Evaluation of Enzyme-Linked Immunosorbent Assays and a PCR Test for Detection of Shiga Toxins for Shiga Toxin-Producing Escherichia coli in Cattle Herds

Abstract

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...

Analysis of the Clonal Relationship of Serotype O26:H11 Enterohemorrhagic Escherichia coli Isolates from Cattle

“…The nucleotide sequences of the A and B subunits of the stx 2 gene of the selected bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. EU700491) were identical to the stx 2 (53). The Stx2 cytotoxicity for Vero cells was also very high in the O26:H11 isolates.…”

Detection of Shiga Toxin-Producing Escherichia coli from Nonhuman Sources and Strain Typing

“…Comparative studies have shown that cytotoxicity tests are more sensitive for detection of Stx than immunological assays (48). The detection limits of any test system can be critical if the samples contain low amounts of Stx or if the assay fails to detect certain subtypes of Stx1 or Stx2 (51,53,54). Cytotoxicity tests can be used for direct Stx testing from fecal samples where immunological methods can fail (53).…”

Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H−/Hnt Isolates from Cattle and Clonal Relationship Analysis

“…Fermentation of sorbitol was detected on sorbitol McConkey agar (SIFIN GmbH, Berlin, Germany) and the production of EHEC hemolysin was detected on enterohemolysin agar (Haipha GmbH, Eppelheim, Germany). The expression of Shiga toxins was monitored by an enzymelinked immunosorbent assay (ELISA) using monoclonal antibodies specific for Stx1 or by the Vero cell neutralization assay described previously (54).…”