The Expression of Cannabinoid and Cannabinoid-Related Receptors on the Gustatory Cells of the Piglet Tongue.

The gustatory system is responsible for detecting and evaluating the palatability of the various chemicals present in food and beverages. Taste bud cells, located primarily on the tongue, communicate with the gustatory sensory neurons by means of neurochemical signals, transmitting taste information to the brain. It has also been found that the endocannabinoid system (ECS) may modulate food intake and palatability, and that taste bud cells express cannabinoid receptors. The purpose of this study was to investigate the expression of cannabinoid and cannabinoid-related receptors in the gustatory cells of the papillae vallatae and foliatae of ten piglets. Specific antibodies against the cannabinoid receptors (CB1R and CB2R), G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) were applied on cryosections of lingual tissue; the lingual tissue was also processed using Western blot analysis. Cannabinoid and cannabinoid-related receptors were found to be expressed in the taste bud cells and the surrounding epithelial cells. The extra-papillary epithelium also showed strong immunolabeling for these receptors. The results showed that these receptors were present in both the taste bud cells and the extra-gustatory epithelial cells, indicating their potential role in taste perception and chemesthesis. These findings contributed to understanding the complex interactions between cannabinoids and the gustatory system, highlighting the role of the ECS within taste perception and its potential use in animal production in order to enhance food intake.

Transcriptomic regulations of heat stress response in the liver of lactating dairy cows.

BACKGROUND: The global dairy industry is currently facing the challenge of heat stress (HS). Despite the implementation of various measures to mitigate the negative impact of HS on milk production, the cellular response of dairy cows to HS is still not well understood. Our study aims to analyze transcriptomic dynamics and functional changes in the liver of cows subjected to heat stress (HS). To achieve this, a total of 9 Holstein dairy cows were randomly selected from three environmental conditions - heat stress (HS), pair-fed (PF), and thermoneutral (TN) groups - and liver biopsies were obtained for transcriptome analysis. RESULTS: Both the dry matter intake (DMI) and milk yield of cows in the HS group exhibited significant reduction compared to the TN group. Through liver transcriptomic analysis, 483 differentially expressed genes (DEGs) were identified among three experimental groups. Especially, we found all the protein coding genes in mitochondria were significantly downregulated under HS and 6 heat shock proteins were significant upregulated after HS exposure, indicating HS may affect mitochondria integrity and jeopardize the metabolic homeostasis in liver. Furthermore, Gene ontology (GO) enrichment of DEGs revealed that the protein folding pathway was upregulated while oxidative phosphorylation was downregulated in the HS group, corresponding to impaired energy production caused by mitochondria dysfunction. CONCLUSIONS: The liver transcriptome analysis generated a comprehensive gene expression regulation network upon HS in lactating dairy cows. Overall, this study provides novel insights into molecular and metabolic changes of cows conditioned under HS. The key genes and pathways identified in this study provided further understanding of transcriptome regulation of HS response and could serve as vital references to mitigate the HS effects on dairy cow health and productivity.

The biological effects of microencapsulated organic acids and botanicals induces tissue-specific and dose-dependent changes to the Gallus gallus microbiota.

BACKGROUND: Microencapsulated organic acids and botanicals have the potential to develop into important tools for the poultry industry. A blend of organic acids and botanicals (AviPlus®P) has previously shown to reduce Salmonella and Campylobacter in chickens; however, changes to the microbiota of the jejunum and ileum have not been evaluated. Microbiota diversity is linked to, but not correlated with, the efficacy of natural products; therefore, understanding the effects on the microbiota is necessary for evaluating their potential as an antibiotic alternative. RESULTS: Ileal and jejunal segments from control and supplement-fed chickens (300 and 500 g/metric ton [MT]) were subjected to alpha diversity analysis including Shannon's diversity and Pielou's Evenness. In both analytics, the diversity in the ileum was significantly decreased compared to the jejunum irrespective of treatment. Similarly, beta diversity metrics including Bray-Curtis dissimilarity index and Weighted Unifrac Distance Matrix, were significant (Q < 0.05) for both tissue and treatments comparisons. Alpha and beta diversity analytics indicated compartmentalization effects between the ileum and jejunum. Additionally, analysis of communities in the microbiota (ANCOM) analysis showed Lactobacilliaceae predominated the total operational taxonomic units (OTU), with a stepwise increase from 53% in the no treatment control (NTC) to 56% in the 300 g/MT and 67% in the 500 g/MT group. Staphylococcaceae were 2% in NTC and 2 and 0% in 300 and 500 g/MT groups. Enterobacteriaceae decreased in the 500 g/MT (31%) and increased in the 300 g/MT (37%) compared to the NTC (35%). Aerococcaceae was 0% for both doses and 7% in NTC. Ruminococcaceae were 0% in NTC and 2 and 1% in the 300 and 500 g/MT. These changes in the microbial consortia were statistically (Q < 0.05) associated with treatment groups in the jejunum that were not observed in the ileum. Least discriminant analysis effect size (LEfSE) indicated different changes directly corresponding to treatment. Enterobacteriaceae demonstrated a stepwise decrease (from NTC onward) while Clostridiaceae, were significantly increased in the 500 g/MT compared to NTC and 300 g/MT (P < 0.05). CONCLUSION: The bioactive site for the microencapsulated blend of organic acids and botanicals was the jejunum, and dietary inclusion enhanced the GIT microbiota and may be a viable antibiotic alternative for the poultry industry.

