Penicillium verrucosum in feed of ochratoxin A positive swine herds

Ochratoxin A contamination of cereal feed grain was monitored during October 1989–September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2–45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (2 ng/ml blood) and ochratoxin A negative herds (<2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p < 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p < 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5% ) of the feed samples collected from the ochratoxin A negative herds.     

Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30 degrees C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 microg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 microg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.     

Vorkommen von ochratoxin A und B in gewürzen

A total of 681 samples of spices, which comprised more than 50 different spice commodities were analysed for the natural occurrence of the mycotoxins ochratoxin A (OTA) and ochratoxin B (OTB). The analytical method involved chloroform extraction, clean-up by immunoaffinity column and HPLC determination of both mycotoxins. OTA and OTB were detected in 143 (21%) and 68 (10%) of the samples, respectively. The highest frequency of occurrence of both mycotoxins detected were in chili (100% for OTA and 55% for OTB), paprika (41% and 15%, respectively) and pepper (23% and 44%, respectively). The toxin concentrations ranged between the detection limit (0.01 ng/g) and 41.8 ng OTA (2.7 ng OTB)/g of chili, 18.9 ng OTA (1.4 ng OTB)/g of paprika and 3.8 ng OTA (4.6 ng OTB)/g of pepper. One sample of a extract of vanilla was found to be positive for OTB at 15 ng/g. However, median values of most samples showed to be below the detection limit. Comparison of the geographical origin of the samples showed that the predominant number of contaminated spices was from Southeast-Asia and India. Highly contaminated paprika samples were found to come from Israel.     

A survey of porcine kidneys and chicken liver for ochratoxin A in Spain

Contamination studies by ochratoxin A on pork kidney and chicken liver has been carried out in Catalonia (Spain). 73% of the pork kidney samples analyzed did not contain an amount of ochratoxin A over our detection limit (0.5 ng/g) whereas only 7% had contamination higher than 1 ng/g. None of the chicken samples analyzed were contaminated by this toxin above the detection limit. All contamination levels found are below the maximum levels accepted by several countries for this kind of material. A confirmative test is necessary before discarding false positive samples.     

Radioimmunoassay of ochratoxin A in barley

A simple and rapid radioimmunoassay was developed for the quantitative determination of ochratoxin A in barley. [14C]ochratoxin A, with a specific activity of 130 Ci/mol, was used as the tracer. Toxin levels below 100 ng/ml required a cleanup step. Three methods (the Association of Official Analytical Chemists cleanup method, the solvent partition method, and the Extrelut 3 column cleanup method) were compared.     

Radioimmunoassay of ochratoxin A in barley.

A simple and rapid radioimmunoassay was developed for the quantitative determination of ochratoxin A in barley. [14C]ochratoxin A, with a specific activity of 130 Ci/mol, was used as the tracer. Toxin levels below 100 ng/ml required a cleanup step. Three methods (the Association of Official Analytical Chemists cleanup method, the solvent partition method, and the Extrelut 3 column cleanup method) were compared.     

Natural contamination with mycotoxins in forage maize and green coffee in Nayarit State (Mexico)

The presence of mycotoxins in forage maize (zearalenone, fumonisin B1, T-2 toxin and diacetoxyscirpenol) and green coffee (ochratoxin A) from Nayarit State (Mexico) has been studied. All maize samples analyzed showed fumonisin B1 contamination, with an average concentration of 2,541 microg/kg. Fifteen percent of the samples contained zearalenone, with an average concentration of 1,610 microg/kg. Only one sample showed T-2 toxin contamination (7 microg/kg), and no diacetoxyscirpenol was detected. Sixty-seven per cent of green coffee samples were contaminated with ochratoxin A, with an average concentration of 30.1 microg/kg. This is the first study about mycotoxins developed in Nayarit and it has shown that mycotoxin contamination is a real problem in both foodstuffs studied.     

Ochratoxin Production by the Aspergillus ochraceus Group and Aspergillus alliaceus

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30°C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 μg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 μg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.     

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability

AIMS: To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP). METHODS AND RESULTS: A total of 321 isolates of P. verrucosum were isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. CONCLUSIONS: Among 184 ochratoxin A-producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.     

Ochratoxin A in pig blood: method of analysis and use as a tool for feed studies

A procedure is presented for screening the quality of feed in respect to ochratoxin A contamination based upon the analysis of ochratoxin A in pig blood. Representative samples from large feed lots may be obtained by using pigs as in vivo sample collectors which enrich the toxin and forms homogeneous samples in the blood. The spectrofluorometric procedure for ochratoxin A analysis (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976) has been adapted to pig blood and has been simplified to involve only three extraction steps. A volume of 2.5 ml of blood or plasma is needed, and the detection limit is 2 ng of ochratoxin A per ml. The disappearance of ochratoxin A from pig blood as a function of time has been studied. A feeding experiment with ochratoxin A has been performed, and the time course of the concentration of ochratoxin A in blood has been followed during the experiment.     

