Roquefortine C in blue-veined and soft-ripened Cheeses in the USA.

Certain fungi can produce secondary metabolites that are toxic, mycotoxins. Two groups of cheeses where fungi are used for ripening are the blue-veined cheeses (Penicillium roqueforti) and the "soft-ripened" cheeses (P. camemberti). An enzyme-linked immunosorbent assay was used to screen for the mycotoxin roquefortine C (ROQC) in 202 samples of cheeses sold in the United States. Of these 152 were blue-veined cheeses, 46 were soft-ripened cheeses and 4 were other varieties of mould-ripened cheeses. ROQC was not detected in any of the soft-ripened cheeses, at a limit of detection of 1.8 µg/kg. ROQC was found in 151 of 152 blue-veined cheeses. The maximum level found was 6,630 µg/kg (median 903 µg/kg, average of positives 1430 µg/kg, limit of quantitation 6.9 µg/kg). These levels are consistent with the levels found previously in blue-veined cheeses in the United Kingdom and Europe, which have generally been considered non-hazardous for human consumption.

Detection of cyclopiazonic acid (CPA) in maize by immunoassay.

Cyclopiazonic acid (α-CPA) is a tremorgenic mycotoxin that is commonly produced by certain species of the aspergilli, in particular Aspergillus flavus, which is more widely known for production of the aflatoxins. Despite the fact that α-CPA may co-occur with aflatoxins, immunoassay-based methods for monitoring for CPA have not been widely developed. We report the development and evaluation of several monoclonal antibodies (mAbs) for α-CPA. Two mAbs in particular were very sensitive, with IC50s of 1.1 and 1 ng/mL (clones 1418 and 1231, respectively). Tolerances to aqueous methanol or acetonitrile were good, which permitted the development of an antigen-immobilized competitive enzyme-linked immunosorbent assay (CI-ELISA) for detection of CPA in maize. Spiked or naturally contaminated maize, extracted with aqueous methanol, was diluted with buffer for analysis. The working range for the assay (IC20 to IC80) was from 5 to 28 µg/kg. Recoveries from maize spiked over the range from 2 to 50 µg/kg averaged 88.6 ± 12.6%. Twenty-eight samples of maize were tested by both the CI-ELISA and a liquid chromatography-fluorescence (LC-FLD) method. For the five samples above the limits of quantitation of both methods, CI-ELISA tended to overestimate CPA content, a difference that we speculate may be due to related metabolites or perhaps "masked" derivatives of CPA. The antibodies developed and the resulting CI-ELISA will be useful tools for further evaluation of the prevalence of this mycotoxin in maize.

Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms.

Immunoassays for deoxynivalenol (DON) that involve binding to DON-specific antibodies have been widely developed. In such assays, the responses of samples are generally compared with calibration curves generated by using DON in competition with labeled reagents such as enzymatic or fluorescent conjugates of the toxin. However, materials that mimic the toxin can also be used, provided that they compete effectively with the labeled reagents for the DON-specific antibodies. Examples include certain types of anti-idiotype antibodies, obtained by the immunization of animals with toxin-specific antibodies. In the present work, anti-idiotype antibodies were developed which mimicked DON in the ability to bind to a DON-specific monoclonal antibody (Mab). Fab fragments of the Mab (Ab1) were used to immunize rabbits. Sera were screened by competitive direct enzyme linked immunosorbent assay (CD-ELISA) for the presence of anti-idiotype antibodies (Ab2). In order to determine the most effective screening format and also the potential efficacy in various forms of biosensors, the sera were further evaluated in biolayer interferometry (BLI) and fluorescence polarization immunoassay (FPIA) formats. All three formats were used to demonstrate the presence of anti-idiotypes capable of binding to the paratope of the DON antibody (subtypes Ab2ß or Ab2γ). Such materials have the potential to replace DON as calibrants in immunoassays for this toxin.

Signal amplification using colloidal gold in a biolayer interferometry-based immunosensor for the mycotoxin deoxynivalenol.

Deoxynivalenol (DON) is a toxin produced by certain species of Fusarium fungi that can infest wheat, barley and corn. The fungi cause diseases in crops worldwide and some of the secondary metabolites, such as DON, can adversely affect animal health and food safety. To monitor DON in wheat rapidly, a biosensor using the principle of biolayer interferometry (BLI) was developed. The signal from the sensor was substantially amplified through the use of a primary antibody-colloidal gold conjugate. The amplification was much greater in the presence of wheat matrix than in buffered solution, suggesting matrix components may have contributed to the enhancement. The improved signal provided by the amplification allowed for the development of rapid qualitative and quantitative assays. The limit of detection of the method was 0.09 mg kg(-1); the limit of quantitation was 0.35 mg kg(-1). Recovery from wheat spiked over the range from 0.2 to 5 mg kg(-1) averaged 103% (RSD = 12%). The quantitative assay compared favourably (r(2) = 0.9698) with a reference chromatographic method for 40 naturally contaminated wheats. The qualitative assay was able to classify accurately the same group of 40 samples as either above or below a 0.5 mg kg(-1) threshold. These results suggest that the BLI technique can be used to measure DON in wheat rapidly.

