Leptospire genomic diversity revealed by microarray-based comparative genomic hybridization.
ABSTRACT: Comparative genomic hybridization was used to compare genetic diversity of five strains of Leptospira (Leptospira interrogans serovars Bratislava, Canicola, and Hebdomadis and Leptospira kirschneri serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced Leptospira reference genomes, those of L. interrogans serovar Copenhageni and L. interrogans serovar Lai. A comparison of genetic contents showed that L. interrogans serovar Bratislava was closest to the reference genomes while L. kirschneri serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that L. interrogans serovars Bratislava and Hebdomadis clustered together first, followed by L. interrogans serovar Canicola, before the two L. kirschneri strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.
Project description:White-tailed deer (Odocoileus virginianus) are commonly exposed to disease agents that affect livestock but environmental factors that predispose deer to exposure are unknown for many pathogens. We trapped deer during winter months on two study areas (Northern Forest and Eastern Farmland) in Wisconsin from 2010 to 2013. Deer were tested for exposure to six serovars of Leptospira interrogans (grippotyphosa, icterohaemorrhagiae, canicola, bratislava, pomona, and hardjo), bovine viral diarrhea virus (BVDV-1 and BVDV-2), infectious bovine rhinotracheitis virus (IBR), and parainfluenza 3 virus (PI3). We used logistic regression to model potential intrinsic (e.g., age, sex) and extrinsic (e.g., land type, study site, year, exposure to multiple pathogens) variables we considered biologically meaningful to exposure of deer to livestock pathogens. Deer sampled in 2010-2011 did not demonstrate exposure to BVDV, so we did not test for BVDV in subsequent years. Deer had evidence of exposure to PI3 (24.7%), IBR (7.9%), Leptospira interrogans serovar pomona (11.7%), L. i. bratislava (1.0%), L. i. grippotyphosa (2.5%) and L. i. hardjo (0.3%). Deer did not demonstrate exposure to L. interrogans serovars canicola and icterohaemorrhagiae. For PI3, we found that capture site and year influenced exposure. Fawns (n = 119) were not exposed to L. i. pomona, but land type was an important predictor of exposure to L. i. pomona for older deer. Our results serve as baseline exposure levels of Wisconsin white-tailed deer to livestock pathogens, and helped to identify important factors that explain deer exposure to livestock pathogens.
Project description:Leptospirosis is a worldwide zoonosis which is responsible for the typical form of Weil's disease. The epidemiological surveillance of the Leptospira species agent is important for host prevalence control. Although the genotyping methods have progressed, the identification of some serovars remains ambiguous. We investigated the multispacer sequence typing (MST) method for genotyping strains belonging to the species Leptospira interrogans, which is the main agent of leptospirosis worldwide. A total of 33 DNA samples isolated from the reference strains of L. interrogans serogroups Icterohaemorrhagiae, Australis, Canicola, and Grippotyphosa, which are the most prevalent serogroups in France, were analyzed by both the variable-number tandem-repeat (VNTR) and MST methods. An MST database has been constructed from the DNA of these reference strains to define the MST profiles. The MST profiles corroborated with the VNTR results. Moreover, the MST analysis allowed the identification at the serovar level or potentially to the isolate level for strains belonging to L. interrogans serovar Icterohaemorrhagiae, which then results in a higher resolution than VNTR (Hunter-Gaston index of 0.94 versus 0.68). Regarding L. interrogans serogroups Australis, Canicola, and Grippotyphosa, the MST and VNTR methods similarly identified the genotype. The MST method enabled the acquisition of simple and robust results that were based on the nucleotide sequences. The MST identified clinical isolates in correlation with the reference serovar profiles, thus permitting an epidemiological surveillance of circulating L. interrogans strains, especially for the Icterohaemorrhagiae serogroup, which includes the most prevalent strains of public health interest.