Ochratoxin A in pig blood: method of analysis and use as a tool for feed studies.

A procedure is presented for screening the quality of feed in respect to ochratoxin A contamination based upon the analysis of ochratoxin A in pig blood. Representative samples from large feed lots may be obtained by using pigs as in vivo sample collectors which enrich the toxin and forms homogeneous samples in the blood. The spectrofluorometric procedure for ochratoxin A analysis (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976) has been adapted to pig blood and has been simplified to involve only three extraction steps. A volume of 2.5 ml of blood or plasma is needed, and the detection limit is 2 ng of ochratoxin A per ml. The disappearance of ochratoxin A from pig blood as a function of time has been studied. A feeding experiment with ochratoxin A has been performed, and the time course of the concentration of ochratoxin A in blood has been followed during the experiment.     

Ochratoxin A production and amplified fragment length polymorphism analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger strains isolated from grapes in Italy

Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than Aspergillus carbonarius within Aspergillus section Nigri to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and beta-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of A. carbonarius produced ochratoxin A (6 to 7,500 microg/liter), 5 of 20 strains of A. tubingensis produced ochratoxin A (4 to 130 microg/liter), 3 of 15 strains of A. niger produced ochratoxin A (250 to 360 microg/liter), and none of the 19 strains of Aspergillus "uniseriate" produced ochratoxin A above the level of detection (4 microg/liter). These findings indicate that A. tubingensis is able to produce ochratoxin and that, together with A. carbonarius and A. niger, it may be responsible for the ochratoxin contamination of wine in Italy.     

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.     

Investigation of the distribution of deoxynivalenol and ochratoxin A contamination within a 26 t truckload of wheat kernels

A study was conducted to determine the distribution of deoxynivalenol (DON) and ochratoxin A (OTA) in a lot of 261 of wheat kernels. Within this study, two different sampling and sample preparation strategies were carried out. On the one hand, following the official commission regulation 401/2006/EC, an aggregate sample out of 100 incremental samples was build, homogenized and prepared for laboratory analysis. On the other hand each individual subsample was investigated for its deoxynivalenol and ochratoxin A content. The determined concentration of DON in the individual samples was in a range from 830 up to 2655 (μg/kg, for OTA results ranged from < 0.2 up to 8.6 μg/kg. Thus, a coefficient of variance of 25% for DON and 200% for OTA was achieved. From this, a spot formation for OTA was observed and the average value of the 100 incremental samples did not correspond to the achieved value for the aggregate sample. While the DON contamination at this concentration range seems to be more even, consequently the result of the aggregate sample was in accordance with the average value. In addition a sample communition study was performed to answer the question whether the time consuming process of grinding of the whole aggregate sample is necessary or not. The results of this study show that contamination of whole wheat kernels with DON is at the same level within a 1 kg sample (CV 16%), while OTA contamination shows high variability (CV 94%). At least for OTA this study indicated that an extensive and complete sample communition of the high volume aggregate sample is necessary.     

Determination of ochratoxin A in urine and faeces of swine by high-performance liquid chromatography

Sensitive methods for the determination of ochratoxin A in urine and faeces of swine are described. The samples were extracted with chloroform at pH <2, and the extracts were cleaned up by a combination of solid-phase extraction and liquid—liquid partition. High-performance liquid chromatography with fluorescence detection was used for detection and determination. The detection limits were 0.3 ng/ml for urine and 1.5 ng/g for faeces. Recoveries of ochratoxin A from spiked samples of urine and faeces were 93% and 60%, respectively. Because of the low detection limit and the fast and relatively easy performance, the method for the determination of ochratoxin A in urine proved suitable for the estimation of possible contamination of live animals.     

赭曲霉毒素A和玉米赤霉烯酮-二联胶体金免疫层析试纸条的制备及应用

【背景】真菌毒素为真菌的有毒次级代谢产物,混合污染时毒性显著增强,可对人类和动物健康造成严重伤害。制备二联胶体金免疫层析试纸条,实现对常见真菌毒素混合污染的快速监测,具有重要意义。【目的】制备赭曲霉毒素A (Ochratoxin A,OTA)和玉米赤霉烯酮(Zeralenone,ZEN)金标单克隆抗体,基于免疫层析原理,采用竞争反应模式,建立二联胶体金免疫层析检测法用于污染样品中OTA和ZEN的同时快速检测。【方法】采用柠檬酸钠还原法制备胶体金颗粒,并标记获得两种真菌毒素金标单克隆抗体,通过优化相关条件,建立稳定的二联胶体金免疫层析检测方法,用于同时检测谷物和饲料样品中的OTA和ZEN。【结果】制备的OTA和ZEN二联胶体金试纸条对OTA和ZEN的检测限分别为0.625 ng/mL和1.25 ng/mL,且与谷物和饲料中其它真菌毒素(黄曲霉毒素B1、伏马毒素B1、桔青霉毒素、展青霉毒素和呕吐毒素)均无交叉反应,人工添加试验结果准确。对天然样本检测结果表明该方法与LC-MS/MS一致性良好。【结论】本研究制备的二联胶体金试纸条可用于实际样品中OTA和ZEN的同时快速筛查。     