Rapid and advanced tools for mycotoxin analysis: a review.

The problems associated with mycotoxin contamination of foods and feeds are well established and, in many cases, have been known for a long time. Consequently, the techniques for detecting known mycotoxins are quite advanced and range from methods for directly detecting the toxins themselves, based upon physical characteristics of the toxins, to methods for indirectly detecting the toxins, such as immunoassays. This review focuses on recent technologies that can be used to detect mycotoxins and, as such, is not a comprehensive review of the mycotoxin analytical literature. Rather, the intent is to survey the range of technologies from those that are instrument intensive such as modern chromatographic methods to those that require no instrumentation, such as certain immunoassays and biosensors. In particular, mass spectrometric techniques using ambient ionization offer the intriguing possibility of non-destructive sampling and detection. The potential application of one such technique, desorption electrospray ionization (DESI), is demonstrated for fumonisin B(1) on maize. While methods for detecting mycotoxins are quite advanced, the need remains for assays with increased throughput, for the exploration of novel detection technologies, and for the comprehensive validation of such technologies as they continue to be developed.

Photoreaction of indole-containing mycotoxins to fluorescent products.

Photochemical reaction of the non-fluorescent mycotoxin cyclopiazonic acid (CPA) to fluorescent products was recently reported. Because CPA contains an indole moiety, believed to contribute to the fluorescence, it was of interest to determine whether the effect might be more generally applicable to indole-containing mycotoxins. Three indole-containing tremorgens (penitrem A, paxilline, verruculogen) that have not previously been reported to be fluorescent were rendered fluorescent by exposure to ultraviolet light in a photoreactor. Naturally fluorescent ergot alkaloids, which also contain an indole-moiety, exhibited a diminished response after exposure. This suggests that the phenomenon may be most useful for detection of indole-containing tremorgens that are non-fluorescent, rather than for the enhancement of materials that are already fluorescent, such as the ergot alkaloids. The extent to which fluorescence enhancement was seen was strongly influenced by the reaction environment, in particular the solvent used and whether cyclodextrins were present. In an HPLC format, placement of the photoreactor post-column allowed for the fluorescence detection of penitrem A, paxilline, and verruculogen. The ability to photoreact indole-containing tremorgens and detect them by fluorescence may open up new avenues for detection of these mycotoxins alone or in combination.

Use of cyclodextrins as modifiers of fluorescence in the detection of mycotoxins.

Cyclodextrins, cyclic oligosaccharides composed of amylose subunits, are known to interact with mycotoxins. The interactions may be useful to analytical chemists by altering the properties of the mycotoxin of interest, namely the chromatographic properties, electrophoretic properties, fluorescence, or absorption of these fungal metabolites. Practical applications of these effects have been the incorporation of cyclodextrins into high-performance liquid chromatography and capillary electrophoresis methods for mycotoxin detection. Specific mycotoxins include those with a native fluorescence such as the aflatoxins, ochratoxin A (OTA) and zearalenone (ZEN) as well as those that can be rendered fluorescent through derivatization, such as T-2 toxin. The literature describing the applications of cyclodextrins in mycotoxin analysis is reviewed and an attempt to extend the use of cyclodextrins to the detection of labelled T-2 toxin is presented. Twenty cyclodextrins were evaluated for their ability to enhance the fluorescence emission of T-2 toxin derivatized with pyrene-1-carbonyl cyanide (T2-Pyr). This evaluation revealed that heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DIMEB), in particular, enhanced T2-Pyr fluorescence. DIMEB was used as a buffer modifier in a capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for detecting T-2 in maize. Because of the effects that certain cyclodextrins have, especially under aqueous conditions, they may make useful additives for a variety of mycotoxin analytical methods.

Development of monoclonal antibodies for the fusarin mycotoxins.