Effects of increasing dietary level of organic acids and nature-identical compounds on growth, intestinal cytokine gene expression and gut microbiota of rainbow trout (Oncorhynchus mykiss) reared at normal and high temperature.

Organic acids (OA) and nature-identical compounds (NIC) such as monoterpenes and aldehydes are well-known growth and health promoters in terrestrial livestock while their application for fish production is recent and their mechanisms of action require further study. Hence, this study tested the increasing dietary level (D0, D250, D500, D1000; 0, 250, 500 and 1000 mg kg feed-1 respectively) of a microencapsulated blend containing citric and sorbic acid, thymol and vanillin over 82 days on rainbow trout to assess the effects on growth, feed utilization, intestine cytokine gene expression and gut microbiota (GM). Furthermore, the effects on intestinal cytokine gene expression and GM were also explored after one week at high water temperature (23 °C). OA and NIC improved specific growth rate (SGR) and feed conversion rate (FCR) during the second half (day 40-82) of the feeding trial, while at the end of the trial protein (PER) and lipid efficiency (LER) increased with increasing dietary level. GM diversity and composition and cytokine gene expression analysis showed no significant differences in fish fed with increasing doses of OA and NIC (82 days) demonstrating the absence of inflammatory activity in the intestinal mucosa. Although there were no statistical differences, GM structure showed a tendency in clustering D0 group separately from the other dietary groups and a trend towards reduction of Streptococcus spp. was observed in the D250 and D1000 groups. After exposure to high water temperature, lower GM diversity and increased gene expression of inflammatory intestinal cytokines were observed for both inclusions (D0 vs. D1000) compared to groups in standard condition. However, the gene up-regulation involved a limited number of cytokines showing the absence of a substantial inflammation process able to compromise the functional activity of the intestine. Despite further study should be conducted to fully clarify this mechanism, cytokines up-regulation seems to be concomitant to the reduction of the GM diversity and, particularly, to the reduction of specific lactic acid bacteria such as Leuconostoc. The application of the microencapsulate blend tested can be a useful strategy to improve growth and feed utilization in rainbow trout under normal temperature conditions. According to the results organic acids and nature-identical compounds did not revert the effects triggered by the increased temperature of water.

Thymol modulates the endocannabinoid system and gut chemosensing of weaning pigs.

