Inactivation of zearalenone (ZEN) and deoxynivalenol (DON) in complete feed for weaned piglets: Efficacy of ZEN hydrolase ZenA and of sodium metabisulfite (SBS) as feed additives.

Female pigs respond sensitive both to DON and ZEN with anorexia and endocrine disruption, respectively, when critical diet concentrations are exceeded. Therefore, the frequent co-contamination of feed by DON and ZEN requires their parallel inactivation. The additive ZenA hydrolyzes ZEN while SBS inactivates DON through sulfonation. Both supplements were simultaneously added (+, 2.5 g SBS and 100 U ZenA/kg) to a control diet (CON-, 0.04 mg DON and < 0.004 mg ZEN/kg; CON+, 0.03 mg DON and < 0.004 mg ZEN/kg) and a Fusarium toxin contaminated diet (FUS-, 2.57 mg DON and 0.24 mg ZEN/kg; FUS+, 2.04 mg DON and 0.24 mg ZEN/kg). The 4 diets were fed to 20 female weaned piglets each (6 kg initial body weight) for 35 days; the piglets were sacrificed thereafter for collecting samples. Supplements improved performance and modified metabolism and hematology independent of dietary DON contamination. The mechanisms behind these changes could not be clarified and require further consideration. SBS reduced DON concentration in feed by approximately 20% and to the same extent in blood plasma and urine suggesting that no further DON sulfonate formation occurred in the digestive tract before absorbing DON in the upper digestive tract or that additionally formed DON sulfonates escaped absorption. DON sulfonates were detected in feces suggesting that unabsorbed DON sulfonates reached feces and/or that unabsorbed DON was sulfonated in the hindgut. The observed reduction rate of 20% was evaluated to be insufficient for feeding practice. Galenic form of SBS added to dry feed needs to be improved to support the DON sulfonation in the proximal digestive tract.ZenA was active in the digestive tract as demonstrated by the presence of its hydrolyzed none-estrogenic reaction products hydrolyzed ZEN (HZEN) and decarboxylated and hydrolyzed ZEN (DHZEN) both in feces, systemic circulation, and urine of group FUS+ compared to group FUS-. The presence of these hydrolysis products was paralleled by a significant decrease in high-estrogenic ZEN concentrations which, in turn, was related to a decrease in relative weights of uteri and ovaries when compared to group FUS-. Thus, ZenA was proven to be effective; both in terms of biomarkers and biological effects.

Zearalenone and Its Emerging Metabolites Promptly Affect the Rumen Microbiota in Holstein Cows Fed a Forage-Rich Diet.

The study investigated the short-term effects of a single oral bolus of zearalenone (ZEN) on the rumen microbiota and fermentation patterns in four rumen-cannulated Holstein cows fed a forage diet with daily 2 kg/cow concentrate. During the baseline day, cows received uncontaminated concentrate, followed by ZEN-contaminated concentrate on the second day, and again the uncontaminated concentrate on day three. Free rumen liquid (FRL) and particle-associated rumen liquid (PARL) were collected at different hours post-feeding on all days to analyze the prokaryotic community composition, absolute abundances of bacteria, archaea, protozoa, and anaerobic fungi, as well as short-chain fatty acid (SCFA) profiles. The ZEN reduced the microbial diversity in FRL but not in the PARL fraction. The abundance of protozoa was higher after ZEN exposure in PARL, which may be related to their strong biodegradation capacity that, therefore, promoted protozoal growth. In contrast, α-zearalenol might compromise anaerobic fungi as indicated by reduced abundances in FRL and fairly negative correlations in both fractions. Total SCFA significantly increased in both fractions after ZEN exposure, while the SCFA profile only changed marginally. Concluding, a single ZEN challenge caused changes in the rumen ecosystem soon after intake, including ruminal eukaryotes, that should be the subject of future studies.

Enzymatic Degradation of Zearalenone in the Gastrointestinal Tract of Pigs, Chickens, and Rainbow Trout.

