Production and characterization of a monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol

A monoclonal antibody was obtained by the fusion of mouse myeloma cells with splenocytes isolated from Balb/c mice, which had been immunized with diacetoxyscirpenol-hemiglutarate (DAS-hemiglutarate) and verrucarol-hemiglutarates covalently bound to ethylenediamine-modified bovine serum albumin. The anti-DAS-antibody that could be induced was of the IgM type with kappa-chains. The titer of the monoclonal anti-DAS-antibody in ascites fluid obtained from mice injected the selected cell line was much higher than those of conventional antisera. An enzyme-linked immunosorbent assay based on the competitive binding principle in which the antibody was applied had a sensitivity of 1 ng DAS per assay. The relative cross-reactivity of the monoclonal antibody in the CI-ELISA with the related trichothecenes such as triacetoxyscirpenol, 15-monoacetoxyscirpenol, diacetylverrucarol, 4-monoacetoxyscirpenol and scirpentriol were found to be 1.8, 0.8, 0.15, 0.02 and less than 0.001, respectively. The trichothecenes verrucarol, T-2 toxin, T-2 tetraol, deoxynivalenol, 3-acetyldeoxynivalenol and trichothecin showed no cross-reactivity.     

A yeast bioassay for trichothecenes

Like all eucaryotic cells, yeasts are sensitive to trichothecenes, especially T-2 toxin and verrucarin A. Based on this sensitivity, a yeast bioassay was developed to evaluate the toxicity of corn samples. The bioassay was optimized using spiked maize extracts. The toxicity of samples was defined as toxicity equivalent to a certain concentration of T-2 toxin standards. The assay can be performed on crude extracts, but the results are more precise after column clean-up. The test can also be used for the screening of trichothecene toxicity in general. The relative standard deviation (RSD) at 85 % growth inhibition (EC85) was 4.5% for the T-2 toxin standards (n = 8). This corresponds to an initial T-2 toxin concentration of approximately 58 ppb in the corn sample. Samples containing 188 and 113 ppb T-2 toxin caused a growth inhibition higher than 85%, whereas samples with toxin concentrations of 56 and 19 ppb had a growth inhibition less than 85%. Therefore the test can be used for the qualitative evaluation of corn samples up to a level of 58 ppb +/- 2.8 ppb. The bioassay is easy to perform with minimum requirements for equipment. Results can be obtained within 24 h and a large number of samples can be analysed daily. The costs are low and the results obtained are repeatable. With some modifications this test can be used for toxicity studies on trichothecene metabolites as well as for extracts with unknown compounds with properties similar to trichothecenes.     

Trichothecene-induced cytotoxicity on human cell lines

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC₅₀). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100–1,000, and the corresponding IC₅₀ values were in the range from 2.2 nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900 nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC₅₀ values were 4.4–10.8 nmol/l for T-2 toxin, 7.5–55.8 mol/l for HT-2 toxin, 600–4,900 nmol/l for DON, 300–2,600 nmol/l for NIV, and 2.2–18.3 nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC₅₀ values ranging from 16.5 nmol/l (T-2 toxin) to 4,500 nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.     

Effects of T-2 toxin and its congeners on membrane functions of cultured human fibroblasts

Cytotoxicity of T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, and T-2 tetraol was compared between normal human fibroblasts and mutant I-cell human fibroblasts, which only produce 10 to 15% of lysosomal hydrolases present in normal fibroblasts. Both cleavage of 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and cell count by hemocytometer were used for evaluations. For all toxins, dose-related effects on both types of cultures were evident. Cytotoxicity of the above mycotoxins on both cell lines were similar, indicating that lysosomal enzymes were not involved in the toxicity of T-2 toxin and its congeners. An inhibitor of lysosomal cysteine proteases (E-64) did not alter the cytotoxicity of T-2 toxin. The decreasing order of toxicity was T-2 toxin, HT-2 toxin, neosolaniol, acetyl T-2 toxin, and T-2 tetraol in both cell lines. When normal human fibroblasts were loaded with the fluorescent dye Lucifer yellow CH (LY), a subsequent treatment of T-2 toxin did not disrupt lysosomal membranes. The uptake of LY was not affected by T-2 toxin, which indicated that T-2 toxin did not interfere with the endocytic pathway. Results indicate that T-2 toxin and its congeners do not exert their primary toxic effect through lysosomal enzymes, membranes, or via the endocytic pathway.     

Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay

A colorimetric MTT (tetrazolium salt) cleavage test was used to evaluate cytotoxicity of twenty-three Fusarium mycotoxins on two cultured human cell lines (K-562 and MIN-GL1) as well as their inhibitory effect on proliferation of phytohemagglutinin-stimulated human peripheral blood lymphocytes. The values of 50% inhibition of lymphocyte blastogenesis were very close to the 50% cytotoxic doses observed with the more sensitive cell line (MIN-GL1). T-2 toxin was the most cytotoxic with CD₅₀ and ID₅₀ values less than 1 ng/ml. Type A trichothecenes were the most cytotoxic followed by the type B trichothecenes; the non-trichothecenes were the least cytotoxic. The MTT cleavage test, in conjunction with cell culture, is a simple and rapid bioassay to evaluate cytotoxicity and immunotoxicity of Fusarium mycotoxins.Abbreviations Ac acetyl - ACU acuminatin - DAS diacetoxyscirpenol - DON deoxynivalenol - FUS fusarenon-X - HT-2 HT-2 toxin - MC mononuclear cell - MTT 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide - NEO neosolaniol - NIV nivalenol - NT-1 4,8-diacetoxy T-2 tetraol - PBS phosphate buffered saline - TAT-2T tetraacetoxy T-2 tetraol - T-2 T-2 toxin     

Metabolic effects of trichothecene T-2 toxin

Cereals and other agricultural products contaminated with trichothecene mycotoxins are unfit for consumption. Until recently, the metabolic effects of T-2 toxin (T-2) were thought to reside in its ability to inhibit protein synthesis. It is now clear that trichothecenes have multiple effects, including inhibition of DNA, RNA, and protein synthesis in several cellular systems, inhibition of in vitro protein synthesis, inhibition of mitochondrial functions, effects on cell division, normal cell shape, and hemolysis of erythrocytes. It is argued that these effects are pleiotropic responses of the cell's biosynthetic network to protein synthesis inhibition. However, in studies with erythrocytes, which lack nuclei and protein synthesis, changes in cell shape and lytic response towards T-2 are observed. Susceptibility to lysis is species dependent and correlates with the presence of phosphatidylcholine. Owing to their amphipathic nature, T-2 and other trichothecenes could exert their cytotoxicity by acting on cell membranes. As for cell energetics, T-2 inhibits the mitochondrial electron transport system, with succinic dehydrogenase as one site of action. Although initial investigations of the metabolic effects of T-2 mediated cytotoxicity suggested the inhibition of protein synthesis as the principal site of action, current thought suggests that the effects of trichothecenes are much more diverse.     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides.

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Crosstalk of JNK1-STAT3 is critical for RAW264.7 cell survival

T-2 toxin, a major compound of trichothecenes, inhibits protein synthesis and induces inflammation and cell apoptosis through the activation of MAPK pathway. The JAK/STAT pathway has recently been shown to be downstream targets of trichothecenes. However, whether there is any crosstalk between JNK and JAK/STAT pathways in trichothecene toxicity has not been studied. In the present study, we explored this potential in RAW264.7 cells treated with T-2 toxin. Our results revealed a crosstalk between JNK1 and STAT3 after T-2 toxin treatment, which was mediated by K-Ras. T-2 toxin treatment resulted in rapid phosphorylation, and more importantly, JNK1-STAT3 signaling pathway was shown to maintain the normal function of the mitochondria and to inhibit T-2 toxin-induced apoptosis. Therefore, this pathway was considered to be a potential cell survival pathway. Breakdown and degranulation of ribosomes in the rough endoplasmic reticulum and swelling of mitochondria were clearly visible after the cells had been incubated with T-2 toxin for 12 h. Our data suggest that T-2 toxin had a Janus face: it induced both apoptotic and cell survival pathways. These results suggest that the crosstalk and the balance between MAPK and JAK/STAT pathway might be involved in T-2 toxin-induced apoptosis in RAW264.7 cells.     

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.     

