Hana Valenta

Effects of graded levels of Fusarium toxin contaminated maize in diets for female weaned piglets

A dose response study was carried out with piglets to examine the effects of increasing amounts of Fusarium toxins in the diet on performance, clinical serum characteristics, organ weights and residues of zearalenone (ZON) and deoxynivalenol (DON) and their metabolites in body fluids and tissues. For this purpose, Fusarium toxin contaminated maize (1.2 mg ZON and 8.6 mg DON per kg maize) was incorporated into a maize based diet for piglets at 0, 6, 12.5, 25 and 50% at the expense of control maize. The experimental diets were tested on 100 female piglets allotted to 20 boxes (five animals per box) covering a body weight range of 12.4 ± 2.2 kg to 32.5 ± 5.6 kg. Voluntary feed intake and, consequently, body weight gain of the animals receiving the highest proportion of Fusarium toxin contaminated maize were significantly decreased while the feed conversion ratio was not affected by the treatment. The mean weight of the uterus related to the body weight of the animals of the same group was increased by almost 100% as compared to the control. For this group, significantly decreased values of total serum protein were determined, while the serum activity of the liver enzyme glutamate dehydrogenase and the serum concentration of the follicle stimulating hormone were decreased for all treatment groups receiving 6% contaminated maize or more in the diet. Serum concentrations of immune-globulins were not consistently altered by the treatment. Corresponding to the dietary exposure, increasing concentrations of ZON and α-zearalenol were detected in the bile fluid, liver and in pooled urine samples. The metabolite β-zearalenol was detected only in bile fluid. The total concentration of ZON plus its metabolites in bile fluid correlated well with the diet contamination (r = 0.844). DON was found in serum, bile fluid and pooled urine samples while de-epoxy-DON was detected only in urine. The serum concentration of DON correlated well with the respective toxin intake 3 - 4 h prior to slaughtering (r = 0.957). For all mentioned analyses of residues it has to be noted that toxin residues were detectable even if negligible concentrations were present in the diet.

On the toxicokinetics and the metabolism of deoxynivalenol (don) in the pig

Eleven castrated male pigs weighing 88.1 ± 3.9 kg on average were adapted to a diet containing DON (4.2 mg DON/kg) over a period of 7 days. Feed was given restrictively with 1.1 kg per meal (two meals per day). On the day of measurement, all pigs were slaughtered at different time intervals following the morning meal containing DON (1, 2, 3, 4, 5, 6, 8, 15, 18 and 24 h after feeding), with the exception of one pig which was slaughtered unfed. DON and de-epoxy-DON were analysed in serum and digesta from consecutive segments of the digestive tract (stomach, small intestine divided into three parts of a similar length, caecum, colon, rectum). DON was rapidly and nearly completely absorbed while passing through the stomach and the proximal small intestine. Maximum serum concentration appeared 4.1 h after the DON-containing meal and half of the systemically absorbed DON was eliminated after 5.8 h. De-epoxy-DON appeared in increasing proportions from the distal small intestine and reached approximately 80% of the sum of DON plus de-epoxy-DON in faeces collected from the rectum. It was concluded that de-epoxydation of DON, which primarily occurs in the hindgut, probably does not contribute much to a detoxification in the pig.

Effects of graded levels of Fusarium toxin contaminated wheat in diets for fattening pigs on growth performance, nutrient digestibility, deoxynivalenol balance and clinical serum characteristics

A dose response study was carried out with pigs in order to examine the effects of increasing dietary deoxynivalenol (DON)-concentrations on performance, clinical serum characteristics, nutrient digestibility and DON-metabolism. For this purpose, wheat contaminated naturally with Fusarium toxins was incorporated into pig diets at increasing proportions to give calculated dietary DON-concentrations of 0, 2.3 and 4.6 mg/kg during the starter period of phase 1 (14 d) of the experiment, and 0/0, 1.2/1.4, 2.3/3.7 mg/kg starter/grower diet during phase 3 (56 d) of the experiment. Each diet was tested on 16 pigs of both sexes with an initial average live weight of approximately 28 kg. A recovery phase (phase 2, 21 d) was intercalated between phase 1 and 3 of the growth experiment where all groups were fed with the uncontaminated control diet since some pigs exposed to the highest dietary DON-concentration during phase 1 nearly completely refused the offered feed. Affected pigs completely recovered during this phase. In phase 3, when diets with lower DON-concentrations were fed, no differences in performance could be detected. Serum clinical characteristics (enzymes indicating liver damage, total protein, immunoglobulins) did not respond to increasing DON-concentration in the diets. DON-concentration in serum increased in a dose-response-related manner as dietary DON-concentration increased. However, this parameter was not or only weakly correlated to any of the examined performance parameters or serum characteristics. Also, nutrient digestibility of the diets and N-retention were not affected by treatments with the exception of crude fat digestibility which was not consistently influenced. Concentration of DON and its metabolite de-epoxy-DON increased in urine with increasing dietary DON-concentration in a strongly linearly related fashion. The proportion of the excretion of de-epoxy-DON of the total urinary excretion of DON plus de-epoxy-DON rose linearly up to approximately 4%. Total recovery of DON plus de-epoxy-DON as percentage of DON-intake varied between 45 and 57% and was not influenced by dietary DON-concentration. Only a very small fraction of approximately 0.1% of ingested DON was recovered in faeces.

