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.

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.

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.

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.

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.

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.

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.

Thyme, Oregano, and Garlic Essential Oils and Their Main Active Compounds Influence Eimeria tenella Intracellular Development.

Coccidiosis poses a significant challenge in poultry production and is typically managed with ionophores and chemical anticoccidials. However, the emergence of drug resistance and limitations on their use have encouraged the exploration of alternative solutions, including botanical compounds and improvements in in vitro screening methods. Prior research focused only on the impact of these alternatives on Eimeria invasion, with intracellular development in cell cultures receiving limited attention. This study assessed the impact of thyme (Thymus vulgaris), oregano (Origanum vulgare), and garlic (Allium sativum) essential oils, as well as their bioactive compounds, on the initial phase of schizogony in Madin-Darby bovine kidney cells, comparing their effectiveness to two commercially used anticoccidial drugs. Using image analysis and quantitative PCR, the study confirmed the efficacy of commercial anticoccidials in reducing invasion and schizont formation, and it found that essential oils were equally effective. Notably, thymol and carvacrol exhibited mild inhibition of intracellular replication of the parasite but significantly reduced schizont numbers, implying a potential reduction in pathogenicity. In conclusion, this research highlights the promise of essential oils and their bioactive components as viable alternatives to traditional anticoccidial drugs for mitigating coccidiosis in poultry, particularly by disrupting the intracellular development of the parasites.

Thymol as an Adjuvant to Restore Antibiotic Efficacy and Reduce Antimicrobial Resistance and Virulence Gene Expression in Enterotoxigenic Escherichia coli Strains.

The continuous spread of antimicrobial resistance is endangering the efficient control of enterotoxigenic Escherichia coli (ETEC), which is mainly responsible for post-weaning diarrhea onset in piglets. Thymol, the key constituent of thyme essential oil, is already used in animal nutrition for its antimicrobial action. The aim of this study was to investigate the potential adjuvant effect of thymol to re-establish antibiotic efficacy against highly resistant ETEC field strains. Secondly, we evaluated the modulation of virulence and antibiotic resistance genes. Thymol showed the capacity to control ETEC growth and, when combined with ineffective antibiotics, it increased their antimicrobial power. In particular, it showed significant effects when blended with colistin and tetracycline, suggesting that the adjuvant effects rely on the presence of complementary mechanisms of action between molecules, or the absence of resistance mechanisms that inactivate antibiotics and target sites. Furthermore, our findings demonstrate that, when added to antibiotics, thymol can help to further downregulate several virulence and antibiotic resistance genes, offering new insights on the potential mechanisms of action. Therefore, in a one-health approach, our study supports the beneficial effects of combining thymol with antibiotics to restore their efficacy, together with the possibility of targeting gene expression as a pioneering approach to manage ETEC pathogenicity.

Dual Antimicrobial Effect of Medium-Chain Fatty Acids against an Italian Multidrug Resistant Brachyspira hyodysenteriae Strain.

The fastidious nature of Brachyspira hyodysenteriae limits an accurate in vitro pre-screening of conventionally used antibiotics and other candidate alternative antimicrobials. This results in a non-judicious use of antibiotics, leading to an exponential increase of the antibiotic resistance issue and a slowdown in the research for new molecules that might stop this serious phenomenon. In this study we tested four antibiotics (tylosin, lincomycin, doxycycline, and tiamulin) and medium-chain fatty acids (MCFA; hexanoic, octanoic, decanoic, and dodecanoic acid) against an Italian field strain of B. hyodysenteriae and the ATCC 27164 strain as reference. We determined the minimal inhibitory concentrations of these substances, underlining the multidrug resistance pattern of the field strain and, on the contrary, a consistent and stable inhibitory effect of the tested MCFA against both strains. Then, sub-inhibitory concentrations of antibiotics and MCFA were examined in modulating a panel of B. hyodysenteriae virulence genes (tlyA, tlyB, bhlp16, bhlp29.7, and bhmp39f). Results of gene expression analysis were variable, with up- and downregulations not properly correlated with particular substances or target genes. Decanoic and dodecanoic acid with their direct and indirect antimicrobial property were the most effective among MCFA, suggesting them as good candidates for subsequent in vivo trials.

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.

Towards Zero Zinc Oxide: Feeding Strategies to Manage Post-Weaning Diarrhea in Piglets.

Zinc oxide (ZnO) at pharmacological doses is extensively employed in the pig industry as an effective tool to manage post-weaning diarrhea (PWD), a condition that causes huge economic losses because of its impact on the most pivotal phase of a piglet's production cycle. In a multifactorial way, ZnO exerts a variety of positive effects along the entire gastrointestinal tract by targeting intestinal architecture, digestive secretions, antioxidant systems, and immune cells. ZnO also has a moderate antibacterial effect against Escherichia coli F4 (K88), the main causative agent of PWD. However, the environmental impact of ZnO and new emerging threats are posing serious questions to the sustainability of its extensive utilization. To work towards a future free from pharmacological ZnO, novel nutritional approaches are necessary, and many strategies have been investigated. This review article provides a comprehensive framework for ZnO utilization and its broad mode of action. Moreover, all the risks related to pharmacological ZnO levels are presented; we focus on European institutions' decisions subsequently. The identification of a novel, complete solution against PWD should be accompanied by the adoption of holistic strategies, thereby combining good management practices to feeding approaches capable of mitigating Escherichia coli F4 (K88) infections and/or lowering ZnO utilization. Promising results can be obtained by adjusting diet composition or employing organic acids, natural identical compounds, polyphenol-rich extracts, prebiotics, and probiotics.

