Effects of feeding a Fusarium toxin-contaminated diet to infectious bursal disease virus-infected broilers on the protein turnover of the bursa of Fabricius and spleen.

Two experiments were carried out to examine the effects of feeding an uncontaminated control diet (CON) or a Fusarium toxin-contaminated diet (FUS; 10.7 mg deoxynivalenol [DON]/kg diet) to growing broilers, which were either uninfected or infected with infectious bursal disease virus (IBDV) beginning at 1 day post hatch. Broilers had been infected at three weeks post hatch with either a classical virulent infectious bursal disease virus (IBDV-IM, Exp. 1) or a very virulent IBDV (vvIBDV, Exp. 2) strain. The effects of the DON-contaminated diet in combination with the virus-infection on the bursa of Fabricius and spleen were determined at 3 and 6-7 days post infection. The transient development of the bursa oedema and the bursa atrophy was not significantly affected by the diet after infection with the different IBDV-strains. The histopathological lesions were more severe in IBDV-IM-infected birds at 6 days post infection when additionally exposed to the FUS diet as compared to the FUS-free feed. Most parameters of the bursa of Fabricius and spleen protein turnover (e.g. fractional protein synthesis rate, protein, DNA and RNA content and derived indices) were significantly and interactively influenced by infection and stage of infection. The vvIBDV-infected birds responded with a more pronounced depressing effect on the fractional protein synthesis rate after feeding the DON-containing FUS diet when compared to their IBDV-IM-infected counterparts, where the opposite effect was observed. It can be concluded that feeding a FUS diet to IBDV-infected broilers might modulate the virulence-dependent pathogenesis of an IBDV infection.

Effects of deoxynivalenol (DON) and related compounds on bovine peripheral blood mononuclear cells (PBMC) in vitro and in vivo.

The Fusarium toxin deoxynivalenol (DON) often co-occurs along with the acetylated derivatives 3-acetyl-DON and 15-acetyl-DON in diets for ruminants. De-epoxy-DON is formed by rumen micro-organisms, while the acetylated DON derivatives might also undergo ruminal metabolism with de-epoxy-DON as an end product. However, despite the fact that de-epoxy-DON is the predominant substance finally absorbed, a complete degradation of the mother compounds can not be assumed for all feeding and metabolic situations of the cow, and thus raising the question of their possible post-absorptive effects. Hence, the aim of the study was to examine the effects of all four compounds on the concanavalin A stimulated proliferation of bovine peripheral blood mononuclear cells (PBMC) using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) as indicator in vitro and ex vivo. Among the DON-related compounds, DON and 15-acetyl-DON resulted in a similar IC50 (i.e. the concentration where the proliferation was inhibited by 50%) of 0.5 µM, whereas 3-acetyl-DON was less toxic (IC50 = 2.6 µM), while actually no IC50 could be estimated for de-epoxy-DON which was characterized by a maximum inhibition of approximately 24% at the highest tested in vitro concentration of 18.29 µM. For the in vivo experiment, 14 Holstein cows were used and fed either an uncontaminated control diet (CON) or a diet contaminated with Fusarium toxins, with DON being the predominating toxin for 18 weeks when blood was collected for PBMC isolation and subsequent proliferation/viability assay. The complete diets for the CON and FUS group contained 0.4 and 4.6 mg DON/kg DM, respectively, at that time. Exposure of dairy cows to the FUS diet resulted in maximum serum de-epoxy-DON levels of 52 ng/ml (0.19 µM), while levels of the unmetabolized DON reached maximum levels of 9 ng/ml (0.03 µM). The PBMC of these cows were slightly less viable, by approximately 18% (p = 0.057), while stimulation capability was not decreased at the same time. Although de-epoxy-DON was characterized by the lowest in vitro toxicity among the tested DON-related compounds, there appeared to be a lower viability of the PBMC isolated from cows fed the FUS diet, which had nearly exclusively de-epoxy DON in serum beside slight traces of unmetabolized DON. Thus, the factors responsible for these apparent discrepancies need to be clarified.

