Ewald Usleber

A survey on the occurrence of mycotoxins in wheat and maize from western Romania

Samples of wheat (n = 25) and maize (n = 30) for animal consumption, collected in 1997 after harvest from western Romania, were analyzed by enzyme immunoassays for mycotoxin contamination. Toxins analyses included deoxynivalenol (DON), 3-acetylDON, 15- acetylDON, fusarenone X (FX), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), zearalenone (ZEA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), ochratoxin A (OA), and citrinin (CT). DON and acetylDONs were the major contaminants in wheat (100%) and maize (46%). Median values for DON, 3-acetylDON, and 15-acetylDON were 880 μg kg-1, 66 μg kg- 1, and 150 μg kg-1 in wheat, and 890 μg kg-1, 180 μg kg-1, and 620 μg kg- 1 in maize, respectively. Additionally, 3,15-diacetylDON was detected in some samples by HPLC-EIA analysis. All samples were negative for FX (<150 μg kg-1). T-2 was found in wheat (n = 6) and maize (n = 1) at levels between 13 and 63 μg kg- 1. DAS (2.6 μg kg-1) was found in one maize sample. ZEA occurred in all wheat and in four maize samples, median values were 10 μg kg-1 and 250 μg kg-1, respectively. One maize sample contained FB1 (140 μg kg-1). All samples were AFB1-negative (<4 μg kg-1). OA was found in one wheat sample (37 μg kg- 1), CT was found in one maize sample (580 μg kg- 1). This first reported natural occurrence of a range of mycotoxins in Romanian feeding stuff shows that DON and acetyl DONs may be present at levels which may affect animal production.

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%: 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.

Immunochemical screening for antimicrobial drug residues in commercial honey

Honey samples (n = 100; origin: various countries from Eurasia, Oceania, and the Americas) were analysed by enzyme immunoassays (EIA) for tetracyclines, streptomycin, and sulfathiazole. Considering antibody specificity, these EIAs are either quantitative (streptomycin) or qualitative (tetracyclines, sulfathiazole) tests. Honey extract purification was achieved by liquid-liquid partition (tetracyclines), and by solid phase extraction-immunoaffinity chromatography (streptomycin, sulfathiazole). Detection limits were 20 micrograms kg-1 (tetracycline equivalents), 10 micrograms kg-1 (streptomycin), and 50 micrograms kg-1 (sulfathiazole equivalents), with mean recoveries of 100-117%. A total of 42% of the samples was found positive by EIA; 25% were positive in one assay, 13% in two, and 3% were positive in all three tests. In the EIA for tetracyclines, 26% were positive, with 12 samples exceeding a level of 50 micrograms kg-1 (tetracycline equivalents). In the EIA for streptomycin, 19% were positive, with a mean concentration of 19 +/- 12 micrograms kg-1. In the sulfathiazole EIA, 16% of the samples were positive, with 13 samples exceeding a level of 100 micrograms kg-1 (sulfathiazole equivalents). However, when samples which were positive in the sulfathiazole EIA were reanalysed for sulfonamides by HPLC, no sulfa drugs could be detected. Experimental heating (40 degrees C) of honey spiked with sulfathiazole indicated that the sulfa drug(s) responsible for positive EIA results could be present a sugar derivatives.

Direct enzyme immunoassay in microtitration plate and test strip format for the detection of saxitoxin in shellfish

Saxitoxin was coupled to horseradish peroxidase via a novel adaptation of the periodate reaction. Based on polyclonal antibodies against saxitoxin, this conjugate was used for the development of two formats of direct enzyme immunoassay (EIA)–a microtitration enzyme‐linked immunosorbent assay (ELISA) and a test strip EIA. The detection of saxitoxin without instrumentation by visual evaluation of the test strip EIA is described. The detection limits for saxitoxin were 7 pg/ml (0·35 pg/assay) in the ELISA and 200 pg/ml in the test strip EIA using visual evaluation. Employing a simple procedure of sample preparation, both ELISA and test strip EIA were applied to the analysis of shellfish. The detection limits for saxitoxin in shellfish tissue of the ELISA and the test strip assay were 3 and 4 ng/g, respectively.

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 μg/kg (DON) and 17 μg/kg (ZEA), maximum concentrations were 1800 μg kg−1 and 120 μg kg−1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 μg kg−1. Only one sample was positive for T-2 (55 μg/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.

Multimycotoxin dipstick enzyme immunoassay applied to wheat

A membrane-based visual dipstick enzyme immunoassay for the simultaneous detection of up to five mycotoxins was developed. Multiple dots of the respective antibodies against aflatoxin B1 (AFB1), T-2 toxin (T-2), 3-acetyldeoxynivalenol (3-AcDON), roridin A (RA), and zearalenone (ZEA) were applied onto a dipstick membrane. The competitive immunoreactions were performed by incubation of the dipstick in a test tube containing sample solution and a mixture of the respective toxin-horseradish peroxidase conjugates. After a second incubation in enzyme substrate/chromogen solution, a complete suppression of the (blue) colour development of the respective dot was scored positive. Visual detection limits for AFB1, T-2, 3-AcDON, RA, and ZEA in buffer solution were 2 ng/ml, 5 ng/ml, 50 ng/ml, 30 ng/ml, and 5 ng/ml respectively. Using a simple extraction procedure, the detection limits of the multimycotoxin assay in artificially contaminated wheat samples were 30 ng/g, 100 ng/g, 600 ng/g, 500 ng/g, and 60 ng/g, respectively.

