A highly specific competitive direct enzyme immunoassay for sterigmatocystin as a tool for rapid immunochemotaxonomic differentiation of mycotoxigenic Aspergillus species.

A simplified method to produce specific polyclonal rabbit antibodies against sterigmatocystin (STC) was established, using a STC-glycolic acid-ether derivative (STC-GE) conjugated to keyhole limpet haemocyanin (immunogen). The competitive direct enzyme immunoassay (EIA) established for STC had a detection limit (20% binding inhibition) of 130 pg ml-1 . The test was highly specific for STC, with minor cross-reactivity with O-methylsterigmatocystin (OMSTC, 0·87%) and negligible reactivity with aflatoxins (<0·02%). STC-EIA was used in combination with a previously developed specific EIA for aflatoxins (<0·1% cross-reactivity with STC and OMSTC), to study the STC/aflatoxin production profiles of reference strains of Aspergillus species. This immunochemotaxonomic procedure was found to be a convenient tool to identify STC- or aflatoxin-producing strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The carcinogenic mycotoxin sterigmatocystin (STC) is produced by several Aspergillus species, either alone or together with aflatoxins. Here, we report a very simple and straightforward procedure to obtain highly sensitive and specific anti-STC antibodies, and their use in the first ever real STC-specific competitive direct enzyme immunoassay (EIA). In combination with a previous EIA for aflatoxins, this study for the first time demonstrates the potential of a STC/aflatoxin EIA pair for what is branded as 'immunochemotaxonomic' identification of mycotoxigenic Aspergillus species. This new analytical tool enhances analytical possibilities for differential analysis of STC and aflatoxins.

Dietary ergot alkaloids as a possible cause of tail necrosis in rabbits.

This study describes the association between tail necrosis in rabbits and mycotoxins in rabbit feed. Clinical cases of tail necrosis were observed in 14 out of 103 rabbits kept in an outdoor group housing, fed with hay and a commercial pelleted feed. The observed clinical symptoms, alopecia, erosions, crusts and necrosis were restricted to the tail area and exclusively occurred in young rabbits aged 113 ± 20 days. Dermatological examination suggested that ischemia had caused necrosis. Analysis of blood samples showed an elevated level of creatine kinase. No weight loss occurred in affected rabbits. Trauma caused by injuries or technopathic lesions was also excluded. Histopathologically, the lesions were characterized by acute muscle fibre degeneration and chronic active dermatitis with granulation tissue formation. Necropsy of one rabbit revealed hepatocellular degeneration and necrosis as remarkable findings. Feed analysis for ergot alkaloids by enzyme immunoassays yielded a mean and maximum ergot alkaloid content of 410 ± 250 µg/kg and 1,700 µg/kg, respectively. Faeces of affected rabbits contained ergot alkaloids at levels up to 200 µg/kg. The mean and maximum dietary intake of total ergot alkaloids were 17 and 71 µg/kg bodyweight, respectively. Fusarium toxins (trichothecenes, zearalenone, fumonisins) were also found in the feed, but at levels which did not explain the observed effects. The results indicate that ergot alkaloids may have been the cause of tail necrosis, which is supported by literature data showing that rabbits are especially sensitive towards these toxins.

Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons.

