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Sample records for leptospira interrogans utilizadas

  1. Global Proteome Analysis of Leptospira interrogans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometr...

  2. The passive diffusion of Leptospira interrogans

    NASA Astrophysics Data System (ADS)

    Koens, Lyndon; Lauga, Eric

    2014-12-01

    Motivated by recent experimental measurements, the passive diffusion of the bacterium Leptospira interrogans is investigated theoretically. By approximating the cell shape as a straight helix and using the slender-body-theory approximation of Stokesian hydrodynamics, the resistance matrix of Leptospira is first determined numerically. The passive diffusion of the helical cell is then obtained computationally using a Langevin formulation which is sampled in time in a manner consistent with the experimental procedure. Our results are in excellent quantitative agreement with the experimental results with no adjustable parameters.

  3. Mouse model for sublethal Leptospira interrogans infection.

    PubMed

    Richer, Luciana; Potula, Hari-Hara; Melo, Rita; Vieira, Ana; Gomes-Solecki, Maria

    2015-12-01

    Although Leptospira can infect a wide range of mammalian species, most studies have been conducted in golden Syrian hamsters, a species particularly sensitive to acute disease. Chronic disease has been well characterized in the rat, one of the natural reservoir hosts. Studies in another asymptomatic reservoir host, the mouse, have occasionally been done and have limited infection to mice younger than 6 weeks of age. We analyzed the outcome of sublethal infection of C3H/HeJ mice older than age 10 weeks with Leptospira interrogans serovar Copenhageni. Infection led to bloodstream dissemination of Leptospira, which was followed by urinary shedding, body weight loss, hypothermia, and colonization of the kidney by live spirochetes 2 weeks after infection. In addition, Leptospira dissemination triggered inflammation in the kidney but not in the liver or lung, as determined by increased levels of mRNA transcripts for the keratinocyte-derived chemokine, RANTES, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-1β, inducible nitric oxide synthase, interleukin-6, and gamma interferon in kidney tissue. The acquired humoral response to Leptospira infection led to the production of IgG mainly of the IgG1 subtype. Flow cytometric analysis of splenocytes from infected mice revealed that cellular expansion was primarily due to an increase in the levels of CD4(+) and double-negative T cells (not CD8(+) cells) and that CD4(+) T cells acquired a CD44(high) CD62L(low) effector phenotype not accompanied by increases in memory T cells. A mouse model for sublethal Leptospira infection allows understanding of the bacterial and host factors that lead to immune evasion, which can result in acute or chronic disease or resistance to infection (protection). PMID:26416909

  4. Mouse Model for Sublethal Leptospira interrogans Infection

    PubMed Central

    Richer, Luciana; Potula, Hari-Hara; Melo, Rita; Vieira, Ana

    2015-01-01

    Although Leptospira can infect a wide range of mammalian species, most studies have been conducted in golden Syrian hamsters, a species particularly sensitive to acute disease. Chronic disease has been well characterized in the rat, one of the natural reservoir hosts. Studies in another asymptomatic reservoir host, the mouse, have occasionally been done and have limited infection to mice younger than 6 weeks of age. We analyzed the outcome of sublethal infection of C3H/HeJ mice older than age 10 weeks with Leptospira interrogans serovar Copenhageni. Infection led to bloodstream dissemination of Leptospira, which was followed by urinary shedding, body weight loss, hypothermia, and colonization of the kidney by live spirochetes 2 weeks after infection. In addition, Leptospira dissemination triggered inflammation in the kidney but not in the liver or lung, as determined by increased levels of mRNA transcripts for the keratinocyte-derived chemokine, RANTES, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-1β, inducible nitric oxide synthase, interleukin-6, and gamma interferon in kidney tissue. The acquired humoral response to Leptospira infection led to the production of IgG mainly of the IgG1 subtype. Flow cytometric analysis of splenocytes from infected mice revealed that cellular expansion was primarily due to an increase in the levels of CD4+ and double-negative T cells (not CD8+ cells) and that CD4+ T cells acquired a CD44high CD62Llow effector phenotype not accompanied by increases in memory T cells. A mouse model for sublethal Leptospira infection allows understanding of the bacterial and host factors that lead to immune evasion, which can result in acute or chronic disease or resistance to infection (protection). PMID:26416909

  5. Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

    PubMed Central

    Eshghi, Azad; Pappalardo, Elisa; Hester, Svenja; Thomas, Benjamin; Pretre, Gabriela

    2015-01-01

    Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins. PMID:25987703

  6. Pathogenic Leptospira interrogans exoproteins are primarily involved in heterotrophic processes.

    PubMed

    Eshghi, Azad; Pappalardo, Elisa; Hester, Svenja; Thomas, Benjamin; Pretre, Gabriela; Picardeau, Mathieu

    2015-08-01

    Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins. PMID:25987703

  7. NEW WILDLIFE HOSTS OF Leptospira interrogans IN CAMPECHE, MEXICO

    PubMed Central

    ESPINOSA-MARTÍNEZ, Deborah V.; SÁNCHEZ-MONTES, Daniel Sokani; LEÓN-PANIAGUA, Livia; RÍOS-MUÑOZ, César A.; BERZUNZA-CRUZ, Miriam; BECKER, Ingeborg

    2015-01-01

    Leptospira interrogans has been identified to cause leptospirosis, a widespread zoonotic disease that has been identified in domestic and wild animals. This work analyzed kidneys from two species of wild rodents from the state of Campeche, Mexico. Analyses were made by PCR using specific primers for detection of Leptospira interrogans DNA. The rodent species that tested positive were Heteromys gaumeri and Ototylomys phyllotis, both of which are new hosts for the bacteria in Southeastern Mexico. These records provide new insights into the disease’s transmission that should be studied carefully in order to identify other potential host species, including humans, which are at risk of becoming infected if they are in contact with infected wildlife. PMID:25923901

  8. New wildlife hosts of Leptospira interrogans in Campeche, Mexico.

    PubMed

    Espinosa-Martínez, Deborah V; Sánchez-Montes, Daniel Sokani; León-Paniagua, Livia; Ríos-Muñoz, César A; Berzunza-Cruz, Miriam; Becker, Ingeborg

    2015-01-01

    Leptospira interrogans has been identified to cause leptospirosis, a widespread zoonotic disease that has been identified in domestic and wild animals. This work analyzed kidneys from two species of wild rodents from the state of Campeche, Mexico. Analyses were made by PCR using specific primers for detection of Leptospira interrogans DNA. The rodent species that tested positive were Heteromys gaumeri and Ototylomys phyllotis, both of which are new hosts for the bacteria in Southeastern Mexico. These records provide new insights into the disease's transmission that should be studied carefully in order to identify other potential host species, including humans, which are at risk of becoming infected if they are in contact with infected wildlife. PMID:25923901

  9. Identification of Cell-Binding Adhesins of Leptospira interrogans

    PubMed Central

    Evangelista, Karen V.; Hahn, Beth; Wunder, Elsio A.; Ko, Albert I.; Haake, David A.; Coburn, Jenifer

    2014-01-01

    Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic Leptospira species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate L. interrogans sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its L. biflexa homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for L. interrogans. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in L. interrogans infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and

  10. Pathogenomic Inference of Virulence-Associated Genes in Leptospira interrogans

    PubMed Central

    Lehmann, Jason S.; Fouts, Derrick E.; Haft, Daniel H.; Cannella, Anthony P.; Ricaldi, Jessica N.; Brinkac, Lauren; Harkins, Derek; Durkin, Scott; Sanka, Ravi; Sutton, Granger; Moreno, Angelo; Vinetz, Joseph M.; Matthias, Michael A.

    2013-01-01

    Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens. PMID:24098822

  11. Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity

    PubMed Central

    Cerqueira, Gustavo M.; Souza, Natalie M.; Araújo, Eduardo R.; Barros, Aline T.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Nascimento, Ana L. T. O.

    2011-01-01

    Background Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. Methodology and Principal Findings A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP) were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA) and Sphingomielynase 2 (sph2) promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. Conclusions The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa. PMID:21445252

  12. Complete genome sequence of Leptospira interrogans serovar Bratislava, strain PigK151

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genus Leptospira contains pathogens serologically classified into over 250 serovars, intermediate pathogens and saprophytes with genetic classification into 21 different species. Worldwide, leptospirosis is one of the most widespread zoonoses. L. interrogans serovar Bratislava has been isolated ...

  13. [Modified EMJH medium for cultivation of Leptospira interrogans serogroup ballum].

    PubMed

    González, A; Borrero, R; Ruiz, J; Batista, N; Fernández, Y; Valdés, Y; González, M

    2006-01-01

    Strains within the Ballum serogroup of spirochete Leptospira show fastidious growth with more exigent nutritional requirements than those of other Leptospira pathogenic strains. The influence of 37 nutritional compounds on the growth of Leptospira interrogans serogroup Ballum was investigated employing the synthetic EMJH medium as the base for the study. Microbial growth was estimated spectrophotometrically and direct counts were performed with a Petroff-Hausser counting chamber. Virulence stability was evaluated by calculating the mean lethal dose in hamsters. Antigenicity stability was evaluated by Western blotting using a specific antiserum. Cell yields commonly obtained in EMJH were triplicated without virulence or antigenicity depletions after culturing in a modified EMJH medium with an increased concentration of Tween 80, and the incorporation of sodium acetate and beef extract. Neither the increased concentration of at least 6 components of EMJH nor the incorporation of a variety of new nutrients stimulated cell yields or the growth rate of the microorganism. The results allow us to make use of an enriched culture medium that promotes high cell yields of this fastidious serogroup most prevalent in humans in Cuba. PMID:17037250

  14. Visual proteomics of the human pathogen Leptospira interrogans

    PubMed Central

    Beck, Martin; Malmström, Johan A.; Lange, Vinzenz; Schmidt, Alexander; Deutsch, Eric W.; Aebersold, Ruedi

    2010-01-01

    Systems biology conceptualizes biological systems as dynamic networks of interacting elements, whereby functionally important properties are thought to emerge from the structure of such networks. Due to the ubiquitous role of complexes of interacting proteins in biological systems, their subunit composition and temporal and spatial arrangement within the cell are of particular interest. ‘Visual proteomics’ attempts to localize individual macromolecular complexes inside of intact cells by template matching reference structures into cryo electron tomograms. Here we have combined quantitative mass spectrometry and cryo electron tomography to detect, count and localize specific protein complexes within the cytoplasm of the human pathogen Leptospira interrogans. We describe a novel scoring function for visual proteomics and assess its performance and accuracy under realistic conditions. We discuss current and general limitations of the approach, as well as expected improvements in the future. PMID:19838170

  15. Crystal structure of homoserine O-acetyltransferase from Leptospira interrogans

    SciTech Connect

    Wang Mingzhu; Liu Lin; Wang Yanli; Wei Zhiyi; Zhang Ping; Li Yikun; Jiang Xiaohua; Xu Hang Gong Weimin

    2007-11-30

    Homoserine O-acetyltransferase (HTA, EC 2.3.1.31) initiates methionine biosynthesis pathway by catalyzing the transfer of acetyl group from acetyl-CoA to homoserine. This study reports the crystal structure of HTA from Leptospira interrogans determined at 2.2 A resolution using selenomethionyl single-wavelength anomalous diffraction method. HTA is modular and consists of two structurally distinct domains-a core {alpha}/{beta} domain containing the catalytic site and a helical bundle called the lid domain. Overall, the structure fold belongs to {alpha}/{beta} hydrolase superfamily with the characteristic 'catalytic triad' residues in the active site. Detailed structure analysis showed that the catalytic histidine and serine are both present in two conformations, which may be involved in the catalytic mechanism for acetyl transfer.

  16. Temperature-Regulated Protein Synthesis by Leptospira interrogans

    PubMed Central

    Nally, Jarlath E.; Timoney, John F.; Stevenson, Brian

    2001-01-01

    Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with L. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection. PMID:11119530

  17. Draft Genome Sequence of Leptospira interrogans Serovar Bataviae Strain LepIMR 22 Isolated from a Rodent in Johor, Malaysia.

    PubMed

    Amran, Fairuz; Mohd Khalid, Mohd Khairul Nizam; Mohamad, Saharuddin; Mat Ripen, Adiratna; Ahmad, Norazah; Goris, Marga G A; Muhammad, Ayu Haslin; Noor Halim, Nurul Atiqah

    2016-01-01

    Leptospira interrogans serovar Bataviae was recently identified as one of the persistent Leptospira serovars in Malaysia. Here, we report the draft genome sequence of the L. interrogans serovar Bataviae strain LepIMR 22 isolated from kidney of a rodent in Johor, Malaysia. PMID:27609924

  18. Molecular characterization of the pL40 protein in Leptospira interrogans.

    PubMed

    Zhao, Wei; Chen, Chun-Yan; Zhang, Xiang-Yan; Lai, Wei-Qiang; Hu, Bao-Yu; Zhao, Guo-Ping; Qin, Jin-Hong; Guo, Xiao-Kui

    2009-06-01

    Leptospirosis is a widespread zoonotic disease caused by pathogenic leptospires. The identification of outer membrane proteins (OMPs) conserved among pathogenic leptospires, which are exposed on the leptospiral surface and expressed during mammalian infection, has become a major focus of leptospirosis research. pL40, a 40 kDa protein coded by the LA3744 gene in Leptospira interrogans, was found to be unique to Leptospira. Triton X-114 fractionation and flow cytometry analyses indicate that pL40 is a component of the leptospiral outer membrane. The conservation of pL40 among Leptospira strains prevalent in China was confirmed by both Western blotting and PCR screening. Furthermore, the pL40 antigen could be recognized by sera from guinea pigs and mice infected with low-passage L. interrogans. These findings indicate that pL40 may serve as a useful serodiagnostic antigen and vaccine candidate for L. interrogans. PMID:19767845

  19. Isolation and Molecular Characterization of Leptospira interrogans and Leptospira borgpetersenii Isolates from the Urban Rat Populations of Kuala Lumpur, Malaysia

    PubMed Central

    Benacer, Douadi; Zain, Siti Nursheena Mohd; Amran, Fairuz; Galloway, Renee L.; Thong, Kwai Lin

