The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Escherichia coli O157 lipopolysaccharide (LPS) was developed with sera from 63 children with confirmed recent E. coli O157 infection and from 256 age-stratified urban controls. The median ELISA values for control and case sera were 0.05 (interquartile range, 0 to 0.20; mean ؎ standard deviation [SD], 0.15 ؎ 0.22) and 1.41 (interquartile range, 1.11 to 1.59; mean ؎ SD, 1.41 ؎ 0.53), respectively (P < 0.001). With a breakpoint of 0.59 (mean ELISA value of the control sera ؉ 2 SDs), the assay had a sensitivity, specificity, and positive and negative predictive values of 95, 94, 80, and 98%, respectively, for recent E. coli O157 infection. The O157 LPS assay and Vero cytotoxin (VT) 1-neutralizing-antibody (NAb) assay were used to compare the relative frequencies of O157 LPS antibodies and VT1-NAbs in an age-stratified urban population from Toronto, Ontario, Canada, and in 216 healthy family members from dairy farms in southern Ontario. The frequency of O157 LPS antibodies was about threefold higher in dairy farm residents (12.5%) than in urban residents (4.7%) (P < 0.01). Similarly, the frequency of VT1-NAbs was about sixfold higher in dairy farm residents (42.0%) than in urban residents (7.7%) (P < 0.001). These findings are consistent with a greater level of exposure of dairy farm residents to VT-producing E. coli (VTEC) strains. The high rate of seropositivity to VT1 in farm residents probably reflects the booster effect of repeated VTEC exposures and argues against a sustained generalized immunosuppressive effect of VT1. Seroepidemiological studies may help in assessing the level of exposure of different populations to VTEC strains.

A PCR assay that uses primers whose sequences were obtained from the published sequence of the cdt-III gene was developed to determine the frequencies of the cdt-I, cdt-II, and cdt-III genes in Escherichia coli isolates from humans and animals. E. coli isolates producing cytolethal distending toxin (CDT) were infrequently detected. The cdt-I gene was preferentially detected in strains with the cnf1 gene, while the cdt-III gene was found in strains carrying the cnf2 gene. The cdt-III genotype was more prevalent in animal isolates, while the cdt-I and cdt-II genotypes were more evident in human isolates. The presence of further cdt gene variants was indicated by the presence of toxin activity in cell culture in the absence of PCR amplification of the cdt-I, cdt-II, or cdt-III gene.Cytolethal distending toxin (CDT) is a potent bacterial exotoxin that has dramatic effects on target cells in culture (23). Intoxication of eukaryotic cells results in blockage of the cell cycle at the G 2 /M transition (2, 22) by a mechanism involving prevention of cdc2 protein kinase dephosphorylation and activation (6). Cellular effects include accumulation of F-actin assemblies resembling stress fibers (2), progressive cell distension, and eventual cell death (14, 15). Epidemiologic studies have not found a statistically significant difference in the incidence of CDT-producing Escherichia coli in children with diarrhea and healthy controls (23). However, animal studies with toxigenic E. coli strains (18) and Campylobacter jejuni CDTknockout mutants (26) suggest that CDT may be a virulence factor in vivo.Three genes, cdtA, cdtB, and cdtC, are required for the production of an active toxin in E. coli (24,28), Shigella dysenteriae (19), Campylobacter spp. (25), Haemophilus ducreyi (7), Helicobacter hepaticus (32), and Actinobacillus actinomycetemcomitans (16). Although these genes appear to be homologous in all bacteria, the degree of relatedness at the genetic level varies widely (23). Three cdt genetic variants, designated cdt-I, cdt-II, and cdt-III, have been identified and cloned from E. coli (22,24,28). However, PCR methods have been developed only for the detection of the cdt-I and cdt-II variants (19). The distribution of the cdt-I and cdt-II genes within E. coli is not well characterized (23), and little is known about the incidence or prevalence of cdt-III. PCR primers were developed for the detection of cdt-III. A number of E. coli isolates from humans and animals were tested for the presence of all three cdt variants.A group of 46 CDT-producing E. coli isolates was collected between 1986 and 1998 from Canadian and international sources. CDT-positive animal isolates collected during this time were also characterized. Enhanced surveillance for cdt genes was accomplished by testing 151 non-Shiga toxin-producing non-O157:H7 E. coli isolates collected between October 1998 and the end of 2000 together with a randomly chosen subset of 53 E. coli O157:H7 isolates. In addition, 72 non-O157 E. coli isolates from a series of consecutive...

Oral inoculation of calves and steers with 10 10 CFU of Escherichia coli O157:H7 induced prompt and sustained increases in serum antibodies to O157 lipopolysaccharide. Neutralizing antibodies to verotoxin 1 also increased rapidly in most steers but more gradually in calves. None of the animals developed neutralizing antibodies to verotoxin 2. These serological responses were not correlated with elimination of infection in calves or steers or protection of calves against reinfection with the same strain.

Investigation of the specificity of an enzyme-linked immunosorbent assay (ELISA) for detection of Escherichia coli O157:H7 in raw meats (EHEC-Tek; Organon Teknika Corp.) revealed that the target antigens of the detection reagent, monoclonal antibody 4E8C12, were present in numerous serotypes of E. coli and that their ELISA reactivity was influenced by bile salts, acriflavine, and heat. The specificity of the ELISA was improved by a modified test protocol incorporating immunocapture.