Escherichia coli 0157:H7 strain 933 contains two distinct toxin-converting phages (933J and 933W). The biologic activities and antigenic relationship between the toxins produced by 933J and 933W lysogens of E. coli K-12, as well as the homology of the genes that encode the two toxins, were examined in this study. The 933J and 933W toxins, like Shiga toxin produced by Shigella dysenteriae type 1, were cytotoxic for the same cell lines, caused paralysis and death in mice, and caused fluid accumulation in rabbit ileal segments. The cytotoxic activity of 933J toxin for HeLa cells was neutralized by anti-Shiga toxin, whereas the activity of 933W toxin was not neutralized by this antiserum. In contrast, an antiserum prepared against E. coli K-12(933W) neutralized 933W toxin but not 933J toxin or Shiga toxin. For E. coli 933, most of the cell-associated cytotoxin was neutralized by anti-Shiga toxin, whereas most of the extracellular cytotoxin was neutralized by anti-933W toxin. However, a mixture of these antisera indicated the presence of both toxins in cell lysates and culture supernatants. Among 50 elevated cytotoxin-producing strains of E. coli, we identified 11 strains isolated from cases of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome that produced cell-associated cytotoxins which were neutralized by the 933W antitoxin. Southern hybridization studies showed that the cloned toxin structural genes from phage 933J hybridized with DNA from phage 933W under conditions estimated to allow no more than 26% base-pair mismatch. These findings indicate that E. coli produces two genetically related but antigenically distinct cytotoxins with similar biologic activities which we propose to name Shiga-like toxins I and II. Strains of E. coli that produce elevated levels of Shiga-like toxin I or Shiga-like toxin II, or both, have been associated with the clinical syndromes of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome.

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Production of Shiga-Like Toxin by Escherichia coli Escherichia coli is one of several agents that cause intestinal disease in humans and animals. Four classes of E. coli have been recognized [1], Enterotoxigenic E. coli (ETEC) strains produce a heat-labile (LT) enterotoxin and/or a heat-stable (ST) enterotoxin. Enteroinvasive E. coli (EIEC) strains, like shigellae, penetrate and multiply within epithelial cells. Enteropathogenic E. coli (EPEC) strains belong to certain serogroups that have been incriminated as pathogens by epidemiological studies. Some EPEC strains have been shown to adhere to cells of the intestinal mucosa and to produce pathognomonic lesions at the site of attachment. Enterohemorrhagic E. coli (EHEC) strains cause a distinct clinical syndrome (hemorrhagic colitis), and serotype 0157:H7 has been associated with this illness. Neither EPEC nor EHEC strains produce classic enterotoxins, nor are they enteroinvasive. Some strains of E. coli produce a cell-associated cytotoxin that is neutralized by antibodies against purified Shiga toxin from Shigella dysenteriae type 1 [2-4]. The cytotoxins purified from one EPEC strain 026:H11 (H30) and from one EHEC strain 0157:H7 (933) have biologic activities (cytotoxicity for HeLa and Vero cells, lethality for mice, and enterotoxicity for ligated ileal segments from rabbits) and subunit structures similar to those of Shiga toxin [5, 6]. The purpose of this study was to determine the frequency and levels of cytotoxin production for a wide variety of E. coli strains. Culture supernatants and sonic lysates of 418 strains isolated from humans, animals, ajid food were examined for cytotoxic effects on HeLa cells and to see whether cytotoxicity could be neutralized by antibodies to Shiga toxin. Materials and Methods Bacterial strains. E. coli strains examined for production of Shiga-like toxin were obtained mainly from the Centers for Disease Control, Atlanta, Georgia. The 418 strains were isolated over a period of 30 years from the following: humans with diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, or no clinical manifestation of disease; calves with diarrhea; piglets with edema dis

Three monoclonal antibodies, designated MAb 16E6, MAb 13C4, and MAb 19G8, were produced which recognize Shiga-like toxin (SLT) from Escherichia coli. All three monoclonal antibodies neutralized the cytotoxicity of E. coli SLT and were able to immunoprecipitate intact labeled toxin with Staphylococcus aureus protein A. The three antibodies were of the Gl heavy and kappa light chain classes. MAb 16E6 bound to the B subunit of SLT in Western blots and also neutralized the lethality of the toxin for mice and the enterotoxicity of the toxin in ligated rabbit ileal loops. The ability of MAb 16E6 to neutralize the cytotoxicity, lethality, and enterotoxicity of E. coli confirms the hypothesis that all three activities are associated with a single toxin. MAb 16E6 and MAb 13C4 also neutralized the cytotoxicity of purified Shiga toxin from ShigeUla dysenteriae type 1 and Shiga-like toxic activities in crude cell extracts from Shigella flexneri, Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella typhimurium. Thus, Shiga toxin and the SLTs from E. coli, Shigellaflexneri, V. cholerae, V. parahaemolyticus, and Salmonella typhimurium share a common B subunit epitope that is involved in neutralization. MAb 13C4 has been successfully used in a colony blot assay for the detection of bacterial colonies which produce high levels of SLT. Sixty-two different strains of bacteria were tested by both the cytotoxicity and colony blot assays for the presence of SLT. The colony blot assay detected all strains of bacteria which produce -10 50% cytotoxic doses of SLT per ml of sonic lysate. There were no false-positive results among the 62 samples tested.

The major obstacle in large-scale epidemiological investigations of the incidence of Shiga-like toxin (SLT)-producing Escherichia coli in diarrheal stools is the lack of a rapid, specific test to detect toxin. Enterohemorrhagic E. coli produces elevated levels of SLT-I, SLT-ll, or both cytotoxins (also called Verotoxins). SLT-I but not SLT-II can be neutralized by antiserum to purified Shiga toxin and by monoclonal antibodies to the B subunit of SLT-I. In this study, monoclonal antibodies were generated against a crude preparation of SLT-II produced by an E. coli K-12 strain lysogenized with the 933W toxin-converting phage of enterohemorrhagic E. coli 933. Hybridoma culture supernatants were screened for anti-SLT-II antibodies by a cytotoxicity neutralization assay and by an enzyme-linked immunosorbent assay (ELISA). Of 53 ELISA-positive lines, 5 were capable of neutralizing the cytotoxicity of SLT-ll but not of SLT-I, Shiga toxin, or a variant of SLT-II produced by E. colu that causes edema disease of swine. All five monoclonal antibodies * Corresponding author.