The mycotoxin deoxynivalenol (DON) can deteriorate vaccination efficacy against porcine reproductive and respiratory syndrome virus (PRRSV) at subtoxic levels.

BACKGROUND: Feedgrain contamination with mycotoxins, including deoxynivalenol (DON, "vomitoxin") is relatively frequently encountered. Pigs are particularly sensitive to the toxicity of DON. To assess the interplay between DON and porcine reproductive and respiratory syndrome virus (PRRSV), we performed an experimental DON exposure-PRRSV vaccination-challenge infection trial. Three-week-old piglets were divided into four groups. Groups I, II and III (10 animals/group) were vaccinated with a PRRSV modified live vaccine and 2 weeks later challenged with a heterologous field strain. While group I was not supplemented with DON, animals in groups II and III received DON for 4 weeks prior to challenge infection at levels that can be encountered in pig feed, employing a low-dose or high-dose regime (group II: 40 µg DON/kg body weight per day; group III: 80 µg DON/kg body weight per day, corresponding to approx. 1 or 2 mg DON/kg feed, respectively). Eight animals (group IV; unvaccinated, not DON exposed) served as control animals for the challenge infection. RESULTS: We assessed clinical signs, virus load in serum and various organs as well as antibody titres in the animals. All vaccinated animals mounted an efficient PRRSV-specific antibody response within 2 weeks, except for 20% of the animals receiving the higher DON dose. Upon virus challenge, the vaccinated animals in group I were protected from clinical signs. Vaccinated DON-exposed animals in group II and III were protected from clinical signs to a lesser extent. Clinical signs in group III receiving the higher dose of DON were as severe as in the (unvaccinated, not DON exposed) control group IV. The animals of group III also displayed lower antibody titres compared with the animals in group I and II. CONCLUSIONS: The experimental vaccination/challenge study therefore revealed that exposure of pigs to DON for a period of 4 weeks deteriorates the efficacy of vaccination against clinical signs of PRRS.

Correction to: Glucuronidation of deoxynivalenol (DON) by different animal species: identification of iso-DON glucuronides and iso-deepoxy-DON glucuronides as novel DON metabolites in pigs, rats, mice, and cows.

The original article can be found online.

On the distribution and metabolism of Fusarium-toxins along the gastrointestinal tract of endotoxaemic pigs.

The aim of this study was to investigate the potential modulatory effect of E. coli lipopolysaccharides (LPS) on residues of deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM-1), zearalenone (ZEN) and its metabolites α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL) and ß-zearalanol (ß-ZAL) after pre- or post-hepatic administration along the gastrointestinal axis. Fifteen barrows were exposed to a naturally mycotoxin contaminated diet (4.59 mg DON/kg feed and 0.22 mg ZEN/kg feed) and equipped with jugular (ju) and portal (po) catheters. On sampling day (day 29), the barrows were infused with LPS or a control fluid (LPS, 7.5 µg/kg body weight; control, 0.9% NaCl) either pre- or post-hepatically, resulting in three infusion groups: CONju-CONpo, CONju-LPSpo and LPSju-CONpo. At 195 min relative to infusion start (210 min post-feeding), pigs were sacrificed and content of stomach and small intestine (proximal, medial and distal part) as well as faeces were collected. In all LPS-infused animals, higher amounts of dry matter were recovered irrespective of LPS entry site suggesting a reduced gastric emptying and a decreased gastrointestinal motility under endotoxaemic conditions. DON metabolism in the gastrointestinal tract (GIT) remained unaltered by treatments and included an increase in the proportion of DOM-1 along the GIT, particularly from distal small intestine to faeces. Variables describing ZEN metabolism suggest a stimulated biliary release of ZEN and its metabolites in LPS-infused groups, particularly in the LPSju-CONpo group. In conclusion, the GIT metabolism of ZEN was markedly influenced in endotoxaemic pigs whereby a jugular induction of an acute phase reaction was more effective than portal LPS infusion hinting at a strong hepatic first-pass effect.

Antibody response of growing German Holstein bulls to a vaccination against bovine viral diarrhea virus (BVDV) is influenced by Fusarium toxin exposure in a non-linear fashion.

