A total of 56 fecal samples collected from free-ranging wild ruminants during the deer hunting season and 20 cumulative fecal samples from wild ruminants in a deer park were examined for the presence of STEC. After selective enrichment, Stx-positive samples were identified by means of ELISA and PCR. Subsequently, STEC were isolated from all positive samples using VT colony immunoblot and characterized by PCR (stx type, ;eae, EHEC hlyA) and serological methods. ill addition, 34 STEC isolates from venison were characterized. 51.8% of the fecal samples from free-ranging deer were positive for Stx. Among the STEC isolated from deer feces and venison, Shiga toxin subtype 2d was found to be predominant. All isolates were eae-negative; EHEC hlyA was found in ca. 1/3 of the isolates. Moreover, a great serovariety was established (11 O serogroups from deer feces, 13 O serogroups from venison). Thus, wild ruminants constitute a reservoir of STEC/EHEC pathogens. In view of their properties, most of these STEC seem to have a low virulence. Nevertheless, meat from wild ruminants should be considered as a potential Source of human EHEC infection.

An optimized method for sensitive detection of Shigatoxin-producing E coli (STEC) in vegetarian foods (apple juice and lettuce) is given. The method is independent from serotype of STEC. Furthermore this procedure is different from those used for investigation of foods originated from animals ( 35 LMBG (L07,18-1) und DIN 10118). One step cultivation for 18 h at +37 degrees C is necessary. The enriched material is used for making a screening test. ELISA and PCR were brought in. When using PCR a modification step for sample preparation is necessary. In case of a Shigatoxin (Stx) or Shigatoxin gene (stx) positive result the specific STEC isolation has to be done by using a VT-colony-immunoblot. The detection limit is 50 cfu/25 g (ml) food.The sensitivity in case of investigation of sprouts from different origin is insufficient. The method describe above should not be used.

Brain samples of 849 wild ruminants (654 roe deer, 189 red deer and 6 chamois) from Bavaria were examined for the occurrence of encephalopathies caused by bacteria, using cultural, serological and genetic methods. In addition, 87 brain samples were investigated histologically for clarification of the pathogenetic relevance of specific microorganisms. Using conventional bacteriological methods, 464 different bacteria were isolated. 229 of them could be differentiated to the genus level and 235 to the species level. Totally, 35 different bacteria species were isolated, most frequently Micrococcus spp., Bacillus spp. and E. coli. Listeria spp. were detected in 43 brain samples (37 from roe deer, 5 from red deer and 1 from chamois). Sixteen strains were identified as L. innocua, 14 as L. monocytogenes, 9 as L. seeligeri and 4 as L. grayi. Serological investigations of L. monocytogenes showed that 9 strains belong to serotype 1/2a and five to 4b. Analysis of the geographical distribution of the Listeria findings indicate a statistically significant (p<0.011) regional aggregation in Unterfranken (prevalence for roe deer: 12.2%, versus 4.5% in Oberbayern-Schwaben, 6.1% in Niederbayern-Oberpfalz and 0% in Oberfranken-Mittelfranken). The histological investigation (HE staining) of 87 tissue samples contaminated with encephalitis relevant bacteria showed inflammation of different severity (mild meningitis and choroiditis (n = 26) to moderate (meningo)encephalitis (n = 13)) in 41 cases.

Mikrobiological controls in the context of the decision 2001/471/EC serve the verification of process controls of individual businesses and aren't designed for the general comparison of individually operating meat plants. The result of the individual meat plants and the continuous check of its good hygienic practice or the improvement in perhaps available hygienic weak points is authoritative. The decision allows besides destructive also non-destructive proceedings for sampling at animal carcases. For the check of the cleaning and disinfection success agar contact plate methods and swab methods can be chosen between. The results of the own checks have to be tested on predefined performance criteria for the mesotrophe aerobe counts and for Enterobacteriaceae of the decision 2001/471/EC.

When the Japanese microbiologist Shiga discovered a bacterium causing dysentery in humans in 1898, the organism was designated Shigella dysenteriae type 1. The toxin produced by the germ was found to have enterotoxic and neurotoxic properties. Later on, it became clear that, in most countries, Shigella dysenteriae type 1 does not play an important role in human infectious diseases. Nevertheless, this microorganism has spread a genetic message among other bacterial species, namely Escherichia coli and Citrobacter freundii.

Shigatoxin-producing E. coli (STEC) and their subgroup, the enterohaemorrhagic E. coli (EHEC) are known since about 25 years. Only EHEC can cause diseases in humans. A molecularbiological method cascade is described in this article. It contains the following steps: preenrichment, enrichment, preparation of samples (DNA extraction), screening PCR, specific STEC isolation by using DNA DNA hybridization, verification of isolates as STEC by using PCR, characterization of STEC isolates. Pulsed field gel electrophoresis and DNA sequencing are additional methods for the detection of clonal correlations. They should be performed in special labs, only. Furthermore, some information about an interlaboratory ring trial within Germany and some conclusions are given.