The fusarins are a group of mycotoxins produced by fungi that commonly infest cereal crops, in particular by the fungus Fusarium verticillioides. This group of compounds is characterized by a substituted 2-pyrrolidone ring attached to a 12-carbon polyunsaturated backbone. Several of the fusarins contain an epoxide substitution on the pyrrolidone ring and are highly mutagenic. This paper describes the development of seven monoclonal antibodies and immunoassays for detecting fusarins C and A. Fusarin C was isolated and conjugated to ovalbumin to produce the immunogen. Competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) were developed based upon the isolated monoclonal antibodies. The concentrations of fusarin C able to inhibit colour development by 50% (IC(50)) in CI-ELISAs were 1.0, 2.0, 3.6, 23.4, 28.9, 31.4, and 66.7 ng ml(-1) for clones 1-38, 1-30, 1-5, 1-7, 1-43, 1-25, and 1-21, respectively. Cross-reactivity with fusarin A was 44.8, 51.4, 41.1, 174.0, 62.6, 78.2, and 98.0% for clones 1-38, 1-30, 1-5, 1-7, 1-43, 1-25, and 1-21, respectively. Given the sensitivity of these antibodies for fusarins it is expected that, with further development, they may be useful for detecting fusarins at relevant levels in foods.

Synthesis and evaluation of molecularly imprinted polymers as sorbents of moniliformin.

Moniliformin is a low molecular weight mycotoxin that has worldwide potential to contaminate cereal grains. Although several traditional methods have been developed to detect moniliformin, the lack of anti-moniliformin antibodies has created a need for materials that recognize moniliformin at the molecular level through a binding mechanism. To address this issue, the authors synthesized molecularly imprinted polymers that bind moniliformin. Imprinted and non-imprinted polymers were evaluated by equilibrium binding assays and moniliformin concentrations were measured by LC analysis using ultraviolet light detection. Successful polymers were imprinted with toxin analogues as the templates; non-imprinted polymers exhibited minimal binding in acetonitrile under the assay conditions. Selected imprinted polymers also bound moniliformin in ethanol, methanol and dimethyl formamide. Significant differences in moniliformin binding by the polymers were dependent on polymer composition, and these differences were highly dependent on the template used to imprint the polymer. Polymers were further evaluated as sorbents for molecularly imprinted solid-phase extraction (MISPE), and an imprinted polymer was used for preconcentration and clean-up of a moniliformin spiked corn extract.

Production and characterization of a monoclonal antibody that cross-reacts with the mycotoxins nivalenol and 4-deoxynivalenol.

Nivalenol is a mycotoxin produced by certain fungi that are pathogenic to important cereal crops, in particular maize, wheat, and barley. This toxin, 3alpha,4beta,7alpha,15-tetrahydroxy-12,13-epoxytrichothec-9-en-8-one, is found worldwide and is closely related to 4-deoxynivalenol (DON or vomitoxin), a mycotoxin associated with outbreaks of Fusarium head blight in North America. The literature on the toxicity of nivalenol suggests it is similar, if not more toxic, than DON. Despite the development of rapid immunologically based assays for detecting DON, such assays have not existed for detecting nivalenol without chemical modification of the analyte. This paper describes the development of a monoclonal antibody using a nivalenol-glycine protein conjugate. The monoclonal antibody was most specific for an acetylated form of DON (3-Ac-DON), but it exhibited sensitivity and cross-reactivity that were useful for detecting nivalenol and DON at relevant levels without the need to modify either toxin chemically. In an competitive indirect ELISA format, the concentrations of toxins able to inhibit colour development by 50% (IC50) were 1.7, 15.8, 27.5, 68.9, and 1740 ng ml(-1) for the mycotoxins 3-Ac-DON, DON, nivalenol, 15-Ac-DON, and fusarenon-X, respectively. The antibody was also used to develop a competitive direct ELISA for DON and nivalenol, with IC50's of 16.5 ng ml(-1) (DON) and 33.4 ng ml(-1) (nivalenol). These assays are capable of detecting both DON and nivalenol simultaneously, a property that may be useful in regions where these toxins co-occur or in formats, such as immunoaffinity columns, where co-isolation of both toxins is desirable.

Fluorescence polarization for mycotoxin determination.

Over the last few years several laboratories have reported fluorescence polarization (FP) immunoassays for mycotoxins. These have included assays for fumonisins, deoxynivalenol and acetylated derivatives, aflatoxins, ochratoxin A, and zearalenone and related metabolites. Sensitivity in the FP assays may change dramatically over time, depending upon the antibody/tracer combination used. An important aspect of these homogeneous assays is the time required to reach an equilibrium endpoint. Although it is counterintuitive, the sensitivity of FP assays can actually be improved with shorter incubation times. However, the need for sensitivity must often be balanced against the need for the analyst to reproducibly time the incubation. The technical acumen of the analyst would be relatively more important in assays where measurements are taken before the system reaches equilibrium. In many cases the desired assays are those which reach equilibrium (and therefore give a stable endpoint) quickly, which may occur at the expense of sensitivity. It is for this reason the FP immunoassays are frequently not as sensitive as traditional ELISAs. Nevertheless, for many of the major mycotoxins rapid FP immunoassays can be developed, provided the appropriate combinations of antibody and tracer are used.