The Fusarium toxin deoxynivalenol (DON) is a frequent contaminant of feedstuffs and is supposed to interfere with immune responses. As the relevance for growing bulls is less clear than for other livestock, the trial was designed according to the dose-response principal with a control group fed a diet with background contamination (CON, 0.36 mg DON per kilogram dry matter [DM]) and three groups with increasing concentrations of DON (mg/kg DM); FUS I, 3.01; FUS II, 5.66; FUS III, 8.31. Half of each treatment group was vaccinated against BVDV at days 1 and 21 of the 70 days lasting experiment. Sequential blood samples were collected for determination of antibody titers to BVDV and for hematological and clinical-chemical traits. Antibody response was strongest in group FUS II while group FUS III responded weakest. This group showed the lowest proportion of CD4+ T cells, but also the highest levels of liver lesion indicating enzyme activities in blood. BVDV-vaccination induced a pronounced decrease in red blood count indices, which occurred dose-dependently at a higher level in the FUS-fed groups. The obvious interactions between DON exposure and BVDV-vaccination require further elucidation.

Detoxification of Fusarium-contaminated maize with sodium sulphite - in vivo efficacy with special emphasis on mycotoxin residues and piglet health.

A feeding experiment with piglets was performed to examine the efficacy of a wet preservation of Fusarium (FUS)-contaminated maize with sodium sulphite (SoS) based on deoxynivalenol (DON) and zearalenone (ZEN) residue levels in urine, bile and liquor and health traits of piglets. For this purpose, 80 castrated male piglets (7.57 ± 0.92 kg BW) were assigned to four treatment groups: CON- (control diet, with 0.09 mg DON and <0.01 mg ZEN/kg diet), CON+ (diet CON-, wet-preserved with 5 g SoS/kg maize; containing 0.05 mg DON and <0.01 mg ZEN/kg diet), FUS- (diet with mycotoxin-contaminated maize; containing 5.36 mg DON and 0.29 mg ZEN/kg diet), and FUS+ (diet FUS-, wet-preserved with 5 g SoS/kg maize; resulting in 0.83 mg DON and 0.27 mg ZEN/kg diet). After 42 d, 40 piglets (n = 10 per group) were sampled. A clear reduction of DON levels by approximately 75% was detected in all specimens of pigs fed diet FUS+. ZEN was detected in all urine, bile and liquor samples, while their metabolites were only detectable in urine and bile. Additionally, their concentrations were not influenced by SoS treatment. Among the health-related traits, feeding of FUS diets increased the total counts of leukocytes and segmented neutrophil granulocytes irrespective of SoS treatment. SoS treatment increased the total blood protein content slightly with a similar numerical trend in albumin concentration. These effects occurred at an obviously lower level in FUS-fed groups. Moreover, SoS treatment recovered the reduction of NO production induced by feeding diet FUS- indicating an effect on the redox level. As this effect only occurred in group FUS+, it is obviously related to the adverse effects of the Fusarium toxins. In conclusion, treatment of FUS-contaminated maize with SoS decreased the inner exposure with DON as indicated by the lower DON levels in various piglet specimens. However, health-related traits did not consistently reflect this decreased exposure.

Effects of oral exposure to sodium sulphite-treated deoxynivalenol (DON)-contaminated maize on performance and plasma concentrations of toxins and metabolites in piglets.

