Leptospira Interrogans Strain Research Articles

Whole Genome Sequencing Allows Better Understanding of the Evolutionary History of Leptospira interrogans Serovar Hardjo.

The genome of a laboratory-adapted strain of Leptospira interrogans serovar Hardjo was sequenced and analyzed. Comparison of the sequenced genome with that recently published for a field isolate of the same serovar revealed relatively high sequence conservation at the nucleotide level, despite the different biological background of both samples. Conversely, comparison of both serovar Hardjo genomes with those of L. borgpetersenii serovar Hardjo showed extensive differences between the corresponding chromosomes, except for the region occupied by their rfb loci. Additionally, comparison of the serovar Hardjo genomes with those of different L. interrogans serovars allowed us to detect several genomic features that may confer an adaptive advantage to L. interrogans serovar Hardjo, including a possible integrated plasmid and an additional copy of a cluster encoding a membrane transport system known to be involved in drug resistance. A phylogenomic strategy was used to better understand the evolutionary position of the Hardjo serovar among L. interrogans serovars and other Leptospira species. The proposed phylogeny supports the hypothesis that the presence of similar rfb loci in two different species may be the result of a lateral gene transfer event.

Leptospira interrogans serovars Bratislava and Muenchen animal infections: Implications for epidemiology and control

Strains of Leptospira interrogans belonging to two very closely related serovars – Bratislava and Muenchen – have been associated with disease in domestic animals, in particular pigs, but also in horses and dogs. Similar strains have also been recovered from various wildlife species. Their epidemiology is poorly understood. Two hundred and forty seven such isolates, from UK domestic animal and wildlife species, were examined by restriction endonuclease analysis in an attempt to elucidate their epidemiology. A representative sub-sample of 65 of these isolates was further examined by multiple-locus variable-number tandem repeat analysis and 22 by secY sequencing. Ten restriction pattern types were identified. The majority of isolates fell into one of three restriction endonuclease analysis pattern types designated B2a, B2b and M2a. B2a was ubiquitous and was isolated from 10 species and represented the majority of the horse and all dog isolates. B2b was very different, being isolated only from pigs, indicating that this type was maintained by pigs. The pattern M2a was reported for the majority of isolates from pigs but also was common in small rodents isolates. Five restriction pattern types were found only in wildlife suggesting that they are unlikely to pose a disease threat to domestic animals. Multiple-locus variable-number tandem repeat analysis identified six clusters. The REA types B2a and B2b were all found in one MLVA cluster while the majority of the M2a strains examined occurred in another cluster. The secY sequencing detected only one sequence type, clustered with other serovars of Leptospira interrogans.

Draft genome of the Leptospira interrogans strains, Acegua, RCA, Prea, and Capivara, obtained from wildlife maintenance hosts and infected domestic animals.

In the present paper, we announce new draft genomes of four Leptospira interrogans strains named Acegua, RCA, Prea, and Capivara. These strains were isolated in the state of Rio Grande do Sul, Brazil, from cattle, dog, Brazilian guinea pig, and capybara, respectively.

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.

Molecular studies on European equine isolates of Leptospira interrogans serovars Bratislava and Muenchen

Strains of Leptospira interrogans belonging to two very closely related serovars – Bratislava and Muenchen – are known to cause widespread infection of the horse population in many parts of the world. Conventional serological typing of isolates has been unable to differentiate between wildlife, pig, dog and possibly horse maintained isolates and therefore has been unable to provide further insight into their diversity and the relationship between them. Twenty-one such European isolates of serovar Bratislava and Muenchen were examined by restriction endonuclease analysis and multiple-locus variable-number tandem repeat analysis in an attempt to elucidate their epidemiology. The restriction pattern types were identified and fell into one of four REA designed pattern types, B1, B2a, M1, M2a. Nine strains from Northern Ireland and two from Germany belonged to B2a, which is a ubiquitous strain being originally isolated from a large number of wild and domestic animal species in the UK. Five strains were identified as B1 and they came from Portugal, The Netherlands, Germany and Northern Ireland; three strains isolated in Germany belong to M1; two strains belonged to M2a. Genotypes B1 and M1 have, with the exception of one hedgehog isolate, been recovered only from horses and it may indicate their adaptation to this species.

