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.

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.

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

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

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

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

Impact of a Blend of Microencapsulated Organic Acids and Botanicals on the Microbiome of Commercial Broiler Breeders under Clinical Necrotic Enteritis.

Previously, the supplementation of a microencapsulated blend of organic acids and botanicals improved the health and performance of broiler breeders under non-challenged conditions. This study aimed to determine if the microencapsulated blend impacted dysbiosis and necrotic enteritis (NE) in broiler breeders. Day-of-hatch chicks were assigned to non-challenge and challenge groups, provided a basal diet supplemented with 0 or 500 g/MT of the blend, and subjected to a laboratory model for NE. On d 20-21, jejunum/ileum content were collected for microbiome sequencing (n = 10; V4 region of 16S rRNA gene). The experiment was repeated (n = 3), and data were analyzed in QIIME2 and R. Alpha and beta diversity, core microbiome, and compositional differences were determined (significance at p ≤ 0.05; Q ≤ 0.05). There was no difference between richness and evenness of those fed diets containing 0 and 500 g/MT microencapsulated blend, but differences were seen between the non-challenged and challenged groups. Beta diversity of the 0 and 500 g/MT non-challenged groups differed, but no differences existed between the NE-challenged groups. The core microbiome of those fed 500 g/MT similarly consisted of Lactobacillus and Clostridiaceae. Furthermore, challenged birds fed diets containing 500 g/MT had a higher abundance of significantly different phyla, namely, Actinobacteriota, Bacteroidota, and Verrucomicrobiota, than the 0 g/MT challenged group. Dietary supplementation of a microencapsulated blend shifted the microbiome by supporting beneficial and core taxa.

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.

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.

Contribution of Starmerella bacillaris and Oak Chips to Trebbiano d'Abruzzo Wine Volatile and Sensory Diversity.

In this study, six fermentation trials were carried out: co-inoculation and sequential inoculation of Saccharomyces cerevisiae and Starmerella bacillaris in the presence and absence of oak chips. Moreover, Starm. bacillaris strain was attached to the oak chips and co-inoculated or sequentially inoculated with S. cerevisiae. Wines fermented with Starm. bacillaris adhered to oak chips showed a higher concentration of glycerol (more than 6 g/L) than the others (about 5 g/L). These wines also showed a higher content of polyphenols (more than 300 g/L) than the others (about 200 g/L). The addition of oak chips induced an increase of yellow color (b* value of about 3). Oak-treated wines were characterized by a higher concentration of higher alcohols, esters and terpenes. Aldehydes, phenols and lactones were detected only in these wines, independently from the inoculation strategy. Significant differences (p < 0.05) were also observed in the sensory profiles. The fruity, toasty, astringency, and vanilla sensations were perceived as more intense in wines treated with oak chips. The white flower descriptor showed a higher score in wines fermented without chips. Oak surface-adhered Starm. bacillaris cells could be a good strategy to improve the volatile and sensory profile of Trebbiano d'Abruzzo wines.

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.

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.

Phenol-Rich Botanicals Modulate Oxidative Stress and Epithelial Integrity in Intestinal Epithelial Cells.

Botanicals are mainly known for their role as antimicrobials and anti-inflammatories. Thus, the dual purpose of the study was to verify the antioxidant potential of the tested botanicals and to evaluate their possible modulation of intestinal barrier integrity. As the effects of various phenol-rich extracts were screened, the human Caco-2 cell line was determined to be most suitable for use as the in vitro model for the intestinal epithelium. The tested botanicals, all approved as feed additives, are ginger essential oil, tea tree oil, grape seed extract, green tea extract, olive extract, chestnut extract, pomegranate extract, thyme essential oil, and capsicum oleoresin. The cells were treated with incremental doses of each botanical, followed by measurements of transepithelial electrical resistance (TEER), gene expression of tight junctions (TJs), and reactive oxygen species (ROS). The results showed how different phenol-rich botanicals could modulate barrier functions and oxidative stress in different ways. Interestingly, all the botanicals tested exerted an antioxidant potential by dropping the cytoplasmatic ROS, while the beneficial effect was exerted at different concentrations for each botanical. Our data support the role of plant extracts and essential oils in controlling gut barrier function and in reducing the negative effects of oxidative stress in intestinal epithelial cells, thereby supporting gut barrier functionality.