Investigations on the kinetics of the concentration of deoxynivalenol (DON) and on spoilage by moulds and yeasts of wheat grain preserved with sodium metabisulfite (Na2S2O5, SBS) and propionic acid at various moisture contents.

Unground wheat kernels contaminated with 2.09 mg deoxynivalenol (DON) per kg dry matter were stored for up to 56 days at moisture contents of 15, 17.5 and 20% to study the alterations of DON concentration when the wheat was stored either unsupplemented or supplemented with 5 g sodium metabisulfite (Na2S2O5, SBS), 10 g propionic acid (PA) or 5 g SBS plus 10 g PA per kg. SBS addition alone or in combination with PA reduced the DON contamination to 1.2-4.3% of the initial DON concentration while DON concentration of unsupplemented and wheat batches supplemented only with PA varied inconsistently or remained unchanged. The SBS-related DON reduction was paralleled by a concomitant increase in the concentration of the non-toxic reaction product DON sulfonate. In contrast to the unsupplemented wet-stored controls, SBS addition prevented the growth of moulds and yeasts when added alone or in combination with PA. In conclusion, for the conditions examined, the wet preservation of DON-contaminated wheat with SBS seems to be promising as an on-farm detoxification measure.

On the effects of the Fusarium toxin deoxynivalenol (DON) administered per os or intraperitoneal infusion to sows during days 63 to 70 of gestation.

Six pregnant sows of 180.6 ± 5.6 kg were fed either a Fusarium-contaminated (4.42 mg DON and 48.3 µg ZON per kg, DON per os, n = 3) or a control diet (0.15 mg DON and 5 µg ZON/kg) in the period of days 63 and 70 of gestation. On day 63 of gestation, sows fed the control diet were implanted with an intraperitoneal osmotic minipump (delivery rate of 10 µL/h, for 7 days) containing 50 mg pure (98%) DON in 2 ml 50% DMSO (DON ip, n = 3). Frequent plasma samples were taken to estimate the kinetics after oral and ip DON exposure. The intended continuous delivery of DON by the intraperitoneal minipump could not be shown, as there was a plasma peak (Cmax) of 4.2-6.4 ng DON/mL either immediately (sow IP-2+3) or 2.5 h (sow IP-1) after implantation of the pump followed by a one-exponential decline with a mean half-time (t1/2) of 1.75-4.0 h and only negligible DON plasma concentrations after 12 h. Therefore, the DON ip exposure has to be regarded as one single dose 1 week before termination of experiment. The DON per os sows showed a mean basis level (after achieving a steady state) of DON plasma concentration of about 6-8 ng/mL, as also indicated by the plasma DON concentration at the termination of the experiment. On day 70, caesarean section was carried out, the fetuses were killed immediately after birth, and samples of plasma, urine, and bile were taken to analyze the concentration of DON and its metabolite de-epoxy-DON. At necropsy there were no macroscopic lesions observed in any organ of either sows or piglets. Histopathological evaluation of sows liver and spleen revealed no alterations. The proliferation rate of peripheral blood mononuclear cells (PBMC) with or without stimulation was not affected by the kind of DON treatment. The exposure of pregnant sows at mid-gestation (days 63-70, period of organogenesis) to a Fusarium toxin-contaminated diet (4.42 mg DON and 0.048 mg ZON per kg) or pure DON via intraperitoneal osmotic minipump did not cause adverse effects on health, fertility, maintenance of pregnancy, and performance of sows and their fetuses. However, DON was detected in fetus plasma, indicating that this toxin can pass the placental barrier and may cause changes in the proportion of white blood cells (lower monocyte and neutrophil and higher lymphocyte proportion in DON per os fetuses).

Effects of increasing concentrations of sodium metabisulphite (Na2S 2O 5, SBS) on deoxynivalenol (DON) concentration and microbial spoilage of triticale kernels preserved without and with propionic acid at various moisture contents.

