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Background: Aflatoxin B1 (AFB1) is a prevalent contaminant in agricultural products, presenting significant risks to animal health. CotA laccase from Bacillus licheniformis has shown significant efficacy in degrading mycotoxins in vitro test. The efficacy of Bacillus CotA laccase in animals, however, remains to be confirmed. A 2 × 2 factorial design was used to investigate the effects of Bacillus CotA laccase level (0 or 1 U/kg), AFB1 challenge (challenged or unchallenged) and their interactions on ducks. The purpose of this study was to evaluate the efficacy of Bacillus CotA laccase in alleviating AFB1 toxicosis of ducks. Results: Bacillus CotA laccase alleviated AFB1-induced declines in growth performance of ducks accompanied by improved average daily gain (ADG) and lower feed/gain ratio (F/G). Bacillus CotA laccase ameliorated AFB1-induced gut barrier dysfunctions and inflammation testified by increasing the jejunal villi height/crypt depth ratio (VH/CD) and the mRNA expression of tight junction protein 1 (TJP1) and zonula occluden-1 (ZO-1) as well as decreasing the expression of inflammation-related genes in the jejunum of ducks. Amino acid metabolome showed that Bacillus CotA laccase ameliorated AFB1-induced amino acid metabolism disorders evidenced by increasing the level of glutamic acid in serum and upregulating the expression of amino acid transport related genes in jejunum of ducks. Bacillus CotA laccase ameliorated AFB1-induced liver injury testified by suppressing oxidative stress, inhibiting apoptosis, and downregulating the expression of hepatic metabolic enzyme related genes of ducks. Moreover, Bacillus CotA laccase degraded AFB1 in digestive tract of ducks, resulting in the reduced absorption level of AFB1 across intestinal epithelium testified by the decreased level of AFB1-DNA adduct in the liver, and the reduced content of AFB1 residues in liver and feces of ducks. Conclusions: Bacillus CotA laccase effectively improved the growth performance, intestinal health, amino acid metabolism and hepatic aflatoxin metabolism of ducks fed AFB1 diets, highlighting its potential as an efficient and safe feed enzyme for AFB1 degradation in animal production. [ABSTRACT FROM AUTHOR]

Fungal HacA/Hac1 transcription factors play a crucial role in regulating the unfolded protein response (UPR). The UPR helps cells to maintain endoplasmic reticulum (ER) protein homeostasis, which is critical for growth, development, and virulence. The Aspergillus flavus hacA gene encodes a domain rich in basic and acidic amino acids (Bsc) and a basic leucine zipper (bZip) domain, and features a non-conventional intron (Nt20). In this study, CRISPR/Cas9 was utilized to dissect the Bsc-coding, bZip-coding, and Nt20 sequences to elucidate the relationship between genotype and phenotype. In the Bsc and bZip experimental sets, all observed mutations in both coding sequences were in frame, suggesting that out-of-frame mutations are lethal. The survival rate of transformants in the Nt20 experiment set was low, at approximately 7%. Mutations in the intron primarily consisted of out-of-frame insertions and deletions. In addition to the wild-type-like conidial morphology, the mutants exhibited varied colony morphologies, including sclerotial, mixed (conidial and sclerotial), and mycelial morphologies. An ER stress test using dithiothreitol revealed that the sclerotial and mycelial mutants were much more sensitive than the conidial mutants. Additionally, the mycelial mutants were unable to produce aflatoxin but still produced aspergillic acid and kojic acid. RNAi experiments targeting the region encompassing Bsc and bZip indicated that transformant survival rates generally decreased, with a small number of transformants displaying phenotypic changes. Defects in the hacA gene at the DNA and transcript levels affected the survival, growth, and development of A. flavus. Thus, this gene may serve as a promising target for future host-induced gene-silencing strategies aimed at controlling infection and reducing aflatoxin contamination in crops. [ABSTRACT FROM AUTHOR]

Aflatoxins (AFs) are highly toxic mycotoxins produced by the fungi, Aspergillus flavus and Aspergillus parasiticus. AFs pose severe health risks owing to their acute toxicity and carcinogenic properties. The control of AF contamination remains significantly challenging despite the extensive efforts toward controlling it. Here, we investigated the potential of mushroom extracts as a source of AF biosynthetic inhibitors. The A. parasiticus mutant strain, NFRI-95, that accumulates an AF biosynthesis intermediate, norsolorinic acid, was used in the bioassay to detect the inhibitory activity against AF biosynthesis. The screening of 195 mushroom extracts revealed that the culture filtrate extract of Chondrostereum purpureum exhibited strong inhibitory activity against AF biosynthesis. Next, large-scale culturing of C. purpureum was performed to isolate the compounds accounting for the inhibitory activity. The culture filtrate was extracted with ethyl acetate, after which the active compound was isolated by silica gel column chromatography and preparative high performance liquid chromatography (HPLC). The active compound was identified as cyclo(Val–Pro) by spectroscopic analyses. Further, four stereoisomers of cyclo(Val–Pro) were synthesized by the condensation of the N -Boc derivatives of d - and l -valine with the methyl esters of d - and l -proline. The naturally isolated compound was identified as cyclo(l -Val- l -Pro) by comparing its retention time with those of synthetic compounds by chiral HPLC analysis and CD spectra. The IC 50 value of cyclo(L-Val-L-Pro) was 2.4 mM, whereas the LD, DL, and DD isomers exhibited weaker activities, with IC 50 values of >5 mM. • Aflatoxin (AF) biosynthesis inhibitors were explored using mushroom extracts. • An extract of Chondrostereum purpureum effectively inhibited AF biosynthesis. • Cyclo(l -valyl- l -proline) was isolated from the C. purpureum culture filtrate. • Four cyclo(Val–Pro) isomers were synthesized. • Cyclo(l -valyl- l -proline) showed the strongest inhibitory activity among the isomers. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) contamination poses a fatal risk to human beings and urgently needs highly sensitive detection for environmental monitoring and food safety. However, the existing challenges are the unsatisfied sensitivity of the immunoassay methods and the complex matrix effect. Rolling circle amplification (RCA) is a promising method for nucleic acid isothermal amplification due to its high specificity and sensitivity. Herein, we constructed a general RCA-based point-of-care test method (RCA−POCT). With biotinylated antibodies, streptavidin, and biotinylated RCA primers, we realized the signal transduction and preliminary signal amplification. In this way, the fluorescent signal of the immunocomplex on the microwells was greatly enhanced. Under optimal conditions, we recorded sensitive detection limits for aflatoxin B1 (AFB1) of 1.94, 16.3, and 37.7 fg/mL (femtogram per microliter), and wide linear ranges with 5 × 10−6 to 5, 5 × 10−5 to 5, and 5 × 10−5 to 5 ng/mL in the irrigation water, field soil, and peanut samples, respectively. Satisfactory recovery, specificity, repeatability, and reproducibility were observed. The RCA−POCT was validated by comparing it to the HPLC method. This work provides a general RCA-assisted detection method for AFB1 in the environment and food. [ABSTRACT FROM AUTHOR]

Natural plant extracts offer numerous health benefits for rabbits, including improved feed utilization, antimycotic and antiaflatoxigenic effect, antioxidants, immunological modulation, and growth performance. The aim of the current study was to investigate the effects of silymarin on the performance, hemato-biochemical indices, antioxidants, and villus morphology. A total of 45 Moshtohor 4 weeks old weaned male rabbits were randomly allocated into three groups (15 rabbit/each) each group with 5 replicates. The first group served as the control group feed on an infected diet by aflatoxin B1 (AFB1) 0.02 mg/kg BW, while the second and third groups received an infected diet by AFB1 (0.02 mg/kg BW) and was treated with Silymarin 20 mg/kg BW/day or 30 mg/kg BW/day, respectively. Regarding the growth performance, silymarin supplementation significantly improved the final body weight compared with the control group. Physiologically, silymarin induced high level of dose-dependent total red blood cell count, hematocrit, eosinophils, high-density lipoprotein cholesterol, superoxid dismutase, catalase activity, total antioxidant capacityand intestinal villi width and length. Moreover, silymarin significantly restricted oxidative stress indicators, malondialdehyde, Alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglyceridein rabbits treated with (AFB1). In conclusion, silymarin supplementation to AFB1 contaminated rabbit diet may mitigate the negative effect of AFB1 on the rabbit performance and health status and increase growth performance, average daily gain, immunological modulation and antioxidants and provide a theoretical basis for the application of silymarin in livestock production. [ABSTRACT FROM AUTHOR]