Prokaryotic expression of tlyA gene of Leptospira interrogans strain and the significance of the expressed protein in hemolysis and inflammation

Objective To construct a prokaryotic expression system for tlyA gene of Leptospira interrogans (L.interrogans) strain and to investigate the effects of the expressed rTlyA protein on the hemolysis of sheep erythrocytes and the secretion of pro-inflammatory cytokines by human THP-1 cells and murine J774A.1 macrophages. Methods The fragment of tlyA gene of L. inetrrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR. The PCR product was sequenced after T-A cloning. A prokaryotic expression system for the tlyA gene was constructed by using pET-42a as the expression vector and E. coli BL21DE3 strain as the host strain. The expression of rTlyA protein was detected by SDS-PAGE. Ni-NTA affinity chromatography was performed for purification. The lytic activity of the rTlyA protein on sheep erythrocytes was determined by plate hemolytic test and spectrophotometry. Real-time fluorescence quantitative PCR and Western blot assay were performed to respectively detect the expression of tlyA gene in THP-1 and J774A.1 cells at mRNA and protein levels after infection with L. interrogans strain Lai. ELISA was performed to evaluate the effects of the rTlyA protein on the secretion of several pro-inflammatory cytokines (IL-β, IL-6 and TNF-α) by THP-1 and J774A.1 cells. Results The nucleotide and amino acid sequences of the cloned tlyA gene were 99.2% and 99.8% identical to those corresponding sequences in GenBank, respectively. The constructed prokaryotic expression system for tlyA gene successfully expressed the rTlyA protein. The rTlyA protein at the concentration of 10 μg/ml showed stronger lytic activity on sheep erythrocytes. The transcription levels of tlyA gene (P<0.05) and the secretion of TlyA protein in THP-1 and J774A.1 cells were significantly increased after infecting with L. interrogans strain Lai for 1 to 8 hours. The rTlyA protein at concentrations of 0.1, 1 and 10 μg/ml significantly enhanced the secretion of IL-1β, IL-6 and TNF-α by THP-1 and J774A.1 cells (P<0.05). Conclusion The TlyA protein, encoded by the tlyA gene of L. interrogans strain, was confirmed to be a hemolysin with the ability to induce macrophages to secret IL-1β, IL-6 and TNF-α. This study suggested that the TlyA protein played an important role in inflammatory responses during leptospirosis. Key words: Leptospira interrogans; tlyA gene; Hemolysin; Expression/Secretion; Pro-inflammatory cytokines

Identification and immunogenicity analysis of predominant T-B combined antigenic epitopes on the outer membrane protein Loa22 of Leptospira interrogans strains

Objective To screen and identify the predominant T- and B-cell (T-B) combined antigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans (L.interrogans) stains and to further analyze their immunogenicity. Methods PCR analysis was used to detect loa22 gene in L. interrogans strains belonging to eight different serogroups or serovars prevalent in China. The PCR products were sequenced after T-A cloning. A prokaryotic expression system for loa22 gene of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was constructed. The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography. The rabbit anti-rLoa22 serum samples and IgG were prepared. The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinformatic softwares. Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B combined antigenic epitopes, respectively. MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γ induced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8% homologies in nucleotide sequences and 93.9%-99.5% homologies in amino acid sequences. The constructed prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein. Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis. The proliferation of CD4+ T cells and the expression of IL-2 (Th1) and IL-4 (Th2) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05). Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L. interrogans. The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic peptide (MAP) vaccines against Leptospira infection. Key words: Leptospira; Outer membrane protein; Loa22; T-B combined antigenic epitope; Immunogenicity

The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response.

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.

Isolation of Leptospira spp. from a man living in a rural area of the Municipality of Cruz Alta, RS, Brazil

The aim of this study was to describe the isolation of a pathogenic strain of Leptospira interrogans from the urine sample of a male human living in the rural area of the County of Cruz Alta, Rio Grande do Sul. An aliquot of each urine sample was sown in a Fletcher and Ellinghausen - McCullough - Johnson - Harris (EMJH) media. Samples in which there was growth of spirochetes were sent to the Leptospirosis Laboratory of the Institute of Pathobiology in the National Institute of Agricultural Technology, Buenos Aires, Argentina and were typified by the Multiple Locus of Variable Number Tandem Repeat technique (MLVA). Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 was isolated, and this is a very important finding that serves as a warning to characterize risk situation of leptospirosis epidemic by a pathogenic strain. Health professionals need to be more committed to the primary health care in Brazil and routinely apply actions of preventive medicine in rural communities in order to get success in the control of leptospirosis and other important zoonoses