Antemortem diagnosis of avian aspergillosis is very challenging. Diagnostic assays using blood samples would aid in an early and more definitive diagnosis. In the current study, detection of anti-Aspergillus antibodies, Aspergillus antigen, and Aspergillus toxin (fumigaclavine A), protein electrophoresis and measurement of acute-phase protein concentrations were performed on serum of 18 adult and plasma of 21 juvenile gyr-saker hybrid falcons (Falco rusticolus x Falco cherrug). Adult (n = 15) and juvenile (n = 18) falcons were experimentally inoculated with different dosages of the same strain of Aspergillus fumigatus and an additional three falcons from each age group were used as uninfected control animals. Blood samples were collected prior to inoculation and at 28 days postinoculation. Of the 33 inoculated falcons, 16 demonstrated clinical signs (vomiting, greenish urates, dyspnea, ruffled feathers) commonly associated with aspergillosis and in 14 falcons necropsy revealed aspergillosis granulomas confirmed by mycology and histopathology. Positive galactomannan results were rare, with only 3/15 positive samples from adult falcons and none in the juvenile birds. Most of the inoculated falcons showed an increase of serum amyloid A (66.7%) and haptoglobin (70.4%), but fumigaclavine A was not detected in the blood from any of the experimental animals. Elevated antibody indices were detected in 96.7% of the inoculated birds, but also in 66.7% of the controls. Significant decreases in albumin:globulin ratio were obvious in 81.5% of the inoculated birds, including 100% of the birds with granulomas. Blood from falcons with granulomas demonstrated significantly increased concentration values of alpha 2 and ß globulins, decreased percentages of prealbumin and albumin, and increased percentages of alpha 2 and ß globulins compared to inoculated falcons without granulomas. In conclusion, acute-phase proteins and the electrophoretic profile of birds challenged with A. fulmigatus show significant alterations, which in combination with other diagnostic procedures, assist in the early diagnosis of avian aspergillosis.

Fumonisin intake of the German consumer.

In order to calculate the dietary fumonisin intake of the German consumer, a large survey was carried out on a variety of potentially contaminated products in the period between December 1998 and July 2001. A total of 1960 food samples comprising all known relevant groups of products were analysed for fumonisins. Furthermore, 272 of these samples were also analysed for hydrolysed fumonisins (HFB). For routine analysis enzyme immunoassay was used, confirmatory and control analyses were performed using HPLC-FLD after precolumn derivatisation, or by LC-MS/MS. Daily intake of fumonisins was calculated by combining fumonisin contamination data obtained in this study with available food consumption data for Germany. In a "mean case" scenario, median fumonisin levels in foods and mean food intake values were used. To generate a "bad case" scenario, the 90(th) percentile of fumonisin levels in foods and mean food intake values were combined. The overall daily fumonisin intake by the German consumer was 1.1 µg in the "mean case" scenario, and 21 µg in the "bad case" scenario. It was concluded that in general there is no increased risk for the German consumer in aspects of exceeding the recommended tolerable daily intake of fumonisins (2 µg/kg body weight). However, certain products (and certain brands of products) were repeatedly found to contain elevated fumonisin levels, which in a "worst case" scenario ("high" food intake of maize-based products) could pose a potential risk for the consumer, in particular concerning foods for infants and young children. High fumonisin levels were found in infant foods in 1999, but contamination levels decreased strongly in the following years. HFBs (mostly HFB1) were frequently found in processed cereals such as corn flakes, but in relatively low concentrations. According to our findings, the new European Union maximum levels for fumonisins are suitable to eliminate peak contamination levels of fumonisins in foods, but would lead to a regular excess of the TDI for infants and young children if these maximum levels would indeed be exhausted.

Airborne mycotoxins in dust from grain elevators.

Workers in grain elevators are exposed to grain dust and may therefore have an increased risk of inhalatory contact with mycotoxins. To study the mycotoxin burden of such environments, settled grain dust samples (n=35) were collected from several locations of a total of 13 grain elevators in Germany, and analysed for ochratoxin A (OTA, detection limit 0.01 ng/g), deoxynivalenol (DON, detection limit 15 ng/g), and zearalenone (ZEA, detection limit 6 ng/g), respectively. Cytotoxicity of these samples was assessed by a MTT bioassay with a swine kidney target cell line. Additionally, the airborne dust concentration of these locations was determined. Nearly all settled dust samples contained OTA (96%), DON (100%), and ZEA (100%) with median concentrations of 0.4 ng/g, 416 ng/g, and 126 ng/g, respectively. Cytotoxic effects in varying degrees from weakly to highly toxic were caused by crude extracts of 86% of the dust samples. However, cytotoxicity did not correlate with mycotoxin levels in these samples and thus indicated the presence of cytotoxic compounds of unknown origin. Based on the mycotoxin findings in settled dust samples and the airborne dust concentrations, the average airborne mycotoxin concentrations were estimated to be 0.002 ng/m(3) (OTA), 2 ng/m(3) (DON), and 1 ng/m(3) (ZEA), respectively. The relevance of these findings for occupational health was assessed by comparison with WHO recommendations for the maximum tolerable daily (oral) intake (TDI). Even in a worst case scenario, the calculated inhalatory intake was far below the TDI values. However, considering the uncertainties resulting from different exposure pathways, namely oral ingestion versus inhalation, further research should primarily address the problem of how adequate assessment criteria for airborne exposure to mycotoxins could be established.