Aspergillus flavus, a fungus known for producing aflatoxins, poses significant threats to agriculture and global health. Flavonoids, plant-derived compounds, inhibit A. flavus proliferation and mitigate aflatoxin production, although the precise molecular and physical mechanisms underlying these effects remain poorly understood. In this study, we investigated three flavonoids—apigenin, luteolin, and quercetin—applied to A. flavus NRRL 3357. We determined the following: (1) glycosylated luteolin led to a 10% reduction in maximum fungal growth capacity; (2) quercetin affected cell wall integrity by triggering extreme mycelial collapse, while apigenin and luteolin caused peeling of the outer layer of cell wall; (3) luteolin exhibited the highest antioxidant capacity in the environment compared to apigenin and quercetin; (4) osmotic stress assays did not reveal morphological defects; (5) flavonoids promoted cell adherence, a precursor for biofilm formation; and (6) RNA sequencing analysis revealed that flavonoids impact expression of putative cell wall and plasma membrane biosynthesis genes. Our findings suggest that the differential effects of quercetin, luteolin, and apigenin on membrane integrity and biofilm formation may be driven by their interactions with fungal cell walls. These insights may inform the development of novel antifungal additives or plant breeding strategies focusing on plant-derived compounds in crop protection. [ABSTRACT FROM AUTHOR]

A magnetic solid phase extraction (M-SPE) based on covalent organic framework materials coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the efficient determination of four aflatoxins (AFs) in tea. The samples were extracted with a mixture of acetonitrile, water and glacial acetic acid (8.0:1.9:0.1, V/V) and centrifuged. The supernatant was evaporated to almost dryness, re-dissolved in water and pH adjusted, extracted M-SPE and filtered, and the filtrate was detected in the positive ion mode. The calibration curves for all analytes were linear in the range of 0.05–50 μg/L with coefficients of determination (R2) > 0.999 2, the spiked recoveries were in the range of 67.6%-103.7%, and the relative standard deviations (RSDs) for intra- and inter-day precisions were less than 9.24%, the limits of detection (LODs) were in the range of 0.01-0.06 μg/kg, and the limits of quantification (LOQs) of the developed method were in the range of 0.05-0.20 μg/kg. The covalent organic framework material prepared in this study has a strong adsorption capacity for AFs, and M-SPE-HPLC-MS/MS is suitable for the rapid, highly sensitive and accurate detection of AFs in tea.

Abstract Background Aflatoxin B1 (AFB1) is a prevalent contaminant in agricultural products, presenting significant risks to animal health. CotA laccase from Bacillus licheniformis has shown significant efficacy in degrading mycotoxins in vitro test. The efficacy of Bacillus CotA laccase in animals, however, remains to be confirmed. A 2 × 2 factorial design was used to investigate the effects of Bacillus CotA laccase level (0 or 1 U/kg), AFB1 challenge (challenged or unchallenged) and their interactions on ducks. The purpose of this study was to evaluate the efficacy of Bacillus CotA laccase in alleviating AFB1 toxicosis of ducks. Results Bacillus CotA laccase alleviated AFB1-induced declines in growth performance of ducks accompanied by improved average daily gain (ADG) and lower feed/gain ratio (F/G). Bacillus CotA laccase ameliorated AFB1-induced gut barrier dysfunctions and inflammation testified by increasing the jejunal villi height/crypt depth ratio (VH/CD) and the mRNA expression of tight junction protein 1 (TJP1) and zonula occluden-1 (ZO-1) as well as decreasing the expression of inflammation-related genes in the jejunum of ducks. Amino acid metabolome showed that Bacillus CotA laccase ameliorated AFB1-induced amino acid metabolism disorders evidenced by increasing the level of glutamic acid in serum and upregulating the expression of amino acid transport related genes in jejunum of ducks. Bacillus CotA laccase ameliorated AFB1-induced liver injury testified by suppressing oxidative stress, inhibiting apoptosis, and downregulating the expression of hepatic metabolic enzyme related genes of ducks. Moreover, Bacillus CotA laccase degraded AFB1 in digestive tract of ducks, resulting in the reduced absorption level of AFB1 across intestinal epithelium testified by the decreased level of AFB1-DNA adduct in the liver, and the reduced content of AFB1 residues in liver and feces of ducks. Conclusions Bacillus CotA laccase effectively improved the growth performance, intestinal health, amino acid metabolism and hepatic aflatoxin metabolism of ducks fed AFB1 diets, highlighting its potential as an efficient and safe feed enzyme for AFB1 degradation in animal production. Graphical Abstract

Mycotoxins-producing fungi can enter the body of animals and humans by contaminating grain and fodder. Aflatoxins are potential carcinogens that can lead to severe health issues such as neurological disorders and liver or kidney failure. In this study, we employed a combination of the QuEChERS method and Dispersive Liquid-Liquid Microextraction (DLLME) for the extraction and determination of aflatoxins B1, B2, G1, and G2 from corn samples, using High-Performance Liquid Chromatography with Fluorescence Detection (HPLC-FLD). We investigated and optimized the key factors affecting both stages of the extraction process. The developed method was validated by assessing parameters such as the linear range, limits of detection and quantification, repeatability, and extraction recovery. The linear range of the technique, with a coefficient of determination exceeding 0.996, spanned from 0.23 to 200 ng/g. The method achieved a low limit of quantification (LOQ) of 0.23-0.38 ng/g, which is below the permissible limits for the target aflatoxins in corn. The method's reproducibility ranged from 3.9% to 7.1%, and extraction recovery was between 60% and 73%. The technique was applied to corn samples to detect aflatoxin B1 in all samples, with concentrations ranging from 1.1 to 11.2 ng/g. Overall, the proposed method is accurate, reliable, and highly efficient for analyzing and identifying aflatoxins in corn samples.

Abstract Natural plant extracts offer numerous health benefits for rabbits, including improved feed utilization, antimycotic and antiaflatoxigenic effect, antioxidants, immunological modulation, and growth performance. The aim of the current study was to investigate the effects of silymarin on the performance, hemato-biochemical indices, antioxidants, and villus morphology. A total of 45 Moshtohor 4 weeks old weaned male rabbits were randomly allocated into three groups (15 rabbit/each) each group with 5 replicates. The first group served as the control group feed on an infected diet by aflatoxin B1 (AFB1) 0.02 mg/kg BW, while the second and third groups received an infected diet by AFB1 (0.02 mg/kg BW) and was treated with Silymarin 20 mg/kg BW/day or 30 mg/kg BW/day, respectively. Regarding the growth performance, silymarin supplementation significantly improved the final body weight compared with the control group. Physiologically, silymarin induced high level of dose-dependent total red blood cell count, hematocrit, eosinophils, high-density lipoprotein cholesterol, superoxid dismutase, catalase activity, total antioxidant capacityand intestinal villi width and length. Moreover, silymarin significantly restricted oxidative stress indicators, malondialdehyde, Alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglyceridein rabbits treated with (AFB1). In conclusion, silymarin supplementation to AFB1 contaminated rabbit diet may mitigate the negative effect of AFB1 on the rabbit performance and health status and increase growth performance, average daily gain, immunological modulation and antioxidants and provide a theoretical basis for the application of silymarin in livestock production.