Unground triticale kernels contaminated with 6.63 mg deoxynivalenol (DON) per kg dry matter were stored for up to 63 days at total moisture contents of 13 and 15% in order to study the time-dependent kinetics of DON concentration in dependence on graded levels of sodium metabisulfite [0, 1, 2, 3, 4 and 5 g Na2S2O5 (SBS) per kg], and in the absence and presence of 10 g propionic acid (PA) per kg. The DON concentration decreased with increasing amounts of supplemented SBS and with increasing duration of the preservation period in a bi-exponential fashion when SBS addition was ≥3 g/kg. Lower SBS concentrations yielded inconsistent results. The maximum measured DON reductions after adding 5 g SBS/kg were 3 and 4% of the initial DON concentration after 63 days in the absence and presence of PA at moisture contents of 15%, while the corresponding recovery for the variants preserved at 13% amounted to 21 and 11%, respectively. The 12 variants preserved without PA supplementation were more frequently contaminated by moulds and yeasts (n = 5) than the corresponding variants stored together with PA (n = 1). The overall results and regressive evaluations do suggest that the highest SBS addition of 5 g/kg triticale at a moisture content of 15% preserved for 63 days would be necessary for a maximum DON reduction. Although PA did not exert a direct decontaminating effect, an additional supplementation together with SBS seemed to be advantageous with regard to the prevention of yeast and mould contamination and favouring the decontamination reaction by the acid milieu.

Effects of a Fusarium toxin-contaminated triticale, either untreated or treated with sodium metabisulphite (Na2S2O5, SBS), on weaned piglets with a special focus on liver function as determined by the 13C-methacetin breath test.

The aim of the present experiment was to test the effects of a wet preservation of triticale contaminated mainly with deoxynivalenol (DON) with sodium metabisulphite (Na2S2O5, SBS) on growth performance, liver function, clinical-chemical plasma parameters and organ histopathology of piglets. For this purpose both the uncontaminated control triticale and the DON contaminated triticale were included in the piglet diet either untreated (CON, FUS) or SBS-treated (CON-SBS, FUS-SBS) and fed for 28 d starting from weaning. The dietary concentrations of DON and DON sulfonate (DONS), the DON derivative resulting from the SBS treatment, amounted to 0.156, 0.084, 2.312 and 0.275 mg DON per kg CON, CON-SBS, FUS and FUS-SBS diet, and to <0.05, <0.05, <0.05 and 1.841 mg/kg diet, respectively. Feeding the FUS diet significantly reduced the feed intake compared to the other three groups as indicated by the significant interactions between triticale source and SBS treatment when the whole experimental period of 28 d was considered (p = 0.014) while live weight gain and feed to gain ratio remained unaffected. The total plasma protein concentration was significantly depressed due to feeding the contaminated diets whereas SBS treatment exerted an increasing effect at the same time (45.4, 49.5, 40.7 and 46.5 g/l for piglets fed the CON, CON-SBS, FUS and FUS-SBS diet, respectively). The liver function was tested by the 13C-methacetin breath test (MBT) allowing evaluation of the cytochrome P4501A2 activity. MBT results, expressed as cumulative percentage dose recovery after 360 min (cPDR360) revealed a slight stimulation of liver function due to SBS treatment (p = 0.052) (37.5, 39.4, 37.4 and 55.1% for piglets fed the CON, CON-SBS, FUS and FUS-SBS diet, respectively). Liver weight and histopathological scoring were only weakly related to the MBT results. Further histopathological examinations of kidneys, pancreas and heart revealed no treatment effects. It was concluded that the SBS treatment of the contaminated triticale restored the performance of piglets to the level of the piglets fed the control diet while the effects on liver function, clinical-chemical plasma parameters - excepting the protein concentration - and organ histopathology were only marginal.

The Fusarium toxin deoxynivalenol disrupts phenotype and function of monocyte-derived dendritic cells in vivo and in vitro.

