Unveiling the emerging role of curcumin to alleviate ochratoxin A-induced muscle toxicity in grass carp(Ctenopharyngodon idella):in vitro and in vivo studies

Background Ochratoxin A(OTA),a globally abundant and extremely hazardous pollutant,is a significant source of contamination in aquafeeds and is responsible for severe food pollution.The developmental toxicity of OTA and the potential relieving strategy of natural products remain unclear.This study screened the substance curcumin(Cur),which had the best effect in alleviating OTA inhibition of myoblast proliferation,from 96 natural products and investigated its effect and mechanism in reducing OTA myotoxicity in vivo and in vitro.Methods A total of 720 healthy juvenile grass carp,with an initial average body weight of 11.06±0.05 g,were randomly assigned into 4 groups:the control group(without OTA and Cur),1.2 mg/kg OTA group,400 mg/kg Cur group,and 1.2 mg/kg OTA+400 mg/kg Cur group.Each treatment consisted of 3 replicates(180 fish)for 60 d.Results Firstly,we cultured,purified,and identified myoblasts using the tissue block culture method.Through preliminary screening and re-screening of 96 substances,we examined cell proliferation-related indicators such as cell viability and ultimately found that Cur had the best effect.Secondly,Cur could alleviate OTA-inhibited myoblast differentiation and myofibrillar development-related proteins(Myo G and MYHC)in vivo and in vitro and improve the growth performance of grass carp.Then,Cur could also promote the expression of OTA-inhibited protein synthesis-related proteins(S6K1 and TOR),which was related to the activation of the AKT/TOR signaling pathway.Finally,Cur could downregulate the expression of OTA-enhanced protein degradation-related genes(murf1,foxo3a,and ub),which was related to the inhibition of the Fox O3a signaling pathway.Conclusions In summary,our data demonstrated the effectiveness of Cur in alleviating OTA myotoxicity in vivo and in vitro.This study confirms the rapidity,feasibility,and effectiveness of establishing a natural product screening method targeting myoblasts to alleviate fungal toxin toxicity.

Molecularly imprinted polymer based on upconversion nanoparticles for highly selective and sensitive determination of Ochratoxin A

A novel molecularly imprinted polymer (MIP) based on upconversion nanoparticles (UCNPs) was successfully synthesized for determination of Ochratoxin A (OTA). The MIP was developed on the silica-coated UCNPs using N-(1-hydroxy-2-naphthoyl amido)-(L)-phenylalanine (HNA-Phe) as the alternative template. The final composite combined the advantages of the high selectivity of MIP with the high fluorescence intensity of UCNPs which was selective and sensitive to OTA. Under the optimal condition, the fluorescence intensity of UCNPs@SiO2@MIP decreases linearly when the concentration of OTA increases from 0.05 to 1.0 mg/L. The detection limit of OTA with the method was 0.031 mg/L. At three spiked concentration levels (50, 100 and 200 μg/kg), the recovery ranges of OTA in corn, rice and feed are 88.0%–91.6%, 80.2%–91.6% and 89.2%–90.4%, respectively.

REVIEW Risk Assessment of the Mycotoxin Ochratoxin A

Ochratoxin A (OA) is a mycotoxin which has been found to occur in foods of plant origin, in edible animal tissues, as well as in human blood sera and tissues. The ability of OA to move up the food chain is aided by its long half-life in certain edible animal species. In this report, an evaluation of the health risks to Canadians due to the presence of OA in food products is presented. The first part of the report deals with the physicochemical aspects, mycology, laboratory production, analytical methods, and natural occurrence in plant products, animal products, and human tissues. The stability of OA in foods and feeds, the effects of food processing, and the removal from foods and feeds by physicochemical means are also discussed. From these data, the worst case estimate for the daily exposure of Canadians to OA, from the consumption of pork-based food products and cereal foods, is approximately 5 ng OA/kg body wt (mean of eaters) for young children, the highest consumption group on a body weight basis. The second part of the report deals with the metabolic disposition as well as the available toxicity database for OA in laboratory animals, farm animals, and humans. The major target for OA toxicity in all mammalian species tested is the kidney, and endemic nephropathies affecting livestock as well as humans have been attributed to OA. OA is also teratogenic, and in the fetus the major target is the developing central nervous system. Recent studies have provided 'clear evidence' of the carcinogenicity of OA in two rodent species. OA was found to be nonmutagenic in various microbial and mammalian gene mutation assays, but weak genotoxic activity to mammalian cells was noted. In addition, OA was found to suppress immune function. Based on the NTP carcinogenicity study with OA in rats, the estimated tolerable daily intake in humans ranges from 0.2 to 4.2 ng OA/kg body wt, depending on the method of extrapolation used. In view of the toxic properties of OA, it is recommended that exposure to OA be kept to a minimum. In Canada, further monitoring programs are required to better define the overall residue profile of OA in cereal grains, animal feeds, animal food products, and human blood. Such data are required to better assess dietary exposure and to ascertain the need for regulatory controls or other control mechanisms. (c)1989 Academic Press, Inc.