    2013-01-01

    Rats are considered the principal maintenance hosts of Leptospira. The objectives of this study were isolation and identification of Leptospira serovars circulating among urban rat populations in Kuala Lumpur. Three hundred urban rats (73% Rattus rattus and 27% R. norvegicus) from three different sites were trapped. Twenty cultures were positive for Leptospira using dark-field microscopy. R. rattus was the dominant carrier (70%). Polymerase chain reaction (PCR) confirmed that all isolates were pathogenic Leptospira species. Two Leptospira serogroups, Javanica and Bataviae, were identified using microscopic agglutination test (MAT). Pulsed-field gel electrophoresis (PFGE) identified two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (85%) and L. interrogans serovar Bataviae (15%). We conclude that these two serovars are the major serovars circulating among the urban rat populations in Kuala Lumpur. Despite the low infection rate reported, the high pathogenicity of these serovars raises concern of public health risks caused by rodent transmission of leptospirosis. PMID:23358635

  20. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    SciTech Connect

    Nascimento, Alessandro S.; Ferrarezi, Thiago; Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A.; Polikarpov, Igor

    2006-07-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

  1. Antibodies against Leptospira interrogans in California sea lion pups from Gulf of California.

    PubMed

    Godínez, C R; Zelaya de Romillo, B; Aurioles-Gamboa, D; Verdugo-Rodríguez, A; Rodríguez-Reyes, E A; De la Peña-Moctezuma, A

    1999-01-01

    One hundred and twenty-five serum samples from California sea lion (Zalophus californianus californianus) pups, and one from an adult female from eight reproductive rookeries located in seven islands in the Gulf of California (Mexico), were collected during the 1994-96 reproductive seasons. These were tested for antibodies to 19 serovars of Leptospira interrogans using a Microscopic Agglutination Test (MAT). Forty-one samples (32%) had antibody levels from 1:20 to 1:320 to one or more serovars. The most frequently detected serotypes were Leptospira interrogans hardjo (n = 13), cynopteri (8), ballum (6), and szwajizak (5). Serovars with the highest prevalence were Leptospira interrogans hardjo and serjoe (1:320), ballum (1:160), and cynopteri, girppotyphosa, and tarassovi (1:80). Based on these results, exposure of sea lions to L. interrogans serovar hardjo seems to be relatively common among colonies located in the islands of the Gulf of California in contrast with those located on the Pacific coast, where the most frequently detected serovar is L. interrogans serovar pomona. PMID:10073358

  2. Molecular characterization of virulent Leptospira interrogans serogroup icterohaemorrhagiae isolated from Cavia aperea.

    PubMed

    Monte, Leonardo G; Jorge, Sérgio; Xavier, Marina A; Leal, Fernanda M A; Amaral, Marta G; Seixas, Fabiana K; Dellagostin, Odir A; Hartleben, Cláudia P

    2013-05-01

    Leptospirosis is a worldwide zoonotic infection caused by pathogenic Leptospira. Synanthropic rodents are recognized carriers of leptospires; however, the role of wild rodents in the epidemiology of the disease is still incipient. In this work, we describe Leptospira strain isolated from Cavia aperea (Brazilian guinea pig). The isolated strain was characterized by partial rpoB gene sequencing, variable-number tandem-repeats and histopathological analysis. The strain was identified as Leptospira interrogans, serogroup Icterohaemorrhagiae and caused clinical signs of leptospirosis in the hamster model, attesting to its virulence. In conclusion, these findings could be useful for elucidating the epidemiological role of C. aperea in leptospirosis. PMID:23435256

  3. Isolation of Leptospira interrogans serovar grippotyphosa from the skin of a dog.

    PubMed

    Anderson, J F; Miller, D A; Post, J E; Johnson, R C; Magnarelli, L A; Andreadis, T G

    1993-12-01

    Leptospira interrogans serovar grippotyphosa was isolated from the skin of a 14-year-old male dog with deteriorating health. Necropsy revealed numerous lesions characteristic of aged dogs, but no evidence of acute hepatitis or nephritis, which are common features of pathogenic Leptospira infections. Antibody to Leptospira was not detected in the dog's serum by microagglutination. Leptospires grew slowly in Barbour-Stoenner-Kelly medium, a medium commonly used to isolate Borrelia, but then grew abundantly in Tween 80-bovine albumin leptospire medium. The isolate was pathogenic to a hamster and was identified by microagglutination and restriction endonuclease analysis. PMID:8288477

  4. Complete Genome Sequence of Leptospira interrogans Serovar Bratislava, Strain PigK151.

    PubMed

    Alt, David P; Wilson-Welder, Jennifer H; Bayles, Darrell O; Cameron, Caroline; Adler, Ben; Bulach, Dieter M; Seemann, Torsten; Lehane, Michael J; Haines, Lee R; Darby, Alistair C; Hall, Neil; Radford, Alan D; Zuerner, Richard L

    2015-01-01

    Leptospira interrogans serovar Bratislava infection occurs in multiple domestic and wildlife species and is associated with poor reproductive performance in swine and horses. We present the complete genome assembly of strain PigK151 comprising two chromosomes, CI (4.457 Mbp) and CII (358 kbp). PMID:26112787

  5. DNA probe for detection of the Leptospira interrogans serovar hardjo genotype hardjo-bovis.

    PubMed Central

    LeFebvre, R B

    1987-01-01

    A DNA probe is described for the diagnostic and taxonomic identification of the North American cattle pathogen Leptospira interrogans genotype hardjo-bovis. The probe is specific for this genotype and does not hybridize to genomic DNA of any other leptospire pathogen commonly found in North America. Images PMID:2826538

  6. First Genome Sequence of Leptospira interrogans Serovar Pomona, Isolated from a Bovine Abortion

    PubMed Central

    Varni, Vanina; Koval, Ariel; Nagel, Ariel; Ruybal, Paula

    2016-01-01

    Leptospirosis is a widespread zoonosis and a re-emergent disease of global distribution with major relevance in veterinary production. Here, we report the whole-genome sequence of Leptospira interrogans serovar Pomona strain AKRFB, isolated from a bovine abortion during a leptospirosis outbreak in Argentina. PMID:27198013

  7. Characterization of an antigenic oligosaccharide from Leptospira interrogans serovar pomona and its role in immunity.

    PubMed Central

    Midwinter, A; Vinh, T; Faine, S; Adler, B

    1994-01-01

    An antigenic oligosaccharide fraction derived from the lipopolysaccharide of Leptospira interrogans serovar pomona was isolated by endo-glycosidase H digestion and column chromatography. The oligosaccharide contained rhamnose, ribose, glucose, and glucosamine and inhibited the binding of opsonic, protective monoclonal antibodies directed against the lipopolysaccharide. When conjugated to diphtheria toxoid, the oligosaccharide elicited the production of agglutinating, opsonic antibodies. Images PMID:7960129

  8. First Genome Sequence of Leptospira interrogans Serovar Pomona, Isolated from a Bovine Abortion.

    PubMed

    Varni, Vanina; Koval, Ariel; Nagel, Ariel; Ruybal, Paula; Caimi, Karina; Amadio, Ariel F

    2016-01-01

    Leptospirosis is a widespread zoonosis and a re-emergent disease of global distribution with major relevance in veterinary production. Here, we report the whole-genome sequence of Leptospira interrogans serovar Pomona strain AKRFB, isolated from a bovine abortion during a leptospirosis outbreak in Argentina. PMID:27198013

  9. Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospira interrogans is the causative agent of leptospirosis, a zoonosis of global significance. Iron is essential for growth of most bacterial species. Since availability of iron is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In ...

  10. Complete Genome Sequence of Leptospira interrogans Serovar Bratislava, Strain PigK151

    PubMed Central

    Alt, David P.; Bayles, Darrell O.; Cameron, Caroline; Adler, Ben; Bulach, Dieter M.; Seemann, Torsten; Lehane, Michael J.; Haines, Lee R.; Darby, Alistair C.; Hall, Neil; Radford, Alan D.; Zuerner, Richard L.

    2015-01-01

    Leptospira interrogans serovar Bratislava infection occurs in multiple domestic and wildlife species and is associated with poor reproductive performance in swine and horses. We present the complete genome assembly of strain PigK151 comprising two chromosomes, CI (4.457 Mbp) and CII (358 kbp). PMID:26112787

  11. Phenotypic and Molecular Characterization of Leptospira interrogans Isolated from Canis familiaris in Southern Brazil.

    PubMed

    Jorge, Sérgio; Monte, Leonardo G; De Oliveira, Natasha R; Collares, Thais F; Roloff, Bárbara C; Gomes, Charles K; Hartwig, Daiane D; Dellagostin, Odir A; Hartleben, Cláudia P

    2015-10-01

    Leptospirosis is a zoonotic disease caused by pathogenic spirochetes from the genus Leptospira, which includes 20 species and more than 300 serovars. Canines are important hosts of pathogenic leptospires and can transmit the pathogen to humans via infected urine. Here, we report the phenotypic and molecular characterization of Leptospira interrogans isolated from Canis familiaris in Southern Brazil. The isolated strain was characterized by variable-number tandem-repeats analysis as L. interrogans, serogroup Icterohaemorrhagiae. In addition, the isolate was recognized by antibodies from human and canine serum samples previously tested by microscopic agglutination test. Ultimately, the expression of membrane-associated antigens (LipL32 and leptospiral immunoglobulin-like proteins) from pathogenic leptospires using monoclonal antibodies was detected by indirect immunofluorescence assay. In conclusion, identification of new strains of Leptospira can help in the diagnosis and control of leptospirosis. PMID:26100241

  12. EFFECTS OF TEMPERATURE ON GENE EXPRESSION PATTERNS IN LEPTOSPIRA INTERROGANS SEROVAR LAI AS ASSESSED BY WHOLE-GENOME MICROARRAYS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The availability of genome sequences for two serovars of Leptospira interrogans, Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor, which is known to affect leptospiral protein expression....

  13. Leptospira interrogans reduces fibrin clot formation by modulating human thrombin activity via exosite I.

    PubMed

    Fernandes, Luis G; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2015-06-01

    Pathogenic bacteria of the genus Leptospira are the etiological agents of leptospirosis, a disease that affects humans and animals worldwide. Although there are an increasing number of studies on the biology of Leptospira, the mechanisms of pathogenesis are not yet understood. We report in this work that Leptospira interrogans FIOCRUZ L1-130 virulent, M20 culture attenuated and the saprophyte L. biflexa Patoc 1 strains do not bind prothrombin. Leptospiral binding to thrombin was detected with the virulent, followed by culture-attenuated M20, and practically none was observed with the saprophyte strain. The interaction of Leptospira with thrombin mostly occurs via exosite I, with a minor participation of catalytic site, as determined by employing the thrombin inhibitors hirugen, hirudin and argatroban. Leptospira interrogans binding to thrombin inhibits its catalytic activity reducing fibrin clot formation in thrombin-catalyzed reaction of fibrinogen. This inhibition was more efficient with the virulent FIOCRUZ L1-130 than with the M20 culture attenuated, while none was seen with the saprophyte strain, suggesting that this binding might be important for bacterial virulence. This is the first study reporting the binding of pathogenic Leptospira to thrombin promoting a decrease in fibrin clotting that could lead to hemorrhage, helping bacteria dissemination. PMID:25834144

  14. Evaluation of pathogenic serovars of Leptospira interrogans in dairy cattle herds of Shahrekord by PCR

    PubMed Central

    Jafari Dehkordi, A; Shahbazkia, HR; Ronagh, N

    2011-01-01

    Background and objectives Leptospirosis is an important zoonotic disease caused by Leptospira interrogans. Leptospirosis leads to economical losses in dairy farm industry. The objective of this study was to evaluate the pathogenic serovars of Leptospira interrogans in dairy cattle herds of Shahrekord by PCR. Materials and Methods Two hundred samples (100 urine and 100 blood) were collected from 100 cows randomly and delivered to the laboratory. Samples were stored at -20 °C. DNA was extracted and purified from the plasma and urine samples and concentrated on diatoms in the presence of guanidine thiocyanate (GuSCN). PCR products were detected and identified as Leptospira by ilumination of the expected size of DNA bands after staining of the agarose gel with ethidium bromide gels. PCR products were purified and sequenced. Results The results showed that 28% of urine samples and 23% of plasma samples were contaminated. The major serotypes were Icterohaemorrhagiae (50%) and Pomona (37.5%). The urine samples of 17 cows were positive for Leptospira without positive plasma samples. This indicated that these cows are reservoirs in dairy herds of Shahrekord and dangerous for human health. The plasma samples of twelve cows were positive for Leptospira without positive urine samples. Conclusions Leptospira serotypes can be maintained in relatively dry regions and must be considered when dealing with leptospirosis in dairy farms of Shahrekord and human health. PMID:22347596

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

    PubMed Central

    Zilber, Anne-Laure; Picardeau, Mathieu; Ayral, Florence; Artois, Marc; Demont, Pierre; Kodjo, Angeli

    2014-01-01

    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. PMID:24478489

  16. Heterogenic colonization patterns by Leptospira interrogans in Rattus norvegicus from urban slums

    PubMed Central

    Santos, Ana Amélia Nunes; Figueira, Cláudio Pereira; dos Reis, Mitermayer Galvão; Costa, Federico; Ristow, Paula

    2015-01-01

    Abstract We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis. PMID:26691476

  17. Heterogenic colonization patterns by Leptospira interrogans in Rattus norvegicus from urban slums.

    PubMed

    Santos, Ana Amélia Nunes; Figueira, Cláudio Pereira; dos Reis, Mitermayer Galvão; Costa, Federico; Ristow, Paula

    2015-01-01

    We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis. PMID:26691476

  18. Leptospira interrogans induces uterine inflammatory responses and abnormal expression of extracellular matrix proteins in dogs.

