Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene

Andrew T Slack,Michael F Dohnt,Lee D Smythe,Meegan L Symonds

doi:10.1186/1471-2180-6-95

Abstract

BackgroundLeptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing.ResultsA conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results.ConclusionThis research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification.

Highlights

Leptospira is the causative genus of the disease, leptospirosis
The grouping of the last three species as opportunistic or possible pathogens is due to the lack of information on the pathogenicity of the species, different phenotypic characteristics compared to the pathogenic Leptospira and the limited number of reports of these species involvement in human leptospirosis
No PCR products were amplified from the representatives of non-pathogenic species L. biflexa, L. meyeri or from the pathogenic/intermediate species such as L. inadai, L. fainei or L. broomi (Figure 1 and 2)

Summary

Introduction

Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radiolabelled isotopes or significant homology between species. Leptospirosis is the zoonotic disease caused by members of the genus, Leptospira. They are motile helical spirochaetes that metabolise long chain fatty acids as their carbon source. There are 17 species of Leptospira as determined by DNA-DNA hybridisation [1,2,3,4] These species can be further divided into pathogenic, non-pathogenic and opportunistic/possibly pathogenic Leptospira with pathogenic (page number not for citation purposes). The grouping of the last three species as opportunistic or possible pathogens is due to the lack of information on the pathogenicity of the species, different phenotypic characteristics compared to the pathogenic Leptospira and the limited number of reports of these species involvement in human leptospirosis

Methods

Results

Conclusion