Aflatoxin-lysine adducts in blood serum of the Malawian rural population and aflatoxin contamination in foods (groundnuts,maize) in the corresponding areas

Aflatoxin-lysine (AFB₁-lys) adduct levels in blood samples collected from 230 individuals living in three districts of Malawi (Kasungu, Mchinji, and Nkhotakota) and aflatoxin B₁ (AFB₁) levels in groundnut and maize samples collected from their respective homesteads were determined using indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) methods. AFB₁-lys adducts were detected in 67% of blood samples, with a mean concentration of 20.5?±?23.4 pg/mg of albumin. AFB₁ was detected in 91% of groundnut samples and in 70% of maize samples, with mean AFB₁ levels of 52.4 and 16.3 μg/kg, respectively. All participants of this study reported consuming maize on a daily basis and consuming groundnuts regularly (mean consumption frequency per week: 3.2?±?1.7). According to regression analysis, a frequency of groundnut consumption of more than four times per week, being female, and being a farmer were significant (p?<?0.05) contributors to elevated AFB₁-lys adduct levels in the blood. This is the first report on AFB₁-lys adducts in blood samples of residents in Malawi. The results reinforce the urgent need for interventions, aiming at a reduction of aflatoxin exposure of the population.     

Aflatoxin B<Subscript>1</Subscript> levels in groundnut products from local markets in Zambia

In Zambia, groundnut products (milled groundnut powder, groundnut kernels) are mostly sold in under-regulated markets. Coupled with the lack of quality enforcement in such markets, consumers may be at risk to aflatoxin exposure. However, the level of aflatoxin contamination in these products is not known. Compared to groundnut kernels, milled groundnut powder obscures visual indicators of aflatoxin contamination in groundnuts such as moldiness, discoloration, insect damage or kernel damage. A survey was therefore conducted from 2012 to 2014, to estimate and compare aflatoxin levels in these products (n = 202), purchased from markets in important groundnut growing districts and in urban areas. Samples of whole groundnut kernels (n = 163) and milled groundnut powder (n = 39) were analysed for aflatoxin B₁ (AFB₁) by competitive enzyme-linked immunosorbent assay (cELISA). Results showed substantial AFB₁ contamination levels in both types of groundnut products with maximum AFB₁ levels of 11,100 μg/kg (groundnut kernels) and 3000 μg/kg (milled groundnut powder). However, paired t test analysis showed that AFB₁ contamination levels in milled groundnut powder were not always significantly higher (P > 0.05) than those in groundnut kernels. Even for products from the same vendor, AFB₁ levels were not consistently higher in milled groundnut powder than in whole groundnut kernels. This suggests that vendors do not systematically sort out whole groundnut kernels of visually poor quality for milling. However, the overall contamination levels of groundnut products with AFB₁ were found to be alarmingly high in all years and locations. Therefore, solutions are needed to reduce aflatoxin levels in such under-regulated markets.     

Qualitative and quantitative detection of aflatoxin B1 in poultry sera by enzyme-linked immunosorbent assay

An indirect competitive inhibition type enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of aflatoxin B₁, in poultry sera. Preincubation of aflatoxin B₁, samples with the antibody prior to competition yielded better results in terms of higher sensitivity. After competition, amount of antibody bound to solid phase was measured by incubation with anti-rabbit immunoglobulins coupled with horse raddish peroxidase. Intensity of colour decreased as the amount of free aflatoxin B₁, increased. Final detection of aflatoxin B₁, was made by (i) visual comparison with standard aflatoxin B₁ using dot-ELISA (qualitative) and (ii) by plate-ELISA, where optical density was measured at 492 nm (quantitative). Plate-ELISA was more sensitive than dot-ELISA, with sensitivity limits being 100 fg and 1 pg per 10 μl, respectively. However, due to ease and speed of performance, dot-ELISA has greater potential as a test for the diagnosis of mycotoxicosis at the field level.     

Spatial and temporal variations in contamination of mycotoxins in sub-humid,semi-arid areas of Eastern Central Tanzania

Studies were conducted to determine the incidences and levels of mycotoxin contamination in household stored maize and groundnuts in Kilosa District, Tanzania. Four villages were chosen for sampling. Seventy-two samples were collected from four villages in 2010, and then tested using a high-performance liquid chromatography method. Levels of Fumonisin B₁ ranged from 63.26 to 213.15 μg/kg in all collected maize samples, while levels of aflatoxin B₁ ranged from 72.97 to 195.17 μg/kg in all collected groundnut samples. Significant differences in levels of fumonisins were observed over time (p < 0.0001) but not among villages (p < 0.3209). Aflatoxin levels differed significantly among villages (p < 0.0491) and over time (p < 0.0001). The association between altitude and the level of aflatoxin contaminations was also significant. This study indicates that stored maize and groundnuts were more prone to mycotoxin contamination.     