In Vitro Assessment of Anticoccidials: Methods and Molecules.

Avian coccidiosis is a disease causing considerable economic losses in the poultry industry. It is caused by Eimeria spp., protozoan parasites characterized by an exogenous-endogenous lifecycle. In vitro research on these pathogens is very complicated and lacks standardization. This review provides a description of the main in vitro protocols so far assessed focusing on the exogenous phase, with oocyst viability and sporulation assays, and on the endogenous phase, with invasion and developmental assays in cell cultures and in ovo. An overview of these in vitro applications to screen both old and new remedies and to understand the relative mode of action is also discussed.

From Acidifiers to Intestinal Health Enhancers: How Organic Acids Can Improve Growth Efficiency of Pigs.

Organic acids have been used successfully in pig production as a cost-effective performance-enhancing option and they continue to be the number one alternative to antibiotic growth promoters. The aim of this review is to provide the biological rationale behind organic acids use in pig production, focusing on their different effects along the gastrointestinal tract of pigs. Organic acids are reviewed for their antimicrobial properties and for their classic use as acidifiers, with particular attention to pH modulation and microflora control. Additional beneficial effects on intestinal health and general metabolism are presented and we explain the advantage of microencapsulation as a tool to deliver organic acids along the intestine.

Nature-Identical Compounds and Organic Acids Reduce E. coli K88 Growth and Virulence Gene Expression In Vitro.

Post-weaning diarrhoea (PWD) is one of the long-standing challenges in pig husbandry. Due to the risks of resistance caused by antibiotics (AB) misuse, conventional treatments against Escherichia coli K88 (E. coli K88), the PWD etiological agent, urgently need to be replaced. Organic acids (OA) and nature-identical compounds (NIC) are currently finding a central role in infection management thanks to their recognized antimicrobial activity. This study investigated the susceptibility of an E. coli K88 field strain to a wide panel of AB, NIC, and OA. Secondly, we evaluated the ability of sub-lethal doses of the most active compounds to modulate the expression of E. coli K88 virulence genes. Results showed that the bacterial strain was resistant to many of the tested antibiotics, but an antimicrobial action was registered for selected NIC and OA. The quantitative PCR analysis revealed that thymol, carvacrol, eugenol, and benzoic acid were able to downregulate (p < 0.05) the expression of bacterial genes related to motility, adhesion to enterocytes, heat-labile (LT) and heat-stable (ST) toxin secretion, quorum sensing, and biofilm formation. Therefore, this study demonstrated that selected OA and NIC not only control E. coli K88 growth but also modulate the expression of many virulence genes at sub-lethal doses, thus offering new insights on their mechanism of action and suggesting a powerful tool to manage PWD.

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.

Butyric acid induces spontaneous adipocytic differentiation of porcine bone marrow-derived mesenchymal stem cells.

Butyric acid (BA) affects the differentiation of mesenchymal stem cells (MSC) through the activation of different transcriptional pathways. The aim of this study was to determine the effects of BA on proliferation and spontaneous differentiation of porcine bone marrow-derived MSC. Second passage MSC (n = 6) were cultured in either a basal medium (BM, DMEM + 10% FBS), or BM + 2.5 mmol/L BA (BA-2.5) or BM + 5 mmol/L BA (BA-5). Cell proliferation was significantly decreased by both BA-2.5 and BA-5 after 48 h and 72 h (- 55% and - 63%, respectively). To assess the impact of BA on spontaneous differentiation, MSC were cultured for 27 d, with complete media changes every 3 d. At day 27, cells were stained for osteocytic, chondrocytic, and adipocytic differentiation. No terminal differentiation was detected in control MSC, while accumulated small drops of lipids were stained by Oil-Red-O in BA-treated cells. The phenotypic changes were associated with changes in gene expression, determined by qPCR. Treatment with BA modulated the expression of adipocytic differentiation markers: peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α were significantly increased by both BA-2.5 and BA-5 throughout the study, while lipoprotein lipase and fatty acid-binding protein 4 were increased by BA-5 at day 3, and decreased by both BA-5 and BA-2.5 later throughout the study. Osteocalcin and aggrecan mRNA was reduced throughout the experiment by both doses of BA (P < 0.05). In conclusion, our data support that BA promotes the spontaneous differentiation of porcine bone marrow-derived MSC toward an adipocytic lineage in the absence of inducing cocktail media.

Cellular stress marker alteration and inflammatory response in pigs fed with an ochratoxin contaminated diet.

Aim of this study was to characterize the effects of an ochratoxin A (181 ± 34 ng/g) contaminated diet on growth performances, blood parameters, systemic cytokine levels, cell stress markers and reactivity of immune system of weaned pigs. Growth performance was not affected by OTA consumption even if OTA levels increased in plasma, kidney and liver. OTA diminished the protein content in the serum and increased levels of TNF-alpha and IL-10 in plasma. HO-1 mRNA, indicative for cells stress, was decreased in the kidney but increased in the liver. Additionally, whole blood of the animals of the OTA-group showed a decreased capacity to respond with cytokine expression (mRNA and protein) to ex vivo challenge with LPS. In conclusion our findings indicate that chronic ingestion with OTA-contaminated feed, even at low level, is hazardous for the animal and virtually for human health, pig being an excellent model for human.