Shiga toxin-producing Escherichia coli (STEC) shedding in a wild roe deer population.

Worldwide infections by Shiga toxin-producing Escherichia coli (STEC) in humans have been reported after consumption of mainly beef, but also deer meat. Not only the consumption of contaminated deer meat represents a risk, but also the transmission of STEC between deer and domestic animals should be considered. Within the framework of a telemetry study of roe deer (Capreolus capreolus) the aim was to analyse the occurrence of STEC. Due to the chance to sample some animals several times it was possible to obtain data on the repeated shedding of STEC in roe deer. In total 124 faeces or rectal swabs of 77 live trapped roe deer were collected. The isolates obtained were characterized for stx subtypes, different virulence genes, the so-called top-five serogroups, phylogenetic groups, PFGE-types and antimicrobial susceptibilities. The majority of roe deer were stx-positive whenever sampled. Twenty-eight animals were sampled more than once and were used to examine the duration of shedding STEC. The time interval of 6 persistently stx-negative tested animals was between 6 and 440d (median 49d, interquartile range (IQR) 17-258d). Ten animals excreted undistinguishable STEC strains in intervals between 4 and 778d (median 42d, IQR 22-79d). Most of the isolates were stx2b-positive, eae-negative and frequently ehlyA-positive. None of the isolates belonged to serogroup O26, O103, O111, O145 and O157, respectively. All isolates were sensitive to the antimicrobial substances tested. Although the duration of each shedding event could not be determined the results indicate long-term excretion of STEC in roe deer. This is an important consideration for the observance of good hygiene practice while field dressing of deer and preparing deer meat.

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.

Antibody Binding Studies Reveal Conformational Flexibility of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) A-Component.

The non-hemolytic enterotoxin complex (Nhe) is supposed to be the main virulence factor of B. cereus causing a diarrheal outcome of food poisoning. This tripartite toxin consists of the single components NheA, -B and -C all of them being necessary for maximum toxicity. In the past, research activities aiming to elucidate the mode-of-action of Nhe were mostly focused on the B- and C-component. In this study the generation of novel monoclonal antibodies (mAb) and their thorough characterization enabled the determination of key features for NheA. By the means of immunoaffinity chromatography it could be shown that NheA does not interact with -B and -C in solution. Additionally, the establishment of a highly sensitive sandwich-EIA now enables the detection of NheA in B. cereus supernatants down to 20 pg ml-1.Peptide-based epitope mapping in combination with partially deleted recombinant NheA fragments allowed the allocation of the binding regions for the three mAbs under study. Furthermore, by different EIA set-ups the conformational flexibility of NheA could be shown. For two of the antibodies under study different mechanisms of NheA neutralization were proven. Due to prevention of complete pore formation by one of the antibodies, NheA could be detected in an intermediate stage of the tripartite complex on the cell surface. Taken together, the results obtained in the present study allow a refinement of the mode-of-action for the Nhe toxin-complex.

Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay.

Simultaneous detection of small and large molecules on microarray immunoassays is a challenge that limits some applications in multiplex analysis. This is the case for biosecurity, where fast, cheap and reliable simultaneous detection of proteotoxins and small toxins is needed. Two highly relevant proteotoxins, ricin (60 kDa) and bacterial toxin staphylococcal enterotoxin B (SEB, 30 kDa) and the small phycotoxin saxitoxin (STX, 0.3 kDa) are potential biological warfare agents and require an analytical tool for simultaneous detection. Proteotoxins are successfully detected by sandwich immunoassays, whereas competitive immunoassays are more suitable for small toxins (<1 kDa). Based on this need, this work provides a novel and efficient solution based on anti-idiotypic antibodies for small molecules to combine both assay principles on one microarray. The biotoxin measurements are performed on a flow-through chemiluminescence microarray platform MCR3 in 18 minutes. The chemiluminescence signal was amplified by using a poly-horseradish peroxidase complex (polyHRP), resulting in low detection limits: 2.9 ± 3.1 µg L(-1) for ricin, 0.1 ± 0.1 µg L(-1) for SEB and 2.3 ± 1.7 µg L(-1) for STX. The developed multiplex system for the three biotoxins is completely novel, relevant in the context of biosecurity and establishes the basis for research on anti-idiotypic antibodies for microarray immunoassays.

Automated regenerable microarray-based immunoassay for rapid parallel quantification of mycotoxins in cereals.

