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.

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.

UV-induced cis-trans isomerization of zearalenone in contaminated maize.

In the literature, it has been shown that the naturally occurring trans-zearalenone (ZEN) is transformed by ultraviolet irradiation to cis-ZEN. However, the practical relevance of this transformation in animal feeding remains unclear. The aim of the present preliminary investigation was to examine the effect of UV-irradiation on the concentration of trans-ZEN in a natural feed matrix at different dry matter contents to simulate the dry and wet feeding techniques usually applied in pig feeding. Four variants, air dry or wet ZEN-contaminated ground maize either irradiated or not were tested and analysed with conventional HPLC-FLD for trans-ZEN changes, which were further examined for cis-ZEN formation by HPLC-MS/MS. In conclusion, it could be shown that, under the investigated wet feed conditions, naturally occurring trans-ZEN was partially converted by ultraviolet irradiation to its cis counterpart. In contrast, the cis/trans isomerization seemed not to be relevant in dry maize. The consequence of this finding for practical liquid feeding systems for pigs requires further investigation. Additionally, an improvement of the analytical method for cis-ZEN determination is needed.

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.

Kinetics and metabolism of the Fusarium toxin deoxynivalenol in farm animals: consequences for diagnosis of exposure and intoxication and carry over.

The knowledge of factors influencing the kinetics, metabolism and bioavailability of the Fusarium toxin deoxynivalenol (DON) is a basic prerequisite for evaluation of the transfer (carry over) of the toxin and its metabolites into edible tissues, and for a physiological specimen-based diagnosis of intoxication. These aspects are addressed in the present review, and potentials and pitfalls of the suitability of analysis of physiological samples for evaluation of the DON exposure as a veterinary tool are discussed. For example, the farm animal species was shown to be a determining factor influencing the metabolic profile and the bioavailability of DON. Although linear relationships were derived between DON exposure of ad libitum and restrictively fed animals and DON or de-epoxy-DON concentration in the blood of pigs, dairy cows and sheep, it has to be considered that individual values might markedly deviate from these relationships, which makes interpretation of measured concentrations of DON and its metabolites difficult. The situation is further complicated by the lack of established relationships between DON residues in physiological matrices and the adverse effects of DON on the health and performance of farm animals.