    PubMed

    Wang, Wei; Gao, Xuejiao; Guo, Mengyao; Zhang, Wenlong; Song, Xiaojing; Wang, Tiancheng; Zhang, Zecai; Jiang, Haichao; Cao, Yongguo; Zhang, Naisheng

    2014-10-01

    Leptospira interrogans (L. interrogans), a worldwide zoonosis, infect humans and animals. In dogs, four syndromes caused by leptospirosis have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups), and also it caused inflammation. Extracellular matrix (ECM) is a complex mixture of matrix molecules that is crucial to the reproduction. Both inflammatory response and ECM are closed relative to reproductive. The aim of this study was to clarify how L. interrogans affected the uterus of dogs, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans. In the present study, 27 dogs were divided into 3 groups, intrauterine infusion with L. interrogans, to make uterine infection, sterile EMJH, and normal saline as a control, respectively. The uteruses were removed by surgical operation in 10, 20, and 30 days, respectively. The methods of histopathological analysis, ELISA, Western blot and qPCR were used. The results showed that L. interrogans induced significantly inflammatory responses, which were characterized by inflammatory cellular infiltration and high expression levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in uterine tissue of these dogs. Furthermore, L. interrogans strongly down-regulated the expression of ECM (collagens (CL) IV, fibronectins (FN) and laminins (LN)) in mRNA and protein levels. These data indicated that strongly inflammatory responses, and abnormal regulation of ECM might contribute to the proliferation of dogs infected by L. interrogans. PMID:25153777

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

    PubMed Central

    Llanes, Alejandro; Restrepo, Carlos Mario; Rajeev, Sreekumari

    2016-01-01

    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. PMID:27442015

  20. A comprehensive survey on isoleucine biosynthesis pathways in seven epidemic Leptospira interrogans reference strains of China.

    PubMed

    Zou, Ying; Guo, Xiaokui; Picardeau, Mathieu; Xu, Hai; Zhao, Guoping

    2007-04-01

    Previous studies have indicated that different species of Leptospira synthesize isoleucine via either pyruvate and/or threonine pathways. Seven epidemic Leptospira interrogans reference strains from China belonging to different serovars, together with three saprophytic strains of Leptospira biflexa and Leptospira meyeri, were analysed. The isoleucine biosynthesis properties were studied firstly by measuring the key enzymes of the two pathways, citramalate synthase (CimA, CE4.1.3.-) and threonine deaminase (IlvA, CE4.2.1.16), from cell extracts of the bacteria. Meanwhile, alpha-isopropylmalate synthase (LeuA, CE4.2.1.12), the key enzyme of leucine biosynthesis, was also measured as a control. It was found that all L. interrogans strains synthesized isoleucine via the pyruvate pathway exclusively, but L. biflexa and L. meyeri used both pathways. Dot-Blot and PCR amplification of both cimA and ilvA genes in the corresponding strains provided additional evidence consistent with the data of enzymatic assays. Although it is evident that leptospires' isoleucine biosynthesis may preferentially adapt either to the pyruvate pathway exclusively for pathogens or to the combination of both pyruvate and threonine pathways for saprophytes, broader sampling with careful genomospecies identification is needed for a solid conclusion. PMID:17227461

  1. Asymptomatic and chronic carriage of Leptospira interrogans serovar Pomona in California sea lions (Zalophus californianus).

    PubMed

    Prager, K C; Greig, Denise J; Alt, David P; Galloway, Renee L; Hornsby, Richard L; Palmer, Lauren J; Soper, Jennifer; Wu, Qingzhong; Zuerner, Richard L; Gulland, Frances M D; Lloyd-Smith, James O

    2013-05-31

    Since 1970, periodic outbreaks of leptospirosis, caused by pathogenic spirochetes in the genus Leptospira, have caused morbidity and mortality of California sea lions (Zalophus californianus) along the Pacific coast of North America. Yearly seasonal epizootics of varying magnitude occur between the months of July and December, with major epizootics occurring every 3-5 years. Genetic and serological data suggest that Leptospira interrogans serovar Pomona is the infecting serovar and is enzootic in the California sea lion population, although the mechanism of persistence is unknown. We report asymptomatic carriage of Leptospira in 39% (33/85) of wild, free-ranging sea lions sampled during the epizootic season, and asymptomatic seroconversion with chronic asymptomatic carriage in a rehabilitated sea lion. This is the first report of asymptomatic carriage in wild, free-ranging California sea lions and the first example of seroconversion and asymptomatic chronic carriage in a sea lion. Detection of asymptomatic chronic carriage of Leptospira in California sea lions, a species known to suffer significant disease and mortality from the same Leptospira strain, goes against widely-held notions regarding leptospirosis in accidental versus maintenance host species. Further, chronic carriage could provide a mechanism for persistent circulation of Leptospira in the California sea lion population, particularly if these animals shed infectious leptospires for months to years. PMID:23419822

  2. Immunological characteristics of the glycolipid antigen of Leptospira interrogans serovar lai.

    PubMed Central

    Masuzawa, T; Nakamura, R; Shimizu, T; Iwamoto, Y; Morita, T; Yanagihara, Y

    1989-01-01

    The protective antigen (PAg), a glycolipid substance, was extracted from Leptospira interrogans serovar lai strain 017 with a chloroform-methanol-water (1:2:0.8 [vol/vol/vol]) solution and partially purified by silica gel column chromatography. The PAg was not detected by Coomassie brilliant blue staining in sodium dodecyl sulfate-polyacrylamide gel electrophoresis but was observed as a smearlike band, which corresponded to a 24- to 30-kilodalton standard protein, by silver staining. The outer envelope (OE) fraction showed the same band, suggesting that the PAg was one of the chemical components of the OE. The immunogenicity and protective activity of the PAg were compared with those of the OE. The PAg as well as the OE and whole cells was able to induce agglutinating antibody against L. interrogans. Furthermore, the immune sera exhibited opsonic activity against L. interrogans, as observed by measurement of chemical luminescence derived from reactive oxygen. The PAg exhibited protective activity in hamsters challenged with lethal doses of L. interrogans. Therefore, the antigen may be useful as a component vaccine against leptospiral infection. Images PMID:2744857

  3. Purification and characterization of a Na+, K+ ATPase inhibitor found in an endotoxin of Leptospira interrogans.

    PubMed Central

    Burth, P; Younes-Ibrahim, M; Gonçalez, F H; Costa, E R; Faria, M V

    1997-01-01

    We showed previously that the glycolipoprotein fraction prepared from Leptospira interrogans inhibited the Na+,K+ ATPase enzyme purified from brain or kidney and in isolated nephron segments (M. Younes-Ibrahim, P. Burth, M. V. Castro Faria, B. Buffin-Meyer, S. Marsy, C. Barlet-Bas, L. Cheval, and A. Doucet, C. R. Acad. Sci. Paris Ser. III 318:619-625, 1995). In the present communication, we have demonstrated that unsaturated fatty acids such as oleic and palmitoleic acids, which are adsorbed to this fraction, are effective inhibitors of the enzyme. PMID:9119504

  4. Adhesins of Leptospira interrogans Mediate the Interaction to Fibrinogen and Inhibit Fibrin Clot Formation In Vitro

    PubMed Central

    Oliveira, Rosane; Domingos, Renan F.; Siqueira, Gabriela H.; Fernandes, Luis G.; Souza, Natalie M.; Vieira, Monica L.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Nascimento, Ana L. T. O.

    2013-01-01

    We report in this work that Leptospira strains, virulent L. interrogans serovar Copenhageni, attenuated L. interrogans serovar Copenhageni and saprophytic L. biflexa serovar Patoc are capable of binding fibrinogen (Fg). The interaction of leptospires with Fg inhibits thrombin- induced fibrin clot formation that may affect the haemostatic equilibrium. Additionally, we show that plasminogen (PLG)/plasmin (PLA) generation on the surface of Leptospira causes degradation of human Fg. The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade. We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins. The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration. The calculated dissociation equilibrium constants (KD) of these reactions ranged from 733.3±276.8 to 128±89.9 nM for rLIC12238 and Lsa33, respectively. The interaction of recombinant proteins with human Fg resulted in inhibition of fibrin clot by thrombin-catalyzed reaction, suggesting that these versatile proteins could mediate Fg interaction in Leptospira. Our data reveal for the first time the inhibition of fibrin clot by Leptospira spp. and presents adhesins that could mediate these interactions. Decreasing fibrin clot would cause an imbalance of the coagulation cascade that may facilitate bleeding and help bacteria dissemination PMID:24009788

  5. Isolation and characterization of Leptospira interrogans from pigs slaughtered in São Paulo State, Brazil

    PubMed Central

    Miraglia, Fabiana; Moreno, Andréa Mike; Gomes, Cleise Ribeiro; Paixão, Renata; Liuson, Esequiel; Morais, Zenaide Maria; Maiorka, Paulo; Seixas, Fabiana Kömmling; Dellagostin, Odir Antonio; Vasconcellos, Silvio Arruda

    2008-01-01

    With the aim of isolating Leptospira spp., blood serum, kidney, liver and genital tract of 137 female swine (40 sows and 97 gilts) and also urine samples from 22 sows were collected in a slaughterhouse in the State of São Paulo, from April 2003 to August 2004. Four isolates were obtained from animals that presented microagglutination test (MAT) titers ≥ 100 for the serovar Pomona and one was obtained from an animal negative by MAT in which Leptospira was isolated from the liver and reproductive tract. The presence of leptospiral DNA was investigated by PCR, and positive results were found in kidneys of 11 females, liver of two, genital tract of two and urine of one of them. Nephrosis, interstitial multifocal nephritis, moderate to severe changing, hyalines cylinders and hemorrhagic focuses, hepatic and uterine horns congestion were histological lesions observed in higher frequency in animals positive for leptospira. The silver impregnation (Warthin Starry) confirmed the presence of spirochetes in renal tubules of four females with positive leptospira cultures from kidneys. The serogroup of the five isolates was identified as Pomona by cross agglutination with reference polyclonal antibodies. Molecular characterization of the isolates was carried out by variable-number tandem-repeats analysis. All the isolates revealed a pattern distinct from the L. interrogans Pomona type strain, but identical to a previously identified pattern from strains isolated in Argentina belonging to serovar Pomona. PMID:24031254

  6. A comparative analysis of the attachment of Leptospira interrogans and L. borgpetersenii to mammalian cells.

    PubMed

    Andrade, Gabrielle I; Brown, Paul D

    2012-06-01

    Leptospirosis, the world's most ubiquitous zoonosis, is caused by pathogenic Leptospira. As microbe-host interactions are specific in pathogenesis, it is likely that there are several molecules mediating the attachment of the Leptospira to mammalian cells. In this study, we analysed the attachment of Leptospira interrogans serovar Portlandvere and Leptospira borgpetersenii serovar Jules to untreated HEp-2 cells or HEp-2 cells treated with the various enzymes, lectins or sugars and to integrins αVβ3 and α5β1, relative to control wells. We found that both serovars bound equally well to HEp-2 cells; however, serovar Jules showed a higher level of attachment to integrins. Both serovars showed an increase in attachment to HEp-2 cells coated with lectins peanut agglutinin, Ulex europaeus agglutinin, soybean agglutinin and Erythrina cristagalli agglutinin (p < 0.05); in the case of Concanavalin A, Jules showed an increase, while Portlandvere showed a significant decrease in attachment. Trypsinizing monolayers resulted in a decrease in attachment for both serovars, while when chondroitinase, neuraminidase and heparinase were used an increase in attachment was recorded. Leptospires coated with sugars showed a decrease in attachment. These results show that serovar Jules' general greater affinity for the mediators examined may suggest a greater potential for virulence over serovar Portlandvere. PMID:22409511

  7. Generation of mammalian host-adapted Leptospira interrogans by cultivation in peritoneal dialysis membrane chamber implantation in rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospira interrogans can infect a myriad of mammalian hosts, including humans (Bharti, Nally et al. 2003, Ko, Goarant et al. 2009). Following acquisition by a suitable host, leptospires disseminate via the bloodstream to multiple tissues, including the kidneys, where they adhere to and colonize th...

  8. Molecular characterization, serotyping, and antibiotic susceptibility profile of Leptospira interrogans serovar Copenhageni isolates from Brazil.

    PubMed

    Miraglia, Fabiana; Matsuo, Minekazo; Morais, Zenaide Maria; Dellagostin, Odir Antonio; Seixas, Fabiana Kömmling; Freitas, Julio César; Hartskeerl, Rudy; Moreno, Luisa Zanolli; Costa, Bárbara Letícia; Souza, Gisele Oliveira; Vasconcellos, Silvio Arruda; Moreno, Andrea Micke

    2013-11-01

    Leptospira interrogans serogroup Icterohaemorrhagiae is the major serogroup infecting humans worldwide, and rodents and dogs are the most significant transmission sources in urban environments. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the epidemiology of leptospirosis. In this study, 20 Leptospira isolates were evaluated by pulsed-field gel electrophoresis (PFGE), variable number tandem-repeat analysis (VNTR), serotyping, and determination of antimicrobial resistance profile. Isolates, originated from bovine, canine, human, and rodent sources, were characterized by microscopic agglutination test with polyclonal and monoclonal antibodies and were identified as L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. MICs of antimicrobials often used in veterinary medicine were determined by broth microdilution test. Most of tested antibiotics were effective against isolates, including penicillin, ampicillin, and ceftiofur. Higher MIC variability was observed for fluoroquinolones and neomycin; all isolates were resistant to trimethoprim/sulfamethoxazole and sulphadimethoxine. Isolates were genotyped by PFGE and VNTR; both techniques were unable to discriminate between serovars Copenhageni and Icterohaemorrhagiae, as expected. PFGE clustered all isolates in 1 pulsotype, indicating that these serovars can be transmitted between species and that bovine, rodent, and dogs can maintain them in the environment endangering the human population. PMID:24054736

  9. Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence.

    PubMed

    Eshghi, Azad; Henderson, Jeremy; Trent, M Stephen; Picardeau, Mathieu

    2015-11-01

    Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model. PMID:26283339

  10. Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

    PubMed Central

    Eshghi, Azad; Henderson, Jeremy; Trent, M. Stephen

    2015-01-01

    Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model. PMID:26283339

  11. Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway.