The estrogenic mycotoxin zearalenone (ZEN) is a common contaminant of animal feed. Effective strategies for the inactivation of ZEN in feed are required. The ZEN-degrading enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) converts ZEN to hydrolyzed ZEN (HZEN), thereby enabling a strong reduction in estrogenicity. In this study, we investigated the efficacy of ZenA added to feed to degrade ZEN in the gastrointestinal tract of three monogastric animal species, i.e., pigs, chickens, and rainbow trout. For each species, groups of animals received (i) feed contaminated with ZEN (chickens: 400 µg/kg, pigs: 200 µg/kg, rainbow trout: 2000 µg/kg), (ii) feed contaminated with ZEN and supplemented with ZenA, or (iii) uncontaminated feed. To investigate the fate of dietary ZEN in the gastrointestinal tract in the presence and absence of ZenA, concentrations of ZEN and ZEN metabolites were analyzed in digesta of chickens and rainbow trout and in feces of pigs. Upon ZenA administration, concentrations of ZEN were significantly decreased and concentrations of the degradation product HZEN were significantly increased in digesta/feces of each investigated animal species, indicating degradation of ZEN by ZenA in the gastrointestinal tract. Moreover, upon addition of ZenA to the diet, the concentration of the highly estrogenic ZEN metabolite α-ZEL was significantly reduced in feces of pigs. In conclusion, ZenA was effective in degrading ZEN to HZEN in the gastrointestinal tract of chickens, pigs, and rainbow trout, and counteracted formation of α-ZEL in pigs. Therefore, ZenA could find application as a ZEN-degrading feed additive for these animal species.

Effects of Heating, Pelleting, and Feed Matrix on Apparent Concentrations of Cereal Ergot Alkaloids in Relation to Growth Performance and Welfare Parameters of Backgrounding Beef Steers.

As the contamination of cereal grains with ergot has been increasing in Western Canada, studies were undertaken to evaluate the impacts of heating (60, 80, 120, or 190 °C) alone or in combination with pelleting on concentrations of ergot alkaloids. Fifteen samples of ergot-contaminated grain from Alberta and Saskatchewan were assayed for R and S epimers of six alkaloids (ergocryptine, ergocristine, ergocornine, ergometrine, ergosine, and ergotamine) using HPLC MS/MS. Five samples with distinct alkaloid profiles were then selected for heating and pelleting studies. Heating resulted in a linear increase (p < 0.05) of total R and total S epimers with increasing temperature, although some individual R epimers were stable (ergometrine, ergosine, ergotamine). Pelleting also increased (p < 0.05) concentrations of total R and total S epimers detected, although ergometrine concentration decreased (p < 0.05) after pelleting. A feeding study arranged in a 2 × 2 factorial structure used 48 backgrounding Angus-cross steers fed four different diets: (1) Control Mash (CM, no added ergot), (2) Control Pellet (CP), (3) Ergot Mash (EM), or (4) Ergot Pellet (EP). Pelleting heated the ergot to 90−100 °C under 4 bars pressure, but the ergot used in the feeding study was not otherwise heated. Alkaloid concentrations of EM and EP varied by up to 1.1 mg/kg depending on the feed matrix assayed. No differences among treatments were noted for growth performance, feed intake, feed conversion, concentrations of serum prolactin and haptoglobin, hair cortisol, or in temperatures of extremities measured by infrared thermography. The only negative impacts of ergot alkaloids were on blood parameters indicative of reduced immune function or chronic inflammation. Pelleting did not heighten the negative clinical outcomes of ergot, although alkaloid concentrations of pelleted feed increased depending on the matrix assayed. It was hypothesized that the heat and pressure associated with pelleting may enhance the recovery of alkaloids from pelleted feed.

Comparative Effects of Deoxynivalenol, Zearalenone and Its Modified Forms De-Epoxy-Deoxynivalenol and Hydrolyzed Zearalenone on Boar Semen In Vitro.

Deoxynivalenol (DON) and zearalenone (ZEN) are described as detrimental factors to sow and boar fertility. In comparison, literature reports on the impact of modified forms of DON and ZEN, such as de-epoxy-DON (DOM-1) and hydrolyzed ZEN (HZEN), on swine reproduction are scarce. The aim of our study was to compare the effects of DON, DOM-1, ZEN and HZEN on boar semen in vitro. To this end, pooled boar semen ejaculates from two adult boars were treated with either 50.6 µM DON, 62.8 µM ZEN or equimolar concentrations of DOM-1 and HZEN, respectively (dilution volume of v/v 0.7% DMSO in all cases). Effects on semen motility, morphology, viability, hypo-osmotic swelling test reaction and DNA integrity were investigated hourly up to four hours of incubation. DON negatively affected particular parameters evaluated with a computer-assisted sperm analysis system (CASA), such as immotile spermatozoa and progressive motile spermatozoa, whereas those effects were absent in the case of DOM-1 treatment. In contrast to HZEN, ZEN affected almost all CASA parameters. Furthermore, only ZEN decreased the proportion of viable spermatozoa and increased the proportion of spermatozoa with abnormalities. In conclusion, DON and ZEN negatively affected boar semen in vitro, whereas equimolar concentrations of DOM-1 and HZEN did not induce harmful effects.

