Cross-protection against challenge by intravenous Escherichia coli verocytotoxin 1 (VT1) in rabbits immunized with VT2 toxoid.

Rabbits challenged intravenously with Escherichia coli verocytotoxin (VT1, Shiga toxin 1, Stx1) die after developing diarrhea and paralysis, and this outcome can be prevented by pre-immunization with VT1 toxoid. In nonimmune rabbits, intravenously administered 125I-VT1 binds to the central nervous system and gastrointestinal tract, whereas in immunized animals, these organs are spared and the toxin localizes in the liver and spleen. In rabbits immunized with either VT1 or VT2 toxoids, both the homologous or heterologous toxins are prevented from binding to target organs. This has lead to the advancement of a hypothesis that cross-protection in vivo can be induced to both toxins by immunization with a toxoid even though these toxins do not exhibit cross-neutralization in vitro. It was shown that rabbits immunized with VT2 were fully protected from the intravenous administration of 10 LD50 and 50 LD50 of VT1, and this correlated directly with the protection from binding of this toxin to target organs. These findings have important implications on the design of the vaccination strategies to prevent human VT-mediated diseases and also validate the concept of testing for immunity to VT by monitoring the inhibition of binding of the 125I-VT to target organs in preference to performing LD50 assays.

Antibody response to Shiga toxins Stx2 and Stx1 in children with enteropathic hemolytic-uremic syndrome.

A Western blot (immunoblot) assay (WBA) for the detection of immunoglobulin G antibodies to Shiga toxins Stx2 and Stx1 in sera from 110 patients with enteropathic hemolytic-uremic syndrome (53 culture confirmed to have Shiga toxin-producing Escherichia coli [STEC] infection) and 110 age-matched controls was established by using a chemiluminescence detection system. Thirty-nine (74%) of the 53 culture-confirmed cases were infections with STEC serotype O157, and 14 (26%) were associated with infection by other STEC serotypes. The frequency of an anti-Stx2 response following infection by a Stx2-producing strain (34 of 48 cases; 71%) was higher than that of an anti-Stx1 response following Stx1-producing STEC infection (4 of 10). Furthermore, the frequency of an anti-Stx2 response in 110 control sera (10%) was significantly higher than the frequency of an anti-Stx1 response (1.8%) (P = 0.0325). For STEC O157 culture-confirmed cases WBA for toxin detection had a diagnostic sensitivity of 71% and a specificity of 90%. Because of its high specificity the assay might be a helpful tool for diagnosing suspected STEC infection when tests of stool samples or serological tests against various lipopolysaccharide antigens are negative. Furthermore, the prevalence of anti-Stx antibodies in healthy controls probably reflects the population immunity to systemic Stx-associated disease. It can thus serve as a basis for comparing immunity levels in different populations and for considering future Stx toxoid immunization strategies.

Escherichia coli Shiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family.

Human intestinal cells lack globotriaosylceramide (Gb(3)), the receptor for Shiga toxin-1 (Stx1) and Shiga toxin-2 (Stx2). Therefore, the role of these toxins in mediating intestinal disease during infection with Shiga toxin-producing Escherichia coli is unclear. The aims of this study were to determine whether Stx1 and Stx2 induce apoptosis in epithelial cells expressing (HEp-2, Caco-2) or lacking (T84) Gb(3) and to characterize the role of the Bcl-2 family. Stx1 (12.5 ng/ml) induced apoptosis in both HEp-2 (21.9 +/- 7.9% vs. 0.8 +/- 0.3%, P = 0.01) and Caco-2 (10.1 +/- 1.2% vs. 3.1 +/- 0.4%, P = 0.006) cells but not in Gb(3)-deficient T84 cells. Toxin-mediated apoptosis of HEp-2 cells was associated with enhanced expression of the proapoptotic protein Bax. Inhibition of caspase activation prevented toxin-stimulated apoptosis. In addition, overexpression of Bcl-2 by transient transfection blocked Stx1-stimulated cell death. These findings indicate that Shiga toxins produced by E. coli signal Gb(3)-expressing epithelial cells to undergo apoptosis in association with enhanced Bax expression, thereby resulting in activation of the caspase cascade.

Evaluation of a microplate latex agglutination method (Verotox-F assay) for detecting and characterizing verotoxins (Shiga toxins) in Escherichia coli.