Objectives: This study aims to evaluate the levels of heavy metals and aflatoxins in Kermanshahi oil and grape pekmez (a concentrated grape product) from the central provinces of Iran, as well as to assess how processing affects their fatty acid profiles.Methods: Twenty samples of Kermanshahi oil and grape pekmez were randomly collected from production centers in Chaharmahal and Bakhtiari and Isfahan provinces in Iran.Results: The aflatoxin levels in both Kermanshahi oil and grape pekmez were within acceptable limits. Lead (Pb) and cadmium (Cd) concentrations in Kermanshahi oil and grape pekmez ranged from 0.001 to 0.1 ppb and 0.001 to 0.04 ppb, respectively. Traditional butter and Kermanshahi oils were found to contain high levels of SFAs, including palmitic (C16) and myristic (C14) acids, as well as unsaturated fatty acids such as oleic acid (C18:1). The composition of saturated, unsaturated, and trans fatty acids in Kermanshahi oils derived from traditional butter was measured at 77.075%, 16.34%, and 4.82%, respectively. Following the addition of grape pekmez to Kermanshahi oil, a decrease in short-chain fatty acids was observed, while palmitic, stearic, oleic, and linolenic acid levels increased. Additionally, the concentration of TFAs decreased.Conclusion: The incorporation of grape pekmez into Kermanshahi oil positively influenced the fatty acid composition, enhancing its overall quality.

This study was conducted in the Plant Pathology Laboratory/ College of Agriculture/ University of Anbar to study and test the effectiveness of plant extracts (turmeric, pomegranate peel, banana peel) in inhibiting the growth of the fungus Aspergillus flavus and test its ability to reduce aflatoxin B1 from liquid media. The plant materials (powders and extracts) were used in three concentrations (10, 15, 20) mg ml-1. The results showed that all plant materials were significantly effective in inhibiting the growth of A. flavus and their ability to reduce aflatoxin B1 from liquid media. Turmeric powder showed its superiority in its inhibitory effect on the growth of fungi on PDA culture medium, as it achieved the highest percentage of inhibition at a concentration of 20 mg ml-1, which amounted to 87.41%. Whereas, banana peel powder gave the lowest inhibition rate at 10mg ml-1 concentration) 64.07% (. While the aqueous extract of turmeric achieved inhibition in the growth of the fungus at a concentration of 20 mg ml-1 by 87.41%, outperformed the aqueous extract of pomegranate and banana, where the rates of inhibition reached 86.66% and 82.96%, respectively at the same concentration. In addition, the alcoholic extract of turmeric showed the highest rate of inhibition of fungi growth at a concentration of 20 mg ml-1, which amounted to 94.07%, followed by the alcoholic extract of banana and pomegranate with a percentage of 93.3% and 88.14%, respectively, at the same concentration.

Oral adsorbents have attracted increasing attention as a potential therapeutic strategy for the removal of toxic substances from the gastrointestinal tract. This review aims to provide a comprehensive overview of the structural and mechanic properties of oral adsorbents such as activated charcoal, medical clays, zeolites, silica-based adsorbents, dietary fibers (carbohydrates alginate, chitosan, pectin, carrageenan etc.), and their physicochemical properties. The review also explores the mechanisms of action of oral adsorbents, including physical adsorption, ion exchange, and specific binding interactions. Furthermore, we provide an overview of the factors that influence the adsorption capacity and selectivity of oral adsorbents. The summary of recent advances in the usage of thiolated adsorbents is discussed, which provides valuable insights into the structural and mechanistic properties of oral adsorbents and their potential as a therapeutic option for the removal of toxic substances from the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effect of bay leaf (Syzygium polyanthum) infusion on preserving the quality of corn during storage. The research employed an experimental approach using a Completely Randomized Design (CRD) with five treatments and four replications. The treatments included A (control corn), B (25% (v/v) bay leaf infusion), C (50% (v/v) bay leaf infusion), D (75% (v/v) bay leaf infusion), and E (100% (v/v) bay leaf infusion). The observed variables included damaged kernels, moldy kernels, moisture content, and aflatoxin content. Corn with high aflatoxin content was immersed in bay leaf infusion according to the respective treatments and then stored for four weeks. The research results showed that soaking corn with high aflatoxin content in bay leaf infusion (had a significant effect (P<0.01) on moisture and aflatoxin content but did not have a considerable impact (P>0.05) on damaged kernels and moldy kernels. Based on the research findings, it was concluded that the application of 100% (v/v) bay leaf infusion improved the quality of corn during a 4 -week storage period, with the percentage of damaged kernels at 4.75%, moldy grains at 0.75%, moisture content at 14.60%, and a decrease in aflatoxin content by 41.11% (150 -88.33 ppb). [ABSTRACT FROM AUTHOR]

Aspergillus flavus is a cosmopolitan saprophytic fungus that infests several foodstuffs and is associated with adverse effects in humans. In Senegal, significant losses of groundnut production are mainly due to contamination caused by this species. This study evaluated in vitro antifungal activities of Cyperus articulatus, Cyperus rotundus and Lippia alba essential oils against A. flavus isolated from peanut seeds. Essential oils obtained by hydrodistillation of rhizomes of the two Cyperus species and leaves of L. alba were analyzed with GC-DIF and GC-MS. The essential oil yields from C. articulatus, C. rotundus and L. alba were 1.1%, 1.3% and 1.7%, respectively. These three samples had the following chemotypes: (i) mustakone (21.4%)/eudesma-4(15)-7-dien-1β-ol (8.8%)/caryophyllene oxide (5.9%), (ii) caryophyllene oxide (25.2%)/humulene epoxyde 2 (35.0%) and (iii) geranial (46.6%)/neral (34.6%). The three oils tested inhibited the growth of A. flavus at concentrations between 100 and 1000 ppm. The L. alba oil was the most effective with total clearance of A. flavus on PDA. For the essential oils of C. rotundus (93.65%) and C. articulatus (78.11%), the highest inhibition rates were obtained with a 1000 ppm dose. Thus, L. alba oil could be used safely as an effective protector of groundnuts against A. flavus. [ABSTRACT FROM AUTHOR]

Approximately 25% of cereal grains present with contamination caused by fungi and the presence of mycotoxins that may cause severe adverse effects when consumed. Maize has been genetically engineered to present different traits, such as fungal or insect resistance and herbicide tolerance. This systematic review compared the observable quantities, via meta-analysis, of four mycotoxins (aflatoxins—AFL, fumonisins—FUM, deoxynivalenol—DON, zearalenone—ZEA) between genetically modified (GM) and conventional maize kernels. This study was conducted following the PRISMA guidelines, with searches performed using PubMed, Web of Science, Scopus, Google Scholar, and CAPES journals databases. Analyses were conducted using RevMan v.5.4 software. Transgenic maize showed a 58% reduction in total mycotoxins (p < 0.001) compared to conventional maize. FUM were the most impacted, with a 59% reduction (p < 0.001) in GM maize. AFL and ZEA levels were also lower in GM maize by 49% (p = 0.02) and 51% (p < 0.001), respectively. On the other hand, DON levels increased by 6% (p < 0.001) in GM maize compared to conventional maize. However, results for ZEA and DON were inconclusive due to the limited research and sample sizes. We conclude that transgenic maize reduces total mycotoxins by over 50%, primarily fumonisin and aflatoxin. Most studies presented maize varieties that were resistant to insects or herbicides, not fungal pathogens, showing a positive collateral effect of these genetic alterations. Therefore, transgenic maize appears to be a safer product for animal and human consumption from a toxicological point of view. Further studies with larger sample sizes are needed to confirm our findings for ZEA and DON in transgenic maize. [ABSTRACT FROM AUTHOR]

