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.

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid phase peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography. This material was pure and identical to native ST by analytical high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography. The formation of the disulfide bonds was critical for biological and immunological activity and were tentatively determined to be between cysteines 5 and 14, 6 and 10, and 9 and 17. This synthetic peptide had full immunological and biological activity when compared to native ST by enzyme-linked immunosorbent assay and the suckling mouse assay respectively.The toxins produced by enterotoxigenic Escherichia coli are known to cause a variety of diarrheal symptoms in man and animals. These range from the inconvenience of travelers' diarrhea to the life-threatening affliction in human infants and high death rate in newborn pigs and cattle [I -51. The two forms of toxin commonly found are termed heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). The heatlabile form [6] is a protein composed of a single 25-kDa subunit (LT-A) in combination with five identical 13-kDa subunits (LT-B). The smaller subunit of LT can be obtained by known methods [6, 71. The heat-stable form of enterotoxin is composed of 18 -20 amino acids, six of which are cysteines [8 -121. Of the sequences known thus far for ST, three were determined by sequencing of the peptide isolated from E. coli strains [8-101 and two were determined from nucleotide sequences [ll, 121. These differ primarily in the N-terminal four residues and the tyrosine and asparagine residues at positions 11 and 18. Natural ST obtained from E. coli and covalently linked to LT-B has been clearly shown in animal studies to be an effective combination for vaccination against the actions of both ST and LT [13]. The difficulty in obtaining sufficient quantities of ST from natural sources for use in conjunction with LT-B, and the recent successful use of synthetic peptides as vaccine agents [14, 151, prompted the synthesis of ST (Fig. 1) for use in the above vaccine. In this paper we report the successful synthesis by the solid phase method [I 61 of ST [9] having full biological and immunological activity. ST prepared in this manner has been successfully used in combination with the B-subunits of LT to immunize against diarrheal symptoms caused by enterotoxigenic E. coli [17 -211. A preliminary report of this synthesis was presented earlier [20].

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