High-Resolution Typing of Leptospira interrogans Strains by Multispacer Sequence Typing

Abstract

Leptospirosis is a worldwide zoonosis which is responsible for the typical form of Weil's disease. The epidemiological surveillance of the Leptospira species agent is important for host prevalence control. Although the genotyping methods have progressed, the identification of some serovars remains ambiguous. We investigated the multispacer sequence typing (MST) method for genotyping strains belonging to the species Leptospira interrogans, which is the main agent of leptospirosis worldwide. A total of 33 DNA samples isolated from the reference strains of L. interrogans serogroups Icterohaemorrhagiae, Australis, Canicola, and Grippotyphosa, which are the most prevalent serogroups in France, were analyzed by both the variable-number tandem-repeat (VNTR) and MST methods. An MST database has been constructed from the DNA of these reference strains to define the MST profiles. The MST profiles corroborated with the VNTR results. Moreover, the MST analysis allowed the identification at the serovar level or potentially to the isolate level for strains belonging to L. interrogans serovar Icterohaemorrhagiae, which then results in a higher resolution than VNTR (Hunter-Gaston index of 0.94 versus 0.68). Regarding L. interrogans serogroups Australis, Canicola, and Grippotyphosa, the MST and VNTR methods similarly identified the genotype. The MST method enabled the acquisition of simple and robust results that were based on the nucleotide sequences. The MST identified clinical isolates in correlation with the reference serovar profiles, thus permitting an epidemiological surveillance of circulating L. interrogans strains, especially for the Icterohaemorrhagiae serogroup, which includes the most prevalent strains of public health interest.

Screening for Leptospira DNA in blood and urine from 30 apparently healthy dogs

“…In our study, all urine samples showing positive PCR results had a quantity of DNA that was notably lower than that reported in the pioneer study describing the MST method [17]. In a previous report [12], serologic testing was shown to be a poor predictor of urinary shedding.…”

“…The amplified fragments were sequenced (see appendix), and the sequences were compared to 16S rRNA sequences available in Genbank 2 . Further characterization was performed using the multispacer sequence typing (MST) method with MST1, MST3 and MST9 primers as described by Zilber et al [17]. For one dog (dog N o 19), a variable-number tandem-repeat (VNTR) analysis was subsequently performed as an alternative molecular approach [18].…”

Molecular studies on European equine isolates of Leptospira interrogans serovars Bratislava and Muenchen

“…For example, this method helped to reveal understanding of serovar Pomona-host relationships and showed a strong tendency to be associated with a specific host (Zuerner and Alt, 2009) or with a specific clinical picture of the infection (Timoney et al, 2011). MLVA's discrimination power for strains belonging to serogroup Australis showed to be more useful than recently developed Multispacer Sequence Typing (MST) based on the comparising of the nucleotide sequences of several intergenic regions (Zilber et al, 2014).…”

Multi-locus sequence analyses reveal a clonal L. borgpetersenii genotype in a heterogeneous invasive Rattus spp. community across the City of Johannesburg, South Africa

“…Therefore, we subsequently sequenced additional loci, secY (~450 bp) (n=13) and lipL41 (~500 bp) (n=5), from a subset of samples to increase the resolution of the molecular typing and allow identi cation of the presumptive serogroup. Similarly, infections in R. norvegicus in Cape Town previously identi ed as L. interrogans by sequencing of the lfb1 amplicon [7] were further typed by sequencing secY (n=3) and intergenic regions MST1, MST3 and MST9 (n=3) [13] to determine whether further genetic resolution was possible. Primer pairs secYFd/secYR3 and lipL41F3/lipL41R3 were used to amplify secY and lipL41 [14] and MST1, MST3 and MST9 were ampli ed using published primers [13] on a Techne TC5000 system (Techne Inc).…”