Antigen capture enzyme-linked immunosorbent assays (ELISAs) for the detection of Stx1 and/or Stx2 in cattle feces were validated in comparison to the Vero cell cytotoxicity neutralization test (as a "gold standard") applied in the course of a monitoring program for Shiga toxin-producing Escherichia coli in German cattle herds as a prescreening test and compared to MK1/MK2 PCR as an alternative prescreening test.Several enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of Shiga toxin-producing Escherichia coli (STEC), some of which use the P1 glycoprotein from hydatid cyst fluid, or Gb3, for Shiga toxin capture (1, 2, 16). Recently, monoclonal antibodies (MAbs) to verocytotoxins Stx1 and Stx2 were used to examine animal feces (3). Mitomycin C can enhance Stx production in E. coli and was applied to increase the ELISA sensitivity (12). We describe the validation of ELISAs for the detection of Stx1 and/or Stx2 in cattle feces. These ELISAs were used in a monitoring program as a prescreening test and compared to MK1/MK2 PCR (13).A total of 100 E. coli field strains isolated from cattle were tested in the STEC-ELISAs, the Vero cell cytotoxicity assay (VCA), and the Vero cell cytotoxicity neutralization assay (VCNA) which served as the "gold standard." The assays were performed as described by Konowalchuk et al. (14), with some modifications (8, 17). The cytopathic effect (CPE) and the neutralization of the CPE were measured by determining cell densities in a Coulter Z2 counter (Coulter Electronics Inc., Miami, Fla.). The specificity of the VCA for the toxin production of E. coli field isolates was determined by neutralization of the CPE on Vero cells (ATCC CRL1587) by MAbs 13C4 (ATCC CRL1794) and 11E10 (ATCC CRL1907).The ELISAs utilized hydatid cyst fluid of Echinococcus granulosus for detecting the Shiga toxins, as described previously (18). A total of 200 l of supernatant of the E. coli field strains or preenriched fecal samples of cattle were directly investigated in this assay. Each sample was tested in quadruplicate. Positive controls (E. coli C600-J1 for Stx1 and E. coli C600-W34 for Stx2) and a negative control (E. coli C600) were tested sixfold on each plate (blanks in each corner). The MAbs 13C4 and 11E10 were used for the specific detection of Stx1 and Stx2. Bound MAbs were detected with goat anti-mouse peroxidase conjugate and 3,3Ј,5,5Јtetramethylbenzidine (TMB; KPL, Gaithersburg, Md.) as the substrate. The reactions were stopped by adding 0.5 M sulfuric acid. Optical densities (ODs) were read at 450 nm with a reference filter at 620 nm. The mean ODs of positive and negative controls and of the samples were calculated from the blank-corrected OD readings (OD value Ϫ blank value), and an index was determined for each sample according to the following equation:index ϭ mean of OD sample Ϫ mean of OD negative control mean of OD positive control Ϫ mean of OD negative controlThe study showed that Stx1-and Stx2-specific ELISAs can be utilized for the detection of these Shiga tox...