Evaluation of Food-Grade Dent Corn Hybrids for Severity of Fusarium Ear Rot and Fumonisin Accumulation in Grain.

Fumonisins produced by Fusarium verticillioides (syn = F. moniliforme) and F. proliferatum have been associated with potentially serious toxicoses of animals and humans. Thus, hybrids with low fumonisin accumulation in grain will be valuable for the production of corn-based human food products. We evaluated 68 food-grade dent corn hybrids for severity of Fusarium ear rot and fumonisin accumulation in grain in inoculated trials in Urbana, IL in 2000 and 2001. Our inoculation technique was successful in initiating fumonisin accumulation that allowed discrimination among hybrids. We identified several hybrids that could have acceptable levels (<4 µg/g) of fumonisin accumulation in Illinois in most years. Twenty-six hybrids with low or high fumonisin accumulation in 2000 were reevaluated in noninoculated trials at three locations in Illinois in 2001. Fumonisin concentration in grain at all three locations was relatively low; thus, separation of hybrids was poor. At two locations, those hybrids with the highest fumonisin concentration in grain also had high concentrations following inoculation. However, one hybrid that had relatively low fumonisin concentration following inoculation had unacceptable levels of fumonisin (5 µg/g) in natural conditions. Therefore, hybrids need to be evaluated by inoculation and further evaluated at locations where the environment favors fumonisin accumulation.

Detection of moniliformin in maize using capillary zone electrophoresis.

Moniliformin is a mycotoxin produced by certain fungi pathogenic to maize. It is capable of causing disease in domestic animals, possibly through inhibition of pyruvate dehydrogenase. Testing for MON commonly involves extraction of maize, isolation of moniliformin using solid-phase extraction columns and detection with high-performance liquid chromatography (HPLC) or gas chromatography. A capillary zone electrophoresis-diode array detection (CZE-DAD) method for determination of moniliformin in maize is reported. The extraction and isolation procedures are similar to those of a commonly used HPLC method, while the detection step requires only 10 min. Sixty-three samples of maize were tested by an established HPLC method using absorbance at 229 nm (HPLC-ultraviolet light) and by the CZE-DAD method. The limit of detection of the CZE-DAD method was 0.1 microg MON g(-1) maize compared with 0.05 microg g(-1) for the HPLC-ultraviolet light method. The CZE-DAD method gave good agreement with the HPLC-ultraviolet light method for samples tested at levels up to 1500 microg g(-1), with a linear regression of r(2) = 0.996.

Sources of resistance to fumonisin accumulation in grain and fusarium ear and kernel rot of corn.

ABSTRACT Fumonisin is a group of homologous mycotoxins produced by several species of Fusarium. Fumonisin has been associated with Fusarium ear and kernel rot of corn (Zea mays) and several toxicoses of animals and humans. Corn inbreds with a high level of resistance to fumonisin production and accumulation in grain have not been identified. The objective of this study was to evaluate a genetically diverse collection of inbreds as potential sources of resistance to fumonisin production and accumulation in grain and Fusarium ear and kernel rot when crossed with a commercial "B73-type" line. F(1) hybrids developed with the inbred FR1064 and 1,589 and 1,030 inbreds were evaluated in inoculated and naturally infected trials, respectively, in 2000. Thirty-five F(1) hybrids with fumonisin concentration in grain of

Evaluation of Inoculation Techniques for Fusarium Ear Rot and Fumonisin Contamination of Corn.

Fumonisins have been associated with potentially serious toxicoses of animals and humans. Prior to initiating a corn (Zea mays) breeding program for resistance to these mycotoxins, an efficient inoculation technique must be developed. Four inoculation techniques were evaluated on 14 commercial corn hybrids in Urbana, IL in 1999 and 2000. The techniques were: injection of inoculum through the ear husk leaves at R2 (blister); silks sprayed with inoculum at R2 and covered with a shoot bag until harvest; silks sprayed with inoculum at R2, covered with a shoot bag, reinoculated 1 week thereafter, and covered with a shoot bag until harvest; and insertion of six Fusarium-colonized toothpicks into the silk channel at R2. Only injection of inoculum through the husk leaves significantly increased the concentration of fumonisin in grain and severity of Fusarium ear rot compared with a control. This technique effectively differentiated hybrids previously identified as resistant or susceptible to Fusarium ear rot. The rank order of hybrids inoculated with this technique did not significantly change in the 2 years of this study. This technique is suitable for efficiently evaluating a large number of corn genotypes for resistance to Fusarium ear rot and fumonisin concentration.

