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. 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.The genus Leptospira consists of a heterogeneous group of pathogenic and saprophytic species belonging to the order Spirochaetales. Pathogenic Leptospira species, currently classified in seven species based on DNA relatedness (2, 25), are the agents of leptospirosis. Transmission to humans occurs through direct or indirect contacts with urine of infected animals. Leptospira interrogans sensu stricto (25) is the main species associated with human leptospirosis. In France, L. interrogans sensu stricto is responsible for about 60% of human cases and for the most severe ones. The intraspecies taxonomy of leptospires is well established and based on antigenic determinants. Since the description of serovars in 1915, about 80 serovars have been identified in L. interrogans sensu stricto (2); among them, 60 serovars are validly described (12). Since each serovar is usually associated with a particular host, identification of serovars is essential to epidemiological studies and strategies for prevention (5). The reference method for serological identification is the microagglutination test, which is a complex and fastidious test since it requires cross adsorption of many rabbit hyperimmune sera (24).Antigenically related serovars are grouped into serogroups. However, a given serogroup is often found in several Leptospira species. For instance, the nine validly described serovars from serogroup Bataviae are distributed among L. interrogans sensu stricto species (two serovars), L. santarosai (four serovars), L. kirschneri (one serovar), L. noguchii (one serovar), and L. borgpetersenii (one serovar). Several studies have thus shown that the system of serogroups is not related to molecular classifications. In contrast, serovars can be characterized by different molecular methods, such as restriction fragment length polymorphism-based methods (15,22), arbitrarily primed PCR (19), and pulsed-field gel electrophoresis (PFGE) (8, 9). However, these techniques are not widely applied, because PFGE and restriction fragment length polymorphism are laborious and require significant volumes of culture and arbitrarily primed PCR results in poor reproducibility and interpretation of results. In add...

Reference strains for each of the 23 serogroups of Leptospira interrogans yielded different pulsed-field gel electrophoresis patterns of NotI digestion products. This was also the case for the 14 serovars belonging to serogroup Icterohaemorrhagiae (with one exception). The NotI restriction patterns of 45 clinical leptospiral isolates belonging to serovar icterohaemorrhagiae were analyzed and compared with those of type strains. No differences were observed between isolates from countries of different continents, namely, France, French Guiana, New Caledonia, and Tahiti. The pattern was indistinguishable from that of the reference strain of serovar icterohaemorrhagiae.

Fingerprints for 72 reference serovar strains of pathogenic Leptospira spp. were obtained by pulsed-field gel electrophoresis (PFGE) following NotI restriction digests of the chromosome. These strains included the serovar reference strains of serogroups Australis, Ballum, Bataviae, Grippotyphosa, Panama, Pomona, and Pyrogenes. Sixty-four serovars could be identified by a unique NotI restriction profile. The remaining serovars were differentiated by chromosomal digestion with SgrAI. These included four serovars from serogroup Australis, two serovars from serogroup Ballum, and two serovars from serogroup Bataviae. Thirteen of 18 recent clinical isolates identified by microagglutination test and cross-adsorption procedure were correctly typed by PFGE. The results indicate that PFGE, which is considerably more rapid than serology, should be useful for identification and epidemiological studies. The order Spirochaetales comprises two families of thin, motile, spiral-shaped eubacteria, the Spirochaetaceae and the Leptospiraceae (5, 21). Within the family Leptospiraceae, pathogenic Leptospira spp. comprise six officially recognized genomic species (32): Leptospira interrogans, L.

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A total of 46 Borrelia burgdorferi sensu lato isolates that were isolated from patients with Lyme borreliosis and infected animals or were extracted from ticks of the genus lxodes were analyzed. Large restriction fragment patterns obtained after cleavage of genomic DNAs with Mlul were analyzed by pulsed-field gel electrophoresis (PFGE). To eliminate the contribution of plasmid DNA, only frmMents greater than 70 kb were used for the analysis. The results indicated that each of the 14 B. burgdorferi sensu stricto isolates were recognized by a band at 135 kbp, each of the 12 Borrelia garinii isolates by two bands (220 and 80 kbp), and each of the 20 Borrelia afzelii isolates by three bands (460, 320, and 90 kbp). Whereas differences in the PFGE patterns among B. burgdorferi sensu stricto isolates and B. garinii isolates were noted, B. afzelii isolates were all similar. Identification of isolates by PFGE correlates with their belonging to a given species within B.

We report the isolation and characterization of 16 Leptospira spp. strains isolated from small rodents captured in 11 different regions of inland Croatia. Large NotI and SgrAI restriction fragment allowed us to assign 10 isolates to the serovar istrica, 5 isolates to the serovar tsaratsovo and 1 isolate to the serovar lora. The phylogenetic analysis conducted from the sequences of the first 330 bp from the 16S rDNA gene revealed that the strains belonged to three different species, L. borgpetersenii, L. kirschneri and L. interrogans. Carrier rates in eight rodent species varied from 0 to 71.4%. Mus musculus showed the highest infection level and confirmed its role as a major reservoir of the serogroup Sejroë. For the first time we reported the occurrence of serovars tsaratsovo and lora in Croatia.

Pulsed-field gel electrophoresis (PFGE) studies performed with leptospiral isolates led us to suspect the existence of a new serovar in the Grippotyphosa serogroup. The results obtained with reference serological techniques used in leptospiral identification, including cross-agglutination absorption and monoclonal antibody techniques, confirmed the existence of a new serovar exemplified by strain Dadas I. Four other isolates from different regions of the world were submitted for identification by PFGE and produced NotI restriction patterns similar to that of strain Dadas I. Our data demonstrate the power of PFGE for identifying leptospiral isolates. The name dadas is proposed for the new serovar.

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.