Induction of fimbriated Vibrio cholerae O139.

Several fimbriated phases of Vibrio cholerae O139 strains were selectively induced and compared immunologically and biochemically with those of V. cholerae O1. Fimbrial antigens were detected on the surfaces of vibrio cells colonizing the epithelial cells of a rabbit small intestine. Convalescent-phase sera from six individuals infected with V. cholerae O139 revealed the development of antibody against the fimbrillin. These findings suggest that the fimbriae of V. cholerae O1 and O139 are expressed in vivo during infection and that consideration must be given to the use of fimbrial antigens as components of vaccines against cholera.

Characterization of filamentous phages of Vibrio cholerae O139 and O1.

We have analyzed our collection of Vibrio cholerae O139 strains to determine whether filamentous phages are produced in their culture supernatants, and whether any replicative form of DNA is detectable in cell lysates. Two types of filamentous phage, designated fs1 (6.4 kb) and fs2 (8.5 kb), were found in strains of Vibrio cholerae O139, fs1 was commonly produced from clinical isolates of Vibrio cholerae O1. Infectious particles (filamentous phages) were inducible by subculture, mitomycin C, and cultivation in a ligated ileal loop of a rabbit. Type 4 fimbriae of Vibrio cholerae O1 sensitive to D-glucose and D-mannose were suggested to be receptors for fs1 and fs2. The genome of fs1 was revealed to encode a potential new enterotoxin homologous to zonula occludens toxin. Clarification of the relation of type 4 fimbriae and these filamentous phages will provide a new understanding of the colonization of Vibrio-cholerae O1 and O139. Thus the presence of a new enterotoxin encoded by the genome of filamentous phage like fs1 may clarify the pathogenesis of cholera toxin negative clinical isolates of Vibrio cholerae O1 and non-O1. Our findings combined with the earlier report by Ehara et al. [Microbio. Immunol. 37 (1993) 679-688] suggest that type 4 fimbriae of Vibrio cholerae O1 are important for the development of an effective vaccine against cholera.

Morphological features of a filamentous phage of Vibrio cholerae O139 Bengal.

A filamentous phage was isolated from carrier strain AI-1841 of Vibrio cholerae 0139 Bengal and thus was termed fs phage. The phage was measured to be approximately 1 microm in length and 6 nm in width. One end of the phage was slightly tapered and had a fibrous appendage. The plaques developed on strain AI-4450 of V. cholerae 0139 were small and turbid. The phage grew in strain AI-4450 and reached a size of 10(8) to 10(9) pfu/ml at 5 hr after infection without inducing any lysis of the host bacteria. The group of phages attached on rod-shaped materials like fimbriae of this bacteria, with their fibrous appendages at the pointed end, were often found in the phage-infected culture. The anti-fimbrial serum effectively inhibited the infection of fs phage to the host strain AI-4450. We thus concluded that the phage can be adsorbed on fimbriae with a fibrous appendage on the pointed end of the phage filament.

The occurrence of macrophage-like cholera toxin uptake cells in the intestinal villi of suckling rats.

An oral administration of cholera toxin (CT. 10m g) caused diarrhea in infant rats ranging in age from 1 to 14 days. After administration of the toxin a time sequence study was carried out using highly sensitive immunohistochemical procedures. CT was exclusively incorporated into a type of macrophage-like (ML) phagocytic cell. These cells were identified within the intestinal epithelium of rats suffering choleraic diarrhea. After 2 hrs cells taking up the toxin markedly increased in number and were found in both the mucosa and the lamina propria mucosae. After 4 hrs a small number of ML cells containing CT were still present in the mucosal epithelium, but were no longer observed in the lamina propria. Two kinds of monoclonal antibodies against rat macrophages were used to gain a clue as to the cytological characteristics of ML cells. ED1- or ED2-positive macrophages were demonstrable in the lamina propria and submucosa of the small intestines from control rats. In CT-treated rats a considerable number of cells positive for CT and ED1, or CT and ED2 antisera, were found within the epithelial cell layer and the lamina propria of intestinal villi. It is suggested that many ML cells responsive to CT, if not all, are ED1 and ED2 macrophages and are resident in the villous lamina propria where they can migrate to uptake CT in the intestinal lumen. CT B-subunit and heat-labile toxin (LT) B'-subunit from a mutant strain Escherichia coli were given to the rats in order to know the onset mechanism of toxin uptake. It seems likely that the toxin receptor, GM1 ganglioside, participates in the initiation of CT-uptake mechanism. A possible role of the intestinal ML cells was discussed.

Cloning and sequencing of the gene encoding Vibrio cholerae O1 fimbrial subunit (fimbrillin).

The gene encoding an 18 kDa fimbrial subunit of Vibrio cholerae O1 was identified in a fimbriate strain Bgd17. Mixed oligoprimers were prepared based on the amino acid sequence of the N-terminus and that from a cyanogen bromide-cleaved fragment of the fimbrillin. A PCR-amplified 185 bp DNA fragment was sequenced. This 185 bp fragment was further extended to 540 bp to 3' and 5' termini by RNA-PCR using a primer containing a random hexamer at its 3' end. This fragment did not contain the stop codons. It was further extended by a gene walking method using Eco RI cassette and its primers. Finally a 660 bp fragment was obtained and sequenced. This fragment contained the complete open reading frame of the structural subunit of the fimbriae, composed of 169 amino acids with a molecular mass of 17435.65 and a leader sequence of 6 or 9 amino acids. The deduced amino acid sequence of the polypeptide encoded by the gene, designated fimA, displayed a highly conserved sequence of MKXXXGFTLI EL of type 4 fimbriae.