[Deoxynivalenol in cereal products from retail outlets in Germany in the year 2006]. / Untersuchungen zu DON-Gehalten in Getreideerzeugnissen aus dem deutschen Einzelhandel im Jahr 2006.

Samples of soft wheat flour (n=78), durum wheat semolina (n=6), and pasta (made from durum wheat, n=49) were purchased in January-April 2006 from retail outlets in Hesse, Germany. Samples were analysed for deoxynivalenol (DON) by enzyme immunoassay. The detection limit of the method was 10 µg/kg, with recoveries of 81-85% (RSDr: 12-17%). DON was detected in 84% of all samples, but the contamination level was low. Median/maximum values for DON in wheat flour, wheat semolina, and pasta were 28µg/kg/217 µg/kg, 38µg/kg/203 µg/kg, and 24µg/kg/119 µg/kg, respectively. Compared with results obtained from previous years, significantly lower DON levels were observed in these commodities.

[Determination of deoxynivalenol and deepoxy-deoxynivalenol in milk]. / Bestimmung von Deoxynivalenol und Deepoxy-Deoxynivalenol in Milch.

A HPLC method with UV/diode array detection for the determination of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) in milk was developed. Milk was incubated with ß-glucuronidase and then defatted. After purification by immunoaffinity chromatography, DON and DOM-1 were separated on a C18 reversed phase column with acetonitril/water (10/90) as the mobile phase and detected at 218 nm. Limits of quantification were 1 µg/l for both toxins, with mean recoveries (1-10 µg/l) of 97% (DON) and 84% (DOM-1), respectively. Milk samples (pasteurized, UHT; n=32) from German retail shops were analysed by this method. Neither DON/DOM-1 nor their glucuronides were found in any sample. These results are consistent with published studies indicating that in lactating cows, DON and DOM-1 are mostly eliminated through urine, and that the carry-over into milk is negligible.

[Deoxynivalenol in food]. / Deoxynivalenol in Lebensmitteln.

Within a joint research project entitled "Analysis and occurrence of importantFusarium toxins (deoxynivalenol and zearalenone) and dietary intake of these toxins by the German consumer", supported by the German Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL), representative analytical data are generated on the contamination level of foods withFusarium mycotoxins. This paper gives a comprehensive summary concerning the contamination of foods from the German market with deoxynivalenol (DON) in the period from August 2001 to April 2004. More than 4700 food samples (mostly cereals and cereal-containing foods) were purchased from food shops in Germany and analysed for DON by enzyme immunoassay, HPLC, and LC-MS/MS, respectively. All analytical methods were validated through intra- and interlaboratory studies and gave mean recoveries of >80% for each matrix. Although DON was detected with high frequency in all cerealcontaining samples, the mean and median levels were in most products well below the recently established maximum permitted limits in Germany.

Application of immunoaffinity chromatography and enzyme immunoassay in rapid detection of aflatoxin B1 in chicken liver tissues.