BACKGROUND: The recent identification of the endocannabinoid system in the gastrointestinal tract suggests a role in controlling intestinal inflammation. In addition, the gut chemosensing system has therapeutic applications in the treatment of gastrointestinal diseases and inflammation due to the presence of a large variety of receptors. The purposes of this study were to investigate the presence of markers of the endocannabinoid system and the chemosensing system in the pig gut and, second, to determine if thymol modulates these markers. One hundred sixty 28-day-old piglets were allocated into one of 5 treatment groups (n = 32 per treatment): T1 (control), T2 (25.5 mg thymol/kg feed), T3 (51 mg thymol/kg feed), T4 (153 mg thymol/kg feed), and T5 (510 mg thymol/kg feed). After 14 days of treatment, piglets were sacrificed (n = 8), and then duodenal and ileal mucosal scrapings were collected. Gene expression of cannabinoid receptors (CB1 and CB2), transient receptor potential vanilloid 1 (TRPV1), the olfactory receptor OR1G1, diacylglycerol lipases (DGL-α and DGL-ß), fatty acid amine hydrolase (FAAH), and cytokines was measured, and ELISAs of pro-inflammatory cytokines levels were performed. RESULTS: mRNAs encoding all markers tested were detected. In the duodenum and ileum, the CB1, CB2, TRPV1, and OR1G1 mRNAs were expressed at higher levels in the T4 and T5 groups compared to the control group. The level of the FAAH mRNA was increased in the ileum of the T4 group compared to the control. Regarding the immune response, the level of the tumor necrosis factor (TNF-α) mRNA was significantly increased in the duodenum of the T5 group, but this increase was not consistent with the protein level. CONCLUSIONS: These results indicate the presence of endocannabinoid system and gut chemosensing markers in the piglet gut mucosa. Moreover, thymol modulated the expression of the CB1, CB2, TRPV1, and OR1G1 mRNAs in the duodenum and ileum. It also modulated the mRNA levels of enzymes involved in the biosynthesis and degradation of endocannabinoid molecules. Based on these findings, the effects of thymol on promoting gut health are potentially mediated by the activation of these receptors.

Nature-Identical Compounds and Organic Acids Ameliorate and Prevent the Damages Induced by an Inflammatory Challenge in Caco-2 Cell Culture.

Bioactive compounds, such as organic acids (OA) and nature-identical compounds (NIC), can exert a role in the protection of intestinal mucosa functionality due to their biological properties. The aim of this study was to understand the role of 2 OA (citric and sorbic acid) and 2 NIC (thymol and vanillin), alone or combined in a blend (OA + NIC), on intestinal barrier functionality, either during homeostatic condition or during an inflammatory challenge performed with pro-inflammatory cytokines and lipopolysaccharides (LPS). The study was performed on the human epithelial cell line Caco-2, a well-known model of the intestinal epithelial barrier. The results showed how OA and NIC alone can improve transepithelial electrical resistance (TEER) and mRNA levels of tight junction (TJ) components, but OA + NIC showed stronger efficacy compared to the single molecules. When an inflammatory challenge occurred, OA + NIC blend was able both to ameliorate, and prevent, damage caused by the pro-inflammatory stimulus, reducing or preventing the drop in TEER and improving the TJ mRNA expression. The data support the role of OA + NIC in modulating gut barrier functionality and reducing the negative effects of inflammation in intestinal epithelial cells, thereby supporting the gut barrier functionality.

Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii Circulation in a Dairy Farm and Sources of Milk Contamination.

Even though dairy cows are known carriers of Arcobacter species and raw or minimally processed foods are recognized as the main sources of human Arcobacter infections in industrialized countries, data on Arcobacter excretion patterns in cows and in milk are scant. This study aimed to identify potentially pathogenic Arcobacter species in a dairy herd and to investigate the routes of Arcobacter transmission among animals and the potential sources of cattle infection and milk contamination. A strategy of sampling the same 50 dairy animals, feed, water, and milk every month for a 10-month period, as well as the sampling of quarter milk, animal teats, the milking environment, and animals living on the farm (pigeons and cats), was used to evaluate, by pulsed-field gel electrophoresis (PFGE), the characteristic patterns in animals, their living environment, and the raw milk they produced. Of the 463 samples collected, 105 (22.6%) were positive for Arcobacter spp. by culture examination. All the matrices except quarter milk and pigeon gut samples were positive, with prevalences ranging from 15 to 83% depending on the sample. Only three Arcobacter species, Arcobacter cryaerophilus (54.2%), A. butzleri (34.2%), and A. skirrowii (32.3%), were detected. PFGE analysis of 370 isolates from positive samples provided strong evidence of Arcobacter circulation in the herd: cattle likely acquire the microorganisms by orofecal transmission, either by direct contact or from the environment, or both. Water appears to be a major source of animal infection. Raw milk produced by the farm and collected from a bulk tank was frequently contaminated (80%) by A. butzleri; our PFGE findings excluded primary contamination of milk, whereas teats and milking machine surfaces could be sources of Arcobacter milk contamination.

Impact of dietary organic acids and botanicals on intestinal integrity and inflammation in weaned pigs.

