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.

Hepatitis E virus in wild rabbits and European brown hares in Germany.

Recently, a change of hepatitis E from being a typical travel-associated disease to an autochthonous zoonosis in Germany was observed. An increasing number of autochthonous infections with the hepatitis E Virus (HEV) have been recognized in developed countries. Venison from wild boar is already known to be a potential source of infection, if not prepared properly by the consumer. In Germany, certain wild animals are known to be a reservoir for HEV. However, current information is missing about European brown hares (Lepus europaeus) and wild rabbits (Oryctolagus cuniculus). Thus, a total of 833 hunting-harvested animals (European brown hares n = 669; wild rabbits n = 164) were tested for the occurrence of HEV RNA and HEV antibodies. For this, liver and blood specimens were taken after hunts in six German federal states. HEV antibodies were found by ELISA in 2.2% (624/14) of European brown hares, but no HEV RNA was detectable by nested real-time RT-PCR. In contrast, a seroprevalence of 37.3% (126/47) was observed for wild rabbits, and 17.1% (164/28) of the samples were HEV RNA positive. Genomic analysis revealed that these partial sequences clustered within the rabbit clade of HEV-3 genotype. In addition, one rabbit sequence segregated into subtype 3g of HEV-3. Highest seroprevalences for hares and rabbits were detected in the federal states of Bavaria and of Schleswig-Holstein, respectively. Comparing urban, rural and insular areas, the highest seroprevalence was shown for wild rabbits in rural areas and for European brown hares on the northern island Fehmarn. This study provides evidence that European brown hares and wild rabbits from Germany can be infected with HEV. The different prevalences indicate that wild rabbits are a potential reservoir for HEV in Germany, whereas European brown hares seem to be only of minor importance for the epidemiology of HEV.

Overall internal exposure to mycotoxins and their occurrence in occupational and residential settings--An overview.

Mycotoxins are toxic secondary metabolites of various fungal species that can contaminate food and feed, as well as indoor environments. Numerous studies have summarized the adverse health effects of mycotoxins and described severe intoxications of humans and animals. The major health concerns are caused via the alimentary route which unambiguously is the main source for human internal exposure; however, the relevance of other pathways under environmental and occupational conditions should also be considered. Thus firstly, this review aims in summarizing literature data on potentially inhalable mycotoxins occurring in dusts or air in residences and in working environments. Secondly, it gives an overview of the overall internal body burden of mycotoxins in humans in an attempt to characterize total human exposure. These data are also discussed in relation to the current toxicologically based values used for risk assessment.

Detection of psychrophilic and psychrotolerant Clostridium spp. in chilled fresh vacuum-packed meat using different PCR methods.

Since 1989, blown pack spoilage has been recognized as a special form of spoilage in vacuum-packed raw and cooked beef. However, only limited information concerning the occurrences of bacteria causing blown pack spoilage on chilled fresh meat is available. In this study, a total of 63 beef and 33 lamb commercially available samples from different countries and without any signs of spoilage were examined for contamination with psychrophilic and psychrotolerant Clostridium spp. using different PCR systems. In total, 34.4% of the chilled fresh vacuum-packed meats were PCR positive. A higher number of lamb samples were identified as PCR positive compared with beef. A geographical relationship between positive results and the origin of the samples could not be determined. PCR system described by Brightwell and Clemens (Development and validation of a real-time PCR assay specific for Clostridium estertheticum and C. estertheticum-like psychrotolerant bacteria. Meat Sci 2012;92:697-703) gave the highest number of positive detections compared with the Broda, Boerema and Bell PCR system (PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats. J Appl Microbiol 2003;94:515-22). Eight clostridia isolates from two German beef and four Welsh lamb samples were isolated overall. Three of these clostridia isolates were identified as Clostridium estertheticum whereas five clostridia isolates remain unidentified. The study shows that psychrophilic and psychrotolerant Clostridium spp. are more prevalent in retail samples than previously suspected.

Stability of bovine spongiform encephalopathy prions: absence of prion protein degradation by bovine gut microbiota.

