Karsten Meyer

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.

Mycophenolic acid in silage.

ABSTRACT We examined 233 silage samples and found that molds were present in 206 samples with counts between 1 × 103 and 8.9 × 107 (mean, 4.7 × 106) CFU/g. Mycophenolic acid, a metabolite of Penicillium roqueforti, was detected by liquid chromatography-mass spectrometry in 74 (32%) of these samples at levels ranging from 20 to 35,000 (mean, 1,400) μg/kg. This compound has well-known immunosuppressive properties, so feeding with contaminated silage may promote the development of infectious diseases in livestock.

Simultaneous determination of type A, B and D trichothecenes and their occurrence in cereals and cereal products

A sensitive LC–MS/MS method for the simultaneous determination of type A, B and D trichothecenes in cereals is presented. The limits of detection ranged between 0.1 and 0.7 µg kg−1 for all analytes. The method was applied to 289 representatively drawn samples of wheat, rye and oat products. Ninety-four percent of the wheat samples (n = 130), 95% of the rye samples (n = 61) and 100% of the oat samples (n = 98) were contaminated with the type A trichothecenes T-2 and HT-2 toxin. Median levels of T-2/HT-2 (sum of the toxins) were 0.91, 0.53 and 8.2 µg kg−1, respectively. Highest levels were found in wheat bran (24 µg kg−1), rye kernels (3.1 µg kg−1) and oat flakes (85 µg kg−1). All wheat and rye samples and 75% of the oat samples were contaminated with the type B trichothecene deoxynivalenol. Median levels of this toxin were 23, 15 and 0.53 µg kg−1, respectively. Highest levels were found in wheat bran (1160 µg kg−1), rye kernels (288 µg kg−1) and oat flakes (55 µg kg−1). The type B trichothecene nivalenol was detected in 67% of the wheat samples, in 3% of the rye samples and in 24% of the oat samples with highest levels in wheat bran (96 µg kg−1), rye kernels (1.8 µg kg−1) and in oat flakes (17 µg kg−1), respectively. Levels of other type A and B trichothecenes played a minor role, although the rates of contamination were often high. Neither macrocyclic type D trichothecenes (satratoxin G and H, verrucarin A, roridin A) nor diacetylverrucarol and verrucarol (type A trichothecenes), were detected in any of the samples.

Phenotypic and genotypic bacterial antimicrobial resistance in liquid pig manure is variously associated with contents of tetracyclines and sulfonamides

Aims:  Antibiotic residues as well as antibiotic‐resistant bacteria in environmental samples might pose a risk to human health. This study aimed to investigate the association between antibiotic residues and bacterial antimicrobial resistance in liquid pig manure used as fertilizer.

Influence of organically or conventionally produced wheat on health, performance and mycotoxin residues in tissues and bile of growing pigs

From 1999 – 2001 three different varieties of wheat [Contur (susceptible to Fusarium), Batis and Petrus (less susceptible to Fusarium)] were cultivated under organic and conventional conditions in order to determine mycotoxin burden. Soil quality, preceding crop and weather conditions were comparable in the different production systems. The wheat batches were analysed for moulds, and the contents of zearalenone (ZEN) and deoxynivalenol (DON). Feeding trials were carried out with growing pigs (n = 96; average initial live weight 22.2 ± 1.5 kg [mean ± SD]) to examine a possible influence on the animal performance and on mycotoxin residues. The data recorded were clinical conditions, performance, biochemical and hematological data. Residues of ZEN, α- and β-zearalenol (ZEL) and of DON were determined in bile, liver and muscle after slaughtering. Conventionally cultivated wheat was more frequently contaminated with Fusarium and contained more frequently ZEN and DON in higher concentrations than the organically produced wheat. Hematological and biochemical parameters of pigs fed with organically cultivated diets were not different from those of conventionally fed pigs. Pigs fed with organically produced wheat showed a slightly higher daily weight gain, but a lower carcass yield than the conventionally fed animals. The highest residues of DON and total-ZEN (ZEN + α-ZEL + β-ZEL) were found in bile. Bile samples of organically fed pigs contained lower concentrations of total-ZEN than those of conventionally fed pigs. Altogether, these data suggest that wheat from an organic farming does not have higher mycotoxin-contamination than wheat from the conventional farming system.

Residue formation of fumonisin B1 in porcine tissues

The residues derived from the uptake of fumonisin B1, a toxic metabolite of Fusarium verticillioides frequently occurring in corn and corn products, were determined in growing pigs. After oral administration of 100 mg FB1/animal/day for 5–11 days, serum, bile, lung, liver, kidney, brain, spleen, pancreas, heart, muscle, eye, and fat samples were collected immediately and analysed by LC-MS. The highest values were measured in kidney (833±1329 µgkg−1, mean±SD), liver (231±163 µgkg−1), lung (170±311 µgkg−1) and spleen (854±2212 µgkg−1). Muscle contained 26±41 µgkg−1, while in fat only 2±3 µgkg−1 were traceable. Despite the potential accumulation over extended feeding periods as well as the large variations in the residue formation of FB1, a carry-over in edible tissues from swine was considered not to be of toxicological relevance.

