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Antibiotics have played a critical role in sustaining and improving livestock production in the past decades, but the emergence of antimicrobial resistance has led several countries to ban or limit their use. Since then, in-feed alternatives have gained a lot of attention but the development of efficacious alternatives implies a better understanding of the mode of action of antibiotic growth promoters (AGP) when administered at subtherapeutic concentrations. In the present study, 120 broiler chickens per group (8 pens/group) were fed for 35 d with either basal feed (control group) or feed supplemented with avilamycin (AGP group; 10 g/1,000 kg of feed). At the end of the trial, the ileum from the small intestine of 5 birds per group was sampled, and RNA were isolated for profiling their transcriptome via RNA sequencing (RNA-Seq). As expected, the growth of chickens in the AGP group was significantly higher than in the control group. Overall, 66 differentially expressed genes (false discovery rate ≤ 0.05 and fold change ≥ 2 or ≤ −2) were found in the ileum of chickens fed avilamycin in comparison with the control group. The functional analysis showed reduced activity of genes related to signaling by interleukins, with IL-22, SOCS3, and certain antimicrobial peptides found multiple times in these pathways in the AGP group at day 35. In addition, higher activity was predicted in a module of genes related to lipid metabolism and transport in the avilamycin group. The use of RNA-Seq allowed a snapshot of the whole transcriptome at day 35 and aimed at delivering additional data on the host-centric hypothesis regarding the mode of action of AGP (i.e. immunomodulation, reduction of the immunological stress).

Phytogenics are plant-based feed additives utilized in animal nutrition to support animal growth and health. Worldwide restrictions and bans on the use of antibiotic growth promoters resulted in an increased demand for in-feed alternatives including phytogenics. However, several challenges remain for technology readiness in animal industry, especially regarding the standardization of the ingredients as well as our knowledge on the cellular mechanisms underlying their biological effects. In the present study, 32 weaned piglets were allocated for 28 days to four experimental diets, a control diet, a phytogenic feed additive (PFA) diet, or the same two diets but with the addition of oxidized oil (OO) at 10%. The last two diets aimed at evaluating the antioxidant properties of PFA. At the end of the trial, the ileum and the liver of the pigs were sampled, and RNA were isolated for profiling their transcriptomeviaRNA sequencing (RNA-Seq). In the ileum, the gene set enrichment analysis showed that the activity of several immune pathways (NF-kB, interferon α/β, antimicrobial peptide, and collagen pathways) was reduced in piglets fed PFA compared to the control piglets. As expected, the addition of OO induced strong effects on the liver transcriptome and most likely accounted for the significant growth impairment. The likelihood ratio test across the four diets revealed a global response driven by the oxidative stress challenge with hundreds of genes associated with fatty acid β-oxidation and peroxisome in the liver. The expression levels of those genes in the piglets fed OO+PFA were much less affected by the challenge. Collectively, the effects seen at day 28 suggest that substances in the PFA formulation provide anti-inflammatory and antioxidant properties. The use of RNA-Seq in animal nutrition allows exploring and deciphering novel mechanisms of natural growth promoters.

The presence of mycotoxins in cereals has led to large economic losses in Europe. In the course of the European project MyToolBox, prevention and control measures to reduce mycotoxin contamination in cereals were developed. This study aimed to estimate the impact of these prevention and control measures on both the reduction in crop losses and the increased volume of crops suitable for food and/or feed. It focused on the following measures: the use of fungicides during wheat cultivation, the use of resistant maize cultivars and/or biocontrol during maize cultivation, the use of real time sensors in storage silos, the use of innovative milling strategies during the pasta making process, and the employment of degrading enzymes during the process of bioethanol and Dried Distillers Grains with Solubles (DDGS) production. The impact assessment was based on the annual volume of cereals produced, the annual levels of mycotoxin contamination, and experimental data on the prevention and control measures collected in the course of the MyToolBox project. Results are expressed in terms of reduced volumes of cereals lost, or as additional volumes of cereals available for food meeting the current European legal limits. Results showed that a reduction in crop losses as well as an increase in the volume of crops suitable as food and/or feed is feasible with each proposed prevention or control measure along the supply chain. The impact was the largest in areas and in years with the highest mycotoxin contamination levels but would have less impact in years with low mycotoxin levels. In further research, the impact assessment may be validated using future data from more years and European sites. Decision makers in the food and feed supply chain can use this impact assessment to decide on the relevant prevention and control strategies to apply.

Affordable and practical tools for farmers and food processors along the chain are required to efficiently reduce the risk of mycotoxin contamination of crops, feeds and foods. Developing new tools and enhancing existing ones was the mission of MyToolBox—a four-year EU-project that included important Chinese partners and joint research efforts. To identify future directions in mycotoxin research and management in China and their role in China–EU relations, a unique stakeholder workshop including group discussions was organized in Beijing. Six related topics: biocontrol, forecasting, sampling and analysis, silo management, detoxification, and the development of safe use options for contaminated materials were covered. The discussions clearly identified a critical need for smart, integrated strategies to address mycotoxin issues to attain safer food and feed, and to minimize losses and export rejections. Managing data on when, where and the size of mycotoxin contamination events and identifying the institution(s) to manage them are complex issues in China. Studies of microbes and novel, genetically-altered enzymes to limit pre-harvest contamination and to manage post-harvest product detoxification and alternate uses of contaminated materials are in the early stages in China. Further efforts are needed to increase the visibility of mycotoxin problems beyond the scientific and research communities.

