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

A 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.This 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.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 [1-4]. These species can be further divided into pathogenic, non-pathogenic and opportunistic/possibly pathogenic Leptospira with pathogenic species. The pathogenic Leptospira include; L. interrogans, L. kirschneri, L. santarosai, L. weilii, L. alexanderi, L. borgpetersenii, L. genomospecies 1 and L. noguchii. The non pathogenic Leptospira include: L. biflexa, L. meyeri, L. wolbachii, L. genomospecies 3, L. genomospecies 4, L. genomospecies 5 and opportunistic/intermediate pathogens Leptospira include L. broomi, L. fainei and L. inadai [3]. 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 also the limited number of reports of these spe