The objective of the present study was to demonstrate the efficiency of the decontamination process applied to deoxynivalenol (DON)-contaminated maize by sodium sulphite (Na2SO3) treatment in vivo. Additionally, in vitro characterisation of the toxicity of the DON sulphonates (DONS 1, 2 and 3 denote structurally different forms), the resulting DON metabolites, on peripheral blood mononuclear cells (PBMC) should substantiate the inactivation of DON. In a piglet experiment, both DON-contaminated maize and -uncontaminated control maize either untreated (DON-, CON-) or Na2SO3-treated (DON+, CON+) were mixed into feed and fed for 42 d starting from weaning. The results showed that feed intake and daily weight gain of animals fed DON- were significantly lower compared to animals fed CON- and CON+, whereas group DON+ reached the control level or even exceeded it. The feed-to-gain ratio was unaffected (p = 0.45). Furthermore, DON concentrations in plasma markedly reflected the diets' DON concentrations. These were < 0.1, < 0.1, 5.4 and 0.8 mg/kg feed for CON-, CON+, DON- and DON+, and amounted to 0.3, 0.4, 33.0 and 9.3 ng/ml in plasma, respectively. Whereas DONS 2 and 3 were detected in the DON+ diet, only DONS 2 was recovered in plasma. Regarding the toxicity of DONS, no or much lower toxicity was found compared to DON. DONS 1 and Na2SO3 did not affect the viability of PBMC. At 32.71µM DONS2 the viability was reduced by 50% and thus this compound was less toxic than DON by a factor of 73. Consequently, wet preservation of maize with Na2SO3 was an effective tool to avoid the adverse effects of DON on performance of piglets.

Glucuronidation of deoxynivalenol (DON) by different animal species: identification of iso-DON glucuronides and iso-deepoxy-DON glucuronides as novel DON metabolites in pigs, rats, mice, and cows.

The Fusarium mycotoxin deoxynivalenol (DON) is a frequent contaminant of cereal-based food and feed. Mammals metabolize DON by conjugation to glucuronic acid (GlcAc), the extent and regioselectivity of which is species-dependent. So far, only DON-3-glucuronide (DON-3-GlcAc) and DON-15-GlcAc have been unequivocally identified as mammalian DON glucuronides, and DON-7-GlcAc has been proposed as further DON metabolite. In the present work, qualitative HPLC-MS/MS analysis of urine samples of animals treated with DON (rats: 2 mg/kg bw, single bolus, gavage; mice: 1 mg/kg bw, single i.p. injection; pigs: 74 µg/kg bw, single bolus, gavage; cows: 5.2 mg DON/kg dry mass, oral for 13 weeks) revealed additional DON and deepoxy-DON (DOM) glucuronides. To elucidate their structures, DON and DOM were incubated with human (HLM) and rat liver microsomes (RLM). Besides the expected DON/DOM-3- and 15-GlcAc, minor amounts of four DON- and four DOM glucuronides were formed. Isolation and enzymatic hydrolysis of four of these compounds yielded iso-DON and iso-DOM, the identities of which were eventually confirmed by NMR. Incubation of iso-DON and iso-DOM with RLM and HLM yielded two main glucuronides for each parent compound, which were isolated and identified as iso-DON/DOM-3-GlcAc and iso-DON/DOM-8-GlcAc by NMR. Iso-DON-3-GlcAc, most likely misidentified as DON-7-GlcAc in the literature, proved to be a major DON metabolite in rats and a minor metabolite in pigs. In addition, iso-DON-8-GlcAc turned out to be one of the major DON metabolites in mice. DOM-3-GlcAc was the dominant DON metabolite in urine of cows and an important DON metabolite in rat urine. Iso-DOM-3-GlcAc was detected in urine of DON-treated rats and cows. Finally, DON-8,15-hemiketal-8-glucuronide, a previously described by-product of DON-3-GlcAc production by RLM, was identified in urine of DON-exposed mice and rats. The discovery of several novel DON-derived glucuronides in animal urine requires adaptation of the currently used methods for DON-biomarker analysis.

Haematological, clinical-chemical and immunological consequences of feeding Fusarium toxin contaminated diets to early lactating dairy cows.

Dairy cows experience a negative energy balance at the onset of lactation which results in an enhanced vulnerability for infectious diseases. Any dietary imbalances, including Fusarium toxin contamination, might therefore exacerbate this situation. The aim of the present investigations was to study the effects of increasing dietary concentrations of deoxynivalenol (DON) and zearalenone (ZEN) on clinical-chemical, haematological and immunological traits up to week 14 of lactation. For this purpose, ten cows each were assigned to a control group (CON; 0.02 mg ZEN and 0.06 mg DON per kg diet at 88 % DM), toxin level 1 (TOX-1; 0.29 mg ZEN and 2.31 mg DON per kg diet at 88 % DM) and toxin level 2 (TOX-2; 0.58 mg ZEN and 4.61 mg DON per kg diet at 88 % DM). The measured values of most parameters were affected by parturition but only a few of them were further modified by dietary treatment. For example, the time-dependent decrease in haemoglobin concentration, haematocrit and erythrocyte counts occurred at a significantly higher level for group TOX-2 while a serum glucose increase was missing in this group. Proportions of CD4+ and CD8+ cells decreased significantly over time solely in group TOX-2 while the CD4+/CD8+ ratio remained uninfluenced. Ability of granulocytes to mount an oxidative burst tended to increase at the end of the study in groups TOX-1 and TOX-2 while the opposite was observed in group CON. The results of this time-limited study indicate that feeding of Fusarium-toxin contaminated diets in early lactation affects health related parameters without compromising milking performance. However, long-term consequences of the observed effects on health need to be addressed in further studies.

