Shiga toxin-producing Escherichia coli infection and antibodies against Stx2 and Stx1 in household contacts of children with enteropathic hemolytic-uremic syndrome

Ninety-five household contacts (aged 2 months to 73 years) of patients with enteropathic hemolytic-uremic syndrome (HUS) were investigated for the presence of immunoglobulin (Ig) G antibodies to Shiga toxins Stx2 and Stx1 by Western blot assay. Thirty-one percent of the household contacts and 19% of 327 controls had anti-Stx2 IgG (heavy and light chain [H + L]), 5 and 8%, respectively, had anti-Stx1 IgG (H + L), and 3 and 2%, respectively, had both anti-Stx2 and anti-Stx1 IgG (H + L). The incidence of infections with Stx-producing Escherichia coli (STEC) was determined based on the following diagnostic criteria: STEC isolation, detection of stx gene sequences, free fecal Stx in stool filtrates, and serum IgM antibodies against E. coli O157 lipopolysaccharide. Evidence of STEC infection was observed in 25 household contacts, of whom 18 (72%) were asymptomatic and represented a potential source of infection. Six of 13 (46%) household contacts with Stx2-producing E. coli O157:H7 in stool culture developed anti-Stx2 IgG (H + L), compared to 71% of Stx2-associated HUS cases. In individuals showing anti-Stx2 IgG (H + L), the antibody response was directed against the B subunit in 69% of household contacts and 71% of controls, in contrast to 28% of HUS patients. In this investigation controls had a significant increase of the median of IgM antibodies to O157 lipopolysaccharide (LPS) with age, up to the fifth decade. The lack of disease in household contacts with B subunit-specific antibodies, as well as the significantly higher median of anti-O157 LPS IgM antibodies in controls beyond 4.9 years of age, suggests a protective role for anti-Stx and anti-O157 LPS antibodies.     

Human Stx2-Specific Monoclonal Antibodies Prevent Systemic Complications of Escherichia coli O157:H7 Infection

Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC), such as E. coli O157:H7. EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits. In piglets and mice, Stx can induce fatal neurological symptoms. Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing E. coli O157:H7 strain 86-24. Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS. Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and kappa light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1kappa A-subunit-specific and 3 IgG1kappa and 1 IgG3kappa B-subunit-specific antibodies. Six IgG1kappa A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1kappa B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of > 95% activity of 1 ng of Stx2 in the presence of 0.04 microg of HuMAb in vitro and significant prolongation of survival of mice given 50 microg of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously. When administered i.p. to gnotobiotic piglets 6 or 12 h after infection with E. coli O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions. The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including E. coli O157:H7.     

Serum antibodies to Escherichia coli serotype O157:H7 in patients with hemolytic uremic syndrome

Sera from 13 patients with hemolytic uremic syndrome (HUS) and 8 healthy control subjects were examined for antibodies specific for bacterial antigens of Eschericia coli serotype O157:H7. Bacterial components, including outer membrane proteins (OMPs), lipopolysaccharide (LPS), and flagella, were reacted with sera by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by enzyme-linked immunosorbent assay. All 13 serum samples from HUS patients contained high-titered antibodies of the immunoglobulin M class against O157 LPS and some OMPs. These same sera reacted weakly with some of the major OMPs, but not the LPS, of non-O157 strains of E. coli. Sera from patients did not contain antibodies to non-O157 LPS or H7 flagella. The possibility of using E. coli serotype O157 LPS in an enzyme-linked immunosorbent assay for the routine diagnostic testing of sera from HUS patients for evidence of O157:H7 infection is discussed.     

Indirect hemagglutination assay for diagnosis of Escherichia coli O157 infection in patients with hemolytic-uremic syndrome.

An indirect hemagglutination assay consisting of sheep erythrocytes coated with lipopolysaccharide (LPS) from Shiga-like toxin-producing Escherichia coli O157 was used for the serological diagnosis of E. coli O157 infections in children with classical (enteropathic) hemolytic-uremic syndrome (HUS). One week after the onset of diarrhea (acute phase of the disease), the E. coli O157 antibody titer was greater than or equal to 1:4,096 in 22 of 27 patients with HUS, compared with 4 of 249 controls, the majority of whom had O157 antibody titers of between 1:4 and 1:256. This antibody response was observed in HUS patients with stool cultures positive and negative for E. coli O157. Selective absorption with homologous LPS and heterologous LPS showed that the antibody response was specific for E. coli O157. Because of its simplicity and ease of interpretation, the indirect hemagglutination assay described in this paper is recommended for the serological diagnosis of E. coli O157 infections in patients with HUS.     

