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An antiflagellin monoclonal antibody (15D8) was used to detect the presence of flagella in nonmotile variants of several pathogenic Escherichia coli serotypes. Of the 48 isolates examined, 15 reacted with monoclonal antibody 15D8 and were culturally confirmed to be motile. Of the 38 clinical strains designated O157:NM or O157:H-, 7 were antibody reactive and motile and agglutinated with anti-H7 sera.

Enterohemorrhagic Escherichia coli (EHEC) causes a variety of clinical conditions, the most important being hemorrhagic colitis and hemolytic uremic syndrome. A curative therapy of EHEC diseases is not yet feasible. This study investigates the antibody reactivity of Lactobin, a standardized immunoglobulin (Ig) preparation, obtained from the colostra of non-immunized cows. Three different batches of Lactobin exhibited equally high titers of specific antibodies against Shiga-like toxins (SLTs, verocytotoxins) and EHEC hemolysin (EHEC-Hly) produced by E. coli O157. In addition, Lactobin blocked the cytotoxic effect of SLT-I and SLT-II on Vero cell monolayers and inhibited the cytolytic effects of EHEC-Hly on human erythrocytes. Since Lactobin contains high levels of antibodies and neutralizing activity against important virulence factors of EHEC O157, this drug has potential use in the treatment of diarrhea and the prevention of EHEC-associated hemolytic uremic syndrome.

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 farm 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.

In June '93, 4 children, aged 1.5-3.5 years, all living in one town, were admitted to our hospital with the diagnosis hemolytic uremic syndrome (HUS) within one week. In cooperation with the local health authorities a common source was searched for. Questionnaires indicated that the single condition shared by all patients was swimming water. The patients were not acquainted, visited different daycares, and had no food resources in common. All 4 patients bathed in the same, shallow, recreational lake within a period of 5 days. During this time the air temperature was high according to Dutch standards (around 27 degrees C), and many people visited the lake, estimated several hundreds a day. The water level was lower than normal. Diarrhea followed 3-11 days after swimming and the first clinical symptoms of HUS developed 6-7 days after the onset of diarrhea. The lake was closed for swimming when the fourth HUS patient was diagnosed and the possibility of transmission by way of the lake was mentioned. E. coli O157: H7 was demonstrated in the fecal samples of 2 index patients. The samples were taken 9-20 days after the start of diarrhea. Antibodies to O157 and verotoxin 2 were strongly positive in all patients. A local outbreak of diarrheal illness was not registered. Of 16 family members who also swam in the same lake, 7 developed symptoms of enteritis, 3 had positive cultures of their fecal samples and 5 had positive serology. Pulsed-field gel electrophoresis of the E. coli isolates of the patients and family members showed an identical pattern. No O157: H7-DNA could be detected in filter concentrated lake water samples using polymerase chain reaction (PCR) enhancement. These samples were, however, taken 16 days after the latest possible date of contamination of our patients, 15 days after decrease of the air temperature to 15-17 degrees C, and 14 days after the inlet from water from the environment. It could thus very well be that the microorganism was no longer present. This third report of swimming water associated HUS should direct environmental surveys in similar cases of local HUS outbreaks.

We examined 30 children with classical hemolytic-uremic syndrome (HUS) for the presence of enterohemorrhagic Escherichia coli (EHEC) strains in stool samples and determined the specific immune response to O157 lipopolysaccharide in acute-phase serum samples from these patients. EHEC O157 strains were isolated from stool samples of 18 (60%) of the patients, and non-O157 EHEC strains were isolated from 5 (17%) of the patients. For O157 strain isolation from stools, we introduced a selective enrichment step using O157-specific antibodies attached to paramagnetic particles (immunomagnetic separation [IMS] method). This procedure allowed the detection of O157 strains at 10(2) CFU/g of stool in the presence of 10(7) coliform background flora organisms. By using IMS followed by plating on sorbitol MacConkey (SMAC) agar and cefixime-tellurite SMAC (CT-SMAC) agar, O157 strains were detected in 18 samples, whereas colony hybridization detected a subset of 12 positive samples and direct culture on CT-SMAC or SMAC agar detected only 7. Three of the 18 O157-positive stools were negative by cytotoxicity assay performed with stool filtrates and by direct PCR with DNA extracted from stools. The IMS technique allowed the isolation of O157 strains from 18 of 20 patients with serological evidence for O157 infection. Apart from the increase in sensitivity in O157 detection compared with that of direct culture, the IMS technique also has the advantage of being less labor-intensive and less time-consuming than the molecular methods. IMS can therefore be considered an efficient method for wide-spread use in the detection of O157 strains in clinical microbiology laboratories. However, because a significant number of HUS cases were attributable to non-O157 EHEC serogroups, the use of additional methods besides IMS in the bacteriological diagnosis of HUS is necessary.

A total of 239 serum samples from 136 persons were used for bacterial agglutination assay (BA) against predominant three O-antigens of VTEC. All VTEC isolates from stools of 30 patients were only O157:H7 serotype (these patients are called group I). The levels of positive BA antibody titers (over 1:160) to O157-antigen were recognized in each patients as follows. The VTEC isolated patients with HUS or without HUS in group I were all of 13 (100%) and 14 (82.4%) in 17 patients, respectively. And 21 (65.6%) patients of group II (HUS patients with stool negative cultures, or stool cultures were not performed in 32 patients), and 6 (15.0%) patients of group III (family members of group I and II; 40 persons), were also recognized. In group IV (patients with diarrhea due to other pathogen than VTEC; 11 patients), and V (clinically healthy persons; 23 persons), none were recognized as positive BA antibody titers. All patients in the group II except one who had a positive BA antibody titer to O111, were not recognized to O111 and O26. A few VTEC-positive patients without gastrointestinal syndrome did not have significant agglutinating titers to O157-antigen on the days after VTEC isolation. However, almost all patients with diarrhea due to VTEC and HUS, and with VTEC but no HUS, had a level of positive BA antibody titer on the 5 day after onset of diarrhea. These results suggest that this serological assay is a very simple and useful tool for diagnosis of VTEC infection when VTEC are not detected by culture method due to antimicrobial treatment, or due to the lapse of many days after onset of diarrhea.