Effects of deoxynivalenol (DON), zearalenone (ZEN), and related metabolites on equine peripheral blood mononuclear cells (PBMC) in vitro and background occurrence of these toxins in horses.

Both deoxynivalenol (DON), zearalenone (ZEN), and their metabolites are known to modulate immune cells in various species whereby viability and proliferation are influenced. Such effects were rarely examined in horses. Therefore, one aim of the present study was to titrate the inhibitory concentrations of DON, 3-acetyl-DON (3AcDON), de-epoxy-DON (DOM-1), ZEN, and α- and ß-zearalenol (ZEL) at which viability and proliferation of equine PBMC were reduced by 50 % (IC50) and 10 % (IC10) in vitro. For evaluation of practical relevance of the in vitro findings, a further aim was to screen horses for the background occurrence of DON, ZEN, and their metabolites in systemic circulation and to relate toxin residues both to the inhibitory toxin concentrations and to hematological and clinical-chemical characteristics.The IC50 (µM) for DON, 3AcDON, ß-ZEL, α-ZEL, and ZEN were determined at 3.09, 25.90, 75.44, 97.44, and 98.15 in unstimulated cells, respectively, while in proliferating cells, the corresponding IC50 values were 0.73, 6.89, 45.16, 75.96, and 82.51. Neither viability nor proliferation was influenced by DOM-1 up to a concentration of 100 µM.The in vivo screening (N = 49) revealed the occurrence of ZEN (N = 24), α-ZEL (N = 3), ß-ZEL (N = 37), DON, and DOM-1 (N = 2). The detected concentrations were much lower than the corresponding IC50 while the IC10 of DON and ß-ZEL for proliferating PBMC corresponded to approximately 26 and 35 ng/mL which might be relevant when contaminated diets are fed.Clinical-chemical and hematological traits were not related to mycotoxin residue levels excepting blood urea nitrogen which was positively correlated to the sum of ß-ZEL, α-ZEL, and ZEN concentration. Whether this reflects simply the feeding history of the horses or renal failures giving rise to a prolonged half-life of the toxins needs to be clarified further.

Fusarium toxin-contaminated maize in diets of growing bulls: effects on performance, slaughtering characteristics, and transfer into physiological liquids.

The present feeding study was carried out to examine the effects of Fusarium toxin-contaminated diets on performance and slaughtering characteristics and on the transfer of the Fusarium toxins zearalenone (ZEN), deoxynivalenol (DON) and their metabolites into physiological matrices. A total of 61 bulls (483 ± 46 kg) were fed with graded proportions of Fusarium toxin-contaminated feed over a period of 10 weeks. The total mixed rations (TMR) consisted of 47 % grass silage, 20 % press pulp silage, and 33 % concentrate on dry matter (DM) basis. Increasing toxin concentrations were achieved by the exchange of control maize with Fusarium toxin-contaminated maize in the concentrates. Thus, dietary toxin concentrations between 0.08 and 0.69 mg ZEN and 0.36 and 8.31 mg DON per kg DM were covered by the four feeding groups. Based on increasing DM intake with increasing mycotoxin contaminations of the diet, the live weight gain and energy intake differed significantly between the groups. No effects were observed on slaughtering characteristics and organ weights. ZEN, α-zeralenol, ß-zeralenol (ß-ZEL), zeralanone, α-zearalanol, ß-zearalanol, DON, and de-deepoxy-DON (de-DON) were simultaneously determined in urine, plasma, and liquor whereby quantifiable concentrations of ZEN, ß-ZEL, DON, and de-DON were found in urine, of DON and de-DON in plasma, and solely of de-DON in liquor. Based on overall results it can be concluded that current EU-guidance values for critical concentrations of DON and ZEN can be regarded as safe levels also for growing bulls. Urine and blood toxin residue levels can be used to assess exposure of bulls.