    PubMed

    Xu, Hai; Zhang, Yuzhen; Guo, Xiaokui; Ren, Shuangxi; Staempfli, Andreas A; Chiao, Juishen; Jiang, Weihong; Zhao, Guoping

    2004-08-01

    Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded alpha-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K(m) (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed beta-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes. PMID:15292141

  12. Heat stability of protective antigen of Leptospira interrogans serovar lai.

    PubMed Central

    Masuzawa, T; Nakamura, R; Shimizu, T; Yanagihara, Y

    1990-01-01

    Protective antigen (PAg; glycolipid antigen; molecular size, 23 to 30 kilodaltons), the serogroup-specific antigen partially purified from leptospiral cells, is one of the most important protective antigens. The heat stability of PAg was compared with that of whole-cell (WC) antigen by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, protective activity, opsonin-inducing activity, agglutinating antibody-inducing activity, and an inhibition test in an enzyme-linked immunosorbent assay. A band of 23 to 30 kilodaltons of PAg, which was seen in untreated PAg and WC, shifted to a position with a molecular size of ca. 20 kilodaltons after heat treatment of PAg at 80 degrees C for 30 min and WC at 100 degrees C for 30 min. In the enzyme-linked immunosorbent assay inhibition test with monoclonal antibody LW2 and a sonicated antigen of WC, the inhibition rate of PAg and WC to sonicated WC was reduced by heat treatment at 80 degrees C for 30 min and at 100 degrees C for 30 min, respectively. Agglutinating antibody-inducing activities and opsonin-inducing activities of PAg and WC in mice were reduced by heat treatment under the same conditions; these activities were assayed by a microscopic agglutination test and by chemical luminescence response in serum from immunized mice, respectively. Protective activity of heated PAg and heated WC in cyclophosphamide-pretreated mice agreed with the results of immunogenicity in mice. These results indicate that the Leptospira PAg is one of the important protective antigens and is altered by heat treatment at 80 degrees C. Furthermore, the immunogenicity and antigenicity of the PAg present in WC are more stable than that of the extracted PAg, and the coexistence of other cellular components with PAg might protect and stabilize PAg from the heat treatment. Images PMID:2332463

  13. Comparative seroprevalence of Leptospira interrogans in Colombian mammals along a climatic gradient.

    PubMed

    Astudillo, Viviana González; Hernández, Dave Wehdeking; Stadlin, Juliana Peña; Bernal, Leonardo Arias; Rodríguez, Dora Adriana Lombo; Hernández, Miryam Astudillo

    2012-12-01

    Leptospirosis is a widespread zoonotic disease with well-established impacts on human health in tropical and subtropical regions. Although Leptospira spp. are known to readily infect many wildlife species, the understanding of interspecies and climatic variability in patterns of infection in Neotropical mammals is limited. To improve the understanding of this interplay, 85 mammals representing 17 species were sampled from four Colombian zoos along a climatic gradient. Prevalence of the 21 primary serovars against Leptospira interrogans was determined using the microagglutination test. Individuals were considered positive for a given serovar if antibodies were observable at a 1:100 dilution or greater. Overall prevalence was 9.52%, with positive titers to serovar hurstbridge in Carnivora (Canidae); serovar sarmin in Primata (Atelidae); and serovars australis, mini, autumnalis, pomona, icterohaemorrhagiae, and seramanga in Primata (Cebidae). Prevalence was positively correlated with humidity and temperature, with significantly higher prevalence at the site characterized by high humidity, severe flooding because of rainfall, and warm weather throughout the year. All positive animals were classified as clinically asymptomatic, meaning that antibodies from a current or past infection were detected but no overt symptoms were apparent. The diversity of serovars observed and the taxon-specific nature of these associations suggest that the epidemiology of Leptospira transmission is likely to be complex and multidimensional. The strong association observed between prevalence and climate suggests that the important role of climate as an indicator of Leptospira infection risk in humans may also be applicable to wildlife. Future studies in both wild and captive populations of Neotropical wildlife will further elucidate this disease interplay. PMID:23272343

  14. Leptospira interrogans serovar hardjo Infection in Cattle in the South Okanagan District of British Columbia

    PubMed Central

    Kingscote, Barbara F.

    1985-01-01

    An outbreak of leptospirosis due to Leptospira interrogans serovar hardjo in the South Okanagan District of British Columbia was investigated. The infection was associated primarily with bulls, but serovar hardjo was isolated from both bulls and cows at slaughter. Kidney and cerebrospinal fluid were found to contain leptospires, independently of the presence and level of serum agglutinins. Treatment of a bull twice in six months with dihydrostreptomycin failed to diminish an agglutinin titer (1/200) which persisted for two years without reexposure of the bull. A serological survey of cull cows sold through a central auction mart revealed the presence of hardjo agglutinins in 15.4% of 1300 sera representing 163 herds in 20 locations. Thirty percent of these herds contained reactor cattle. The number of premises from which reactor cattle came in a given locality varied from 4% to 67.7%. Measures to control leptospirosis in the study are suggested. PMID:17422584

  15. Multiple-locus variable-number tandem repeat analysis of Leptospira interrogans and Leptospira borgpetersenii isolated from small feral and wild mammals in East Asia.

    PubMed

    Koizumi, Nobuo; Izumiya, Hidemasa; Mu, Jung-Jung; Arent, Zbigniew; Okano, Shou; Nakajima, Chie; Suzuki, Yasuhiko; Mizutani Muto, Maki; Tanikawa, Tsutomu; Taylor, Kyle R; Komatsu, Noriyuki; Yoshimatsu, Kumiko; Thi Thu Ha, Hoang; Ohnishi, Makoto

    2015-12-01

    Leptospira spp. are the causative agents of a worldwide zoonosis, leptospirosis, maintained by various mammals. Each Leptospira serovar is frequently associated with a particular maintenance host, and recently, Leptospira genotype-host association has also been suggested to limit serovars to restricted areas. We investigated the molecular characteristics of L. interrogans and L. borgpetersenii which were isolated from small feral and wild animals in four East Asian states using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA using 11 loci was performed on 110 L. interrogans serogroups from Japan (79 strains of 5 serogroups from 3 animal species), Philippines (21; 3; 2), Taiwan (7; 2; 3), and Vietnam (3; 1; 1). A MLVA method using 4 loci for L. borgpetersenii was established and performed on 52 isolates from Japan (26; 3; 7), Philippines (13; 1; 2), and Taiwan (13; 1; 3). In L. interrogans, serogroups Autumnalis and Hebdomadis appeared more genetically diverse than serogroups Bataviae, Grippotyphosa, Icterohaemorrhagiae, Pomona, or Pyrogenes. The former serogroup strains with the exception of one Hebdomadis strain were isolated from Apodemus speciosus while all the latter serogroup strains with the exception of Grippotyphosa were isolated from Rattus norvegicus. L. borgpetersenii was isolated from at least 11 animal species while L. interrogans was isolated from five species, which might suggest a wider host range for L. borgpetersenii. Broad host preference in a single genotype was also observed, which colonized not only different species of the same genera but also multiple animal genera. This study demonstrates that there may be variability in the range of genetic diversity among different Leptospira serogroups, which may be attributed to maintenance host animals and environmental factors. PMID:26296603

  16. Whole-Genome Sequence of Leptospira interrogans Serovar Hardjo Subtype Hardjoprajitno Strain Norma, Isolated from Cattle in a Leptospirosis Outbreak in Brazil.

    PubMed

    Cosate, M R V; Soares, S C; Mendes, T A; Raittz, R T; Moreira, E C; Leite, R; Fernandes, G R; Haddad, J P A; Ortega, J Miguel

    2015-01-01

    Leptospirosis is caused by pathogenic bacteria of the genus Leptospira spp. This neglected re-emergent disease has global distribution and relevance in veterinary production. Here, we report the whole-genome sequence and annotation of Leptospira interrogans serovar Hardjo subtype Hardjoprajitno strain Norma, isolated from cattle in a livestock leptospirosis outbreak in Brazil. PMID:26543126

  17. Whole-Genome Sequence of Leptospira interrogans Serovar Hardjo Subtype Hardjoprajitno Strain Norma, Isolated from Cattle in a Leptospirosis Outbreak in Brazil

    PubMed Central

    Soares, S. C.; Mendes, T. A.; Raittz, R. T.; Moreira, E. C.; Leite, R.; Fernandes, G. R.; Haddad, J. P. A.; Ortega, J. Miguel

    2015-01-01

    Leptospirosis is caused by pathogenic bacteria of the genus Leptospira spp. This neglected re-emergent disease has global distribution and relevance in veterinary production. Here, we report the whole-genome sequence and annotation of Leptospira interrogans serovar Hardjo subtype Hardjoprajitno strain Norma, isolated from cattle in a livestock leptospirosis outbreak in Brazil. PMID:26543126

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

    PubMed

    Arent, Z; Frizzell, C; Gilmore, C; Allen, A; Ellis, W A

    2016-07-15

    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. PMID:27283852

  19. Evidence of Leptospira interrogans infection in California sea lion pups from the Gulf of California.

    PubMed

    Acevedo-Whitehouse, Karina; de la Cueva, Horacio; Gulland, Frances M D; Aurioles-Gamboa, David; Arellano-Carbajal, Fausto; Suarez-Güemes, Francisco

    2003-01-01

    Forty-two urine and 96 blood and serum samples were obtained from California sea lion (Zalophus californianus) pups from the Gulf of California during the 2000 reproductive season. Antibody prevalence to 13 serovars of Leptospira interrogans was determined by microagglutination tests (MAT); presence of pathogenic leptospires was detected by polymerase chain reaction (PCR). Samples with antibody titers > or = 1:25 or 115 bp fragments on ethidium bromidestained 1.5% agarose gels were considered positive. Antibody prevalence was 54% overall with highest prevalence against serovar cynopteri (50% of all positive reactions). Highest antibody titers (1:50) were detected against serovars cynopteri and pomona. Polymerase chain reaction products were observed in two of 42 urine samples, six of 96 blood samples, and one of 96 serum samples. Presence of PCR products in blood and serum was demonstrated in pups that were seronegative. Kruskall-Wallis tests and corresponding post hoc Tukey tests (alpha = 0.05) showed that prevalence of leptospirosis was significantly different among all rookeries. The high seroprevalence (54%), low antibody titers (maximum 1:50), absence of pups showing clinical signs indicative of the disease, and lack of recent reports of increased mortality of sea lions in the Gulf of California are suggestive of the presence of enzootic host-adapted serovars. Crowding in rookeries as well as the presence of bats and rodents on some of the islands may explain infection by L. interrogans (sensu lato) and some of the differences in seroprevalence among reproductive rookeries. PMID:12685078

  20. Geographical dissemination of Leptospira interrogans serovar Pomona during seasonal migration of California sea lions.

    PubMed

    Zuerner, Richard L; Cameron, Caroline E; Raverty, Stephen; Robinson, John; Colegrove, Kathleen M; Norman, Stephanie A; Lambourn, Dyanna; Jeffries, Steven; Alt, David P; Gulland, Frances

    2009-05-28

    Leptospirosis is one of the most widespread bacterial zoonoses in the world and affects most mammalian species. Although leptospirosis is well documented and characterized in terrestrial species, less information is available regarding the distribution and impact of leptospirosis in marine mammals. Additionally, the role of animal migrations on the geographical spread of leptospirosis has not been reported. Periodic epizootic outbreaks of acute leptospirosis among California sea lions (Zalophus californianus) have been reported since 1971. In this study, we collected samples from California sea lions stranded along the Pacific coast of North America during the most recent epidemic in 2004, and maintained leptospirosis surveillance of the California sea lion population along the California coast through 2007. Several isolates of Leptospira interrogans serovar Pomona were obtained from kidney and urine samples collected during this study, a finding consistent with serological evidence that California sea lions are persistently exposed to this leptospiral serovar. Combined, these data support a model whereby California sea lions are maintenance hosts for L. interrogans serovar Pomona, yet periodically undergo outbreaks of acute infection. During the 2004 outbreak, the incidence of new leptospirosis cases among California sea lions coincided with the seasonal movement of male sea lions from rookeries along the coast of central and southern California north as far as British Columbia. These data show that seasonal animal movement contributes to the distribution of leptospirosis across a large geographical region. PMID:19186009

  1. Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans

    PubMed Central

    Malmström, Johan; Beck, Martin; Schmidt, Alexander; Lange, Vinzenz; Deutsch, Eric W.; Aebersold, Ruedi

    2009-01-01

    Mass spectrometry based methods for relative proteome quantification have broadly impacted life science research. However, important research directions, particularly those involving mathematical modeling and simulation of biological processes, also critically depend on absolutely quantitative data, i.e. knowledge of the concentration of the expressed proteins as a function of cellular state. Until now, absolute protein concentration measurements of a significant fraction of the proteome (73%) have only been derived from genetically altered S. cerevisiae cells 1, a technique that is not directly portable from yeast to other species. In this study we developed and applied a mass spectrometry based strategy to determine the absolute quantity i.e. the average number of protein copies per cell in a cell population, for a significant fraction of the proteome in genetically unperturbed cells. Applying the technology to the human pathogen Leptospira interrogans, a spirochete responsible for Leptospirosis 4, we generated an absolute protein abundance scale for 83% of the mass spectrometry detectable proteome, from cells at different states. Taking advantage of the unique cellular dimensions of L. interrogans, we used cryo electron tomography (cryoET) morphological measurements to verify at the single cell level the average absolute abundance values of selected proteins determined by mass spectrometry on a population of cells. As the strategy is relatively fast and applicable to any cell type we expect that it will become a cornerstone of quantitative biology and systems biology. PMID:19606093

  2. Purification, crystallization and preliminary crystallographic studies on 2-dehydro-3-deoxygalactarate aldolase from Leptospira interrogans

    SciTech Connect

    Li, Xu; Huang, Hua; Song, Xiaomin; Wang, Yanli; Xu, Hang; Teng, Maikun; Gong, Weimin

    2006-12-01

    Preliminary crystallographic studies on 2-dehydro-3-deoxygalactarate aldolase from L. interrogans. 2-Dehydro-3-deoxygalactarate (DDG) aldolase is a member of the class II aldolase family and plays an important role in the pyruvate-metabolism pathway, catalyzing the reversible aldol cleavage of DDG to pyruvate and tartronic semialdehyde. As it is a potential novel antibiotic target, it is necessary to elucidate the catalytic mechanism of DDG aldolase. To determine the crystal structure, crystals of DDG aldolase from Leptospira interrogans were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution using a Cu Kα rotating-anode X-ray source. The crystal belonged to space group C2, with unit-cell parameters a = 293.5, b = 125.6, c = 87.6 Å, β = 100.9°. The V{sub M} is calculated to be 2.4 Å{sup 3} Da{sup −1}, assuming there to be 12 protein molecules in the asymmetric unit.

  3. Molecular basis of the substrate specificity and the catalytic mechanism of citramalate synthase from Leptospira interrogans.