Carboxyfluorescein diacetate succinimidyl ester labeling method to study the interaction between Leptospira and macrophages

Leptospirosis, which is caused by pathogenic species of the genus Leptospira, has emerged as one of the most widespread zoonotic diseases in the world. The exact mechanism of pathogenesis remains unknown, and the interaction between Leptospira and macrophages is not well understood. In this study, we report that carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) can efficiently label different Leptospira interrogans strains without affecting bacterial motility, viability, or virulence. Following co-incubation, CFDA-SE-labeled leptospires associated with macrophages were quantified by flow cytometry or confocal microscopy. In addition, we showed that trypan blue efficiently quenched the extracellular fluorescence from the adherent leptospires, which enabled intracellular and extracellular bacteria to be distinguished.

ATCC 43642 replaces ATCC 23581 as the type strain of Leptospira interrogans (Stimson 1907) Wenyon 1926. Opinion 91. Judicial Commission of the International Committee on Systematics of Prokaryotes.

The Judicial Commission affirms that, according to information presented to it, the type strain of Leptospira interrogans (Stimson 1907) Wenyon 1926 designated on the Approved Lists of Bacterial Names (ATCC 23581) has been shown not to represent an authentic culture of strain RGA (a member of the serovar Icterohaemorrhagiae) and ATCC 43642, derived from an authentic strain of strain RGA, a member of the serovar Icterohaemorrhagiae, is designated the type strain of Leptospira interrogans (Stimson 1907) Wenyon 1926.

Decay-accelerating factor 1 deficiency exacerbates leptospiral-induced murine chronic nephritis and renal fibrosis.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira, which can colonize the proximal renal tubules and persist for long periods in the kidneys of infected hosts. Here, we characterized the infection of C57BL/6J wild-type and Daf1−/− mice, which have an enhanced host response, with a virulent Leptospira interrogans strain at 14 days post-infection, its persistence in the kidney, and its link to kidney fibrosis at 90 days post-infection. We found that Leptospira interrogans can induce acute moderate nephritis in wild-type mice and is able to persist in some animals, inducing fibrosis in the absence of mortality. In contrast, Daf1−/− mice showed acute mortality, with a higher bacterial burden. At the chronic stage, Daf1−/− mice showed greater inflammation and fibrosis than at 14 days post-infection and higher levels at all times than the wild-type counterpart. Compared with uninfected mice, infected wild-type mice showed higher levels of IL-4, IL-10 and IL-13, with similar levels of α-smooth muscle actin, galectin-3, TGF-β1, IL-17, IFN-γ, and lower IL-12 levels at 90 days post-infection. In contrast, fibrosis in Daf1−/− mice was accompanied by high expression of α-smooth muscle actin, galectin-3, IL-10, IL-13, and IFN-γ, similar levels of TGF-β1, IL-12, and IL-17 and lower IL-4 levels. This study demonstrates the link between Leptospira-induced murine chronic nephritis with renal fibrosis and shows a protective role of Daf1.

Immunomodulatory treatment with thalidomide in experimental leptospirosis in Golden Syrian hamsters (Mesocricetus auratus)

The benefit of antibiotics in leptospirosis is limited when treatment is started four days after symptoms appear, and new adjuvant therapeutic options are urgently needed. Hamsters (Mesocricetus auratus) were infected by Leptospira interrogans strain L1-130, and groups were assigned based on no treatment (NONE), thalidomide only (TAL), ampicillin only (AMP) or both (AMP-TAL). Treatment was started two days after the onset of symptoms (experiment 1) and immediately after detection of the first death (experiment 2). Experiment 1: all hamsters from the groups AMP and AMP-TAL survived (n=8), while all hamsters from groups NONE (n=6) and TAL (n=8) died. The AMP and the AMP-TAL groups showed no renal or liver pathology and absent or very low leptospiral burden in target organs. Experiment 2: lethal outcome was observed in 6/6 hamsters in the NONE group, 8/8 in the TAL group, and 6/8 in both the AMP and AMP-TAL groups. Thalidomide showed no survival benefit when compared to hamsters treated with ampicillin alone. The TAL, AMP and AMP-TAL groups had very low tissue leptospiral counts. Thalidomide had minimal impact on survival in the late treatment of leptospirosis hamster model.