Characterization and Comparison of Ochratoxin A-Ovalbumin(OTA-OVA) Conjugation by Three Methods

In order to generate an antibody against a small hapten molecule, the hapten is cross-linked with carrier protein to make it immunogenic. In this study, the hapten （ochratoxin A, OTA） was coupled to ovalbumin （OVA） by an active ester reaction. To develop a technique for detecting the conjugation, the hapten-protein conjugate （OTA-OVA） was characterized thoroughly by immunoarray technology, ultraviolet （UV） spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF-MS）, respectively. The molecular weight of OTA-OVA was 50 350.141 Da, and the molecular weight of OVA was 44 887.506 Da, which were determined by MALDI-TOF-MS, respectively. In OTA-OVA, the molecular coupling ratio was 13：1 by MALDI-TOF-MS while the molecular coupling ratio was 10：1 by UV. In this experiment, UV and MALDI-TOF-MS were selected as the efficient methods to evaluate the cross-linking effect and calculate the molecular coupling ratio.

Aptamer based detection and separation platforms for ochratoxin A:A systematic review

Ochratoxin A(OTA),one of the most dangerous mycotoxins for human health,has been subjected to numerous studies for separation and detection in minimal amounts.Aptamers as novel recognition elements have been employed to fabricate ultrasensitive biosensors for the detection of OTA and designing delicate analytical tools.This review attempted to comprehensively examine all reported aptamer-based detection and separation platforms for ochratoxin.The most relevant databases were considered to discover all specific aptamers for dealing with OTA.Aptamer-based detection and separation devices specified for OTA were searched for,analyzed,discussed,and classified based on their specifications.The optical aptasensors have gathered a higher interest than electrochemical aptasensors,which can achieve a lower limit of detections.Moreover,some extraction platforms based on these aptamers were also found.However,aptamer-based devices seem to have some challenges in their application.

Ochratoxin A induces abnormal tryptophan metabolism in the intestine and liver to activate AMPK signaling pathway

Background Ochratoxin A(OTA)is a mycotoxin widely present in raw food and feed materials and is mainly pro-duced by Aspergillus ochraceus and Penicillium verrucosum.Our previous study showed that OTA principally induces liver inflammation by causing intestinal flora disorder,especially Bacteroides plebeius(B.plebeius)overgrowth.However,whether OTA or B.plebeius alteration leads to abnormal tryptophan-related metabolism in the intestine and liver is largely unknown.This study aimed to elucidate the metabolic changes in the intestine and liver induced by OTA and the tryptophan-related metabolic pathway in the liver.Materials and methods A total of 30 healthy 1-day-old male Cherry Valley ducks were randomly divided into 2 groups.The control group was given 0.1 mol/L NaHCO3 solution,and the OTA group was given 235μg/kg body weight OTA for 14 consecutive days.Tryptophan metabolites were determined by intestinal chyme metabolomics and liver tryptophan-targeted metabolomics.AMPK-related signaling pathway factors were analyzed by Western blot-ting and mRNA expression.Results Metabolomic analysis of the intestinal chyme showed that OTA treatment resulted in a decrease in intesti-nal nicotinuric acid levels,the downstream product of tryptophan metabolism,which were significantly negatively correlated with B.plebeius abundance.In contrast,OTA induced a significant increase in indole-3-acetamide levels,which were positively correlated with B.plebeius abundance.Simultaneously,OTA decreased the levels of ATP,NAD+and dipeptidase in the liver.Liver tryptophan metabolomics analysis showed that OTA inhibited the kynurenine metabolic pathway and reduced the levels of kynurenine,anthranilic acid and nicotinic acid.Moreover,OTA increased the phosphorylation of AMPK protein and decreased the phosphorylation of mTOR protein.Conclusion OTA decreased the level of nicotinuric acid in the intestinal tract,which was negatively correlated with B.plebeius abundance.The abnormal metabolism of tryptophan led to a deficiency of NAD+and ATP in the liver,which in turn activated the AMPK signaling pathway.Our results provide new insights into the toxic mechanism of OTA,and tryptophan metabolism might be a target for prevention and treatment.