An Ultra-Sensitive Monoclonal Antibody-Based Competitive Enzyme Immunoassay for Sterigmatocystin in Cereal and Oil Products

Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B₁, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B₁, B₂, G₁, G₂, and M₁. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL⁻¹) ELISA format, in which the antibody was diluted to 1∶80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g⁻¹ for wheat, 0.06 ng·g⁻¹ for maize, and 0.1 ng·g⁻¹ for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products.     

Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B₁ complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B₁ remained bound. Nonviable bacteria retained the highest amount of aflatoxin B₁. Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B₁ from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B₁ to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B₁. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B₁ released. Binding of aflatoxin B₁ appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.     

Immunomagnetic bead-based recovery and real time quantitative PCR (RT iq-PCR) for sensitive quantification of aflatoxin B(1)

Aflatoxin B₁ is an unavoidable natural mycotoxin that enters the food chain by contamination of food grains and feedstuffs, potentially posing carcinogenic risks to animal and human health. Immuno-PCR methods have the potential to address the need of meeting the regulatory limits by detecting trace levels of toxins present in food and animal feeds. This paper describes a real-time immuno-quantitative PCR (RT-iqPCR) assay for quantification of aflatoxin B₁ suspended in methanol:water solution that can also serve as an extraction solvent. Immuno-PCR approaches were examined including direct vs. indirect sandwich assays using monoclonal vs. polyclonal antibodies. Our best approach was obtained using monoclonal antibodies to capture aflatoxin in solution prior to immobilizing the F_c portion of the capture antibodies onto to protein G magnetic beads. This was followed by the addition of a polyclonal ‘signal antibody’ tethered with an oligonucleotide template for a subsequent PCR assay. The RT-iqPCR assay described herein leads to the sensitive detection and quantification of aflatoxin B₁ from 10 ppb down to 0.1 ppb with high correlation (r² = 0.97) and efficiency (99.5%). The approach also detected the high-dose ‘hook effect’ phenomenon (excess antigen) which was overcome by the use of dilution protocols to eliminate false negatives that may occur at levels above quantification limits of the assay. The RT-iqPCR approach discussed here is presented as a model system that could easily be adapted for aflatoxin detection in a variety of food or animal feed samples using a simple methanol:water solution as an extraction solvent.     

Mechanism of action of aflatoxin B1

The inhibitory effects of aflatoxin B₁ were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0.05% w/v) or Tween-80 (0.05 % v/v) addition accentuated the aflatoxin B₁ growth inhibition inSalmonella typhi andEscherichia coli at different pH values. The inhibition of lipase production was only 5–20 % inPseudomonas fluorescence ca. 25–48% inStaphylococcus aureus andBacillus cereus at different aflatoxin B₁ concentrations (4–16μg/ml).However, inhibition of α-amylase induction was complete in1Bacillus megaterium whereas the inhibition was partial inPseudomonas fluorescence (27–40%) at 32μg aflatoxin B₁ concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B₁ concentration     

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

Fumonisin B₁ (FMB₁) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB₁ (anti-FMB₁ mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB₁ (FMB₁-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB₁ mAb has about 10 ppb of minimum FMB₁ detection concentration and 220 ppb of 50% inhibition concentration (IC₅₀). Much lower cross-reactivity of anti-FMB₁ mAb on ochratoxin A, aflatoxin B₁ and deoxynivalenol provided that anti-FMB₁ mAb was specific for FMB₁. The gene coding single chain variable fragment against FMB₁ (anti-FMB₁ scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB₁ 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-FMB₁ scFv on FMB₁-BSA was obtained in comparison with that of the parental mAb.     