Nachweis und Vorkommen von Mykophenolsäure und Citrinin in Rotwein

Due to infections with moulds already in the vineyards, the formation of mycotoxins is possible under certain circumstances during the process of red wine making. At this, metabolites ofPenicillium spp. are of major importance, as this species are to be found frequently on grapes. Beside the nephrotoxic citrinin, which is often co-occurring with ochratoxin A, the occurrence of mycophenolic acid (MPA), a substance of immunosuppressive action, was investigated since it is formed by a great number of Penicillium-species. The detection of these compounds was carried out by means of ELISA and LC-MS. As testing material 44 red wine samples of different provenience and vintages were used. Mycophenolic acid could be detected in 91 % of the samples. The maximum content amounted to 130 ng/ml, yet most of the samples resulted in much lower concentrations of between 3 and 20 ng/ml. The extent of contamination seems to depend rather on the origin of the wine than on the vintage. In particular samples from Southern Europe were most contaminated. This could be due to different practises in wine-making. Citrinin was not detectable in any sample (< 0,2 ng/ml). Regarding the detected concentrations of MPA and citrinin, there is probably no concern for consumers’ health. However, the degree of contamination of wine with MPA may well serve as an indicator for hygiene in production.     

Mould and mycotoxin contamination of pig feed in northwest Croatia

The aim of this study was to investigate the contamination of pig feed with moulds and the occurrence of mycotoxins. A total of 30 feed samples were collected at different animal feed factories in the north-western part of Croatia. Mycological analysis showed that the total number of moulds ranged from 1?×?10³ to 1?×?10⁵?cfu/g with samples contaminated with Aspergillus spp. (63?%), Penicillium spp. (80?%), and Fusarium spp. (77?%). A determination of aflatoxin B1 (AFB₁), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), T-2 toxin (T-2) and fumonisin (FUM) concentration was done using the validated ELISA method. The mean concentrations of AFB₁ (0.5?±?0.6???g/kg), OTA (1.53?±?0.42???g/kg) and FUM (405?±?298???g/kg) were below the maximum levels or recommended values in the EU in all the investigated samples. The observed results indicated an increased contamination of pig feed with Fusarium mycotoxins DON and ZEA with mean concentrations of 817?±?447 and 184?±?214???g/kg, higher than recommended in 40 and 17?% of the analysed samples, respectively.     

Development of stable isotope dilution assays for ochratoxin A in blood samples

Two new stable isotope dilution assays were developed for the quantification of ochratoxin A in human blood samples for exposure studies. The methods based on two different sample extraction and cleanup procedures including liquid–liquid extraction with following immunoaffinity chromatography (IA) as well as a dispersive solid-phase extraction (DSPE) method. For detection, LC–MS/MS was applied. For the first time, exact quantitation of the reference compound ochratoxin A was performed by quantitative NMR spectroscopy (qNMR). Additionally, a comparison of different blood-drawing procedures revealed no differences for heparin plasma and serum whereas citrate plasma gave significantly lower results for the mycotoxin. Limits of detection (LOD: 0.02 ng/g (IA) vs 0.03 ng/g (DSPE)), limits of quantification (LOQ: 0.07 ng/g (IA) vs 0.08 ng/g (DSPE)), relative recovery (?94%), precision, and linearity indicated excellent performance of the developed methods.     

Natural occurrence of ochratoxin A and citrinin in food stuffs in Egypt

Natural occurrence of ochratoxin A (OA) and citrinin in cereals (274 samples) and animal tissues (250 samples) have been investigated during a period of more than 2 years. OA was found in cereals and animal tissues while citrinin was found in cereals only. The highest level of OA (up to 80.0 μg/kg) was found in yellow corn, 52.8% of contaminated samples while respectively 55.9% and 39.4% of barley and rice samples were contaminated with citrinin, with the highest level up to 100.0 and 27.92 μg/kg for barley and rice respectively. The frequent contamination of animal kidney with OA (28% positive out of 150 tested) average concentration 12.33 μg/kg. 2% of liver and 4% of muscles tissue were observed.