Efficacy of Fumonisin Esterase in Piglets as Animal Model for Fumonisin Detoxification in Humans: Pilot Study Comparing Intraoral to Intragastric Administration.

Fumonisins, a group of highly prevalent and toxic mycotoxins, are suspected to be causal agents of several diseases in animals and humans. In the animal feed industry, fumonisin esterase is used as feed additive to prevent mycotoxicosis caused by fumonisins. In humans, a popular dosage form for dietary supplements, with high patient acceptance for oral intake, is capsule ingestion. Thus, fumonisin esterase provided in a capsule could be an effective strategy against fumonisin intoxication in humans. To determine the efficacy of fumonisin esterase through capsule ingestion, two modes of application were compared using piglets in a small-scale preliminary study. The enzyme was administered intraorally (in-feed analogue) or intragastrically (capsule analogue), in combination with fumonisin B1 (FB1). Biomarkers for FB1 exposure; namely FB1, hydrolysed FB1 (HFB1) and partially hydrolysed forms (pHFB1a and pHFB1b), were measured both in serum and faeces using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and toxicokinetic parameters were calculated. Additionally, the serum sphinganine/sphingosine (Sa/So) ratio, a biomarker of effect, was determined using LC-MS/MS. A significantly higher Sa/So ratio was shown in the placebo group compared to both esterase treatments, demonstrating the efficacy of the esterase. Moreover, a significant decrease in serum FB1 area under the concentration-time curve (AUC) and an increase of faecal HFB1 AUC were observed after intraoral esterase administration. However, these effects were not observed with statistical significance after intragastric esterase administration with the current sample size.

Effects of Fusarium metabolites beauvericin and enniatins alone or in mixture with deoxynivalenol on weaning piglets.

The impact of the Fusarium-derived metabolites beauvericin, enniatin B and B1 (EB) alone or in combination with deoxynivalenol (DON) was investigated in 28-29 days old weaning piglets over a time period of 14 days. The co-application of EB and DON (EB + DON) led to a significant decrease in the weight gain of the animals. Liver enzyme activities in plasma were significantly decreased at day 14 in piglets receiving the EB + DON-containing diet compared to piglets receiving the control diet. All mycotoxin-contaminated diets led to moderate to severe histological lesions in the jejunum, the liver and lymph nodes. Shotgun metagenomics revealed a significant effect of EB-application on the gut microbiota. Our results provide novel insights into the harmful impact of emerging mycotoxins alone or with DON on the performance, gut health and immunological parameters in pigs.

Effects of Deoxynivalenol and Fumonisins Fed in Combination to Beef Cattle: Immunotoxicity and Gene Expression.

We evaluated the effects of a treatment diet contaminated with 1.7 mg deoxynivalenol and 3.5 mg fumonisins (B1, B2 and B3) per kg ration on immune status and peripheral blood gene expression profiles in finishing-stage Angus steers. The mycotoxin treatment diet was fed for a period of 21 days followed by a two-week washout period during which time all animals consumed the control diet. Whole-blood leukocyte differentials were performed weekly throughout the experimental and washout period. Comparative profiles of CD4+ and CD8+ T cells, along with bactericidal capacity of circulating neutrophils and monocytes were evaluated at 0, 7, 14, 21 and 35 days. Peripheral blood gene expression was measured at 0, 7, 21 and 35 days via RNA sequencing. Significant increases in the percentage of CD4-CD8+ T cells were observed in treatment-fed steers after two weeks of treatment and were associated with decreased CD4:CD8 T-cell ratios at this same timepoint (p ≤ 0.10). No significant differences were observed as an effect of treatment in terms of bactericidal capacity at any timepoint. Dietary treatments induced major changes in transcripts associated with endocrine, metabolic and infectious diseases; protein digestion and absorption; and environmental information processing (inhibition of signaling and processing), as evaluated by dynamic impact analysis. DAVID analysis also suggested treatment effects on oxygen transport, extra-cellular signaling, cell membrane structure and immune system function. These results indicate that finishing-stage beef cattle are susceptible to the immunotoxic and transcript-inhibitory effects of deoxynivalenol and fumonisins at levels which may be realistically encountered in feedlot situations.

