Sven Dänicke

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.

In vivo and in vitro effects of the mycotoxins zearalenone and deoxynivalenol on different non-reproductive and reproductive organs in female pigs: A review

This review summarizes the toxicological data on the effects of the mycotoxins zearalenone (ZON), its metabolites, and deoxynivalenol (DON) on different parameters relating to reproductive and non-reproductive organs in female pigs. In vivo, 22 mg ZON kg−1 in the diet cause alterations in the reproductive tract of swine such as in the uterus, and affects follicular and embryo development. ZON and its metabolites have been shown to bind competitively to oestrogen receptors in an in vitro system. The feeding of pigs with a 9 mg DON kg−1-contaminated diet can act on protein synthesis, humoral and cellular immune response depending on dose, exposure and timing of functional immune assay, and affect liver and spleen cell structures. Beside these effects, reproductive alterations were observed in pigs, too. Both in vivo and in vitro exposure to DON decreased oocyte and embryo development. In vitro application of DON to uterine cells inhibits their proliferation rate and modulates the process of translation at a different molecular level when compared with the in vivo application. The histopathological results provide evidence of spleen and liver dysfunction in the absence of clinical signs, especially in pigs fed higher concentrations of Fusarium toxin-contaminated wheat. Prepuberal gilts react more sensitively to DON > ZON feeding compared with pregnant sows. In the liver, histopathological changes such as glycogen decrease and interlobular collagen uptake were only observed in prepuberal gilts, whereas enhancement of haemosiderin was found in both perpuberal gilts and pregnant sows. This review presents some of the current knowledge on the biological activities of ZON and DON in pig. Altogether, ZON affects reproduction of pigs most seriously because it possesses oestrogenic activity. However, DON affects reproduction in pigs via indirect effects such as reduced feed intake, resulting in reduced growth or impairment of function in vital organs such as liver and spleen.

Proposal of a comprehensive definition of modified and other forms of mycotoxins including “masked” mycotoxins

As the term “masked mycotoxins” encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, “modified mycotoxins” into “biologically modified” and “chemically modified” with all variations of metabolites of the former and dividing the latter into “thermally formed” and “non-thermally formed” ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term “modified mycotoxins” should be used in the future and the term “masked mycotoxins” to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.

The Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) in animal feeding.

The contamination of cereal grains with toxic secondary metabolites of fungi, mycotoxins, is a permanent challenge in animal nutrition as health and performance of the animals may be compromised as well as the quality of animal derived food. Therefore the present article reviews the issue of mycotoxins in animal nutrition. As the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) are of particular importance under the production conditions in central Europe and Germany, with respect to their frequent occurrence in toxicologically relevant concentrations, special emphasis is layed on those mycotoxins. The effects of DON and ZON on susceptible animals as well as management strategies to cope with the contamination of grain with those toxins are reviewed.

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.

Progression of mycotoxin and nutrient concentrations in wheat after inoculation with Fusarium culmorum.

The objective of this study was to follow the mycotoxin formation and changes in nutrient composition of wheat (cv. Ritmo) artificially inoculated with Fusarium culmorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on monitoring the progression of the contamination of the wheat kernels with deoxynivalenol (DON) and zearalenone (ZON). Both the uncontaminated control kernels and the contaminated kernels were examined also for the presence of zearalenone-4-beta-D-glucopyranoside and several trichothecenes at harvest. Furthermore, the impact of the Fusarium inoculation on some nutrients as starch, crude protein, amino acid composition, crude ash, non starch polysaccharides (NSP) as well as viscosity and thousand seed weight (TSW) was examined. Also proteolytic and amylolytic activity as well as the NSP-degrading enzyme activities of inoculated and control samples were analysed at the time of harvest. DON was detected in higher concentrations and in earlier stages, while ZON was found later and in smaller amounts. On average 7.79 mg/kg DM of DON and 100 μg/kg DM of ZON were found in the inoculated kernels at the time of harvest. Neither in the contaminated nor in the control samples glucose conjugates of ZON (Zearalenone-4-beta-D-glucopyranoside) were detected. Moreover, the infection with Fusarium culmorum had pronounced effects on some quality parameters. The crude protein content of the inoculated kernels showed significantly higher values over the whole period compared to the control kernels. The protein content of the inoculated kernels amounted 13.9% DM at harvest, while only a concentration of 12.5% DM was detected in the control samples. Similarly, in almost all stages of development the crude ash content of inoculated samples was higher than in control samples. These distinct differences in kernel composition resulted possibly from the changes of the thousand seed weight. In the present work the grain harvested from the control plots showed a significantly higher TSW (24.2 g) as compared to their inoculated counterparts (15.5 g). Despite lower extract viscosity of inoculated samples at time of harvest, the content of soluble NSP of inoculated plots was higher than in control samples at the same time. Moreover, inoculation resulted in markedly increased activities of protease, amylase and several NSP-degrading enzyme activities. This would suggest that the cell wall penetrating properties of the fungus itself and/or that the fungus induced alterations of the metabolic activity of the embryo or other constituents of the wheat kernel could be responsible.

Vulnerability of polarised intestinal porcine epithelial cells to mycotoxin deoxynivalenol depends on the route of application.

Background and Aims Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated. Methods A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity. Results Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL. Conclusions Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.

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.

Mycotoxin deoxynivalenol (DON) mediates biphasic cellular response in intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2

The Fusarium derived mycotoxin deoxynivalenol (DON) is frequently found in cereals used for human and animal nutrition. We studied effects of DON in non-transformed, non-carcinoma, polarized epithelial cells of porcine small intestinal origin (IPEC-1 and IPEC-J2) in a low (200 ng/mL) and a high (2000 ng/mL) concentration. Application of high DON concentrations showed significant toxic effects as indicated by a reduction in cell number, in cellular reduction capacity measured by MTT assay, reduced uptake of neutral red (NR) and a decrease in cell proliferation. High dose toxicity was accompanied by disintegration of tight junction protein ZO-1 and increase of cell cycle phase G2/M. Activation of caspase 3 was found as an early event in the high DON concentration with an initial maximum after 6-8 h. In contrast, application of 200 ng/mL DON exhibited a response pattern distinct from the high dose DON toxicity. The cell cycle, ZO-1 expression and distribution as well as caspase 3 activation were not changed. BrdU incorporation was significantly increased after 72 h incubation with 200 ng/mL DON and NR uptake was only transiently reduced after 24 h. Low dose effects of DON on intestinal epithelial cells were triggered by mechanisms different from those responsible for the high dose toxicity.