The performance of a commercial microplate latex agglutination assay, the Verotox-F assay, was compared with that of the Vero cell assay for the detection and characterization of Escherichia coli verocytotoxins (VTs). Culture filtrates of 68 VT-positive E. coli strains (65 human isolates [33 of serotype O157:H7/H-, 32 of non-O157 serotypes] and 3 reference strains) and 104 VT-negative strains (100 human isolates and 4 reference strains) were investigated. The toxin phenotypes and genotypes of the 68 VT-positive isolates were VT1 only (18 strains), VT2 and/or VT2c (33 strains), and VT1 plus VT2 (17 strains). The Verotox-F assay involved incubation of serial dilutions of culture filtrates with equal volumes of latex particles sensitized with anti-VT1 antibody or anti-VT2 antibody in 96-well microtiter plates with appropriate controls and examination for latex agglutination after 20 to 24 h. Compared to the results of the Vero cell assay, the Verotox-F assay was 100% sensitive and 100% specific for the detection of VTs in culture filtrates and correctly identified the toxin types of all 68 VT producers. By checkerboard titration with purified toxins, the sensitivity of the Verotox-F assay was found to be 14 pg (0.7 ng/ml) for VT1, 12 pg (0.6 ng/ml) for VT2, and 350 pg (17.5 ng/ml) for VT2c; this sensitivity is comparable to that of the bioassay. The anti-VT2 latex reagent detected both VT2 and VT2c and did not cross-react with VT1. The anti-VT1 reagent showed a low-level cross-reaction with VT2c only at levels (>/=4.5 microg/ml) that were about 1,000-fold higher than those found in culture filtrates. We conclude that the Verotox-F assay is highly sensitive and specific for the detection and characterization of VTs in culture filtrates of human E. coli isolates. The test is rapid, reliable, and easy to perform; its results are easy to interpret; and it should allow testing for VT to become more widely performed.

Isolation and characterization of sorbitol-fermenting Shiga toxin (Verocytotoxin)-producing Escherichia coli O157:H- strains in the Czech Republic.

Two sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) O157:H- strains were isolated from patients with hemolytic-uremic syndrome in the Czech Republic in 1995. Their phenotypic and genotypic characteristics and genomic DNA fingerprints were identical or closely related to those of SF STEC O157:H- strains isolated in Germany in 1988 to 1997. This indicates that the Czech isolates belong to the SF STEC O157 clone which is widespread in Germany. It is the first finding of the clone outside Germany.

Risk factors for infection with verocytotoxigenic Escherichia coli in cattle on Ontario dairy farms.

Risk factors for prevalent infection with verocytotoxigenic Escherichia coli (VTEC) were studied in a random sample of 886 cows and 592 calves under 3 months of age on 80 randomly selected dairy farms in southern Ontario. Fecal-culture supernatants from each animal were screened for verocytotoxicity using a Vero cell assay (VCA) and for verocytotoxin (VT) genes by a polymerase chain reaction (PCR) procedure. Up to 20 F. colt isolates from positive samples were tested for VT production using VCA and PCR. VTEC isolates were serotyped. Farm managers were interviewed using a standardized questionnaire to obtain information on farm- and individual animal-level management practices and characteristics. There was a significant (P < 0.001) positive association between age of calves and their VTEC infection status, and calves were significantly more likely to be infected than cows. The proportion of calves infected on the farm was positively associated with both the use of regular pails for feeding calves (as opposed to nipple bottles or nipple pails) and bringing new animals into the herd in the previous year.

Inhibition of Helicobacter pylori and Helicobacter mustelae binding to lipid receptors by bovine colostrum.

Helicobacter pylori, the etiologic agent of chronic-active gastritis and duodenal ulcers in humans, and Helicobacter mustelae, a gastric pathogen in ferrets, bind to phosphatidylethanolamine (PE), a constituent of host gastric mucosal cells, and to gangliotetraosylceramide (Gg4) and gangliotriaosylceramide (Gg3). The effect of a bovine colostrum concentrate (BCC) on the interaction of H. pylori and H. mustelae to their lipid receptors was examined. BCC blocked attachment of both species to Gg4, Gg3, and PE. Partial inhibition of binding was observed with native bovine and human colostra. BCC lacked detectable antibodies (by immunoblotting) to H. pylori surface proteins (adhesins). However, colostral lipid extracts contained PE and lyso-PE that bound H. pylori in vitro. These results indicate that colostrum can block the binding of Helicobacter species to select lipids and that binding inhibition is conferred, in part, by colostral PE or PE derivatives. Colostral lipids may modulate the interaction of H. pylori and other adhesin-expressing pathogens with their target tissues.