Ochratoxin A (OTA), a mycotoxin that is of utmost concern in food and feed safety, is produced by fungal species that mainly belong to the Aspergillus and Penicillium genera. The development of mitigation strategies to reduce OTA content along the supply chains is key to ensuring safer production of food and feed. Enzyme-based strategies are among the most promising methods due to their specificity, efficacy, and multi-situ applicability. In particular, some enzymes are already known for hydrolyzing OTA into ochratoxin alpha (OT&alpha, ) and phenylalanine (Phe), eventually resulting in detoxification action. Therefore, the discovery of novel OTA hydrolyzing enzymes, along with the advancement of an innovative approach for their identification, could provide a broader basis to develop more effective mitigating strategies in the future. In the present study, a hybrid in silico/in vitro workflow coupling virtual screening with enzymatic assays was applied in order to identify novel OTA hydrolyzing enzymes. Among the various hits, porcine carboxypeptidase B was identified for the first time as an effective OTA hydrolyzing enzyme. The successful experimental endorsement of findings of the workflow confirms that the presented strategy is suitable for identifying novel OTA hydrolyzing enzymes, and it might be relevant for the discovery of other mycotoxin- mitigating enzymes.

As animal feed is prone to infestation with mycotoxin-producing fungi, mycotoxin contamination of feed should be monitored. Here, we report a multi-mycotoxin survey of feed samples from Africa. We determined the concentrations of aflatoxins, fumonisins, deoxynivalenol, T-2 toxin, zearalenone and ochratoxin A in 1,045 samples of finished feed and feed raw materials (maize, maize silage, other cereals, etc.) from South Africa and 318 samples from Algeria, Tunisia, Morocco, Senegal, Côte d’Ivoire, Nigeria, Ghana, Namibia, Uganda, Kenya, Tanzania, Zambia and Madagascar. We compared the measured mycotoxin concentrations to regulatory limits or guidance values that are in effect in the European Union and analysed the co-occurrence of these mycotoxins. To determine the occurrence of other fungal secondary metabolites, a subset of the samples was analysed using a multi-analyte liquid chromatography tandem mass spectrometry-based method for the simultaneous detection of over 700 fungal metabolites. We found that 33.3% of maize samples and 54.4% of finished feed samples from Senegal, Côte d’Ivoire, Nigeria, Ghana, Namibia, Uganda, Kenya and Tanzania exceeded the European regulatory limit of 20 ng/g aflatoxins. The Fusarium mycotoxins zearalenone, fumonisins and deoxynivalenol were prevalent in all commodities from all countries, but concentrations were in most cases below European guidance values. Concentrations of deoxynivalenol and zearalenone were correlated. Several other Fusarium metabolites occurred frequently (e.g. moniliformin, beauvericin, aurofusarin) or in high concentrations (e.g. aurofusarin, fusaproliferin). Furthermore, high levels of diplodiatoxin were occasionally detected in samples from South Africa and the Alternaria metabolite tenuazonic acid was prevalent and reached high concentrations. In conclusion, aflatoxins frequently occurred in African feed samples in potentially unsafe concentrations. While Fusarium mycotoxins mostly occurred in concentrations below European guidance values, a correlation between deoxynivalenol and zearalenone concentrations suggests that toxicological interactions of these compounds deserve attention. Several less investigated fungal secondary metabolites occurred frequently or reached high concentrations.

International audience; Ingestion of deoxynivalenol (DON), one of the most common mycotoxin contaminants of cereals, leads to adverse effects for animal and human health. Bacterial biotransformation is a strategy to mitigate the toxicity of this mycotoxin. The present study aims to evaluate the toxicity of two bacterial biotranformation products of DON: 3-epi-deoxynivalenol (3-epi-DON) and de-epoxy-deoxynivalenol (DOM-1) through zootechnical, hematological, histological and immunological assays. Twenty-four 4weeks-old piglets received a control diet or a diet contaminated with 3 mg kg-1 DON, DOM-1, or 3-epi-DON for 7 days. Sample tissues were collected for histomorphometrical analysis, expression of cytokines and cell protein junctions. The zootechnical and hematological parameters were not modulated by any treatment. Ingestion of DON induced histological alterations in the intestine, liver and lymphoid organs, as well as an overexpression of pro-inflammatory cytokines, E-cadherin and occludin. These changes were not observed in piglets receiving the DOM-1 and 3-epi-DON contaminated diets. Pigs fed 3-epi-DON contaminated diet showed an increase in IgM levels in comparison with other diets, while no change was observed in IgA and IgG levels among the diets. Our results indicate that DOM-1 and 3-epi-DON are not toxic for piglets; thus bacterial biotransformation seems to be a sustainable alternative to reduce mycotoxin toxicity.