Bovine immune response to shiga-toxigenic Escherichia coli O157:H7

Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx- E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+ O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx- O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+ O157 was significantly higher than that of Stx- O157 or the control. Three weeks after the second inoculation, all calves were challenged with Stx2+ O157. Following the challenge, levels of fecal shedding of Stx2+ O157 were similar in all three groups. Both groups inoculated with an O157 strain developed antibodies to O157 LPS. Calves initially inoculated with Stx- O157, but not those inoculated with Stx2+ O157, developed statistically significant lymphoproliferative responses to heat-killed Stx2+ O157. These results provide evidence that infections with STEC can suppress the development of specific cellular immune responses in cattle, a finding that will need to be addressed in designing vaccines against E. coli O157:H7 infections in cattle.     

Escherichia coli O157:H7 and O157:H− Strains That Do Not Produce Shiga Toxin: Phenotypic and Genetic Characterization of Isolates Associated with Diarrhea and Hemolytic-Uremic Syndrome

We have isolated one sorbitol-nonfermenting (SNF) Escherichia coli O157:H7 isolate and five sorbitol-fermenting (SF) E. coli O157:H(-) isolates that do not contain Shiga toxin (Stx) genes (stx). Isolates originated from patients with diarrhea (n = 4) and hemolytic-uremic syndrome (HUS) (n = 2). All isolates harbored a chromosomal eae gene encoding gamma-intimin as well as the plasmid genes E-hly and etp. The E. coli O157:H7 isolate was katP and espP positive. Respective sera obtained from the patient with HUS contained antibodies to the O157 lipopolysaccharide antigen. The stx-negative E. coli O157:H7 isolate is genetically related to stx-positive SNF E. coli O157:H7. All stx-negative SF E. coli O157:H(-) isolates belong to the same genetic cluster and are closely related to stx-positive SF E. coli O157:H(-) isolates. Our data indicate that stx-negative E. coli O157:H7/H(-) variants may occur at a low frequency and cannot be recognized by diagnostic methods that target Stx.     

Antibody responses to Escherichia coli O157 and other lipopolysaccharides in healthy children and adults

In Mexico, diarrheal disease due to different serotypes of Escherichia coli is highly prevalent, with only sporadic isolation of O157 non-H7 strains. This could be due to exposure to the O157 or related E. coli lipopolysaccharide (LPS), such as O7 or O116, at an early age. By using enzyme-linked immunosorbent assay (ELISA) and Western blotting, the present study analyzed 605 serum samples from Mexican adults and infants without clinical symptoms of disease for the presence of antibodies to these three E. coli LPSs. The bactericidal activities of homologous and heterologous rabbit and human serum samples against O7, O116, and O157 E. coli LPSs were also determined. By using a cutoff point of 0.7, it was found by the ELISAs that 28 of 562 (5%) of the serum samples from adolescents and adults and 2 of 43 (5%) of the serum samples from infants less than 1 year of age reacted with the O157 LPS. By using cutoff points between 0.4 and 0.699, the proportion of serum samples from both age groups that reacted with the O157 LPS increased to 20%. Western blotting analysis of selected serum samples that showed an intermediate response against the O157 LPS by the ELISAs showed that 61 of 88 (69%) reacted with the same LPS. A similar result was observed for maternal milk samples. The bactericidal activities of rabbit serum samples against the O7, O116, and O157 LPSs showed that they were positive for both homologous and heterologous antigens. Similar results were observed with the human serum samples. O157 non-H7 strains were identified in only 10% of the E. coli strains isolated from 263 Mexican children with and without diarrhea over the past 15 years. This absence of O157:H7 strains in Mexico may be associated with the presence of antibodies against O157 or related E. coli LPSs.     

Shiga toxin 2 and lipopolysaccharide induce human microvascular endothelial cells to release chemokines and factors that stimulate platelet function

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1alpha (SDF-1alpha) or SDF-1beta and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1alpha at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1alpha, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.     