Haematological and immunological adaptations of non-pregnant, non-lactating dairy cows to a high-energetic diet containing mycotoxins.

Diet change and fatness are supposed to challenge the immune system of the cow. Therefore, immunological and haematological consequences of adaptation to and continued feeding of a high-energy diet were studied in eight non-pregnant, non-lactating Holstein cows over 16 weeks. Blood haptoglobin concentration remained unaltered, suggesting that an acute phase reaction was not induced. Stimulation ability of peripheral blood mononuclear cells and stimulated oxidative burst capacity of granulocytes increased significantly in the course of the experiment after an initial drop. While total leucocyte counts increased, the proportion of granulocytes increased and that of lymphocytes decreased at the same time as the ratio of CD4(+)/CD8(+) lymphocytes did. Capability of rumen microbes to detoxify the immune-modulating mycotoxin deoxynivalenol (DON) was not compromised as indicated by the exclusive presence of de-DON as the detoxified DON metabolite in blood. In conclusion, both diet change and prolonged positive energy balance influenced the bovine immune system.

Studies on the bioavailability of deoxynivalenol (DON) and DON sulfonate (DONS) 1, 2, and 3 in pigs fed with sodium sulfite-treated DON-contaminated maize.

Deoxynivalenol (DON) exposure of pigs might cause serious problems when critical dietary toxin concentrations are exceeded. As DON contamination of agricultural crops cannot be completely prevented, detoxification measures are needed. Wet preservation with sodium sulfite resulted in a significant DON reduction of naturally-contaminated maize in previous experiments. The preserved material had a characteristic DON sulfonates (DONS) pattern. DONS is known to be less toxic than DON but its stability was shown to depend on pH, which gives rise to the question if a back-conversion to DON occurs in vivo. Therefore, the toxicokinetics and bioavailability of DON and DONS were studied in pigs. After the administration of a single oral or intravenous bolus of DON or DONS, serial blood samples were collected and subsequently analyzed. DONS was not detectable after oral administration of DONS mixtures. The results showed further that the bioavailability of DONS as DON in pigs fed maize preserved wet with sodium sulfite was significantly decreased compared to untreated control maize (DON), indicating that DONS obviously did not convert back to DON to a large extent in vivo. Moreover, the fact that DONS was not detectable in systemic blood requires further investigations regarding their ingestive and/or metabolic fate.

Invited review: Diagnosis of zearalenone (ZEN) exposure of farm animals and transfer of its residues into edible tissues (carry over).

The aim of the review was to evaluate the opportunities for diagnosing the zearalenone (ZEN) exposure and intoxication of farm animals by analyzing biological specimens for ZEN residue levels. Metabolism is discussed to be important when evaluating species-specific consequences for the overall toxicity of ZEN. Besides these toxicological facts, analytics of ZEN residues in various animal-derived matrices requires sensitive, matrix-adapted multi-methods with low limits of quantification, which is more challenging than the ZEN analysis in feed. Based on dose-response experiments with farm animals, the principle usability of various specimens as bio-indicators for ZEN exposure is discussed with regard to individual variation and practicability for the veterinary practitioner. ZEN residue analysis in biological samples does not only enable evaluation of ZEN exposure but also allows the risk for the consumer arising from contaminated foodstuffs of animal origin to be assessed. It was compiled from literature that the tolerable daily intake of 0.25 µg ZEN/kg body weight and day is exploited to approximately 8%, when a daily basket of animal foodstuffs and associated carry over factors are assumed at reported ZEN contamination levels of complete feed.

Development of a multi-toxin method for investigating the carryover of zearalenone, deoxynivalenol and their metabolites into milk of dairy cows.