International audience; Deoxynivalenol (DON) is the most abundant trichothecene in food and feed. It causes both acute and chronic disorders of the human and animal intestine, liver and the immune system. The structural basis for the toxicity of DON has not been fully elucidated. Using the pig as a target and a model species for human, the toxicity of DON and its deepoxy-metabolite (DOM-1) were compared. Animals were exposed by gavage to 1 and 0.5 nmol toxin/kg bw./day for two and three weeks respectively. Whatever the dose/duration, DOM-1 was less toxic than DON in terms of weight gain and emesis. In the three week experiment, animals were vaccinated with ovalbumin, and their immune response was analyzed in addition to tissue morphology, biochemistry and hematology. DON impaired the morphology of the jejunum and the ileum, reduced villi height, decreased Ecadherin expression and modified the intestinal expression of cytokine. Similarly, DON induced hepatotoxicity as indicated by the lesion score and the blood biochemistry. By contrast, DOM-1 only induced minimal intestinal toxicity and did not trigger hepatotoxicity. As far as the immune response was concerned, the effects of ingesting DOM-1 were similar to those caused by DON, as measured by histopathology of lymphoid organs, PCNA expression and the specific antibody responses. Taken together, these data demonstrated that DOM-1, a microbial detoxification product of DON, was not toxic in the sensitive pig model but retained some immunemodulatory properties of DON, especially its ability to stimulate a specific antibody response during a vaccination protocol.

Fumonisins (FB) are among the most frequently detected mycotoxins in feedstuffs and finished feed, and recent data suggest that the functions of the gastrointestinal tract (GIT) in poultry species might be compromised at doses ranging from 10 to 20 mg/kg, close to field incidences and below the US and EU guidelines. Strategies are therefore necessary to reduce the exposure of poultry to FB. In the present study, we assessed the efficacy of fumonisin esterase FumD (EC 3.1.1.87, commercial name FUMzyme®) to cleave the tricarballylic acid side chains of FB, leading to the formation of non-toxic hydrolyzed fumonisins in the GIT of broiler chickens. Broiler chickens were fed for 14 d (7 to 21 d of age) 3 different diets (6 birds/cage, 6 cages/diet), i) control feed (negative control group), ii) feed contaminated with 10 mg FB/kg (FB group), and iii) feed contaminated with 10 mg FB/kg and supplemented with 100 units of FUMzyme®/kg (FB+FUMzyme® group). To determine the degree of reduction of FB in the GIT, 2 characteristics were analyzed. First, the sphinganine-to-sphingosine ratio in the serum and liver was determined as a biomarker of effect for exposure to FB. Second, the concentration of fumonisin B1 and its hydrolyzed forms was evaluated in the gizzard, the proximal and distal parts of the small intestine, and the excreta. Significantly reduced sphinganine-to-sphingosine ratios in the serum and liver of the FB+FUMzyme® group (serum: 0.15 ± 0.01; liver: 0.17 ± 0.01) compared to the FB group (serum: 0.20 ± 0.01; liver: 0.29 ± 0.03) proved that supplementation of broiler feed with FUMzyme® was effective in partially counteracting the toxic effect of dietary FB. Likewise, FB concentrations in digesta and excreta were significantly reduced in the FB+FUMzyme® group compared to the FB group (P < 0.05; up to 75%). FUMzyme® furthermore partially counteracted FB-induced up-regulation of cytokine gene expression (IL-8 and IL-10) in the jejunum. The FB group showed significantly higher gene expression of IL-8 and IL-10 compared to the negative control group (IL-8: fold change = 2.9 ± 1.1, P < 0.05; IL-10: fold change = 3.6 ± 1.4, P < 0.05), whereas IL-8 and IL-10 mRNA levels were not significantly different in the FB+FUMzyme®® group compared to the other 2 groups. In conclusion, FUMzyme® is suitable to detoxify FB in chickens and maintain gut functions.

Deoxynivalenol (DON), a trichothecene produced by various Fusarium species, is one of the most prevalent food- and feed-associated mycotoxins. The effects of DON and deepoxy-deoxynivalenol (DOM-1) were assessed in five different cell lines from different tissues and species starting from the first line of defense, the trout gill (RTgill-W1) and pig intestinal cells (IPEC-1 and IPEC-J2) over immune cells, as second line of defense (mouse macrophages RAW 264.7) to human liver cells (HepG2). Viability was assessed with a WST-1 assay, except for RTgill-W1, where a neutral red (NR) and sulforhodamine B (SRB) assay was performed. Additionally, more sensitive parameters, such as interleukin-, nitric oxide (NO)-, and albumin-release were determined. Viability was affected by DON at concentrations starting at 10 μmol/L (RTgill-W1), 0.9 μmol/L (IPEC-1), 3.5 μmol/L (IPEC-J2), and 0.9 μmol/L (HepG2), whereas DOM-1 did not have such an effect. Additionally, NO was decreased (0.84 μmol/L DON), whereas interleukin (IL)-6 was increased (0.42 μmol/L DON) in lipopolysaccharide (LPS)-stimulated DON-, but not DOM-1-treated RAW cells. Tumor necrosis factor (TNF)-α release, however, was not affected. Interestingly, albumin secretion of HepG2 cells was decreased by both DON and DOM-1 but at a much higher concentration for DOM-1 (228 versus 0.9 μmol/L for DON). 98.9% of DOM-1 was retrieved by liquid chromatography tandem mass spectrometry at the end of the experiment, proving its stability. In this study, IL-6 was the most sensitive parameter, followed by NO and albumin release and viability for HepG2 and IPEC-1.

