Diagnosis of intoxications of piglets fed with Fusarium toxin-contaminated maize by the analysis of mycotoxin residues in serum, liquor and urine with LC-MS/MS.

Concentrations of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL), ß-zearalanol (ß-ZAL) and de-epoxy-deoxynivalenol (de-DON) in serum, liquor and urine of female piglets fed diets containing 0.01, 0.05, 0.08, 0.17 and 0.29 mg ZEN/kg and 0.03, 0.59, 1.27, 2.01 and 4.52 mg DON/kg during 29 days of treatment were analysed. After 1, 3, 8, 15, 22 and 29 days, four piglets per group were slaughtered. The simultaneous determination of all analytes was carried out using a sensitive and selective in-house-validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method after sample preparation with Oasis™ HLB columns. ZEN, α-ZEL, DON and de-DON were detected in serum, whereas in liquor only ZEN, DON and de-DON were found at lower concentrations. In urine, all analytes were detected in considerably higher concentrations as in serum and liquor, whereby α- and ß-ZAL could only be detected sporadically. Apart from ZEN in liquor and α- and ß-ZAL in urine, the mycotoxin concentrations increased with increasing concentrations of Fusarium toxins in the diet. The toxin intake per kg body weight 3-4 h prior to slaughtering correlated well with the DON and the sum of DON and de-DON concentrations in all three specimens as well as with the ZEN, α-ZEL and the sum of ZEN and metabolite concentrations in urine. Due to the high correlation between the dietary DON concentration and the DON (r = 0.855) and the sum of DON and de-DON (r = 0.870) concentration in serum, the exposure to DON can be evaluated. Moreover, serum levels of these toxins indicative of an exceeding of the guidance value in feed can be established using the corresponding regression equations. Strictly speaking, these relationships are only valid for the experimental conditions of the underlying experiment. For practical application of these relationships, the individual variation needs to be additionally considered. Effects of the duration of toxin exposure within the feeding groups were observed for ZEN, DON and de-DON in all specimens as well as for α-ZEL, ß-ZEL and ZAN in urine.

Effects of increasing concentrations of sodium sulfite on deoxynivalenol and deoxynivalenol sulfonate concentrations of maize kernels and maize meal preserved at various moisture content.

Under moderate climatic conditions, deoxynivalenol (DON) contamination occurs frequently on cereals. Detoxification measures are required to avoid adverse effects on farm animals. In the present study, a wet preservation method with sodium sulfite (Na2SO3) and propionic acid was tested to titrate the optimum Na2SO3-dose for maximum DON reduction of contaminated maize kernels and meal and to examine the interaction between dose and moisture content in dependence on the preservation duration. The DON concentration decreased with increasing amounts of supplemented Na2SO3 and with increasing duration of the preservation period in a bi-exponential fashion. Additionally, the feed structure and moisture content had a significant influence on the decontaminating effect. Variants with 30% moisture content favored higher DON reduction rates compared to 14% moisture, but especially at low moisture contents, DON reduction was more pronounced in maize kernels than in maize meal. In addition to the decrease of DON, a concomitant formation of three different DON sulfonates was observed which differed in their formation pattern over the time course of preservation. The overall results and statistical analysis clarified that Na2SO3 addition of 10 g/kg maize at 30% moisture for eight days was necessary to obtain a complete DON reduction.

Development of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of zearalenone, deoxynivalenol and their metabolites in pig serum.

A sensitive and selective liquid chromatography tandem mass spectrometry method using negative electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites α-zearalenol, ß-zearalenol, zearalanone, α-zearalanol, ß-zearalanol and de-epoxy-deoxynivalenol in pig serum. For method development, different sample preparation columns were tested for their suitability for extraction and clean up. Finally, preparation of serum samples was carried out using Oasis™ HLB solid-phase extraction (SPE) columns. The analyte concentrations were determined by the use of isotopically labelled internal standards (IS). The method was in-house validated for all analytes. Calibration graphs (0.3-480 ng/ml) were prepared and high degree of linearity was achieved (r ≥ 0.99). Results for method precision ranged between 2.7 and 21.5 % for inter-day and between 1.1 and 11.1 % for intra-day. The recoveries were in the range of 82-131 %. Limits of detection and quantification ranged 0.03-0.71 and 0.08-2.37 ng/ml, respectively. The method has been successfully used for quantitative determination of ZEN, DON and their metabolites in pig serum from a feeding trial with practically relevant ZEN and DON concentrations. This method is precise and reproducible and can be used as a multi-biomarker method to assess animal exposure to these mycotoxins and for diagnosis of intoxications.