    PubMed

    Ma, Jun; Zhang, Peng; Zhang, Zilong; Zha, Manwu; Xu, Hai; Zhao, Guoping; Ding, Jianping

    2008-10-01

    Leptospira interrogans is the causative agent for leptospirosis, a zoonotic disease of global importance. In contrast with most other micro-organisms, L. interrogans employs a pyruvate pathway to synthesize isoleucine and LiCMS (L. interrogans citramalate synthase) catalyses the first reaction of the pathway which converts pyruvate and acetyl-CoA into citramalate, thus making it an attractive target for the development of antibacterial agents. We report here the crystal structures of the catalytic domain of LiCMS and its complexes with substrates, and kinetic and mutagenesis studies of LiCMS, which together reveal the molecular basis of the high substrate specificity and the catalytic mechanism of LiCMS. The catalytic domain consists of a TIM barrel flanked by an extended C-terminal region. It forms a homodimer in the crystal structure, and the active site is located at the centre of the TIM barrel near the C-terminal ends of the beta-strands and is composed of conserved residues of the beta-strands of one subunit and the C-terminal region of the other. The substrate specificity of LiCMS towards pyruvate against other alpha-oxo acids is dictated primarily by residues Leu(81), Leu(104) and Tyr(144), which form a hydrophobic pocket to accommodate the C(2)-methyl group of pyruvate. The catalysis follows the typical aldol condensation reaction, in which Glu(146) functions as a catalytic base to activate the methyl group of acetyl-CoA to form an enolated acetyl-CoA intermediate and Arg(16) as a general acid to stabilize the intermediate. PMID:18498255

  4. Dual nuclease activity of a Cas2 protein in CRISPR-Cas subtype I-B of Leptospira interrogans.

    PubMed

    Dixit, Bhuvan; Ghosh, Karukriti Kaushik; Fernandes, Gary; Kumar, Pankaj; Gogoi, Prerana; Kumar, Manish

    2016-04-01

    Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system. PMID:26950513

  5. Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans

    PubMed Central

    Narayanavari, Suneel A.; Lourdault, Kristel; Sritharan, Manjula; Haake, David A.; Matsunaga, James

    2015-01-01

    Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity

  6. Immunological and molecular characterization of Leptospira interrogans isolated from a bovine foetus.

    PubMed

    Monte, Leonardo Garcia; Ridieri, Karine Forster; Jorge, Sérgio; Oliveira, Natasha Rodrigues; Hartwig, Daiane Drawanz; Amaral, Marta Gonçalves; Hartleben, Cláudia Pinho; Dellagostin, Odir Antonio

    2015-06-01

    Cattle are commonly infected with pathogenic leptospires, and similarly to rodents, they excrete the bacteria in their urine and can transmit the pathogen from animal to animal or animal to human. Thus, surveillance and monitoring systems for detection of new Leptospira serovars are important for the control of leptospirosis. Here, we report the isolation of a spirochete from a stillborn bovine foetus and its characterization by immunological and molecular techniques. A variable number tandem repeat profile using seven discriminatory primers identified the spirochete as belonging to species Leptospira interrogans serogroup Australis serovar Muenchen. A phenotypic analysis using monoclonal antibodies (mAbs) against leptospiral membrane-associated proteins confirmed the expression of important virulence and pathogenicity factors (LipL32 and LigBrep). Out of 120 reference sera tested, 22 positive (36.66%) and 9 negative (15%) also reacted with the new isolate. Furthermore, the serovar Muenchen isolate was virulent in hamster model. The animal inoculated developed acute lethal infection characterized by hepatic, pulmonary and renal lesions. Local isolates exhibited unique characteristics that differed from those of reference strains; therefore, isolation of leptospires is useful in the surveillance of local pathogenic serovars. In conclusion, the data obtained from this study can contribute to the epidemiological understanding and control of leptospirosis in southern Brazil. PMID:25976319

  7. Protection of guinea pigs against Leptospira interrogans serovar Lai by LipL21 DNA vaccine.

    PubMed

    He, Han Jiang; Wang, Wen Yu; Wu, Zhong Dao; Lv, Zhi Yue; Li, Jun; Tan, Li Zhi

    2008-10-01

    In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1(+). The guinea pigs were immunized with pcDNA3.1(+)-lipL21, pcDNA3.1(+) or PBS. Six weeks after the second immunization, the splenocytes were isolated to detect their proliferative ability by lymphocyte transformation experiments. In addition, microscopic agglutination test was used for quantitative detection of specific antibodies. The rest guinea pigs were challenged intraperitoneally with L. interogans sorevar Lai. Then, protective effect was evaluated on the basis of survival and histopathological lesions in the kidneys, lungs, and liver. The lipL21 gene was successfully expressed in COS-7 cells through recombinant pcDNA3.1(+)-lipL21. The titer of specific antibodies substantially increased, and the stimulation index of splenocytes increased significantly. Hence, the pcDNA3.1(+)-lipL21 could protect the immunized guinea pigs from homotypic Leptospira infection. Furthermore, no obvious pathologic changes were observed in the pcDNA3.1(+)-lipL21 immunized guinea pigs. The results showed that the protective effect with pathogenic strains of Leptospira was shared by LipL21 mediated through a plasmid vector. Consequently, these results indicated that the lipL21 DNA vaccine was a promising candidate for the prevention of leptospirosis. PMID:18954563

  8. Post-translational Modification of LipL32 during Leptospira interrogans Infection

    PubMed Central

    Witchell, Timothy D.; Eshghi, Azad; Nally, Jarlath E.; Hof, Rebecca; Boulanger, Martin J.; Wunder, Elsio A.; Ko, Albert I.; Haake, David A.; Cameron, Caroline E.

    2014-01-01

    Background Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin. Methodology/Principal Findings Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32. Conclusions/Significance The exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although

  9. First isolation of Leptospira interrogans from Lycalopex griseus (South American gray fox) in Argentina shows new MLVA genotype.

    PubMed

    Scialfa, Exequiel; Brihuega, Bibiana; Venzano, Agustín; Morris, Winston Eduardo; Bolpe, Jorge; Schettino, Mateo

    2013-01-01

    To identify carriers of Leptospira spp. in Argentina, wild animals were trapped in Buenos Aires Province during three nights, capturing 12 Didelphis albiventris (white-eared opossum), six Chaetophractus villosus (big hairy armadillo), five Lycalopex griseus (South American gray fox), and two Conepatus chinga (Molina's hog-nosed skunk). All were tested by microscopic agglutination test, and five (two gray foxes, two armadillos, and one skunk) were positive for Leptospira interrogans serovars Canicola and Icterohaemorrhagiae, L. borgpetersenii serovar Castellonis, and L. kirschneri serovar Grippotyphosa, at titers of 1:50 and 1:100. Kidney tissue from all animals was cultured, and one isolate of L. interrogans from a gray fox was obtained. Hamsters inoculated with the isolate died after 6 days with no macroscopic lesions at necropsy. However, histologic examination revealed glomerulonephritis, interstitial nephritis, and pneumonia. The Leptospira strain from the South American gray fox was analyzed serologically and its pathogenicity was established. Genotyping through multiple-locus variable-number tandem repeat analysis showed that the strain was a new genotype related to the L. interrogans serogroup Icterohaemorrhagiae. PMID:23307384

  10. Seroprevalence and risk factors associated with within-flock transmission of Leptospira interrogans in transhumant farming systems in Mexico.

    PubMed

    Arteaga-Troncoso, G; Jiménez-Estrada, J M; Montes De Oca-Jimenez, R; López-Hurtado, M; Luna-Alvarez, M; Hernandez-Andrade, L; Moreno-Alfaro, A; Galan-Herrera, J F; Guerra-Infante, F M

    2015-10-01

    A number of recent reports emphasize the risk of zoonotic diseases and the high degree of prevalence of asymptomatic animals infected with Leptospira interrogans. This report sought to assess the prevalence of antibodies to certain serovars of L. interrogans, and to describe the association between seropositivity and risk factors associated with within-flock transmission in a mountainous region of Mexico. Overall seroprevalence to L. interrogans was 54·5% (95% confidence interval 48·3-60·7); the most frequent serovar was Icterohaemorrhagiae. The accumulation of placentas and fetuses at a site close to lambing paddocks can play a significant role as a risk factor for within-flock transmission of L. interrogans in transhumant farming systems in the municipality of Xalatlaco. The high prevalence of L. interrogans antibodies supports the hypothesis that natural foci of this zoonosis are present in sheep flocks in this area. These findings emphasize the need for planning and implementation of control programmes for ovine leptospirosis in Mexico and elsewhere. PMID:25600318

  11. Iron metabolism in hamsters experimentally infected with Leptospira interrogans serovar Pomona: influence on disease pathogenesis.

    PubMed

    Sobroza, Ânderson O; Tonin, Alexandre A; Da Silva, Alekandro S; Dornelles, Guilherme L; Wolkmer, Patrícia; Duarte, Marta M M F; Hausen, Bruna S; Sangoi, Manuela B; Moresco, Rafael N; Stefani, Lenita M; Mazzantti, Cinthia M; Lopes, Sonia T A; Leal, Marta L R

    2014-12-01

    The aim of this study was to analyze the classic iron markers associated to the storage process in hamsters experimentally infected by Leptospira interrogans serovar Pomona. Four groups with six hamsters each were used; two were negative controls (C7 and C14) and two were composed by infected animals (T7 and T14). Blood samples were collected on the seventh (C7 and T7) and fourteenth days (C14 and T14) post-inoculation. Iron availability was determined in sera samples through the assessment of iron, ferritin, transferrin, and iron binding capacity, whereas the bone marrow was also evaluated for the presence of iron by Pearl's reaction. Additionally, the total antioxidant capacity (TAC) and total oxidant status (TOS) were assessed, along with hepcidin and IL-6 levels. Based on the results, it was possible to observe the onset of an anemic profile, predominantly hemolytic and regenerative. Also, The other parameters showed an increase in seric iron (P<0.01) and ferritin (P<0.01), and a positive Pearl's reaction in T7 and T14, when compared with the control groups. Transferrin levels decreased (P<0.05) in animals of T14 with saturation index. TAC was increased in both periods (P<0.01), while TOS was increased only on T14 (P<0.05). Hepcidin and IL-6 were increased on T7 and T14 (P<0.01). Therefore, it was observed that the serum profile from infected animals showed a strong hemolytic pattern, with some demonstration of ferric tissue sequestration when the infection tended to become chronic. The results show that iron metabolism is activated in hamsters infected by L. interrogans serovar Pomona. PMID:25449998

  12. Kinetics of Leptospira interrogans Infection in Hamsters after Intradermal and Subcutaneous Challenge

    PubMed Central

    Coutinho, Mariana L.; Matsunaga, James; Wang, Long-Chieh; de la Peña Moctezuma, Alejandro; Lewis, Michael S.; Babbitt, Jane T.; Aleixo, Jose Antonio G.; Haake, David A.

    2014-01-01

    Background Leptospirosis is a zoonosis caused by highly motile, helically shaped bacteria that penetrate the skin and mucous membranes through lesions or abrasions, and rapidly disseminate throughout the body. Although the intraperitoneal route of infection is widely used to experimentally inoculate hamsters, this challenge route does not represent a natural route of infection. Methodology/Principal Findings Here we describe the kinetics of disease and infection in hamster model of leptospirosis after subcutaneous and intradermal inoculation of Leptospira interrogans serovar Copenhageni, strain Fiocruz L1-130. Histopathologic changes in and around the kidney, including glomerular and tubular damage and interstitial inflammatory changes, began on day 5, and preceded deterioration in renal function as measured by serum creatinine. Weight loss, hemoconcentration, increased absolute neutrophil counts (ANC) in the blood and hepatic dysfunction were first noted on day 6. Vascular endothelial growth factor, a serum marker of sepsis severity, became elevated during the later stages of infection. The burden of infection, as measured by quantitative PCR, was highest in the kidney and peaked on day 5 after intradermal challenge and on day 6 after subcutaneous challenge. Compared to subcutaneous challenge, intradermal challenge resulted in a lower burden of infection in both the kidney and liver on day 6, lower ANC and less weight loss on day 7. Conclusions/Significance The intradermal and subcutaneous challenge routes result in significant differences in the kinetics of dissemination and disease after challenge with L. interrogans serovar Copenhageni strain Fiocruz L1-130 at an experimental dose of 2×106 leptospires. These results provide new information regarding infection kinetics in the hamster model of leptospirosis. PMID:25411782

  13. Multiple-locus variable-number tandem repeat analysis and clinical characterization of Leptospira interrogans canine isolates.

    PubMed

    Koizumi, Nobuo; Muto, Maki Mizutani; Izumiya, Hidemasa; Suzuki, Motoi; Ohnishi, Makoto

    2015-03-01

    Canine leptospirosis occurs worldwide; however, information on the relationship between Leptospira serotypes/genotypes and virulence in dogs remains limited. We investigated the molecular characteristics of Leptospira interrogans canine isolates belonging to three serogroups using multiple-locus variable-number tandem repeat analysis (MLVA) and the effects of each serotype/genotype on the clinical characteristics of leptospirosis in dogs. MLVA using 11 loci of the three major L. interrogans serogroups in Japan, Australis (32 strains from 21 dogs), Autumnalis (12; 7) and Hebdomadis (66; 39), revealed more divergent genetic heterogeneity within each serogroup than multilocus sequence typing (MLST), and they formed two, three and five clusters (CLs), respectively. Lethal infections were caused by all Leptospira serogroup isolates (70.3 % with Hebdomadis, 83.3 % with Australis and 100 % with Autumnalis) or Leptospira isolates belonging to all the CLs (57.1-100 %) without any significant differences. A significant difference in hyperaemia and haemorrhage of mucus membrane was observed between serogroups Australis and Autumnalis (P = 0.03). Leptospira isolates of Australis CL2 caused no hyperaemia and haemorrhage from mucus membrane, whereas those of Australis CL1, Autumnalis CL3 and Hebdomadis CL1 and CL3 did (P<0.05). Significant differences in creatinine (Cre) levels were observed between serogroups Australis and Hebdomadis (P = 0.02). In addition, significant differences in blood urea nitrogen levels were observed between serogroups Australis and Hebdomadis (P = 0.004) and Australis and Autumnalis (P = 0.02). Based on MLVA types, a significant difference in Cre levels was observed between Hebdomadis CL1 and CL4 (P = 0.0018). Our results indicated that MLVA had a higher discriminatory power and was more concordant with serotyping than MLST. Although all Leptospira serotypes and genotypes caused lethal infections in dogs, the L. interrogans

  14. Generation of Mammalian Host-adapted Leptospira interrogans by Cultivation in Peritoneal Dialysis Membrane Chamber Implantation in Rats

    PubMed Central

    Grassmann, André Alex; McBride, Alan John Alexander; Nally, Jarlath E.; Caimano, Melissa J.