Feed samples are frequently contaminated by a wide range of chemically diverse natural products, which can be determined using highly sensitive analytical techniques. Next to already well-investigated mycotoxins, unknown or unregulated fungal secondary metabolites have also been found, some of which at significant concentrations. In our study, 1141 pig feed samples were analyzed for more than 800 secondary fungal metabolites using the same LC-MS/MS method and ranked according to their prevalence. Effects on the viability of the 28 most relevant were tested on an intestinal porcine epithelial cell line (IPEC-J2). The most frequently occurring compounds were determined as being cyclo-(L-Pro-L-Tyr), moniliformin, and enniatin B, followed by enniatin B1, aurofusarin, culmorin, and enniatin A1. The main mycotoxins, deoxynivalenol and zearalenone, were found only at ranks 8 and 10. Regarding cytotoxicity, apicidin, gliotoxin, bikaverin, and beauvericin led to lower IC50 values, between 0.52 and 2.43 &micro, M, compared to deoxynivalenol (IC50 = 2.55 &micro, M). Significant cytotoxic effects were also seen for the group of enniatins, which occurred in up to 82.2% of the feed samples. Our study gives an overall insight into the amount of fungal secondary metabolites found in pig feed samples compared to their cytotoxic effects in vitro.

Enzymatic detoxification has become a promising approach for control of mycotoxins postharvest in grains through modification of chemical structures determining their toxicity. In the present study fumonisin esterase FumD (EC 3.1.1.87) (FUMzyme&reg, BIOMIN, Tulln, Austria), hydrolysing fumonisin (FB) mycotoxins by de-esterification, was utilised to develop an enzymatic reduction method in a maize kernel enzyme incubation mixture. Efficacy of the FumD FB reduction method in &ldquo, low&rdquo, and &ldquo, high&rdquo, FB contaminated home-grown maize was compared by monitoring FB1 hydrolysis to the hydrolysed FB1 (HFB1) product utilising a validated LC-MS/MS analytical method. The method was further evaluated in terms of enzyme activity and treatment duration by assessing enzyme kinetic parameters and the relative distribution of HFB1 between maize kernels and the residual aqueous environment. FumD treatments resulted in significant reduction (&ge, 80%) in &ldquo, (&ge, 1000 U/L, p &lt, 0.05) and &ldquo, (100 U/L, p &lt, 0.05, &ge, 0.0001) FB contaminated maize after 1 h respectively, with an approximate 1:1 &micro, mol conversion ratio of FB1 into the formation of HFB1. Enzyme kinetic parameters indicated that, depending on the activity of FumD utilised, a significantly (p &lt, 0.05) higher FB1 conversion rate was noticed in &ldquo, FB contaminated maize. The FumD FB reduction method in maize could find application in commercial maize-based practices as well as in communities utilising home-grown maize as a main dietary staple and known to be exposed above the tolerable daily intake levels.

The mycotoxin zearalenone (ZEN) poses a risk to animal health because of its estrogenic effects. Diagnosis of ZEN-induced disorders remains challenging due to the lack of appropriate biomarkers. In this regard, circulating microRNAs (small non-coding RNAs) have remarkable potential, as they can serve as indicators for pathological processes in tissue. Thus, we combined untargeted and targeted transcriptomics approaches to investigate the effects of ZEN on the microRNA expression in porcine uterus, jejunum and serum, respectively. To this end, twenty-four piglets received uncontaminated feed (Control) or feed containing 0.17 mg/kg ZEN (ZEN low), 1.46 mg/kg ZEN (ZEN medium) and 4.58 mg/kg ZEN (ZEN high). After 28 days, the microRNA expression in the jejunum remained unaffected, while significant changes in the uterine microRNA profile were observed. Importantly, 14 microRNAs were commonly and dose-dependently affected in both the ZEN medium and ZEN high group, including microRNAs from the miR-503 cluster (i.e. ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-503 and ssc-miR-542-3p). Predicted target genes for those microRNAs are associated with regulation of gene expression and signal transduction (e.g. cell cycle). Although the effects in serum were less pronounced, receiver operating characteristic analysis revealed that several microRNA ratios were able to discriminate properly between non-exposed and ZEN-exposed pigs (e.g. ssc-miR-135a-5p/ssc-miR-432-5p, ssc-miR-542-3p/ssc-miR-493-3p). This work sheds new light on the molecular mechanisms of ZEN, and fosters biomarker discovery.

