Analysis of multiple mycotoxins-contaminated wheat by a smart analysis platform.

This study describes a smart analysis platform capable of quantitative measurements using a multiplex lateral flow strip. Using the multi-mycotoxin strip, five fungal toxins were simultaneously and quantitatively detected in naturally contaminated wheat. First, a matrix-based standard curve was established for the detection of aflatoxin B1 (AFB1), fumonisin B1 (FB1), T-2, deoxynivalenol (DON), and zearalenone (ZEN). Established on an open android system, the platform is able to read 6 lines on the strip simultaneously. The platform is equipped with a Quick Response code scanning model, which reads the established standard curves, and then rapidly quantify mycotoxins in naturally contaminated wheat. All the data and sample information are stored on a central server through the platform which is linked to the cloud. The limits of detection (LOD) for AFB1, FB1, T-2, DON, and ZEN in wheat were 4, 20, 10, 200, and 40 µg/kg and the visual cut off values was 20, 1000, 200, 4000, and 400 µg/kg, separately. To validate the platform and the multi-mycotoxin detection method, 10 wheat samples were analyzed and the results were in a good agreement with those obtained by LC-MS/MS. The platform will be a powerful tool for crop monitoring services.

Single-chain variable fragment antibody-based immunochromatographic strip for rapid detection of fumonisin B1 in maize samples.

A rapid and sensitive immunochromatographic strip (ICS) based on a single-chain variable fragment (scFv) was developed for detecting fumonisin B1 (FB1). The ICS was based on a competitive reaction for colloidal gold-labeled scFv between FB1 and FB1-BSA, which was used along with sheep anti-mouse IgG as capture reagents immobilized at test and control lines, respectively, on a nitrocellulose membrane of the strip. The limit of detection of the ICS was 2.5 ng/mL (25 µg/kg) FB1 in buffer, and the sensitivity was eight times higher than that of monoclonal antibodies for the preparation of the scFv. The cross-reactivity of the scFv with common mycotoxins was determined by ICS, the results showed that the scFv were not against other mycotoxins. Eight naturally contaminated maize samples were analyzed with the scFv-based ICS and by LC-MS/MS. The results of analysis obtained with the strip assay showed good agreement with those obtained by LC-MS/MS.

Colour-encoded lateral flow immunoassay for the simultaneous detection of aflatoxin B1 and type-B fumonisins in a single Test line.

A multiplex Lateral Flow Immunoassay was developed based on the use of a single Test line and multicolour gold nanoparticles (GNPs) as signal reporters. Red and blue GNPs were linked to antibodies directed towards two different analytes and included in a typical lateral flow immunoassay configuration, in which the Test line was formed by the mixture of two antigens. As a result of the immunoreactions occurring at the Test zone, diverse combinations of red and blue GNPs labels were captured. Therefore, the Test line assumed different colours depending on which - and how much - analyte is present in the sample. The multiplexing capability of the 'colour-encoded assay' is illustrated by the simultaneous detection of aflatoxin B1 (AFB1) and type-B fumonisins (FMs) in wheat and food products that made with wheat. Reproducible detection of AFB1 and FMs contamination in raw and processed food was achieved with visual cut-off levels at 1 ng mL-1 and 50 ng mL-1, respectively. The contaminant was identified based on the colour of the label according with a specific colour code. Furthermore, strips images were acquired by means of a common smartphone and analysed through RGB data analysis providing semi-quantitative detection of the two mycotoxins.

Combining E-Nose and Lateral Flow Immunoassays (LFIAs) for Rapid Occurrence/Co-Occurrence Aflatoxin and Fumonisin Detection in Maize.

The aim of this study was to evaluate the potential use of an e-nose in combination with lateral flow immunoassays for rapid aflatoxin and fumonisin occurrence/co-occurrence detection in maize samples. For this purpose, 161 samples of corn have been used. Below the regulatory limits, single-contaminated, and co-contaminated samples were classified according to the detection ranges established for commercial lateral flow immunoassays (LFIAs) for mycotoxin determination. Correspondence between methods was evaluated by discriminant function analysis (DFA) procedures using IBM SPSS Statistics 22. Stepwise variable selection was done to select the e-nose sensors for classifying samples by DFA. The overall leave-out-one cross-validated percentage of samples correctly classified by the eight-variate DFA model for aflatoxin was 81%. The overall leave-out-one cross-validated percentage of samples correctly classified by the seven-variate DFA model for fumonisin was 85%. The overall leave-out-one cross-validated percentage of samples correctly classified by the nine-variate DFA model for the three classes of contamination (below the regulatory limits, single-contaminated, co-contaminated) was 65%. Therefore, even though an exhaustive evaluation will require a larger dataset to perform a validation procedure, an electronic nose (e-nose) seems to be a promising rapid/screening method to detect contamination by aflatoxin, fumonisin, or both in maize kernel stocks.