The climate-change-coupled fungal burden in crop management and the need to reduce chemical pesticide usage highlight the importance of finding sustainable ways to control Aspergillus flavus. This study examines the effectiveness of 50 Pseudomonas isolates obtained from corn rhizospheres against A. flavus in both solid and liquid co-cultures. The presence and quantity of aflatoxin B1 (AFB1) and AFB1-related compounds were determined using high-performance liquid chromatography–high resolution mass spectrometry analysis. Various enzymatic- or non-enzymatic mechanisms are proposed to interpret the decrease in AFB1 production, accompanied by the accumulation of biosynthetic intermediates (11-hydroxy-O-methylsterigmatocystin, aspertoxin, 11-hydroxyaspertoxin) or degradation products (the compounds C16H10O6, C16H14O5, C18H16O7, and C19H16O8). Our finding implies the upregulation or enhanced activity of fungal oxidoreductases and laccases in response to bacterial bioactive compound(s). Furthermore, non-enzymatic reactions resulted in the formation of additional degradation products due to acid accumulation in the fermented broth. Three isolates completely inhibited AFB1 or any AFB1-related compounds without significantly affecting fungal growth. These bacterial isolates supposedly block the entire pathway for AFB1 production in the fungus during interaction. Apart from identifying effective Pseudomonas isolates as potential biocontrol agents, this work lays the foundation for exploring new bacterial bioactive compounds. [ABSTRACT FROM AUTHOR]

Food crops around the world are commonly contaminated with Aspergillus flavus, which can produce the carcinogenic mycotoxin aflatoxin B1 (AFB1). The objective of this study is to test an X-ray irradiation sterilization method for studying AFB1 in contaminated maize samples in the laboratory. Maize that had been naturally contaminated with 300 ppb AFB1 by the growth of aflatoxigenic A. flavus was ground and then irradiated at 0.0, 1.0, 1.5, 2.0, 2.5, and 3.0 kGy. A. flavus was quantified by dilution plating on potato dextrose agar (PDA) and modified Rose Bengal media (MDRB) for viability and qPCR for gene presence. AFB1 was quantified by HPLC and ELISA. A. flavus viability, but not gene copies, significantly decreased with increasing doses of radiation (PDA: p < 0.001; MDRB: p < 0.001; qPCR: p = 0.026). AFB1 concentration did not significantly change with increasing doses of radiation (HPLC: p = 0.153; ELISA: p = 0.567). Our results imply that X-ray irradiation is an effective means of reducing viable A. flavus without affecting AFB1 concentrations. Reducing the hazard of fungal spores and halting AFB1 production at the targeted dose are important steps to safely and reproducibly move forward research on the global mycotoxin challenge. [ABSTRACT FROM AUTHOR]

Aspergillus flavus produces aflatoxin, a carcinogenic fungal toxin that poses a threat to the agricultural and food industries. There is a concern that the distribution of aflatoxin-producing A. flavus is expanding in Japan due to climate change, and it is necessary to understand what types of strains inhabit. In this study, we sequenced the genomes of four Aspergillus strains isolated from agricultural fields in the Ibaraki prefecture of Japan and identified their genetic variants. Phylogenetic analysis based on single-nucleotide variants revealed that the two aflatoxin-producing strains were closely related to A. flavus NRRL3357, whereas the two non-producing strains were closely related to the RIB40 strain of Aspergillus oryzae, a fungus widely used in the Japanese fermentation industry. A detailed analysis of the variants in the aflatoxin biosynthetic gene cluster showed that the two aflatoxin-producing strains belonged to different morphotype lineages. RT-qPCR results indicated that the expression of aflatoxin biosynthetic genes was consistent with aflatoxin production in the two aflatoxin-producing strains, whereas the two non-producing strains expressed most of the aflatoxin biosynthetic genes, unlike common knowledge in A. oryzae, suggesting that the lack of aflatoxin production was attributed to genes outside of the aflatoxin biosynthetic gene cluster in these strains. [ABSTRACT FROM AUTHOR]

Non-genetic variation limits the identification of novel maize germplasm with genetic markers for reduced Aspergillus flavus infection and aflatoxin contamination. Aflatoxin measurements can vary substantially within fields containing the same germplasm following inoculation with A. flavus. While some variation is expected due to microenvironmental differences, components of field screening methodologies may also contribute to variability in collected data. Therefore, the objective of this study is to test the effects of three different shelling methods (whole ear (WE), ear end removal (EER), and inoculation site-surrounding (ISS)) to obtain bulk samples from maize on aflatoxin measurements. Five ears per row of three inbred lines and two hybrids were inoculated with A. flavus, then shelled using the three different methods, and aflatoxin was quantified. Overall, EER and ISS resulted in reduced coefficients of variance (CVs) in comparison to WE for both inbred and hybrid maize lines, with two exceptions. Susceptible B73 showed increased CVs with both EER and ISS compared to WE, and resistant Mp719's EER CVs marginally increased compared to WE. While WE is the standard practice for most breeding programs due to its technical simplicity, EER and ISS may allow for finely phenotyping parental lines for further breeding applications. [ABSTRACT FROM AUTHOR]

Chemical pesticides help reduce crop loss during production and storage. However, the carbon footprints and ecological costs associated with this strategy are unsustainable. Here, we used three in vitro models to characterize how different Trichoderma species interact with two aflatoxin producers, Aspergillus flavus and Aspergillus parasiticus, to help develop a climate-resilient biological control strategy against aflatoxigenic Aspergillus species. The growth rate of Trichoderma species is a critical factor in suppressing aflatoxigenic strains via physical interactions. The dual plate assay suggests that Trichoderma mainly suppresses A. flavus via antibiosis, whereas the suppression of A. parasiticus occurs through mycoparasitism. Volatile organic compounds (VOCs) produced by Trichoderma inhibited the growth of A. parasiticus (34.6 ± 3.3%) and A. flavus (20.9 ± 1.6%). The VOCs released by T. asperellum BTU and T. harzianum OSK-34 were most effective in suppressing A. flavus growth. Metabolites secreted by T. asperellum OSK-38, T. asperellum BTU, T. virens OSK-13, and T. virens OSK-36 reduced the growth of both aflatoxigenic species. Overall, T. asperellum BTU was the most effective at suppressing the growth and aflatoxin B1 production of both species across all models. This work will guide efforts to screen for effective biological control agents to mitigate aflatoxin accumulation. [ABSTRACT FROM AUTHOR]

Aflatoxin intoxication has been reported in most species; however, it is infrequently documented in sheep. In this report, three 7-month-old Romane ewe lambs were found with severe icterus, tachypnea, slight fever, diarrhea, anorexia, and lethargy. Two of them were culled, and one recovered after treatment. Necropsy findings showed jaundice, ascites, diffuse hemorrhage on the peritoneum, gall bladder enlargement, and alteration in the color and texture of the liver and kidney. Histopathological evaluation revealed hepatocellular necrosis, megalocytosis, centrilobular to bridging fibrosis in the liver, and hemoglobinuria without nephrotic degeneration in the kidney. The polymerase chain reaction test for leptospirosis as the main differential diagnosis was conducted, and the result was negative. Similarly, the Warthin Starry special staining for spirochetes was negative. Clinical signs and characteristics of microscopic lesions were suggestive of aflatoxicosis in the lambs described in this investigation. As sheep flocks with intensive rearing systems are becoming more common in Iran, the emergence of aflatoxicosis due to fungi-contaminated feedstuff can be a serious health concern in such farms. [ABSTRACT FROM AUTHOR]

Aspergillus flavus is notorious for contaminating food with its secondary metabolite—highly carcinogenic aflatoxins. In this study, we found that exogenous nitric oxide (NO) donor could influence aflatoxin production in A. flavus. Flavohemoglobins (FHbs) are vital functional units in maintaining nitric oxide (NO) homeostasis and are crucial for normal cell function. To investigate whether endogenous NO changes affect aflatoxin biosynthesis, two FHbs, FHbA and FHbB, were identified in this study. FHbA was confirmed as the main protein to maintain NO homeostasis, as its absence led to a significant increase in intracellular NO levels and heightened sensitivity to SNP stress. Dramatically, FHbA deletion retarded aflatoxin production. In addition, FHbA played important roles in mycelial growth, conidial germination, and sclerotial development, and response to oxidative stress and high-temperature stress. Although FHbB did not significantly impact the cellular NO level, it was also involved in sclerotial development, aflatoxin synthesis, and stress response. Our findings provide a new perspective for studying the regulatory mechanism of the development and secondary mechanism in A. flavus. [ABSTRACT FROM AUTHOR]