BACKGROUND: Organic acids, such as citric and sorbic acid, and pure plant-derived constituents, like monoterpens and aldehydes, have a long history of use in pig feeding as alternatives to antibiotic growth promoters. However, their effects on the intestinal barrier function and inflammation have never been investigated. Therefore, aim of this study was to assess the impact of a microencapsulated mixture of citric acid and sorbic acid (OA) and pure botanicals, namely thymol and vanillin, (PB) on the intestinal integrity and functionality of weaned pigs and in vitro on Caco-2 cells. In the first study 20 piglets were divided in 2 groups and received either a basal diet or the basal diet supplemented with OA + PB (5 g/kg) for 2 weeks post-weaning at the end of which ileum and jejunum samples were collected for Ussing chambers analysis of trans-epithelial electrical resistance (TER), intermittent short-circuit current (I SC), and dextran flux. Scrapings of ileum mucosa were also collected for cytokine analysis (n = 6). In the second study we measured the effect of these compounds directly on TER and permeability of Caco-2 monolayers treated with either 0.2 or 1 g/l of OA + PB. RESULTS: Pigs fed with OA + PB tended to have reduced I SC in the ileum (P = 0.07) and the ileal gene expression of IL-12, TGF-ß, and IL-6 was down regulated. In the in vitro study on Caco-2 cells, TER was increased by the supplementation of 0.2 g/l at 4, 6, and 14 days of the experiment, whereas 1 g/l increased TER at 10 and 12 days of treatment (P < 0.05). Dextran flux was not significantly affected though a decrease was observed at 7 and 14 days (P = 0.10 and P = 0.09, respectively). CONCLUSIONS: Overall, considering the results from both experiments, OA + PB improved the maturation of the intestinal mucosa by modulating the local and systemic inflammatory pressure ultimately resulting in a less permeable intestine, and eventually improving the growth of piglets prematurely weaned.

Organic acid blend with pure botanical product treatment reduces Escherichia coli and Salmonella populations in pure culture and in in vitro mixed ruminal microorganism fermentations.

Foodborne pathogenic bacteria can live in the intestinal tract of food animals and can be transmitted to humans via food or indirectly through animal or fecal contact. Organic acid blend products have been used as nonantibiotic modifiers of the gastrointestinal fermentation of food animals to improve growth performance efficiency. However, the impact of these organic acid products on the microbial population, including foodborne pathogens, remains unknown. Therefore, this study was designed to examine the effects of a commercial organic acid and botanical blend product (OABP) on populations of the foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. Pure cultures (2×10(6) colony-forming units [CFU]/mL) of each pathogen were added to tubes that contained water-solubilized OABP added at concentrations of 0, 0.1, 0.5, 1, 2, 5, and 10% (vol/vol; n=3). Water-solubilized OABP reduced (p<0.05) the growth rate and final populations of E. coli O157:H7 and Salmonella Typhimurium in pure culture at concentrations >2%. E. coli O157:H7 and Salmonella Typhimurium were added (2×10(5) and 3×10(6) CFU/mL, respectively) to in vitro mixed ruminal microorganism fermentations that contained water-solubilized OABP at concentrations of 0, 1, 2, 5, and 10% (vol/vol; n=3) that were incubated for 24 h. OABP addition reduced (p<0.05) final populations of E. coli O157:H7 and Salmonella Typhimurium in the ruminal fluid at concentrations ≥5%. The acetate-to-propionate ratios from the in vitro fermentations were reduced (p<0.05) by OABP treatment ≥5%. Treatments to reduce foodborne pathogens must be economically feasible to implement, and results indicate that organic acid products, such as OABP, can enhance animal growth efficiency and can be used to reduce populations of pathogenic bacteria.

Microencapsulated sorbic acid and pure botanicals affect Salmonella Typhimurium shedding in pigs: a close-up look from weaning to slaughter in controlled and field conditions.