The trichothecene mycotoxin deoxynivalenol (DON) causes systemic immuno-suppression in pigs and possibly also in humans after chronic dietary exposure. Since the outcome of every immune response is largely controlled by dendritic cells (DC), we hypothesised that a direct influence of DON on DC function might play a role in mediating DON immunotoxicity. To test this hypothesis, a 2x2 factorial design study was performed. Pigs were fed a control diet or a diet containing DON (DON-diet); monocyte-derived DC (MoDC) from these pigs were then treated with DON in vitro or left untreated. Phenotype and function of the MoDC were analysed. In vitro DON-treatment of MoDC from pigs fed the control diet resulted in a down-regulation of CD80/86 and CD40. This was associated with an activation of the mitogen-associated protein kinases ERK1/2 and JNK. The endocytic activity of MoDC was decreased after in vitro DON-exposure while their T cell stimulatory capacity was not altered. MoDC derived from pigs that had been fed the DON-diet failed to up-regulate MHC-II in response to LPS/TNFalpha. Dietary exposure of pigs to DON inhibited endocytosis of FITC-dextran by MoDC, but did not influence T cell stimulatory capacity. ERK1/2 and JNK were constitutively activated in MoDC from pigs fed the DON-diet. If MoDC derived from pigs fed the DON-diet were exposed to DON in vitro, this resulted in an up-regulation of MHC-II and CD80/86, but not CD40. In comparison to untreated MoDC from pigs fed DON-diet, endocytic capacity was further down-regulated, whereas mitogen-activated protein kinase activation was increased. In summary, DON disrupts porcine DC function in vitro and in vivo, which might contribute to the immunosuppressive effects of this mycotoxin.

Practically relevant concentrations of deoxynivalenol in diets for growing-finishing pigs offered as mash or pellets.

A complete 2 x 3 two factorial design was applied to investigate the effects of Fusarium-infected wheat (2.5 mg DON/kg, 0, 25 and 50% of the diets), feed processing (mash and pellets) and the interactions thereof on fattening pigs (96, n= 16/group). Feed-to-gain ratio was significantly increased by contaminated wheat (2.65; 2.62 and 2.73 kg/kg for diets containing 0, 25 and 50% Fusarium-infected wheat, respectively) while digestibility of nutrients and metabolizable energy were not affected by the wheat batch. The feed processing also resulted in significant differences in feed-to-gain ratio but was accompanied by significant effects on the digestibility of organic matter and crude fat and on the metabolizable energy. Clinical chemical parameters were not significantly altered by the inclusion of the infected wheat. The lymphocyte proliferation capacity was not significantly affected by any of the experimental factors. A contribution of the feed processing to the variation of the deoxynivalenol (DON) effect may not be deduced from the present results.

On the specific and unspecific effects of a polymeric glucomannan mycotoxin adsorbent on piglets when fed with uncontaminated or with Fusarium toxins contaminated diets.

A 35-day feeding experiment was performed with weaned piglets (initial live weight of 7.7 +/- 1 kg) to test the effects of a Fusarium toxin contamination of the diet (4.44 mg deoxynivalenol [DON] per kg diet) compared to an uncontaminated control diet. Both diet types were fed either unsupplemented or supplemented with a polymeric glucomannan mycotoxin adsorbent (GMA) to examine the claimed efficacy of detoxification of this feed additive through unspecific performance parameters and the specific proof of the prevention of DON absorption. Feeding of the mycotoxin-contaminated diets resulted in a decrease in feed intake and live weight gain by 28% and 14% when compared to the control group. These effects were independent of GMA addition. Based on the lack of significant interactions between diet mycotoxin-contamination and GMA supplementation for the performance data, and because of the inefficiency of GMA in decreasing DON absorption, it was concluded that the supplementation of DON-contaminated piglet diets with GMA cannot be recommended as an effective tool for alleviation of adverse DON effects.

Carry-over of Fusarium toxins (deoxynivalenol and zearalenone) from naturally contaminated wheat to pigs.

