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

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

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

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 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

Effects of Microencapsulated Blend of Organic Acids and Pure Botanicals on the Ruminal Microbiota in an In Vitro Dual-Flow Continuous Culture System

The objective of the study was to evaluate the effects of dietary supplementation of a microencapsulated blend of organic acids and pure botanicals (mOAPBs) on the solid- and liquid-associated microenvironment (SAM and LAM, respectively) of the ruminal microbiome using an in vitro dual-flow continuous culture system. Ruminal content was incubated in eight fermenters and the basal diet was supplemented with increasing levels of mOAPBs (0; 0.12; 0.24; or 0.36% DM) which contained 55.6% hydrogenated and refined palm oil, 25% citric acid, 16.7% sorbic acid, 1.7% thymol, and 1% vanillin. All diets had a similar nutritional composition (16.1 CP, 30.9 NDF, and 32.0 starch, % DM basis). After 7 days of adaptation, a pooled sample across the days was collected in each period for identification of the microbiome of SAM and LAM. There was no effect of mOAPB on alpha-, beta-diversity, and microbial abundance. The SAM had a greater bacterial diversity and the principal component analysis demonstrated that it had a divergent bacterial profile from LAM. Additionally, SAM had an increased abundance of carbohydrate-degrading microorganisms. In summary, mOAPBs did not modulate the ruminal microbiome. The microenvironment microbiome of solid- and liquid-associated microenvironments were different, with SAM having a greater carbohydrate-degrading microorganism population

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 sub-clinical 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 3h to assess the beneficial effect of two 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 two 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

The use of thymol, carvacrol and sorbic acid in microencapsules to control Salmonella Heidelberg, S. Minnesota and S. Typhimurium in broilers

Introduction: The control of Salmonella spp. in poultry involves different biosecurity actions and lately has been complicated by the emergence of multidrug resistant serovars. The application of organic acids and essential oils has been used with different approaches due to the antibacterial properties as food preservatives. The use of these molecules in animal feed to control enteric pathogens is a major interest within the poultry industry. Methods: The use of a blend containing nature-identical compounds of sorbic acid (25%), thymol (9.5%) and carvacrol (2.5%) microencapsulated in a lipid matrix, was investigated in the present work, for the control of three Salmonella serovars (S. ser. Typhimurium, S. ser. Heidelberg and S. ser. Minnesota). Commercial broilers were challenged at 3 or at 33 days of age. Groups SH-1, SM-1 and ST-1, received treatment in the feed, at 2 kg/ton from 1–21 days of age and at 1 kg/ton from 35–42 days of age (last week), while groups SH-2, SM-2 and ST-2, were treated only during the last week receiving 2 kg/ton. Each treated group had an untreated control group, that was challenged at the same moment with the respective serovar (groups PCH, PCM and PCT). The challenge strains were enumerated in liver and cecal contents, weekly after challenge, at 7, 14, 21, 28, 35 and 42 days-of-age. Results and discussion: Significant reduction was noticed at 7 and 14 days of age in all groups that received treatment during the initial phase (p < 0.05). Moreover, the body weight was significantly higher at the last experimental day (p < 0.05) in chickens that received treatment at the initial and at the final growth stages

Antimicrobial Power of Organic Acids and Nature-Identical Compounds against Two Vibrio spp.: An In Vitro Study

Vibrosis is one of the major threats in aquaculture farming, and due to the increasing antimicrobial resistance of different Vibrio species, there is an urgent need to replace conventional treatments with more sustainable solutions. Antimicrobial molecules such as organic acids (OA) and nature-identical compounds (NIC) are currently finding a central role in the infection management of terrestrial livestock, but little is known about their usage in aquaculture. The aim of this study was to perform a preliminary screening of the in vitro antimicrobial activity of a wide panel of OA and NIC against 2 Vibrio species characteristic of the Mediterranean area, Vibrio harveyi and Vibrio anguillarum, through minimal inhibitory/bactericidal concentration tests. The active principles that showed the best effective pathogen control were medium-chain fatty acids, sorbic and benzoic acid among OA and phenolic monoterpenoids (thymol, carvacrol and eugenol) and aromatic monoterpene aldehydes (vanillin and cinnamaldehyde) among NIC. These results showed how the usage of OA and NIC can open promising perspectives in terms of Vibrio spp. load control in aquaculture. Natural or nature-identical feed additives can make aquaculture production not only more efficient by reducing the need for medicated treatments, but also more sustainable

A microencapsulated feed additive containing organic acids, thymol, and vanillin increases in vitro functional activity of peripheral blood leukocytes from broiler chicks

During the first week after hatch, young chicks are vulnerable to pathogens as the immune system is not fully developed. The objectives of this study were to determine if supplementing the starter diet with a microencapsulated feed additive containing citric and sorbic acids, thymol, and vanillin affects in vitro functional activity of peripheral blood leukocytes (PBLs). Day-old chicks (n = 800) were assigned to either a control diet (0 g/metric ton [MT]) or a diet supplemented with 500 g/MT of the microencapsulated additive. At 4 D of age, peripheral blood was collected (100 birds per treatment), and heterophils and monocytes isolated (n = 4). Heterophils were assayed for the ability to undergo degranulation and production of an oxidative burst response while nitric oxide production was measured in monocytes. Select cytokine and chemokine mRNA expression levels were also determined. Statistical analysis was performed using Student t test comparing the supplemented diet to the control (P ≤ 0.05). Heterophils isolated from chicks fed the microencapsulated citric and sorbic acids, thymol, and vanillin had higher (P ≤ 0.05) levels of degranulation and oxidative burst responses than those isolated from chicks on the control diet. Heterophils from the supplemented chicks also had greater (P ≤ 0.05) expression of IL10, IL1β, and CXCL8 mRNA than those from control-fed chicks. Similarly, nitric oxide production was significantly (P ≤ 0.05) higher in monocytes isolated from birds fed the supplement. The cytokine and chemokine profile in monocytes from the supplement-fed chicks showed a significant (P ≤ 0.05) drop in IL10 mRNA expression while IL1β, IL4, and CXCL8 were unchanged. In conclusion, 4 D of supplementation with a microencapsulated blend made up of citric and sorbic acids, thymol, and vanillin enhanced the in vitro PBL functions of degranulation, oxidative burst, and nitric oxide production compared with the control diet. Collectively, the data suggest feeding broiler chicks a diet supplemented with a microencapsulated blend of citric and sorbic acids, thymol, and vanillin may prime key immune cells making them more functionally efficient and acts as an immune-modulator to boost the inefficient and undeveloped immune system of young chicks