Identification of various T-2 toxin metabolites in chicken excreta and tissues

Gas chromatography-mass spectrometry was used to identify various T-2 toxin metabolites in chicken excreta and organs 18 h after intraperitoneal injection of the toxin. No trichothecenes were detected in the heart and kidneys, and only trace amounts were detected in the lungs. Most of the T-2 metabolites were found in the excreta, although considerable amounts were also found in the liver. In addition to the previously identified T-2 metabolites in chicken excreta (HT-2 toxin, 15 acetoxy T-2 tetraol, and T-2 tetraol), we found 3'-hydroxy HT-2 toxin (the major metabolite in excreta and organs), 3'-hydroxy T-2 toxin, 4-acetoxy T-2 tetraol, and trace amounts of 8-acetoxy T-2 tetraol, 3-acetoxy-3'hydroxy HT-2 toxin, and T-2 triol. Unmetabolized T-2 toxin and an unidentified isomer of T-2 tetraol monoacetate were also detected in the excreta. Most of the metabolites in the chicken are similar to those encountered in cultures of fungal species producing T-2 toxin. A comparison with T-2 toxin metabolism in the cow is also reported.     

Identification of various T-2 toxin metabolites in chicken excreta and tissues.

Gas chromatography-mass spectrometry was used to identify various T-2 toxin metabolites in chicken excreta and organs 18 h after intraperitoneal injection of the toxin. No trichothecenes were detected in the heart and kidneys, and only trace amounts were detected in the lungs. Most of the T-2 metabolites were found in the excreta, although considerable amounts were also found in the liver. In addition to the previously identified T-2 metabolites in chicken excreta (HT-2 toxin, 15 acetoxy T-2 tetraol, and T-2 tetraol), we found 3'-hydroxy HT-2 toxin (the major metabolite in excreta and organs), 3'-hydroxy T-2 toxin, 4-acetoxy T-2 tetraol, and trace amounts of 8-acetoxy T-2 tetraol, 3-acetoxy-3'hydroxy HT-2 toxin, and T-2 triol. Unmetabolized T-2 toxin and an unidentified isomer of T-2 tetraol monoacetate were also detected in the excreta. Most of the metabolites in the chicken are similar to those encountered in cultures of fungal species producing T-2 toxin. A comparison with T-2 toxin metabolism in the cow is also reported.     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil.

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

Immunochemical analysis of trichothecenes produced by various fusaria

The production of type A trichothecene mycotoxins by 19 Fusaria, including 12Fusarium sporotrichioides, 4F. chlamydosporum and 3F. graminearum at 15°C and 25°C over a 35-day period was analyzed by ELISA using antibodies cross-reactive with most type A trichothecenes after conversion to T-2 tetraol tetraacetate. The toxin production peaked at 20–25 days of incubation with maximum yield between 4–6 mg type A trichothecene/ml of culture medium for 5F. sporotrichioides cultures and between 1 to 2 mg/ml for 6F. sporotrichioides cultures. OneF. sporotrichioides produced 700 µg type A trichothecenes/ml of culture medium. Detectable type A trichothecene was also found in the culture extracts ofF. chlamydosporum andF. graminearum, but the yield was very low (less than 100 µg/ml). Quantitative determination of individual trichothecenes was achieved by separation of different toxin in HPLC and followed by ELISA analysis. Eight to 10 immunoreactive peaks, corresponding to various type A trichothecenes, were detected in all the fungal extracts. T-2 tetraol (T-2-4ol), 4-acetyl-T-2 tetraol (4-Ac-T-2-4ol), neosolaniol (NEOS), diacetoxyscirpenol (DAS), HT-2 and T-2 toxin accounted for more than 85% of the total toxins. In general, low temperature was preferred for total type A trichothecene production. More T-2-4ol, 4-Ac-T-2-4ol, HT-2 and DAS were produced at 25°C. In contrast, more T-2 toxin and NEOS were produced at 15°C. Transformation of T-2 toxin and NEOS to polar metabolites such as T-2-4ol, 4-acetyl-T-2-4ol and HT-2 by various strains were observed at both temperatures after 25 days incubation.     

Trichothecene Production in Liquid Stationary Cultures of Fusarium tricinctum NRRL 3299 (Synonym: F. sporotrichioides): Comparison of Quantitative Brine Shrimp Assay with Physicochemical Analysis

