Determination of aflatoxin and fumonisin levels through ELISA and HPLC,on tilapia feed in Nayarit,Mexico

A survey of fungal contamination and presence of aflatoxins (AFs) and fumonisins (FBs) in 30 feed samples collected from 10 tilapia farms during three seasons in Nayarit State, located in north-western Mexico, was carried out using ELISA as screening and High-Performance Liquid Chromatography (HPLC) as confirmatory method. Mycobiota included Aspergillus flavus and Fusarium spp. AFs were detected in 63.3% of samples using ELISA, but confirmation by HPLC revealed that all samples were under the detection limit. Regarding to FBs, positive samples were detected using both methods, with 19 positive samples (60% of total) by ELISA and 14 positive samples (46.6% of total) by HPLC and levels ranging from 0.148 to 2.587 mg/kg. Correlation was observed between both methods (r = 0.516, p = 0.004) for FBs results. No sample exceeded the European maximum levels for any of the mycotoxins. Water activity of samples ranged from 0.345 to 0.655, suggesting that mycotoxin occurrence is probably related to raw material contamination.     

Risk of dietary and breastmilk exposure to mycotoxins among lactating women and infants 2–4 months in northern India

Mycotoxins are carcinogenic secondary metabolites of fungi that have been linked to infant growth faltering. In this study, we quantified co‐occurring mycotoxins in breast milk and food samples from Haryana, India, and characterized determinants of exposure. Deterministic risk assessment was conducted for mothers and infants. We examined levels of eight mycotoxins (Aflatoxin B₁, B₂, G₁, G₂, M₁, M₂; Ochratoxin A, B) in 100 breast milk samples (infants 2–4 months) using ultra‐high‐performance liquid chromatography tandem mass spectrometry. Aflatoxin B₁ (AFB₁), fumonisin B₁ (FB₁) and deoxynivalenol (DON) were detected in several food items (n = 298) using enzyme‐linked immunosorbent assays. We report novel data on the presence of mycotoxins in breast milk samples from India. Whereas breast milk concentrations (AFM₁ median: 13.7; range: 3.9–1200 ng/L) remain low, AFM₁ was detected above regulatory limits in 27% of animal milk samples. Additionally, 41% of infants were above provisional maximum tolerable daily intake (PMTDI) limits for AFM₁ due to consumption of breast milk (mean: 3.04, range: 0.26–80.7 ng kg⁻¹ bw day⁻¹). Maternal consumption of breads (p < 0.05) was associated with breast milk AFM₁ exposure. AFB₁ (μg/kg) was detected in dried red chilies (15.7; 0–302.3), flour (3.13; 0–214.9), groundnuts (0; 0–249.1), maize (56.0; 0–836.7), pearl millet (1.85; 0–160.2), rice (0; 0–195.6), wheat (1.9; 0–196.0) and sorghum (0; 0–63.5). FB₁ (mg/kg) was detected in maize (0; 0–61.4), pearl millet (0; 0–35.4) and sorghum (0.95; 0–33.2). DON was not detected in food samples. Mothers in our study exceeded PMTDI recommendations for AFB₁ due to consumption of rice and flour (mean: 75.81; range: 35.2–318.2 ng kg⁻¹ bw day⁻¹). Our findings show the presence of Aflatoxin B₁ and M₁ at various levels of the food chain and in breast milk, with estimated intakes exceeding PMTDI recommendations. Aflatoxins are known carcinogens and have also been linked to stunting in children. Their presence across the food system and in breast milk is concerning, thus warranting further research to replicate and expand on our findings and to understand implications for maternal and child health.     

Variation of Aflatoxin Levels in Stored Edible Seed and Oil Samples and Risk Assessment in the Local Population

Five hundred and twenty samples of edible seeds and oilseeds (sunflower, palm, peanut, sesame, cotton, and grapeseed) were purchased from markets, farmers, and superstores in the central cities of Punjab, Pakistan. A total of 125 (48.1%) edible seed samples from a 6 ≤ months storage period, and 127 (48.8%) from a 2 ≥ years storage period were found to be infested with AFs. The average elevated amount of AFB₁ and total AFs was observed in a 2 ≥ years storage period, i.e., 28.6 ± 4.5 and 51.3 ± 10.4 µg/kg, respectively, in sesame seeds. The minimum amount of AFB₁ and total AFs was observed in palm seed samples with a storage period of 6 ≤ months, i.e., 9.96 ± 2.4, and 11.7 ± 1.90 µg/kg, respectively. The maximum amount of AFB₁ and total AFs were observed in peanut oil samples, i.e., 21.43 ± 2.60 and 25.96 ± 4.30 µg/kg, respectively, with a storage period of 2 ≥ years. Therefore, the maximum dietary intake of 59.60 ng/kg/day was observed in oil samples stored at a ≥ 2 years storage period. The results of the present study concluded that a significant difference was found in the amounts of total AFs in edible seed samples stored at 6 ≤ months and 2 ≥ years storage periods (p < 0.05).     