    2015-01-01

    Leptospira interrogans can infect a myriad of mammalian hosts, including humans (Bharti et al., 2003; Ko et al., 2009). Following acquisition by a suitable host, leptospires disseminate via the bloodstream to multiple tissues, including the kidneys, where they adhere to and colonize the proximal convoluted renal tubules (Athanazio et al., 2008). Infected hosts shed large number of spirochetes in their urine and the leptospires can survive in different environmental conditions before transmission to another host. Differential gene expression by Leptospira spp. permits adaption to these new conditions. Here we describe a protocol for the cultivation of Leptospira interrogans within Dialysis Membrane Chambers (DMCs) implanted into the peritoneal cavities of Sprague-Dawley rats (Caimano et al., 2014). This technique was originally developed to study mammalian adaption by the Lyme disease spirochete, Borrelia burgdorferi (Akins et al., 1998; Caimano, 2005). The small pore size (8,000 MWCO) of the dialysis membrane tubing used for this procedure permits access to host nutrients but excludes host antibodies and immune effector cells. Given the physiological and environmental similarities between DMCs and the proximal convoluted renal tubule, we reasoned that the DMC model would be suitable for studying in vivo gene expression by L. interrogans. In a 20 to 30 min procedure, DMCs containing virulent leptospires are surgically-implanted into the rat peritoneal cavity. Nine to 11 days post-implantation, DMCs are explanted and organisms recovered. Typically, a single DMC yields ~109 mammalian host-adapted leptospires (Caimano et al., 2014). In addition to providing a facile system for studying the transcriptional and physiologic changes pathogenic L. interrogans undergo within the mammal, the DMC model also provides a rationale basis for selecting new targets for mutagenesis and the identification of novel virulence determinants. Caution: Leptospira interrogans is a BSL-2

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

    PubMed

    Arent, Zbigniew; Gilmore, Colm; Brem, Siegfried; Ellis, William A

    2015-08-01

    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. PMID:26165505

  16. Leptospira interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues.

    PubMed

    Davis, J Muse; Haake, David A; Ramakrishnan, Lalita

    2009-01-01

    Leptospirosis is an extremely widespread zoonotic infection with outcomes ranging from subclinical infection to fatal Weil's syndrome. Despite the global impact of the disease, key aspects of its pathogenesis remain unclear. To examine in detail the earliest steps in the host response to leptospires, we used fluorescently labelled Leptospira interrogans serovar Copenhageni to infect 30 hour post fertilization zebrafish embryos by either the caudal vein or hindbrain ventricle. These embryos have functional innate immunity but have not yet developed an adaptive immune system. Furthermore, they are optically transparent, allowing direct visualization of host-pathogen interactions from the moment of infection. We observed rapid uptake of leptospires by phagocytes, followed by persistent, intracellular infection over the first 48 hours. Phagocytosis of leptospires occasionally resulted in formation of large cellular vesicles consistent with apoptotic bodies. By 24 hours, clusters of infected phagocytes were accumulating lateral to the dorsal artery, presumably in early hematopoietic tissue. Our observations suggest that phagocytosis may be a key defense mechanism in the early stages of leptospirosis, and that phagocytic cells play roles in immunopathogenesis and likely in the dissemination of leptospires to specific target tissues. PMID:19547748

  17. Relationships between prevalence of Leptospira interrogans in cattle, and regional, climatic, and seasonal factors.

    PubMed

    Miller, D A; Wilson, M A; Beran, G W

    1991-11-01

    On the basis of serologic test results and isolation of leptospires from mature cattle, distribution and prevalence of Leptospira interrogans serovars and genotypes were compared by state and region of the United States. Relationships between isolation rate and month of sample collection, mean regional temperature, and mean regional precipitation were examined. Isolation rate and seroprevalence were significantly (P less than 0.001) higher for southeastern, south central, and Pacific coastal regions than for other regions of the United States. Isolates of genotypes hardjo-bovis A and kennewicki A and B, and of serovar grippotyphosa appeared to be randomly distributed. Genotype hardjo-bovis B isolates came from a southern area of the country that extends from Georgia to New Mexico. To the authors' knowledge, this is the first recorded isolation of serovar hardjo from Hawaii. Although significant relationship was not documented between isolation rate and month or season of the year, seroprevalence for summer, fall, and winter was significantly (P less than 0.001) higher than that for spring. Regional isolation rate was related more to mean temperature (r = 0.83; P less than 0.05) than to mean precipitation amount (r = 0.34; P greater than 0.50). PMID:1785720

  18. Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant

    PubMed Central

    Monaris, D.; Sbrogio-Almeida, M. E.; Dib, C. C.; Canhamero, T. A.; Souza, G. O.; Vasconcellos, S. A.; Ferreira, L. C. S.

    2015-01-01

    Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigAC) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigAC, either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigAC or LigAC coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

  19. Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant.

    PubMed

    Monaris, D; Sbrogio-Almeida, M E; Dib, C C; Canhamero, T A; Souza, G O; Vasconcellos, S A; Ferreira, L C S; Abreu, P A E

    2015-08-01

    Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

  20. Prevalence of antibodies to Leptospira interrogans serovar hardjo in bulk tank milk from unvaccinated irish dairy herds

    PubMed Central

    2004-01-01

    Bulk tank milk samples, collected from 347 herds throughout the Republic of Ireland using a sampling frame based on seven milk-recording organisations, were tested by ELISA for antibodies to Leptospira interrogans serovar hardjo. These herds, which had not been vaccinated against leptospirosis within the previous five years, were categorised according to their province, milk-recording organisation and size. Two-hundred-and-seventy-three herds (79%) had a positive ELISA titre. Both the probability of a herd being seropositive and the antibody level in the herd milk sample were affected by the province (P < 0.05 and P < 0.01, respectively) and the herd size category (P < 0.05 and P < 0.01, respectively). Larger herds were significantly more likely to have positive reactions and higher mean concentrations of antibody. It was concluded that a high proportion of unvaccinated Irish dairy herds have been exposed to infection with Leptospira hardjo. PMID:21851657

  1. Molecular and serological characterization of Leptospira interrogans serovar Canicola isolated from dogs, swine, and bovine in Brazil.

    PubMed

    Miraglia, Fabiana; de Morais, Zenaide M; Dellagostin, Odir A; Seixas, Fabiana K; Freitas, Julio C; Zacarias, Francielle G S; Delbem, Adina C; Ferreira, Thaís S P; Souza, Gisele O; Hartskeerl, Rudy A; Vasconcellos, Silvio A; Moreno, Andrea M

    2013-01-01

    The identification of Leptospira clinical isolates through genotyping and serotyping, besides the recognition of its reservoirs, are important tools for understanding the epidemiology of leptospirosis, and they are also keys for identifying new species and serovars. Fourteen clinical isolates from animals were characterized by means of single enzyme amplified length polymorphism, variable number of tandem repeat analysis, pulsed field gel electrophoresis, and serotyping. All isolates were identified as Leptospira interrogans, serovar Canicola. Infections by this serovar occur in urban regions, where dogs represent the main maintenance hosts, whereas bovine and swine may act as reservoirs of serovar Canicola in rural areas. Both urban and rural aspects of leptospirosis, and the role of domestic animals as maintenance hosts, cannot be neglected in developing and developed countries. PMID:22610538

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

    PubMed Central

    Kremer, Frederico S; Eslabão, Marcus R; Jorge, Sérgio; Oliveira, Natasha R; Labonde, Julia; Santos, Monize NP; Monte, Leonardo G; Grassmann, André A; Cunha, Carlos EP; Forster, Karine M; Moreno, Luísa Z; Moreno, Andrea M; Campos, Vinicius F; McBride, Alan JA; Pinto, Luciano S; Dellagostin, Odir A

    2016-01-01

    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. PMID:27074260

  3. Variable Nucleotide Tandem-Repeat Analysis Revealing a Unique Group of Leptospira interrogans Serovar Pomona Isolates Associated with California Sea Lions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospira interrogans serovar Pomona is commonly isolated from a variety of wildlife and domesticated livestock. It is difficult to assess whether disease outbreaks with serovar Pomona in given animal populations are due to endemic infections or accidental exposure. Unlike many leptospiral serovars...

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

    PubMed

    Kremer, Frederico S; Eslabão, Marcus R; Jorge, Sérgio; Oliveira, Natasha R; Labonde, Julia; Santos, Monize N P; Monte, Leonardo G; Grassmann, André A; Cunha, Carlos E P; Forster, Karine M; Moreno, Luísa Z; Moreno, Andrea M; Campos, Vinicius F; McBride, Alan J A; Pinto, Luciano S; Dellagostin, Odir A

    2016-04-01

    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. PMID:27074260

  5. Management practices as risk factors for the presence of bulk milk antibodies to Salmonella, Neospora caninum and Leptospira interrogans serovar hardjo in Irish dairy herds.

    PubMed

    O' Doherty, E; Berry, D P; O' Grady, L; Sayers, R

    2014-06-01

    A survey of management practices in 309 Irish dairy herds was used to identify risk factors for the presence of antibodies to Salmonella, Neospora caninum and Leptospira interrogans serovar hardjo in extensively managed unvaccinated dairy herds. A previous study documented a herd-level seroprevalence in bulk milk of 49%, 19% and 86% for Salmonella, Neospora caninum and leptospira interrogans serovar hardjo, respectively in the unvaccinated proportion of these 309 herds in 2009. Association analyses in the present study were carried out using multiple logistic regression models. Herds where cattle were purchased or introduced had a greater likelihood of being positive to leptospira interrogans serovar hardjo (P<0.01) and Salmonella (P<0.01). Larger herds had a greater likelihood of recording a positive bulk milk antibody result to leptospira interrogans serovar hardjo (P<0.05). Herds that practiced year round calving were more likely to be positive to Neospora caninum (P<0.05) compared to herds with a spring-calving season, with no difference in risk between herds that practiced split calving compared to herds that practiced spring calving. No association was found between presence of dogs on farms and prevalence of Neospora caninum possibly due to limited access of dogs to infected materials including afterbirths. The information from this study will assist in the design of suitable control programmes for the diseases under investigation in pasture-based livestock systems. PMID:24661904

  6. Carriage of Leptospira interrogans among domestic rats from an urban setting highly endemic for leptospirosis in Brazil.

    PubMed

    de Faria, Marcos Tucunduva; Calderwood, Michael S; Athanazio, Daniel A; McBride, Alan J A; Hartskeerl, Rudy A; Pereira, Martha Maria; Ko, Albert I; Reis, Mitermayer G

    2008-10-01

    A survey was conducted to identify reservoirs for urban leptospirosis in the city of Salvador, Brazil. Sampling protocols were performed in the vicinity of households of severe leptospirosis cases identified during active hospital-based surveillance. Among a total of 142 captured Rattus norvegicus (Norwegian brown rat), 80.3% had a positive culture isolate from urine or kidney specimens and 68.1% had a positive serum sample by microscopic agglutination test (MAT) titre of > or = 1:100. Monoclonal antibody-based typing of isolates identified that the agent carried by rats was Leptospira interrogans serovar Copenhageni, which was the same serovar isolated from patients during hospital-based surveillance. Leptospira spp. were not isolated from 8 captured Didelphis marsupialis (Opossum), while 5/7 had a positive MAT titre against a saprophytic serogroup. R. rattus were not captured during the survey. The study findings indicate that the brown rat is a major rodent reservoir for leptospirosis in this urban setting. Furthermore, the high carriage rates of L. interrogans serovar Copenhageni in captured rats suggest that there is a significant degree of environmental contamination with this agent in the household environment of high risk areas, which in turn is a cause of transmission during urban epidemics. PMID:18721789

  7. In vivo cell aggregations of a recent swine biofilm-forming isolate of Leptospira interrogans strain from Argentina.

    PubMed

    Brihuega, Bibiana; Samartino, Luis; Auteri, Carmelo; Venzano, Agustín; Caimi, Karina

    2012-01-01

    Leptospirosis is a zoonosis of ubiquitous distribution caused by spirochetes. Leptospires exist either as saprophytic water-associated organisms or as animal pathogens that can survive in water. Previous works have demonstrated that both saprophytic and pathogenic leptospires are able to produce functional biofilms, which consist of a community of bacteria embedded in an extracellular matrix attached to a surface. This structure is believed to provide protection from environmental aggressiveness. In the present study, we analyzed the capacity of biofilm formation both of a a recent field isolate of Leptospira interrogans serovar Pomona obtained from an aborted swine fetus and of the saprophytic Leptospira biflexa serovar Patoc. We used light microscopy, immunofluorescence, and scanning electron microscopic examinations on glass and polystyrene plate models to evaluate the process in vitro. The ability to form bacterial aggregations in vivo was tested using pregnant guinea pigs infected with both strains. We obtained biofilms both on glass and plastic surfaces. Scanning electron microscopic analysis showed differences in the biofilm structure formed by both strains. L. interrogans serovar Pomona cell aggregations were observed in placental tissues by light microscopy. Biofilms and cell aggregations are consistent with the life of saprophytic strains in water and could help pathogenic strains to colonize the host and lead to abortion in pregnant animals. PMID:23102459

  8. Leptospira interrogans at the human-wildlife interface in northern Botswana: a newly identified public health threat.

    PubMed

    Jobbins, S E; Sanderson, C E; Alexander, K A

    2014-03-01

    Leptospirosis is the most widespread zoonosis in the world. In northern Botswana, humans live in close proximity to a diversity of wildlife and peridomestic rodents and may be exposed to a variety of zoonotic pathogens. Little is known regarding the occurrence and epidemiology of L. interrogans in Africa despite the recognized global importance of this zoonotic disease and the threat it poses to public health. In Botswana, banded mongooses (Mungos mungo) live in close proximity to humans across protected and unprotected landscapes and may be a useful sentinel species for assessing the occurrence of zoonotic organisms, such as L. interrogans. We utilized PCR to screen banded mongoose kidneys for leptospiral DNA and identified 41.5% prevalence of renal carriage of L. interrogans (exact binomial 95% CI 27.7-56.7%, n = 41). Renal carriage was also detected in one Selous' mongoose (Paracynictis selousi). This is the first published confirmation of carriage of L. interrogans in either species. This is also the first report of L. interrogans occurrence in northern Botswana and the only report of this organism in a wildlife host in the country. Pathogenic Leptospira are usually transmitted indirectly to humans through soil or water contaminated with infected urine. Other avenues, such as direct contact between humans and wildlife, as well as consumption of mongooses and other wildlife as bushmeat, may pose additional exposure risk and must be considered in public health management of this newly identified zoonotic disease threat. There is a critical need to characterize host species involvement and pathogen transmission dynamics, including human-wildlife interactions that may increase human exposure potential and infection risk. We recommend that public health strategy be modified to include sensitization of medical practitioners to the presence of L. interrogans in the region, the potential for human infection, and implementation of clinical screening. This study

  9. Profiling of Leptospira interrogans, L. santarosai, L. meyeri and L. borgpetersenii by SE-AFLP, PFGE and susceptibility testing--a continuous attempt at species and serovar differentiation.