High-Resolution Typing of Leptospira interrogans Strains by Multispacer Sequence Typing

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.

Identification of Collagenase as a Critical Virulence Factor for Invasiveness and Transmission of Pathogenic Leptospira Species

Leptospirosis is a global zoonotic disease. Transmission of Leptospira from animals to humans occurs through contact with water contaminated with leptospire-containing urine of infected animals. However, the molecular basis for the invasiveness of Leptospira and transmission of leptospirosis remains unknown. Activity of Leptospira interrogans strain Lai colA gene product (ColA) to hydrolyze different collagenic substrates was determined by spectrophotometry. Expression and secretion of ColA during infection were detected by reverse-transcription quantitative polymerase chain reaction and Western blot assay. The colA gene-deleted (ΔcolA) and colA gene-complemented (CΔcolA) mutants were generated to determine the roles of ColA in transcytosis in vitro and virulence in hamsters. Recombinant or native ColA hydrolyzed all the tested substrates in which type III collagen was the favorite substrate with 2.16 mg/mL Km and 35.6 h(-)(1) Kcat values. Coincubation of the spirochete with HUVEC or HEK293 cells directly caused the significant elevation of ColA expression and secretion. Compared with wild-type strain, ΔcolA mutant displayed much-attenuated transcytosis through HEK293 and HUVEC monolayers, and less leptospires in blood, lung, liver, kidney and urine and 25-fold-decreased 50% lethal dose and milder histopathological injury in hamsters. The product of colA gene is a collagenase as a crucial virulence factor in the invasiveness and transmission of L. interrogans.

The serological response of working farm dogs to a vaccine containing Leptospira interrogans serovars Copenhageni and Pomona, and L. borgpetersenii serovar Hardjo

AIMS: To evaluate the serological response in dogs to a commercial vaccine for use in cattle containing cultured strains of Leptospira interrogans serovars Copenhageni and Pomona, and L. borgpetersenii serovar Hardjo.METHODS: Blood samples were obtained from 67 working farm dogs on 12 farms, and the microscopic agglutination test (MAT) was used to measure titres to the Leptospira spp. serovars Pomona, Hardjo, and Copenhageni. Samples with a titre of <1:25 were defined as seronegative. Dogs that were seronegative to both Pomona and Hardjo (n=33) were randomised to either the vaccination (n=20) or control (n=13) groups. Seven of these dogs were seropositive to Copenhageni. Vaccinated dogs were given the three-component vaccine subcutaneously on two occasions, 4 weeks apart. MAT titres were measured again in both groups 2 weeks after the second vaccination.RESULTS: Of the vaccinated dogs, 20/20 (100%) developed titres to serovar Pomona, and 16/20 (80%) had titres >100; for serovar Hardjo, 19/20 (95%) dogs had titres, with 18/20 (90%) being >100; and for serovar Copenhageni, 15/17 (88%) dogs that were initially seronegative had titres, with 6/17 (35%) being >100. The median titres for Pomona and Hardjo (200 (95% CI=179–359) and 200 (95% CI=176–379), respectively) were lower than for Copenhageni (50 (95% CI=26–124)) in dogs originally seronegative (p<0.001). There was no association between titres to the different serovars. Of the 13 unvaccinated dogs, two developed titres to serovar Pomona, and three to Hardjo. All titres were <100.CONCLUSIONS: The tested vaccine was effective in raising antibodies to the three serovars, although the titres do not guarantee protection.CLINICAL RELEVANCE: There has been a recent increase in cases of leptospirosis attributed to the serovar Pomona in dogs in New Zealand, but the vaccines licensed for use in dogs in New Zealand for the prevention of leptospirosis only protect against Copenhageni/Icterohaemorrhagiae. The vaccine tested in this study produced titres to Pomona and Hardjo that suggest the vaccine could be useful for reducing disease caused by these serovars in working dogs in New Zealand.