Food and feed can be naturally contaminated by several mycotoxins, and concern about the hazard of exposure to mycotoxin mixtures is increasing. In this study, more than 800 metabolites were analyzed in 524 finished pig feed samples collected worldwide. Eighty-eight percent of the samples were co-contaminated with deoxynivalenol (DON) and other regulated/emerging mycotoxins. The Top 60 emerging/regulated mycotoxins co-occurring with DON in pig feed shows that 48%, 13%, 8% and 12% are produced by Fusarium, Aspergillus, Penicillium and Alternaria species, respectively. Then, the individual and combined toxicity of DON and the 10 most prevalent emerging mycotoxins (brevianamide F, cyclo-(L-Pro-L-Tyr), tryptophol, enniatins A1, B, B1, emodin, aurofusarin, beauvericin and apicidin) was measured at three ratios corresponding to pig feed contamination. Toxicity was assessed by measuring the viability of intestinal porcine epithelial cells, IPEC-1, at 48-h. BRV-F, Cyclo and TRPT did not alter cell viability. The other metabolites were ranked in the following order of toxicity: apicidin &gt, enniatin A1 &gt, DON &gt, beauvericin &gt, enniatin B &gt, enniatin B1 &gt, emodin &gt, aurofusarin. In most of the mixtures, combined toxicity was similar to the toxicity of DON alone. In terms of pig health, these results demonstrate that the co-occurrence of emerging mycotoxins that we tested with DON does not exacerbate toxicity.

Fumonisin esterase FumD (EC 3.1.1.87), FUMzyme® (BIOMIN, Austria), effectively detoxifies fumonisin B mycotoxins (FB) by hydrolysis and removal of the tricarballylic acid groups. The current study evaluated FumD detoxification of total FB (FBT) in commercial maize utilising an experimental dry milling plant by introducing the enzyme during the kernel conditioning stage. Total FB and the hydrolysed product of FB1, HFB1, in maize and milling products were determined by LC-MS/MS. During maize conditioning of 4 h 10 min substantial FB1 hydrolysis was achieved between 1 and 4 U FumD/100 g maize. Complete conversion into HFB1 was delayed and only achieved at the highest enzyme concentration (32 U/100 g maize) reaching a 1:1 M conversion ratio. Dry milling of maize containing 3.29 ± 0.20 μmole FBT/kg (2354 ± 140 μg/kg) in the absence of FumD, resulted in a 2.5-fold increase in the FBT concentration in total hominy feed (8.34 ± 0.22 μmol/kg) (5979 ± 158 μg/kg) compared to the levels that prevail in Super (0.52 ± 0.07 μmol/kg) (347 ± 48 μg/kg) and Special (1.70 ± 0.01 μmol/kg) (1213 ± 8 μg/kg) maize meal, and Semolina (1.07 ± 0.14 μmol/kg) (765 ± 100 μg/kg) milling products. Introduction of FumD (40 U/kg) mainly impacted the total hominy feed product (germ + hominy milling fractions), constituting up to 30% of the reconstituted whole maize. A 99% reduction in FBT was obtained in total hominy feed, 48% in Semolina, 7% in Special maize meal, whereas no reduction was recorded in Super maize meal. FB1 reduction rates depend on the contamination level, kernel moisture and the diffusion rate from inner kernel layers to the kernel surface/aqueous interface. Risk modelling in children and adults indicated that FumD-treated whole maize and the resultant Semolina milling product intended for human consumption reduces the risk of exposure to FBT. However, no reduction in the exposure risk was observed when considering the Super and Special maize meal milling products. FB reduction in total hominy feed could open up new applications, such as its dietary incorporation as a source of fibre, minerals and bioactive plant constituents in maize-based food. In addition, the animal feed industry and subsistence maize farming communities using rudimentary milling processes, could also benefit.

The mycotoxin deoxynivalenol (DON) contaminates agricultural commodities worldwide, posing health threats to humans and animals. Associated with DON are derivatives, such as deepoxy-deoxynivalenol (DOM-1), produced by enzymatic transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Using differentiated porcine intestinal epithelial cells (IPEC-J2), we provide the first multi-parameter comparative cytotoxicity analysis of DON and DOM-1, based on the parallel evaluation of lysosomal activity, total protein content, membrane integrity, mitochondrial metabolism and ATP synthesis. The study investigated the ability of DON and-for the first time of its metabolite DOM-1-to induce apoptosis, mitogen-activated protein kinase (MAPK) signalling, oxidative events and alterations of mitochondrial structure in porcine intestinal epithelial cells (IECs). The degree of DON toxicity strongly varied, depending on the cytotoxicity parameter evaluated. DON compromised viability according to the parameters of lysosomal activity, total protein content and membrane integrity, but increased viability according to assays based on mitochondrial metabolism and ATP synthesis. DON induced expression of cleaved caspase-3 (maximum induction 3.9-fold) and MAPK p38 and p42/p44 (maximum induction 2.51- and 2.30-fold, respectively). DON altered mitochondrial morphology, but did not increase intracellular ROS. DOM-1-treated IPEC-J2 remained unaffected at equimolar concentrations in all assays, thereby confirming the safety of feed additives using DON- to DOM-1-transforming bacteria. The study additionally highlights that an extensive multi-parameter analysis significantly contributes to the quality of in vitro data.