The aim of this study was to assess the efficacy of a combination of sorbic acid, thymol, and carvacrol in reducing the prevalence and shedding level of Salmonella Typhimurium in pigs either in a controlled challenge environment or in a production setting. In the first study, 24 weaned piglets were separated in 4 isolation units (6 piglets/isolation unit). Each unit received either a basal diet (no treatment) or a microencapsulated mixture of sorbic acid, thymol, and carvacrol at 1, 2, or 5 g/kg of feed. After 21 d, pigs were orally challenged with 6 log10 colony-forming units of Salmonella Typhimurium. Blood samples and feces from rectal ampullae were collected every week. On d56 of the study, pigs were euthanized and necropsied to collect intestinal contents (jejunum through colon) and ileocecal lymph nodes. Samples were analyzed for Salmonella Typhimurium and serological analysis was also conducted. In the second study, an all-in-all-out multisite pig farm that was positive for monophasic Salmonella Typhimurium was followed throughout a production cycle from weaning to slaughter. Pigs received either a basal diet or the basal diet including 5 g/kg of the microencapsulated additive. Environmental, fecal, and blood samples were collected monthly, and cecal contents and ileocecal lymph nodes were collected at slaughter to isolate and enumerate Salmonella. The results indicate that the additive at 5 g/kg tended to reduce Salmonella fecal prevalence in both a controlled challenge (p=0.07) and in production conditions (p=0.03). Nevertheless, the additive did not reduce the number of pigs seropositive for Salmonella, nor it reduced the Salmonella prevalence at slaughter. The data indicate that these additives are not effective alone but must be used in conjunction with appropriate containment measures at lairage in order to prevent reinfection in pigs and to reduce the number of pigs carrying Salmonella entering the food chain.

Dietary supplementation of microencapsulated botanicals and organic acids enhances the expression and function of intestine epithelial digestive enzymes and nutrient transporters in broiler chickens.

Organic acids and botanicals have shown protective effects on gut barrier and against inflammation in broilers. However, their effects on intestinal digestive enzymes and nutrients transporters expression and functions have not been fully studied. The objective of this study was to understand how a microencapsulated blend of botanicals and organic acids affected intestinal enzyme activities and nutrient transporters expression and functions in broilers. A total of 288 birds were assigned to a commercial control diet or diet supplemented with 500 g/MT (metric ton) of the microencapsulated additive. Growth performance was recorded weekly. At d 21 and d 42, jejunum and ileum were isolated for enzyme (maltase, sucrase, and aminopeptidase) and transporter (SGLT1, GLUT2, GLUT1, EAAT3, B0AT1, and PepT1) analyses. Jejunum specific nutrients (glucose, alanine, and glutamate) transport activities were evaluated by Ussing chamber. Protein expression of nutrient transporters in small intestine were measured in mucosa and brush-border membrane (BBM) samples by western blot. Intestinal gene expression of the transporters was determined by RT-PCR. Statistical analysis was performed using Student's t-test comparing the supplemented diet to the control. The feed efficiency was significantly improved through the study period in the supplemented group (P ≤ 0.05). Significant changes of intestinal histology were shown in both jejunum (P ≤ 0.10) and ileum (P ≤ 0.05) after 21 d of treatment. At d21, jejunal maltase activity was upregulated (P ≤ 0.10). The Ussing chamber transport of glucose and alanine was increased, which was in line with increased gene expression (GLUT2, GLUT1, EAAT3, and B0AT1) (P ≤ 0.10 and P ≤ 0.05, respectively) and BBMV protein levels (B0AT1, P < 0.10). At d21, ileal sucrase and maltase activities were upregulated (P ≤ 0.05). Increased expressions of GLUT1, EAAT3, and B0AT1 were observed in both mRNA and protein levels (P ≤ 0.05). Similar pattern of changes was also shown at d42 of age. Our results suggest that feeding microencapsulated additives improves intestinal nutrient digestion and transporter expression and function in broilers, thereby enhancing feed efficiency.

A microencapsulated blend of botanicals supports weaning piglets during a lipopolysaccharide challenge by modulating liver inflammation and intestinal integrity.