The frequent contamination of grain with the Fusarium toxins, deoxynivalenol (DON) and zearalenone (ZON), is an important issue in animal and human nutrition. However, data on the exposure of humans to these toxins through consumption of animal tissues exposed to Fusarium toxins (carry-over) are fragmentary. Therefore, residues of DON, ZON and their metabolites were determined in tissues and body fluids of pigs (female and castrated male) from a fattening trial. Pigs were fed a control (n = 6, 0.24 mg DON and 0.009 mg ZON per kg diet as fed) or a Fusarium toxin-contaminated diet (n = 12, 6.68 mg DON and 0.056 mg ZON per kg diet as fed) either ad libitum or for restrictive consumption for 12 weeks. After slaughter (96.3 +/- 11.6 kg live weight), the concentrations of DON and its metabolite, de-epoxy-DON, were measured in serum, bile, liver, kidney, musculus longissimus and back fat, while ZON and its metabolites, alpha- and beta-zearalenol (alpha-/beta-ZOL), were determined in serum, bile and liver. The mean carry-over factor of DON + de-epoxy-DON, defined as the concentration of both substances in the tissue/fluid divided by the DON concentration in the diet, for all pigs decreased from bile (0.1046 +/- 0.0653) >> kidney (0.0151 +/- 0.0070) > liver (0.0057 +/- 0.0043) > serum (0.0023 +/- 0.0018) > muscle (0.0016 +/- 0.0016) >> back fat (0.0002 +/- 0.0004). The time interval between the end of feeding and slaughter had no consistent effect on DON + de-epoxy-DON concentrations in the analysed specimen of Fusarium toxin-exposed pigs fed restrictively. No transfer of ZON and its metabolites could be observed into serum of pigs, while the mean carry-over factors of ZON + alpha-ZOL + beta-ZOL were 0.0094 +/- 0.0123 and 4.0 +/- 2.2 for liver and bile, respectively. Therefore, it can be concluded that serum is a reliable indicator for DON exposure, but an inappropriate parameter to deduce ZON exposure, which is better represented by bile concentration of ZON + alpha-ZOL + beta-ZOL. However, the exposure risk to humans by consumption of edible tissues of animals exposed to Fusarium toxins is negligible compared to the direct consumption of grain-based food.

Effects of the Fusarium toxin deoxynivalenol from naturally contaminated wheat given subchronically or as one single dose on the in vivo protein synthesis of peripheral blood lymphocytes and plasma proteins in the pig.

Trichothecenes, such as deoxynivalenol (DON), are known to inhibit the protein synthesis in vitro by binding at the 60S subunit of eukaryotic ribosomes. Therefore, cells and tissues with high protein turnover, such as lymphocytes and the liver (albumin and fibrinogen synthesis), were suggested to react most sensitively to DON. However, to the author's knowledge this observation was not proven in vivo in pigs, which were regarded as the farm animals most susceptible to DON. A total of 31 castrated male, crossbred German Landrace x Pietrain pigs weighing approx. 40 kg were fed a DON contaminated diet (5.7 mg/kg) either acutely (one single dose) or subchronically (4 weeks) or a control diet (0.1 mg/kg). In addition, one group received an intravenous injection of 53 microg DON/kg LW. One hour after feeding, a "flooding dose" of the stable isotope l-[(2)H(5)]-phenylalanine (125 mg/kg LW) was given and frequent blood samples (permanent catheter) were collected over a 60 min period. The molar percent excess (MPE) of plasma free and protein-bound phenylalanine were measured by GC/MS. No differences could be observed in the plasma concentrations of total protein, albumin, fibrinogen and serum enzymes between the groups. On the other hand, fractional synthesis rates (FSR, %/d) of albumin were significantly decreased by 43%, 45% and 26% and FSR of lymphocytes declined by 27%, 19% and 24%, whereas fibrinogen was not significantly affected after subchronic or one single oral and intravenous DON exposure, respectively. Additionally, the absolute synthesis rate (ASR, g/d) of albumin and the proportion of albumin to total body protein synthesis were reduced in the same manner, whereas the albumin secretion time ranged between 6.8 and 34.4 min and was not affected by treatment. In conclusion, the flooding dose technique appeared to be suitable for distinguishing DON-related effects on the protein synthesis, while determination of plasma protein concentrations seemed not to be an appropriate parameter.