Simultaneous determination of aflatoxin B1, fumonisin B1 and deoxynivalenol in beer samples with a label-free monolithically integrated optoelectronic biosensor.

A label-free optical biosensor for the fast simultaneous determination of three mycotoxins, aflatoxin B1 (AFB1), fumonisin B1 (FB1) and deoxynivalenol (DON), in beer samples is presented. The biosensor is based on an array of ten Mach-Zehnder interferometers (MZIs) monolithically integrated along with their respective broad-band silicon light sources onto a single chip. Multi-analyte determination is accomplished by functionalizing the sensing arms of individual MZIs with mycotoxin-protein conjugates. Assay is performed by pumping over the chip mixtures of calibrators or samples with a mixture of specific monoclonal antibodies, followed by reaction with a secondary anti-mouse IgG antibody. Reactions are monitored in real-time by continuously recording the MZI output spectra, which are then subjected to Discrete Fourier Transform to convert spectrum shifts to phase shifts. The detection limits achieved for AFB1, FB1 and DON were 0.8, 5.6 and 24 ng/ml, respectively, while the assay duration was 12 min. Recovery values ranging from 85 to 115% were determined in beer samples spiked with known concentrations of the three mycotoxins. In addition, beers of different types and origin were analysed with the biosensor developed and the results were compared with those provided by established laboratory methods, further supporting the accuracy of the proposed device.

Development of a nanoarray capable of the rapid and simultaneous detection of zearalenone, T2-toxin and fumonisin.

Fusarium mycotoxins such as trichothecenes, zearalenone and fumonisins occur on a worldwide basis in cereal grains, animal feeds and forages. Practical solutions for multiple mycotoxin determination in samples are required by industry and regulators for cost effective screening purposes. The feasibility of developing a novel multiplex nanoarray for the simultaneous and semi-quantitative detection of three regulated mycotoxins: zearalenone (ZEA), T2-toxin (T2) and fumonisin B1 (FUM) was examined. Additionally, the assay was also able to detect HT2 toxin and fumonisin B2 and B3 due to the cross reactivity profiles of the antibodies used. Individual mycotoxin conjugates specific to the three mycotoxins were nano-spotted onto wells of a microtitre plate. Optimisation of assay parameters and antibodies was undertaken with both individual and multiplex calibration curves generated. A competitive assay format was employed enabling a calibration curve for concentration analysis and duplicate results for up to 40 samples in 70min for the three target mycotoxins. The characteristics and performance of the nanoarray were evaluated including sensitivity and specificity for each target. Additionally, intra and inter spotting precision, cross reactivity, matrix effects and sample analysis in maize and wheat (n=8) was performed. Sensitivity, determined as the concentration causing 50% inhibition, was 70.1, 2.8 and 90.9ppb in PBS, 172.4, 3.2 and 129.3ppb in methanol, 197.4, 0.7 and 216.7ppb in wheat and 43.6, 0.5 and 25.9ppb in maize for ZEA, T2 and FUM respectively. Intra spotting precision was 6%, 11% and 10% for PBS and 5%, 11% and 12% for methanol for ZEA, T2 and FUM respectively. Inter spotting precision was 4%, 14% and 6% for PBS and 3%, 9% and 16% for methanol for ZEA, T2 and FUM respectively. The feasibility of the nanoarray as an easy to use sensitive screening tool in the 96 well format has been demonstrated for the multiplex detection of three regulated mycotoxins. Improvements in automated image and data analysis software for novice end users are required to improve the overall rapidity of analysis.

A sensitive immunochromatographic assay using colloidal gold-antibody probe for rapid detection of fumonisin B1 in corn.