Detection of human leptospirosis as a cause of acute fever by capture ELISA using a Leptospira interrogansserovar Copenhageni (M20) derived antigen

BackgroundLeptospirosis is a potentially lethal zoonosis mainly affecting low-resource tropical countries, including Peru and its neighbouring countries. Timely diagnosis of leptospirosis is critical but may be challenging in the regions where it is most prevalent. The serodiagnostic gold standard microagglutination test (MAT) may be technically prohibitive. Our objective in this study was to assess the sensitivity, specificity, and predictive value of an IgM antibody capture enzyme-linked immunoassay (MAC-ELISA) derived from the M20 strain of Leptospira interrogans serovar Copenhageni (M20) by comparison to MAT, which was used as the gold standard method of diagnosis.MethodsAcute and convalescent sera from participants participating in a passive febrile surveillance study in multiple regions of Peru were tested by both IgM MAC-ELISA and MAT. The sensitivity, specificity, positive and negative predictive value (PPV, NPV) of the MAC-ELISA assay for acute, convalescent and paired sera by comparison to MAT were calculated.ResultsThe sensitivity, specificity, PPV and NPV of the MAC-ELISA assay for acute sera were 92.3%, 56.0%, 35.3% and 96.6% respectively. For convalescent sera, the sensitivity, specificity, PPV and NPV of the MAC-ELISA assay were 93.3%, 51.5%, 63.6% and 89.5% respectively. For paired sera, the sensitivity, specificity, PPV and NPV of the MAC-ELISA assay were 93.6%, 37.5%, 59.2%, 85.7% respectively.ConclusionsThe M20 MAC-ELISA assay performed with a high sensitivity and low specificity in the acute phase of illness. Sensitivity was similar as compared with MAT in the convalescent phase and specificity remained low. Paired sera were the most sensitive but least specific by comparison to MAT serodiagnosis. NPV for acute, convalescent and paired sera was high. The limited specificity and high sensitivity of the MAC-ELISA IgM suggests that it would be most valuable to exclude leptospirosis in low-resource regions that lack immediate access to definitive reference laboratory techniques such as MAT.

Prozone effects in microscopic agglutination tests for leptospirosis in the sera of mice infected with the pathogenic Leptospira interrogans serovar Canicola

Mice experimentally infected with a pathogenic strain of Leptospira interrogans serovar Canicola produced false negative results (prozone effect) in a microscopic agglutination test (MAT). This prozone effect occurred in several serum samples collected at different post-infection times, but it was more prominent in samples collected from seven-42 days post-infection and for 1:50 and 1:100 sample dilutions. This phenomenon was correlated with increased antibody titres in the early post-infection phase. While prozone effects are often observed in serological agglutination assays for the diagnosis of animal brucellosis and human syphilis, they are not widely reported in leptospirosis MATs.

Detection of leptospires from infected urine and tissue samples in vitro by modified Fontana silver stain

INTRODUCTION: The microbiological diagnosis of leptospirosis comprises bacteriological and serological methods. The former ones allow the direct detection of leptospires and are considered presumptive with the exception of culture. Therefore, they constitute invaluable tools for rapid diagnosis, mainly in samples from deceased subjects. OBJECTIVE: To evaluate a modified Fontana silver staining method in experimentally infected samples. MATERIAL AND METHODS: Human and animal (hamster) urine samples were experimentally infected with different strains of Leptospira interrogans sensu lato. Liquid culture medium, leptospira cultures, experimentally infected and non-infected human urine samples, clarified and non-clarified imprints, and clarified and non-clarified suspension smears from tissues of experimentally infected and non-infected hamsters were applied for the ass essment of silver staining. The analytical sensitivity of the assay was compared with dark field microscopy and culture. Other bacterial and fungi species were also used. RESULTS: The modified Fontana silver staining allowed the accurate observation of the well-defined leptospire helical structure. On leptospire cultures from infected human samples, we could observe until (1-10) × 10³ leptospires/ml, higher sensitivity in comparison with direct dark field microscopy and lower in comparison with culture. The best results in tissues were obtained on clarified imprints and non-clarified suspension smears. Morphological and stainable structures compatible with leptospires were not observed in the samples without them. CONCLUSION: This procedure allowed differentiating the characteristic morphology of leptospires. As its application suggests, it consists of a simple and easily conducted procedure with stable reagents.

Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool

Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.