GTPase Rac Regulates Conidiation,AFB1 Production and Stress Response in Pathogenic Fungus Aspergillus flavus

As a member of the Rho family, Rac plays important roles in many species, including proliferation, differentiation, apoptosis, DNA damage responses, metabolism, angiogenesis, and immunosuppression. In this study, by constructing Rac-deleted mutants in Aspergillus flavus, it was found that the deletion of Rac gene led to the decline of growth and development, conidia production, AFB1 toxin synthesis, and seed infection ability of A. flavus. The deletion of Rac gene also caused the disappearance of A. flavus sclerotium, indicating that Rac is required for sclerotium formation in A. flavus. The sensitivity of Rac-deficient strains responding to cell wall stress and osmotic pressure stress increased when compared to A. flavus WT. The Western blot result showed that mitogen-activated serine/threonine-protein kinase Slt2 and mitogen-activated protein kinase Hog1 proteins were no longer phosphorylated in Rac-deficient strains of A. flavus, showing that Rac may be used as a molecular switch to control the Slt2-MAPK cascade pathway and regulate the osmotic Hog-MAPK cascade pathway in A. flavus in response to external stress. Altogether, these results indicated that Rac was involved in regulating the growth and development, conidia formation and AFB1 synthesis, and response to cell wall stress and osmotic pressure stress in A. flavus.     

Relaxant effects of aflatoxins on isolated guinea pig trachea.

Dyspnea is one of the symptoms of acute aflatoxicosis. Contrary to expectations, we observed that naturally occurring aflatoxins (AF) AFB(1), AFB(2), AFG(1), and AFG(2) and their major metabolites AFM(1), AFM(2), AFP(1), AFQ(1), and AFG(2a) relaxed carbachol (C) precontracted guinea pig trachea to different degrees. The efficacies but not the potencies of AFB(1), AFB(2), AFG(1), and AFG(2) were similar to that of the beta-agonist, isoprenaline, whose activity was potentiated by the AF. Their mechanism of action is not clearly understood but several mechanistic indications were obtained with AFB(1): 1) its effect was not influenced by the beta-blocker, timolol, indicating that a direct interaction with beta(2)-adrenergic receptors was not involved. 2) AFB(1) potentiated PGE(1) and PGE(2), two relaxant prostaglandins, and its activity was reduced by indomethacin. 3) The cAMP level in the guinea pig trachea relaxed by AFB(1) increased, possibly due to inhibition of phosphodiesterase; direct interaction with PG receptors; and/or interaction with A(2) adenosinic receptors, suggested by the inhibitory activity of XAC, a specific antagonist. 4) Finally, since tetrodotoxin reduced the relaxant activity of AFB(1), it is speculated that this mycotoxin could stimulate inhibitory nonadrenergic, noncholinergic nerves (i-NANC). In conclusion, the symptoms of acute aflatoxicosis do not seem to be due to a direct activity on the tracheal muscle, but rather, to the well-known pro-inflammatory activity of the aflatoxins, which are capable of releasing arachidonic acid from cell membranes.     

Contamination by Aflatoxins B/G in Food and Commodities Imported in Southern Italy from 2017 to 2020: A Risk-Based Evaluation

This study reports the results of aflatoxins B/G monitoring in food of vegetal origin, imported in Southern Italy from extra-European Union countries. From 2017 to 2020, we analyzed 1675 samples using an accredited HPLC method with fluorescence detection. We found out 295 samples (17.6%) were contaminated by aflatoxin B1, 204 by aflatoxins B/G (12.2%), while 75 (4.5%) resulted non-compliant to maximum limits set by the European Union law. Most of the batches tested were unprocessed food; the distribution of contamination levels, incidence of non-compliant samples, inference for different kinds of food are reported. The study focuses on the food more susceptible to contamination by aflatoxins; nuts are the food more controlled, showing the higher number of non-compliant samples. Our study confirms that pistachio nuts, hazelnuts and almonds are the major sources of exposure for consumers. Still, other products, such as chili pepper and Brazil nuts, need to get more information about their contamination levels. The study’s findings are discussed in the perspective of the last opinion by EFSA about chronic exposure to aflatoxins. A case study to evaluate not compliance of a composed food to the European Union law is reported.     