Deepoxy-deoxynivalenol (DOM-1), a derivate of deoxynivalenol (DON), exhibits less toxicity on intestinal barrier function, Campylobacter jejuni colonization and translocation in broiler chickens.

BACKGROUND: Intestinal epithelial cells are challenged by mycotoxins and many bacterial pathogens. It was previously shown that the mycotoxin deoxynivalenol (DON) as well as Campylobacter (C.) jejuni have a negative impact on gut integrity. Recently, it was demonstrated that DON increased the load of C. jejuni in the gut and inner organs. Based on this finding, it was hypothesized the DON metabolite (deepoxy-deoxynivalenol, DOM-1) should be able to reduce the negative effects of DON on colonization and translocation of C. jejuni in broilers, since it lacks the epoxide ring, which is responsible for the toxicity of DON. METHODS: A total of 180 broiler chickens were housed in floor pens on wood shavings with feed and water provided ad libitum. Birds were divided into six groups (n = 30 with 5 replicates/group): 1. Control, 2. DOM-1, 3. DON, 4. DOM-1 + C. jejuni, 5. DON + C. jejuni, 6. C. jejuni. At day 14, birds of groups 4, 5 and 6 were orally inoculated via feeding tube (gavage) with 1-ml of a PBS suspension containing 1 × 108 CFU of C. jejuni NCTC 12744. The performance parameters: body weight (BW), body weight gain (BWG), and feed intake of the birds were determined. At 7, 14, and 21 days post infection, samples from liver, spleen, duodenum, jejunum and cecum were aseptically collected and processed for bacteriological investigations. Finally, at each killing time point, segments of duodenum, jejunum and cecum were harvested and prepared for Ussing chamber studies to measure the paracellular mannitol fluxes. RESULTS: A significant decrease in body weight was observed for chickens receiving the DON diet with or without C. jejuni compared to the other groups. Furthermore, it was found that the co-exposure of birds to DON and C. jejuni resulted in a higher C. jejuni load not only in the gut but also in liver and spleen due to increased paracellular permeability of the duodenum, jejunum and cecum. On the contrary, DOM-1 supplementation in the feed improved the birds' performance and led to a better feed conversion ratio throughout the trial. Furthermore, DOM-1 did not negatively affect gut permeability and decreased the C. jejuni counts in the intestine and internal organs. CONCLUSION: Altogether, the presence of DOM-1 in the feed as a result of the enzymatic biotransformation of DON leads to a lower C. jejuni count in the intestine and better feed conversion ratio. Moreover, this study demonstrates that the detoxification product of DON, DOM-1, does not have negative effects on the gastrointestinal tract and reduces the Campylobacter burden in chickens and also the risk for human infection.

The efficacy and effect on gut microbiota of an aflatoxin binder and a fumonisin esterase using an in vitro simulator of the human intestinal microbial ecosystem (SHIME®).

Mycotoxin intoxication is in general an acknowledged and tackled issue in animals. However, in several parts of the world, mycotoxicoses in humans still remain a relevant issue. The efficacy of two mycotoxin detoxifying animal feed additives, an aflatoxin bentonite clay binder and a fumonisin esterase, was investigated in a human child gut model, i.e. the in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). Additionally, the effect of the detoxifiers on gut microbiota was examined in the SHIME. After an initial two weeks of system stabilisation, aflatoxin B1 (AFB1) and fumonisin B1 (FB1) were added to the SHIME diet during one week. Next, the two detoxifiers and mycotoxins were added to the system for an additional week. The AFB1, FB1, hydrolysed FB1 (HFB1), partially hydrolysed FB1a and FB1b concentrations were determined in SHIME samples using a validated ultra-performance liquid chromatography-tandem mass spectrometry method. The short-chain fatty acid (SCFA) concentrations were determined by a validated gas chromatography-mass spectrometry method. Colonic bacterial communities were analysed using metabarcoding, targeting the hypervariable V1-V3 regions of the 16S rRNA genes. The AFB1 and FB1 concentrations significantly decreased after the addition of the detoxifiers. Likewise, the concentration of HFB1 significantly increased. Concentrations of SCFAs remained generally stable throughout the experiment. No major changes in bacterial composition occurred during the experiment. The results demonstrate the promising effect of these detoxifiers in reducing AFB1 and FB1 concentrations in the human intestinal environment, without compromising the gastrointestinal microbiota.