Existing physicochemical analytical methods for the determination of aflatoxins in animal tissues are expensive, cumbersome, and hazardous. To offer an alternative to these methods, a novel and highly sensitive immunochemical method for the rapid detection of aflatoxin B1 (AFB1) in chicken liver tissues is described in this study. Liver tissues were homogenized with cold methanol-acetone (50:50), followed by AFB1 extraction with methanol-acetone-PBS (25:25:50). The tissue extracts were, with or without further purification by immunoaffinity chromatography (IAC), applied to a highly sensitive direct ELISA for determination of AFB1. The detection limits for this assay were 15 +/- 0.77 pg/mL when standards and samples were dissolved in methanol-PBS (10:90) and 17 +/- 2.0 pg/mL when methanol-acetone-PBS (5:5:90) solution was used. The average recoveries of AFB1 were 54.3 to 65.5% in artificially contaminated tissue samples at 1 to 5 ng/g. In samples spiked with AFB1 at 1 ng/g, the method had diagnostic sensitivity and specificity of 100% for samples processed with IAC and 91.7 and 100%, respectively, for samples without IAC purification. The test was successfully applied to the detection of AFB1 in liver tissues from chickens that were experimentally dosed with AFB1. It is hoped that this test will be applicable in rapid detection of aflatoxins in poultry meats and in diagnosis of aflatoxicosis in chicken.

[Determination of deoxynivalenol in bread and beer]. / Bestimmung von Deoxynivalenol in Brot und Bier.

Analytical procedures for the determination of deoxynivalenol (DON) in bread and beer, using enzyme immunoassay (EIA) and HPLC methods, were developed. For determination of DON by EIA, aqueous raw extracts of bread or degassed beer were extracted by liquid-liquid partitioning with ethyl acetate, the organic solvent evaporated, and the residue redissolved in phosphate buffered saline (PBS) for analysis. For determination by HPLC (UV detection at 218 nm), DON in bread extracts or beer was purified on immunoaffinity chromatographic columns. In bread, detection limits for DON of 15 µg/kg (EIA) and 7 µg/kg (HPLC) were achieved, with mean recoveries of 81%. In beer, the detection limit for DON was 2 µg/l both in EIA and HPLC, with recoveries of 91-93%. Both methods showed good agreement of the results for naturally contaminated sample materials, with r(2)=0.993 for bread and r(2)=0.823 for beer, respectively.

[Not Available]. / Deoxynivalenol in lebensmitteln: Ergebnisse einer Pilotstudie.

Cereal food products (n=333) were purchased in retail stores from Germany in 2001 and analysed for deoxynivalenol (DON), either by enzyme immunoassay or by HPLC after immunoaffinity chromatographic cleanup. Detection limits were dependent of the sample matrix and varied from 20-100 µg/kg. The overall DON incidence was 53%, with mean and median levels for positives of 251 µg/kg and 142 µg/kg, respectively. The contamination with DON (mean/median value, µg/kg) as found for bread (90/87), wheat flour (161/124), and noodles (472/297) indicate that the levels of DON in cereal foods were significant in view of the tolerable daily intake (1 µg/kg body weight) as established by the European Union scientific committee on food.

[Not Available]. / Aufreinigung von Fumonisinen und ihren Hydrolysaten über ein neuartiges Festphasenextraktions-Material.

Tests with various clean-up materials after optimisation of different parameters showed that the use of Oasis® material resulted in matrixless chromatograms in HPLC-FLD. The selectivity and detection limit of the method was improved by using LC-MS/MS as the detection system. Mean recovery was 100%, and no negative food matrix effects could be observed.

Immunoassay methods for paralytic shellfish poisoning toxins.