Aflatoxin B1-lysine adduct in dried blood spot samples of animals and humans

Dried blood spots (DBS) were proposed as potentially viable method for exposure assessment of environmental toxicants in infant and young children. For this study, we validated an experimental protocol to quantify AFB₁-lysine adduct in DBS samples of AFB₁-treated F344 rats, as well as samples from human field study. Significant dose-response relationships in AFB₁-lysine adduct formation were found in DBS samples of rats treated with single- and repeated-dose AFB₁. AFB₁-lysine levels in DBS samples were highly correlated with corresponding serum sample levels. The Person coefficients were 0.997 for the single-dose exposure, and 0.996 for the repeated-dose exposure. Levels of AFB₁-lysine adduct had also good agreement between DBS and serum samples as shown by Bland-Altman plot analysis. For human field study samples (n = 36), a Pearson correlation coefficient of 0.784 was found between AFB₁-lysine adduct levels of DBS and corresponding serum samples. Bland-Altman plots showed the distribution of the log differences between DBS and serum AFB₁-lysine levels are within 95% confidence intervals. These results showed AFB₁-lysine adduct levels in DBS cards and serum samples from animals and human samples are comparable, and the DBS technique and analytical protocol is a good means to assess AFB₁ exposure in infant and children populations.     

检测黄曲霉毒素生物合成相关基因芯片的研制

目的 建立比较成熟的可用于分析黄曲霉毒素生物合成相关基因的芯片技术.方法 采用反转录PCR和芯片检测对比的方法.实验所用探针来源于寄生曲霉,待测样品选择黄曲霉菌株.结果 芯片点阵后依次通过温浴2 h,650 mJ/cm<'2>紫外交联30 S,80℃烘烤2 h,预杂交45 min,清洗,干燥等步骤,最后与待测样品在42℃杂交16 h,获得了低背景高质量的芯片.芯片扫描显示荧光信号稳定,与反转录PCR扩增结果一致,并且无背景干扰.结论 探针设计合理,实验方法可靠.初步建立了用芯片检测与黄曲霉毒素生物合成相关基因的技术平台.     

稀释法结合LC-MS/MS检测在10种中药材污染黄曲霉毒素高通量筛查中的应用研究

该研究运用稀释法,克服了基质效应对10种中药材污染黄曲霉毒素B₁,B₂,G₁,G₂的液质联用检测的影响,高、中、低3个浓度水平的基质效应检测结果在80.23%~115.5%,符合准确定量的要求。为增强检测结果的特异性和准确性,研究结合保留时间窗、定量离子与定性离子的峰面积比值2个指标对83批中药材的检测进行色谱峰的辨认,并以苦杏仁为基质溶液配制的3个浓度的质控样品对检测体系的稳定性进行考察。检测结果显示医院和药店的药材污染率分别为13.89%和17.02%,其中以使君子和红枣的污染率较高。质控样品的RSD<6.6%,表明该分析方法可用于这10种中药材的高通量的筛查和检测,具有经济、快速的优点。     

Toxigenic Potential of Aspergillus Species Occurring on Maize Kernels from Two Agro-Ecological Zones in Kenya

Two agro-ecological zones in Kenya were selected to compare the distribution in maize of Aspergillus spp. and their toxigenicity. These were Nandi County, which is the main maize growing region in the country but where no human aflatoxicoses have been reported, and Makueni County where most of the aflatoxicosis cases have occurred. Two hundred and fifty-five households were sampled in Nandi and 258 in Makueni, and Aspergillus was isolated from maize. Aspergillus flavus and A. parasiticus isolates were tested for the presence of aflD and aflQ genes. Positive strains were induced to produce aflatoxins on yeast extract sucrose and quantified using liquid chromatography-tandem mass spectrometry (LCMSMS). Aspergillus flavus was the most common contaminant, and the incidence of occurrence in Nandi and Makueni was not significantly different (82.33% and 73.26%, respectively). Toxigenic strains were more prevalent than non-toxigenic strains. All the toxigenic strains from Makueni were of the S-type while those from Nandi belonged to the L-type. Quantitative differences in aflatoxin production in vitro between isolates and between strains were detected with S strains producing relatively larger amounts of total aflatoxins, B toxins and lower values for G toxins. This was in accord with the frequent aflatoxicosis outbreaks in Makueni. However some L strains produced considerable amounts of B toxins. Given the widespread distribution of toxigenic strains in both regions, the risk of aflatoxin poisoning is high when favorable conditions for toxin production occur.