Development of a direct competitive enzyme-linked immunosorbent assay for parathion residue in food samples

A heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) for parathion residue determination is described based on a monoclonal antibody and a new competitor. The effects of several physicochemical factors, such as methanol concentration, ionic strength, pH value, and sample matrix, on the performance of the ELISA were optimized for the sake of obtaining a satisfactory assay sensitivity. Results showed that when the assay medium was in the optimized condition (phosphate buffer solution [PBS] containing 10% [v/v] methanol and 0.2 mol/L NaCl at a pH value of 5.0), the sensitivity (estimated as the IC₅₀ value) and the limit of detection (LOD, estimated as the IC₁₀ value) were 1.19 and 0.08 ng/ml, respectively. The precision investigation indicated that the intraassay precision values all were below 10% and that the interassay precision values ranged from 4.89 to 19.12%. In addition, the developed ELISA showed a good linear correlation (r² = 0.9962) to gas chromatography within the analyte’s concentration range of 0.1 to 16 ng/ml. When applied to the fortified samples (parathion adding level: 5-15 μg/kg), the developed ELISA presented mean recoveries of 127.46, 122.52, 91.92, 124.01, 129.72, 99.37, and 87.17% for tomato, cucumber, banana, apple, orange, pear, and sugarcane, respectively. Results indicated that the established ELISA is a potential tool for parathion residue determination.     

A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice

A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B₁ (AFB₁) in rice. The immunosensor was based on an indirect competitive format between free AFB₁ and aflatoxin B₁-bovine serum albumin (AFB₁-BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB₁ antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB₁-BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB₁ in real samples.     

Preharvest aflatoxin contamination of groundnuts subjected to terminal drought stress in postrainy season

Groundnuts grown in the postrainy season under terminal drought stress imposed by withholding irrigation, or under a water-deficit gradient created by line-source sprinkler irrigation, were examined for preharvest aflatoxin contamination. High levels of aflatoxin B₁ were found in damaged seeds in both situations. When grown under continuous drought-stress, toxin levels in damaged seed samples ranged from 1480 to 2467 ?g/kg in the 1990/91, and 1.3 to 2000 ?g/kg in the 1991/92 postrainy seasons. Aflatoxin B₁ contamination in all damaged seed samples increased with increasing water deficit; toxin levels ranged from 26 to 850 ?g/kg across the water deficit gradient. Aflatoxin was either absent or almost negligible (1–2 ?g/kg) in apparently undamaged seed samples. Low risk of aflatoxin contamination in apparently undamaged seeds of groundnuts grown in postrainy seasons is indicated, even when there is terminal drought stress.     

Occurrence of aflatoxins and its management in diverse cropping systems of central Tanzania

The staple crops, maize, sorghum, bambara nut, groundnut, and sunflower common in semi-arid agro-pastoral farming systems of central Tanzania are prone to aflatoxin contamination. Consumption of such crop produce, contaminated with high levels of aflatoxin B₁ (AFB₁), affects growth and health. In this paper, aflatoxin contamination in freshly harvested and stored crop produce from central Tanzania was examined, including the efficacy of aflatoxin mitigation technologies on grain/kernal quality. A total of 312 farmers were recruited, trained on aflatoxin mitigation technologies, and allowed to deploy the technologies for 2 years. After 2 years, 188 of the 312 farmers were tracked to determine whether they had adopted and complied with the mitigation practices. Aflatoxigenic Aspergillus flavus and aflatoxin B1 contamination in freshly harvested and stored grains/kernels were assessed. A. flavus frequency and aflatoxin production by fungi were assayed by examining culture characteristics and thin-layer chromatography respectively. AFB₁ was assayed by enzyme-linked immunosorbent assay. The average aflatoxin contamination in freshly harvested samples was 18.8 μg/kg, which is above the acceptable standard of 10 μg/kg. Contamination increased during storage to an average of 57.2 μg/kg, indicating a high exposure risk. Grains and oilseeds from maize, sorghum, and sunflower produced in aboveground reproductive structures had relatively low aflatoxin contamination compared to those produced in geocarpic structures of groundnut and bambara nut. Farmers who adopted recommended post-harvest management practices had considerably lower aflatoxin contamination in their stored kernels/grains. Furthermore, the effects of these factors were quantified by multivariate statistical analyses. Training and behavioral changes by farmers in their post-harvest practice minimize aflatoxin contamination and improve food safety. Moreover, if non-trained farmers receive mitigation training, aflatoxin concentration is predicted to decrease by 28.9 μg/kg on average.     

The presence of aflatoxin in kernels from five years groundnut crops and ofAspergillus flavus isolates from kernels and soils

Summary In T.L.C. tests for 605 samples of groundnut kernels from 5 years' yield, the percentage of fresh kernels in which aflatoxin was present was very low (up to 6.4%), while that of stored kernels ranged from 0 to 32.0%. But the intensity of toxicity was invariably very low (up to 125 ppb). Of 1626Aspergillus flavus isolates from groundnut kernels rhizosphere and geocarposphere, and from soil in which groundnuts grew, about 90% were found capable of forming aflatoxin. In quantitative tests with 750 isolates 60% of the isolates produce aflatoxin in excess of 25,000 ppb. This research is supported by Grant Number FG-Is-161 of the United States Department of Agriculture to whom the author is indebted.     