Fungal infections were observed on the coffee plant, this study aims to determine the types of fungi causing this pollution and the possibility of producing mycotoxins or not. so we make this study to isolate and identify the contaminating fungi that find in three types of Arabica coffee plants, which are common in the city of Jazan in the Kingdom of Saudi Arabia, namely (Khawlani, Luqmati and Harari) by using the dilution method on Potato Dextrose Agar media (PDA). The results showed that some coffee samples were contaminated with fungi, which belong to 5 fungal genera, most of which belong to the Ascomycetes fungi. The species Aspergillus niger had the highest percentage of appearance in the coffee samples, followed by Aspergillus flavus and then Penicillium sp. The ammonia detection results showed that most Aspergillus flavus and A. ochrous isolates produced mycotoxins, while all isolates of Aspergillus niger and Penicillium sp. do not produce aflatoxins. [ABSTRACT FROM AUTHOR]

Foodborne diseases can be attributed not only to contamination with bacterial or fungal pathogens but also their associated toxins. Thus, to maintain food safety, innovative decontamination techniques for toxins are required. We previously demonstrated that an atmospheric-pressure dielectric-barrier discharge (APDBD) plasma generated by a roller conveyer plasma device is effective at inactivating bacteria and fungi in foods. Here, we have further examined whether the roller conveyer plasma device can be used to degrade toxins produced by foodborne bacterial pathogens, including aflatoxin, Shiga toxins (Stx1 and Stx2), enterotoxin B and cereulide. Each toxin was spotted onto an aluminum plate, allowed to dry, and then treated with APDBD plasma applied by the roller conveyer plasma device for different time periods. Assessments were conducted using a competitive enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). The results demonstrate a significant time-dependent decrease in the levels of these toxins. ELISA showed that aflatoxin B1 concentrations were reduced from 308.6 µg/mL to 74.4 µg/mL within 1 min. For Shiga toxins, Stx1 decreased from 913.8 µg/mL to 65.1 µg/mL, and Stx2 from 2309.0 µg/mL to 187.6 µg/mL within the same time frame (1 min). Enterotoxin B levels dropped from 62.67 µg/mL to 1.74 µg/mL at 15 min, and 1.43 µg/mL at 30 min, but did not display a significant decrease within 5 min. LC-MS/MS analysis verified that cereulide was reduced to below the detection limit following 30 min of APDBD plasma treatment. Taken together, these findings highlight that a range of foodborne toxins can be degraded by a relatively short exposure to plasma generated by an APDBD using a roller conveyer device. This technology offers promising advancements in food safety, providing a novel method to alleviate toxin contamination in the food processing industry. [ABSTRACT FROM AUTHOR]

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BackgroundAflatoxin (AF) exposure is associated with child growth faltering in cross-sectional studies, with limited findings from longitudinal studies.ObjectivesTo evaluate the relationship between maternal AF B1-lysine adduct concentration, child AF B1-lysine adduct concentration, and child growth in the first 30 mo of life.MethodsAF B1-lysine adduct was measured in mother-child dyad plasma samples using isotope dilution mass spectrometry. Using linear regression, we assessed the relationship between AF B1-lysine adduct concentration and child weight, height, and head and mid-upper arm circumferences at 1 wk, 6, 12, 18, 24, and 30 mo of age.ResultsIn adjusted models, maternal prenatal AF B1-lysine adduct (pg/μL) was positively associated with newborn anthropometric outcomes; largest beta coefficients for associations between standardized values were for newborn weight-for-age z-score [β = 0.13; 95% confidence interval (CI): 0.02, 0.24; P < 0.05 and β = 0.11; 95% CI: 0.00, 0.22; P < 0.05 for second and third trimester AF, respectively]. Child AF B1-lysine adduct (pg/μL) at 6 mo was negatively associated with head circumference-for-age z-score at 6, 18, 24, and 30 mo, with beta coefficients ranging from β = -0.15; 95% CI: -0.28, -0.02 to β = -0.17; 95% CI: -0.31, -0.03; P < 0.05); 18-mo AF was negatively associated with anthropometric outcomes at 18, 24, and 30 mo, most consistently with length-for-age z-score (β = -0.18; 95% CI: -0.32, -0.04, β = -0.21; 95% CI: -0.35, -0.07, β = -0.18; 95% CI: -0.32, -0.03 at 18, 24 and 30 mo, respectively).ConclusionsChild AF exposure was associated with impaired child growth, but maternal AF exposure was not. Exposure during infancy was linked to persistent deficit in head circumference, implying reduced brain size lasting beyond the age of 2 years. Exposure at 18 mo was linked to persistent linear growth deficit. Further research should elucidate mechanisms through which AF affects child growth.

Rapid identification of peanut seed quality is crucial for public health. In this study, we present a terahertz wave imaging system using a convolutional neural network (CNN) machine learning approach. Terahertz waves are capable of penetrating the seed shell to identify the quality of peanuts without causing any damage to the seeds. The specificity of seed quality on terahertz wave images is investigated, and the image characteristics of five different qualities are summarized. Terahertz wave images are digitized and used for training and testing of convolutional neural networks, resulting in a high model accuracy of 98.7% in quality identification. The trained THz-CNNs system can accurately identify standard, mildewed, defective, dried and germinated seeds, with an average detection time of 2.2 s. This process does not require any sample preparation steps such as concentration or culture. Our method swiftly and accurately assesses shelled seed quality non-destructively.

The potential of Raman hyperspectral imaging with a 785 nm excitation line laser was examined for the detection of aflatoxin contamination in corn kernels. Nine-hundred kernels were artificially inoculated in the laboratory, with 300 kernels each inoculated with AF13 (aflatoxigenic) fungus, AF36 (nonaflatoxigenic) fungus, and sterile distilled water (control). One-hundred kernels from each treatment were subsequently incubated for 3, 5, and 8 days. The mean spectra of single kernels were extracted from the endosperm side and the embryo area of the germ side, and local Raman peaks were identified based upon the calculated reference spectra of aflatoxin-negative and -positive categories separately. The principal component analysis-linear discriminant analysis models were established using different types of variable inputs including original full spectra, preprocessed full spectra, and identified local peaks over kernel endosperm-side, germ-side, and both sides. The results of the established discriminant models showed that the germ-side spectra performed better than the endosperm-side spectra. Based upon the 20 ppb-threshold, the best mean prediction accuracy of 82.6% was achieved for the aflatoxin-negative category using the original spectra in the combined form of both kernel sides, and the best mean prediction accuracy of 86.7% was obtained for the -positive category using the preprocessed germ-side spectra. Based upon the 100 ppb-threshold, the best mean prediction accuracies of 85.0% and 89.6% were achieved for the aflatoxin-negative and -positive categories separately, using the same type of variable inputs for the 20 ppb-threshold. In terms of overall prediction accuracy, the models established upon the original spectra in the combined form of both kernel sides achieved the best predictive performance, regardless of the threshold. The mean overall prediction accuracies of 81.8% and 84.5% were achieved with the 20 ppb- and 100 ppb-thresholds, respectively.