A dose-response study was carried out to examine the carryover of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites into bovine milk. Therefore, a feeding trial with 30 dairy cows fed with three different levels of Fusarium (FUS) toxin-contaminated maize was performed. A control group (0.02 mg ZEN kg(-1) dry matter (DM) and 0.07 DON kg(-1) DM) was compared with two groups fed contaminated diets. The first diet contained 0.33 mg ZEN kg(-1) DM and 2.62 mg DON kg(-1) DM (group FUS-50) and the second diet contained 0.66 mg ZEN kg(-1) DM and 5.24 mg DON kg(-1) DM (group FUS-100). For milk sample analysis, a new cost-efficient sample preparation method was developed for the simultaneous determination of ZEN, DON and their metabolites. The method comprised the separation of the milk fat followed by an SPE clean-up on Oasis HLB and a LC-MS/MS measurement. The less toxic metabolite de-epoxy-DON had the highest detected concentration (5.6 ng ml(-1) milk) in the milk samples obtained from the feeding trial. Additionally, ZEN (up to 0.29 ng ml(-1)), α-zearalenol (up to 0.17 ng ml(-1)), ß-zearalenol (up to 0.95 ng ml(-1)) and DON (up to 2.5 ng ml(-1)) were detected in these samples. The milk toxin concentrations of cows fed the control diet were significantly lower compared with cows fed the contaminated diet. The calculated carryover rates ranged between 0 and 0.0075 for ZEN and metabolites and between 0 and 0.0017 for DON independent of exposure. It can be concluded that dietary toxin concentrations in the feed below or close to the current guidance values do not pose a risk for consumers due to negligible carryover rates.

Description of a bovine model for studying digestive and metabolic effects of a positive energy balance not biased by lactation or gravidity.

Physiological consequences of adaptation to and continued feeding of a high-energetic diet were studied in eight non-pregnant, non-lactating dairy Holstein cows over a period of 16 weeks. The first six weeks served as an adaptation period from the low energetic straw-based diet (3.8 MJ NEL/kg DM) to the high-energetic ration (7.5 MJ NEL/kg DM). Intake of dry matter (DM) increased with dietary energy concentration from 9 to 20 kg/d up to week 9 to 12 and decreased thereafter. The initial live weight (LW) of 550 ± 60 kg was increased linearly and corresponded to an average daily LW gain of 2.3 ± 0.3 kg. Energy balance increased approximately nine-fold to a maximum of 114 MJ NEL/d in week 10. Ruminal fermentation pattern was completely changed from an acetate dominating profile to a propionate based one, which was paralleled by a marked increase in the rumen fluid endotoxin concentration. Unlike blood glucose concentration, which increased continuously, that of cholesterol and triglycerides started to increase after an initial stagnation. In conclusion, both ruminal adaptation to a high-energetic diet and the continued feeding of such a diet induced digestive and metabolic adaptations in non-pregnant, non-lactating cows characterised by a progressing positive energy balance.

Residues of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites in plasma of dairy cows fed Fusarium contaminated maize and their relationships to performance parameters.

A feeding trial with dairy cows fed graded proportions of a Fusarium toxin contaminated maize containing mainly deoxynivalenol (DON) was carried out to relate the plasma levels of DON, zearalenone (ZEN) and their metabolites to the performance. German Holstein cows (n=30) were divided into three groups (n=10 in each): CON (0.02mgZEN and 0.07mgDON, per kg dry matter, DM), FUS-50 (0.33mg ZEN and 2.62mgDON, per kg DM), FUS-100 (0.66mgZEN and 5.24mgDON, per kg DM). The average performance level was characterised by daily DM intake, energy balance and milk yield which were not affected by the DON and ZEN levels in feed. DON, de-epoxy-DON (de-DON) and ZEN were detected simultaneously in all plasma samples. A linear relationship between toxin intake and plasma levels could be established. Moreover, a linear relationship between DON and de-DON concentration could be derived. It was concluded that DON and ZEN intake of 0.5mgZEN/kg and 5mgDON/kg (current guidance values) had no considerable effects on the performance parameter of dairy cows. Furthermore, increased plasma concentrations of ZEN, DON and de-DON may hint on toxin exposure through the diets.