ZusammenfassungFiir den Nachweis von VTEC in Lebensmitteln und F~izes wird ein Verfahren vorgestellt, das nach optimierter Anreicherung sowohl einen sensitiven VT-Screening-Schritt (alternativ ELISA oder PCR) als auch eine Methode zur gezielten Isolierung der VTEC beinhaltet. Die VT-positiven Isolate miissen dann nach biochemischer Best~itigung als E. coli und Serologie-molekularbiologisch auf zus~itzliche Virulenzmarker untersucht werden. Als geeignetes Anreicherungsmedium fiir Lebensmittel und F~izes stellte sich mTSB heraus. Das Sch/.itteln der Kulturen und der Mitomycin-C-Zusatz erh6hten die Nachweisempfindlichkeit fiir VT erheblich. Fiir das VT-Screening aus der 18h-Kultur sind der selbstentwickelte VT-Rezeptor-ELISA,,BgVV,,, der kommerzielle Premier-EHEC und die PCR (MKI/MK2) alternativ einsetzbar. SummaryA procedure for the detection of VTEC in foods and faeces A procedure for the detection of VTEC in foods and faeces is presented. This procedure includes an optimized enrichment, sensitive VT-screening tests by using ELISA or PCR and a specific isolation of VTEC/EHEC. The VT-positive isolates must be confirmed as E. coti by means of biochemical methods. Furthermore serotype and other virulence factors have to be detected.A suitable nutrient medium for VTEC-enrichment in foods and faeces is mTSB. Shaking and addition of Mitomycin C to the nutrient medium increase the sensitivity of VT-detection considerably. The self-developed VT-receptor-ELISA,,BgVV,,, the commercial available Premier-EHEC and PCR (MKI/MK2) are alternatively applicable for VTscreening after enrichment. In case of VT-positive samples a specific serovare-independent isolation by using of VT-Colony-Immunoblot is following. The isolation rate is more than 90 %. A combination of this method with the immunomagnetic separation (IMS) is recommended in case of EHTEC O157. The detection limit of the whole procedure is approximately 10 CfU/25 ml or 25 g of foods. Reproducible results are also found after deepfreezing or storing samples in refrigerator before testing (milk > 8 d at -20 °C, minced meat > 14 d at-20 °C, feacaI samples > 8 d at +4 °C). Soft cheeses and raw sausages have to be examined immediately without storage.
ZusammenfassungWir haben in vom Tier stammenden Lebensmitteln, wie roher Milch (einschliefflich Vorzugsmilch), rohem oder unzureichend gegartem Hackfleisch und rindfleischhaltiger Rohwurst STEC nachgewiesen, isoliert und charakterisiert. We-.gen ihrer geringen Anzahl im Vergleich zur Gesamtkeimflora 1st ein spezielles Verfahren hierzu erforderlich. Die STECIsolate wurden mittels PCR-Verfahren auf das Vorhandensein yon Genen ffir zehn Virulenzfaktoren (stxl, stx2, eae, hlyA, katP, espP, etpD, astA, colD157 und SummaryDetection of shigatoxin producing Escbericbia coli (STEC) in foods and characterization of isolates We detected STEC in foods of animal origin like raw milk including certified milk, raw or undercooked minced beef and beef containing raw sausage. Furthermore we isolated and characterized the STEC. The number of STEC in the total bacterial count is low. A multistep procedure was to be performed including two-step enrichment, sensitive screening tests, methods for specific STEC isolation and characterization. The STEC isolates were checked for the existence of genes of encoding ten virulence factors (stxl, stx2, eae, hlyA, katP, espP, etpD, astA, colD157, and ileX) by using PCR. All genes of these virulence factors could be detected in isolates from raw milk and with exception of katP in raw minced beef, too. The genes katP, etpD, colD157 and ileX could not be detected in isolates from raw sausage. One isolate from this matrix belonged to serogroup O22:H8 and had the gene combination eae, hlyA, and espP. 15,2 % of the investigated STEC isolates from raw milk had the stx-genes and besides the factors hlyA, katP, espP, and etpD. In addition, 9,1% of these isolates showed the genes eae and colD157. One isolate from minced beef had the combination stx2, hlyA; espP, etpD, and eae. All these isolates belonged to serogroup O157.Furthermore, we investigated 28 EHEC strains belonging to serogroup O157. These isolates were obtained from patients suffering from HUS. The combination eae, hlyA, etpD, katP, and espP was found in 18 cases (64 %). EHEC strains belonging to other serogroups isolated from stool samples of HUS patients showed different combinations of virulence factor genes. In the serogroups 026, O103 and O111 the combination eae, hlyA, and etpD was frequent. The genes for the virulence factors were distributed sporadically in ten EHEC isolates of different serogroups from patients suffering from diarrhoea and in 17 isolates from carriers showing no symptoms.Einleitung
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