Verocytotoxin inhibits mitogenesis and protein synthesis in purified human glomerular mesangial cells without affecting cell viability: evidence for two distinct mechanisms.

Acute renal failure is one of the hallmarks of the hemolytic uremic syndrome (HUS). Infection with a verocytotoxin (VT)- or Shiga-like toxin (SLT)-producing Escherichia coli has been strongly implicated in the etiology of the epidemic form of HUS. The functional receptor for these closely related toxins appears to be a glycosphingolipid, globotriaosylceramide (Gb3). Endothelial damage in the glomeruli and arterioles of the kidney induced by VT is believed to play a crucial role in the pathogenesis of HUS. However, little information is available regarding the effects of VT on mesangial cells, which also play an important role in glomerular function. In this study, the effects of VT on human mesangial cells in vitro were investigated. Mesangial cells were enriched by collecting hillock-shaped outgrowths derived from adult human glomeruli and subsequently purified by elimination of contaminating epithelial cells by immunoseparation with ulex europaeus lectin-I (UEA-I)-coated dynabeads. The obtained and subcultured mesangial cell populations were >98% pure. Their mesangial nature was established by the presence of a-smooth muscle cell actin in highly confluent cultures and the absence of cytokeratin or platelet/endothelial cell adhesion molecule-1. Mesangial cells bound VT to bands of Gb3 and a closely related glycolipid, which is similar to a glycolipid involved in the VT-dependent cytokine production in monocytes. VT did not induce the release of cytokines or chemokines in mesangial cells. In VT-susceptible cells, binding of VT to Gb3 causes cell death by the inhibition of protein synthesis. Although protein synthesis was inhibited in mesangial cells, all cells remained viable, both under basal and tumor necrosis factor-alpha-stimulated conditions. However, the marked reduction in protein synthesis may impair a proper response of the cells in conditions of increased demand of newly synthesized proteins. Furthermore, VT markedly inhibited DNA synthesis and proliferation of mesangial cells. The inhibition of mitogenesis was also found with the B-subunit of VT-1 alone, albeit to a lesser extent, without a significant effect on protein synthesis. Because the inhibition of protein synthesis involves the A-subunit, this suggests that two distinct mechanisms contribute to the effects of VT on protein synthesis and mitogenesis. Intracellular routing of VT (A- and B-subunits) may vary between cell types and result in differential effects on human mesangial cells when compared with other cell types.

Interobserver variation in the sonographic estimation of renal volume in children.

OBJECTIVE: To quantify the interobserver variability in the sonographic estimation of renal volume in children. MATERIALS AND METHODS: Three observer pairs measured the lengths and diameters of 176 kidneys in 90 children. Renal volumes were estimated from the formula for a prolate ellipsoid. The mean and standard deviation of the absolute and percentage differences between observers were calculated. The 95 % limits of agreement for each pair of observers were derived. Log-transformed data were used to derive overall limits of agreement. RESULTS: Absolute interobserver variation increased with renal volume. Percentage variation was independent of renal size. There is a 95 % probability that a measurement of renal volume by one observer will be between 0.69 and 1.44 times a measurement made by a different observer. CONCLUSION: The interobserver variation in the sonographic estimation of renal volume represents approximately 2-3 years' normal growth in children aged over 2 years.

Localization of intravenously administered verocytotoxins (Shiga-like toxins) 1 and 2 in rabbits immunized with homologous and heterologous toxoids and toxin subunits.

Rabbits challenged intravenously with Shiga toxin or with Escherichia coli verocytotoxin 1 or 2 (VT1 or VT2) are known to develop diarrhea, paralysis, and death, which can be prevented by immunization with a toxoid. The pathological effects of VT1 in the central nervous system and the gastrointestinal tract of unimmunized rabbits correlate with the localization of 125I-VT1 in these tissues, whereas in immunized animals, localization of 125I-VT1 in target tissues is inhibited and labeled toxin is cleared by the liver and spleen. By using the approach described above in this study, rabbits immunized with VT1 toxoid, VT2 toxoid, or with the A or B subunit of each toxin were challenged with intravenous 125I-VT1 or 125I-VT2. After 2 h, the animals were sacrificed, and selected tissues were analyzed for uptake of labeled toxin. It was found that animals immunized with either VT1 toxoid or VT2 toxoid were protected from target tissue uptake of both 125I-VT1 and 125I-VT2. Rabbits immunized with either the VT1 A or VT2 A subunit were also protected from target tissue uptake of both the homologous and heterologous 125I-labeled holotoxins. In contrast, in animals immunized with the toxin B subunits, protection extended only against challenge by the homologous toxin. These results provide evidence of VT1 and VT2 cross-neutralization in vivo in the rabbit model and indicate that the in vivo cross-neutralization is a function, mainly, of antibodies directed to the VT A subunits. This suggests that the VT1 A or VT2 A subunit may be a suitable immunogen for immunizing humans against systemic VT-mediated disease.