The aim of this work was to assess the effect of dietary viable or heat inactivated probiotic forms (PF) combined or not with avilamycin (AV) used as a growth promoter, on broiler growth performance, nutrient digestibility, digestive enzyme activities, and expression of immune response related genes.Depending on the type of PF (i.e., no addition, viable, inactivated) and AV addition (no/yes), 450 one-day-old Cobb male broilers were allocated in the following 6 treatments according to a 3 × 2 factorial arrangement with 5 replicates of 15 broilers each for 6 wk: CoN: diet without any addition; CoN+A: combination of CoN with AV; ViP: viable PF - no AV; ViP+A: combination of ViP with AV; InP: inactivated PF - no AV; InP+A: combination of InP with AV.There were no interactions (P > 0.05) for overall performance parameters. In contrast, PF or AV addition improved BW gain (PPF= 0.015; PAV

Zearalenone (ZEA) is an oestrogenic mycotoxin produced by several Fusarium species, and it frequently contaminates cereals used for food or animal feed. A ZEA-lactonase of Gliocladium roseum was previously described to hydrolyse ZEA to an unstable intermediate, which spontaneously decarboxylates to non-oestrogenic, decarboxylated hydrolysed ZEA (DHZEN). We expressed a codon-optimised version of the ZEA-lactonase (ZHD101) gene of G. roseum MA 918 with a secretion leader in Pichia pastoris and purified the recombinant enzyme from culture supernatant by His-tag mediated affinity chromatography. After incubation of the enzyme with ZEA, we detected the previously elusive primary reaction product hydrolysed ZEA (HZEN) by liquid chromatography tandem mass spectrometry, purified it by preparative high-performance liquid chromatography, and confirmed its postulated structure ((E)-2,4-dihydroxy-6-(10-hydroxy-6-oxo-1-undecen-1-yl)benzoic acid) by nuclear magnetic resonance techniques. Spontaneous decarboxylation to DHZEN ((E)-1-(3,5-dihydroxy-phenyl)-10-hydroxy-1-undecen-6-one), but not to a previously reported isomer, was observed. Biomass resuspensions of G. roseum strains MA 918 and the strains used for previous work, NBRC 7063 and ATCC 8684, all converted ZEA to HZEN, DHZEN, and further unknown metabolites. We studied partitioning of HZEN and DHZEN between aqueous phases and organic solvents, and found that HZEN did not partition into chloroform as extraction solvent, under the conditions used by previous authors. In contrast, extraction with ethyl acetate at pH 2.0 was suitable for simultaneous extraction of HZEN and DHZEN. The detection of HZEN and its availability as an analytical standard may assist further work towards possible application of ZEA-lactonase (e.g. determining kinetic parameters) for detoxification of ZEA.

Lipopolysaccharides (LPS), also termed endotoxins, are the major component of the outer membrane of Gram-negative bacteria. In general, endotoxins in the intestine are considered harmless in healthy animals. However, different stressors, such as heat stress, can lead to a compromised gut barrier, resulting in endotoxin translocation. Chickens are considered to be less sensitive to the effects of LPS compared with other species, for example, humans, pigs, or calves, probably because of the lack of the functional-specific TRAM-TRIF signalling pathway (MyD88-independent). Therefore, six LPS preparations (three different strains with two different preparation methods each) were compared in murine macrophages and characterized according to their MyD88-dependent pathway activation. All tested LPS preparations induced a strong inflammatory response after 4 and 24 h on a murine macrophage cell line. However, there was a similar strong response in the gene expression profile as well as production of nitrite oxide and TNF-alpha from LPS of different strains and preparation methods. On the basis of the results of the in vitro study, one LPS preparation was chosen for the subsequent in vivo study with broilers to assess the effect of an oral LPS bolus (E. coli O55:B5 phenol extracted, 2 mg/kg b.w.) during heat stress conditions (10 h, 36 &deg, C). The most pronounced effects were seen in broilers receiving the oral LPS bolus during heat stress conditions. The endotoxin activity in the intestine as well as the serum concentration of the 3-OH C14 (part of LPS) were increased. In addition, an increased expression of genes related to inflammation and stress response (e.g., IL-6, IL-1beta, HSP70) was observed, whereas the expression of genes associated with gut health (e.g., MUC2, FABP2) was decreased. To conclude, an increase of intestinal LPS combined with heat stress can pose a risk to animal health.

Deoxynivalenol (DON) is one of the most prevalent mycotoxins, contaminating cereals and cereal-derived products. Its derivative deepoxy-deoxynivalenol (DOM-1) is produced by certain bacteria, which either occur naturally or are supplemented in feed additive. DON-induced impairments in protein synthesis are particularly problematic for highly proliferating immune cells. This study provides the first comparison of the effects of DON and DOM-1 on the concanavalin A-induced proliferation of porcine, chicken, and bovine peripheral blood mononuclear cells (PBMCs). Therefore, isolated PBMCs were treated with DON (0.01–3.37 µM) and DOM-1 (1.39–357 µM) separately, and proliferation was measured using a bromodeoxyuridine (BrdU) assay. Although pigs are considered highly sensitive to DON, the present study revealed a substantially higher sensitivity of bovine (IC50 = 0.314 µM) PBMCs compared to chicken (IC50 = 0.691 µM) and porcine (IC50 = 0.693 µM) PBMCs. Analyses on the proliferation of bovine T-cell subsets showed that all major subsets, namely, CD4+, CD8β+, and γδ T cells, were affected to a similar extent. In contrast, DOM-1 did not affect bovine PBMCs, but reduced the proliferation of chicken and porcine PBMCs at the highest tested concentration (357 µM). Results confirm the necessity of feed additives containing DON-to-DOM-1-transforming bacteria and highlights species-specific differences in the DON sensitivity of immune cells.

