Identification of Variable-Number Tandem-Repeat Loci in Leptospira interrogans Sensu Stricto

Abstract

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...

Multiple-locus variable-number tandem repeat analysis and clinical characterization of Leptospira interrogans canine isolates

“…In this study, we characterized the isolates at the serogroup level and were unaware if each cluster represented a serovar. A previous study demonstrated that MLVA for L. interrogans was useful for identifying serovars (Majed et al, 2005), whereas other studies indicated considerable diversity of genotypes defined by MLVA within the same serovar (Slack et al, 2006;Zuerner & Alt, 2009). We performed MLVA on 25 serovar strains and compared their profiles with those of the isolates (Table S1).…”

“…MLVA was performed using 11 loci: variable number tandem repeat (VNTR)-4, -7, -10 (Salaün et al, 2006), -19 (Majed et al, 2005), -23 (Zuerner & Alt, 2009), -31 (Majed et al, 2005), -27, -29, -30, -36 and -51 (Slack et al, 2006 for the isolates. The 25 Leptospira serovar strains were also subjected to MLVA as reference.…”

“…On the other hand, the same sequence types determined by MLST were found in multiple serogroups, indicating that MLST was not suitable for serovar (or serogroup) identification. Recently, multiple-locus variable-number tandem repeat analysis (MLVA) of L. interrogans was demonstrated as a useful tool for identifying serovars or discriminating strains within the same serovar (Majed et al, 2005;Slack et al, 2006;Zuerner & Alt, 2009).…”

Isolation of Leptospira spp. from a man living in a rural area of the Municipality of Cruz Alta, RS, Brazil

“…The MLVA typing was done using two sets of oligonucleotides specifi c for the species Leptospira interrogans, L. borgpetersenii and L. kirschneri. The loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31 were used to discriminate L. interrogans strains (MAJED et al, 2005) and VNTR4, VNTR7, VNTR10, LB4 and LB5 were used to L. kirschneri, L. borgpetersenii and L. interrogans (SALAÜN et al, 2006). The fi nal volume (50µL) of each reaction mixture containing RCP buffer (20mM Tris-HCL, pH 8.4, 50mM of KCl), 200µm of deoxinucleoside triphosphates, 2µm of each corresponding primer, 2mM of MgCl2, 1.25U of Taq DNA polymerase (Invitrogen) and 5uL of DNA model.…”

“…From the DNA formers used in the typing procedures MLVA performed with the primers described by MAJED et al (2005) and SALAÜN et al (2006), 100µL from the culture sample were incubated at 100°C during 10 minutes. The MLVA typing was done using two sets of oligonucleotides specifi c for the species Leptospira interrogans, L. borgpetersenii and L. kirschneri.…”