Metabolism of Zearalenone in the Rumen of Dairy Cows with and without Application of a Zearalenone-Degrading Enzyme.

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feed and is well known for its estrogenic effects in animals. Cattle are considered less sensitive to ZEN than pigs. However, ZEN has previously been shown to be converted to the highly estrogenic metabolite α-zearalenol (α-ZEL) in rumen fluid in vitro. Here, we investigate the metabolism of ZEN in the reticulorumen of dairy cows. To this end, rumen-fistulated non-lactating Holstein Friesian cows (n = 4) received a one-time oral dose of ZEN (5 mg ZEN in 500 g concentrate feed) and the concentrations of ZEN and ZEN metabolites were measured in free rumen liquid from three reticulorumen locations (reticulum, ventral sac and dorsal mat layer) during a 34-h period. In all three locations, α-ZEL was the predominant ZEN metabolite and ß-zearalenol (ß-ZEL) was detected in lower concentrations. ZEN, α-ZEL and ß-ZEL were eliminated from the ventral sac and reticulum within 34 h, yet low concentrations of ZEN and α-ZEL were still detected in the dorsal mat 34 h after ZEN administration. In a second step, we investigated the efficacy of the enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) to degrade ZEN to the non-estrogenic metabolite hydrolyzed zearalenone (HZEN) in the reticulorumen in vitro and in vivo. ZenA showed a high ZEN-degrading activity in rumen fluid in vitro. When ZenA was added to ZEN-contaminated concentrate fed to rumen-fistulated cows (n = 4), concentrations of ZEN, α-ZEL and ß-ZEL were significantly reduced in all three reticulorumen compartments compared to administration of ZEN-contaminated concentrate without ZenA. Upon ZenA administration, degradation products HZEN and decarboxylated HZEN were detected in the reticulorumen. In conclusion, endogenous metabolization of ZEN in the reticulorumen increases its estrogenic potency due to the formation of α-ZEL. Our results suggest that application of zearalenone hydrolase ZenA as a feed additive may be a promising strategy to counteract estrogenic effects of ZEN in cattle.

The Mycotoxin Deoxynivalenol (DON) Promotes Campylobacter jejuni Multiplication in the Intestine of Broiler Chickens With Consequences on Bacterial Translocation and Gut Integrity.

Deoxynivalenol (DON) is one of the major health concern in poultry production as it targets epithelial cells of the gastrointestinal tract and contributes to the loss of the epithelial barrier function. It is well-documented that DON severely compromises various important intestinal functions in coincidence with aggravated clinical symptoms in livestock. In addition, a prolonged persistence of intestinal pathogens (e.g., Salmonella, Clostridium) in the gut has also been reported in pigs and chickens, respectively. Similar to DON, recent studies demonstrated that an experimental Campylobacter infection has severe consequences on gut health. Through experimental infection, it was found that Campylobacter (C.) jejuni negatively affects the integrity of the intestine and promotes the translocation of bacteria from the gut to inner organs. So far, no data are available investigating the simultaneous exposure of DON and C. jejuni in broilers albeit both are widely distributed. Thus, the aim of the present study was to explore the interaction between DON and C. jejuni which is of a significant public and animal health concern as it may affect the prevalence and the ability to control this pathogen. Following oral infection of birds at 14 days of age with C. jejuni NCTC 12744, we show that the co-exposure to DON and C. jejuni has a considerable consequence on C. jejuni loads in chicken gut as well as on gut permeability of the birds. A reduced growth performance was found for DON and/or C. jejuni exposed birds. Furthermore, it was found that the co-exposure of DON and C. jejuni aggravated the negative effect on paracellular permeability of the intestine already noticed for the bacteria or the mycotoxin alone by the Ussing chamber technique at certain times or intestinal segments. Furthermore, the increased paracellular permeability promotes the translocation of C. jejuni and E. coli to inner organs, namely liver and spleen. Interestingly, C. jejuni loads in the intestine were higher in DON-fed groups indicating a supportive growth effect of the mycotoxin. The actual study demonstrates that co-exposure of broiler chickens to DON and C. jejuni has not only considerable consequences on gut integrity but also on bacterial balance. These findings indicate that the co-exposure of broiler chickens to DON and C. jejuni could have a significant impact on gut health and bacteria translocation leading to an increased risk for public health.

Sodium sulfite (SoS) as decontamination strategy for Fusarium-toxin contaminated maize and its impact on immunological traits in pigs challenged with lipopolysaccharide (LPS).

