First Isolation and Molecular Typing of Pathogenic and Intermediate Leptospira Species from Urine of Symptomatic Dogs

Ivana Piredda,Aureliana Pedditzi,Giuseppe Benvenuto,Pierangela Pintore,Valentina Chisu,Loris Bertoldi,Bruna Palmas

doi:10.3390/vetsci8120304

Abstract

Aim of this study was to evaluate, the presence and diversity of Leptospira spp. in blood and urine samples collected from 175 owned-dogs from Sardinia, Italy. After determination of leptospiral infection by microscopic agglutination test (MAT), urine from MAT-positive dogs were examined by real-time polymerase chain reaction (lipL32 rt-PCR) and then isolated by culture. In order to characterize obtained serovars, positive cultures were then subjected to 16S rRNA and secY sequencing, phylogenetic analysis and Multilocus Sequence Typing (MLST). Results showed that seven dogs (4%; 95% CI: 0–55) had Leptospira DNAs in their urine and five strains were isolated from urine cultures. The three different sequence types (ST17, ST198 and ST24) belonging to Leptospira interrogans genomospecies identified by MLST analyses in this study, confirmed that the leptospiral infection was widespread in Sardinian dogs. We also reported the first characterization of a new Leptospira spp. isolated from urine of one dog living in the study area. Whole genome sequencing and phylogenetic analysis, confirmed that this genospecies was closely related to Leptospira hovindhougenii, an intermediate Leptospira spp. with unknown pathogenicity previously isolated from a rat in Denmark. Further studies are required to clarify whether healthy dogs that shed leptospires in their urine could represent a zoonotic risk for humans in this region.

Highlights

Leptospirosis is a globally distributed zoonotic disease caused by infection with bacterial pathogens of the genus Leptospira [1]
In order to improve the scientific knowledge about Leptospira serovars circulating in Sardinian dogs, this study aimed to: (a) identify canine Leptospira serovars by serological and molecular techniques, (b) isolate Leptospira strains from urine samples of dogs that resulted positive after serological exams, (c) characterize the new Leptospira serovars by phylogenetic analysis of the newly identified sequences
Concerning the physical examination, dogs were classified in two groups: healthy dogs (Nr. = 150) if no clinical abnormalities were detected and unhealthy dogs (Nr. = 25) if they showed clinical symptoms that consisted of increased body temperature, oral lesions, anorexia, vomiting, and icterus) relevant to leptospirosis

Summary

Introduction

Leptospirosis is a globally distributed zoonotic disease caused by infection with bacterial pathogens of the genus Leptospira [1]. Leptospira species have been described worldwide and many of which are well-established threats to domestic animals and livestock [2,3,4,5]. A wide range of Leptospira reservoirs have been described in the last few years, rats represent the commonly recognized reservoir of Leptospira species and the primary animal source of human outbreaks worldwide [6,7]. Humans and domestic animals can become infected through direct contact with urine of infected animals or indirectly via the contaminated environments [10,11]. Since some infected dogs do not show any signs of illness, specific examination is crucial in this context, and an early diagnosis of leptospirosis could help readily initiate antibiotic therapy and decide the more effective treatment for the patient

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