A sensitive immunochromatographic assay (ICA) using a colloidal gold-antibody probe for the rapid detection of fumonisin B1 (FB1) in corn samples was developed. The colour density of the test line correlated with the concentration of FB1 in the range 2-40 ng ml(-1) by the assay, and the detection limit for FB1 was 2 ng ml(-1). The linear range for FB1 was 50-1000 µg kg(-1), and the visual limit detection of the test was 1000 µg kg(-1) in corn samples. The ICA to detect FB1 is sensitive, specific and rapid. Specific anti-FB1 monoclonal antibody (mAb) and FB1-ovalbumin (FB1-OVA) conjugate antigen were prepared. FB1 mAb, labelled with colloidal gold, was used as the probe on the immunochromatographic strip. FB1-OVA and goat-anti-mouse IgG were coated onto a nitrocellulose (NC) membrane as test lines and control lines, respectively. FB1 in samples will competitively combines the FB1 mAb with the FB1-OVA in an NC membrane and the results are directly observed by the colour of the detection and quality control lines. The concentrations of FB1 mAb labelled with colloidal gold, detecting antigen and goat anti-mouse IgG, were optimised. The results indicate that the test strip is specific for FB1, with no cross-reactivity to other toxins. The strip assay for FB1 was simple, only needing one step without complicated assay performance and expensive equipment, and the total time for visual evaluation was less than 10 min. A survey of 24 corn samples from Hefei, China, was performed with the test strip and HPLC, and the detection results showed that the developed ICA and the HPLC were in excellent agreement. Hence, the developed ICA can be used as a method for rapid detection of FB1 in corn samples.

Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal.

A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB1 was developed. The anti-idiotypic nanobody-alkaline phosphatase (Ab2ß-Nb-AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB1 were 2.69 and 0.35 ng mL(-1), respectively, with a linear range of 0.93-7.73 ng mL(-1). The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL(-1), and the IC50 was 0.89 ± 0.09 ng mL(-1) with a linear range of 0.29-2.68 ng mL(-1). Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2ß-Nb-AP fusion protein based one-step competitive immunoassay for monitoring FB1 contamination in cereals. The Ab2ß-Nb-AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems.

Urchin-like gold nanoparticle-based immunochromatographic strip test for rapid detection of fumonisin B1 in grains.

An immunochromatographic strip (ICS) using urchin-like gold nanoparticles (UGNs) for sensitive detection of fumonisin B1 (FB1) was developed to meet the requirement for rapidly monitoring FB1 in grain samples. The sensitivity of the ICS was 5.0 ng/mL, which represents a fourfold increase in sensitivity over conventional strip preparation using colloidal gold as the antibody-labeled probe. Analysis of FB1 in grain samples showed that data obtained from the strip tests were in a good agreement with those obtained from HPLC and enzyme-linked immunosorbent assays (ELISAs). This qualitative test did not require any specialized equipment, and the detection time was less than 5 min, which is suitable for on-site testing of FB1 in grain samples. Overall, to our knowledge, this is the first report of using a UGN as the antibody-labeled probe for sensitive detection of FB1 in grains using an ICS. Graphical Abstract Preparation of ICS using conventional colloidal gold and urchin-like gold nanoparticle, respectively.

Anti-idiotypic nanobody: A strategy for development of sensitive and green immunoassay for Fumonisin B1.

Nanobodies that are small and thermally stable, as well as have high expression level, are leading alternative to produce anti-idiotypic antibodies. These antibodies have the advantage of replacing mycotoxins and their conjugates for immunoassays. In this work, anti-fumonisin B1 (FB1) monoclonal antibody (mAb) was used as the target for biopanning from a naïve alpaca nanobody (Nb) phage display library. After three cycles of panning, one anti-idiotypic nanobody (Ab2ß Nb) was isolated and subjected to a Nb-ELISA for the detection of FB1. Surface plasmon resonance was used to analyze the reaction kinetics between the Ab2ß Nb and anti-FB1 mAb. The developed assay showed a half inhibitory concentration (IC50) of 0.95±0.12 ng/mL, a limit of detection of 0.15 ng/mL, a linear range of 0.27-5.92 ng/mL, and a low cross-reactivity toward FB2 of 4.93%. The sensitivity was enhanced approximately 20-fold compared with that of the chemosynthetic FB1-BSA conjugates. The equilibrium dissociation constant (KD) measured for Ab2ß Nb: anti-FB1 mAb was 164.6 nM. The assay was compared with conventional ELISA (the commercial ELISA kit), and the results indicated the reliability of Ab2ß Nb replacing the antigen-carrier protein conjugates. The use of biotechnology in developing the surrogate is an ideal strategy for replacing conventional synthesized antigens.

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 µM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples.

Application of mimotope peptides of fumonisin b1 in Peptide ELISA.

