Direct Detection and Differentiation of Pathogenic Leptospira Species Using a Multi-Gene Targeted Real Time PCR Approach

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.

Genetic diversity of pathogenic leptospires from wild, domestic and captive host species in Portugal

“…Four isolates (INIAV 17433 Z1, INIAV 18078 Z6, UTAD Rim283 and UTAD Rim156) were identified as L. borgpetersenii and clustered together in the secY phylogenetic tree under cluster B (Table , Figure ). Two of these isolates (INIAV 17433 Z1 and INIAV 18078 Z6) were obtained from M. musculus captured in the Lisbon zoo, in 2012 (Ferreira et al, ), while the third (UTAD Rim283) was also obtained from a M. musculus , however the latter was captured in Vila Real, far north of Portugal, in 2009. All these three isolates belonged to serogroup Ballum, serovars Castellonis (INIAV 17433 Z1 and INIAV 18078 Z6) and Ballum (UTAD Rim283).…”

“…The species assignment of the isolates was confirmed by a multi‐gene targeted real‐time PCR assay using species‐specific TaqMan ® probes as described by Ferreira et al (). This assay allows the differentiation between the pathogenic L. interrogans , L. borgpetersenii , L. kirschneri and L. noguchii .…”

“…Portuguese Leptospira isolates are highlighted in bold and the GenBank accession numbers are indicated for the remaining sequences. Letters A -G identify the clades containing Portuguese isolates probes as described by Ferreira et al (2014). This assay allows the differentiation between the pathogenic L. interrogans, L. borgpetersenii, L. kirschneri and L. noguchii.…”

Neuroleptospirosis: Aseptic Meningoencephalitis or Invasion into the Central Nervous System?

“…Electron microscopy findings typically show a helically wound spiral shaped bacterium (0.01-0.02 µm in diameter) with characteristic hooked ends [1]. A TaqMan-based multi-gene targeted real-time PCR has a specificity of up to 100% and can detect a variety of pathogenic and non-pathogenic Leptospira strains [7].…”

<b>Pesquisa de <i>Leptospira</i> spp. em fêmeas bovinas pertencentes ao município de Novo Repartimento - Pará</b>

“…No presente trabalho houve a detecção do DNA da bactéria em amostras de rim e/ou urina de algumas fêmeas bovinas não reagentes na SAM. A associação da sorologia com a PCR pode ser muito útil na identificação de animais portadores de leptospiras, que usualmente, eliminam a bactéria na urina por longos períodos, contaminando o ambiente e outros animais do rebanho (LILENBAUM, 1996;LILENBAUM et al, 2009 (VICTORIA et al, 2008;PEREZ;GOARANT, 2010;FERREIRA et al, 2014;HAMOND et al, 2015a;HAMOND et al, 2015b;LOUREIRO et al, 2016;MIOTTO et al, 2016).…”