Effect of the Fusarium toxin deoxynivalenol (DON) on IgA, IgM and IgG concentrations and proliferation of porcine blood lymphocytes.

An important effect of the trichothecene mycotoxins is the impairment of the immune function, but immunotoxicity studies have mainly been conducted on the mouse model. In the present study, the effect of deoxynivalenol (DON) on the proliferation of ConA stimulated porcine peripheral blood lymphocytes (PBL) was assessed in vitro after adding of 70-560 ng DON per ml medium, and in vivo after chronic and acute (one single dose) dietary DON exposure (5.7 mg/kg). Immunoglobulin (IgA, IgG, IgM) concentrations were measured by ELISA in supernatants and serum of pigs. The proliferation rate was estimated with two different assays (BrdU incorporation and MTT cleavage). In vitro the ConA stimulated proliferation was inhibited to 50% (IC50) at 200 and 309 ng DON/ml for the BrdU and MTT assay, respectively, indicating a higher sensitivity of DNA synthesis to DON. Immunoglobulin concentrations in the supernatant after in vitro proliferation of PBL with increasing DON concentrations for 72 h were significantly decreased, with IC50 values of 120.6, 84.1 and 71.7 ng DON/ml for IgA, IgM and IgG, respectively. In vivo significant inhibition of lymphocyte proliferation was observed only in the DON acute group using the MTT assay, but values tended to be decreased in the BrdU assay and after chronic DON exposure. Immunoglobulins (IgA, IgM and IgG) in the supernatant of cultured lymphocytes were not significantly affected after dietary DON exposure. Serum IgA of pigs showed no significant differences between the groups, whereas IgM and IgG were significant increased in the DON acute group.

Bioavailability of the Fusarium toxin deoxynivalenol (DON) from naturally contaminated wheat for the pig.

Experiments were carried out with 16 castrated male pigs (41.5 +/- 2.0 kg) to examine the toxicokinetics of deoxynivalenol (DON) from naturally contaminated wheat (16.6 mg DON/kg) after chronic exposure or one single oral dose (acute). The systemic absorption (bioavailability) of DON was estimated based on the area under the curves (AUC) after oral (chronic or acute) and intravenous application of pure DON (53 microg/kg live weight). Additionally, a balance study was conducted to quantitatively trace the DON metabolism. After intravenous (IV) DON application (n = 5), serum DON concentrations decreased biphasically with terminal elimination half-lives (t(1/2)beta) of between 4.2 and 33.6h. DON was rapidly absorbed following oral exposure and reached maximal plasma concentrations (C(max)) of 21.79 and 15.21 ng DON/ml serum after (t(max)) 88.4 and 99.1 min in the chronic (n = 5) and acute (n = 6) fed group, respectively. Thereafter serum DON levels declined slowly with an elimination half-life (t(1/2)beta) of 6.28 and 5.32 h for both oral groups. The mean bioavailability (F) of DON was 89% for the chronic group and 54% for the acute oral group. DON was highly distributed in all groups, with an apparent volume of distribution (V(d)) higher than the total body water. Glucuronide conjugation of deoxynivalenol was found in serum samples after oral exposure, but not after intravenous application. Dietary DON caused a significant increase in DON concentrations of urine and faeces, whereby the metabolite de-epoxy-DON was found only in the trials with a pre-period of longer than 4 weeks. The total recovery was 66.6 +/- 39.0% and 54.0 +/- 9.7% for the control and the chronic DON groups, respectively, with urine being the main excretory route. In conclusion, orally administered DON was quickly absorbed to an extent of over 50%, highly distributed and only poorly metabolized. Twenty-four hours following oral dosing, DON could not be detected in the serum, except in one chronically fed pig at the level of the detection limit.