The results presented comprise the detection of a pathogenic island termed 'Locus of Proteolysis Activity' (LPA) in STEC by using PCR. Key genes for LPA are esp I, iha and btu B. We investigated 34 stx 2d positive isolates from fecal samples of wild life population (fallow deer, roe deer and red deer). All key genes for LPA could be detected in 23 (67,3 %) strains. 10 (29,4 %) isolates showed an iba gene, only. 1 (2,9 %) strain owned no LPA key gene at all. - Furthermore we investigated 25 STEC strains isolated from meat of wild life. All owned the stx 2d gene, too. The 3 key genes of LPA were detected in 15 (60 %) strains. 4 (16 %) isolates showed only an iha gene whereas 6 (24 %) strains did not show any LPA key gene at all. Other Stx producer Stx 1, Stx 2, Stx 2c, Stx 2f independent of the content of an eae gene did not show LPA key genes except 5 strains having an iha. It is known that LPA is integrated in sel C similar to LEE (Locus of Enterocyte Effacement). So it is impossible for strains having LPA to take up an LEE, too.

Fourteen, O157 serogroup Escherichia coli strains, isolated from humans, cattle, and pigs in Poland and in Germany, were investigated by means of molecular biological methods. The presence of genes encoding the production of H7 flagellar antigen, Shiga toxins, intimin (including all five of its variants), translocated intimin receptor, and enterohemolysin, was investigated by PCR. It was demonstrated that only 3 strains had the H7 genes; therefore, they were classified as O157:H7. The Shiga toxin markers were detected in all 10 but one strains isolated from humans and from cattle, whereas none of the four O157 isolates of pig origin was stx-positive. The eaeA intimin (all-gamma variant) and ehly enterohemolysin genes were found in 10 and in 11 strains, respectively, whereas the tir marker was detected in 7 isolates. The genetic relatedness of the strains was examined by the amplification of DNA sequences located between the repetitive element IS3 by the IS-PCR method. The electrophoretic picture of the amplicons obtained was analysed by the UPGMA test with the Jaccard coefficient, and it was demonstrated that the E. coli strains tested were classified into 4 distinct clonal lineages. A close-genetic correlation between bacteria isolated in Poland and in Germany was also observed. The results obtained showed that IS-PCR is a powerful tool for revealing the clonal nature and genetic differences among E. coli O157 strains isolated from different sources.

The results presented comprise the detection of a pathogenic island termed 'Locus of Proteolysis Activity' (LPA) in STEC by using PCR. Key genes for LPA are esp 1, iha and btu B. We investigated 34 stx 2d positive isolates from fecal samples of wild life population (fallow deer, roe deer and red deer). All key genes for LPA could be detected in 23 (67,3 % ) strains. 10 (29,4 %) isolates showed an iha gene, only. 1 (2,9 %) strain owned no LPA key gene at all. - Furthermore we investigated 25 STEC strains isolated from meat of wild life. All owned the stx 2d gene, too. The 3 key genes of LPA were detected in 15 (60 %) strains. 4 (16 %) isolates showed only an iha gene whereas 6 (24 %) strains did not show any LPA key gene at all. Other Stx producer Stx 1, Stx 2, Stx 2c, Stx 2f independent of the content of an eae gene did not show LPA key genes except 5 strains having an iha. It is known that LPA is integrated in sel C similar to LEE (Locus of Enterocyte Effacement). So it is impossible for strains having LPA to take up an LEE, too.

The occurrence of Shiga toxin-producing Escherichia coli (STEC) was studied on four cattle farms. STEC were detected in 29–82% of the cattle. STEC with additional EHEC markers were detected on all farms. The occurrence of the complete virulence marker pattern (stx1 and/or stx2, eae, EHEChlyA, katP, espP) was correlated with the presence of known STEC serotypes. STEC O26[ratio ]H11 and O165[ratio ]H25 with the complete pattern of virulence markers were the most prevalent. STEC O157 (H7/H-) STEC O103[ratio ]H2 and STEC O145[ratio ]H- were found sporadically. Five clonal subgroups of the STEC O26[ratio ]H11 isolates were identified by pulsed-field gel electrophoresis. STEC O26[ratio ]H11 were present in three groups of cattle. This serotype was detected in a single group over the entire fattening period. Most STEC O26[ratio ]H11 with the complete pattern of potential virulence markers were found in clinically healthy cattle. These animals may represent a risk factor for farmers and consumers.

The existence of a High-Pathogenicity Island (HPI) in 62 eae-positive Shigatoxin-producing Escherichia coli (STEC) was checked. The HPI was detected in 2 of 18 STEC-strains isolated from foods, in 5 of 15 STEC-strains from fecal samples of cattle an in 5 of 29 EHEC-strains isolated from human stool samples. All HPI-positive strains showed the beta-eae-gene and belonged to the serogroups O 26 and O 118. (The O-Group of 5 HPI-positive strains was not typable.).

