Multi-locus sequence typing for species/serovar identification of clinical isolates of Leptospira spp

K Justin Davis

doi:10.51966/jvas.2021.52.3.238-244

Abstract

Leptospirosis is an emerging zoonotic disease endemic in Kerala and close monitoring of emerging serovars is essential to adopt appropriate control strategies. Multi-Locus Sequence Typing (MLST) was reported to be less expensive compared to other cumbersome methods like whole genome sequencing. The present study was conducted to obtain isolates of Leptospira from infected animals and rats and for the identification of serovars using MLST. A total of 205 blood samples (dog, cat, cattle, goat), 43 urine samples (dog, cattle) and post-mortem kidney samples from various animals such as dog (n=12), cattle (n=2) and rat (n=25) were collected and subjected to polymerase chain reaction (PCR) using G1/G2 primers to identify the pathogenic Leptospira. Fifteen samples were found to be positive, these samples when inoculated in the Ellinghausen- McCullough-Johnson-Harris (EMJH) semi-solid medium to obtain ten isolates. These ten isolates were further subjected to secY, icdA and GyraseB PCR and sequenced. The obtained sequences were analysed using BLAST and were fed into specified MLST database of Leptospira scheme-3, the allelic profile and sequence type were generated. The MLST results obtained in the study indicated that the isolates S24 and S33 belonged to serovar Canicola, S40 and 47 were Sejroe and S19, S27, S55, S69 and S71 were Bataviae, Autumnalis, Pomona, Icterohaemorraghiae and Australis, respectively. It was concluded that MLST is a convenient method for identifying leptospiral serovars.

Highlights

Leptospirosis is an emerging zoonotic disease endemic in Kerala and close monitoring of emerging serovars is essential to adopt appropriate control strategies
A total of 205 blood samples were collected from dogs (n=139), cats (n=11), cattle (n=29) and goats (n=26) presented to Teaching Veterinary Clinical Complex, College of Veterinary Animal Sciences, Mannuthy with clinical signs indicating to acute leptospirosis, during the study period of March 2019 to
Whole blood samples collected in heparin and ethylene diamine tetra-acetic acid (EDTA) vials were used for isolation of Leptospira and to perform polymerase chain reaction (PCR), respectively

Summary

Introduction

Leptospirosis is an emerging zoonotic disease endemic in Kerala and close monitoring of emerging serovars is essential to adopt appropriate control strategies. Alternative DNA based methods such as Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), Pulsed Field Gel Electrophoresis (PFGE), Variable Number Tandem Repeat (VNTR), and Multi-Locus Sequence Typing (MLST) were used for identification of leptospiral serovars (Ahmed et al, 2006). When compared to all these DNA based methods, MLST was preferred because of its high reproducibility and discriminatory power to differentiate leptospiral serovars (Ahmed et al, 2011).

Results

Conclusion