Toxicity and immunogenicity of a verotoxin 1 mutant with reduced globotriaosylceramide receptor binding in rabbits.

The verotoxins (VT1 and VT2), produced by strains of enterohemorrhagic Escherichia coli, have been implicated in the pathogenesis of hemorrhagic colitis and the hemolytic uremic syndrome. To better understand the role of globotriaosylceramide (Gb3) receptor binding by the verotoxins in disease production, we examined the clinicopathologic effects of an intravenously (i.v.) administered verotoxin 1 mutant holotoxin (Phe30Ala) in rabbits. The substitution of alanine for phenylalanine 30 in the VT1 B subunit has been shown previously to reduce both Gb3 binding affinity and capacity in vitro. This reduction in receptor binding corresponded to a 10(5)-fold reduction in the toxic activity of VT1 on a Vero cell monolayer. In this study, purified 125I-labeled Phe30Ala was administered i.v. to rabbits to determine its specific distribution in rabbit tissues. In contrast to the rapid elimination of i.v. administered 125I-VT1 from the bloodstream, 125I-Phe30Ala had a 52-fold-longer half-life in serum and failed to localize preferentially in the gastrointestinal tract and central nervous system (CNS). Rabbits challenged with Phe30Ala at a dose equivalent to 10 times the 50% lethal dose (LD50) of VT1 showed no visible clinical symptoms typical of VT effect after 7 days. Administration of Phe30Ala at a dose equivalent to 100 times the LD50 of VT1, however, caused both clinical and histopathologic features indistinguishable from VT1 toxemia in rabbits, although the onset of symptoms was delayed. Rabbits were immunized with Phe30Ala and challenged i.v. with either 125I-VT1 or 125I-VT2. The specific uptake of 125I-VT1 in the gastrointestinal tract and CNS was totally inhibited in Phe30Ala immune rabbits. Only a partial decrease in target organ uptake was observed in Phe30Ala immune rabbits challenged with 125I-VT2. From this study, we conclude that Gb3 binding is responsible for target organ localization of VT1 and disease production in the rabbit. The ability of Phe30Ala to induce both strong antibody and protective responses against VT1 suggests that VT mutants with reduced receptor binding properties may be useful in vaccine strategies. A further reduction in the toxicity of Phe30Ala would be required for its use as a natural toxoid to protect against human verotoxigenic E. coli infections.

Comparison of the western blot assay with the neutralizing-antibody and enzyme-linked immunosorbent assays for measuring antibody to verocytotoxin 1.

A Western blot (immunoblot) assay (WBA) was developed to detect immunoglobulin G (IgG) antibodies against Escherichia coli Verocytotoxin 1 (VT1) by using a chemiluminescence detection system. The assay was compared with a VT1-neutralizing-antibody (VT1-NAb) assay and an anti-VT1 IgG enzyme-linked immunosorbent assay (ELISA). When four human serum samples that were known to be positive by VT1-NAb assay and ELISA were titrated to the endpoint by the three assays, the WBA gave endpoint titers that were up to 8-fold higher than those by ELISA and up to 256-fold higher than those by the VT1-NAb assay. Of 32 serum samples that were known to be positive by VT1-NAb assay and ELISA, 31 (97%) were positive by WBA; the one sample with a discrepant result gave borderline results by the VT1-NAb assay and ELISA. Of 52 serum samples that were known to be negative by the VT1-NAb assay and ELISA, 50 (96%) were negative and 2 (4%) were positive by WBA. Of 44 serum samples that gave discrepant results by the VT1-NAb assay and ELISA, neither of the latter correlated with the results of WBA. In an investigation of 19 pairs of acute- and convalescent-phase serum samples from patients with hemolytic-uremic syndrome, 10 pairs that were positive by the VT1-NAb assay were also WBA positive, while 9 pairs that were NAb negative were also WBA negative. The WBA is inherently more specific and sensitive than either the NAb assay or the ELISA and may be used as a "gold standard" to detect IgG antibodies to VT1. Like the NAb assay and the ELISA for detecting antibodies to VT1, the WBA has little to offer in the diagnostic setting but is expected to play an important role in seroepidemiological studies.