Determnation of ochratoxin A in grain by monoclonal antibody－based enzyme－linked immunosorbent assay

The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on immunomicroplate. Direct FLIAS was found to be less timeconsuming than indirect ELISA. For direct FLISA, recovery of 1 -500 ppb OA added to wheat was78.9-100.0% and rice was 88.9- 120.0%. For indirect EI.IAS, recovery of 1-500 ppb OA addedto wheat was 79.0- 110.0% and rice was 82.0 120.0%. The minimal detection level for OA was Ippb. Analyses of 31 samples that caused humanintoxicant for OA showed that the ELISA resultsagreed wtll with those obtained by thin-layer chromatogrdphy.

Ochratoxin A in dried figs, raisings, apricots, dates on Iranian retail market

Ochratoxin A (OTA) is among the most important mycotoxins, and the International Agency for Research on Cancer (IARC) classifies it as possibly carcinogenic to humans (group 2B). A total of 121 samples of dried fruits from the central zone of Iran were analyzed for OTA by enzyme linked immunosorbent assay (ELISA) technique. The recovery percentages of OTA in spiked dried fruit samples at concentrations of 5, 10 and 20 ng/g were found to be 84.9%, 89.3% and 90.4% as mean, respectively. OTA was found in 20.7% of the analyzed samples by average concentration of 6.7 ± 3.9 ng/g. The incidence rates of OTA contamination in dried fig, raising, apricots, and date samples were 10.4%, 44.7%, 6.7% and 10.0%, respectively. The concentrations of OTA in 7.9% of contaminated dried raising samples and 2.1% of dried fig samples were higher than maximum tolerance limit accepted by European Union (10 ng/g). This value reflects that the analyzed samples have a minimal contribution to toxicological risk. To our best knowledge, the present study is the first report of the presence of OTA by ELISA in dried fruit samples in Iran.

Hepatocellular carcinoma and food contamination: Ochratoxin A as a great prompter

Ochratoxin A (OTA) is a secondary metabolite of Asper-gillus and Penicillium, microorganisms that can be haz-ardous to health when present as food contaminants. OTA is a potent member of a group of mycotoxins. Prolonged exposure to mycotoxins in the diet is related to cancer, among other diseases. Hepatocellular car-cinoma (HCC) accounts for 70%-90% of primary liver cancers and is the third leading cause of cancer-related deaths worldwide. In a recent study, Ibrahim et al proposed a correlation between the incidence of HCC and contamination with OTA. Analysis of OTA in serum samples showed that HCC patients had the highest incidence of OTA of the subjects examined (5-fold higher than that of the control group). OTA levels were significantly increased in HCC patients. This study demonstrates that chronic exposure to high levels of OTA may be associated with a high risk of liver cancer development. Future epidemiologic studies of HCC should focus on good practices in food preparation, food storage and the consumption of OTA-containing foods.

Intercalation of Phenylalanine, Isocoumarin and Ochratoxin A (OTA) into LDH’s

Phenylalanine, isocoumarin and Ochratoxin A (OTA) have been intercalated within the interlayer space of layered double hydroxides. Synthesis of these nanocompounds was achieved via co-precipitation. Their physicochemical properties were studied by element chemical analysis, powder X-ray diffraction, infrared spectroscopy and thermal analyses. The presence of OTA in the interlayer is demonstrated by the study of LC-FD Analysis. On the other hand, these studies allow to check how some of the toxin is on the surface of the nanocomposite.