    PubMed

    Moreno, Luisa Z; Miraglia, Fabiana; Lilenbaum, Walter; Neto, José S F; Freitas, Julio C; Morais, Zenaide M; Hartskeerl, Rudy A; da Costa, Barbara L P; Vasconcellos, Silvio A; Moreno, Andrea M

    2016-01-01

    Leptospirosis is a widespread systemic zoonosis, considered as reemerging in certain developing countries. Although the cross agglutinin absorption test is still considered the standard method for Leptospira identification, it presents several disadvantages. The aim of this study was to characterize Leptospira spp. isolated from various hosts by genotyping and broth microdilution susceptibility testing in an attempt to differentiate Leptospira species, serogroups and serovars. Forty-seven isolates were studied. They were previously serotyped, and species confirmation was performed by 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) and pulsed-field gel electrophoresis (PFGE) analysis enabled the distinction of L. interrogans from L. santarosai, L. meyeri and L. borgpetersenii in two main clusters. Among L. interrogans, it was possible to differentiate into two new clusters the serogroup Icterohaemorrhagiae from the serogroups Canicola and Pomona. L. santarosai isolates presented higher genetic variation than the other species in both techniques. Interestingly, the minimum inhibitory concentration (MIC) cluster analysis also provided Leptospira serogroup differentiation. Further studies are necessary regarding serovar Bananal isolates, as they presented the highest MIC values for most of the antimicrobials tested. All studied techniques successfully distinguished Leptospira species and serogroups. Despite being library-dependent methods, these approaches are less labor intensive and more economically viable, particularly SE-AFLP, and can be implemented in most reference laboratories worldwide to enable faster Leptospira typing. PMID:26956446

  10. Profiling of Leptospira interrogans, L. santarosai, L. meyeri and L. borgpetersenii by SE-AFLP, PFGE and susceptibility testing—a continuous attempt at species and serovar differentiation

    PubMed Central

    Moreno, Luisa Z; Miraglia, Fabiana; Lilenbaum, Walter; Neto, José SF; Freitas, Julio C; Morais, Zenaide M; Hartskeerl, Rudy A; da Costa, Barbara LP; Vasconcellos, Silvio A; Moreno, Andrea M

    2016-01-01

    Leptospirosis is a widespread systemic zoonosis, considered as reemerging in certain developing countries. Although the cross agglutinin absorption test is still considered the standard method for Leptospira identification, it presents several disadvantages. The aim of this study was to characterize Leptospira spp. isolated from various hosts by genotyping and broth microdilution susceptibility testing in an attempt to differentiate Leptospira species, serogroups and serovars. Forty-seven isolates were studied. They were previously serotyped, and species confirmation was performed by 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) and pulsed-field gel electrophoresis (PFGE) analysis enabled the distinction of L. interrogans from L. santarosai, L. meyeri and L. borgpetersenii in two main clusters. Among L. interrogans, it was possible to differentiate into two new clusters the serogroup Icterohaemorrhagiae from the serogroups Canicola and Pomona. L. santarosai isolates presented higher genetic variation than the other species in both techniques. Interestingly, the minimum inhibitory concentration (MIC) cluster analysis also provided Leptospira serogroup differentiation. Further studies are necessary regarding serovar Bananal isolates, as they presented the highest MIC values for most of the antimicrobials tested. All studied techniques successfully distinguished Leptospira species and serogroups. Despite being library-dependent methods, these approaches are less labor intensive and more economically viable, particularly SE-AFLP, and can be implemented in most reference laboratories worldwide to enable faster Leptospira typing. PMID:26956446

  11. Isolation of Salmonella enterica and serologic reactivity to Leptospira interrogans in opossums (Didelphis virginiana) from Yucatán, México.

    PubMed

    Ruiz-Pina, Hugo Antonio; Puc-Franco, Miguel Angel; Flores-Abuxapqui, Javier; Vado-Solis, Ignacio; Cardenas-Marrufo, María Fidelia

    2002-01-01

    The presence of Salmonella enterica and serologic evidence of infection by Leptospira interrogans, were detected in the opossum Didelphis virginiana in a semi-urban locality of the Yucatán State, México. Ninety-one opossums were captured during the period April 1996 and May 1998. From a total of 17 feces samples, four Salmonella enterica subsp. enterica serotypes (Sandiego, Newport, Anatum, and Minnesota), and one Salmonella enterica subsp. arizonae serovar O44:Z4,Z23:- were isolated. Some opossums presented mixed infections. From 81 sera samples, four (4.9%) were positive to antibodies to Leptospira serovars pomona and wolfii. Both animals infected with Salmonella enterica and those serologically positive to Leptospira interrogans were captured in peridomestic habitat. Opossums infected with Salmonella enterica, were captured in dry season, and those seropositive to Leptospira interrogans during the rainy season. The implications of infection and reactivity of these zoonotic pathogens in D. virginiana in the Yucatan state are briefly discussed. PMID:12219118

  12. A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans.

    PubMed

    Caimi, Karina; Varni, Vanina; Melendez, Yamil; Koval, Ariel; Brihuega, Bibiana; Ruybal, Paula

    2012-08-01

    Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context. PMID:22850955

  13. Real-Time PCR Reveals Rapid Dissemination of Leptospira interrogans after Intraperitoneal and Conjunctival Inoculation of Hamsters.

    PubMed

    Wunder, Elsio A; Figueira, Claudio P; Santos, Gisele R; Lourdault, Kristel; Matthias, Michael A; Vinetz, Joseph M; Ramos, Eduardo; Haake, David A; Picardeau, Mathieu; Dos Reis, Mitermayer G; Ko, Albert I

    2016-07-01

    The pathogen Leptospira interrogans is a highly motile spirochete that causes acute and fulminant infections in humans and other accidental hosts. Hematogenous dissemination is important for infection by the pathogen but remains poorly understood because few animal model studies have used sensitive tools to quantify the bacteria. We evaluated the kinetics of leptospiral infection in Golden Syrian hamsters by a sensitive quantitative real-time PCR (TaqMan) with lipl32 as the target gene. The dissemination and bacterial burden were measured after intraperitoneal infection with a high dose (10(8)) or low dose (2.5 × 10(2)) of leptospires. We also examined the conjunctival challenge route to mimic the natural history of infection. Quantification of leptospires in perfused animals revealed that pathogens were detected in all organs of intraperitoneally infected hamsters, including the eye and brain, within 1 h after inoculation of 10(8) virulent L. interrogans bacteria. Peaks of 10(5) to 10(8) leptospires per gram or per milliliter were achieved in blood and all tissues between day 4 and day 8 after intraperitoneal inoculation of high- and low-dose challenges, respectively, coinciding with macroscopic and histological changes. The conjunctival route resulted in a delay in the time to peak organ burden in comparison to intraperitoneal infection, indicating that although infection could be established, penetration efficiency was low across this epithelial barrier. Surprisingly, infection with a large inoculum of high-passage-number attenuated L. interrogans strains resulted in dissemination to all organs in the first 4 days postinfection, albeit with a lower burden, followed by clearance from the blood and organs 7 days postinfection and survival of all animals. These results demonstrate that leptospiral dissemination and tissue invasion occur. In contrast, development of a critical level of tissue burden and pathology are dependent on the virulence of the infecting

  14. Seasonal prevalence of antibodies to Leptospira interrogans in Antillean manatees from a landlocked lake in Tabasco, Mexico.

    PubMed

    Aragón-Martínez, Arianna; Olivera-Gómez, León D; Jiménez-Domínguez, Darwin

    2014-07-01

    Factors that alter the dynamics of ecologic systems can influence transmission of infectious diseases and may lead to decreases in natural populations. Leptospirosis is a cosmopolitan disease of zoonotic importance that affects most mammals. At the southern Gulf of Mexico, Antillean manatees (Trichechus manatus manatus) inhabit highly variable environments, with extended floods during the rainy season and drought conditions during the dry season that affect food availability and the thermal environment for manatees. We tested for changes in prevalence and titers of antibodies to 12 serovars of Leptospira interrogans in manatees between dry and rainy seasons. We determined titers for L. interrogans through microscopic agglutination tests (MAT) from 10 manatees, six during the dry season (DS), and six during the rainy season (RS) in Laguna de las Ilusiones, a landlocked lake hosting a population of about 20 manatees. All individuals were antibody positive (titers ≥ 100) to at least one serovar. The serovars bataviae, bratislava, canicola, and icterohaemorrhagiae had overall prevalences ≥ 50%; bataviae, bratislava, and canicola had prevalences ≥ 50% during both seasons. Serovars icterohaemorrhagiae and pyrogenes had prevalences ≥ 50% during DS and pomona, tarassovi, wolfii, and autumnalis during RS. Significant differences in prevalence between seasons were found for pomona, tarassovi, and autumnalis. Titers of tarassovi, wolfii, autumnalis, and bataviae were significantly higher during RS. There was a high prevalence of L. interrogans during the RS independent of high availability of plant foods, coinciding with the epizootiology of the bacteria that are endemic to tropical regions. Another factor possibly influencing prevalence is high anthropogenic pressure at the lake, causing an increase in potential sources of infection. Because of possible cross-reaction in MAT, further research is needed on the molecular discrimination of serovars in animals in the

  15. Response of Leptospira interrogans to Physiologic Osmolarity: Relevance in Signaling the Environment-to-Host Transition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transmission of pathogenic Leptospira between mammalian hosts usually involves dissemination via soil or water contaminated by the urine of reservoir hosts. The ability of Leptospira to adapt to the variety of conditions found inside and outside of the host is reflected in the relatively large geno...

  16. Post-translational modification of LipL32 during Leptospira interrogans infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world’s most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize ...

  17. Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans

    PubMed Central

    Schons-Fonseca, Luciane; da Silva, Josefa B.; Milanez, Juliana S.; Domingos, Renan H.; Smith, Janet L.; Nakaya, Helder I.; Grossman, Alan D.; Ho, Paulo L.; da Costa, Renata MA

    2016-01-01

    We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e.g. nucleotide excision repair uvrABC, recombinases recBCD and resolvases ruvABC) were not induced. Genes involved in various metabolic pathways were down regulated, including genes involved in cell growth, RNA metabolism and the tricarboxylic acid cycle. From ChIP-seq data, we observed 24 LexA1 binding sites located throughout chromosome 1 and one binding site in chromosome 2. Expression of many, but not all, genes near those sites was increased following DNA damage. Binding sites were found as far as 550 bp upstream from the start codon, or 1 kb into the coding sequence. Our findings indicate that there is a shift in gene expression following DNA damage that represses genes involved in cell growth and virulence, and induces genes involved in mutagenesis and recombination. PMID:26762976

  18. Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans.

    PubMed

    Schons-Fonseca, Luciane; da Silva, Josefa B; Milanez, Juliana S; Domingos, Renan H; Smith, Janet L; Nakaya, Helder I; Grossman, Alan D; Ho, Paulo L; da Costa, Renata M A

    2016-02-18

    We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e.g. nucleotide excision repair uvrABC, recombinases recBCD and resolvases ruvABC) were not induced. Genes involved in various metabolic pathways were down regulated, including genes involved in cell growth, RNA metabolism and the tricarboxylic acid cycle. From ChIP-seq data, we observed 24 LexA1 binding sites located throughout chromosome 1 and one binding site in chromosome 2. Expression of many, but not all, genes near those sites was increased following DNA damage. Binding sites were found as far as 550 bp upstream from the start codon, or 1 kb into the coding sequence. Our findings indicate that there is a shift in gene expression following DNA damage that represses genes involved in cell growth and virulence, and induces genes involved in mutagenesis and recombination. PMID:26762976

  19. Leptospira interrogans survey by PCR in wild rodents coming from different urban areas of Palermo, Italy.

    PubMed

    Vitale, Maria; Di Bella, Carobelo; Agnello, Stefano; Curro, Victoria; Vicari, Domenico; Vitale, Fabrizio

    2007-01-01

    DNA extracted from the kidneys of rodents captured in different urban areas of Palermo, Italy, had been analysed for the presence of pathogenic L. interrogans sensu latu DNA. PCR analysis had shown that in rodents captured close to green areas and small river up to 40 % animals give positive PCR results. Not many cases of human leptospirosis are reported in Sicilian island in which hot season is usually dry. But considering climate change toward subtropical aspect in Sicily, with hot humid summer and sudden thunderstorm, screening for L. interrogans sensu latu prevalence can be useful for leptospirosis risk analysis on human population. PMID:23427420

  20. Molecular basis of the inhibitor selectivity and insights into the feedback inhibition mechanism of citramalate synthase from Leptospira interrogans.