Detection of Bordetella pertussis in clinical specimens by PCR and a microtiter plate-based DNA hybridization assay.

In order to improve detection of Bordetella pertussis in nasopharyngeal aspirates (NPAs) in our laboratory, a PCR-based assay was optimized, and a study was designed (i) to compare results obtained by PCR to those obtained by culture and (ii) to evaluate a novel microtiter plate-based DNA hybridization assay (PCR-plate) by comparing it to agarose gel electrophoresis (PCR-gel) for detection of the PCR product. DNA for the PCR was extracted with a guanidine thiocyanate buffer and used in a PCR mixture containing primers directed against a reiterated gene sequence in B. pertussis (Q. He, J. Mertsola, H. Soini, M. Skurnik, O. Ruuskanen, and M. K. Viljanen, J. Clin, Microbiol. 31:642-645, 1993). Of 96 NPAs submitted from a targeted study group, 23 were positive by culture, 27 were positive by PCR-gel, and 31 were positive by PCR-plate. All culture-positive specimens were also positive by PCR. Of nine patients with culture-negative-PCR-positive results, six had discharge diagnoses of pertussis. Thus, PCR with plate-based product detection is a sensitive method for the laboratory detection of B. pertussis in NPAs. Additional advantages of the plate assay include rapidity, objectivity in reading results, specificity, and the capability of being adapted to a high-volume, automated system.

Human Escherichia coli O157:H7 infection associated with the consumption of unpasteurized goat's milk.

A cluster of four cases of haemolytic uraemic syndrome in children occurred in Northern Bohemia, Czech Republic, between 15 June and 7 July, 1995. All the cases had significantly elevated titres of anti-O157 lipopolysaccharide (LPS) antibodies as detected by the indirect haemagglutination assay. All but one of them had drunk unpasteurized goat's milk from the same farm within the week before the disease. Evidence of E. coli O157 infection was subsequently found in 5 of 15 regular drinkers of the farm's raw goat's milk; four of them were asymptomatic, 1 had mild diarrhoea at the end of June. Verocytotoxin 2-producing E. coli O157:H7 strains of phage type 2 and of identical pulsed-field gel electrophoresis patterns were isolated from 1 of 2 farm goats and from 1 of the asymptomatic goat's milk drinkers. The frequency of anti-O157 LPS antibodies found among regular drinkers of the farm's raw goat's milk (33%; 5 of 15) was significantly higher than that found in control population (0%; none of 45) (P = 0.0005; Fisher's exact test). Our findings indicate that goats may be a reservoir of E. coli O157:H7 and a source of the infection for humans; raw goat's milk may serve as a vehicle of the pathogen transmission.

Translocation of verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium.

Verotoxin-producing Escherichia coli (VTEC) are pathogenic bacteria associated with diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Verotoxins (VTs) elaborated by these organisms produce cytopathic effects on a restricted number of cell types, including endothelial cells lining the microvasculature of the bowel and the kidney. Because human intestinal epithelial cells lack the globotriaosylceramide receptor for VT binding, it is unclear how the toxin moves across the intestinal mucosa to the systemic circulation. The aims of this study were to determine the effects of VT-1 on intestinal epithelial cell function and to characterize VT-1 translocation across monolayers of T84 cells, an intestinal epithelial cell line. VT-1 at concentrations up to 1 microgram/ml had no effect on the barrier function of T84 monolayers as assessed by measuring transmonolayer electrical resistance (102 +/- 8% of control monolayers). In contrast, both VT-positive and VT-negative VTEC bacterial strains lowered T84 transmonolayer resistance (45 +/- 7 and 38 +/- 6% of controls, respectively). Comparable amounts of toxin moved across monolayers of T84 cells, exhibiting high-resistance values, as monolayers with VTEC-induced decreases in barrier function, suggesting a transcellular mode of transport. Translocation of VT-1 across T84 monolayers paralleled the movement of a comparably sized protein, horseradish peroxidase. Immunoelectron microscopy confirmed transcellular transport of VT-1, since the toxin was observed within endosomes and associated with specific intracellular targets, including the Golgi network and endoplasmic reticulum. These data present a mode of VT-1 uptake by toxin-insensitive cells and suggest a general mechanism by which bacterial toxins lacking specific intestinal receptors can penetrate the intestinal epithelial barrier.