Project description:<h4>Background</h4>Leptospirosis is a re-emerging bacterial zoonosis caused by spirochetes of the genus Leptospira. Severe disease has been reported in dogs in Europe despite vaccination with bivalent Leptospira vaccines. Recently, a tetravalent canine Leptospira vaccine (Nobivac® L4) was licenced in Europe. The goal of this study was to investigate clinical signs, microscopic agglutination test (MAT) titres, haematology, blood biochemistry, cardiac (c) Troponin I levels and echocardiography before and after vaccination with this tetravalent vaccine. Forty-eight healthy dogs were prospectively enrolled and vaccinated twice, 3-4 weeks apart (T0 and T1). Before vaccination (T0) and 16-31 days after the second vaccination (T2), MAT (n = 48), haematology (n = 48), blood biochemistry (n = 36) and cTroponin I measurements (n = 29) were performed, and MAT was repeated 347-413 days after the second vaccination (T3, n = 44). Echocardiography was performed before the first and second vaccination (T0 and T1, n = 24).<h4>Results</h4>Mild and transient clinical signs within 5 days following the first and second vaccination occurred in 23% and 10% of the dogs, respectively. Before the first vaccination (T0), all dogs showed negative MAT titres for the tested serovars except for Canicola (50% with titres 100-400). At T2, positive MAT titres to the serovars Canicola (100%), Australis (89%), Grippotyphosa (86%), Bratislava (60%), Autumnalis (58%), Copenhageni (42%), Pomona (12%), Pyrogenes (8%) and Icterohaemorrhagiae (2%) were found. Median to high titres (? 400) were most common to the serovar Canicola (92%) and less common to the serovars Australis (41%), Grippotyphosa (21%), Bratislava (12%), Autumnalis (4%), Pyrogenes (4%) and Pomona (2%). At T3, positive MAT titres (titre range: 100-400) were found in 2-18% of the dogs to serovars of the vaccine serogroups and in 2-18% of the dogs to the non-vaccine serovars Pomona, Autumnalis, Pyrogenes and Ballum. Haematology, blood biochemistry, cTroponin I levels and echocardiography results did not change significantly following vaccination.<h4>Conclusions</h4>Clinical signs following vaccination with Nobivac® L4 were transient and mild in all cases. Seroconversion differed considerably among individual dogs and among the vaccine serogroups.
Project description:Rats are known to be the most important reservoirs and transmission sources of leptospirosis. However, the status of leptospirosis in the Philippines regarding reservoirs and transmission remains unknown. A survey was conducted in Metro Manila and Laguna that analyzed samples obtained from 106 rats. Using the microscopic agglutination test, we found that 92% of rat serum samples were positive for anti-Leptospira antibodies; the most common infecting serovars were Manilae, Hebdomadis, and Losbanos. On the basis of pulsed-field gel electrophoresis and gyrase B gene sequence analyses, four groups of rat kidney isolates were found: L. interrogans serovar Manilae, serovar Losbanos, and serogroup Grippotyphosa, and L. borgpetersenii serogroup Javanica. Most isolates were lethal after experimental infection of golden Syrian hamsters. Results showed that these four Leptospira serovars and serogroups are circulating among rats, and that these animals may be one of the possible transmission sources of leptospirosis in the Philippines.
Project description:Over 230 serovars of Leptospira interrogans have been identified; however few have been completely characterised. The aim of this study was to characterise the proteome of serovar Canicola and to compare this against the serovars of Copenhageni and Pomona. 2D-LC/MS analysis identified 1653 Leptospira proteins in serovar Canicola; 60 of these proteins were common to Copenhageni and Pomona, 16 of which are known to be immunogenic. This study provides the first reported proteome for serovar Canicola and suggests that proteomic comparison of different serovars could be used as a tool for identification of novel target molecules for vaccine development.
Project description:Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.