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile (LT) and heat-stable (STI or STII) enterotoxins. Differentiation between ETEC and other pathogenic and non-pathogenic E. coli as well as other Gram-negative bacteria responsible for induction of diarrhoea, requires isolation, biochemical identification and determination of toxins (or their genes--elt, estI, estII). A multiplex polymerase chain reaction (PCR) system for the rapid and specific detection of enterotoxin-gene-positive E. coli was developed. The primers described by other authors, specific for the universal stress protein A (UspA) of E. coli and enterotoxin genes were used and allowed a simultaneous amplification of the E. coli-specific uspA and the respective toxin genes. The specificity of this multiplex PCR system was confirmed by testing ETEC, non-ETEC and other non-E. coli bacteria. The specific 884 bp uspA gene and 280 bp (eltI), 166 bp (estI) or 278 bp (estII) amplification products were generated with the respective ETEC strains whereas no amplification was detected with non-E. coli bacteria. The multiplex PCR developed allowed the rapid and specific identification of enterotoxin-producing E. coli colonies directly grown from faecal samples of pigs with diarrhoea. The test may be used as a method for the determination of ETEC among other pathogenic groups of E. coli and other Gram-negative enteric isolates.

Fecal samples from 67 3-5-months-old calves with diarrhea were screened for the presence of shiga toxin-producing Escherichia coli (STEC). Several accessory virulence factors genes were also tested. Among 192 E.coli isolates tested, 15 (7.6%) were found to harbour the shiga toxin 1 or 2 (stx1 or stx2) genes. The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that stx2-positive bacteria mainly possessed the stx2e shiga toxin type gene. The enterohemolysin (hlyA) and intimin (ene) genes were found in seven (46.7%) STEC strains whereas the cytotoxic necrotizin factor 1 and 2 or the P fimbrial genes were detected in two isolates only. This study confirmed that calves are a reservoir of STEC strains (with all pathogenicity genes) that may be virulent for humans. (C) 2000 Elsevier Science Ltd. All rights reserved.

We subtyped eae-genes in 19 isolates from raw or undercooked samples (milk, meat, sausage, cheese) by using PCR. Furthermore we investigated 17 isolates from faecal samples from cattle and 30 isolates from stool samples from HUS-and enteritis patients and from carriers without symptoms for comparison. We detected the gamma-eae type first and foremost in 20 isolates belonging to serogroups O157:H7 or O157:H-.In addition it was found in 5 strains belonging to serogroup O145 and in 1 isolate of serogroup O111. beta-eae was detected in 7 isolates belonging to serogroup O26 and in 2 strains of serogroups O118 and O5. It was impossible to estimate the serogroup of 6 other isolates containing the beta-type. alpha-eae was detected in 5 isolates of different serogroups but the epsilon-type was found in 10 isolates belonging to serogroup O103:H2. It was impossible to detect differences in occurrence of eae-types between isolates from foods, faeces or stool samples. Furthermore we used different PCR systems for investigation of the insertion of locus of enterocyte effacement (LEE) in sel C. We detected that a LEE, containing the epsilon-eae is not integrated in sel C. But a LEE with the alpha-eae disruptes the sel C. All isolates containing the gamma-type showed only a positive PCR result by using primer pair K295/K296 indicating an insertion in sel C. But PCR results by using primer pair K255/K260 were negative. All beta-eae containing strains showed no PCR signal independent of the primer types. Further investigations by using DNA-Sequencing are necessary for detection of other LEE-insertion loci like phe U.

Zusammenfassung Die Diagnostik einer EHEC-Infektion des Menschen, einer seit 1998 bundesweit meldepflichtigen Infektionskrankheit, ist gegenwärtig nicht verbindlich geregelt. Dabei bestehen insbesondere zum Einsatz von genotypischen und phänotypischen Screeningverfahren noch unterschiedliche Auffassungen. Es gilt als gesichert, dass sie auf der Basis der Shigatoxinogenität erfolgen müssen. Anhand von künstlich kontaminierten Stuhlproben und vier unterschiedlichen EHEC-Teststämmen wurde mithilfe von Kulturmedien und ELISA-Kits ein zeit- und kostengünstiges Verfahren überprüft. Ziel war es, bereits einen Tag nach Proben-Eingang eine positive Verdachtsdiagnose vorliegen zu haben. Dieser ersten Stufe der EHEC-Diagnostik muss als zweiten Stufe zur Bestätigung der Verdachtsdiagnose die Isolierung des entsprechenden Ätiologischen Agens erfolgen (ggf. Speziallabor). Eine dritte Stufe der EHEC-Diagnostik umfasst die weitere Subdifferenzierung der Isolate durch die Genotypie, Lysotypie, die Virulenzmusterbestimmung, insbesondere für epidemiologische Zwecke, die in das Aufgabenspektrum des Nationalen Referenzzentrums für Salmonellen und andere Enteritiserreger fallen.