Deoxynivalenol (DON) is one of the most frequently occurring mycotoxins in cereal crops worldwide. DON poses a risk to human and animal health due to its wide range of adverse effects. Recently, novel insights into the metabolism of DON enabled the use of the biomarker approach for human exposure assessment. In certain subpopulations, a considerable proportion of tested individuals were found to exceed the maximum tolerable intake for DON. Since the masked mycotoxin deoxynivalenol-3-β- d -glucoside (DON-3-Glc) is partly cleaved during mammalian digestion, liberation of DON might further increase the total mycotoxin burden. However, progress in better understanding detoxification strategies along the food and feed chain has been made and the first product for DON detoxification in feed was approved in the EU.

Aflatoxins are a class of mycotoxins that impair poultry health and performance. Some clays have the ability to adsorb aflatoxins. In our study, mineralogical composition of the tested clays was confirmed by powder X-ray diffraction and simultaneous thermal analysis and their properties like pH, electrical conductivity, exchangeable cations, cation exchange capacity and clay content were determined. For their in vitro assessment regarding aflatoxin B1 (AFB1) adsorption, adsorption tests under ‘intensified conditions’ (low adsorbent and high toxin concentration) were carried out in buffers at various pH values and in real gastric juice followed by isothermal analysis in phosphate buffer. In vivo we used a completely randomised design with 4 replicate pens of 5 chicks assigned to each dietary treatment from hatch to 21 days. Dietary treatments included a negative and a positive control diet (2 mg/kg AFB1), and treatment groups receiving 2 mg/kg AFB1 and 0.5% of one of 8 adsorbents. Results of in vitro experiments ranked the adsorbents as ‘good’ (R, MB, B7, M32, M34, M5; 6 bentonites containing a cis-vacant smectite), ‘average’ (bentonite C2 containing a trans-vacant smectite) or ‘poor’ (zeolite Z08, containing clinoptilolite). The addition of AFB1 significantly reduced feed intake and/or body weight gain of the chicks compared to the negative control and to the treatment groups, except for C2 and Z08. Except Z08, all adsorbents numerically decreased the liver weight compared to the AFB1-fed group. Kidney weights were significantly increased by the addition of AFB1 compared to the control and most adsorbents, except C2 and Z08. Similarly, ‘good’ adsorbents significantly prevented the decrease in serum albumin and serum total protein. The ability of adsorbents to ameliorate AFB1 toxicity in poultry basically correlated with the in vitro findings meaning that ‘poor’ and ‘average’ adsorbents did not (Z08) or only partially (C2) protect against AFB1 in vivo.

Ergot alkaloids are frequent contaminants of cereal crops. Strategies for their inactivation include the use of microorganisms or enzymes as feed additives capable of degrading ergot alkaloids. Recently, an ergopeptine-degrading Rhodococcus erythropolis strain MTHt3 (DSM 25948) has been isolated from soil and the involved enzymes ErgA and ErgB have been identified. The aim of the current study was to characterise the metabolites formed by degradation of various ergopeptines with the MTHt3 strain, its lysate and the purified enzyme ErgA. Using preparative HPLC, 1H-, 13C- and 2D-NMR as well as HR-MS measurements, two main groups of metabolites formed during microbial and enzymatic degradation of ergopeptines were identified: diketopiperazines (cyclic dipeptides, DKPs) and unstable ergine hydroxy carboxylic acids. However, degradation by strain, lysate and enzyme yielded different end-products. Whereas DKPs were transient and lysergic acid the only final product upon incubation of ergopeptines with the R. erythropolis strain, incubation with the lysate resulted in formation of lysergic acid and two isomeric DKPs at different ratios. Enzymatic degradation by ErgA yielded only one DKP isomer and ended at the stage of the ergine hydroxy carboxylic acids which then spontaneously degraded to ergine. In conclusion, we succeeded in identification of metabolites formed by microbial and enzymatic degradation of ergot alkaloids which is a crucial step in the future development of feed additives for gastro intestinal detoxification of ergopeptines in farm animals.

Extensive research over the last couple of decades has made it obvious that mycotoxins are commonly prevalent in majority of feed ingredients. A worldwide mycotoxin survey in 2013 revealed 81% of around 3,000 grain and feed samples analyzed had at least 1 mycotoxin, which was higher than the 10-year average (from 2004 to 2013) of 76% in a total of 25,944 samples. The considerable increase in the number of positive samples in 2013 may be due to the improvements in detection methods and their sensitivity. The recently developed liquid chromatography coupled to (tandem) mass spectrometry allows the inclusion of a high number of analytes and is the most selective, sensitive, and accurate of all the mycotoxin analytical methods. Mycotoxins can affect the animals either individually or additively in the presence of more than 1 mycotoxin, and may affect various organs such as gastrointestinal tract, liver, and immune system, essentially resulting in reduced productivity of the birds and mortality in extreme cases. While the use of mycotoxin binding agents has been a commonly used counteracting strategy, considering the great diversity in the chemical structures of mycotoxins, it is very obvious that there is no single method that can be used to deactivate mycotoxins in feed. Therefore, different strategies have to be combined in order to specifically target individual mycotoxins without impacting the quality of feed. Enzymatic or microbial detoxification, referred to as “biotransformation” or “biodetoxification,” utilizes microorganisms or purified enzymes thereof to catabolize the entire mycotoxin or transform or cleave it to less or non-toxic compounds. However, the awareness on the prevalence of mycotoxins, available modern techniques to analyze them, the effects of mycotoxicoses, and the recent developments in the ways to safely eliminate the mycotoxins from the feed are very minimal among the producers. This symposium review paper comprehensively discusses the above mentioned aspects.

