Characterization of a flagellar sheath protein of Vibrio cholerae.

A flagellar sheath protein of Vibrio cholerae CA401 (Inaba) was characterized. Purity of the preparation was indicated by a single band on polyacrylamide gel electrophoresis gels and on Ouchterlony plates prepared with antibody against crude sheath material. The sheath protein was composed of three polypeptides with minimal molecular weights of 61,500, 60,000, and 56,500. The presence of sheath protein on the flagellum as well as on the outer membrane of the cell was demonstrated by ferritin labeling experiments with antiserum. Sheath protein antibody reacted similarly in labeling experiments and agglutination tests with a classical Ogawa strain and two nonagglutinating V. cholerae isolates, indicating that the sheath protein may represent the common Vibrio H antigen. Antibody specific for lipopolysaccharide labeled the cell but not the sheathed flagellum, which demonstrated that the sheath is not a simple extension of the outer membrane of the cell.

Evaluation of surface components of Vibrio cholerae as protective immunogens.

Surface components of a motile Inaba strain (CA401) were removed from washed cells by low-speed shearing. Flagella contaminated with a vesicular material (designated as crude flagella [CFA1) were obtained by differential centrifugation of the shear fluid. Vesicles were obtained from a nonflagellated mutant by the same procedure. Homogeneous small vesicles were obtained in diminished yield from CsCl gradients of CF preparations. Treatment of CF with sodium deoxycholate removed the vesicular material and flagellar sheaths and yielded naked flagella (NF). The ability of these preparations of passively protect infant mice suckled by CFW mothers that had been immunized at the time of mating was compared, on a dry-weight basis, with commercial vaccine (CV). Eight-day-old mice were challenged orally with more than 1,000 50% lethal doses of either the homologous or a heterologous (Ogawa Ca411) strain. The most effective immunogen was CF, which provided complete protection at 1 microng against both challenges. CF and vesicles provided 50- to 100-fold greater protection than CV against homologous challenge. With heterologous challenge, vesicles were 10-fold more protective than CV, markedly less protective than CF. The NF offered only slightly greater protection than CV against both challenges. Immunoelectrophoresis revealed an antigen in CF distinct from vesicles, cell wall lipopolysaccharide or NF. This antigen is not present in the nonflagellated mutant and is apparently associated with motility,

Use of fluorescent antibody in studies of immunity to cholera in infant mice.

Infant mice 8 days of age were infected orally with virulent, motile, classical or El Tor strains of Vibrio cholerae and with nonmotile mutants of low virulence derived from the same strains. At intervals of 8 and 12 h postinfection, frozen thin sections of the ileum were prepared, stained with fluorescein isothiocyanate-labeled rabbit anti-vibrio antibody, and examined with the fluorescence microscope. The motile organisms were present in larger numbers, especially at 12h, and had penetrated the intervillous spaces and crypts of Lieberkuhn more completely than nonmotile vibrios. Dilution counts were made on various regions of the intestines of infant mice challenged orally 12 h previously with either motile or nonmotile strains of V. cholerae. Greater numbers of organisms were found, especially in the upper intestinal regions, when motile organisms were used. Low numbers of vibrios, limited mostly to the lumen, were seen in the ileum of infant mice infected with motile organisms when the infants were the offspring of mothers that had been immunized with crude flagellar vaccine or a vesicular preparation derived from the vibrio cell surface. The distribution of vibrios in this case was similar to that found in infected infants of unvaccinated mothers challenged with nonmotile organisms. Motility appears to enable the bacteria to better populate the upper regions of the intestinal tract and to avoid the washing effects of secretions and peristalsis. Antibacterial immunity may function, at least in part, by making it impossible for motile vibrios to accomplish this widespread distribution within the ileum.

Virulence factors involved in the intraperitoneal infection of adult mice with Vibrio cholerae.

Nonmotile mutants of Vibrio cholerae isolated from Ogawa, Inaba, and El Tor strains were less virulent than parent wild types when administered to adult mice intraperitoneally. The cells were suspended in 5% hog gastric mucin. Antitoxic immunity did not protect mice against this type of challenge, but a ribosomally derived vaccine did. Intraperitoneal injection of 10 50% lethal doses of enterotoxin (based on intravenous doses) was without toxic manifestations as were 10(10) heat-killed vibrios similarly administered, regardless of strain. Virulent organisms killed with formalin or ultraviolet irradiation were significantly lethal at a dose of 10(10) cells. Mice made tolerant to endotoxin were protected from death caused by an injection of 3 X 10(10) boiled cells, but they did not survive an injection of formalin-killed cells. It is believed that the cause of death in this animal model of cholera is dependent, at least in part, on a toxic heat-labile moiety closely associated with the vibrios.