Anti-fumonisin B(1) (FB(1)) McAb 1D11 was used as the target for biopanning from a phage random loop-constrained heptapeptide library. After three cycles of panning, seven phages with three mimotope peptides were selected to mimic the binding of FB(1) to 1D11. After the identification of phage ELISA, the phage clone that showed the best linear range of detection was chosen for further research. One peptide with the inserted peptide sequence of the phage was synthetized, named CT-452. An indirect competitive ELISA (peptide ELISA) for detecting FB(1) was established using the CT-452-bovine serum albumin conjugate as coating antigen. The linear range of the inhibition curve was 1.77-20.73 ng/mL. The half inhibitory concentration (IC50) was 6.06 ng/mL, and the limit of detection was 1.18 ng/mL. This method was compared with conventional indirect ELISA (commercial ELISA kit) and high-performance liquid chromatography (HPLC), and the results showed the reliability of the peptide ELISA for the determination of FB(1) in cereal samples. The relationship between the CT-452 and FB(1) standard concentrations in peptide ELISA was evaluated. The results indicated that synthetic peptide CT-452 can replace the FB(1) standard to establish an immunoassay free of FB(1).

A flow cytometry based competitive fluorescent microsphere immunoassay (CFIA) system for detecting up to six mycotoxins.

BACKGROUND: Exposure to multiple mycotoxins through the food chain represents a major potential health hazard to both humans and livestock. They can cause a variety of severe acute as well as chronic diseases. Eliminating mycotoxins from various grain crops is a global health priority. According to the Food and Agriculture Organization (FAO), world food production needs to double by 2050. Innovative solutions will be required to sustain toxin free grain supplies worldwide. METHODS: A competitive flow cytometry based multiplexed assay with fluorescent microspheres has been developed. The new multiplexed method can analyze simultaneously any one or all six major mycotoxins. They include: Ochratoxin A (OTA), Aflatoxin B1 (AFB1), Fumonisin B1 (FB1), T-2 toxin (T-2), Deoxynivalenol (DON) and Zearalenone (ZEA), which are all potential human health hazards. The CFIA described here includes a simplified single extraction step for mycotoxins from specimens and a comprehensive post acquisition software module. The new assay system was developed with a FACSArray™ BD Bioanalyzer flow cytometer (BD Biosciences, Belgium). RESULTS: The CFIA performs favourably when compared to commercial ELISA. Sensitivity range with CFIA increased between 13% and 100% with an average improvement of 50% for the six mycotoxins. CONCLUSIONS: The multiplexed assay presented here has the unique capacity to quantify up to six mycotoxins simultaneously from a single specimen extraction. CFIA's poly-mycotoxin detection sensitivity exceeds standard ELISA. CFIA may be part of a comprehensive assay system that will provide reliable and effective safeguard for agricultural commodities to be free of mycotoxins.

Fumonisins: probable role as effectors in the complex interaction of susceptible and resistant maize hybrids and Fusarium verticillioides.

Fusarium verticillioides is best known for its worldwide occurrence on maize resulting in highly variable disease symptoms, ranging from asymptomatic to severe rotting and wilting and fumonisin production. The aim of this study was to investigate the influence of hybrid genotypes in the early stages of F. verticillioides infection, and the role of fumonisins as effectors in the outcome of this complex interaction. Disease symptoms, growth parameters, root morphology, and fungal colonization were evaluated at 7, 14, and 21 days after planting in seedlings from maize seeds of resistant (RH) and susceptible (SH) hybrids inoculated with F. verticillioides or watered with solutions of fumonisins. F. verticillioides induced growth enhancement or retardation depending on the plant genetic background and the fungal colonization rate, while fumonisins caused severe reduction in biomass and fitness. Seedlings watered with high fumonisin concentrations displayed lesions similar to those seen in F. verticillioides maize seedling disease, and also elicited inhibitory effects on root growth and morphology and on functional properties. In summary, these data strongly suggest a dual role for fumonisins in the F. verticillioides-maize interaction, acting as pathogenic factors at high concentrations, or triggering the plant detoxification mechanisms at low levels.

Development and application of a quantitative lateral flow immunoassay for fumonisins in maize.

A quantitative lateral flow immunoassay for measuring fumonisins in maize was developed. Strip preparation and assay parameters were optimized to obtain a dipstick usable outside the laboratory with different samples, and which shows performance comparable with that of other screening methods, as confirmed by the intra- and the inter-day precision of data (RSD 5-16%). Quantification was obtained by an external calibration curve, which can be stored and used for measurements made with strips of the same batch in different days and at varying temperatures (22-37°C). Limit of detection (120 µgL(-1)) and dynamic range (200-5000 µgL(-1)) allow the direct assessment of fumonisin contamination at all levels of regulatory relevance. Twenty-seven maize samples were analyzed after a simple sample preparation which avoids the use of organic solvent. Linear correlation was observed (y=1.071x-0.2, r(2)=0.990) when data was compared with that obtained through a reference LC-MS/MS method, across a wide range of fumonisin contamination.

Sensitive enzyme-linked immunosorbent assay and rapid one-step immunochromatographic strip for fumonisin B1 in grain-based food and feed samples.