The main objective of this study was to evaluate the effects of sodium sulfite (SoS) treatment of maize and its impact on the porcine immune system in the presence of an LPS-induced systemic inflammation. Control maize (CON) and Fusarium-toxin contaminated maize (FUS) were wet-preserved (20% moisture) for 79 days with (+) or without (-) SoS and then included at 10% in a diet, resulting in four experimental groups: CON-, CON+, FUS-, and FUS+ with deoxynivalenol (DON) concentrations of 0.09, 0.05, 5.36, and 0.83 mg DON/kg feed, respectively. After 42-day feeding trial (weaned barrows, n = 20/group), ten pigs per group were challenged intraperitoneally with either 7.5 µg LPS/kg BW or placebo (0.9% NaCl), observed for 2 h, and then sacrificed. Blood, mesenteric lymph nodes, and spleen were collected for phenotyping of different T cell subsets, B cells, and monocytes. Phagocytic activity and intracellular formation of reactive oxygen species (ROS) were analyzed in both polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) using flow cytometry. Our results revealed that the impact of DON was more notable on CD3+CD4+CD8+ T cells in lymphoid tissues rather than in blood T cells. In contrast, SoS treatment of maize altered leukocyte subpopulations in blood, e.g., reduced the percentage and fluorescence signal of CD8high T cells. Interestingly, SoS treatment reduced the amount of free radicals in basal ROS-producing PMNs only in LPS-challenged animals, suggesting a decrease in basal cellular ROS production (pSoS*LPS = 0.022).

Individual and Combined In Vitro Effects of Deoxynivalenol and Zearalenone on Boar Semen.

Mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) can negatively affect pig health. However, little is known about their effects on boar semen. We assessed the individual and combined effects of DON and ZEN on boar semen in vitro. In a pretrial, we determined the minimum dose (MiD) of each mycotoxin that induces a significant alteration of sperm progressive motility, as investigated using computer-assisted semen analysis (CASA). In the main trial, the individual and combined effects of each mycotoxin's MiD on sperm motility and kinetics (CASA analysis), morphology (SpermBlue staining), viability (calcein-propidium iodide staining), membrane functional status (hypoosmotic swelling test), and chromatin integrity (acridine orange staining) were analyzed. Pretrial results suggested a MiD of 50.6 µM and 62.8 µM for DON and ZEN, respectively. In the main trial, DON and ZEN administered at MiD significantly affected CASA parameters (e.g., increase of immotile spermatozoa, reduction of progressive motile spermatozoa), decreased sperm viability, and affected sperm morphology (head abnormalities) and membrane functional status. DON and ZEN showed less than additive effects on most parameters tested and a synergistic effect on viability and on two CASA parameters. In conclusion, DON and ZEN showed individual and combined toxic effects on boar semen in vitro.

Evaluation of the Efficacy of Mycotoxin Modifiers and Mycotoxin Binders by Using an In Vitro Rumen Model as a First Screening Tool.

Ruminal microbiota of cattle are not able to detoxify all mycotoxins. In addition, detoxification can be hampered by adverse ruminal conditions (e.g., low ruminal pH). Hence, in the cattle husbandry, mycotoxin binders and modifiers could be used to prevent animal exposure to mycotoxins. In this study, an in vitro rumen model, including feed matrix, was established as first screening tool to test the efficacy of five products claiming to detoxify mycotoxins. The detoxifiers had different modes of action: (a) binding (three products); (b) enzymatic detoxification of zearalenone (ZEN; one product, ZenA); and (c) bacterial transformation of trichothecenes (one product, BBSH 797). For the mycotoxin binders, the binding to the mycotoxins enniatin B (ENN B), roquefortine C (ROQ-C), mycophenolic acid (MPA), deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) were tested at a dose recommended by the manufacturers. The in vitro model demonstrated that all binders adsorbed ENN B to a certain extent, while only one of the binders also partially adsorbed ROQ-C. The binders did not change the concentrations of the other mycotoxins in the ruminal fluid. The enzyme ZenA detoxified ZEN very quickly and prevented the formation of the more toxic metabolite α-zearalenol (α-ZEL), both at normal (6.8) and low ruminal pH (5.8). The addition of BBSH 797 enhanced detoxification of DON and NIV, both at normal and low ruminal pH. The in vitro rumen model demonstrated that the addition of ZenA seems to be a very promising strategy to prevent estrogenic effects of ZEN contaminated feed, and BBSH 797 is efficient in the detoxification of trichothecenes.

