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...

The phylogenetic relationships of 20 relapsing fever (RF) Borrelia spp. were estimated on the basis of the sequences of rrs genes. Complete sequences were aligned and compared with previously published sequences, and the similarity values were found to be 97.7 to 99.9%. Phylogenetic trees were constructed by using the three neighbor-joining, maximum-parsimony, and maximum-likelihood methods. The results of the comparative phylogenetic analysis divided the RF Borrelia spp. into three major clusters. One cluster included Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis, and Borrelia hispanica. Another cluster comprised tow main branches with Borrelia coriaceae, Borrelia lonestari, and Borrelia miyamotoi on one side and Borrelia parkeri, Borrelia turicatae, and Borrelia hermsii on the other side. Borrelia anserina constituted the third cluster. The phylogenetic position of Borrelia persica was more uncertain. These results suggested that the taxonomy of these spirochetes should be revised. To overcome the problems of culturing the spirochetes, RF Borrelia primers were defined. Following PCR amplification of the rrs gene, restriction length fragment polymorphism could be used to distinguish between RF Borrelia strains.

Human Lyme borreliosis (LB) is the most prevalent arthropod-borne infection in temperate climate zones around the world and is caused by Borrelia spirochetes. We have identified 10 variable-number tandem repeat (VNTR) loci present within the genome of Borrelia burgdorferi and subsequently developed a multiple-locus VNTR analysis (MLVA) typing system for this disease agent. We report here the successful application of MLVA for strain discrimination among a group of 41 globally diverse Borrelia isolates including B. burgdorferi, B. afzelii, and B. garinii. PCR assays displayed diversity at these loci, with total allele numbers ranging from two to nine and Nei's diversity (

We describe a nested polymerase chain reaction for the identification of Borrelia species from serum of patients with unidentified fevers. This technique, based on single nucleotide polymorphisms of the 16S ribosomal RNA gene, was used to test blood samples from 7,750 patients, 33 of whom were diagnosed with spirochete infections. Borrelia crocidurae was the only species identified.

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