Aflatoxin contamination of groundnuts in Sudan

Groundnut samples, collected soon after harvest, from different districts in the irrigated region (Central Sudan) were free from aflatoxins with the method used. Samples collected from the rainfed region (Western Sudan) showed variable levels of aflatoxin ranging from 100% sample contamination in El Hamdi to only 10% in Casgeal.Damaged pods were highly contaminated with A. flavus and accumulated large amounts of aflatoxins. However, sound intact pods, recorded lower fungal contamination and were almost free of aflatoxins. Groundnut products collected from Khartoum North (Bahri) have higher levels of aflatoxins than those collected from Khartoum and Umdorman. Gray and red roasted pods showed higher amounts of aflatoxins, while the groundnut paste was the least contaminated.None of the three varieties of groundnuts tested in this work was completely resistant to aflatoxin production. A temperature of 30°C and 86.3% relative humidy (RH) are the optimum conditions for both A. flavus growth and aflatoxin production in groundnuts.     

Production and characterization of antibody against aflatoxin Q1.

Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization with AFQ2a-bovine serum albumin bound most effectively with AFQ2a. AFQ2a antibody was selected for the subsequent direct and indirect ELISA for the detection of AFQ1 in biological fluids. When AFQ2a-peroxidase and AFQ2a antibody were used, direct ELISA was able to detect as low as 2 ppb (ng/ml) of AFQ1 spiked in the urine samples that had been subjected to a Sep-Pak cleanup treatment. In indirect ELISA in which the antigen (AFQ2a-bovine serum albumin) was coated to the solid phase followed by reaction with rabbit antibody and goat anti-rabbit immunoglobulin G-peroxidase conjugate, 50-fold less antibody was used without sacrificing sensitivity. Recoveries of AFQ1 added to urine samples (2 to 40 ppb) were 46.3 to 73% and 65.8 to 85.8% for direct and indirect ELISA, respectively.     

Production and characterization of antibody against aflatoxin Q1

Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization with AFQ2a-bovine serum albumin bound most effectively with AFQ2a. AFQ2a antibody was selected for the subsequent direct and indirect ELISA for the detection of AFQ1 in biological fluids. When AFQ2a-peroxidase and AFQ2a antibody were used, direct ELISA was able to detect as low as 2 ppb (ng/ml) of AFQ1 spiked in the urine samples that had been subjected to a Sep-Pak cleanup treatment. In indirect ELISA in which the antigen (AFQ2a-bovine serum albumin) was coated to the solid phase followed by reaction with rabbit antibody and goat anti-rabbit immunoglobulin G-peroxidase conjugate, 50-fold less antibody was used without sacrificing sensitivity. Recoveries of AFQ1 added to urine samples (2 to 40 ppb) were 46.3 to 73% and 65.8 to 85.8% for direct and indirect ELISA, respectively.     

Mycotoxigenic potential ofAspergillus flavus strains isolated from groundnuts growing in Israel

Two hundred strains of the Aspergillus flavus group isolated from groundnuts (peanuts) growing in Israel were examined for their ability to produce mycotoxins in potato dextrose (PD) broth. Almost 77% of the isolates produced aflatoxin; aflatoxins B₁ and B₂ were formed by most of the isolates. Simultaneous production of aflatoxins of groups B and G was detected in only 0.5% of the isolates. Microscopic examination revealed that 98% of the isolates wereA. flavus and only 2%A. parasiticus. Cyclopiazonic acid (CPA) was detected in 22.5% of the isolates, including 3.5% that produced only CPA. Sterigmatocystin was detected in only 2% of the isolates and only one isolate produced aflatoxin simultaneously with CPA and sterigmatocysin. The dry weight (DW) of mycelium, 7 days after inoculating the medium, was between 71–110 mg/30 ml medium in more than 70% of the isolates. A general decrease in the pH was observed and 75% of the isolates reduced the pH to 4.5 or below. After 14 days, a small increase in DW and an increase in the pH toward neutrality was observed. On PD agar, 30% of the isolates produced sclerotia, including 5% that produced them profusely. No correlation between mycelial growth, changes in pH of the medium, sclerotium formation, and aflatoxin accumulation could be observed. The mycotoxigenic potential of theA. flavus strains isolated from groundnuts seems to be relatively high and may present a potential threat to human and animal health.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel. No. 3559-E.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.