SUMMARY: The poor quality and safety of poultry feed in developing countries pose significant concerns as they negatively impact poultry performance and present potential health risks to human consumers. Aflatoxicosis, resulting from postharvest contamination of feed ingredients by certain mold species, severely affects birds' overall health and productivity. To address these challenges, various feed additives have become increasingly important in poultry nutrition. Phytogenics, natural plant-derived products, have gained popularity for their ability to mitigate aflatoxicosis by their rich composition of bioactive compounds that offer beneficial effects on physiological and immunological functions. In light of the growing significance of aflatoxicosis in poultry nutrition, this review provides an overview of aflatoxins and their harmful effects on poultry health. It highlights the role of phytogenic compounds as growth and health stimulators, emphasizing their potential application in poultry nutrition. Furthermore, the review explores the antioxidant and immunological mechanisms through which phytogenics mitigate aflatoxicosis in poultry, including specific compounds and pathways involved. The findings reveal that aflatoxins induce inflammation, and oxidative stress in poultry, resulting in cellular damage and immune dysfunction. Phytogenics play a crucial role in ameliorating the harmful effects of aflatoxins in birds by countering aflatoxin-induced oxidative stress, reducing inflammation, and restoring gut health and immune functions. In conclusion, phytogenics offer an invaluable tool for the mitigation of aflatoxicosis in poultry and serve as potent natural and safe alternatives to antibiotics and synthetic antioxidants, promoting sustainable production practices.

Aflatoxins, potent carcinogens produced by Aspergillus species (A. flavus and A. parasiticus), are primarily consumed through dietary sources. Aflatoxin management using non-aflatoxigenic Aspergillus to outcompete their aflatoxigenic congeneric to achieve low residual toxin levels is a promising biological control (BC) field intervention. A prerequisite for the BC program is the accurate delineation of aflatoxigenic fungi from their non-aflatoxigenic relatives. In this study, we use three methods: Chemical (Dichlorvos-ammonia), Metabolic (ELISA coupled live cultures), and Molecular (10 aflatoxin biosynthetic genes) to determine the aflatoxigenicity of Aspergillus spp. previously collected from Eastern Kenya, an aflatoxin hotspot. Thirty-six different Aspergillus isolates were identified and studied microscopically from a total of 314 fungal cultures. The chemical method reported 21 aflatoxigenic and 15 non-aflatoxigenic isolates, while the metabolic technique distinguished 20 aflatoxigenic and 16 non-aflatoxigenic isolates. A large proportion (97.22 %) of the isolates harboured the aflR, aflO, and aflM genes, while 91.67 % of the isolates possessed the aflS gene. All isolates (100 %) harboured the aflD, aflQ, aflP, and aflK genes. ELISA-coupled live cultures positively correlated with Dichlorvos-ammonia (p > 0.01), only differing in one sample, indicating a strong concurrence between the two methods at segregating toxigenic A. flavus isolates. Cluster analysis based on Multiple Correspondence revealed that the non-aflatoxigenic group exhibited similar characteristics correlating with the absence of aflR, aflS, aflI, and aflO genes. These findings indicate the reliability of the Chemical and Metabolic methods and can function as confirmatory assays to complement the molecular detection approach in determining aflatoxigenicity among Aspergillus spp.

Abstract Aflatoxin, mainly produced by Aspergillus flavus, is one of the most notorious mycotoxin for its toxicity and carcinogenicity. Despite extensive efforts, effective strategies to control A. flavus and AFB1 contamination remain elusive. Here, we investigate the potential of p‐anisaldehyde (AS), an aldehyde derived from plant essential oils, as a natural antifungal agent against A. flavus and its ability to modulate AFB1 biosynthesis. We found that AS exhibited broad‐spectrum antifungal activities against Aspergillus spp. and effectively inhibited A. flavus asexual development, AFB1 production, and pathogenicity. AS treatment disrupted the cell surface structure and membrane integrity, as observed by scanning electron microscopy and PI staining. RNA‐sequencing analysis revealed that AS significantly altered the expression of genes involved in redox homeostasis, plasma membrane function, and cell cycle progression. Further investigation demonstrated that AS induced a reduction in mitochondrial membrane potential (Δψm) and accumulation of reactive oxygen species (ROS), leading to cell cycle arrest at the G2/M phase. The AS‐induced ROS accumulation was found to be mitigated by the superoxide dismutase‐mediated antioxidant system, which is regulated by transcriptional factor Ap1. Notably, the Ap1‐regulatory ROS detoxification system was also found to be involved in A. flavus pathogenicity and AFB1 production. Overall, these findings provide valuable insights into the inhibitory mechanism of AS against A. flavus, paving the way for its potential application as a natural strategy to mitigate AFB1 contamination in both food and agriculture crops.

Abstract Maize (Zea mays), a major food crop worldwide, is susceptible to infection by the saprophytic fungus Aspergillus flavus that can produce the carcinogenic metabolite aflatoxin (AF) especially under climate change induced abiotic stressors that favor mold growth. Several studies have used “-omics” approaches to identify genetic elements with potential roles in AF resistance, but there is a lack of research identifying the involvement of small RNAs such as microRNAs (miRNAs) in maize-A. flavus interaction. In this study, we compared the miRNA profiles of three maize lines (resistant TZAR102, moderately resistant MI82, and susceptible Va35) at 8 h, 3 d, and 7 d after A. flavus infection to investigate possible regulatory antifungal role of miRNAs. A total of 316 miRNAs (275 known and 41 putative novel) belonging to 115 miRNA families were identified in response to the fungal infection across all three maize lines. Eighty-two unique miRNAs were significantly differentially expressed with 39 miRNAs exhibiting temporal differential regulation irrespective of the maize genotype, which targeted 544 genes (mRNAs) involved in diverse molecular functions. The two most notable biological processes involved in plant immunity, namely cellular responses to oxidative stress (GO:00345990) and reactive oxygen species (GO:0034614) were significantly enriched in the resistant line TZAR102. Coexpression network analysis identified 34 hubs of miRNA-mRNA pairs where nine hubs had a node in the module connected to their target gene with potentially important roles in resistance/susceptible response of maize to A. flavus. The miRNA hubs in resistance modules (TZAR102 and MI82) were mostly connected to transcription factors and protein kinases. Specifically, the module of miRNA zma-miR156b-nb – squamosa promoter binding protein (SBP), zma-miR398a-3p – SKIP5, and zma-miR394a-5p – F-box protein 6 combinations in the resistance-associated modules were considered important candidates for future functional studies.

Abstract Background Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. Results Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. Conclusion In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

ABSTRACT Aflatoxin, produced by Aspergillus flavus and Aspergillus paraciticus, commonly contaminates agricultural and processed products. This study aims to analyze aflatoxin levels in fried onions produced in Palu through high-performance liquid chromatography (HPLC) using an immunoaffinity column an a fluorescent detector at an excitation wavelength of 360 nm and an emission wavelength of 440 nm. The standard working chromatogram of aflatoxin gave a peak response at retention times of 28.618 minutes for aflatoxin B1, 21.985 minutes for aflatoxin B2, 17.625 minutes for aflatoxinn G1, and 13.719 minutes for aflatoxin G2. The limit of detection (LOD) values for each standard were : aflatoxin B1 : 0.2607 ng/ml; aflatoxin B2 : 0.0600 ng/ml; aflatoxin G1 : 0.1986 ng/ml; and aflatoxin G2 : 0.0310 ng/ml. The chromatogram results of the 10 fried onion samples analyzed showed no peak response with the same retention time as the standard aflatoxin retention time, so that the 10 samples can be concluded as meet requirements. Keywords: Aflatoxin, HPLC, Immunoaffinity colum, P Palu fried shallot

Objectives: This investigation aimed to assess the growth response of Nubian goats to the inclusion of Nigella sativa (NS) seeds in a ration contaminated with aflatoxin (AF). Materials and Methods: Twenty healthy, growing male goats were randomly assigned to five groups in a completely randomized design. The control group was provided with a basic ration, whereas the treatment groups were subjected to an identical ration contaminated with 150 ppb of AF (AF + NS-0), supplemented with crushed NS seeds at levels of 2% (AF + NS-2), 4% (AF + NS-4), and 6% (AF + NS-6) of total dry matter. Daily feed intake and weekly body weight were recorded to evaluate growth parameters such as dry matter intake (DMI), average daily gain (ADG), feed conversion ratio (FCR), and protein efficiency ratio (PER). Results: AF contamination significantly suppressed DMI, ADG, FCR, and PER, whereas NS seed supplementation improved ADG, FCR, and PER. The weekly DMI did not differ significantly between the control group and those supplemented with NS seeds in the AF-contaminated ration. Over the 6-week experimental period, NS seed supplementation exhibited a positive impact on growth performance, with the 6% NS seed supplementation showing superior effects. The study highlighted a strong correlation between growth parameters and NS levels, supported by higher R2 values. Conclusion: The findings recommend a 6% NS seed supplementation in goat rations as a potential strategy to mitigate suspected AF contamination. Further examinations are necessary to explore the potential protective effects of NS seeds on different animal species exposed to rations contaminated with AF. [Vet. Res. Notes 2024; 4(3.000): 30-37]