BACKGROUND: Maize contaminated with mycotoxin fumonisin B1 poses a global threat to agricultural production. In this study, polyclonal antibodies (pAb) specific to fumonisin B1 were generated from rabbits immunised with fumonisin B1-keyhole limpet haemocyanin (KLH). These antibodies were used to establish a sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and gold nanoparticle immunochromatographic strip for detecting fumonisin B1 levels in maize-based foods and feeds. RESULTS: In cdELISA, fumonisins B1, B2 and B3 at concentrations of 0.42, 0.58 and 81.5 ng mL(-1) respectively caused 50% inhibition (IC(50)) of binding of fumonisin B1-horseradish peroxidase (HRP) to the antibodies. Effective on-site detection of fumonisin B1 was achieved by developing a rapid and sensitive pAb-based gold nanoparticle immunochromatographic strip. This strip had a detection limit of 5 ng mL(-1) for fumonisin B1 in maize-based samples. Additionally, the whole analytical process could be completed within 10 min. Close examination of 15 maize-based samples by cdELISA revealed that 11 were fumonisin-positive, with a mean concentration of 435 +/- 20.1 ng g(-1). These results correlated well with those obtained by immunochromatographic strip. CONCLUSION: Both cdELISA and immunochromatographic strip methods established in this study are sensitive for rapid detection of fumonisins in agricultural commodities.

Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1.

Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb.

[Development of monoclonal antibody against fumonisin B1 and ELISA-kit of quantitative analysis for fumonisin B1].

OBJECTIVE: To detect the contamination level of fumonisin B1 in grain, and to develop rapid detection kit that possess patent of China. METHODS: Hybridoma cell line excreting monoclonal antibody against fumonisin B, was produced using B cell hybridoma technique and develop a rapid, sensitive, quantitative ELISA-kit for detection fumonisin B1. RESULTS: The monoclonal antibody used in the ELISA-kit were tested for subtype as IgG1 and its affinity constant was 8.3 x 10(-8) mol/L. The monoclonal antibody obtained in the present study was highly specific to fumonisin B1 , because no cross reactions between the monoclonal antibodies against fumonisin B1 with the analogues of fumonisin B, were found. The limited concentration of the ELISA-kit was 5 ng/ml, linear range was 50-500 ng/ml, the linear equation was Y = -0.582 x +1.793( = 0.99, P < 0.05). The recovery rate of maize on the level of 50n g/ml, 200 ng/ml and 500 ng/ml was among 71.89%-112.95%. The kit can be stored at normal tempertature in ten months at least. The coefficient of variant winthin-laboratory and between-laboratory was less than 20%.

Production of a single-chain variable fragment antibody against fumonisin B1.

The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free fumonisin B1 and fumonisin-biotin conjugate. Also fumonisin B2 was bound by the scFv. Modeling of both scFv and fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentially be used for developing a rapid and affordable immunoassay for detection of food contamination and can be applied in immunoaffinity chromatography, usually carried out prior to HPLC analysis of mycotoxin-contaminated food and feed.

Immunobiological effects of AFB1 and AFB1-FB1 mixture in experimental subchronic mycotoxicoses in rats.

Maize co-contamination with aflatoxin B1 (AFB1) and fumonisin B1 (FB1) is frequently found in several countries. Although the alterations on nutritional and immunologic parameters induced by these mycotoxins, when administered individually, are partially characterised, little is known about the effects induced in animals by a subchronic administration of both toxins mixtures. We have studied the nutritional and immunological alterations induced in rats fed during 90 days with a diet without mycotoxins, containing 40 ppb AFB1, and with a diet containing a mixture of 40 ppb AFB1 and 100 ppm FB1. Animals fed with the mixture of toxins obtained lower body weight than the control ones. The mitogenic response of spleen mononuclear cells (SMC) in vivo was higher in animals fed with AFB1. In in vitro studies, lower proliferations of SMC pre-exposed to AFB1 and to the mixture of toxins were detected. The SMC of animals fed with AFB1 produced lower levels of IL-2, higher of IL-4 and equal levels of IL-10. The SMC of animals fed with both toxins produced higher levels of IL-4, lower of IL-10 and equal levels of IL-2. The SMC preincubated with an AFB1-FB1 mixture produced higher concentrations of IL-4, lower of IL-10 and equal levels of IL-2. The peritoneal macrophages of animals that consumed AFB1 released less H(2)O(2), while animals fed with the mixture of toxins produced higher levels. In in vitro studies, macrophages pre-exposed to the mixture of toxins released less H(2)O(2). These results show different immunobiological effects produced by a mixture of mycotoxins in comparison to the individual action of the same toxins.