First-time isolation and characterization of a bacteriophage encoding the Shiga toxin 2c variant, which is globally spread in strains of Escherichia coli O157

A bacteriophage encoding the Shiga toxin 2c variant (Stx2c) was isolated from the human Escherichia coli O157 strain CB2851 and shown to form lysogens on the E. coli K-12 laboratory strains C600 and MG1655. Production of Stx2c was found in the wild-type E. coli O157 strain and the K-12 lysogens and was inducible by growing bacteria in the presence of ciprofloxacin. Phage 2851 is the first reported viable bacteriophage which carries an stx(2c) gene. Electron micrographs of phage 2851 showed particles with elongated hexagonal heads and long flexible tails resembling phage lambda. Sequence analysis of an 8.4-kb region flanking the stx(2c) gene and other genetic elements revealed a mosaic gene structure, as found in other Stx phages. Phage 2851 showed lysis of E. coli K-12 strains lysogenic for Stx phages encoding Stx1 (H19), Stx2 (933W), Stx (7888), and Stx1c (6220) but showed superinfection immunity with phage lambda, presumably originating from the similarity of the cI repressor proteins of both phages. Apparently, phage 2851 integrates at a different chromosomal locus than Stx2 phage 933W and Stx1 phage H19 in E. coli, explaining why Stx2c is often found in combination with Stx1 or Stx2 in E. coli O157 strains. Diagnostic PCR was performed to determine gene sequences specific for phage 2851 in wild-type E. coli O157 strains producing Stx2c. The phage 2851 q and o genes were frequently detected in Stx2c-producing E. coli O157 strains, indicating that phages related to 2851 are associated with Stx2c production in strains of E. coli O157 that were isolated in different locations and time periods.     

Serum antibodies to verotoxin-producing Escherichia coli (VTEC) strains in patients with haemolytic uraemic syndrome.

Serological evidence of infection with verotoxin-producing Escherichia coli (VTEC) was sought in 28 patients suffering from haemolytic uraemic syndrome (HUS) and 25 age- and sex-matched controls. ELISA was used to detect anti-lipopolysaccharide (LPS) antibodies to E. coli strains O157, O111, O26 and NCTC 10418, a non-VTEC strain, and Shigella dysenteriae O1. Sera from 19 of the HUS patients but from none of the 25 controls had significant antibody levels to the verotoxin-producing bacteria. Sera from 13 patients reacted with only one LPS of the four verotoxin-producing bacteria; sera from six reacted with more than one LPS antigen but not with LPS of E. coli NCTC 10418. Paired sera taken 2-3 weeks apart were obtained from 20 HUS patients; 14 of these had high levels of antibody in the acute phase sample. Analysis of antibody levels in the convalescent sera showed that one patient had an increase, one was unchanged and 12 patients had a decrease in antibody to the verotoxin-producing bacteria.     

Localization of Shiga toxins of enterohaemorrhagic Escherichia coli in kidneys of paediatric and geriatric patients with fatal haemolytic uraemic syndrome.

Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopenia and renal failure. Infection with enterohaemorrhagic Escherichia coli (EHEC), mainly O157:H7, has been strongly implicated as the major cause of HUS in children. The pathogenesis of HUS caused by the infection is not well understood and the defined sites of Stx in kidney of EHEC-infected humans has not been clearly demonstrated. The aim of this study was to investigate and compare the locations of Stx deposition in kidneys of paediatric and geriatric patients who died from enterohaemorrhagic E. coli O157 (EHEC) associated HUS, using an immunoperoxidase staining of the tissues. The study revealed that binding of Stx was relatively less and limited only to the renal tubules of an adult case (81 years old), while more binding was found at both renal tubules and glomeruli of an infant case (21 months old). The Stx binding in the infant's glomeruli was at podocytes, mesangial and endothelial cells. It has been known that young children are more susceptible than adults to HUS. One possibility for this is that the more extensive binding of the Stx to the kidney tissue of the paediatric patient might be due to the higher synthesis and expression of Stx receptors, i.e. Gb(3), in infants and less so in the aged individuals. However, other alternatives are possible, for example, the difference in stage of HUS in individual patients. Thus it is too early to draw any conclusion on this enigma and further investigation is required.     

Antibody-Based Protection of Gnotobiotic Piglets Infected with Escherichia coli O157:H7 against Systemic Complications Associated with Shiga Toxin 2

Hemolytic-uremic syndrome (HUS) is a serious disease in children, attributable in the majority of cases to infection with Shiga toxin (Stx)-producing Escherichia coli. Using gnotobiotic piglets orally infected with E. coli O157:H7, which develop Stx-related cerebellar lesions and fatal neurological symptoms, we show that administration of Stx2-specific antiserum well after challenge protected, in a dose-response fashion, against these symptoms for at least 24 h after bacterial challenge. Twenty-six of 30 piglets given Stx2 antiserum survived the challenge, compared to only 4 of 16 animals given control serum or saline. Given our observations in piglets, Stx antibody of human origin may likewise prevent HUS in children.     