Peculiarities of feed contamination with citrinin and ochratoxin A

Occurrence of citrinin and ochratoxin A in different feed ingredients and compound feeds was screened by accredited methods based on the indirect competitive enzyme-linked immunosorbent assay. High frequency co-occurrence of both toxins was found in wheat grain and processed sunflower seeds. Citrinin levels exceeded those of ochratoxin A in the majority of co-contaminated feed samples, and the ratio of (1.1 - 10):1 proved to be the most frequent. A possible role of Aspergillus and Penicillium fungi in separate and simultaneous OTA and CIT occurrence in feeds is also discussed.

Efficacy of Organo-Modified Nano Montmorillonite to Protect against the Cumulative Health Risk of Aflatoxin B₁and Ochratoxin A in Rats

The aim of the current study was to prepare organo-modified nano montmorillonite (OMNM) and to evaluate its chemopreventive effects against the hapatonephrotoxicity induced by aflatoxin B1 (AFB1) and ochratoxin A (OA) singly or in combination in rats. OMNM was prepared using Cetyltrimethylammoniumbromide (CTAB) as organic modifier. Eighty male Sprague Dawley were divided into 8 groups and treated for 8 weeks as follow: the control group;the group treated orally with AFB1 (80 μg/kg b.w.);the group treated with OA (100 μg/kg b.w.);the group treated with AFB1 plus OA, the group treated with OMNM (5 g/kg diet) and the groups treated with AFB1 and/or OA plus OMNM. At the end of treatment period, blood and tissue samples were collected from all animals for biochemical and histological analysis. The results revealed that the expansion in the basal spacing of the montmorillonite due to the intercalation of CTAB was 7.20?&#197 and the average particle size of OMNM was 120 nm. The in vivo results indicated that treatment with both AFB1 and OA singly or in combination resulted in a significant increase in liver and kidney function parameters, oxidative stress and tumor markers accompanied with a significant decrease in antioxidant enzyme activities and significant histological changes in liver and kidney tissues. These changes were severe in the group received the combined treatment of AFB1 and OA. OMNM alone did not show any toxic effect and it succeeded to prevent or at least diminish the toxic effects and the histological changes in liver and kidney. It can be concluded that treatment with AFB1 and OA has a synergistic toxic effects and OMNM is safe and it is a promise candidate as an additive to protect against the exposure to multi-mycotoxins in high risk population.

Fate of ochratoxin A in dried red chilies during roasting process

Dried red chilies are widely used globally and are susceptible to contamination by fungi and fungal toxins.Roasting is a common way of processing dried red chilies.This study explored the effects of Aspergillus niger and ochratoxin A(OTA)contamination on the quality of roasted chilies,and the fate of OTA during the roasting process.Three optimum roasting conditions(140°C×8 min,160°C×6 min,and 180°C×4 min)were screened out by a combination of instrumental and manual sensory evaluations.Under these roasting conditions,A.niger and OTA contamination diminished the quality and taste of roasted chilies.With increasing roasting temperature and time duration,OTA content and mold counts gradually decreased,together with the DNA degradation of OTA biosynthesis-related genes of A.niger in roasted chilies.The roasting condition at 180°C×4 min showed the greatest decomposition effect on OTA,while also maintaining good sensory quality of roasted chilies.This study shed light on the fate of OTA during the chili roasting process.

Dietary curcumin alleviates intestinal damage induced by ochratoxin A in juvenile grass carp(Ctenopharyngodon idella):Necroptosis and inflammatory responses

