MLVA has proved a powerful tool for identifying L. interrogans
). In this study, we extended this molecular approach to other pathogenic species of Leptospira
by defining new primer pairs and selecting new VNTR loci. As a first step, we suggest using VNTR-4, VNTR-7, and VNTR-10 for discriminating between the serovars of Leptospira
strains. This first screen discriminates about 92% and 90% of L. interrogans
and L. kirschneri
serovars, respectively. As a second step, for strains in which only VNTR-10 is amplified, we propose further analysis with primer pairs for VNTR-Lb4 and VNTR-Lb5 markers. This second screen discriminates about 60% of L. borgpetersenii
The redefinition of the primer pairs for amplifying VNTR-4, VNTR-7, and VNTR-10 allowed us to type L. interrogans
and L. kirschneri
strains. This revealed that these two species are closely related, as suggested by DNA-DNA hybridization analysis and 16S rRNA gene sequencing (30
) and by the recent description of L. kirschneri
as a genomospecies. It also suggests that gene order is conserved among these two species, at least for the genes flanking these VNTR loci.
VNTR-10 was the only VNTR marker that could be used to identify L. borgpetersenii
. VNTR-10 is located within the rfb
locus, which encodes proteins involved in lipopolysaccharide O-antigen biosynthesis. The rfb
locus has a different genetic organization in L. interrogans
and L. borgpetersenii
). However, the gene organization around VNTR-10 seems to be conserved, irrespective of the serovar and the species considered.
Although searching for tandem repeats in the L. borgpetersenii genome revealed many fewer VNTRs than in the L. interrogans genomes, we identified two loci (VNTR-Lb4 and VNTR-Lb5) as useful markers.
MLVA of the Leptospira noguchii serovar Panama strain CZ214K shows that this strain harbors degenerate repeats at several VNTR loci (data not shown). The analysis of the whole genome of the saprophyte Leptospira biflexa strain Patoc I (M. Picardeau, C. Bouchier, C. Boursaux-Eude, and C. Médigue, unpublished data) using Tandem Repeats Finder showed that this strain does not have obvious VNTR loci, except within the infB1 gene (corresponding to the locus VNTR-Lb5). Variations in the number of repeats at the VNTR-Lb5 locus does not change the coding sequence frame of infB1, irrespective of the species considered: L. interrogans (39-bp-long repeats), L. kirschneri (39-bp-long repeats), L. borgpetersenii (36-bp-long repeats), L. noguchii (two 36-bp-long repeats in strain CZ214K), or L. biflexa (six 42-bp-long repeats in strain Patoc I) (data not shown).
The analysis of Leptospira collection strains showed that unilocus genetic diversity is higher among serovars in L. interrogans than in L. kirschneri and L. borgpetersenii species. As our strain collection was isolated from different hosts and from different geographic origins, this should not be a biased finding. We determined the validity of the VNTR loci for Leptospira spp. typing by studying clinical strains isolated from different hosts and from different geographic origins (Table ). The selected VNTR loci were useful for typing L. interrogans and L. kirschneri strains. However, we should test more strains and, if possible, more VNTR loci to validate the usefulness of MLVA for typing L. borgpetersenii.
MLVA also showed that there is a genetic diversity within a few serovars and that the serovars of the collection strains underestimate the variety of isolates in natural populations. For example, the clinical strains identified by PFGE as belonging to serovar Grippotyphosa showed different VNTR patterns, suggesting that these strains are genetically heterogeneous. However, the serovar determination of these strains was by PFGE and not by a serological method. PFGE is useful for characterizing leptospiral serovars (9
), but discrepancies between PFGE and serological methods have also been shown for serovars belonging to serogroups Grippotyphosa, Pomona, and Pyrogenes (11
). Strains of the serovar Bogvere isolated in Guadeloupe show two slightly different patterns, one of which is similar to that seen for the collection strain LT60-69. These strains are found either in humans or in rats. In contrast, we were unable to determine the serovars for L. interrogans
strains of the serogroup Pomona by comparing the VNTR patterns for collection strains. Analysis of clinical strains belonging to the same serogroup also showed that only one serovar is found among strains isolated from the same geographic area. The strain isolated in Far Eastern Russia in 1939 is interesting because the strains of serovar Schueffneri (serogroup Canicola) have been described in Japan and Indonesia (20
). This is consistent with the east Asian origin of this strain belonging to the serovar Schueffneri. This also reflects the high stability of the serovars and that few changes are observed over time and geographical distribution. This was also observed among L. interrogans
strains analyzed by PFGE from French overseas territories (10
We confirmed MLVA as a powerful method for epidemiological purposes through a retrospective study in New Caledonia, a region of high leptospirosis incidence located in the South Pacific (22
) (e.g., in 1999, the incidence of leptospirosis in New Caledonia was 122 cases per 100,000 inhabitants). Leptospirosis is endemic in the South Pacific and is a veterinary health problem in Australia (38
) and New Zealand (35
). It has also been reported in Pacific archipelagos such as Fiji (4
) and Vanuatu (27
). Our study showed that MLVA can be easily used for the large-scale typing of clinical isolates. It also showed that, as seen throughout the world, the most encountered serogroup is Icterohaemorrhagiae, confirming that rats (Rattus rattus
, Rattus exulans
, and more rarely, Rattus norvegicus
) are the main reservoirs of leptospirosis in New Caledonia. The occurrence of serogroups Ballum and Pomona in human isolates indicates that, as well as rodents, cattle-breeding and swine-breeding activities are important risk factors (2
). For each serogroup, we found only one serovar. The only exception was one strain of the serogroup Pyrogenes, which had a slightly different VNTR pattern and may be a different genotype rather than a different serovar. The VNTR pattern of strains belonging to the serogroup Pyrogenes is not found among collection strains, suggesting that a new Pyrogenes serovar may be present in New Caledonia. Surprisingly, the L. interrogans
serovar Australis is present in Australia, being one of the main serovars, and in French Polynesia and other South Pacific archipelagos, whereas it is rare in New Caledonia.
In conclusion, MLVA is useful for epidemiologic investigations of leptospirosis in regions of high endemicity. MLVA provides a suitable tool for identifying circulating genotypes of slow-growing bacteria such as Leptospira spp. Further improvements need to be made to the method so that MLVA can be applied directly to biological (serum, blood, or urine of human and animals) and environmental samples, thus avoiding culturing of the pathogen. This would allow epidemiological studies in developing countries where it is not always possible to culture Leptospira spp., allowing a better surveillance of this underestimated disease.