The amount of crude Campylobacter jejuni enterotoxin present in culture products was quantitated by comparing the response of these preparations with that of pure Escherichia coli heat-labile toxin (LT) in the Chinese hamster ovary assay and in enzyme-linked immunosorbent assays that used GM ganglioside or antisera to LT or both. Maximum C. jejuni enterotoxin production was achieved by growth at 42°C for 24 b under agitation in supplemented GC medium. Adding polymyxin separately to either the broth supernatant or the cells enhanced the recovery of toxin; the yield from cell lysates was much lower. The quantity of C. jejuni enterotoxin produced by clinical isolates obtained locally or provided from Mexico varied widely, over a spectrum from none to large amounts; quantitative values for the amount of C. jejuni enterotoxin determined by the Chinese hamster ovary and enzyme-linked immunosorbent assays correlated with the degree of secretory potency of this material in ligated rat ileal loops. The cytotonic activity of C. jejuni enterotoxin in Chinese hamster ovary cells was abolished by heating at 96°C for 10 min and by preincubation either with GM ganglioside or with LT or cholera toxin antisera. The secretory activity of C. jejuni enterotoxin in ligated rat ileal loops was passively neutralized by antiserum to LT, and immunizing rats with either LT or its B subunit significantly (P < 0.001) reduced fluid response to active challenge with C. jejuni enterotoxin in ligated ileal loops. These observations indicate that strains of C. jejuni vary in their capacity to elaborate a heat-labile enterotoxin that has close immunological homology with LT and cholera toxin.

The pathogenic properties of 20 strains of Campylobacter jejuni isolated from persons with clearly defined clinical manifestations were determined. Cell-free broth filtrates were examined for (i) enterotoxin production by Chinese hamster tissue culture assay and an enzyme-linked immunosorbent assay (ELISA) employing GM1 ganglioside and affinity-purified antiserum to Escherichia coli heat-labile toxin, (ii) cytotoxin production by Vero and HeLa cell tissue culture lines, and (iii) their ability to cause fluid secretion in rat ligated ileal loops. Viable bacteria were examined for invasive properties by an ELISA with the immunoglobulin fraction of antiserum to Formalin-killed bacteria of an invasive strain, and by their effect on fluid secretion and morphology in rat ligated ileal loops. None of the eight isolates obtained from asymptomatic carriers had any detectable pathogenic properties. All six strains isolated from persons with bloody invasive-type diarrhea elaborated a cytotoxin; their viable bacteria had high titers in the ELISA for invasive properties and caused fluid secretion in ligated ileal loops, although consistent morphologic abnormalities and evidence of mucosal invasion, examined by immunofluorescence techniques, were not detected. All six strains isolated from persons with watery secretory-type diarrhea produced an enterotoxin, one elaborated a cytotoxin, and broth filtrates of all strains caused fluid secretion in ligated ileal loops; viable bacteria had low titers in the ELISA for invasive properties and evoked fluid secretion in ligated loops by means of enterotoxin production. These observations show (i) that a correlation exists between the pathogenic properties of the infective C. jejuni strain and gastrointestinal manifestations in the infected host, and (ii) that these pathogenic properties can be identified by in vitro assays, including ELISAs.

Synthetically produced Escherichia coli heat-stable toxin (ST) was conjugated to the nontoxic B subunit of the heat-labile toxin (LT) by the carbodiimide reaction. Modifying the molar ratio of toxins mixed and the ratio of carbodiimide added to the toxins permitted synthesis of conjugates with any desired degree of proportional antigenicity for each toxin component. Immunization of rats by the parenteral/peroral routes with cross-linked vaccine containing 39% ST and 61% B subunit antigenicity, with 0.06% residual ST toxicity, evoked fourfold to sevenfold increases over control values of serum IgG and mucosal secretory IgA antitoxin titers to each of the component toxins, thus providing significant (P less than 0.001) protection against challenge with either LT or ST or with viable heterologous strains which produce these toxins. These observations show that cross-linking synthetic ST to the B subunit results in a nontoxic vaccine that provides protection against all types of enterotoxigenic E. coli.

The protective effect of active immunization by different routes with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile toxin was evaluated in rats. Immunized animals were challenged by placing toxin into ligated ileal loops at dosages which produced either 50% or the maximum secretory response in unimmunized rats. Immunization exclusively by the parenteral route yielded significant protection. Rats were also protected when parenteral priming was followed by boosting given either directly into the duodenum or perorally 2 h after intragastric cimetidine, but not when the peroral boosts were given with bicarbonate. Immunization administered entirely by the peroral route with cimetidine yielded protection but only when the immunizing dosage was fivefold greater than that found effective in the parenteral-peroral approach. Rats immunized exclusively by the parenteral route and those boosted perorally with cimetidine were also tested, and found to be protected, against challenge with viable organisms of strains that produce either heat-labile toxin alone or both heat-labile and heat-stable toxin, but they were not protected against a strain which produces just heat-stable toxin. Geometric mean serum antibody titers were increased by 16-fold or more over control values in those groups of rats in which protection was achieved, with the exception of those immunized exclusively by the peroral route. These observations demonstrate that (i) active immunization with purified E. coli heat-labile toxin results in significant protection against both this toxin as well as viable organisms which produce it, but not against viable strains which produce heat-stable toxin only, and (ii) concomitant ablation of gastric secretion by the use of cimetidine renders the peroral route of immunization effective. They suggest that prophylactic immunization against diarrheal disease caused by heat-labile toxin-producing strains of E. coli may be feasible in humans.

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