Fluorescence polarization as a tool for the determination of deoxynivalenol in wheat.

The mould Fusarium graminearum is found worldwide as a pathogen of cereal grains, in particular of wheat and maize, and it produces a mycotoxin known as deoxynivalenol (DON or vomitoxin). Each year, the presence of this compound and related trichothecenes causes substantial losses to agricultural productivity. Rapid methods for the measurement of the toxin in grains are required to monitor and divert effectively contaminated grain from the food supply. A fluorescence polarization (FP) immunoassay using a previously described monoclonal antibody for DON was developed. The assay was based on the competition of unlabeled DON from a sample with a fluorescently tagged DON, DON-fluorescein (DON-FL), for a DON-specific monoclonal antibody in solution. The FP of the tagged DON was increased upon binding with the antibody. In the presence of free toxin, less of the DON-FL was bound and the polarization signal was decreased. The assays were very simple to perform, requiring only mixing of an aqueous extract of wheat with the DON-FL and antibody. The sensitivity of the assay was strongly dependent upon the time between mixing of the sample with the tracer and measurement of the fluorescence polarization, with midpoints for the competition curves ranging from 0.03 microg ml(-1) with a 15-s incubation to >1 microg ml(-1) with a 12-min incubation. Samples of wheat naturally contaminated with DON were evaluated by FP and by an HPLC-UV method, with a good correlation (r2 = 0.97). Although the FP method tended to overestimate DON slightly in the wheat samples, by approxiamtely 20%, the assay was easy to use and very useful for the screening of wheat.

Fluorescence polarization as a means for determination of fumonisins in maize.

Fumonisins, mycotoxins produced by certain species of Fusaria, are commonly found worldwide as contaminants in maize. This paper reports the development of a rapid, portable fluorescence polarization-based assay for fumonisins in maize. The assay was based on the competition of unlabeled fumonisin, from a sample, with a fluorescently tagged fumonisin (FB(1)-FL) for a fumonisin-specific monoclonal antibody in solution. The fluorescence polarization (FP) of the tagged fumonisin was increased upon binding with the antibody. In the presence of free toxin, less of the FB(1)-FL was bound and the polarization signal was decreased. The assays were very simple to perform, requiring only mixing of an aqueous extract of maize with the tagged fumonisin and antibody, and required <2 min per sample, excluding extraction time. Two permutations of the assay were tested, one with each sample matrix serving as its own blank, and the other with all of the samples compared relative to a PBS blank with normalization of the data similar to an ELISA. The limit of detection, defined as the toxin content associated with a fluorescence polarization signal 5 standard deviations from that of a fumonisin-free control, was 0.5 microg of FB(1)/g in spiked maize. Recoveries from spiked maize over the range of 0.5-20 ppm averaged 94.3 +/- 13.8%. Forty-eight samples of field-contaminated maize were tested by the FP and an established HPLC method, with a good correlation between the two (r(2) = 0.85-0.88). For these samples, the two variations of the FP assay also compared well to one another (r(2) = 0.97), suggesting the assay principle is very robust. The results, combined with the speed and ease of use for the assay, suggest that this technology has substantial potential as a screening tool for mycotoxins in foods.

Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels.

Maize (Zea mays) and wheat (Triticum aestivum) collected in the foothills of the Nepal Himalaya Mountains were analyzed for Fusarium species and mycotoxins: fumonisins, nivalenol (NIV), and deoxynivalenol (DON). Predominant species were Gibberella fujikuroi mating population A (F. moniliforme) in maize and F. graminearum in maize and wheat; G. fujikuroi mating population D (F. proliferatum), F. acuminatum, F. avenaceum, F. chlamydosporum, F. equiseti, F. oxysporum, F. semitectum, and F. torulosum were also present. Strains of G. fujikuroi mating population A produced fumonisins, and strains of F. graminearum produced NIV or DON. By immunoassay or high-performance liquid chromatography, fumonisins were >1000 ng/g in 22% of 74 maize samples. By immunoassay or fluorometry, NIV and DON were >1000 ng/g in 16% of maize samples but were not detected in wheat. Fumonisins and DON were not eliminated by traditional fermentation for producing maize beer, but Nepalese rural and urban women were able to detoxify contaminated maize by hand-sorting visibly diseased kernels.

Fusarium species from nepalese rice and production of mycotoxins and gibberellic acid by selected species.

Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.