This study examined the action of a blend of botanicals (BOT) against lipopolysaccharide (LPS)-induced inflammation on cultured hepatocytes and weaning piglets. In vitro studies examined HepG2 cells treated with BOT and challenged with Escherichiacoli LPS for 8 d. BOT treatment reduced IL-6 concentration in cell culture media across time (P < 0.05) and decreased pro-inflammatory cytokine expression on days 1 and 8 of experiment (TNFα, IL-1ß; P < 0.05). BOT also increased the expression of antioxidant enzymes (GPX-2, SOD, CAT) on day 8 (P < 0.05), which was supported by lowered reactive oxygen species concentration after LPS challenge (P < 0.1). The in vivo study was conducted with 72 weaning pigs, allotted into 24 pens and divided into 3 groups: a negative control (CTR-, basal diet), a challenged control (CTR+) that received an intraperitoneal injection of E. coli O55:B5 LPS on days 14 and 16, and a challenged treated group which received a diet containing 1.5 g/kg of microencapsulated BOT (BOT+) for the whole duration of the study. Growth performance was determined weekly and, on days 21 (1 animal per pen) and 28 (remaining animals), pigs were sacrificed to collect liver and jejunal tissues. After the challenge, BOT+ pigs had increased BW on days 21 (P < 0.05) and 28 (P < 0.1) compared to CTR+. Similar improvements in average daily gain and FCR on days 14 to 21 (P < 0.05) and 21 to 28 (P < 0.1) were also seen in BOT+ group. In the liver, compared to CTR+ pigs, BOT+ pigs had downregulated expression of TLR-4, IL-6, IFN-γ on day 21 (P < 0.05), and TLR-4, TNF-α, IL-8 on day 28 (P < 0.05). BOT+ also increased GPX-2 expression on days 21 and 28 (P < 0.05), while also upregulating SOD-1 and SOD-2 on day 21 (P < 0.05) and CAT on day 28 (P < 0.05) compared to CTR+. In the jejunum, BOT+ reduced inflammation by affecting cytokine expression (P < 0.05) and increasing the expression of tight-junction proteins, ZO-1 on day 21 and CLD-1 on day 28 (P < 0.05). Furthermore, BOT+ pigs had lower crypt depth on days 21 (P < 0.1) and 28 (P < 0.05), and increased villi-to-crypt ratio on days 21 and 28 (P < 0.05). By day 28, BOT+ intestinal measurements were restored to values similar to the CTR-. Finally, BOT+ also reduced mast cell activation on day 21 (P < 0.05) compared to CTR+. Considering all the findings, BOT controlled inflammatory activation and oxidative stress in liver cells, enhanced intestinal integrity, and as a result improved the growth performance of weaning piglets challenged with LPS.

Piglets are particularly susceptible to stress due to the abrupt changes they face during weaning. These stressors cause a surge of oxidation and inflammation, particularly in the intestinal tract. Inflammation in the intestine causes a loss in its barrier function and facilitates the translocation of harmful compounds. Of particular concern is the translocation of lipopolysaccharide (LPS), which elicits an immune response in the liver, diverting energy from growth to inflammatory processes. Exposure to LPS also has the potential to have long-lasting detrimental effects on piglets' health. Research has identified the potential of many botanicals to minimize weaning stress through diverse modes of action. This study investigated the efficacy of a blend of botanicals (BOT) to help hepatocytes control inflammatory stress in vitro and to ameliorate the effects of an LPS challenge in piglets in vivo. Our in vitro and in vivo models successfully generated an inflammatory state. In vitro, BOT decreased inflammation and oxidation, and similar effects were seen in vivo, where BOT supplementation modulated the expression of cytokines in the liver and maintained intestinal integrity. These effects validate BOT ability to improve the performance of LPS-challenged piglets and support its utilization as a feed supplement to mitigate weaning stress.

Botanicals as a zinc oxide alternative to protect intestinal cells from an Escherichia coli F4 infection in vitro by modulation of enterocyte inflammatory response and bacterial virulence.

Pharmacological doses of zinc oxide (ZnO) have been widely used in pig industry to control post-weaning diarrhea (PWD) symptoms exacerbated by enterotoxigenic Escherichia coli F4 infections. Because of environmental issues and regulatory restrictions, ZnO is no longer sustainable, and novel nutritional alternatives to manage PWD are urgently required. Botanicals represent a wide class of compounds employed in animal nutrition because of their diverse beneficial functions. The aim of this study was to investigate the in vitro protective action of a panel of essential oils and natural extracts on intestinal Caco-2 cells against an E. coli F4 infection. Moreover, we explored the potential mechanisms of action of all the botanicals compared to ZnO. Amongst the others, thyme essential oil, grape seed extract, and Capsicum oleoresin were the most effective in maintaining epithelial integrity and reducing bacterial translocation. Their mechanism of action was related to the modulation of cellular inflammatory response, the protection of tight junctions' expression and function, and the control of bacterial virulence, thus resembling the positive functions of ZnO. Moreover, despite their mild effects on the host side, ginger and tea tree essential oils provided promising results in the control of pathogen adhesion when employed during the challenge. These outcomes support the advantages of employing selected botanicals to manage E. coli F4 infections in vitro, therefore offering novel environmentally-friendly alternatives to pharmacological doses of ZnO capable to modulate host-pathogen interaction at different levels during PWD in pigs.

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.