Tetracycline in Liquid Manure Selects for Co-occurrence of the Resistance Genes tet(M) and tet(L) in Enterococcus faecalis

Causal relations between antibiotic use and selection of antibiotic resistance have been widely discussed. However, appropriate examinations have been mainly performed on phenotypic level, whereas genetic investigations, as well as researches under realistic conditions, are scarce. Therefore, the present field study aimed to accomplish a particular description of how an antibiotic contaminated environment influences microorganisms on both a phenotypic and a genetic level, using analytical, phenotypical and molecular biological methods. For this purpose, concentrations of tetracycline, chlortetracycline, oxytetracycline and doxycycline were analysed in liquid manure samples (n=179) from Bavarian (Germany) pig farms. All detected tetracyclines found in each manure sample were summed up and referred to as total tetracycline concentrations (TET). Phenotypic doxycycline resistance of Enterococcus faecalis isolated from these manure samples was determined by means of the microdilution method. After that, doxycycline resistant (n=147) and susceptible (n=32) E. faecalis were screened for tet(L), tet(M), tet(S) and tet(O) by using PCR. If despite doxycycline resistance no respective gene was detected, tet(A/B/C/D/K/L/M/W/Z) were additionally tested. The most frequent resistance determinant was tet(M) (n=128), followed by tet(L) (n=95). Tet(S) and tet(O) were present in 12 and 7 isolates; the remaining tet-genes were not detected. A correlation between the TET concentration in manure and the occurrence of tet(M) and tet(L) could be shown. In particular, strains that contained neither tet(M) nor tet(L) (n=44) were isolated from manure samples with mean TET of 0.35 mg/kg. If tet(M) was the only tetracycline resistance gene (n=40), mean concentrations were 0.51 mg/kg, and, if tet(L) was the only tet-gene (n=7), 1.18 mg/kg, respectively. On the other hand, if co-occurrence of tet(M) and tet(L) was detected (n=88, including 1 susceptible isolate), mean TET in the referring manure samples was 4.08 mg/kg. The present study demonstrates that high tetracycline concentrations in manure lead to higher doxycycline minimum inhibitory concentrations (MICs) in E. faecalis, genetically based on co-occurrence of tet(M) and tet(L).

Distribution and elimination of fumonisin analogues in weaned piglets after oral administration of Fusarium verticillioides fungal culture

The distribution and elimination of fumonisins after oral administration of 50 mg FB1, 20 mg FB2 and 5 mg FB3 per animal day–1 for 22 days was studied in weaned barrows. At the end of the trial, the lung, heart, liver, kidney, spleen, brain, serum, bile, muscle, fat, urine and faeces samples were collected and their content of fumonisins (FB1, FB2) determined by LC-MS. The highest FB1 concentrations were found in the liver (99.4 ± 37.5 ng g–1) and kidneys (30.6 ± 10.1 ng g–1), whilst the highest average amount of FB2 was in the liver (1.4 ± 2.3 ng g–1) and fat (2.6 ng g–1 ± 4.8) samples. Comparing the FB1/FB2 ratio in different organs (19/1), it was found that the ratio in the abdominal and subcutaneous fat samples (4/1) was markedly different from those in all other tissues, namely the relative proportion of FB2 was higher in latter cases. Of the total quantity of FB1, the 13% taken up during 5 days was excreted unchanged with the faeces and urine. On average, in the urine and faeces, FB1 was detected in nine- and 14-fold quantities, as compared with FB2.

In vitro microbial metabolism of fumonisin B1

There is a lack of information on the effect of swine caecal microbiota on fumonisin metabolism. In this in vitro study, the biotransformation of fumonisin B1 (FB1) by the gut microbiota of adult, healthy pigs was examined. Suspensions of caecal contents and McDougall buffer solution were incubated anaerobically with pure FB1 for 0, 12, 24, 48 and 72 h. After 48 h, the conversion of FB1 to partially hydrolysed FB1 (46%) was nearly equal to the percentage ratio of FB1, while by 72 h it was 49%. In vitro, the conversion of fumonisin B1 to aminopentol was less than 1%. The results show that the caecal microbiota are capable of transforming fumonisin B1 to the above metabolites. Further studies on FB1 metabolism in the small intestine are clearly justified.

Detection of melengestrol acetate residues in plasma and edible tissues of heifers

The aim of this study was to gain knowledge of residue formation after the use of melengestrol acetate (MGA) as a growth-promoting agent. Two Holstein-Friesian heifers each received a daily dose through the feed of 0, 0.5 mg (2 heifers with and without withdrawal each), 1.5 mg or 5.0 mg MGA for 8 weeks. MGA residues in plasma were screened by enzyme immuno-assay (EIA). Concentrations in kidney, liver, and muscle were quantified by liquid-chromatography-mass spectrometry (LC-MS), and in fat by gas chromatography-mass spectrometry (GC-MS). MGA levels in plasma were 40, 128, and 280 ng/L, respectively. Residues accumulated in muscle and kidney (5-fold), liver (20-to-40-fold), and fat (200-fold). After administration of 1.5 mg per day the mean MGA concentration in fat was 29 micrograms/kg and thus violated USA regulations which specify a limit of 25 ppb. Therefore the labelled use of MGA (0.5 mg per day) has to be officially controlled.