The effects of four mycotoxins on the mitogen stimulated proliferation of bovine peripheral blood mononuclear cells in vitro

The effects of four mycotoxins, T-2 toxin, deoxynivalenol (DON), ochratoxin A (OTA) and fumonisin B1 (FB1) on the response of bovine peripheral blood mononuclear cells (PBM) in vitro to the mitogens concanavalin A (Con A), phytohaemagglutinin A (PHA) and pokeweed mitogen (PWM) were assayed after 4 days' incubation using 3H-thymidine uptake and the MTT bioassay. The concentrations of mycotoxin required to reduce the proliferative response of PBM by 50% for Con A, PHA and PWM as measured by 3H-thymidine incorporation was for T-2 toxin 0.30, 0.40 and 0.18 ng ml-1; for DON 0.07, 0.09 and 0.04 μg ml-1; for OTA 0.10, 0.20 and 0.15 μg ml- 1, and for FB1 35, 18 and 11 μg ml-1M by 50% for Con A, PHA and PWM as measured by the MTT bioassay were for T-2 toxin 2.0, 2.0 and 1.0 mg ml-1; for DON 0.70, 0.50 and 0.50 μg ml-1; OTA 1.5, 1,5 and 1.5 μg ml- 1; and FB1 >50, >50 and 20 μg ml-1 respectively. Further cytotoxicity assays including the LDH bioassay and Trypan blue exclusion were performed only on Con A-stimulated PBM cells after 72 h incubation. With the LDH-bioassay the 50% inhibition levels were T-2 toxin 0.3 ng ml-1, DON 0.4 μg ml- 1, OTA 1.4 μg ml-1 and FB1 3.5 μg ml-1; for Trypan blue uptake the 50% inhibition levels were T-2 toxin 5 ng ml-1, DON 2.3 μg ml-1 and OTA 4 μg ml-1 respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence of type A,B and D trichothecenes in barley and barley products from the Bavarian market

Fifty-nine samples of barley and barley products were analysed for 18 trichothecene mycotoxins by a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (detection limits 0.062-0.70 μg/kg) after sample extract clean-up on MycoSep®-226 columns. The samples were collected in 2009 from barley processing facilities (mills and malt houses) and at wholesale and retail stage from the Bavarian market. The predominant toxins were T-2 toxin (T-2), HT-2 toxin (HT-2) and deoxynivalenol (DON). For all samples, the mean levels of T-2 and HT-2 were 3.0 μg/kg and 6.8 μg/kg with rates of contamination of 63% and 71%, respectively. The maximum values were 40 μg/kg for T-2 and 47 μg/kg for HT-2. The rate of contamination with DON was high (95%) with a low mean level of 23 μg/kg. The DON levels ranged between 3.4 to 420 μg/kg. For T-2 tetraol, a mean level of 9.2 μg/kg and a maximum level of 51 μg/kg with a rate of contamination of 71% were determined. NIV was detected in 69% of the samples with a mean level of 11 μg/kg and a maximum level of 72 μg/kg. Other type A and B trichothecenes were detected only in traces. Type D trichothecenes, fusarenon-X, verrucarol and 4,15-diacetylverrucarol were not detected in any sample. Winter barley and malting barley were the most contaminated groups of all samples in this study. In malting barley, the highest levels of contamination with type A trichothecenes were found. In contrast, winter barley showed the highest contamination with type B trichothecenes. The lowest mycotoxin concentrations were found in de-hulled and naked barley and in pearl barley.     

Airborne mycotoxins in dust from grain elevators

Workers in grain elevators are exposed to grain dust and may therefore have an increased risk of inhalatory contact with mycotoxins. To study the mycotoxin burden of such environments, settled grain dust samples (n=35) were collected from several locations of a total of 13 grain elevators in Germany, and analysed for ochratoxin A (OTA, detection limit 0.01 ng/g), deoxynivalenol (DON, detection limit 15 ng/g), and zearalenone (ZEA, detection limit 6 ng/g), respectively. Cytotoxicity of these samples was assessed by a MTT bioassay with a swine kidney target cell line. Additionally, the airborne dust concentration of these locations was determined. Nearly all settled dust samples contained OTA (96%), DON (100%), and ZEA (100%) with median concentrations of 0.4 ng/g, 416 ng/g, and 126 ng/g, respectively. Cytotoxic effects in varying degrees from weakly to highly toxic were caused by crude extracts of 86% of the dust samples. However, cytotoxicity did not correlate with mycotoxin levels in these samples and thus indicated the presence of cytotoxic compounds of unknown origin. Based on the mycotoxin findings in settled dust samples and the airborne dust concentrations, the average airborne mycotoxin concentrations were estimated to be 0.002 ng/m³ (OTA), 2 ng/m³ (DON), and 1 ng/m³ (ZEA), respectively. The relevance of these findings for occupational health was assessed by comparison with WHO recommendations for the maximum tolerable daily (oral) intake (TDI). Even in a worst case scenario, the calculated inhalatory intake was far below the TDI values. However, considering the uncertainties resulting from different exposure pathways, namely oral ingestion versus inhalation, further research should primarily address the problem of how adequate assessment criteria for airborne exposure to mycotoxins could be established. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

A practical method for measuring deoxynivalenol, nivalenol, and T-2 + HT-2 toxin in foods by an enzyme-linked immunosorbent assay using monoclonal antibodies

We have developed and tested an enzyme-linked immunosorbent assay system for individual measurement of deoxynivalenol, nivalenol, and T-2 + HT-2 toxin using monoclonal antibodies for 3,4,15-triacetyl-nivalenol, for both 3,4,15-triacetyl-nivalenol and 3,15-diacetyl-deoxynivalenol, and for acetyl-T-2 toxin. The assay system comprised three kits (desinated the DON + NIV kit, the NIV kit, and the T-2 + HT-2 kit). The practical performance of the enzyme-linked immunosorbent assay system was assessed by assaying trichothecene mycotoxins in wheat kernels. The enzyme-linked immunosorbent assay system meets all the requirements for use in a routine assay in terms of sensitivity (detection limit: deoxynivalenol 80 ng/g, nivalenol 80 ng/g, T-2 toxin 30 ng/g), reproducibility (total coefficient of variation: 1.9-6.2%), accuracy (recovery: 93.8-112.0%), simplicity and rapidity (time required: <2 h), mass handling (>42 samples/assay), and a good correlation with gas chromatography-mass spectrometry (r=0.9146-0.9991). Components derived from the wheat extract did not interfere with the assay kits. The enzyme-linked immunosorbent assay system is a useful alternative method to gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or liquid chromatography-ultraviolet absorption for screening cereals and foods for trichothecene mycotoxin contamination.     

Mycotoxins in horse feed

A total of 62 samples of commercial horse feed preparations (complementary feeds) containing cereal mixtures (“muesli” or mash, n = 39; pelleted feeds, n = 12), and plain horse feed grains (maize, n = 5; oats, n = 4; barley, n = 2) were purchased from 21 different producers/distributors from the German market. All samples were analysed by competitive enzyme immunoassays (EIA) for six different mycotoxins (mycotoxin groups). Analytes (detection limit, mean recovery) were: deoxynivalenol (DON, 10 μg/kg, 84%), zearalenone (ZEA, 5 μg/kg, 93%), fumonisin B₁ (FB₁, 2 μg/kg, 113%), T-2 toxin (T-2, 0.1 μg/kg, 71%), sum of T-2 + HT-2 toxin (T-2/HT2, 0.2 μg/kg, 97%), ochratoxin A (OTA, 0.2 μg/kg, 67%), and total ergot alkaloids (Generic Ergot Alkaloids “GEA”, 30 μg/kg, 132%). All samples contained DON (16–4,900 μg/kg, median 220 μg/kg), T-2/HT-2 (0.8–230 μg/kg, median 24 μg/kg), and T-2 (0.3–91 μg/kg, median 7 μg/kg). ZEA was detected in 98% of the samples (7–310 μg/kg, median 61 μg/kg). Most samples (94%) were positive for FB₁ (2–2,200 μg/kg, median 27 μg/kg). Ergot alkaloids were detected in 61% of samples (28–1,200 μg/kg, median 97 μg/kg), OTA was found in 42% of samples (0.2–4 μg/kg, median 0.35 μg/kg). The results demonstrate that a co-contamination with several mycotoxins is very common in commercial horse feed from the German market. The toxin concentrations were in most cases well below the levels which are usually considered as critical or even toxic. The highest mycotoxin concentrations were mostly found in single-grain cereal feed: the maximum values for DON and FB₁ were found in maize, the highest T-2/HT-2 toxin concentrations were found in oats, and the highest concentration of ergot alkaloids was found in barley. In composed feeds, no correlation between cereal composition and mycotoxin levels could be found.     

Trichothecenes and Mycoflora in Wheat Harvested in Nine Locations in Buenos Aires Province, Argentina

A total of 120 freshly harvested wheat samples from the 2004 season in nine locations from Northern Buenos Aires Province, Argentina, were analysed for trichothecene natural occurrence and associated mycoflora, and for determining the influence of commonly used fungicide field treatment and the cultivar type on trichothecene contamination. The trichothecenes T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), diacetoxyscirpenol (DAS), nivalenol (NIV), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) were analysed by gas chromatography and electron capture detection. Detection limits ranged from 4 to 20 μg/kg. The isolation frequencies of species were calculated. Alternaria alternata, Fusarium graminearum, Fusarium poae and Fusarium semitectum were the predominant fungal species identified as endogenous mycoflora. The type of cultivar and the fungicide field treatment did not affect significantly the trichothecene contamination. The trichothecenes type A detected were HT-2 and T-2 triol toxins and the type B were DON, NIV and 3-ADON. Based on 120 samples the incidences were 21.7% for 3-ADON, 22.5% for HT-2, 27.5% for T-2 triol and 85% for DON. NIV was confirmed in one sample. Mean levels of trichothecene positive samples were between 7 and 2788 μg/kg.     

Simultaneous occurrence of fumonisin B1 and other mycotoxins in moldy corn collected from the People's Republic of China in regions with high incidences of esophageal cancer.

A total of 31 corn samples collected from households in the counties of Cixian and Linxian of the People's Republic of China, where high incidences of esophageal cancer have been reported, were analyzed for fumonisin B1 (FB1), aflatoxin, and total trichothecene mycotoxins. High levels of FB1 (18 to 155 ppm; mean, 74 ppm) were found in 16 of the samples that showed heavy mold contamination. FB1, at lower levels (20 to 60 ppm; mean, 35.3 ppm), was also found in 15 samples, collected from the same households, that did not show any visible mold contamination. The levels of aflatoxin in the samples were low (1 to 38.4 ppb; mean, 8.61 ppb). High levels of total type-A trichothecenes were also found in the moldy corn samples (139 to 2,030 ppb; mean, 627 ppb). Immunochromatography of selected samples revealed that these samples contained T-2 toxin, HT-2 toxin, iso-neosolaniol, monoacetoxyscirpenol, and several other type-A trichothecenes. The concentration of total type-B trichothecenes in 15 moldy corn samples was in the range of 470 to 5,826 ppb (mean, 2,359 ppb). High levels (3.7 to 5.0 mg/g) of FB1 were produced in corn in the laboratory by five Fusarium moniliforme strains isolated from the moldy corn. These fungi were also capable of forming various nitrosamines (5 to 16 micrograms per flask) in the presence of nitrate and precursor amines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Natural Occurrence of 16 Fusarium Toxins in Grains and Feedstuffs of Plant Origin from Germany

A total of 220 samples comprising cereals, cereal byproducts, corn plants and corn silage as well as non-grain based feedstuffs was randomly collected during 2000 and 2001 from sources located in Germany and analysed for 16 Fusarium toxins. The trichothecenes scirpentriol (SCIRP), 15-monoacetoxyscirpenol (MAS), diacetoxyscirpenol (DAS), T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), neosolaniol (NEO), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivealenol (15-ADON), nivalenol (NIV) and fusarenon-X (FUS-X) were determined by gas chromatography/mass spectrometry. Zearalenone (ZEA) and α- and β-zearalenol (α- and β-ZOL) were analysed by high performance liquid chromatography with fluorescence and UV-detection. Detection limits ranged between 1 and 19 μg/kg. Out of 125 samples of a group consisting of wheat, oats, corn, corn byproducts, corn plants and corn silage only two wheat samples did not contain any of the toxins analysed. Based on 125 samples the incidences were at 2–11% for DAS, NEO, T-2 Triol, FUS-X, α- and β-ZOL, at 20–22% for SCIRP, MAS, T-2 tetraol and 3-ADON, at 44–74% for HT-2, T-2, 15-ADON, NIV and ZEA, and at 94% for DON. Mean levels of positive samples were between 6 and 758 μg/kg. Out of 95 samples of a group consisting of hay, lupines, peas, soya meal, rapeseed meal and other oilseed meals, 64 samples were toxin negative. DAS, T-2 triol, NEO and FUS-X were not detected in any sample. The incidences of DON and ZEA were at 14 and 23% respectively, those of the other toxins between 1–4%, mean levels of positive samples were between 5 and 95 μg/kg.     

T-2 Toxin Contamination in Grains and Selenium Concentration in Drinking Water and Grains in Kaschin–Beck Disease Endemic Areas of Qinghai Province

It has been strongly suggested that two factors are involved in the development of Kaschin?CBeck Disease (KBD), namely grains contamination with T-2 toxin and selenium deficiency. So our team undertook a survey about grains and drinking water in three rural KBD endemic villages and one non-KBD village in Qinghai Province. The level of T-2 toxin contamination in 364 grain samples was assayed using an ELISA kit. The selenium concentration in these grains and 15 drinking water samples from three KBD endemic villages were determined using the 2,3-diaminonaphthalene fluorometric assay. The results revealed that the level of T-2 toxin contamination in the samples from three KBD endemic villages was relatively high with an average level of 78.91?ng/g in wheat and 47.47?ng/g in flour. The T-2 toxin level in samples from the non-KBD village (12.23?ng/g) was significantly lower than that of local grains from the three KBD endemic villages. The average selenium content in wheat and flour from KBD areas was 0.0045 and 0.0067???g/g, respectively. The selenium concentration in local grain samples was significantly lower than that in samples from the non-KBD village (0.0604???g/g). In addition, the selenium concentration in drinking water from three KBD endemic villages was also low (0.156???g/L). These results support a potential role of T-2 toxin contamination and selenium deficiency in KBD. Compared with non-KBD endemic areas, health hazards in grains and in the environment of KBD endemic areas were observed.     

Field incidence of mycotoxins in commercial popcorn and potential environmental influences

Popcorn ear damage by insects and mycotoxin levels in kernels were monitored in several commercial popcorn fields in central Illinois over a 4-year period. Aflatoxin was rare, but fumonisin and deoxynivalenol (DON) were commonly encountered each year, and occurred at mean levels in fields up to 1.7 mg/kg (sample max. 2.77 mg/kg) and 1.9 mg/kg (sample max. 2.66 mg/kg), respectively. Neither fumonisin nor DON levels were significantly correlated with the percent of ears with visibly moldy insect-damaged kernels. Significant correlations were noted for the percent of ears with early caterpillar damage and both fumonisin and DON levels overall for some years and at specific sites in other years. Fumonisin levels were generally more highly correlated with insect damage than DON levels. Insect damaged kernels had 100- to 500-fold or greater levels of fumonisin compared to noninsect-damaged kernels, while DON levels were closer to 10- to 30-fold higher in insect damaged versus nondamaged kernels. A high percentage of DON-contaminated kernels were not insect damaged in 2007 and 2008. In some cases, differing mycotoxin levels for the same hybrid and same year planted at different locations appeared to be due to the prior crop. Higher DON levels in 2008 than other years were most likely associated with higher levels of rainfall and cooler temperatures than average during ear fill. While kernel sorters are reported to remove mycotoxin-contaminated popcorn kernels to acceptible levels, consideration of environmental factors that promote mycotoxins in popcorn should result in more effective control measures in the field.     

Occurrence of deoxynivalenol and zearalenone in brewing barley grains from Brazil

Barley (Hordeum vulgare L.) is an important cereal crop for food and represents one of the main ingredients in beer production. Considering the importance of barley and its derived products, the knowledge about the mycotoxin contamination in the barley production is essential in order to assess its safety. In this study, the levels of deoxynivalenol (DON) and zearalenone (ZEN) in brewing barley were determined using a LC-MS/MS method. A survey was conducted in 2015 to estimate the mycotoxin levels in these products (n?=?76) from four crop regions in Brazil. The results showed high levels of DON and ZEN in the analyzed samples, with contamination levels of 94 and 73.6%, respectively. The mean levels of DON and ZEN ranged from 1700 to 7500 μg/kg and from 300 to 630 μg/kg, respectively. Barley samples from regions 1 and 2 presented higher levels of ZEN and DON, respectively, and those from region 4 presented lower levels of both. Co-occurrence of DON and ZEN was seen in the majority of the barley grain samples, and the mycotoxin content was above the maximum levels established by the Brazilian and European regulations.     

Fusarium toxins in cereals grown in Switzerland

770 cereal samples of Swiss origin which were collected in various feed mills and cereal collection centres in the years 2000 – 2002 were assayed for Deoxynivalenol (DON) and zearalenone (ZEA). 137 samples were also assayed for T-2 toxin. The prevalence of DON and ZEA contamination was higher in cereals harvested in the rainy summer 2002 than in the previous years. T-2 toxin levels exceeding 100 μg/kg were found only in three oats samples. High levels ofFusarium toxins do not frequently occur in Swiss cereals. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Detection and quantitation of T-2 mycotoxin with a simplified protein synthesis inhibition assay.

We describe a simple, rapid, and sensitive bioassay for the detection and quantitation of T-2 mycotoxin by using a protein synthesis assay in cultured cells. Increased sensitivity of the cells to the mycotoxin occurred with time up to ca. 60-min. Time and dose response curves show that an average of 10 to 20 ng of T-2 per ml was sufficient to cause 50% inhibition of protein synthesis in tissue culture cells. A wide range of tissue culture cells with varied type, tissue, and species sources and growth characteristics were tested by this system. All showed approximately the same sensitivity to the mycotoxin. A slight modification of the procedure was used for suspended cultures of mitogen-stimulated lymphocytes, which also showed an equal degree of sensitivity to the mycotoxin. By simply changing the labeled precursor, the inhibition of RNA, DNA, and protein synthesis by T-2 mycotoxin can be compared. Although T-2 mycotoxin had little effect on RNA synthesis, DNA and protein synthesis were equally inhibited. Because of its sensitivity and its capacity to quickly assay a large number of samples, this technique has been a valuable tool in screening samples for the presence of active toxin and has been used to help establish laboratory safety standards for the inactivation of T-2 mycotoxin by chemical agents. It is presently being used in studies of mycotoxin mechanism of action and approaches toward in vivo neutralization of the toxic effects of mycotoxins.     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination.

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

Determination of trichothecenes, zearalenone and zearalenols in commercially available corn-based foods in Spain

Among the main Spanish commercially available trademarks, we have selected a total of 25 samples of corn-based foods, which have the highest consume rate, to carry out the analysis of deoxynivalenol (DON), T-2 toxin, zearalenone (ZEA) and zearalenols (ZOL). The contents of mycotoxins were determined by gas chromatography with flame ionization detection, and those of ZEA were confirmed by HPLC with fluorescence detection. Of the 25 analyzed samples, the incidence of DON, ZEA and alfa-ZOL was 68, 44 and 24%, respectively; levels detected ranged from 29-195, 34-216, and 36-71 microg/kg, respectively. T-2 toxin was only detected in one sample (<50 microg/kg). Beta-ZOL was not present in excess of the detection limit in the investigated samples. The results suggest a risk for consumers of corn products and the need to monitor the final products before consumption. This is the first report in Spain on natural contamination with these mycotoxins in corn-based foods.     

The effect of vitamin E supplementation on reduction of lymphocyte DNA damage induced by T-2 toxin and deoxynivalenol in weaned pigs

The objective of the present study was to establish the effect of deoxynivalenol (DON) and T-2 toxin on lipid peroxidation, lymphocyte DNA fragmentation and immunoglobulin production in weaned pigs, and furthermore, to evaluate the potential of vitamin E (α-tocopheryl acetate) in prevention of toxin mediated changes. Forty-eight weaned castrated male crossbred pigs (mean live weight at the beginning of the experimental period was 11.7 kg) were randomly assigned to five experimental groups: control (without toxin and vitamin E), T-2 (3 mg/kg T-2 toxin), T-2 + E (3 mg/kg T-2 toxin + 100 mg/kg vitamin E), DON (4 mg/kg DON) and DON + E (4 mg/kg DON + 100 mg/kg vitamin E). After 14 days of treatment blood was collected for analysis. Lipid peroxidation was studied by assays of malondialdehyde (MDA), total antioxidant status (TAS) of plasma and erythrocyte glutathione peroxidase (GPx). DNA damage in lymphocytes was measured by comet assay. Serum immunoglobulin levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and the hepatotoxicity was studied by measuring plasma liver enzyme levels (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl-transferase (GGT). Production parameters of both DON groups were significantly impaired in comparison to the control. DON significantly increased the amount of DNA damage in lymphocytes by 28%. Moreover, the levels of TAS were lowered by addition of DON. T-2 toxin significantly impaired daily live weight gain and feed conversion, increased the amount of DNA damage in lymphocytes by 27%, decreased total serum IgG and did not alter plasma TAS. Plasma and 24-h urinary malondialdehyde (MDA) excretion rate and erythrocyte Gpx levels did not differ among the groups. Supplementation with vitamin E did not improve production parameters impaired by DON and T-2 toxin and only partially protected lymphocyte DNA from toxin impact. To our knowledge, these are the first data on genotoxic effects of moderate doses of DON and T-2 toxin on pig lymphocytes. The effect of DON and T-2 toxin on the immune system was reflected as a change in immunoglobulin synthesis, which might be toxin and species specific. According to other results no major induction of oxidative stress could be proven. Enhancement of antioxidant status with vitamin E in the case of DON and T-2 toxin intoxication can be beneficial for remaining the lymphocyte DNA integrity.     

Occurrence of trichothecenes, zearalenone and ochratoxin A in cereals and mixed feed from central Lithuania

A total of 92 samples — 23 winter wheat, 12 summer barley, 5 oats and 52 mixed feed — were collected from a state factory in Kaunas, Lithuania and were analysed for the presence of trichothecenes, zearalenone (ZEN) and ochratoxin A (OA) using gas chromatography with electron capture detection and immunoaffinity column/high performance liquid chromatography with fluorescence and UV detections. Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin and HT-2 toxin were detected at concentrations above 10 μg/kg in 68%, 48%, 38% and 8% of cereal samples, respectively, and in 98%, 88%, 12% and 8% of samples of mixed feed for swine and poultry. More than 10 μg/kg of zearalenone and ochratoxin A were found in 58% and 92% of the mixed feed samples, respectively. The highest concentrations of all analysed trichothecenes in Lithuanian mixed feed and cereal grains, with an exception of T-2 toxin in one oat lot and one sample of mixed feed and OA in two mixed feed samples, were lower than those reported as Lithuanian advisory or tolerance limits.     

Production and characterization of a generic antibody against group A trichothecenes

An antibody against group A trichothecenes was produced after immunization of rabbits with an immunogen prepared by conjugation of T-2 toxin to bovine albumin at the C-8 position. T-2 toxin was first converted to 3-acetylneosolaniol (3-Ac-NEOS) and then to its hemisuccinate (HS) before conjugation to the protein. The rabbits showed a quick immune response after immunization of the new conjugate. The antibody produced bound with tritiated T-2 toxin, T-2 tetraol tetraacetate, and diacetoxyscirpenol (DAS) and showed good cross-reactivities with most of the group A trichothecenes. The concentrations causing 50% inhibition of binding of 3H-T-2 toxin to the new antibody by unlabeled T-2, acetyl-T-2, 3'-OH-T-2, DAS, 3-Ac-NEOS-HS, 3'-OH-Ac-T-2, T-2 tetraol tetraacetate, iso-T-2, 3-Ac-NEOS, Ac-DAS, and 3,4,15-triacetyl-7-deoxynivalenol were found to be 0.34, 0.34, 0.6, 2.5, 4, 10, 18, 24, 100, 200, and 300 ng/assay, respectively; for HT-2, T-2 triol, and T-2 tetraol, the concentration was greater than 1000 ng/assay. Nivalenol, deoxynivalenol (DON), 15-acetyl-DON, and triacetyl-DON, did not inhibit the binding at 1000 ng/assay. The practical application of using this new antibody for radioimmunoassay (RIA) of trichothecene was tested by spiking T-2 toxin to corn. T-2 toxin was then extracted with acetone, subjected to a simple Sep-Pak C-18 reversed-phase treatment, and analyzed by RIA. The overall recovery for 18 samples spiked with 10 to 50 ppb of T-2 toxin was 94.22%.     

Natural multi-occurrence of mycotoxins in rice from Niger State,Nigeria

Twenty-one rice samples from field (ten), store (six) and market (five) from the traditional rice-growing areas of Niger State, Nigeria were analysed for aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisin B₁ (FB₁) and B₂ (FB₂), and patulin (PAT) by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) respectively. T-2 toxin was determined using TLC only. AFs were detected in all samples, at total AF concentrations of 28–372 μg/kg. OTA was found in 66.7% of the samples, also at high concentrations (134–341 μg/kg) that have to be considered as critical levels in aspects of nephrotoxicity. ZEA (53.4%), DON (23.8), FB₁ (14.3%) and FB₂ (4.8%) were also found in rice, although at relatively low levels. T-2 toxin was qualitatively detected by TLC in only one sample. Co-contamination with AFs, OTA, and ZEA was very common, and up to five mycotoxins were detected in a single sample. The high AF and OTA levels as found in rice in this study are regarded as unsafe, and multi-occurrences of mycotoxins in the rice samples with possible additive or synergistic toxic effects in consumers raise concern with respect to public health.