MOLECULAR TYPING METHOD OF LEPTOSPIRA INTERROGANS SEROVARS

Josefa Bezerra da Silva (Instituto Butantan); Eneas de Carvalho (Instituto Butantan); Zenaide Maria de Morais (USP); Regiane Degan Favaro (Instituto Butantan); Sílvio Arruda Vasconcellos (USP); Paulo Lee Ho (Instituto Butantan)

Leptospirosis is the most widespread zoonotic disease in the world. There are about 200 pathogenic serovars of Leptospira, the bacteria that cause leptospirosis. The serovar classification is based on antigenic determinants, mostly related to lipopolysaccharides (LPS). Nevertheless, other classification methods are currently being proposed. Since the difference in the LPS composition observed among Leptospira serovars can be a result of the differences present in their LPS biosynthesis loci, the amplification of these genes can be a powerful tool to associate the serological and the molecular classifications. In this work, we investigated if the amplification of the LPS biosynthesis loci can provide adequate information for Leptospira typing. The total genomic DNA was isolated from L. interrogans serovars Copenhageni, Canicola, Hardjo, Bataviae, Lai, Naam, Pyrogenes, Australis, Autumnalis, Smith and Mwogolo. Paired primers were designed based on the nucleotide sequence of the rfb loci from diverse leptospires serovars. The polymorphic regions were chosen for primer design, to promote differential amplifications among the serovars. An in silico analysis was used to confirm if the fragments amplified will, indeed, differ among some Leptospira serovars. The polymerase chain reaction (PCR) was carried out using single pairs primer or using multiplexed primers. The annealing cycle was performed in two conditions: 45º and 52º C.  Several nonspecific amplifications were observed in the two annealing conditions. Even so, the amplifications using four selected primer pairs (at 45º C) were efficient to differentiate three serovars and also to differentiate three groups of serovars from all others. The serovars and groups of serovars that can be differentiated were: (1) Hardjo; (2) Australis; (3) Bataviae; (4) Copenhageni, Mwogolo, Smith and Lai; (5) Naam and Autumnalis; (6) Canicola and Pyrogenes. However, when these primers were multiplexed in a single PCR (using Copenhageni as the template DNA), a nonspecific fragment, that was not present in the reactions using single primers, was unexpected amplified. Thus the use of primers that can amplify the LPS biosynthesis loci is a method able to link the serological classification to a molecular one. Nevertheless, the amplification of nonspecific fragments was a difficulty not yet transposed step.