Unit: Spirochetes (Laboratory of)
Director: Isabelle SAINT GIRONS
The use of a transposon of the mariner family has allowed to successfully introduce foreign DNA into Leptospira biflexa and to obtain mutants. The typing of leptospires, by use of mini satellites (VNTR), proved to be quite efficient to identify quickly most of the Leptospira interrogans serovars. For Lyme borreliosis, the focus was on two fundamental aspects: the clinical incidence of the human disease and the study of the vector density (the Ixodes ricinus tick) and its infection rate by Borrelia burgdorferi.
Random insertional mutagenesis of Leptospira biflexa using a mariner transposon (H. Louvel, P. Bourhy, I. Saint Girons and M. Picardeau)
A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. We have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. The use of Himar1 in vivo transposon mutagenesis in L. biflexa has allowed to screen mutants that were impaired in amino acid biosynthesis, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, L. biflexa transposon mutants were screened onto medium with and without hemin, thus allowing the identification of hemin-requiring mutants and the putative genes responsible for this phenotype were identified. Some mutants had distinct insertions in a gene encoding a protein which shares homology with TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Our results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.
Identification of variable-number of tandem repeats loci in Leptospira interrogans sensu stricto (Z. Majed, C. Pourcel, D. Postic, G. Baranton and M. Picardeau, in the frame of the Transversal Research Program n° 139)
Leptospira interrogans sensu stricto is responsible for the most frequent and severe cases of human leptospirosis. The epidemiology and clinical features of leptospirosis are usually associated with the serovars and serogroups of Leptospira. Because of the difficulties associated with serological identification of Leptospira strains, we evaluated a novel PCR-based method for typing L. interrogans serovars in collaboration with the CNR of Leptospires. Based upon the genome sequence of L. interrogans serovar Lai type strain 5660, 44 loci were analyzed by PCR for their variability in size due to the presence of variable-number tandem repeats (VNTR). Seven VNTR loci were found to be powerful markers for serovar identification, epidemiology, and phylogenetic studies of L. interrogans. This rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.
CNR of Leptospires, WHO and FAO Collaborating Center for Leptospirosis (G. Baranton, D. Postic, E. Bellenger , E. Fournié-Amazouz, MA. Gollety and N. Sertour, with the collaboration of D. Portnoï)
In 2003, it has been (wrongly) thought that an unusual heat wave could lead to a leptospirosis increase. In 2004, leptospirosis endemicity is rather low in continental France: about 190 cases detected by the sole CNR in continental France as compared to 242 in 2003, 246 in 2002 and 226 in 2001. Concerning overseas France, only Guadeloupe data are available at this moment. They show significant increase: almost 150 cases as compared to the 130 cases recorded in 2003 which have already been considered as exceptional. This could be due to both increase awareness from physicians and unusual frequency of hurricanes and tropical depressions on West Indies in 2004.
A proposal from the Health Ministry lead G. Baranton to chair a working group on upgraduation of recommendations for prevention of leptospirosis. Concerning vaccination, new indications target risk activities rather than occupational exposition.
Another working group in Aquitaine showed that cases reporting from laboratories was more efficient than from physicians. A computer based system for alert, initiated by Y. Le Strat (Institut de Veille Sanitaire), has been tested and could become automatic in 2005.
A specific diagnosis by Real Time PCR for pathogenic leptospires has also been studied on demand of Pasteur Cerba Laboratory.
Consult also: http://www.pasteur.fr/recherche/Leptospira/LeptospiraF.html
CNR of Borrelia (D. Postic, E. Ferquel, M. Garnier and N. Sertour, with the collaboration of C. Pérez-Eid and D. Portnoï)
Our interest within the framework of the activity of the Borrelia CNR focused on two fundamental aspects of Lyme disease. The first one relates to the clinical incidence of the human disease; the second one relates to the evaluation of the contamination risk by studying the vector density and its infestation rate by B. burgdorferi sensu lato (sl).
The clinical study of Lyme disease cases observed in liberal medicine was undertaken in the department of Meuse where we estimated the incidence at 83.4 cases for 100 000 inhabitants. In hospital area, we benefited from the participation of two important hospitals, one located in Paris, the other in Rennes. On the whole, we noted that erythema migrans represents 65% of the clinical forms and the neurological manifestations nearly 24%. The other symptoms (articular, cardiac and cutaneous) are much less observed.
Moreover in 2004, we undertook the setting of a surveillance network of the disease in the Auvergne and Limousin areas, in collaboration with the Regional Intervention Cells in Epidemiology (CIRE), Pasteur Cerba laboratory and the federations of professional medical training.
On the other hand, we carried out an ecological study of the vector in the department of Meuse and in three cantons of the Alsace region for which, thanks to the study of the CIRE East, we got clinical data. This work showed a density of the vector Ixodes ricinus and an infestation rate by B. burgdorferi sl very important in Alsace, more moderate in Meuse. Important local disparities were highlighted. Indeed, if the density of ticks varies from 100 to 200 ticks by 100m2 in the two cantons of Munster and Guebwiller in July, on the opposite the density in the canton very close of Dannemarie is only of 29,4 ticks. Similarly, the tick infestation rate is very high, ranging from 7 to 36% according to the tick stage, the collecting place and the period of the year. On average over the year, 13% of the ticks were found infected at Guebwiller and 20% at Munster. Thus, the density of the ticks infected in Alsace is quite comparable to the one observed in the neighbouring countries with a very strong endemicity of Lyme disease. These ecological data are in perfect correlation with the data of the disease incidence.
The species of B. burgdorferi sl most frequently found in the ticks in Alsace are B. afzelii (for nymphs) and B. garinii (for adults). B. burgdorferi sensu stricto (ss) is much more unusual. The nonpathogenic species B. valaisiana is often identified. On the other hand, B. lusitaniae, a potential pathogenic species, was never found during this study in Alsace.
At the same time, we are working on the development of a RT-PCR allowing us, in only one step, to detect and identify the Borrelia species present in a sample, with a sufficient sensitivity to be used in diagnosis. This method will also be used for detection and identification of B. burgdorferi sl in ticks.
Consult also: http://www.pasteur.fr/recherche/borrelia/Welcome.html
Keywords: Spirochetes, Leptospira, Borrelia, epidemiology, genetics, iron, mutagenesis, transposition, VNTR