Toxicokinetics of Hydrolyzed Fumonisin B1 after Single Oral or Intravenous Bolus to Broiler Chickens Fed a Control or a Fumonisins-Contaminated Diet.

The toxicokinetics (TK) of hydrolyzed fumonisin B1 (HFB1) were evaluated in 16 broiler chickens after being fed either a control or a fumonisins-contaminated diet (10.8 mg fumonisin B1, 3.3 mg B2 and 1.5 mg B3/kg feed) for two weeks, followed by a single oral (PO) or intravenous (IV) dose of 1.25 mg/kg bodyweight (BW) of HFB1. Fumonisin B1 (FB1), its partially hydrolyzed metabolites pHFB1a and pHFB1b, and fully hydrolyzed metabolite HFB1, were determined in chicken plasma using a validated ultra-performance liquid chromatography-tandem mass spectrometry method. None of the broiler chicken showed clinical symptoms of fumonisins (FBs) or HFB1 toxicity during the trial, nor was an aberration in body weight observed between the animals fed the FBs-contaminated diet and those fed the control diet. HFB1 was shown to follow a two-compartmental pharmacokinetic model with first order elimination in broiler chickens after IV administration. Toxicokinetic parameters of HFB1 demonstrated a total body clearance of 16.39 L/kg·h and an intercompartmental flow of 8.34 L/kg·h. Low levels of FB1 and traces of pHFB1b were found in plasma of chickens fed the FBs-contaminated diet. Due to plasma concentrations being under the limit of quantification (LOQ) after oral administration of HFB1, no toxicokinetic modelling could be performed in broiler chickens after oral administration of HFB1. Moreover, no phase II metabolites, nor N-acyl-metabolites of HFB1 could be detected in this study.

Feeding of deoxynivalenol increases the intestinal paracellular permeability of broiler chickens.

In recent years, the deleterious effects attributed to mycotoxins, in particular on the intestine, faced increased attention and it was shown that deoxynivalenol (DON) causes adverse effects on gut health. In this context, it has been repeatedly reported that DON can alter the intestinal morphology, disrupt the intestinal barrier and reduce nutrient absorption. The underlying mechanism of a compromised intestinal barrier caused by DON in chickens has yet to be illustrated. Although, DON is rapidly absorbed from the upper parts of the small intestine, the effects on the large intestine cannot be excluded. Additionally, a damaging effect of DON on the gut epithelium might decrease the resistance of the gut against infectious agents. Consequently, the objectives of the present studies were: (1) to investigate the impact of DON on the epithelial paracellular permeability by demonstrating the mucosal to serosal flux of 14C-mannitol in the small and large intestine applying Ussing chambers and (2) to delineate the effects of DON on the colonization and translocation of Escherichia coli. Both parameters are well suited as potential indicators for gut barrier failure. For this, a total of 75 one-day-old Ross 308 broiler chickens were housed in floor pens on wood shavings with feed and water provided ad libitum. Birds were randomly allocated to three different groups (n = 25 with 5 replicates/group) and were fed for 5 weeks with either contaminated diets (5 or 10 mg DON/kg feed) or basal diets (control). Body weight (BW) and BW gain of birds in the group fed with 10 mg/kg DON were significantly lower than in group with 5 mg/kg DON and the control group. Moreover, the mannitol flux in jejunum and cecum was significantly (P < 0.05) higher in DON-fed groups compared to control birds. Consistent with this, DON enhanced the translocation of E. coli with a higher number of bacteria encountered in the spleen and liver. Altogether, the actual results verified that DON can alter the intestinal paracellular permeability in broiler chickens and facilitates the translocation of enteric microorganisms such as E. coli to extra-intestinal organs. Considering that moderate levels of DON are present in feed, the consumption of DON-contaminated feed can induce an intestinal breakdown with negative consequences on broiler health.

Hydrolyzed fumonisin B1 induces less inflammatory responses than fumonisin B1 in the co-culture model of porcine intestinal epithelial and immune cells.

