Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes

Chuanling Xu,Ashutosh Verma,Chengming Wang,Amanda Loftis,Bernhard Kaltenboeck,Sudhir K. Ahluwalia,Dongya Gao,Odir A. Dellagostin

doi:10.1371/journal.pone.0089507

Abstract

Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis.

Highlights

Leptospirosis is a zoonotic disease caused by pathogenic leptospires that infect a wide range of animals around the world [1,2,3]
We obtained all available lig gene sequences for canine pathogenic leptospires in GenBank for alignment and identified a target region that is highly conserved among these taxa (Fig. 1), but does not exist in saprophytic leptospires
The lig fluorescence resonance energy transfer (FRET)-PCR amplified genomic DNA extracted from L. interrogans Pomona with high sensitivity and specificity

Summary

Introduction

Leptospirosis is a zoonotic disease caused by pathogenic leptospires that infect a wide range of animals around the world [1,2,3]. Quantitative real-time PCR assays targeting the genes secY, lfb, lipL32, lig, and using either SYBR green or hydrolysis probes (such as TaqMan) for detection, have been widely used for the diagnosis of leptospirosis [6,7,8,9,10,11,12,13]. Before 1989, the genus Leptospira was divided into two species, L. interrogans, including all pathogenic leptospires, and L. biflexa, including all saprophytic leptospires. The leptospiral genomes are highly fluid and contain a high number of transposases to mediate multiple genome rearrangements [2], which makes it challenging to design highly sensitive and specific real-time PCR assays to accurately quantitate and differentiate all pathogenic and saprophytic leptospiral strains. Serovars Canicola and Icterohaemorrhagiae are the primary pathogenic strains associated with canine leptospiral disease [1]

Methods

Results

Conclusion