Bovine spongiform encephalopathy (BSE) is transmitted by the oral route. However, the impacts of anaerobic fermentation processes in cattle on the stability of BSE-associated prion protein (PrP(Sc)) are still unresolved. In this study, experiments were designed to assess the ability of complex ruminal and colonic contents of bovines to degrade BSE-derived PrP(Sc). No significant decrease in PrP(Sc) levels in BSE brain homogenates was detected by Western blotting after up to 66 h of co-incubation with intestinal fluids. These results indicate that BSE-associated PrP(Sc) survive gastrointestinal digestion processes in cattle and might be excreted via faeces.

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.

Infectivity of scrapie prion protein (PrPSc) following in vitro digestion with bovine gastrointestinal microbiota.

The influence of a complex microflora residing in the gastrointestinal tract of cattle on the prion protein plays a crucial role with respect to early pathogenesis and the potential infectivity of faeces resulting in contamination of the environment. It is unknown whether infectious prion proteins, considered to be very stable, are inactivated by microbial processes in the gastrointestinal tract of animals during digestion. In our previous study it was shown that the scrapie-associated prion protein was degraded by ruminal and colonic microbiota of cattle, as indicated by a loss of anti-prion antibody 3F4 immunoreactivity in Western blot. Subsequently, in this study hamster bioassays with the pre-treated samples were performed. Although the PrP(Sc) signal was reduced up to immunochemically undetectable levels within 40 h of pre-treatment, significant residual prion infectivity was retained after degradation of infected hamster brain through the gastrointestinal microflora of cattle. The data presented here show that the loss of anti-prion antibody 3F4 immunoreactivity is obviously not correlated with a biological inactivation of PrP(Sc). These results highlight the deficiency of using Western blot in transmissible spongiform encephalopathies inactivation assessment studies and, additionally, point to the possibility of environmental contamination with faeces containing PrP(Sc) following an oral ingestion of prions.

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.

[25 years Mycotoxin workshop]. / 25 Jahre Mykotoxin-Workshop.

Mycotoxin research has a long tradition in Germany and is documented by a series of annual meetings which started 25 years ago. This paper gives an historical review on these Mycotoxin-Workshops. The first mycotoxin workshop in 1979 at the Federal Centre for Meat Research in Kulmbach was initiated by the former Federal Ministry of Agriculture and mainly thought to bring together scientists from the Federal research facilities. Main topics at that early time of mycotoxin research were food and feed safety, the mycology of toxin producers, the analysis and toxicology of mycotoxins. In the following years the Mycotoxin Workshop was influenced not only by working groups from the Federal research facilities but also from universities, state laboratories, other organisations and research scientists from outside Germany and with different disciplines. The number of participants increased from 19 at the beginning to more than 150 up to now and in order to organise these annual meetings at varying locations, in 1997 the society for mycotoxin research was founded. Since that time the Society for Mycotoxin Research (www.mykotoxin.de) is responsible for the organisation of the Mycotoxin Workshops.In addition the Society for Mycotoxin Research organizes the Brigitte Gedek science award, endowed with ¢ 10,000, and the Münchner Mycotoxin fellowship program, both intented to promote scientific research in mycotoxinology.

[Meat and potential risks]. / Fleisch und Risikopotential.

