examined cytokine gene expression in cIEC at 24 hpi and found that AMP, BEO, OEO, TEO, carvacrol (CAR), and thymol (THY) significantly reduced interleukin (IL)8 expression, with CAR also reducing expression of IL1ß and IL6 compared to the infected control. In addition, this work investigated the morphology of E. tenella sporozoites treated with anticoccidial drugs and EO using a scanning electron microscope. All the treatments induced morphological anomalies, characterized by a reduction of area, perimeter and length of sporozoites. SAL had a significant impact on altering sporozoite shape only at 24 h, whereas CAR and THY significantly compromised the morphology already at 2 hpi, compared to the untreated control. OEO and GAR showed the most significant alterations among all the treatments. The findings of this study highlight the potential of EO as an alternative to traditional anticoccidial drugs in controlling E. tenella invasion and in modulating primary immune response.

A mixture of organic acids and thymol protects primary chicken intestinal epithelial cells from Clostridium perfringens infection in vitro.

Necrotic enteritis causes economic losses estimated to be up to 6 billion US dollars per year. Clinical and subclinical infections in poultry are also both correlated with decreased growth and feed efficiency. Moreover, in a context of increased antibiotic resistance, feed additives with enhanced antimicrobial properties are a useful and increasingly needed strategy. In this study, the protective effects of a blend of thymol and organic acids against the effects of Clostridium perfringens type A (CP) on chicken intestinal epithelial cells were investigated and compared to bacitracin, a widely used antibiotic in poultry production. Primary chicken intestinal epithelial cells were challenged with CP for a total time of 3 h to assess the beneficial effect of 2 doses of citric acid, dodecanoic acid, and thymol-containing blend, and compare them with bacitracin. During the challenge, different parameters were recorded, such as transepithelial electrical resistance, cell viability, mRNA expression, and reactive oxygen species production. CP induced inflammation with cytokine production and loss of epithelial barrier integrity. It was also able to induce reactive oxygen species production and increase the caspase expression leading to cellular death. The high dose of the blend acted similarly to bacitracin, preventing the disruptive effects of CP and inducing also an increase in zonula occludens-1 mRNA expression. The low dose only partially prevented the disruptive effects of CP but successfully reduced the associated inflammation. This study shows that the usage of thymol combined with 2 organic acids can protect primary chicken intestinal epithelial cells from CP-induced damages creating a valid candidate to substitute or adjuvate the antibiotic treatment against necrotic enteritis.

Assessing Intestinal Health. In Vitro and Ex vivo Gut Barrier Models of Farm Animals: Benefits and Limitations.

Animal performance is determined by the functionality and health of the gastrointestinal tract (GIT). Complex mechanisms and interactions are involved in the regulation of GIT functionality and health. The understanding of these relationships could be crucial for developing strategies to improve animal production yields. The concept of "gut health" is not well defined, but this concept has begun to play a very important role in the field of animal science. However, a clear definition of GIT health and the means by which to measure it are lacking. In vitro and ex vivo models can facilitate these studies, creating well-controlled and repeatable conditions to understand how to improve animal gut health. Over the years, several models have been developed and used to study the beneficial or pathogenic relationships between the GIT and the external environment. This review aims to describe the most commonly used animals' in vitro or ex vivo models and techniques that are useful for better understanding the intestinal health of production animals, elucidating their benefits and limitations.

Isolation, culture, and characterization of chicken intestinal epithelial cells.

BACKGROUND: Enterocytes exert an absorptive and protective function in the intestine, and they encounter many different challenging factors such as feed, bacteria, and parasites. An intestinal epithelial in vitro model can help to understand how enterocytes are affected by these factors and contribute to the development of strategies against pathogens. RESULTS: The present study describes a novel method to culture and maintain primary chicken enterocytes and their characterization by immunofluorescence and biomolecular approaches. Starting from 19-day-old chicken embryos it was possible to isolate viable intestinal cell aggregates that can expand and produce a self-maintaining intestinal epithelial cell population that survives until 12 days in culture. These cells resulted positive in immunofluorescence to Cytokeratin 18, Zonula occludens 1, Villin, and Occludin that are common intestinal epithelial markers, and negative to Vimentin that is expressed by endothelial cells. Cells were cultured also on Transwell® permeable supports and trans-epithelial electrical resistance, was measured. This value gradually increased reaching 64 Ω*cm2 7 days after seeding and it remained stable until day 12. CONCLUSIONS: Based on these results it was confirmed that it is possible to isolate and maintain chicken intestinal epithelial cells in culture and that they can be suitable as in vitro intestinal model for further studies.

Thymol and Carvacrol Downregulate the Expression of Salmonella typhimurium Virulence Genes during an In Vitro Infection on Caco-2 Cells.

Salmonella typhimurium is one of the major bacteria responsible for gastroenteritis in humans caused by foodborne pathogens. As pork is one of the main routes of transmission, bioactive compounds used as feed additives may be an important strategy to control Salmonella typhimurium. The aim of this study was to assess the antimicrobial activity of several organic acids and nature identical compounds against Salmonella typhimurium ATCC®® 6994™. Moreover, the effect of sub-lethal concentrations of thymol and carvacrol in counteracting a Salmonella typhimurium in vitro infection on Caco-2 cells was evaluated, focusing on the maintenance of the epithelial barrier and the alteration of Salmonella virulence genes. The results showed a protective effect of the compounds on the integrity of the intestinal monolayer, improving transepithelial electrical resistance and bacterial translocation compared to the non-treated cells. A real-time PCR study highlighted a significant downregulation of the main virulence genes of Salmonella (hilA, prgH, invA, sipA, sipC, sipD, sopB, sopE2). These findings indicate that thymol and carvacrol could be good candidates for the control of Salmonella typhimurium in pigs.

In vitro anticoccidial activity of thymol, carvacrol, and saponins.

The anticoccidial activity of thymol, carvacrol, and saponins was assessed in an in vitro model of coccidiosis. Eimeria spp. sporozoites were collected from field samples, characterized, and used for 2 different invasion assays on Madin-Darby Bovine Kidney cells (MDBK). The cells were challenged with 5 × 104 sporozoites without (control) or with various treatments: saponins (10 ppm), thymol, and carvacrol (7 ppm each) or a combination of saponins, thymol, and carvacrol at 2 doses; MIX 1 (saponins 5 ppm, thymol 3.5 ppm, and carvacrol 3.5 ppm) and MIX 2 (saponins 10 ppm, thymol 7 ppm, and carvacrol 7 ppm). The treated cells were incubated at 37°C for 24 h (invasion assay 1) and for 2, 24, and 48 h (invasion assay 2). The efficiency of invasion was determined by counting the sporozoites left in the supernatant that were not able to invade the cells, whereas intracellular Eimeria DNA was detected by qPCR to confirm the data. Data were analyzed with ANOVA, and differences were considered significant when P value was ≤0.05. Data from invasion assay 1 showed that the thymol and carvacrol-containing blends significantly reduced invasion, especially in combination with saponins at the highest dose. Saponins alone did not have a strong inhibiting activity but acted synergistically with the other molecules. Interestingly, in invasion assay 2, it was found that the effect of the highest dose of the blend of saponins, thymol, and carvacrol was already visible at 2 h postinfection, whereas the other treatments were significantly successful at 24 h postinfection. The invasion assay protocol was designed to screen molecules in vitro starting from field fecal samples, and it can represent a potential tool in Eimeria research. Moreover, this study shows that invasion in MDBK cells by Eimeria sporozoites is inhibited in presence of thymol, carvacrol, and saponins, thus highlighting the anticoccidial potential of these compounds.