Purification and characterization of a protein cryoprotective for Vibrio cholerae extracted from the prawn shell surface.

A substance cryoprotective for Vibrio cholerae on the prawn shell surface was purified by ammonium sulfate precipitation and gel filtration. It was a protein of 81 kDa and called cryoprotective protein (CPP). The cryoprotective activity of this protein for V. cholerae was sensitive to heat at 100 C and trypsin treatment. In the presence of Mg ion the protein can bind to the bacterial cell surface. V. cholerae can adhere to the shell surface of the prawn. The number of adhered bacteria was reduced by treating the shell with anti-CPP serum, heat or by trypsin. The presence of Mg ion promoted the adherence. These results suggest that the CPP could serve as an adherence site for V. cholerae on the shell surface.

Immunogenicity of Vibrio cholerae O1 fimbriae in animal and human cholera.

Parenteral immunization with either formalin-fixed whole cells of the fimbriate Bgd17 strain or purified fimbriae protected against Vibrio cholerae O1 infection in rabbits, independent of biotype and serotype. Parenteral immunization of adult rabbits with purified fimbriae prior to V. cholerae O1 challenge resulted in a reduction of 2 to 3 orders of magnitude in the number of bacteria recovered from the small intestines of immunized rabbits in comparison to non-immunized controls. IgG and IgA antibodies against fimbrillin of V. cholerae O1 were detected in the convalescent sera of patients with cholera; however, little fimbrial antigen was detected in the commercially available cholera vaccines when examined by polyclonal and monoclonal antibodies against fimbriae. These data suggest that fimbrial hemagglutinin is a major adhesin of V. cholerae O1 and that parenteral immunization with fimbriae generates a specific immune response in the gut that may serve as one means of mitigating subsequent V. cholerae O1 gut infection.

Extraction from prawn shells of substances cryoprotective for Vibrio cholerae.

Substances cryoprotective for Vibrio cholerae were detected from prawn shells immersed in phosphate-buffered saline. This cryoprotective activity was heat resistant and sensitive to treatment with trypsin. For the exhibition of its full activity, the presence of Mg ion was indispensable. The cryoprotective activity of this substance was more active than that of other known cryoprotectants, like glycerol or serum.

Effects of chitin and its soluble derivatives on survival of Vibrio cholerae O1 at low temperature.

Chitin concentrations greater than 0.04% (wt/wt) protected cholera vibrios against killing at low temperature. This protective effect was detected with both the soluble form of chitin, glycol chitin, and the insoluble particulate form of chitin. Some amino acids or peptides also showed the same protective effect.

The characterization of Vibrio cholerae isolated in Kenya in 1983.

A total of 245 strains of Vibrio cholerae 01 and two strains of V. cholerae non-01 were isolated and collected from diarrhoeal patients in Homa Bay District Hospital and the other medical facilities in Nyanza Province, Kenya in 1983. The majority of V. cholerae 01 tested were Ogawa type (with the exception of nine Inaba type), biotype E1 Tor (except one untypable strain) and Celebes original type (except one cured type). Haemolytic activity to sheep red blood cells was detected in 75.5% of isolates. Out of 245 strains of V. cholerae 01, 184 were resistant to tetracycline, streptomycin and ampicillin. All were sensitive to chloramphenicol and nalidixic acid. Only one strain of V. cholerae 01 was sensitive to all five antimicrobial agents tested. An environmental cholera survey was done after the cholera outbreak subsided. Twenty strains of V. cholerae non-01 were isolated from water samples in Nyanza Province but none of V. cholerae 01 was isolated.

Lysogenicity and prophage type of the strains of Vibrio cholerae O-1 isolated mainly from the natural environment.

Attention has recently been given to the increasingly frequent detection of atypical Vibrio cholerae O-1 in the natural environments throughout the world. Lysogenicity of V. cholerae O-1, mainly isolated from environmental sources in nine areas, including the United States, was studied by electron microscopy and a cross-lysis test between bacteria and the culture supernatants. A total of 38 strains isolated in Texas in 1973 and in Louisiana in 1978 were lysogenic, whereas there were no lysogenic strains among those isolated in Louisiana in 1979. Because these phages were identical in terms of serology and host range, the phage in them was named T-L phage. T-L phage differed in host range from the k type phage in the epidemic strain of the seventh cholera pandemic. Although the T-L phage was neutralized by antiserum to the k type phage and vice versa, the two phage types were not serologically identical. By a prophage typing method, strains of V. cholerae O-1 isolated from the environment in Guam, Bangladesh, and Japan were classified as either of the Celebes type (epidemic strain) or the Classic-Ubon type. All strains isolated in Brazil, the Chesapeake Bay, and England were nonlysogenic and classified as Classic-Ubon type.

New method for the detection of a lethal factor in vibrios.

When vibriocinogenic vibrios were mixed with an indicator strain, "lacunas" (plaquelike clearings) were observed, thus providing a new method for detection of vibriocins.