Abstract

Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of Leptospira. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. In this work we describe a novel and simple TaqMan-based multi-gene targeted real-time PCR approach able to detect and differentiate Leptospira interrogans, L. kirschneri, L. borgpeteresenii and L. noguchii, which constitute the veterinary most relevant pathogenic species of Leptospira. The method uses sets of species-specific probes, and respective flanking primers, designed from ompL1 and secY gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal β-actin and the leptospiral lipL32 genes was implemented. The analytical sensitivity of all primer and probe sets was estimated to be <10 genome equivalents (GE) in the reaction mixture. Application of the amplification reactions on genomic DNA from a variety of pathogenic and non-pathogenic Leptospira strains and other non-related bacteria revealed a 100% analytical specificity. Additionally, pathogenic leptospires were successfully detected in five out of 29 tissue samples from animals (Mus spp., Rattus spp., Dolichotis patagonum and Sus domesticus). Two samples were infected with L. borgpetersenii, two with L. interrogans and one with L. kirschneri. The possibility to detect and identify these pathogenic agents to the species level in domestic and wildlife animals reinforces the diagnostic information and will enhance our understanding of the epidemiology of leptopirosis.

Highlights

  • Leptospirosis is a growing and underestimated public health and veterinary concern, caused by pathogenic spirochetes belonging to the family Leptospiracea, genus Leptospira [1,2]

  • Analytical specificity and sensitivity Execution of the PCRs on DNA extracted from various bacteria, revealed a highly specific amplification from any of the pathogenic strains belonging to the respective target Leptospira spp., i.e. L. interrogans, L. kirschneri, L borgpetersenii and L. noguchii

  • The same LOD was estimated for the lipL32-targeted probe/primers when used in duplex amplification reactions with the mammal b-actin probe

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Summary

Introduction

Leptospirosis is a growing and underestimated public health and veterinary concern, caused by pathogenic spirochetes belonging to the family Leptospiracea, genus Leptospira [1,2]. Its transmission requires circulation of the agents among domestic and wild animal reservoirs, with rodents recognized as the most important sources that establish persistent renal carriage and urinary shedding of Leptospira. Conventional classification of Leptospira is based on serological criteria, using the serovar as the basic taxon. The serological classification system is complemented by a genotypic one, in which 21 genetic species are currently recognized, including pathogenic, intermediate and non-pathogenic (or saprophytic) species [7,8,9,10]. Genetic species boundaries hardly correlate with the serological classification [8]

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