An automated flow-through multi-mycotoxin immunoassay using the stand-alone Munich Chip Reader 3 platform and reusable biochips was developed and evaluated. This technology combines a unique microarray, prepared by covalent immobilization of target analytes or derivatives on diamino-poly(ethylene glycol) functionalized glass slides, with a dedicated chemiluminescence readout by a CCD camera. In a first stage, we aimed for the parallel detection of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisins in cereal samples in a competitive indirect immunoassay format. The method combines sample extraction with methanol/water (80:20, v/v), extract filtration and dilution, and immunodetection using horseradish peroxidase-labeled anti-mouse IgG antibodies. The total analysis time, including extraction, extract dilution, measurement, and surface regeneration, was 19 min. The prepared microarray chip was reusable for at least 50 times. Oat extract revealed itself as a representative sample matrix for preparation of mycotoxin standards and determination of different types of cereals such as oat, wheat, rye, and maize polenta at relevant concentrations according to the European Commission regulation. The recovery rates of fortified samples in different matrices, with 55-80 and 58-79%, were lower for the better water-soluble fumonisin B1 and deoxynivalenol and with 127-132 and 82-120% higher for the more unpolar aflatoxins and ochratoxin A, respectively. Finally, the results of wheat samples which were naturally contaminated with deoxynivalenol were critically compared in an interlaboratory comparison with data obtained from microtiter plate ELISA, aokinmycontrol® method, and liquid chromatography-mass spectrometry and found to be in good agreement.

Detection and characterization of Shiga toxin-producing Escherichia coli in faeces and lymphatic tissue of free-ranging deer.

Shiga toxin-producing Escherichia coli (STEC) have led to outbreaks worldwide and are considered emerging pathogens. Infections by STEC in humans have been reported after consumption of mainly beef, but also deer. This study investigated the occurrence of STEC in deer in Germany. The virulence genes eae, e-hlyA and saa, the stx subtypes, pulsed-field gel electrophoresis (PFGE) patterns and serovars were studied. In total, 120 samples of 60 animals were screened by real-time polymerase chain reaction (PCR). The PCR results showed a high detection rate of stx genes (83%). Mainly faecal samples, but also some lymphatic tissue samples, tested stx-positive. All isolates carried stx2, were eae-negative and carried e-hlyA in 38% and saa in 9% of samples. Serovars (O88:[H8], O174:[H8], O146:H28) associated with human diseases were also identified. In some animals, isolates from lymphatic tissue and faecal samples showed undistinguishable PFGE patterns. The examined deer were shown to be relevant reservoirs of STEC with subtype stx2b predominating.

Short communication: Streptococcus species isolated from mastitis milk samples in Germany and their resistance to antimicrobial agents.

Mastitis is one of the most frequent infectious diseases in dairy cattle and is a reason for antimicrobial drug usage in dairy cows. The bacteria involved in bovine mastitis are mainly Streptococcus spp., Staphylococcus spp., and coliforms. The aim of this study was to determine antimicrobial resistance among Streptococcus spp. isolated from bovine mastitis milk. Antimicrobial resistance in Strep. uberis (n=227), Strep. dysgalactiae (n=49), and Strep. agalactiae (n=3) was determined for 9 antimicrobial agents using the broth microdilution method in accordance with Clinical and Laboratory Standards Institute recommendations. Of all Streptococcus spp., 13% were multidrug resistant. The rate of multidrug resistance was higher among Strep. uberis (15%) than among Strep. dysgalactiae (6%) and Strep. agalactiae (0%). Resistance to tetracycline was the most common, followed by resistance to erythromycin, pirlimycin, and gentamicin. Resistance rates were higher on farms with more than 80 cows compared with those with fewer than 20 cows. ß-Lactams should remain the drugs of choice in the treatment of streptococcal mastitis. The slightly elevated minimum inhibitory concentrations determined for these antibiotics may indicate, however, the emergence of resistant streptococci. To identify such changes in susceptibility as early as possible, antimicrobial resistance in streptococci should be surveyed regularly.

Seroprevalence of anti-hepatitis E virus and anti-Salmonella antibodies in pigs at slaughter in Switzerland.

Hepatitis E virus (HEV) and Salmonella bacteria are zoonotic pathogens that can be acquired by foodborne transmission because food animals, for example pigs, are recognized as a reservoir. The objectives of this study were to determine the seroprevalence of anti-HEV immunoglobulin G (IgG) and anti-Salmonella antibodies from healthy pigs at slaughter in Switzerland, a country with a good health status of pig herds (e.g., eradication of enzootic pneumonia) compared with those of many countries in the European Union, and a rate of importation of live pigs that is very low (<1%). Based on pooled (diaphragm muscles from 3 to 5 animals per producer) meat juice samples, 120 (60%) of 200 and 8 (4%) of 200 samples were positive for anti-HEV IgG and anti-Salmonella antibodies, respectively. HEV seems to be highly prevalent among fattening pigs in Switzerland, whereas the low seroprevalence of anti-Salmonella IgG has not changed in recent years.

Occurrence of Fusarium T-2 and HT-2 toxins in oats from cultivar studies in Germany and degradation of the toxins during grain cleaning treatment and food processing.