Stationary liquid cultures of Fusarium tricinctum NRRL 3299 (synonym: F. sporotrichioides) produce T-2 toxin, neosolaniol, diacetoxyscirpenol, and HT-2 toxin when cultured on peptone-enriched Czapek Dox medium. At 15 and 27°C, maximum T-2 toxin yield (265 and 50 μg/ml) was found after 10 to 14 and 7 days, respectively. The T-2 toxin in the culture medium was metabolized rapidly at 27°C and slowly at 15°C. Addition of 0.025% (wt/vol) sorbic acid to the medium resulted in an increased production of trichothecenes at 15°C (400 μg of T-2 per ml after 14 days). Trichothecenes in the culture liquid were determined by the brine shrimp bioassay and physicochemical analysis. The brine shrimp assay was improved by using modern bioassay equipment, including tissue culture trays and multipipettes, and by a standardized approach with positive and negative controls. The physicochemical analysis was based on adsorption of the trichothecenes onto Amberlite XAD-2 columns, derivatization with trifluoroacetic anhydride followed by capillary gas chromatography, and identification by mass spectrometry (as many as 17 trichothecenes were detected in the culture medium). The brine shrimp assay offers an interesting monitoring system for the quantitation of T-2 toxin and should be useful for studies on production of this toxin in culture. Specific information on less toxic trichothecenes, however, requires a more time-consuming chemical analysis.     

Large-scale production of selected type A trichothecenes: the use of HT-2 toxin and T-2 triol as precursors for the synthesis of <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-T-2 and <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-HT-2 toxin

The type A trichothecenes T-2 and HT-2 toxins are toxic secondary metabolites produced by fungi of the Fusarium genus. Their occurrence in cereals, especially in oats, implies health risks for the consumer. Therefore, it is an important task to develop selective and sensitive methods for the analysis of T-2 and HT-2 toxins, and to undertake further studies on their stability and toxicity. Although most toxins are commercially available, their high prices are the limiting factor on the realization of these experiments. Thus, we developed a method for large-scale production of T-2 and HT-2 toxin as well as T-2 triol and T-2 tetraol. T-2 toxin was obtained in gram quantities by biosynthetic production with cultures of F. sporotrichioides. As HT-2 toxin was only formed as a by-product, and T-2 triol and T-2 tetraol were not generated, these compounds were produced by alkaline hydrolysis of T-2 toxin. Separation and isolation of crude toxins was achieved by fast centrifugal partition chromatography (FCPC), which is an efficient tool for the large-scale purification of natural products. Using this fast and yield effective technique, several hundred milligrams of HT-2 toxin, T-2 triol, and T-2 tetraol were obtained. Subsequent, HT-2 toxin and T-2 triol were used for the large-scale synthesis of isotope-labeled T-2 and HT-2 toxin, respectively. Using these standards, an isotope dilution-(ID)-HPLC-MS/MS method for the quantification of T-2 and HT-2 toxin in different matrices was developed.     

Production and characterization of antibody against deoxyverrucarol.

Immunization of rabbits with deoxyverrucarol (DOVE) conjugated to bovine serum albumin resulted in antibodies bound with either tritiated DOVE or diacetoxyscirpenol (DAS), but not with T-2 toxin. The affinity of antibodies with DOVE was found to be much higher than with DAS. When [3H] DOVE was used as a marking ligand in the competitive radioimmunoassay, the concentrations causing 50% inhibition of binding radioactivities by unlabeled DOVE, verrucarol, verrucarin A, and 4-monoacetoxyscirpenol were found to be 0.32, 1,070, 9,500, and 10,000 ng per assay, respectively. T-2 toxin, 15-monoacetoxyscirpenol, and deoxynivalenol gave less than 20% inhibition at 10 micrograms per assay. However, when [3H] DAS was used as the marking ligand, the concentrations causing 50% inhibition by DOVE, DAS, and verrucarol were found to be in the 50 to 60 ng per assay range. The antibodies are thus highly specific to DOVE rather than a common trichothecene backbone. The possible use of this antiserum for assay of macrocyclic trichothecenes is discussed.     

Screening fusarium strains isolated from overwintered Canadian grains for trichothecenes

A survey of 38 samples of Canadian overwintered grains showed that 14 (37 %) contained viableFusarium. Of a total of 38Fusarium isolates, cultured on autoclaved corn, 20 (from 7 grain samples) showed toxicity to brine shrimp larvae and 12 (from 5 samples) produced levels of trichothecenes detectable by thin layer chromatography. The principal trichothecene found was T-2 toxin, produced by 10 strains and accompanied in half of these by neosolaniol; some of these strains were identified asF. sporotrichioides Sherbakoff. Two strains ofF. poae (Peck) Wollenw. formed small amounts of diacetoxyscirpenol. T-2 toxin was the most toxic of 8 trichothecenes tested on brine shrimp larvae; the wide range of toxicities limits the usefulness of this bioassay as a general screening method for trichothecenes.