Analysis of deoxynivalenol and de-epoxy-deoxynivalenol in animal tissues by liquid chromatography after clean-up with an immunoaffinity column

A method for the determination of deoxynivalenol (DON) and its metabolite de-epoxy-deoxynivalenol (de-epoxy-DON) in blood serum, urine, faeces/digesta and bile fluid is described. Liquid samples (urine and bile after incubation with ß-glucuronidase) were extracted with ethyl acetate on a ChemElut column. Freeze dried faeces and digesta were extracted with a mixture of acetonitrile and water. For clean-up, serum and urine extracts could be directly applied to an immunoaffinity column (IAC). A pre-treatment prior to the IAC clean-up was necessary in the case of bile and faeces/digesta. DON and de-epoxy-DON were determined by high performance liquid chromatography (HPLC) with diode-array detection (DAD).Because of the clean extracts, low detection limits in the range of 4 ng/ml (serum) to 20 ng/g (dried faeces/digesta) were achieved. The recovery of DON and de-epoxy-DON was in the range of 75–89% and 64–85%, respectively. DON was found in blood serum, urine, bile, digesta and faeces of pigs from several feeding studies with DON contaminated feed; de-epoxy-DON could be detected only in digesta, faeces and urine.

Effects of Fusarium toxin-contaminated wheat and feed intake level on the biotransformation and carry-over of deoxynivalenol in dairy cows

An experiment was carried out to examine the effects of feeding Fusarium toxin-contaminated wheat (8.21 mg deoxynivalenol (DON) and 0.09 mg zearalenone (ZON) per kg dry matter) at different feed intake levels on the biotransformation and carry-over of DON in dairy cows. For this purpose, 14 ruminal and duodenal fistulated dairy cows were fed a diet containing 60% concentrate with a wheat portion of 55% (Fusarium toxin-contaminated wheat (mycotoxin period) or control wheat (control period)) and the ration was completed with maize- and grass silage (50 : 50) on a dry matter basis. Daily DON intakes ranged from 16.6 to 75.6 mg in the mycotoxin period at dry matter intakes of 5.6–20.5 kg. DON was almost completely biotransformed to de-epoxy DON (94–99%) independent of the DON/feed intake, and the flow of DON and de-epoxy DON at the duodenum related to DON intake ranged from 12 to 77% when the Fusarium toxin-contaminated wheat was fed. In the serum samples, de-epoxy DON was detected in the range of 4–28 ng ml−1 in the mycotoxin period, while concentrations of DON were all below the detection limit. The daily excretion of DON and de-epoxy DON in the milk of cows fed the contaminated wheat varied between 1 and 10 µg and between 14 and 104 µg, respectively. The total carry-over rates as the ratio between the daily excretion of DON and de-epoxy DON into milk and DON intake were in the ranges of 0.0001–0.0002 and 0.0004–0.0024, respectively. Total carry-over rates of DON as DON and de-epoxy DON into the milk increased significantly with increasing milk yield. In the urine samples, de-epoxy DON was the predominant substance as compared with DON with a portion of the total DON plus de-epoxy DON concentration to 96% when the Fusarium toxin-contaminated wheat was fed, whereas the total residues of DON plus de-epoxy DON in faeces ranged between 2 and 18% of DON intake in the mycotoxin period. The degree of glucuronidation of de-epoxy DON was found to be approximately 100% in serum. From 33 to 80% of DON and from 73 to 92% of de-epoxy DON, and from 21 to 92% of DON and from 86 to 100% of de-epoxy DON were glucuronidated in the milk and urine, respectively. It is concluded that DON is very rapidly biotransformed to de-epoxy DON in the rumen and only negligible amounts of DON and de-epoxy DON were transmitted into the milk within the range of 5.6–20.5 kg day−1 dry matter intake and milk yields (fat corrected milk) between 10 and 42 kg day−1.