International audience; Mycotoxins are secondary fungal metabolites produced mainly by Aspergillus, Penicillium and Fusarium. As evidenced by large-scale surveys, humans and animals are simultaneously exposed to several mycotoxins. Simultaneous exposure could result in synergistic, additive or antagonistic effects. However, most toxicity studies addressed the effects of mycotoxins separately. We present the experimental designs and we discuss the conclusions drawn from in vitro experiments exploring toxicological interactions of mycotoxins. We report more than 80 publications related to mycotoxin interactions. The studies explored combinations involving the regulated groups of mycotoxins, especially aflatoxins, ochratoxins, fumonisins, zearalenone and trichothecenes, but also the "emerging" mycotoxins beauvericin and enniatins. Over 50 publications are based on the arithmetic model of additivity. Few studies used the factorial designs or the theoretical biology-based models of additivity. The latter approaches are gaining increased attention. These analyses allow determination of the type of interaction and, optionally, its magnitude. The type of interaction reported for mycotoxin combinations depended on several factors, in particular cell models and the tested dose ranges. However, synergy among Fusarium toxins was highlighted in several studies. This review indicates that well-addressed in vitro studies remain valuable tools for the screening of interactive potential in mycotoxin mixtures.

During an 8-year period, 17 316 samples of feed and feed raw materials from all over the world were analysed for contamination with aflatoxins, ochratoxin A, zearalenone, deoxynivalenol and fumonisins. Overall, 72% of the samples tested positive for at least one mycotoxin and 38% were found to be co-contaminated. Mycotoxin concentrations were generally low and the majority of the samples were compliant with the most stringent EU guidance values or maximum levels for mycotoxins in feed. However, in their present state these regulations do not address co-contamination and associated risks. Long-term trends are difficult to establish as strong yearly variations were observed regarding mycotoxin prevalence and contamination levels. In some cases unusual weather conditions can be linked with high observed mycotoxin loads. An exception to this rule is South-East Asia, where a steady increase of aflatoxin prevalence has been observed. The percentage of aflatoxin-positive samples in this region rose from 32% in 2005 to 71% in 2011.

The development of liquid chromatography-mass spectrometry (LC-MS)/mass spectrometry (MS) methods for the simultaneous detection and quantification of a broad spectrum of mycotoxins has facilitated the screening of a larger number of samples for contamination with a wide array of less well-known "emerging" mycotoxins and other metabolites. In this study, 83 samples of feed and feed raw materials were analysed. All of them were found to contain seven to 69 metabolites. The total number of detected metabolites amounts to 139. Fusarium mycotoxins were most common, but a number of Alternaria toxins also occurred very often. Furthermore, two so-called masked mycotoxins (i.e., mycotoxin conjugates), namely deoxynivalenol-3-glucoside (75% positives) and zearalenone-4-sulfate (49% positives), were frequently detected. Although the observed median concentrations of the individual analytes were generally in the low μg/kg range, evaluating the toxicological potential of a given sample is difficult. Toxicity data on less well-known mycotoxins and other detected metabolites are notoriously scarce, as an overview on the available information on the most commonly detected metabolites shows. Besides, the possible synergistic effects of co-occurring substances have to be considered.

The human, animal and plant pathogen Fusarium, which contaminates agricultural commodities worldwide, produces numerous secondary metabolites. An example is the thoroughly-investigated deoxynivalenol (DON), which severely impairs gastrointestinal barrier integrity. However, to date, the toxicological profile of other Fusarium-derived metabolites, such as enniatins, beauvericin, moniliformin, apicidin, aurofusarin, rubrofusarin, equisetin and bikaverin, are poorly characterized. Thus we examined their effects—as metabolites alone and as metabolites in combination with DON—on the intestinal barrier function of differentiated intestinal porcine epithelial cells (IPEC-J2) over 72 h. Transepithelial electrical resistance (TEER) was measured at 24-h intervals, followed by evaluation of cell viability using neutral red (NR) assay. Enniatins A, A1, B and B1, apicidin, aurofusarin and beauvericin significantly reduced TEER. Moniliformin, equisetin, bikaverin and rubrofusarin had no effect on TEER. In the case of apicidin, aurofusarin and beauvericin, TEER reductions were further substantiated by the addition of otherwise no-effect DON concentrations. In all cases, viability was unaffected, confirming that TEER reductions were not due to compromised viability. Considering the prevalence of mycotoxin contamination and the diseases associated with intestinal barrier disruption, consumption of contaminated food or feed may have substantial health implications.

Deoxynivalenol (DON), produced by the plant pathogens Fusarium graminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways.