Method for detecting production of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol by Fusarium isolates

Three methods for detecting toxigenic fusaria in culture were compared by using known producers of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol. Moist, autoclaved rice cultures of known toxigenic isolates grown in 20-ml tubes yielded oily extracts containing compounds which interfered with qualitative and quantitative analysis for the mycotoxins. Vermiculite moistened with nutrient broth in 20-ml tubes yielded a much cleaner extract. Growing the fungi on a liquid medium required a shorter incubation period, but yields of T-2 toxin and deoxynivalenol were low and variable, and the method required greater space in the incubator. Screening of the extracts by thin-layer chromatography with colorimetric spray reagents to detect the presence of these toxins permitted reduction in the number of extracts quantified by the more lengthy gas-liquid chromatographic method. Culturing in nutrient broth on vermiculite in tubes coupled to a qualitative screen before quantitation proved to be a convenient, inexpensive, and relatively rapid method that enabled reliable screening of a large number of Fusarium isolates for toxin production as compared with prior methods.     

Method for detecting production of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol by Fusarium isolates.

Three methods for detecting toxigenic fusaria in culture were compared by using known producers of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol. Moist, autoclaved rice cultures of known toxigenic isolates grown in 20-ml tubes yielded oily extracts containing compounds which interfered with qualitative and quantitative analysis for the mycotoxins. Vermiculite moistened with nutrient broth in 20-ml tubes yielded a much cleaner extract. Growing the fungi on a liquid medium required a shorter incubation period, but yields of T-2 toxin and deoxynivalenol were low and variable, and the method required greater space in the incubator. Screening of the extracts by thin-layer chromatography with colorimetric spray reagents to detect the presence of these toxins permitted reduction in the number of extracts quantified by the more lengthy gas-liquid chromatographic method. Culturing in nutrient broth on vermiculite in tubes coupled to a qualitative screen before quantitation proved to be a convenient, inexpensive, and relatively rapid method that enabled reliable screening of a large number of Fusarium isolates for toxin production as compared with prior methods.     

A yeast bioassay for T-2 toxin

A disk diffusion type bioassay was developed for T-2 toxin using the yeast Kluyveromyces fragilis. The lower limit of detection for this in 0.2 μg of T-2 toxin. The growth of this yeast was sensitive to other trichothecenes such as verrucarin A (0.01 μg). Aflatoxin B₁ (50 μg) and zearalanone (20 μg) did not inhibit the growth of this yeast.     

Immunoenzyme assay for detection of T-2 toxin in contaminated grain]

Based on indirect solid-phase competitive enzyme immunoassay, a method for determination of T-2 toxin in grain was designed. Determination errors were measured on samples of contaminated grain. The method makes allows determination of the toxin levels ranging from 30 to 1000 ng/g.     

Immunochromatographic assay of T-2 toxin using labeled anti-species antibodies

A new scheme of immunochromatographic assay was developed for the highly sensitive detection of low-molecular-weight analytes. This scheme includes the following two steps: the formation of complexes of free specific antibodies with an antigen and their detection by anti-species antibodies conjugated to gold nanoparticles as the label. This scheme was tested with mycotoxin T-2 toxin in maize extracts. The use of specific antibodies and a label as two individual components made it possible to independently vary their concentrations with a simultaneous decrease in the detection limit and an increase in the color intensity. The assay did not require additional reagents and manipulations. The instrumental and visual detection limits of the designed test system were 0.1 and 5.0 ng/mL, respectively (2 and 90 ng per gram of analytes), which are two orders of magnitude lower compared to conventional immunochromatography using the same reagents.     

Monoclonal antibody-based enzyme-linked immunosorbent assay of Fusarium T-2 and zearalenone toxins in cereals.

Direct, competitive enzyme-linked immunosorbent assays (ELISAs) with monoclonal antibodies have been developed for quantitative determination of trichothecene T-2 toxin (T-2), and zearalenone (F-2) from different cereals: Among the several extraction solvents tried, 89% acetonitrile with additives was chosen. The extracts were then used without cleanup in the ELISA. With appropriate dilution of the samples (1:25 or 1:50), the matrix effects caused by lipid and/or protein content of the samples can be diminished to the extent that the assay is no longer impaired. The mean recoveries from cereals infected with 100 to 2,000 ng of T-2 and 50 to 500 ng of F-2 per g were 85 and 91%, respectively. The measuring range of the T-2 test is 100 to 2,000 ng/g, and that of the F-2 test is 25 to 400 ng/g. The mean within-assay and interassay coefficients of variation of standard curves are both less than 10%. According to recovery results with artificially infected cereals, our tests proved to be suitable for rapid screening of food and feed samples for the presence of T-2 and F-2 toxins.     

A monoclonal antibody to T-2 toxin

A specific, high affinity monoclonal antibody to T-2 toxin of Fusarium has been produced. The monoclonal antibody was conjugated to horse-radish peroxidase and employed to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the toxin. The sensitivity of the ELISA was 10 ng/ml with a working range up to 500 ng/ml. The antibody cross-reacted with HT-2 toxin (25%) but did not bind to any other trichothecene tested.     

Repetitive hit-and-run fluoroimmunoassay for T-2 toxin

A monoclonal antibody for T-2 toxin is converted to a Fab'-fluorescein derivative. The latter is specifically complexed onto a T-2 agarose gel. Fifteen successive doses of T-2 ranging from 1 to 50 ng are then repetitively and linearly detected using a column packed with a small volume (0.2 ml) of this gel without recharging with Fab'-fluorescein. For these assays the effluent from the column is monitored with a spectrofluorometer.     

小麦中T—2毒素的污染调查

采用酶联免疫吸附测定法(ELlSA),调查了我国部分地区1990年国产小麦T—2毒素的污染状况。调查结果表明,在330份样品中,有264份检出T—2毒素,占样品总数的80％。样品中毒素含量最高达1122．Oppb,平均含量为53．3ppb。在引起食物中毒的48份小麦样品中,检出高含量的T—2毒素(84．5—1064．4Ppb)。我国小麦中T—2毒素的污染水平与小麦赤霉病的流行密切相关。     

Production of zearalenone,T-2 toxin,and deoxynivalenol by Fusarium spp. isolated from plant materials grown in North Carolina

Fusarium spp. isolated from plant materials grown in the hot, humid climate of North Carolina were tested for production of mycotoxins. Isolates of F. acuminatum, F. graminearum, F. moniliforme, F. oxysporum, and F. solani produced zearalenone while isolates of F. equiseti and F. graminearum produced T-2 toxin and deoxynivalenol, respectively. This is the first report of zearalenone production by F. solani. The toxins were identified by capillary gas chromatography-mass spectrometry. These findings suggest that there are toxigenic strains of Fusarium indigenous to the warmer regions of the USA and that fasariotoxicoses of animals in this region are not necessarily the result of importing toxic grains from the cooler, upper midwestern USA.Paper No. 8953 of the Journal Series of North Carolina Agricultural Research Service, Raleigh, North Carolina. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the product named nor criticism of similar ones not mentioned.     

Comparison of in vitro cytotoxicity of Fusarium mycotoxins,deoxynivalenol, T-2 toxin and zearalenone on selected human epithelial cell lines

Three human epithelial cell lines (CaCo-2, HEp-2 and HeLa) implicated as potential targets for three Fusarium toxins were tested for the extent of survival on exposure to increasing toxin concentration and incubation periods. Cytotoxicity assay using 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was carried out with deoxynivalenol (DON), T-2 toxins and zearalenone (ZON) on CaCo-2, HEp-2 and HeLa cell lines. Of the three cell lines used, HeLa was the most sensitive, eliciting cell death after 2 days exposure at 100 ng ml^–1with T-2 toxin. HeLa was the only cell line to exhibit cytotoxicity towards ZON showing cell death at 1000 ng ml^–1after 2 days which increased to 4 days, showing substantial cell death at 200 ng ml^–1. HEp-2 was sensitive to DON showing cell death after 2 days (100 ng ml^–1) with complete cell death occurring at 200 ng ml^–1 after 4 days of exposure. Substantial cytoxicity of T-2 towards HEp-2 occurred after 2 days at 1000 ng ml^–1 and complete cell death occurred with 100 ng ml^–1 at day 4. The CaCo-2 cell line was generally resistant to the mycotoxins tested between 100 and 1000 ng ml^–1. This study shows that cytotoxicity of Fusarium toxins to epithelium cell lines is concentration- and time- dependant and results from ZON–HeLa interaction indicate possible cell type-mycotoxin specificity.     

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.     

New process for T-2 toxin production.

Strains of Fusarium produced high levels of T-2 toxin when cultured on certain media absorbed into vermiculite. Modified Gregory medium was nutritionally complex (2% soya meal, 0.5% corn steep liquor, 10% glucose) and, when inoculated with the appropriate fungal strain, yielded maximum T-2 toxin within 24 days of incubation at 19 degrees C. On Vogel synthetic medium N (H. J. Vogel, Microb. Genet, Bull. 13:42-43, 1956) supplemented with 5% glucose, optimal toxin levels were synthesized after incubation for 12 to 14 days at 15 degrees C. Fusarium tricinctum T-340 produced 714 and 353 mg/liter on modified Gregory medium and Vogel synthetic medium N plus 5% glucose, respectively. Improved analytical procedures were developed and involved aqueous methanol extraction, purification by liquid-liquid partitions, and gas-chromatographic quantitation.     

Cross-reactivity of antibodies against T-2 with deepoxide T-2 toxin

Two types of antibodies raised against T-2 toxin, namely anti-T-2-HS-BSA and anti-3 -Ac -NEOS-HS -BSA, showed good cross-reactivity with deepoxy T-2 toxin. Our results indicate that the epoxide is not an important epitope for the production of antibody against T-2 toxin     

T-2 toxin degradation by micromycetes

The biodegradation of T-2 toxin was studied by strains of micromycetes which were isolated from the environment. The 26 tested strains were divided into three groups. Group contains strains which degraded T-2 toxin very fast. This toxin could not be chromatographically determined in the medium even after 48 hours of incubation and the antifungal activity of residua against Kluyveromyces fragilis CCY-51-1-2 was low or zero. There were strains of Alternaria sp., Ulocladium sp., Aspergillus candidus, Cladosporium cladosporioides, Rhodotorula sp., Aspergillus flavus and Cladosporium macrocarpum. Group II contains with a low activity and in group III the results were variable and non stable.     

Detection of T-2 toxin in Fusarium sporotrichioides-infected corn by enzyme-linked immunosorbent assay.

A competitive enzyme-linked immunosorbent assay was used to screen for T-2 toxin in Fusarium sporotrichioides -infected corn. The assay detected T-2 toxin in diluted methanol extracts of corn samples at concentrations of 0.05 ng/ml. In infected corn samples, enzyme-linked immunosorbent assay and gas-liquid chromatography estimations of T-2 toxin concentrations were similar.     

A microbiological assay for sodium azide

A reproducible and sensitive method is presented for quantitating sodium azide (NaN₃) that exploits the fact that NaN₃ inhibits Escherichia coli RNA synthesis. A linear correlation is observed between incorporation of [³H]uridine into a trichloroacetic acid-precipitable form and NaN₃ concentration over a 31- to 2000-μg range of azide. This technique was used to determine the azide content of a complex enzyme solution where established colorimetric azide determinations proved to be unworkable. This technique when properly controlled should be applicable to a variety of similar solutions.     

Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell viability assay

The neutral red (NR) cell viability assay was used with various cell types of human origin to quantitate the potency of T-2 mycotoxin and its metabolites. The human melanoma SK-Mel/27 cell line was the most sensitive, with a midpoint cytotoxicity value of 2.8 ng of T-2 per ml. With the human hepatoma cell line, HepG2, the sequence of potency for a series of mycotoxins was T-2 greater than HT-2 greater than T-2 triol greater than T-2 tetraol.