Effects of a Fusarium toxin-contaminated maize treated with sodium metabisulphite, methylamine and calcium hydroxide in diets for female piglets.

Deoxynivalenol (DON) and zearalenone (ZEN) contaminated maize was hydrothermally treated in the presence of sodium metabisulphite (SBS), methylamine and calcium hydroxide (Ca(OH)2) and included into diets for female piglets to evaluate effects on performance, organ weights, development of hyperestrogenism, serum biochemical parameters, stimulation of peripheral blood mononuclear cells and toxin residues in serum. For this purpose, both uncontaminated maize (CON) and Fusarium toxin-contaminated maize (FUS) were included into diets either untreated (-) or treated (+) according to a 2 by 2-factorial design. One-hundred female weaned piglets were assigned to one of the four treatment groups (n = 25) CON-, CON+, FUS- and FUS+ with DON/ZEN concentrations of 0.43/0.03, 0.04/0.0, 3.67/0.32 and 0.36/0.08 mg per kg diet, respectively. After a feeding period of 27 days, 20 piglets (n = 5) were slaughtered. Performance parameters such as feed intake, live weight gain and feed-to-gain ratio remained unaffected by the treatments. Uterus weights were significantly reduced in group FUS+ compared to FUS- (p = 0.028), while visceral organ weights were not influenced. Vulva width in relation to body weight was highest in group FUS- at the end of the trial, while hydrothermal treatment significantly reduced the parameter (p < 0.01). The highest toxin and toxin metabolite concentrations in serum were detected in group FUS-, whereas ingestion of diet FUS+ reduced the concentrations to the level of the control groups. Serum biochemical and haematological parameters were mainly within the given reference ranges and showed no treatment-related alterations. Stimulation of peripheral blood mononuclear cells was not affected. An effective detoxification of maize by hydrothermal treatment in the presence of SBS, methylamine and Ca(OH)2 could be demonstrated by means of serum toxin analyses. No undesired side effects of the treated-feed stuff or the chemicals themselves on the health of piglets were detected.

Hydrothermal treatment of naturally contaminated maize in the presence of sodium metabisulfite, methylamine and calcium hydroxide; effects on the concentration of zearalenone and deoxynivalenol.

Fusarium toxin-contaminated ground maize was hydrothermally treated in the presence of different combinations of chemicals in order to simultaneously reduce zearalenone (ZEA) and deoxynivalenol (DON) concentrations. Treatments were carried out in a laboratory conditioner at 80 °C and 17 % moisture. Six different treatments were performed, consisting of 3 doses of methylamine (MMA; 2.5, 5 and 10 g/kg maize) at a constant dose of 5 g sodium metabisulfite (SBS)/kg, either with or without the addition of 20 g calcium hydroxide (Ca(OH)2)/kg. The used maize was contaminated with approximately 45.99 mg DON/kg and 3.46 mg ZEA/kg. Without the addition of Ca(OH)2, DON reductions reached approximately 82% after 1-min treatment and the toxin disappeared nearly completely after 10 min when 2.5 or 5 g MMA were applied. ZEA concentrations were only marginally affected. In the presence of Ca(OH)2, reductions in DON concentrations were lower, but were enhanced by increasing doses of MMA. ZEA concentrations were reduced by 72, 85 and 95% within the first 5 min of the treatment at MMA dosages of 2.5, 5 and 10 g/kg maize, respectively. The application of SBS in combination with a strong alkaline during hydrothermal treatment seems to be a promising approach to simultaneously decontaminate even high amounts of DON and ZEA in ground maize and may contribute to reduce the toxin load of diets.