A blend of selected botanicals maintains intestinal epithelial integrity and reduces susceptibility to Escherichia coli F4 infection by modulating acute and chronic inflammation in vitro.

In the pig production cycle, the most delicate phase is weaning, a sudden and early change that requires a quick adaptation, at the cost of developing inflammation and oxidation, especially at the intestinal level. In this period, pathogens like enterotoxigenic Escherichia coli (ETEC) contribute to the establishment of diarrhea, with long-lasting detrimental effects. Botanicals and their single bioactive components represent sustainable well-recognized tools in animal nutrition thanks to their wide-ranging beneficial functions. The aim of this study was to investigate the in vitro mechanism of action of a blend of botanicals (BOT), composed of thymol, grapeseed extract, and capsicum oleoresin, in supporting intestinal cell health during inflammatory challenges and ETEC infections. To reach this, we performed inflammatory and ETEC challenges on Caco-2 cells treated with BOT, measuring epithelial integrity, cellular oxidative stress, bacterial translocation and adhesion, gene expression levels, and examining tight junction distribution. BOT protected enterocytes against acute inflammation: while the challenge reduced epithelial tightness by 40%, BOT significantly limited its drop to 30%, also allowing faster recovery rates. In the case of chronic inflammation, BOT systematically improved by an average of 25% the integrity of challenged cells (p < 0.05). Moreover, when cells were infected with ETEC, BOT maintained epithelial integrity at the same level as an effective antibiotic and significantly reduced bacterial translocation by 1 log average. The mode of action of BOT was strictly related to the modulation of the inflammatory response, protecting tight junctions' expression and structure. In addition, BOT influenced ETEC adhesion to intestinal cells (-4%, p < 0.05), also thanks to the reduction of enterocytes' susceptibility to pathogens. Finally, BOT effectively scavenged reactive oxygen species generated by inflammatory and H2O2 challenges, thus alleviating oxidative stress by 40% compared to challenge (p < 0.05). These results support the employment of BOT in piglets at weaning to help manage bacterial infections and relieve transient or prolonged stressful states thanks to the modulation of host-pathogen interaction and the fine-tuning activity on the inflammatory tone.

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.

Effects of microencapsulated blend of organic acids and botanicals on growth performance, intestinal barrier function, inflammatory cytokines, and endocannabinoid system gene expression in broiler chickens.

With restricted usage of growth-promoting antibiotics, identifying alternative feed additives that both improve intestinal barrier function and reduce inflammation is the center to improve chickens' health. This study examined the effects of a microencapsulated feed additive containing citric acid, sorbic acids, thymol, and vanillin on intestinal barrier function and inflammation status. A total of 240 birds were assigned to either a commercial control diet or control diet supplemented with 500 g/MT of the microencapsulated additive product. Birds were raised by feeding a 2-phase diet (starter, d 1 to d 21; and grower, d 15 to d 42). Growth performance was recorded weekly. At d 21 and d 42, total gastrointestinal tract permeability was evaluated by FITC-dextran (FD4) oral gavage. Jejunum-specific barrier functions were evaluated by Ussing chamber. Intestinal gene expression of selected epithelial cell markers, tight junction (TJ) proteins, inflammatory cytokines, and endocannabinoid system (ECS) markers were determined by RT-PCR. Statistical analysis was performed using Student t test. Results showed significant improvement of feed efficiency in the birds supplemented with the blend of organic acids and botanicals. At d 21, both oral and jejunal FD4 permeability were lower in the supplemented group. Jejunal transepithelial resistance was higher in the supplemented birds. At d 21, expression of TJs mRNA (CLDN1 and ZO2) was both upregulated in the jejunum and ileum of supplemented birds, while CLDN2 was downregulated in cecum. Proliferating cell marker SOX9 was higher expressed in jejunum and ceca. Goblet cell marker (MUC2) was upregulated, while Paneth cell marker (LYZ) was downregulated in the ileum. Proinflammatory cytokine expressions of IL1B, TNFA, and IFNG were downregulated in jejunum, while anti-inflammatory IL10 expression was higher in jejunum, ileum, cecum, and cecal tonsil. The ECS markers expressions were upregulated in most intestinal regions. Together, these results demonstrated that the blend of organic acids and botanical supplementation reduced inflammation, improved the TJs expression and intestinal barrier function, and thus improved chicken feed efficiency. The activated ECS may play a role in reducing intestinal tissue inflammation.

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.

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.