For the Fusarium trichothecene mycotoxins T-2 and HT-2, a combined (T-2 + HT-2) temporary tolerable daily intake (tTDI) of 0.06 microg kg(-1) body weight day(-1) was proposed at the European level in 2001 (Opinion of the Scientific Committee on Food). In the near future, maximum levels for these trichothecenes will be regulated by the European Commission as announced in EU (VO) 1881/2006. For the implementation of these maximum levels, more data on occurrence and behaviour of T-2 and HT-2 toxins in primary agricultural products as well as during cleaning treatment and food processing are needed. In the current work, we determined the T-2/HT-2 concentrations in four oat cultivars (Aragon, Dominik, Ivory, Pergamon) from ten different agricultural sites in Germany, grown in cultivar studies in 2007. The grains were de-hulled, oat meal was prepared, and bread with 20% oat meal and 80% wheat flour was baked. In the cereal-processing chain, samples were taken at various steps and subsequently analysed for their T-2/HT-2 content. We employed liquid chromatography-mass spectrometry (LC-MS) and an immunological screening method (enzyme-linked immunoabsorbent assay (ELISA)) for T-2/HT-2 determination. Detection limits were between 1 and 10 microg kg(-1) in different matrices. T-2/HT-2 concentrations determined by ELISA in oat samples from ten different agricultural sites in Germany were between 9 and 623 microg kg(-1). The median and 90th percentile were 48 and 191 microg kg(-1) T-2/HT-2, respectively. One site showed six times higher T-2/HT-2 levels than the other sites, where concentrations ranged from 322 to 623 microg kg(-1). In 80% of the samples the cultivars Pergamon and Ivory had the lowest concentration of T-2 and HT-2 toxins. Using LC-MS for T-2/HT-2 determination, cleaning of the raw material did not lead to significant reductions of T-2 and HT-2 levels, whereas de-hulling led to a reduction of over 90%. Boiling of oat meal produced from cleaned raw material to yield 'porridge' resulted in varying T-2/HT-2 levels in experimental replicates. No major reduction of T-2/HT-2 levels in cooked porridge was obtained. Standardized baking experiments using 20% oat meal showed that T-2 and HT-2 toxins are relatively stable during the baking process, probably due to their temperature stability.

Cellular prion protein in mammary gland and milk fractions of domestic ruminants.

The present study shows that PrP(c) is expressed in the mammary gland and milk fractions of domestic ruminants in a species-specific manner. By applying immunohistochemistry, Western blot and ELISA, clear expression differences between bovine, ovine and caprine mammary gland, skimmed milk, acid whey and cream could be demonstrated, the highest relative PrP(c) levels being associated with the cream fraction. In the bovine gland PrP(c) was preferentially detectable at the basolateral surface of mammary gland epithelial cells, whereas in ovine and caprine samples the prion protein was more homogeneously distributed. Moreover, in ovine and caprine bovine mammary gland epithelial cells, apocrine secretory vesicles were strongly stained. Ovine and caprine milk proved to contain PrP(c) in all fractions with an additional truncated form at 12kDa in Western blot. This truncated isoform is the predominate one in caprine acid whey. These results support the hypothesis that the apocrine secretion mode of milk fat globules is a major way of PrP(c) transport into the milk.

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.

Prion protein expression in bovine podocytes and extraglomerular mesangial cells.

The cellular form of the prion protein (PrP(c)) is thought to be a substrate for an abnormal isoform of the prion protein (PrP(sc)). One emerging hypothesis is that the proposed conversion phenomenon takes place at the site at which the infectious agent meets PrP(c). PrP(c) is abundant in the central nervous system, but little is known about the cell-type-specific distribution of PrP(c) in non-neuronal tissues of cattle. We have studied whether PrP(c), a protein found predominantly in neurons, also exists in bovine podocytes, since neurons and podocytes share a large number of similarities. We have therefore examined the expression of PrP(c) by immunohistochemistry, reverse transcription/polymerase chain reaction and enzyme-linked immunosorbent analysis. Immunostained serial sections and specific antibodies against PrP(c) have revealed that PrP(c) is selectively localized in podocytes and is particularly strongly expressed in extraglomerular mesangial cells but not in endothelial or intraglomerular mesangial cells. The selective expression of PrP(c) in podocytes is of special importance, as it suggests that these cells represent possible targets for peripheral infection with prions and demonstrates that PrP(c) can be added to the list of neuronal factors expressed in mammalian podocytes.

[Zearalenone in food]. / Zearalenon in Lebensmitteln.

The research project "Methods of Analysis and occurrence of important Fusarium toxins (deoxynivalenol and zearalenone) as well as the intake of these toxins by the German consumer" supported by the Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL) is processed by the institutions mentioned above. This work represents a comprehensive summary of the contamination of food by zearalenone (ZEA).

[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.

Nuclease fluorescence assay for the detection of verotoxin genes in raw milk.

AIMS: To develop a rapid, high throughput PCR method for the detection of verotoxigenic Escherichia coli (VTEC) in raw milk based on TaqMan PCR. METHODS AND RESULTS: Two TaqMan PCR systems for the detection of verotoxin genes 1 and 2, respectively, have been established. A total of 74 bacterial strains, among them 15 VTEC, were used to characterize the PCR tests. No false negative and no false positive reactions were observed. When artificially contaminated raw milk samples of 25 ml were cultured in enrichment broth for 24 h, inocula of 10(-1) cells ml-1 could be detected. CONCLUSIONS: The TaqMan PCR systems are feasible for the detection of VTEC in raw milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The TaqMan PCR offers a rapid semiautomated alternative to conventional PCR methods for the detection of VTEC in raw milk.

[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.