Project description:Leptospira interrogans serovar Canicola is one of the most important pathogenic serovars for the maintenance of urban leptospirosis. Even though it is considered highly adapted to dogs, serovar Canicola infection has already been described in other animals and even a few human cases. Here, we present the genomic characterisation of two Brazilian L. interrogans serovar Canicola strains isolated from slaughtered sows (L0-3 and L0-4) and their comparison with human strain Fiocruz LV133. It was observed that the porcine serovar Canicola strains present the genetic machinery to cause human infection and, therefore, represent a higher risk to public health. Both human and porcine serovar Canicola isolates also presented sequences with high identity to the Chinese serovar Canicola published plasmids pGui1 and pGui2. The plasmids identification in the Brazilian and Chinese serovar Canicola strains suggest that extra-chromosomal elements are one more feature of this serovar that was previously unnoticed.
Project description: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.
Project description:BACKGROUND:Leptospirosis has gained much attention in Sri Lanka since its large outbreak in 2008. However, most of the cases were clinically diagnosed and information on Leptospira genotypes and serotypes currently prevailing in the country is lacking. METHODOLOGY/PRINCIPAL FINDINGS:We retrospectively analyzed 24 Leptospira strains from human patients as well as isolated and characterized three Leptospira strains from black rats using the microscopic agglutination test with antisera for 19 serovars and multilocus sequence typing. The isolates were identified as Leptospira borgpetersenii sequence types (STs) 143 and 144; L. interrogans STs 30, 34, 43, 44, 74, 75, 80, 308, 313, 314, 316, and 317; and L. kirschneri ST318. Six of the 15 STs were identified for the first time in this study. Five serogroups such as Autumnalis, Grippotyphosa, Hebdomadis, Javanica, and Pyrogenes were detected among the isolates. Contrary to previous studies, various genotypes including novel STs were isolated during an outbreak in Southern Province. L. borgpetersenii serogroup Javanica ST143 was isolated both from a human and black rat. CONCLUSIONS/SIGNIFICANCE:This study revealed that genetically diverse Leptospira strains currently circulate in Sri Lanka: some genotypes have been circulating and others have emerged recently, which may explain the recent surge of leptospirosis patients with varying clinical manifestations and frequent outbreaks of leptospirosis. Black rats were identified as the source of infection for humans, but reservoir animals for other genotypes remain unknown.
Project description:Leptospirosis is a neglected zoonosis with worldwide distribution. The causative agents are spirochete bacteria of the Leptospira genus, displaying huge diversity of serovars, the identity of which is critical for effective diagnosis and vaccination purposes. Among many other mammalian species, Leptospira infects cattle, eliciting acute signs in calves, and chronic disease in adult animals often leading to abortions. In South America, and including in Uruguay, beef and dairy export are leading sources of national income. Despite the importance of bovine health, food safety, and bovine-related dissemination of leptospirosis to humans, extremely limited information is available as to the identity of Leptospira species and serovars infecting cattle in Uruguay and the South American subcontinent. Here we report a multicentric 3-year study resulting in the isolation and detailed characterization of 40 strains of Leptospira spp. obtained from infected cattle. Combined serologic and molecular typing identified these isolates as L. interrogans serogroup Pomona serovar Kennewicki (20 strains), L. interrogans serogroup Canicola serovar Canicola (1 strain), L. borgpetersenii serogroup Sejroe serovar Hardjo (10 strains) and L. noguchii (9 strains). The latter showed remarkable phenotypic and genetic variability, belonging to 6 distinct serogroups, including 3 that did not react with a large panel of reference serogrouping antisera. Approximately 20% of cattle sampled in the field were found to be shedding pathogenic Leptospira in their urine, uncovering a threat for public health that is being largely neglected. The two L. interrogans serovars that we isolated from cattle displayed identical genetic signatures to those of human isolates that had previously been obtained from leptospirosis patients. This report of local Leptospira strains shall improve diagnostic tools and the understanding of leptospirosis epidemiology in South America. These strains could also be used as new components within bacterin vaccines to protect against the pathogenic Leptospira strains that are actually circulating, a direct measure to reduce the risk of human leptospirosis.