Ochratoxin A(OTA)is one of the most common pollutants in aquatic feed.As a first line of defense,intestinal barriers could be utilized against OTA in order to prevent disorders.Natural product supplementation is one of the most popular strategies to alleviate toxicity induced by mycotoxins,but there is a lack of knowledge about how it functions in the teleost intestine.In this study,720 juvenile grass carp of about 11 g were selected and four treatment groups(control group,OTA group,curcumin[Cur]group,and OTA+Cur group)were set up to conduct a 60-day growth test.After the test,the growth performance and intestinal health related indexes of grass carp were investigated.The addition of dietary Cur could have the following main results:(1)inhibit absorption and promote efflux transporters mRNA expression,reducing the residuals of OTA,(2)decrease oxidative stress by reducing oxidative damage and enhancing the expression of antioxidant enzymes,(3)promote mitochondrial fusion proteins to inhibit the expression of mitotic proteins and mitochondrial autophagy proteins and enhance mitochondrial function,(4)reduce necroptosis-related gene expression through inhibiting the tumor necrotic factor receptor-interacting protein kinase/mixed lineage kinase domain-like pathway,(5)reduce the expression of pro-inflammatory factors by inhibiting the Toll-like receptor 4/nuclear factor-κB signaling pathway to alleviate the intestinal inflammatory response.In summary,the results suggested that Cur could alleviate OTA-induced intestinal damage by enhancing antioxidant capacity and mitochondrial function as well as reducing necroptosis and inflammation in the grass carp intestine.This study provided a theoretical basis and production implications for dietary Cur that could improve growth performance and alleviate the intestinal damage induced by OTA in fish.

Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin A and zearalenone in corn, wheat, and feed samples

A one-step dual flow immunochromatographic assay （DICGA）, based on a competitive format, was de- veloped for simultaneous quantification of ochratoxin A （OTA） and zearalenone （ZEN） in corn, wheat, and feed sam- ples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53-12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06-39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry （LC-MS/MS） and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.

A Label-free Fluorescent Aptasensor for Turn-on Monitoring Ochratoxin A Based on AIE-active Probe and Graphene Oxide

A label-free fluorescent aptasensor for specific and ultrasensitive monitoring ochratoxin A（OTA） was developed using the specific aptamer of OTA（OSA） as recognition dement, an aggregation-induced emission（AIE） molecule（a 9,10-distyrylanthracene with two ammonium groups, DSAI） as a fluorescent probe, and graphene oxide（GO） as a quencher. In the absence of OTA, the AIE probe DSAI and OSA complex（DSAI/OSA） is adsorbed on the GO surface, and the fluorescence of DSAI will be quenched efficiently via the fluorescence resonance energy transfer（FRET） from DSAI to GO. Upon the OTA addition, a more stable complex（OSA-OTA） is formed and released from GO. Meanwhile, DSAI and OSA-OTA can form a new complex（DSAI/OSA-OTA）, then the fluorescent signal of DSAI recovers gradually. Therefore, by introducing GO and DSAI, the fluorescence signal of DSAI can be easily turned from ＂off＂ to ＂on＂ after the addition of OTA, and the ultrasensitive detection of OTA by monitoring the change of the fluorescence signal of DSAI can be readily realized. The detection limit of the assay can reach 0.324 nmol/L with a linear detection range of 10-200 nmol/L. And the aptasensor exhibits high selectivity for OTA against other analogues. Moreover, it has been successfully applied for the detection of OTA in red wine samples.

Ochratoxin A:Carryover from animal feed into livestock and the mitigation strategies

This review aims to highlight the effects of ochratoxin A(OTA)in the feed of meat-producing animals.The accumulation of OTA in feed and its distribution in various farm animals were compared and evaluated.Primarily,the oral administration of OTA-contaminated feed and the predisposition in an animal's vital organ were critically examined in this work.The collated reports show that OTA directly associated with endemic nephropathy and its high concentration leads to degeneration of liver cells,and necrosis of intestinal and lymphoid tissues.At present,limited reports are available in the recent liter-ature on the problems and consequences of OTA in feed.Therefore,this review focused on the OTA carryover from feed to farm animals and the interaction of its secondary metabolites on their biochemical parameters.Hence,this report provides greater insights into animal health related to OTA residues in meat and meat products.This article also explores mitigation strategies that can be used to prevent the carryover effects of OTA in livestock feeds and the effects in the food chain.