AbstractThe traditional cooking of dry maize grain for preparing either boiled maize (nifro) or roasted maize (qollo) in Ethiopia is preceded by soaking dry maize in water for a certain time. This study aimed to assess the practice of soaking dry maize grain and determine the effect of soaking water volume (0, 200 or 300 ml per 100 g), soaking time (12 or 48 h), soaking water temperature (warm or cold water) and type of cooking method (boiling and roasting) on the aflatoxin content of traditional Ethiopian cooked maize, nifro and qollo. The survey and laboratory experiments used semi-structured interview questionnaire and fractional factorial design, respectively. Aflatoxins were extracted from aflatoxigenic Aspergillus flavus spores inoculated onto raw maize, soaked in water and finally cooked by boiling or roasting of the soaked maize grain. Cleanup was done using Immuno Affinity Column and analyzed using Ultra High Performance Liquid Chromatography with fluorescence detector. Soaking dry maize grain in excess water (300 ml water/100 g maize) varying the soaking water temperature and duration before boiling and roasting had resulted in significant total aflatoxin reduction (p

Aims: The current review aims to outline and summarize the latest research on aflatoxin, with research studies describing natural, herbal and chemical compound applications in animal (pig) models and in vitro cellular studies. Aflatoxin, a carcinogenic toxin metabolite, is produced by Aspergillus flavus in humid environments, posing a threat to human health and crop production. The current treatment involves the prevention of exposure to aflatoxin and counteracting its harmful toxic effects, enabling survival and research studies on an antidote for aflatoxin. Objectives: To summarize current research prospects and to outline the influence of aflatoxin on animal forage in farm production, food and crop processing. The research application of remedies to treat aflatoxin is undergoing development to pinpoint biochemical pathways responsible for aflatoxin effects transmission and actions of treatment. Significance: To underline the environmental stress of aflatoxin on meat and dairy products; to describe clinical syndromes associated with aflatoxicosis on human health that are counteracted with proposed treatment and preventive interventions. To understand how to improve the health of farm animals with feed conditions. [ABSTRACT FROM AUTHOR]

Maize (Zea mays), a major food crop worldwide, is susceptible to infection by the saprophytic fungus Aspergillus flavus that can produce the carcinogenic metabolite aflatoxin (AF) especially under climate change induced abiotic stressors that favor mold growth. Several studies have used "-omics" approaches to identify genetic elements with potential roles in AF resistance, but there is a lack of research identifying the involvement of small RNAs such as microRNAs (miRNAs) in maize-A. flavus interaction. In this study, we compared the miRNA profiles of three maize lines (resistant TZAR102, moderately resistant MI82, and susceptible Va35) at 8 h, 3 d, and 7 d after A. flavus infection to investigate possible regulatory antifungal role of miRNAs. A total of 316 miRNAs (275 known and 41 putative novel) belonging to 115 miRNA families were identified in response to the fungal infection across all three maize lines. Eighty-two unique miRNAs were significantly differentially expressed with 39 miRNAs exhibiting temporal differential regulation irrespective of the maize genotype, which targeted 544 genes (mRNAs) involved in diverse molecular functions. The two most notable biological processes involved in plant immunity, namely cellular responses to oxidative stress (GO:00345990) and reactive oxygen species (GO:0034614) were significantly enriched in the resistant line TZAR102. Coexpression network analysis identified 34 hubs of miRNA-mRNA pairs where nine hubs had a node in the module connected to their target gene with potentially important roles in resistance/susceptible response of maize to A. flavus. The miRNA hubs in resistance modules (TZAR102 and MI82) were mostly connected to transcription factors and protein kinases. Specifically, the module of miRNA zma-miR156b-nb – squamosa promoter binding protein (SBP), zma-miR398a-3p – SKIP5, and zma-miR394a-5p – F-box protein 6 combinations in the resistance-associated modules were considered important candidates for future functional studies. [ABSTRACT FROM AUTHOR]

Background: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. Results: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. Conclusion: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research. [ABSTRACT FROM AUTHOR]

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus. [ABSTRACT FROM AUTHOR]

Introduction: Identification of chemical toxins from complex or highly processed foods can present 'needle in the haystack' challenges for chemists. Metagenomic data can be used to guide chemical toxicity evaluations by providing DNA-based description of the wholistic composition (eukaryotic, bacterial, protozoal, viral, and antimicrobial resistance) of foods suspected to harbor toxins, allergens, or pathogens. This type of information can focus chemistry-based diagnostics, improve hazard characterization and risk assessment, and address data gaps. Additionally, there is increasing recognition that simultaneously co-occurring mycotoxins, either from single or multiple species, can impact dietary toxicity exposure. Metagenomic data provides a way to address data gaps related to cooccurrence of multiple fungal species. Methods: Paired metagenomic and chemical data were used to evaluate aflatoxin-contaminated kibble with known levels of specific mycotoxins. Kibble was ground to a fine powder for both chemical and molecular analyses. Chemical analyses were performed with Liquid Chromatography Mass Spectrometry (LCMS) and according to the AOAC Official method 2005.08: Aflatoxins in Corn, Raw Peanuts, and Peanut Butter using Liquid Chromatography with Post-Column Photochemical Derivatization. Metagenomes were created from DNA extracted from ground kibble and sequenced on an Illumina NextSeq 2000 with an average sequence depth of 180 million reads per replicate. Results and discussion: Metagenomic data demonstrated that the abundance of DNA from putative aflatoxigenic Aspergillus spp. correlated with the levels of aflatoxin quantified by LCMS. Metagenomic data also identified an expansive range of co-occurring fungal taxa which may produce additional mycotoxins. DNA data paired with chemical data provides a novel modality to address current data gaps surrounding dietary mycotoxin exposure, toxigenic fungal taxonomy, and mycotoxins of emerging concern. [ABSTRACT FROM AUTHOR]

Aspergillus flavus is a ubiquitous pathogen that can infect many foods and grains, and it produces large amounts of aflatoxins during their storage. Aflatoxins are considered highly toxic and carcinogenic to humans, and they cause great damage to crop production, food security, and human health. Thus, controlling A. flavus and aflatoxins in grains presents a great challenge to humans worldwide. Over the past decade, many strategies have been demonstrated to be useful in controlling A. flavus and aflatoxins during food storage. These methods involve physical agents, chemical agents, biological agents, etc. Some of these methods are currently used in actual production. In this review, we summarize the reported methods for controlling A. flavus and aflatoxins during food storage in the past ten years and elucidate their advantages and disadvantages. The methods discussed include irradiation technology; low oxygen atmospheres; chemical fungicides (benzalkonium chloride, iodine, ammonium bicarbonate, and phenolic and azole compounds); biological agents from plants, animals, and micro-organisms; and aflatoxin elimination methods. We expect that this review will promote the applications of current strategies and be useful for the development of novel technologies to prevent or eliminate A. flavus and aflatoxins in food and feed during storage. [ABSTRACT FROM AUTHOR]