Widespread occurrence of low levels of alternariol in apple and tomato products, as determined by comparative immunochemical assessment using monoclonal and polyclonal antibodies.

This study investigated the production of polyclonal (pAB) antibodies and the first time production of monoclonal (mAB) antibodies against the mycotoxin alternariol, and their implementation in enzyme immunoassay (EIA) for the rapid determination of alternariol in foods. Both EIAs were highly sensitive, with detection limits (IC₂₀) of 35 ± 6.9 pg/mL (mAb EIA) and 59 ± 16 pg/mL (pAb EIA). Food products (n = 109; apple and tomato products, white wine) from German retail shops were analyzed. At a detection limit of 1-2 μg/kg, alternariol at 1-13 μg/kg was found with high frequency in apple (67%) and tomato (93%) products. Tomatoes with visible signs of Alternaria infection, stored at room temperature for up to 4 weeks, contained alternariol at levels up to 50 mg/kg, as determined by EIA and HPLC-FLD. It is concluded that the alternariol immunoassays present a versatile screening tool which could facilitate food control for Alternaria toxins.

Immunochemical detection of antibiotics and sulfonamides.

To control the maximum residue limits (MRLs) for residues of veterinary drugs in food of animal origin, according to EU regulations, a broad spectrum of sensitive analytical methods is required. One effective approach is the development of immunoassays, particularly for screening purposes. Strategies for the production of specific polyclonal and monoclonal antibodies against beta-lactams, tetracyclines, streptomycin, chloramphenicol, sulfonamides and trimethoprim, are outlined, as well as methods for the synthesis of the respective enzyme-labelled antigens. The sensitivity and the specificity of the antibodies were characterized, and the immunochemical test systems were designed as quantitative routine tests (microtitre plate format) and as rapid qualitative tests (membrane-based assay formats). The detection limits of the assays were found to be well below the regulatory limits. The range of recovery, for the analysis of artificially contaminated samples, was between 68 and 104%. In principle, the enzyme immunoassays for antimicrobial drugs showed the advantage of sensitivity and speed together with the simplicity of manipulations involved in the procedure. However, because of the results of the specificity studies, as well as the possibility of false positive results owing to unspecific inhibition of the assay, confirmation of immunoassay results is still required for all legal and statutory purposes.

Enterobacteriaceae in dehydrated powdered infant formula manufactured in Indonesia and Malaysia

To determine the occurrence of Salmonella and Shigella in infant formula from Southeast Asia, 74 packages of dehydrated powdered infant follow-on formula (recommended age, > 4 months) from five different manufacturers, four from Indonesia and one from Malaysia, were analyzed. None of the 25-g test portions yielded Salmonella or Shigella. However, further identification of colonies growing on selective media used for Salmonella and Shigella detection revealed the frequent occurrence of several other Enterobacteriaceae species. A total of 35 samples (47%) were positive for Enterobacteriaceae. Ten samples (13.5%) from two Indonesian manufacturers yielded Enterobacter sakazakii. Other Enterobacteriaceae isolated included Pantoea spp. (n = 12), Escherichia hermanii (n = 10), Enterobacter cloacae (n = 8), Klebsiella pneumoniae subsp. pneumoniae (n = 3), Citrobacter spp. (n = 2), Serratia spp. (n = 2), and Escherichia coli (n = 2). To our knowledge, this is the first report to describe the contamination of dehydrated powdered infant formula from Indonesia with E. sakazakii and several other Enterobacteriaceae that could be opportunistic pathogens. Improper preparation and conservation of these products could result in a health risk for infants in Indonesia.

The potential of monoclonal antibodies against ampicillin for the preparation of a multi-immunoaffinity chromatography for penicillins†

Monoclonal antibodies (Mab) against ampicillin were prepared by immunization of mice with an ampicillin-keyhole limpet hemocyanin conjugate coupled by a glutaraldehyde method. Sensitivity and specificity of these antibodies were tested in a direct competitive enzyme immunoassay, in which an ampicillin-horseradish peroxidase conjugate prepared by a carbodiimide method served as the labelled antigen. According to their cross-reactivities with the other beta-lactam antibiotics, the Mabs could be divided into two groups, which are represented by the clones designated 1D1 and 3B5. While Mab 3B5 (IgG1) showed no major cross-reactions with the other penicillins frequently used in veterinary medicine except for amoxicillin (108%), Mab 1D1 (IgG2a) had marked cross-reactivities with most of the 17 tested beta-lactam antibiotics (e.g., amoxicillin 187%, penicillin G 31%, cloxacillin 30%, dicloxacillin 44%, and oxacillin 14%). The detection limits for ampicillin, calculated from the antibiotic concentration giving 30% binding inhibition, were 11.7 (Mab 3B5) and 16.6 ng ml-1 (Mab 1D1). To prepare multi-immunoaffinity chromatography columns, Mab 1D1 and a previously described antibody against cloxacillin (Mab 1F7) were each coupled to CNBr activated sepharose. The capacity of the resulting immunosorbents was approximately 6.6 and 5.4 micrograms ml-1 gel for ampicillin and cloxacillin, respectively. Recoveries of amoxicillin, ampicillin, cloxacillin, dicloxacillin, penicillin G and oxacillin (in buffer solutions) from the produced immunoaffinity columns were in the range from 67 to 100%.