Occurrence of deoxynivalenol and zearalenone in commercial fish feed: an initial study.

The control of mycotoxins is a global challenge not only in human consumption but also in nutrition of farm animals including aquatic species. Fusarium toxins, such as deoxynivalenol (DON) and zearalenone (ZEN), are common contaminants of animal feed but no study reported the occurrence of both mycotoxins in fish feed so far. Here, we report for the first time the occurrence of DON and ZEN in samples of commercial fish feed designed for nutrition of cyprinids collected from central Europe. A maximal DON concentration of 825 μg kg−1 feed was found in one feed whereas average values of 289 μg kg−1 feed were noted. ZEN was the more prevalent mycotoxin but the concentrations were lower showing an average level of 67.9 μg kg−1 feed.

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, α- and β-zearalenol (α-/β-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 + α-ZOL + β-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 + α-ZOL + β-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.

In vitro studies on the evaluation of mycotoxin detoxifying agents for their efficacy on deoxynivalenol and zearalenone

A simple in vitro system was developed to study the efficacy of commercially available mycotoxin detoxifying agents and adsorbing substances as feed additives to detoxify deoxynivalenol (DON) and zearalenone (ZON) in situ. The in vitro model simulates the conditions (pH, temperature and transit time) of the porcine gastrointestinal tract, as pigs react most sensitively to these mycotoxins. The commercially available products were not effective in detoxifying DON and ZON under the applied conditions, while activated carbon was able to bind both toxins and cholestyramine, and a modified aluminosilicate showed good adsorption abilities for ZON. Data obtained in dose dependency studies showed an estimated adsorption capacity of cholestyramine and the modified aluminosilicate of 11.7 and 5.7 g ZON/kg detoxifying agent. The in vitro system deployed in the present study was demonstrated to be a simple, helpful tool in screening substances for their ability to detoxify DON and ZON under the simulated conditions of the porcine gastrointestinal tract. Nonetheless in vivo experiments are indispensable to proof the efficacy.

Excretion kinetics and metabolism of zearalenone in broilers in dependence on a detoxifying agent

Two experiments were carried out with male broilers to examine excretion kinetics of zearalenone (ZON) and its metabolites and their occurrence in blood plasma and bile fluid after a single oral dose of ZON (approximately 6 μg/kg BW) from naturally contaminated wheat (406 μg ZON per kg). In addition, this ZON bolus was administered either in the absence or presence of a detoxifying agent (Mycofix®‐Plus, Biomin GmbH, Herzogenburg, Austria). Specimens were sampled after administration of the zearalenone bolus at different times of up to 48 h. Excretion of zearalenone and α‐zearalenol as the only detectable metabolite of ZON peaked at approximately 6.5 h after administration of the bolus. Cumulative excretion of both substances amounted to approximately 58% of ZON intake after 48 h, when a plateau was achieved. The incomplete recovery could have been due to a partial total degradation of ZON in the digestive tract, undetected sulfate conjugates of ZON or its metabolites, to other unknown and undetected metabolites or to incomplete analytical recovery from the matrix, and needs to be examined further. Peak concentrations of zearalenone and a‐zearalenol in bile were detected in the time period of approximately 2 to 6 h after bolus, whereas ZON and metabolite concentrations in blood plasma were around or lower than the detection limits. Mycofix®‐Plus supplementation seemed to have only minor or no effects on the parameters examined.

Determination of ochratoxin A in regional samples of cow's milk from Germany

Samples (121) of cows milk from a northern region of Germany were analysed for the occurrence of ochratoxin A by means of a sensitive high-performance liquid chromatographic (HPLC) method. The samples were extracted with a mixture of chloroform and methanol at pH < 2. The extracts were cleaned up by solid-phase extraction on silica gel cartridges. The detection limit was 0.01 ng/ml, the quantitation limit was estimated at 0.03 ng/ml. The mean recovery from spiked samples was 84 +/- 7% in the concentration range of 0.03-0.5 ng/ml. An enzyme-linked immunosorbent-assay (ELISA) was shown to be suitable for the confirmation of ochratoxin A levels down to the detection limit of the HPLC method. No ochratoxin A was detected in the samples analysed, either by HPLC or by ELISA.