Novel flaB gene variants of Leptospira interrogans detected in leptospirosis patient samples from the Western province of Sri Lanka

Background Leptospirosis caused by pathogenic spirochaetes of Leptospira spp remains the most widespread zoonotic disease in the world. Clinical status in leptospirosis patients varies from asymptomatic infection to mild illness to severe/fatal outcomes. The objective of this study was to determine the species of Leptospira that cause mild and severe infections, from patient blood samples obtained from the Western Province in Sri Lanka. Methods DNA extracted from 60 blood samples of previously confirmed leptospirosis patients by Lipl32 based Real Time PCR, and clinically characterized as mild and severe (n = 30 each) were used to perform nested PCR with primers designed from fla B gene sequence. Results Of the 45 samples that gave a 725 bp fragment in fla B nested PCR, 23 and 22 were from severe and mild leptospirosis patients respectively. Of these fla B PCR positive samples, 16 which exhibited strong bands (8 severe cases and 8 mild) were selected for Sanger’s dideoxy sequencing. The sequences obtained from 16 samples were deposited in GenBank. Those partial fla B gene sequences showed highest similarity with L. interrogans sequences available in GenBank database. Phylogenetic analysis was performed with 82 other randomly selected Leptospira fla B gene sequences (from the GenBank) by Neighbor-Joining method using MEGA 11 tool. According to dendrogram, 16 partial fla B gene sequences obtained in this study clustered together with L. interrogans . Three sequences, one from a severe leptospirosis patient and two from mild leptospirosis patients exhibited novel mutations. Computational analysis of the predicted amino acid substitutions in these three variants deduced from mutant DNA sequences with that of the wild type FlaB indicate possible functional changes in the flagellar protein. However, the impact of these novel mutations on the flagella assembly and motility could not be determined without functional confirmation. This highlights the need for further functional conformational studies to elucidate possible impact on motility which may influence the pathogenicity of different isolates of L. interrogans.