Double Magnetic Separation-assisted Fluorescence Method for Sensitive Detection of Ochratoxin A

A double magnetic separation-assisted fluorescence method was developed to rapidly detect ochratoxin A(OTA). The OTA aptamer functionalized magnetic nanomaterial(Fe3O4-Aptanier) and complementary DNA conjugated nitrogen-doped graphene quantum dots(NGQDs-cDNA) were used in this assay. Aptamer could hybridize with cDNA, which induced tlie NGQDs-cDNA to bind onto Fe3O4-Aptamer, and resulted in the fluorescence quenching of NGQDs. After the addition of OTA, the NGQDs-cDNA could release into the solution, and resulted in the recovery of fluorescence signal of NGQDs consequently. By utilizing the magnetic separation, the unbonded NGQDs-cDNA and residual Fe3O4-Aptamer were removed, which significantly increased the fluorescence signal intensity. OTA could be detected in the linear range of 10 nmol/L to 2000 nmol/L, with a limit of detection as 0.66 mnol/L. The advantages of this method include simple operation, good selectivity and high sensitivity, and this method can be used for the rapid detection of ochratoxin A in wheat and com.

Occurrence of mycotoxins in feed ingredients and complete feeds obtained from the Beijing region of China

Background：The current study was carried out to provide a reference for the control of mycotoxin contamination in feed ingredients and complete feeds for swine.Methods：A total of 55 feed ingredients,including 14 corn,13 wheat bran,11 soybean meal and 17 dried distillers grains with solubles（DDGS） as well as 76 complete swine feeds including 7 creep feeds,14 starter feeds,14 grower feeds,18 grower-finisher feeds,10 gestating sow feeds,and 13 lactating sow feeds were randomly collected from15 swine farms located in the Beijing region of China from July to August 2011.Immunoaffinity clean-up,using High-Performance Liquid Chromatography（HPLC） in combination with UV or Fluorescence Detection,was used for quantitative analysis of aflatoxin B,（AFB,）,deoxynivalenol（DON）,zearalenone（ZEA） and ochratoxin A（OTA） in the ingredients and complete feeds.Results：DON and ZEA were the most prevalent mycotoxins found.DON was detected at percentages of 93,92,54,100 and 97%with a mean level of 1.01,0.44,0.05,1.36 and 0.65 ppm in the samples of corn,wheat bran,soybean meal,DDGS and complete feeds,respectively.The detected percentages of ZEA were 100,100,54,100 and 100 with mean levels of 109.1,14.9,9.2,882.7 and 58.9 ppb in the same samples.In the wheat bran and soybean meal samples,the content of all four mycotoxins were below the maximum limits set by Chinese regulations while the percentage of samples that exceeded regulatory limits were 7,57 and 7%for corn,and 7,14 and 3%for the complete feeds for AFB,,DON and OTA respectively.DDGS showed the most serious mycotoxin contamination and the percentage of samples that exceeded regulatory limits were 6,88 and 41%,for AFB,,DON and ZEA,respectively.Conclusions：This paper is the first to present data on the natural occurrence of AFB,,DON,ZEA and OTA in ingredients and complete feeds obtained from swine farms in China＇s Beijing region.The data shows that feed ingredients and complete swine feeds obtained from these farms are most often contaminated with DON followed by contamination with AFB,and ZEA.

Ochratoxigenic Black Aspergilli Isolated from Dried Agricultural Products in Yogyakarta, Indonesia

Black aspergilli, the potential ochratoxin A （OTA） producers, were predominant fungi in fermented cocoa bean, coffee bean and dried cassava in Yogyakarta. Identification of black aspergilli at species level will be useful to make clear link between OTA contamination on food product and the toxin producer. The objective of this study was to identify the species of the black aspergilli producing OTA which contaminated dried agriculture products. In this study, 16 isolates were obtained, and four isolates among of them （YAC-9, YAK-6, YAK-12 and YAG-2） were found as OTA producing-strains, with the highest OTA found on Yeast Extract Sucrose （YES） solid medium of 57.68 ppb. Based on morphological characters, 16 isolates can be grouped into four species, after confirmation by molecular data based on PCR method, the groups were identified as A. carbonarius, A. niger, A. tubingensis and A. aculeatus. OTA producing-strains were identified as A. carbonarius and A. niger, meanwhile, A. tubingensis and A. aculeatus were found as non-OTA producing-strains.