By the term "meat" the consumer understands muscle meat, that is skeletal muscle. The german regulations, the "Leitsätze für Fleisch und Fleischerzeugnisse" as well as the Codex Alimentarius define meat in a much broader sense to include all edible parts of slaughtered or shot warm-blooded animals (skeletal muscles, fat, heart, gut, liver etc). Therefore, a differentiated procedure is required when the risk of BSE transmission by meat has to be estimated. This must be based on knowledge of occurrence, amount, and persistence of BSE agents in the organs of animals. The risk evaluation has to include further factors: differences between animal species, age of the animal at the time of slaughter, the possibility of contamination during the slaughtering and cutting process, and--as far as meat products are concerned--the processing technology. To date there are no indications for the existence of transmissible spongiform encephalopathy in pigs, poultry and fish, even though BSE has been transmitted to pigs experimentally by intracranial administration. Muscle meat of these species can be considered safe. Muscle meat of experimentally infected cattle in the preclinical stage and even of animals with clinically manifest BSE has not shown infectivity in homologous and autologous bioassays performed so far. This finding justifies the assumption that the risk of BSE exposure by the consumption of beef can be classified as extremely low. Nevertheless, the absence of a proof of infectivity can at present not be equated with absence of BSE agents. This is because of the limits of sensitivity of the bioassays and because muscle meat does contain non-muscle tissue such as connective tissue collagen, nerve and lymph tissue and blood vessels.

Ochratoxin A in Brewer's Yeast Used as Nutrient Supplement.

Brewer's yeast comprises different strains ofSaccharomyces cerevisiae used for beermaking. It is additionally used as a nutrient supplement to increase the intake of B vitamins and is recommended primarily for children in growth, women during pregnancy and lactation and persons during convalescence. A total of 51 samples of brewer's yeast from the German market were analysed for the occurrence of ochratoxin A (OTA) by means of immunoaffinity clean up and HPLC with fluorescence detection. Thirty-two samples (63%) were found to be naturally contaminated with OTA in the range from the detection limit (0.03) to 1.53 ng/g. Mean values of the positive samples varied between 0.10 ng/g (powder) and 1.2 ng/g (dragees). In a worst case scenario, the consumption of brewer's yeast could enhance the calculated daily intake for the German population by 10 to 14 ng OTA/day and person and increase the intake particularly for children from 1.3 up to about 1.9 ng/kg body weight.Thus, the results document that food supplements consisting of natural brewer's yeast from the brewing process are a yet unknown source for the intake of ochratoxin A and a potential exposure risk. The screening of brewer's yeast food supplements for OTA is therefore recommended in the context of food safety and quality control.

Analyses of red fermented rice (angkak) and report of a newMonascus metabolite.

A HPLC-based method for the analysis of red fermented rice and results obtained with it are presented. Formation of citrinin, red, orange and yellow pigments byMonascus depends on the culture substrate. Citrinin and some pigments are decomposed by heat. A newMonascus metabolite, monascodilone, its structure and preliminary data on its toxicity are reported.

A simple HPLC method for the determination of the mycotoxins ochratoxin A and B in blood serum of swine.

This paper presents a simple method for the determination of ochratoxins A (OTA) and B (OTB) in pig blood serum. The method includes serum acidification (pH < 1.6) and precipitation of protein with 15% trichloroacetic acid, liquid partitioning with dichloromethane and fluorescence detection. The estimated detection limits were 0.1 ng OTA/ml and 0.2 ng OTB/ml. The mean recoveries from artificially contaminated samples (n = 6 replicates/mycotoxin) spiked at 0.3, 1 and 3ng OTA and OTB/ml, respectively, were 86.8% (s.d. = 8.4) for OTA and 90.0% (s.d. = 9.8) for OTB. Forty-nine Romanian pig blood serum samples (94% of 52 analysed) were found to be naturally contaminated with OTA in the range 0.1-13.4 ng/ml. No sample was found positive for OTB. The method is technically simple, specific, cost effective, suitable for large sample throughput and requires small amount of sample and reagents. It fulfils the criteria for a routine method and could be a suitable toolfor surveying OTA in pig herds and in slaughtered pigs.

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.

Preliminary results on Ochratoxin A concentrations in blood of patients with various kidney diseases in Germany.