The current status of immunochemical techniques for analysis of paralytic shellfish poisoning (PSP) toxins is summarized. Important aspects regarding production of the biological reagents necessary for immunochemical methods, the characteristics of polyclonal and monoclonal antibodies against saxitoxin and neosaxitoxin, and the importance of test sensitivity and specificity are discussed. Applications of immunochemical techniques for PSP toxins include microtiter plate enzyme immunoasays and enzyme-linked immunofiltration assays for toxin detection, and immunoaffinity chromatography (IAC) for sample extract cleanup. A major advantage of enzyme immunoassay (EIA) is simplicity and rapidity of the test procedure, and higher sensitivity than other methods. However, quantitative agreement between EIA and mouse bioassay is dependent on antibody specificity and the toxin profile in the shellfish; thus, both over- and underestimation of total toxicity may occur. For screening purposes, however, EIAs offer major advantages over the mouse bioassay, which is criticized in Europe because of animal welfare. A major application of antibodies against PSP toxins is their use for extract cleanup by IAC, which gives highly purified extracts, thereby enhancing determination of PSP toxins by conventional physicochemical methods such as liquid chromatography. IAC can also be used to isolate PSP toxins for preparation of analytical standard solutions.

Survey of Romanian slaughtered pigs for the occurrence of mycotoxins ochratoxins A and B, and zearalenone.

Blood serum, kidney, liver and muscle sample per animal were collected from slaughtered pigs (n = 52). The samples were analysed for ochratoxin A (OTA) and B (OTB) by HPLC methods. Zearalenone (ZEA) in serum was analysed by enzyme immunoassay. A total of 98% serum samples were OTA positive in the range of 0.05-13.4 ng/ml and 85% contained under 5 ng OTA/ml. The incidences of OTA in kidney and liver were very similar (79%, 75%) with mean levels of 0.54 ng/g and 0.16 ng/g, respectively. The lowest incidence (17%) and the lowest mean level contamination (0.15 ng/g) were in muscle samples. The mean distribution in tissues followed the pattern serum > kidney > liver > muscle (100%; 0.26%; 8.5%; 2.57%). No kidney, liver or muscle sample was found OTA positive above the maximum admitted limit in Romania (5 ng/g). No sample was found to be positive for OTB. A very similar OTA contamination (mean = 4.19 ng/ml, coefficient of variation = 34.4%) was observed in the serum samples (n = 10) collected from the same farm. A possible difference in regional distribution of OTA in Romania is suggested. Zearalenone was detected only in 17.3% of the serum samples with a maximum concentration of 0.96 ng/ml. This study shows the presence of OTA and ZEA in Romanian slaughtered pigs at levels comparable to those reported in other countries.

Production of ultrasensitive antibodies against aflatoxin B1.

AIMS: To produce specific antibodies against the haptenic fungal toxin aflatoxin B1 (AFB1) and apply these antibodies in immunochemical assays for aflatoxins. METHODS AND RESULTS: Rabbits were immunized using an AFB1-bovine serum albumin conjugate and serum titres determined by double-antibody enzyme immunoassay. High titres of antibodies with very high affinity for AFB1 were obtained 15 and 4 weeks after the initial immunization and the first booster immunization respectively. The antibodies were employed in enzyme immunoassay (EIA) and immunoaffinity chromatography (IAC) methods for aflatoxins. With a detection limit of 15.8 pg ml(-1) for AFB1, the EIA employing these antibodies is the most sensitive test for AFB1 described so far. In IAC columns, these antibodies provided high binding capacity for all major aflatoxins, including AFB1, AFB2, AFG1 and AFG2. CONCLUSION: The antibodies described here are useful for the analysis of trace levels of aflatoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyclonal antibody-based EIA and IAC methods for aflatoxin analysis offer a suitable alternative to the more expensive monoclonal antibody-based methods.

Development and application of an enzyme-linked immunosorbent assay for the analysis of hydrolyzed fumonisins.

