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  Cholvib


  Director : FOURNIER Jean-Michel (fournier@pasteur.fr)


  abstract

 

The Unit's contribution to the fight against cholera covers four areas: (1) bacteriological diagnosis and molecular epidemiology of cholera vibrios; (2) the design of a chemically defined glycoconjugate cholera vaccine; (3) bacteriological identification of non cholera vibrios; (4) molecular study of non cholera vibrios of medical interest. The French Ministry of Health has designated our laboratory as the " National Reference Center for Vibrios and Cholera ", due to its expertise in this field.



  report

cale

Vibrios are gram-negative bacteria that naturally colonize the marine environment. Vibrios are classified into "cholera vibrios", including isolates belonging to serogroups O1 and O139 of Vibrio cholerae, and "non cholera vibrios", including (i) isolates belonging to serogroups other than O1 and O139 of V. cholerae – known as V. cholerae non-O1/non-O139 – and (ii) isolates belonging to other Vibrio species. Our aims are to increase understanding of the ways in which cholera vibrios spread in countries in which cholera outbreaks occur, to design a new vaccine against cholera, and improve the molecular identification of cholera and non cholera vibrios isolated from seafood, to make it easier to evaluate the risk associated with these products.

1. Diagnosis and molecular epidemiology of cholera vibrios (Marie-Laure QUILICI, Jean-Michel FOURNIER)

Cholera remains a major individual and public health problem. According to the WHO, the number of individuals susceptible to cholera due to socioeconomic problems has dramatically increased worldwide, creating conditions favorable for a global cholera crisis. During 2000, 56 countries officially notified the WHO of a total of 137,071 cases and 4,908 deaths. However, according to the WHO, the actual global figures are likely to be higher, due to underreporting and other limitations of surveillance systems.

France is not directly concerned by cholera epidemics as, like most developed countries, it has a high level of hygiene. However, imported cases of cholera are regularly identified in travelers. Such imported cholera cases are notified to the WHO. In 2001, 8 cholera cases were reported on the Mayotte Island, a French territory in the Indian Ocean. Most of these cases concerned individuals not vaccinated during the vaccination campaign in 2000. Two cholera cases were reported in France. These two cases were imported from Indonesia and Togo. No deaths occurred among these 10 cases.

Cholera epidemics do not recognize national borders. We therefore collaborate with biologists from other countries reporting cholera outbreaks, with institutes from the International Network of Instituts Pasteur and with humanitarian nongovernmental organizations. In 2001, we studied 73 V. cholerae O1 strains received from 11 foreign countries. These collaborations make it possible for us to carry out molecular epidemiology studies of cholera and new cholera vibrio strains that might be imported into France. Two molecular typing methods, ribotyping and PFGE, have been used to study the spread of the cholera vibrio strains responsible for cholera outbreaks in Africa since the start of the 7th pandemic. To date, 347 strains isolated from 34 countries have been studied. We are currently analyzing the results obtained.

2. New vaccine against cholera (Alain BOUTONNIER, Bruno DASSY, Jean-Michel FOURNIER)

The long-term control of cholera depends on improving hygiene by providing safe drinking water and appropriate sewage disposal, and by changing personal hygiene behavior. However, these control measures are expensive and it is difficult to change people's hygiene and water-use behavior. Hence, there is an urgent need for cholera vaccines that confer reliable and long-term protection in all age groups, including children aged <5 years.

The starting point of our research, which began in 1991, was to determine which antibodies protect against cholera. We have shown that immunoglobulin G (IgG) monoclonal antibodies directed against the polysaccharide moiety of the lipopolysaccharide (LPS) of V. cholerae O1, are immunoprotective in an experimental model of cholera in neonatal mice. We have also shown that IgG antibodies injected intravenously into adult mice are transferred from blood vessels to the lumen of the intestine. These results encouraged us to start, in 1995, the development of chemically defined vaccines inducing protective serum IgG directed against the polysaccharide moiety of the LPS of V. cholerae O1 and V. cholerae O139. These vaccines will consist of glycoconjugates of the purified polysaccharides of the LPSs covalently linked to a carrier protein.

Serogroup O1 of V. cholerae consists of two serotypes, Ogawa and Inaba. The LPS of the Ogawa serotype contains a specific antigenic determinant. Another antigenic determinant is common to Ogawa and Inaba serotypes. LPSs from these two serotypes were purified and their antigenic determinants studied, using immunochemical and physicochemical methods. By studying in cristallography the binding of anti-Ogawa monoclonal antibodies to synthetic oligosaccharides fragments mimicking the Ogawa O-specific polysaccharide, we showed that the terminal monosaccharide, bearing a 2-O-methyl group, is the determinant for the Ogawa serotype. We then showed that both the core and the O-specific polysaccharide of the LPS are involved in the antigenic determinant common to the Ogawa and Inaba serotypes. These results will help us to prepare glycoconjugates containing these O1 polysaccharides.