Simple Summary: Mycotoxins, which are secondary metabolites of fungi, have several adverse effects on both animal and human organisms. Because of climate change, mycotoxins are gradually spreading from the south to the north and west in Europe, causing a growing risk for feed and food. The toxic influences of mycotoxins are intensively studied in domestic animals, but much less is known about the game species. Reproductive abnormalities have been observed in a valuable game species, the fallow deer. We supposed mycotoxin contamination to be one of the possible causes because it is known to be dangerous even at low toxin levels, especially for young animals. As the mycotoxin exposure of the fallow deer living in a forest–agriculture complex habitat is not known, our goals were (i) to determine the mycotoxin presence in hinds; (ii) to detect the spatial and individual variability in mycotoxin levels; and (iii) to reveal the mycotoxin concentration in the fetuses in relation to their mothers. A total of 72 fallow deer embryos and their mothers were sampled in seven forested regions in Hungary in the 2020/2021 hunting season. Mycotoxin metabolites were analyzed by ELISA from the liver tissue. We detected the studied mycotoxins (Aflatoxin, Zearalenone, Fumonisin B1, DON, T2/HT2-toxin) in 41–96% of fallow deer hinds and 3–98% of the fetuses. All mycotoxins were passed into the fetus, but only Fumonisin B1 rarely passed. The individual variability of mycotoxin levels was extremely high, which obscured the spatial differences among the study sites. We concluded a possible accumulation of ZEA and DON in the fetuses because of the higher concentrations in the fetus than in the hind. These results reflect the potential threats of mycotoxins to the population dynamics and reproduction of wild fallow deer. Reproductive abnormalities have been observed in fallow deer populations in Hungary. We supposed mycotoxin contamination to be one of the possible causes because multi-mycotoxin contamination is known to be dangerous even at low toxin levels, especially for young animals. We investigated the spatial pattern of mycotoxin occurrences and the relationship between maternal and fetal mycotoxin levels. A total of 72 fallow deer embryos and their mothers were sampled in seven forested regions in Hungary in the 2020/2021 hunting season. We analyzed Aflatoxin (AF), Zearalenone (ZEA), Fumonizin B1 (FB1), DON, and T2-toxin concentrations in maternal and fetal livers by ELISA. AF was present in 70% and 82%, ZEA in 41% and 96%, DON in 90% and 98%, T2-toxin in 96% and 85%, and FB1 in 84% and 3% of hind and fetus livers, respectively. All mycotoxins passed into the fetus, but only Fumonizin B1 rarely passed. The individual variability of mycotoxin levels was extremely high, but the spatial differences were moderate. We could not prove a relation between the maternal and fetal mycotoxin concentrations, but we found an accumulation of ZEA and DON in the fetuses. These results reflect the possible threats of mycotoxins to the population dynamics and reproduction of wild fallow deer. [ABSTRACT FROM AUTHOR]

Velvet (VeA), a light-regulated protein that shuttles between the cytoplasm and the nucleus, serves as a key global regulator of secondary metabolism in various Aspergillus species and plays a pivotal role in controlling multiple developmental processes. The gene vepN was chosen for further investigation through CHIP-seq analysis due to significant alterations in its interaction with VeA under varying conditions. This gene (AFLA_006970) contains a Septin-type guanine nucleotide-binding (G) domain, which has not been previously reported in Aspergillus flavus (A. flavus). The functional role of vepN in A. flavus was elucidated through the creation of a gene knockout mutant and a gene overexpression strain using a well-established dual-crossover recombinational technique. A comparison between the wild type (WT) and the ΔvepN mutant revealed distinct differences in morphology, reproductive capacity, colonization efficiency, and aflatoxin production. The mutant displayed reduced growth rate; dispersion of conidial heads; impaired cell wall integrity; and decreased sclerotia formation, colonization capacity, and aflatoxin levels. Notably, ΔvepN exhibited complete growth inhibition under specific stress conditions, highlighting the essential role of vepN in A. flavus. This study provides evidence that vepN positively influences aflatoxin production, morphological development, and pathogenicity in A. flavus. [ABSTRACT FROM AUTHOR]

Aflatoxin contamination remains a major global food safety challenge, with serious implications on agriculture and public health. Kenya has frequently experienced outbreaks of acute and chronic aflatoxin poisoning over the years with adverse human health effects. Farmers respond to incidences of aflatoxin contamination using a myriad of control practices. This study evaluates the choice of aflatoxin contamination control practices among smallholder maize farmers in Kilifi County, an aflatoxin hotspot in the Coast region of Kenya. A Poisson regression model was employed on a sample of 270 farmers selected using a multistage sampling technique. The aflatoxin contamination control practices used by maize farmers in Kilifi County can broadly be categorized into pre-harvest practices (timely planting, pest control, use of improved seed varieties, and early harvesting) and postharvest practices (sorting, proper drying, and use of storage pesticides). The Poisson regression estimates reveal that aflatoxin awareness, access to extension services, wealth status, and gender were the most important drivers of farmers choice of aflatoxin contamination control practices in Kilifi County. Thus, policy initiatives that improve farmers awareness and access to information are recommended in improving maize farmers aflatoxin contamination knowledge and uptake of management practices. To be effective, such interventions must address gender and wealth differences. [ABSTRACT FROM AUTHOR]

ABSTRACT: An experiment was conducted to assess the crop yield, nutritional composition, and mycotoxin contamination in different corn hybrids. The impact of these variables on the feed formulation cost of starter diets for broilers was also evaluated. A total of 150 samples from 50 corn hybrids was obtained from a field experiment. Nutrients were predicted by NIRS and mycotoxins were analyzed by HPLC-MS/MS. Data were submitted to analysis of variance and corn hybrids were grouped using Scott-Knott test at 5% significance. Pearson correlation analysis was performed among the main variables. Most of the variables were different among corn hybrids (P < 0.05), with exception of available phosphorus (Av. P), damaged grains, and DON and ZEA contaminations (P > 0.05). Crop yield had a positive correlation (P < 0.05) with feed formulation cost (r = 0.35) and a negative correlation (P < 0.05) with crude protein (-0.42), digestible (dig.) Thr (-0.42), and dig. Met+Cys (-0.42). The feed cost correlated positively (P < 0.05) with FUM (0.33) and ZEA (0.29), and negatively (P < 0.05) with crude protein (-0.74). Different corn hybrids vary in their productivity, nutritional content and mycotoxins contamination. It was demonstrated that an increase in the crop yield might be related to a reduction in corn nutritional content and quality, resulting in an increase in feed formulation cost.

The target of rapamycin (TOR) signaling pathway is highly conserved and plays a crucial role in diverse biological processes in eukaryotes. Despite its significance, the underlying mechanism of the TOR pathway in Aspergillus flavus remains elusive. In this study, we comprehensively analyzed the TOR signaling pathway in A. flavus by identifying and characterizing nine genes that encode distinct components of this pathway. The FK506-binding protein Fkbp3 and its lysine succinylation are important for aflatoxin production and rapamycin resistance. The TorA kinase plays a pivotal role in the regulation of growth, spore production, aflatoxin biosynthesis, and responses to rapamycin and cell membrane stress. As a significant downstream effector molecule of the TorA kinase, the Sch9 kinase regulates aflatoxin B1 (AFB1) synthesis, osmotic and calcium stress response in A. flavus, and this regulation is mediated through its S_TKc, S_TK_X domains, and the ATP-binding site at K340. We also showed that the Sch9 kinase may have a regulatory impact on the high osmolarity glycerol (HOG) signaling pathway. TapA and TipA, the other downstream components of the TorA kinase, play a significant role in regulating cell wall stress response in A. flavus. Moreover, the members of the TapA-phosphatase complexes, SitA and Ppg1, are important for various biological processes in A. flavus, including vegetative growth, sclerotia formation, AFB1 biosynthesis, and pathogenicity. We also demonstrated that SitA and Ppg1 are involved in regulating lipid droplets (LDs) biogenesis and cell wall integrity (CWI) signaling pathways. In addition, another phosphatase complex, Nem1/Spo7, plays critical roles in hyphal development, conidiation, aflatoxin production, and LDs biogenesis. Collectively, our study has provided important insight into the regulatory network of the TOR signaling pathway and has elucidated the underlying molecular mechanisms of aflatoxin biosynthesis in A. flavus.