Polyclonal antibodies against hydrolyzed fumonisin B1 (HFB1) were prepared by immunization of rabbits with a keyhole limpet hemocyanin conjugate coupled by glutaraldehyde. Sensitivity and specificity of these antibodies were tested in a direct competitive enzyme-linked immunosorbent assay (ELISA), in which a HFB1-horseradish peroxidase conjugate prepared by reductive alkylation served as the labeled antigen. A direct competitive ELISA for the quantitative determination of HFB1 in corn-based food samples was developed with this antibody. The 50% inhibition level was used for the determination of cross-reactivity. The cross-reactivities of the antibodies for HFB2 and HFB3 were 4.3% and 14.4%, respectively, whereas the parent compound fumonisin B1 (FB1) showed no cross reactivity. The detection limit of the EIA calculated from the HFB1-concentration given a 30% binding inhibition was 0.25 ng/ml for the standard solutions and 10 ng/g for the analyzed food samples e.g. tortilla chips, nachos and cornflakes. Mean recoveries of HFB1 for artificially contaminated food samples were in the range of 96 to 98%. This test offers a rapid and economic opportunity for the HFB1-screening of food samples.

[Not Available]. / Entwicklung eines hochempfindlichen Enzymimmuntests zum Nachweis von Ochratoxin A.

Antibodies against ochratoxin A were produced in rabbits after immunization with an ochratoxin A-keyhole limpet hemocyanine conjugate. The immunogen was found to be very efficient, and high antibody titers were detected in the sera of all immunized rabbits. In a competitive enzyme immunoassay using ochratoxin A-horseradish peroxidase as the labelled antigen, low levels of ochratoxin A in buffer solution could be detected. The mean standard curve detection limit and 50% inhibition level of the optimized assay were at 15 pg/ml and 50 pg/ml, respectively. Relative cross-reactivity of ochratoxin B was found to be 2%. With these characteristics, this novel enzyme immunoassay should be useful for the routine detection of ochratoxin A in food and in biological samples at levels well below 1 ng/g.

[Not Available]. / Stachybotrys-Toxine in einer Münchner Wohnung mit Wasserschaden.

This paper presents a case report of indoor Stachybotrys toxins in Germany. In the summer of 2000, heavy rainfall and a leakage of the gutter system of an appartement house in Munich, Germany, caused a severe water damage in one of the appartments. The appartment was at that time occupied by two of the authors (ES, EU). A wall (covered with wallpaper) in one room in particular was most strongly affected, about 6-8 m(2) of the wallpaper became very wet. Large parts of this wet wallpaper were within days infested by multiple circumscribed round, grey-black colonies (0.5-5 cm in diameter) of presumtiveStachybotrys spp.. At the same time, the air in this room became very damp and oppressive. Several persons reported burning sensations on the mucous membranes of the eye, nose, and larynx, followed by headache symptoms, as soon as a few minutes after they had entered this room. Inadvertent skin contact with one of the fungal colonies by one person resulted in burning sensations immediately, but symptoms disappeared after thorough rinsing of the skin with water. The infested room was evacuated, and samples were taken from the wet wallpaper at areas (1) in the center of the most heavy fungal colony growth, (2) several cm away from colony growth, and (3) from locations which were wet but about 1 m away from visible colony growth. The samples were extracted with methanol, the extract diluted with buffer solution and analysed by competitive enzyme immunoassay (EIA) for roridin A. Due to cross-reactivity, this EIA detects several macrocyclic trichothecenes produced byStachybotrys spp.. Heavily infected pieces of wallpaper were strongly contaminated with toxins, with maximum concentrations of 1.1 microgram per cm(2) (Roridin A equivalents). Wet but visibly not infected samples of wallpaper were negative in the EIA. Considering a EIA cross-reactivity of satratoxin H (the major Stachybotrys-toxin) of 15%, the actual maximum toxin concentration would correspond to 6 microgram of satratoxin H per cm(2). As far as we know, this is the first documented case of indoor Stachybotrys toxins in Germany. The toxin concentration by far exceeds the minimum toxin level required for skin toxicity, and was considered as unhealthy. Health authorities in Munich were informed but were unable to provide assistance, probably because awareness of the importance and the possible health risks caused by indoor mycotoxins is not yet widespread in Germany. Reporting and monitoring programs for mycotoxins in water-damaged buildings seem to be necessary to provide insight into the occurrence of similar cases as the one described here.