The polysaccharide moiety of the LPS of V. cholerae O139 was purified and conjugated to tetanus toxoid. The immunogenicity of the conjugate was assessed in mice. The unconjugated polysaccharide moiety elicited mostly IgM antibodies, whereas only low levels of anti-LPS IgG antibodies were detected. The conjugate elicited high levels of IgG antibodies, peaking 3 months after the first immunization and declining slowly during the following 5 months. Conjugation of the O139 polysaccharide, therefore, enhanced its immunogenicity and conferred T-dependent properties on this polysaccharide. Clinical evaluation of this V. cholerae O139 glycoconjugate is planned.

3. Bacteriological identification of non cholera vibrios (Marie-Laure QUILICI, Jean-Michel FOURNIER)

The genus Vibrio is known to contain more than 50 species, including 7 species frequently isolated from human clinical samples: V. cholerae, V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. fluvialis, V. hollisae, and V. mimicus. Non cholera vibrio infections in humans may result in gastro-enteritis, skin and soft tissue infections and septicemia, and miscellaneous extraintestinal infections, such as ear infections. Patients with underlying immunosuppressive disease are at high risk of the rapid spread of infections with these microorganisms. Most of these infections are associated with contact with seawater or seafood consumption, and their occurrence is correlated with the warmer months of the year.

In 2001, 11 cases of infections due to non cholera vibrios were identified in France by the National Reference Center. Four cases of septicemia or gastroenteritis were caused by V. cholerae non-O1/non-O139. Four cases of septicemia or ear infections were caused by V. alginolyticus, 2 cases of septicemia and gastroenteritis were caused by V. fluvialis and 1 case of septicemia and soft tissue infection was caused by V. vulnificus. Eleven clinical isolates from Romania were also studied. Within the context of the surveillance of seafood products imported into France, we studied 228 Vibrio strains. Forty-seven environmental isolates were also studied.

4. Characterization of non cholera vibrios by molecular methods (Annick ROBERT- PILLOT, Marie-Laure QUILICI)

Ecological modifications to marine and estuarine environments, due to global climate change or local industrial activities, favor the proliferation and the dissemination of vibrios, which may then colonize humans, either by direct contact with seawater or following the consumption of seafood. Increasing international trade and the consumption of raw seafood, together with an increase in the number of susceptible individuals with underlying immunosuppressive disease, are leading to concern that the incidence of infections due to non cholera vibrios may increase. Public health concern has stressed the importance of developing molecular methods for the detection and identification of two vibrio species, V. cholerae and V. parahaemolyticus, present both in seawater and in seafood, and for the characterization of their virulence factors.

Within this context, we have developed a rapid and efficient method, based on colony hybridization assays, for the detection of V. cholerae in seawater. This method uses a 22-nucleotide sequence of the 16S-23S rDNA intergenic spacer region as a probe. Efforts have also focused on the identification of V. parahaemolyticus isolated from the environment. We have shown that biochemical tests are not accurate enough to differentiate this species from closely related species such as V. alginolyticus and have demonstrated that polymerase chain reaction (PCR) with the R72H V. parahaemolyticus-specific sequence is a powerful tool for the reliable identification of this species.

Environmental strains of V. cholerae constitute a reservoir of virulence genes and of potential epidemic strains that represent a hazard for human populations. We are monitoring the evolution of environmental V. cholerae by the subspecies typing of strains isolated from environmental samples and their comparison with strains isolated from clinical samples. To date, 86 strains of clinical origin and 185 strains of environmental origin have been characterized by ribotyping.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

BREGEAT Annick, abregeat@pasteur.fr

DASSY Bruno, bdassy@pasteur.fr

FOURNIER Jean-Michel, fournier@pasteur.fr

QUILICI Marie-Laure, quilici@pasteur.fr

ROBERT-PILLOT Annick, arp@pasteur.fr

MASIP Montserrat

DENIS Loïc

ROBERT Quentin

BOUTONNIER Alain, Engineer IP, abouto@pasteur.fr

GUENOLE, Alain,Technician IP, aguenole@pasteur.fr

OUZILLEAU Betty, Technician IP

Shared with the Laboratory of Listeria :

BERTEL Arnaud, Laboratory Assistant IP

DELAIRE Marie-Claire, Laboratory Agent IP

TESSAUD Nathalie, Laboratory Assistant IP


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