Comparative analysis of the abilities of Shiga toxins 1 and 2 to bind to and influence neutrophil apoptosis

Hemolytic-uremic syndrome (HUS), the life-threatening complication following infection by the intestinal pathogen Escherichia coli O157:H7, is due to the ability of the pathogen to produce toxins in the Shiga toxin (Stx) family. Activated neutrophils are observed in HUS patients, yet it is unclear whether Stx exerts a direct effect on neutrophils or whether the toxin acts indirectly. The effect of Stx1 and Stx2 on human neutrophils was examined. Neither Stx1 nor Stx2 altered the rate of neutrophil apoptosis. Minimal binding of either toxin to neutrophils was observed, and the toxin was easily eluted from the cells. Stx1 and Stx2 were found to circulate in the plasma of mice following intravenous injection, and both toxins were cleared rapidly from the blood. Together these results suggest that neither Stx1 nor Stx2 interacts directly with neutrophils.     

Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157.

Identification of the O157 antigen is an essential part of the detection of Escherichia coli O157:H7, which is recognized as a major etiologic agent of hemorrhagic colitis. However, polyclonal antibodies produced against E. coli O157:H7 lipopolysaccharide (LPS) may react with several other bacteria including Brucella abortus, Brucella melitensis, Yersinia enterocolitica O9, Escherichia hermannii, and Stenotrophomonas maltophilia. We produced eight monoclonal antibodies (MAbs) specific for the LPS of E. coli O157. Western blots (immunoblots) of both the phenol phase (smooth) and the aqueous phase (rough) of hot phenol-water-purified LPS indicated that three of the MAbs were specific for the O antigen and five were reactive with the LPS core. The eight MAbs could be further differentiated by their reactivities to Salmonella O30 LPS (group N), which is reported to be identical to the E. coli O157 antigen. All eight MAbs reacted strongly to all of the 64 strains of E. coli O157 tested, which included 47 isolates of O157:H7 and 17 other O157 strains. None of the eight MAbs cross-reacted with any of the 38 other E. coli serotypes tested, which consisted of 29 different O-antigen serotypes, or with 38 strains (22 genera) of non-E. coli gram-negative enteric bacteria.     

Patients with haemolytic uraemic syndrome caused by Escherichia coli O157: absence of antibodies to Vero cytotoxin 1 (VT1) or VT2.

Serum samples from 30 patients with haemolytic uraemic syndrome (HUS), caused by Escherichia coli O157, and 30 apparently healthy volunteers, were used to examine the immune response of patients to Vero cytotoxins (VT) 1 and VT2. Patients' sera could not be differentiated from control sera using ELISA; and using immunoblotting, none of the sera had antibodies reactive with either the A or B subunits of VT1 or VT2. Examination of sera for antibodies to VT1 and VT2 seems to be of little value in the serodiagnosis of HUS caused by Vero cytotoxin-producing E coli O157.     

Antibodies to Escherichia coli O157 in patients with haemorrhagic colitis and haemolytic uraemic syndrome.

Twenty four sera from patients with haemolytic uraemic syndrome or haemorrhagic colitis and healthy controls were examined for antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157. Faecal specimens from these patients were also examined for Vero cytotoxin producing E coli (VTEC) by DNA probes, and for faecal Vero cytotoxin. Eight patients with faecal E coli O157:H7 gave a strong antibody response to O157 LPS, shown by immunoblotting and an enzyme linked immunosorbent assay. Six symptomatic patients without evidence of faecal VTEC also gave a strong antibody response to O157 LPS. Sera from the remaining five patients and five healthy controls did not contain antibodies to E coli O157. The results suggest that the testing of sera from patients with haemorrhagic colitis or haemolytic uraemic syndrome by ELISA or immunoblot would prove valuable in addition to the established procedures for detecting VTEC, using DNA probes and testing for faecal Vero cytotoxin.     

Inhibition of Water Absorption and Selective Damage to Human Colonic Mucosa Are Induced by Subtilase Cytotoxin Produced by Escherichia coli O113:H21

Shiga toxin-producing Escherichia coli O157:H7 (STEC) is by far the most prevalent serotype associated with hemolytic uremic syndrome (HUS) although many non-O157 STEC strains have been also isolated from patients with HUS. The main virulence factor of STEC is the Shiga toxin type 2 (Stx2) present in O157 and non-O157 strains. Recently, another toxin, named subtilase cytotoxin (SubAB), has been isolated from several non-O157 strains and may contribute to the pathogenesis of HUS. Here, we have demonstrated that an O113:H21 STEC strain expressing SubAB and Stx2 inhibits normal water absorption across human colon and causes damage to the surface epithelium, necrosis, mononuclear inflammatory infiltration, edema, and marked mucin depletion. This damage was less marked, but nevertheless significant, when purified SubAB or E. coli O113:H21 expressing only SubAB was assayed. This is the first study showing that SubAB may directly participate in the mechanisms of diarrhea in children infected with non-O157 STEC strains.     