Ochratoxin A (OTA) is supposed to induce renal diseases in man and animals and a correlation between renal diseases and OTA concentration in blood is suspected. Therefore, we measured OTA concentrations in blood of subjects suffering from various renal diseases as e.g. interstitial nephritis or mesangial proliferating glomerular nephritis (GN) and compared them with the blood concentration of healthy individuals. We found OTA in 87% of all samples. There was no significant difference between OTA concentrations of healthy individuals and patients but some renal diseases (e.g. chronic glomerular nephritis) showed increased numbers of samples containing more than 1.5 nmol/l OTA in sera. In contrast, in samples from patients suffering from membranous or focal-sclerotic glomerular nephritis no concentrations above 1.5 nmol/l were found. Our preliminary results show that OTA is abundant in nearly all serum samples but some renal diseases show increased numbers of samples with high (>1.5 nmol/l) OTA concentrations.

[Relevance of mycotoxin contaminated feed for farm animals and carryover of mycotoxins to food of animal origin]. / Mykotoxinbelastung der Nutztiere über Futtermittelaufnahme und der Verbraucher durch Lebensmittel tierischer Herkunft.

Contaminated feed is the main source for mycotoxin infestation of farm animals. The oral intake of fungal metabolites with feed results in a negative impact on all relevant parameters of animal production. Moreover, under experimental conditions mycotoxins and/or their metabolites can be traced in meat, edible tissues, milk and eggs. However due to the high concentrations of toxins involved, such findings are rare in the daily practice. In Germany today only aflatoxins (aflatoxin M1 in milk) and ochratoxin A (in blood, meat and edible tissues from swine) are of practical relevance from the view of food hygiene and food safety. Other mycotoxins at present discussed like toxins of Fusaria (trichothecenes, zearaleone, fumonisins) and ergot alkaloids are of no importance as possible contaminants in food from animal origin although they could have a negative impact on animal production.

Chemical characterisation of cheese associated fungi.

Recent work in our laboratory has demonstrated that the most common contaminating fungi on different types of cheese are;Penicillium commune, P. nalgiovense, P. solitum, P. discolor, P. roqueforti, P. crustosum, P. nordicum andAspergillus versicolor. On blue cheese a new speciesP. caseifulvum has been discovered as a surface contaminant. A large number of known and unknown metabolites have been described from the above mentioned cheese associated fungi from both synthetic media and real samples. Based on chemotaxonomy our laboratory has discovered thatP. roqueforti should be divided into three species:P. roqueforti (from cheese),P. carneum (from meat) andP. paneum (from bread). SimilarlyP. verrucosum should be divided intoP. verrucosum (from cereals) andP. nordicum (from cheese and meat products). Both species produce ochratoxins, however, only the former species produce citrinin.

Dust of grains and malts as a source of ochratoxin A exposure.

Ochratoxin A (OTA) was found more frequently and in higher concentrations (range 0.05-9.9 µg/kg) in dust samples than in corresponding samples of barley (n=23) and malt (n=9), which contained only trace amounts. Feed pellets and meals, prepared out of malt rootlets and dust fractions, contained OTA up to 6.01 µg/kg. Serum samples from seven malt factory workers showed OTA levels between 0.13 and 2.6 ng/ml. Higher levels in autum (median 0.58 ng/ml) than in summer (median 0.34 ng/ml) indicate that handling of barley in past-harvest time and an increased exposure to dusts might be responsible for these findings.

Comparison of ELISA and HPLC for the determination of histamine in cheese.

A competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for histamine in cheese was compared with a reversed-phase liquid chromatography (RP-HPLC) method. Cheese was homogenized with phosphate-buffered saline (PBS), centrifuged, and filtered, and the supernatant was diluted with PBS for CD-ELISA. For RP-HPLC, biogenic amines (histamine, tyramine, putrescine, and cadaverine) were derivatized with 9-fluorenylmethylchloroformate, followed by reversed-phase chromatography and fluorescence detection. Detection limits and mean recoveries (10-1000 mg/kg) were 2 mg/kg and 93% for CD-ELISA and 1 mg/kg and 99% for RP-HPLC, respectively. Analysis of 50 commercial cheeses according to both methods showed good agreement for histamine (r = 0.979; concentration range = 2-1800 mg/kg). At a threshold level of 10 mg/kg, the ELISA gave no false-negative and three false-positive results. The results show that the ELISA is suitable for the determination of histamine in cheese.