Fumonisin B1 (FB1), mainly produced by Fusarium verticillioides and Fusarium proliferatum, can be converted to the less toxic metabolite hydrolyzed FB1 (HFB1) by enzymatic degradation. The application of an FB1degrading enzyme as a feed additive is a strategy to reduce fumonisin exposure of animals. However, the difference between the effect of FB1 and HFB1 on porcine intestinal immunity is poorly documented. We investigated the toxic effects of FB1 and HFB1 exposure on porcine gut barrier function and intestinal immunity by using a co-culture model of intestinal porcine epithelial cells (IPEC-J2) and porcine peripheral blood mononuclear cells (PBMCs). First, we confirmed that Fusarium mycotoxin (deoxynivalenol; DON), in the presence of an endotoxin (lipopolysaccharide: LPS), disrupted gut permeability of IPEC-J2 and induced inflammatory response in the co-culture system. FB1 induced additional damage to gut barrier function and promoted pro-inflammatory responses in the presence of LPS and DON compared to only LPS/DON treatment. In the co-culture system, FB1/LPS/DON induced increased cell death of PBMCs and pro-inflammatory cytokines than LPS/DON treatment. In contrast, the application of HFB1 resulted in reduced levels of chemokines and pro-inflammatory cytokines together with marginal immune cell death compared to FB1/LPS/DON in the IPEC-J2/PBMC co-culture system. These findings suggest that FB1 aggravates LPS/DON-induced intestinal inflammation, and HFB1 showed less toxicity to immune response. Therefore, enzymatic degradation of FB1 to HFB1 could be an effective strategy to reduce intestinal inflammation in pigs.

Effects of a Fusarium Toxin-Contaminated Maize Treated with Sodium Sulfite on Male Piglets in the Presence of an LPS-Induced Acute Inflammation.

We investigated the effects of feeding sodium sulfite (SoS) treated uncontaminated and Fusarium contaminated maize in a porcine lipopolysaccharide (LPS) challenge model. Eighty piglets (7.59 ± 0.92 kg body weight [BW]) were equally assigned to one of four experimental diets containing 10% maize, either uncontaminated and untreated (CON-, 0.09 mg deoxynivalenol [DON]/kg diet) or uncontaminated and SoS-treated (CON+, wet-preserved with 5 g SoS/kg maize; 0.05 mg DON/kg diet), or prepared with 10% of a Fusarium contaminated maize containing mainly deoxynivalenol (DON), either contaminated and untreated (FUS-, 5.36 mg DON/kg diet), or contaminated and SoS-treated (FUS+, wet-preserved with 5 g SoS/kg maize; 0.83 mg DON/kg diet). At day 42 of experiment, ten pigs of each group were injected intraperitoneally with either 7.5 µg LPS/kg BW or placebo (0.9% NaCl). At 120 min after injection, blood samples were collected to analyse TNF-α, hematological profile, clinical biochemistry as well as the redox status. A significant increase in body temperature and cytokine TNF-α concentration was observed in the LPS-injected piglets. Results for hematology, clinical chemistry and redox status indicate no effects of SoS treatment, with exception of neutrophil counts being significantly more pronounced after feeding the SoS treated FUS maize. In conclusion, SoS treatment of maize did not modulate the LPS-induced acute inflammation.

Biomarker Evaluation and Toxic Effects of an Acute Oral and Systemic Fumonisin Exposure of Pigs with a Special Focus on Dietary Fumonisin Esterase Supplementation.

The mycotoxin fumonisin B1 (FB1) is a frequent contaminant of feed. It causes a disruption of sphingolipid metabolism and pulmonary, hepatic, and immunological lesions in pigs depending on the exposure scenario. One sensitive biomarker for FB1 exposure is the sphinganine (Sa) to sphingosine (So) ratio in blood. The fumonisin esterase FumD, which can be used as a feed additive, converts FB1 into the much less toxic metabolite hydrolyzed FB1 (HFB1). We conducted a single-dose study with barrows allocated to one of five treatments: (1) control (feed, 0.9% NaCl intravenously iv), (2) 139 nmol FB1 or (3) HFB1/kg BW iv, (4) 3425 nmol FB1/kg BW orally (po), or (5) 3321 nmol FB1/kg BW and 240 U FumD/kg feed po. The Sa/So ratio of iv and po FB1 administered groups was significantly elevated in blood and Liquor cerebrospinalis, but no fumonisin-associated differences were reflected in other endpoints. Neither clinical lung affections nor histopathological pulmonary lesions were detected in either group, while some parameters of hematology and clinical biochemistry showed a treatment⁻time interaction. FumD application resulted in Sa/So ratios comparable to the control, indicating that the enzymatic treatment was effectively preventing the fumonisin-induced disruption of sphingolipid metabolism.