Antibody Responses to Escherichia coli O157 and Other Lipopolysaccharides in Healthy Children and Adults

In Mexico, diarrheal disease due to different serotypes of Escherichia coli is highly prevalent, with only sporadic isolation of O157 non-H7 strains. This could be due to exposure to the O157 or related E. coli lipopolysaccharide (LPS), such as O7 or O116, at an early age. By using enzyme-linked immunosorbent assay (ELISA) and Western blotting, the present study analyzed 605 serum samples from Mexican adults and infants without clinical symptoms of disease for the presence of antibodies to these three E. coli LPSs. The bactericidal activities of homologous and heterologous rabbit and human serum samples against O7, O116, and O157 E. coli LPSs were also determined. By using a cutoff point of 0.7, it was found by the ELISAs that 28 of 562 (5%) of the serum samples from adolescents and adults and 2 of 43 (5%) of the serum samples from infants less than 1 year of age reacted with the O157 LPS. By using cutoff points between 0.4 and 0.699, the proportion of serum samples from both age groups that reacted with the O157 LPS increased to 20%. Western blotting analysis of selected serum samples that showed an intermediate response against the O157 LPS by the ELISAs showed that 61 of 88 (69%) reacted with the same LPS. A similar result was observed for maternal milk samples. The bactericidal activities of rabbit serum samples against the O7, O116, and O157 LPSs showed that they were positive for both homologous and heterologous antigens. Similar results were observed with the human serum samples. O157 non-H7 strains were identified in only 10% of the E. coli strains isolated from 263 Mexican children with and without diarrhea over the past 15 years. This absence of O157:H7 strains in Mexico may be associated with the presence of antibodies against O157 or related E. coli LPSs.     

The use of serodiagnosis in the retrospective investigation of a nursery outbreak associated with Escherichia coli O157:H7.

AIMS: To use serology to investigate an outbreak of verocytotoxin (VT) producing Escherichia coli O157 in a hospital nursery, following the detection of faecal E coli O157 (phage type 49) producing VT type 2. METHODS: ELISA and immunoblotting techniques, based on lipopolysaccharide (LPS) purified from E coli O157; diagnostic bacteriology; serotyping and phage typing; DNA probes for VT. RESULTS: 29 of 126 sera contained antibodies to the LPS of E coli O157: 10 were from children, three were from staff, and 11 were from hospital kitchen staff. Five parents of children attending the nursery were antibody positive. Sixty four sera from other hospital staff and controls did not contain antibodies to the LPS of E coli O157. CONCLUSIONS: Serology detected evidence of infection with E coli O157 in 23% of sera examined. By bacteriology alone, only a single case of infection with E coli O157 would have been detected. Serology is valuable in providing evidence of infection with E coli O157.     

The kinetics of antibody production to antigens of Escherichia coli O157 in a pregnant woman with haemolytic uraemic syndrome

Sequential blood samples taken from a pregnant woman with haemolytic uraemic syndrome caused by verocytotoxin (VT)-producing Escherichia coli O157 were used to examine the kinetics of serum antibody production to E. coli O157 lipopolysaccharide (LPS), intimin and the conserved region of the translocated intimin receptor (Tir-M). Umbilical cord blood and two samples of blood from the newborn baby were also examined for antibodies to these antigens. In the mother, antibodies of the IgM class, specific for E. coli O157 LPS, were produced in the initial stages of the infection, reaching a peak at 9 days after onset of diarrhoea and subsiding 3 days later. High levels of IgG class antibodies, specific for E. coli O157 LPS, were detected 8 days after the onset of diarrhoea and were present at high titres on day 18. Serum antibodies of the IgA class to E. coli O157 LPS were not detected. Antibodies binding to Tir-M were detected 8 days after the onset of diarrhoea and high titres of these antibodies were still present on day 18. Serum antibodies to intimin were not detected in the mother and no antibodies to any of the antigens tested were detected in either the baby's blood or cord blood. This study describes for the first time the kinetics of serum antibody production during pregnancy, to selected antigens expressed by E. coli O157.