Director: GUISO Nicole
Bacteria from the Bordetella genus are responsible for respiratory infections in humans (Bordetella pertussis and Bordetella parapertussis and whooping cough) and in animals (Bordetella bronchiseptica and atrophic rhinitis and kennel cough). The researches developed in our laboratory are aimed to characterize the determinants involved in the pathogenicity of these bacteria, analyse the immune responses induced in the host after infection or vaccination, surveillance of polymorphism of clinical isolates and setting up of new prevention and new diagnosis for whooping cough and other bordetellosis.
I INTERACTIONS OF BORDETELLA PERTUSSIS, PARAPERTUSSIS AND BRONCHISEPTICA WITH HOST CELLS Interaction of Bordetella pertussis, parapertussis and bronchiseptica with human tracheal epithelial cells. [L. Bassinet, in collaboration with the group of J.M. Cavaillon]
We continued the analysis of the three Bordetella species interactions with human tracheal epithelial cells. Although B. pertussis and B. parapertussis human isolates are not cytotoxic for these cells, some of B. bronchiseptica isolates are. Bacteria can persist inside the cells but do not multiply. B. pertussis isolates stimulate IL6 secretion by the host cells but only when they express toxins and adhesins. Preliminary results indicate that the expression of one of the toxin could be responsable of this secretion.
Interaction de Bordetella pertussis, parapertussis and bronchiseptica withmast cells [I. Tchou, L. Guillemot, N. Guiso, E. Njamkepo, G. Soubigou in collaboration with the group of S. Mecheri]
As for epithelial cells, B. pertussis and B. parapertussis human isolates are not cytotoxic for mastocytes whereas some B. bronchiseptica isolates are. A correlation between the pathogenicity in vitro and in vivo using the murine respiratory model is under an analysis.
II Polymorphism OF Bordetella pertussis, parapertussis AND bronchiseptica isolates Analyse of the evolution of Bordetella pertussis population [V. Caro, K. Dalet, L. Guillemot, N. Guiso, E. Njamkepo, and G. Soubigou]
Analysis, by typing technique or sequencing of toxin and adhesin structural genes, of isolates circulating in France, a vaccinated country since more than thirty five years with a whole-cell vaccine, confirmed that B. pertussis isolates circulating before and after introduction of generalized vaccination are different. However, a precise analysis suggests that B. pertussis population could vary regularly every three years. The impact of the observed polymorphism is low in this country where the population of the isolates circulating is clonal.
Analysis of the sequences of the genes encoding major toxins and adhesins of B. pertussis indicate that those encoding pertussis toxin and adenylate cyclase hemolysin do not vary. The gene encoding pertactin varies. Using a murine respiratory model, we have tested the immunity of the pertussis vaccine used in France. Variations at the level of pertactin do not seem to effect vaccin efficacy.
Analysis of some clinical isolates allowed us to visualize plasticity of the genome. In fact, the modifications of the restriction profiles of the genomic DNA of the some of these isolates might be linked to a duplication or a deletion of a genome fragment. In particular, an isolate harbouring a duplicaton of the adenylate cyclase-hemolysin operon was studied.
Analysis of the evolution of the Bordetella parapertussis population [V. Caro, D. Foureau, N. Guiso]
Analysis of B. parapertussis isolates collected between 1950 and 2003 showed a great homogeneity of the population. All isolates carry the genes encoding pertussis toxin but none express it. They express major adhesins and toxins which encoding genes don't vary.
Analysis of Bordetella bronchiseptica isolates [L. Guillemot, Nicole Guiso, E. Njamkepo, G. Soubigou]
B. bronchiseptica is a respiratory pathogen for many mamals species, including human. In human, B. bronchiseptica is considered as an opportunist pathogen for immunodepressed patient or patient with respiratory problems. We previously described that this bacterium can induce chronic infections. We confirmed the zoonotic character of this infection. The analysis of different B. bronchiseptica isolates indicate a higher polymorphism of isolates collected on dogs than collected on pigs. No isolate express pertussis toxin but they all express AC-Hly, FHA, PRN and a flagellum. However, pig isolates which express identical AC-Hly and PRN have very different pathogenicity in a murine model suggesting that other factors play a role on B. bronchiseptica pathogenicity.
III DEVELOPMENT OF MICROARRAYS [V. Caro, M. Arrive in collaboration with the group of Y. Lemoine and< C. Locht of the Institut Pasteur in Lille]
In collaboration with the Institute Pasteur in Lille, we participate in the development of DNA microarrays in order to analyse the polymorphism of the three bordetella species pathogenic for humans and their regulation of gene expression. In parallel we are developing quantitative RT-PCR technique to study the expression of some genes by clinical isolates.
IV DURATION OF HUMORAL IMMUNITY IN CHILDREN VACCINATED WITH DIFFERENT PERTUSSIS< VACCINES [N. Guiso, E. Njamkepo]
Analysis of duration of humoral immunity after vaccination with different pertussis vaccines in children has been continued in order to help health authorities to adapt vaccine calendar. Our preliminary results indicate the duration of humoral immunity after vaccination with whole-cell vaccine or acellular vaccine composed of three pertussis antigens is similar at least 6-8 years after the 16-18 months booster.
V DEVELOPMENT OF BORDETELLA PERTUSSIS DIAGNOSIS BY REAL TIME PCR [V. Caro, L. Ormières]
A new molecular diagnosis based on the amplification of the promoter region of the pertussis toxin was developed in Real Time PCR, including an internal controll developed by "Overlap Extension". This new specific diagnosis is under validation.
VI CENTRE NATIONAL DE REFERENCE [L. Guillemot, N. Guiso, E. Njamkepo, G. Soubigou]
The missions of this Centre is to confirm the identification of Bordetella isolates collected in France, to maintain the collection, to teach French bacteriologists, to analyse isolates by Pulsed-Field-Gel Electrophoresis, as well as the toxins and adhesins they express, to set up sensitive and specific biological diagnosis techniques and to be part of the National surveillance net, RENACOQ, coordinated by the Ministery of Health.
Furthermore, we participated to different collaborative studies with PHLS (London) as experts < for Bordetella serotyping.
VII QUALITY INSURANCE [F. Fouque]
The set up of procedures and quality insurance in the laboratory was pursued.
Keywords: Whooping cough, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, vaccine, immunity, chronic infection"