Impact of Saccharomyces cerevisiae and non-Saccharomyces yeasts to improve traditional sparkling wines production.

In this study the effect of a co-inoculum of S. cerevisiae (F6789) with Torulaspora delbrueckii (TB1) or Starmerella bacillaris (SB48) on the oenological and aroma characteristics of sparkling wines obtained with the Champenoise method was investigated. The autolytic outcome and the sensory profile of sparkling wines were also evaluated. The secondary fermentations were completed by all mixed and single starter cultures with the only exception of those guided by Starm.bacillaris. Sparkling wines produced with S. cerevisiae F6789+Starm.bacillaris SB48 showed the highest amounts of glycerol (6.51 g/L). The best autolytic potential was observed in sparkling wines produced with +Starm.bacillaris (81.98 mg leucin/L) and S. cerevisiae+T. delbrueckii (79.03 mg leucin/L). The lowest value was observed for sparkling wines obtained with S. cerevisiae F6789 (53.96 mg leucin/L). Sparkling wines showed different aroma and sensory profiles. Esters were mainly present in sparkling wines obtained with S. cerevisiae F6789 (88.09 mg/L) followed by those obtained with S. cerevisiae+T. delbrueckii (87.20 mg/L), S. cerevisiae +Starm.bacillaris (81.93 mg/L). The content of esters decreased over time, and that might be related to the adsorption on lees and chemical hydrolysis. The highest concentrations of higher alcohols were found in sparkling wines produced with S. cerevisiae+T. delbrueckii (27.50 mg/L). Sparkling wines obtained with S. cerevisiae +Starm.bacillaris were well differentiated from the others due to their high score for the descriptor for spicy, bread crust, freshness and floral. Tailored strains with different autolytic potential might represent an interesting strategy to improve traditional sparkling wine production and favour their differentiation.

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.

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 microencapsulated organic acids and botanicals reduces necrotic enteritis via specific signaling pathways in broilers.

Necrotic enteritis (NE) is a devastating disease that has seen a resurgence of cases following the removal of antibiotics from feed resulting in financial loss and significant animal health concerns across the poultry industry. The objective was to evaluate the efficacy of a microencapsulated blend of organic (25% citric and 16.7% sorbic) acids and botanicals (1.7% thymol and 1% vanillin [AviPlusP]) to reduce clinical NE and determine the signaling pathways associated with any changes. Day-of-hatch by-product broiler breeder chicks were randomly assigned to a control (0) or supplemented (500 g/MT) diet (n = 23-26) and evaluated in a NE challenge model (n = 3). Birds were administered 2X cocci vaccine on d 14 and challenged with a cocktail of Clostridium perfringens strains (107) on d 17 to 19. On d 20 to 21 birds were weighed, euthanized, and scored for NE lesions. Jejunal tissue was collected for kinome analysis using an immuno-metabolism peptide array (n = 5; 15/treatment) to compare tissue from supplement-fed birds to controls. Mortality and weight were analyzed using Student's t test and lesion scores analyzed using F-test two-sample for variances (P < 0.05). The kinome data was analyzed using PIIKA2 peptide array analysis software and fold-change between control and treated groups determined. Mortality in the supplemented group was 47.4% and 70.7% in controls (P = 0.004). Lesions scores were lower (P = 0.006) in supplemented birds (2.47) compared to controls (3.3). Supplement-fed birds tended (P = 0.19) to be heavier (848.6 g) than controls (796.2 g). Kinome analysis showed T cell receptor, TNF and NF-kB signaling pathways contributed to the improvements seen in the supplement-fed birds. The following peptides were significant (P < 0.05) in all 3 pathways: CHUK, MAP3K14, MAP3K7, and NFKB1 indicating their importance. Additionally, there were changes to IL6, IL10, and IFN- γ mRNA expression in tissue between control- and supplement-fed chickens. In conclusion, the addition of a microencapsulated blend of organic acids and botanicals to a broiler diet reduced the clinical signs of NE that was mediated by specific immune-related pathways.

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.

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.

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

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

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.

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.