    PubMed

    Zhang, Peng; Ma, Jun; Zhang, Zilong; Zha, Manwu; Xu, Hai; Zhao, Guoping; Ding, Jianping

    2009-07-01

    LiCMS (Leptospira interrogans citramalate synthase) catalyses the first reaction of the isoleucine biosynthesis pathway in L. interrogans, the pathogen of leptospirosis. The catalytic reaction is regulated through feedback inhibition by its end product isoleucine. To understand the molecular basis of the high selectivity of the inhibitor and the mechanism of feedback inhibition, we determined the crystal structure of LiCMSC (C-terminal regulatory domain of LiCMS) in complex with isoleucine, and performed a biochemical study of the inhibition of LiCMS using mutagenesis and kinetic methods. LiCMSC forms a dimer of dimers in both the crystal structure and solution and the dimeric LiCMSC is the basic functional unit. LiCMSC consists of six beta-strands forming two anti-parallel beta-sheets and two alpha-helices and assumes a betaalphabeta three-layer sandwich structure. The inhibitor isoleucine is bound in a pocket at the dimer interface and has both hydrophobic and hydrogen-bonding interactions with several conserved residues of both subunits. The high selectivity of LiCMS for isoleucine over leucine is primarily dictated by the residues, Tyr430, Leu451, Tyr454, Ile458 and Val468, that form a hydrophobic pocket to accommodate the side chain of the inhibitor. The binding of isoleucine has inhibitory effects on the binding of both the substrate, pyruvate, and coenzyme, acetyl-CoA, in a typical pattern of K-type inhibition. The structural and biochemical data from the present study together suggest that the binding of isoleucine affects the binding of the substrate and coenzyme at the active site, possibly via conformational change of the dimer interface of the regulatory domain, leading to inhibition of the catalytic reaction. PMID:19351325

  1. A Methylated Phosphate Group and Four Amide-linked Acyl Chains in Leptospira interrogans Lipid A. The Membrane Anchor of an Unusual Lipopolysaccharide that Activates TLR2*

    PubMed Central

    Que-Gewirth, Nanette L. S.; Ribeiro, Anthony A.; Kalb, Suzanne R.; Cotter, Robert J.; Bulach, Dieter M.; Adler, Ben; Girons, Isabelle Saint; Werts, Catherine; Raetz, Christian R. H.

    2008-01-01

    Leptospira interrogans differs from other spirochetes in that it contains homologs of all the Escherichia coli lpx genes required for the biosynthesis of the lipid A anchor of lipopolysaccharide (LPS). LPS from L. interrogans cells is unusual in that it activates TLR2 rather than TLR4. The structure of L. interrogans lipid A has now been determined by a combination of matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR spectroscopy, and biochemical studies. Lipid A was released from LPS of L. interrogans serovar Pomona by 100 °C hydrolysis at pH 4.5 in the presence of SDS. Following purification by anion exchange and thin layer chromatography, the major component was shown to have a molecular weight of 1727. Mild hydrolysis with dilute NaOH reduced this to 1338, consistent with the presence of four N-linked and two O-linked acyl chains. The lipid A molecules of both the virulent and nonvirulent forms of L. interrogans serovar Icterohaemorrhagiae (strain Verdun) were identical to those of L. interrogans Pomona by the above criteria. Given the selectivity of L. interrogans LpxA for 3-hydroxylaurate, we propose that L. interrogans lipid A is acylated with R-3-hydroxylaurate at positions 3 and 3′ and with R-3-hydroxypalmitate at positions 2 and 2′. The hydroxyacyl chain composition was validated by gas chromatography and mass spectrometry of fatty acid methyl esters. Intact hexa-acylated lipid A of L. interrogans Pomona was also analyzed by NMR, confirming the presence a β-1′,6-linked disaccharide of 2,3-diamino-2,3-dideoxy-D-glucopyranose units. Two secondary unsaturated acyl chains are attached to the distal residue. The 1-position of the disaccharide is derivatized with an axial phosphate moiety, but the 4′-OH is unsubstituted. 1H and 31P NMR analyses revealed that the 1-phosphate group is methylated. Purified L. interrogans lipid A is inactive against human THP-1 cells but does stimulate tumor necrosis factor production by

  2. The multifunctional LigB adhesin binds homeostatic proteins with potential roles in cutaneous infection by pathogenic Leptospira interrogans.

    PubMed

    Choy, Henry A; Kelley, Melissa M; Croda, Julio; Matsunaga, James; Babbitt, Jane T; Ko, Albert I; Picardeau, Mathieu; Haake, David A

    2011-01-01

    Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9-11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react

  3. Characterization of Leptospira interrogans Serovars by Polymorphism Variable Number Tandem Repeat Analysis

    PubMed Central

    Rezasoltani, Sama; Dabiri, Hossein; Khaki, Pejvak; Rostami Nejad, Mohammad; Karimnasab, Nasim; Modirrousta, Shiva

    2015-01-01

    Background: Leptospirosis is recognized as a re-emerging infectious disease; therefore, understanding the epidemiology of the disease is vital for designing intervention programs and diminishing its transmission. Recently, Multilocus variable number tandem repeat analysis (MLVA) is used for segregating and identifying Leptospira serovars. The method has potential application in investigating the molecular epidemiology of Leptospira. Objectives: The propose of this study was genomic identification of pathogenic Leptospires in Iran by MLVA. Materials and Methods: Leptospira serovars were obtained from National Reference Laboratory of Leptospira at Razi Vaccine and Serum Research Institute, Karaj, Iran. Serovars were cultured into the liquid EMJH medium and incubated at 28˚C for 7 days. DNA of serovars was extracted using the phenol-chloroform method. PCR was performed with 5 selected variable number tandem repeat analysis (VNTR) loci. The amplified products were analyzed by agarose gel electrophoresis. The size of the amplified products was estimated by 100 bp ladder and sequencing analysis. Results: The saprophytic serovar showed no amplified fragments. PCR products in all pathogenic serovars were observed. The 12 reference serovars used for the development of technique displayed distinct patterns. Conclusions: Results showed that MLVA technique with its range of polymorphism is a good marker for identification of pathogenic serovars. Some VNTR loci are more powerful than the other ones with regard to differentiation. Serovars from the same geographical area have more genetic similarity than same serovars from different places. MLVA is a suitable technique for epidemiological survey. PMID:26568805

  4. Asymptomatic and chronic carriage of Leptospira interrogans serovar Pomona in California sea lions (Zalophus californianus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since 1970, periodic outbreaks of leptospirosis, caused by pathogenic spirochetes in the genus Leptospira, have caused morbidity and mortality of California sea lions (Zalophus californianus) along the Pacific coast of North America. Yearly seasonal epizootics of varying magnitude occur between the ...

  5. Comparative genomic analysis of eight Leptospira strains from Japan and the Philippines revealing the existence of four putative novel genomic islands/islets in L. interrogans serovar Lai strain 56601.

    PubMed

    Youn, Jung-Ho; Hayashida, Kyoko; Koizumi, Nobuo; Ohnishi, Makoto; Sugimoto, Chihiro

    2014-12-01

    Leptospirosis is one of the most widespread zoonotic diseases worldwide and can be considered an emerging health problem to both human and animal. Despite the importance of the disease, complete genome sequences are currently available for only three Leptospira interrogans strains: 56601, Fiocruz L1-130, and IPAV. Therefore, intra- and inter-species comparative genomic analyses of Leptospira are limited. Here, to advance current knowledge of the genomic differences within Leptospira species, next-generation sequencing technology was used to examine the genomes of eight L. interrogans strains belonging to six different serogroups isolated from humans and dogs in Japan and the Philippines. The genomic sequences were mapped to that of the reference strain, L. interrogans serovar Lai strain 56601. The results revealed the presence of four novel genomic islands/islets (GIs) in strain 56601. This study provides a deeper insight into the molecular basis and evolutionary perspective of the virulence of leptospires. PMID:25449997

  6. Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis.

    PubMed

    Lourdault, Kristel; Aviat, Florence; Picardeau, Mathieu

    2009-05-01

    The dynamics of leptospirosis infection have been poorly studied. The purpose of this study was to determine the LD(50), rate of bacterial dissemination, histopathology and antibody responses against leptospira following inoculation with the highly virulent Leptospira interrogans Fiocruz L1-130 strain in a guinea pig model of leptospirosis. Three routes of infection (intraperitoneal, conjunctival and subcutaneous inoculation) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in the blood and tissues of infected animals were determined over a 1 week course of infection using quantitative real-time PCR (qPCR). Bacteraemia peaked at day 5 post-infection reaching more than 5x10(4) leptospires ml(-1). The highest spirochaetal load was found in the liver and kidneys, and was associated with alterations in organ tissues and a decline in liver and kidney functions. In contrast, lesions and bacteria were not detected in guinea pigs infected with an avirulent strain derived from a high-passage-number in vitro-passaged variant of the Fiocruz L1-130 strain. The use of qPCR supports the findings of earlier studies and provides an easy and reliable method for the quantification of L. interrogans in the tissues of infected animals. qPCR will be used in future studies to evaluate the efficacy of vaccine candidates against leptospirosis and the virulence of selected L. interrogans mutants relative to the parental strain. PMID:19369528

  7. Distribution of Leptospira interrogans by Multispacer Sequence Typing in Urban Norway Rats (Rattus norvegicus): A Survey in France in 2011-2013

    PubMed Central

    Bicout, Dominique J.; Kodjo, Angeli; Artois, Marc; Djelouadji, Zoheira

    2015-01-01

    Background Urban leptospirosis has increasingly been reported in both developing and developed countries. The control of the disease is limited because our understanding of basic aspects of the epidemiology, including the transmission routes of leptospires among rat populations, remains incomplete. Through the ability to distinguish among Leptospira strains in rats, multispacer sequence typing (MST) could provide a modern understanding of Leptospira epidemiology; however, to our knowledge, the distribution of Leptospira strains among urban rat colonies has not been investigated using MST. Aims and Methodology The objective of this study was to identify the Leptospira strains present in rats (Rattus norvegicus) in Lyon (France) using MST and to characterize their spatial distribution. Kidneys and urine were collected from rats trapped live in seven locations in the city and in one suburban location. Each location was considered to represent a rat colony. Bacterial cultures and quantitative polymerase chain reaction (qPCR) assays were performed, and the L. interrogans DNA identified was then genotyped using MST. The distributions of Leptospira strains were spatially described. Key Results Among 84 wild rats, MST profiles were obtained in 35 of 37 rats that had a positive result for L. interrogans by bacterial culture and/or qPCR analyses. All of the MST profiles were related to reference strains previously isolated from human patients that belong to the serogroup Icterohaemorrhagiae and the serovars [strain(s)] Copenhageni [Wijinberg or M20] (n = 26), Icterohaemorrhagiae [CHU Réunion] (n = 7), Icterohaemorrhagiae [R1] (n = 1) and Copenhageni [Shibaura 9] (n = 1). Each colony was infected with leptospires having the same MST profile. Major Conclusions This study demonstrated that MST could be used for the purpose of field studies, either on culture isolates or on DNA extracted from kidneys and urine, to distinguish among L. interrogans isolates in rats. MST could

  8. Features of Two New Proteins with OmpA-Like Domains Identified in the Genome Sequences of Leptospira interrogans

    PubMed Central

    Teixeira, Aline F.; de Morais, Zenaide M.; Kirchgatter, Karin; Romero, Eliete C.; Vasconcellos, Silvio A.; Nascimento, Ana Lucia T. O.

    2015-01-01

    Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG)-interacting proteins, capable of generating plasmin (PLA) and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis. PMID:25849456

  9. Occurrence of antibodies anti -Toxoplasma gondii, Neospora caninum and Leptospira interrogans in a captive deer herd in Southern Brazil.

    PubMed

    Zimpel, Cristina Kraemer; Grazziotin, Ana Laura; de Barros Filho, Ivan Roque; Guimaraes, Ana Marcia de Sa; dos Santos, Leonilda Correia; de Moraes, Wanderlei; Cubas, Zalmir Silvino; de Oliveira, Marcos Jose; Pituco, Edviges Maristela; Lara, Maria do Carmo Custódio de Souza Hunold; Villalobos, Eliana Monteforte Cassaro; Silva, Lília Marcia Paulin; Cunha, Elenice Maria Sequetin; Castro, Vanessa; Biondo, Alexander Welker

    2015-01-01

    A large number of Brazilian zoos keep many endangered species of deer, however, very few disease surveillance studies have been conducted among captive cervids. Blood samples from 32 Brazilian deer (Blastocerus dichotomus, Mazama nana and Mazama americana) kept in captivity at Bela Vista Biological Sanctuary (Foz do Iguaçu, Brazil) were investigated for 10 ruminant pathogens, with the aims of monitoring deer health status and evaluating any potential zoonotic risk. Deer serum samples were tested for Brucella abortus, Leptospira (23 serovars), Toxoplasma gondii, Neospora caninum, bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, foot-and-mouth disease virus, western equine encephalitis virus, eastern equine encephalitis virus and Venezuelan equine encephalitis virus. Antibodies against T. gondii (15.6%), N. caninum (6.2%) and L. interrogans serogroup Serjoe (3.1%) were detected. The serological results for all other infectious agents were negative. The deer were considered to be clinically healthy and asymptomatic regarding any disease. Compared with studies on free-ranging deer, the prevalences of the same agents tested among the captive deer kept at the Sanctuary were lower, thus indicating good sanitary conditions and high-quality management practices at the zoo. PMID:26689185

  10. Risk factors associated with prevalence of antibodies to Leptospira interrogans in a metapopulation of black-tailed prairie dogs in Mexico.

    PubMed

    Montiel-Arteaga, Ana; Atilano, Daniel; Ayanegui, Alejandra; Ceballos, Gerardo; Suzán, Gerardo

    2015-01-01

    Interest in the study of infectious diseases of wildlife has grown in recent decades and now focuses on understanding host-parasite dynamics and factors involved in disease occurrence. The black-tailed prairie dog (Cynomys ludovicianus) is a useful species for this type of investigation because it lives in heterogeneous landscapes where human activities take place, and its populations are structured as a metapopulation. Our goal was to determine if colony area, density, and proximity to human settlements are associated with prevalence of antibodies to Leptospira interrogans in black-tailed prairie dogs of northwestern Chihuahua State, Mexico. We captured 266 prairie dogs in 11 colonies in 2009 and analyzed 248 serum samples with the microscopic agglutination test (MAT) for antibody to any of the 12 pathogenic serovars of L. interrogans. Serologically positive test results for only serovars Bratislava, Canicola, Celledoni, and Tarassovi were considered for statistical analysis. Almost 80% of sera were positive for at least one pathogenic serovar (MAT titer ≥1∶80). The highest recorded antibody prevalences were to serovars Bratislava and Canicola. Correlation analysis showed a negative relationship between L. interrogans antibody prevalence and colony area (r = -0.125, P<0.005), suggesting that animals living in larger colonies were at a lower risk than those in smaller colonies. The correlation between the serovar Canicola and distance was negative (r = -0.171, P<0.007), and this relationship may be explained by the presence of domestic dogs associated with human dwellings. This is the first study of Leptospira spp. antibody prevalence in prairie dogs, and it provides valuable insights into the dynamics of leptospirosis in threatened wildlife species. Further studies are needed to evaluate the impact of Leptospira serovars in metapopulations of prairie dogs and other domestic and wild mammals in grassland communities. PMID:25380365

  11. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    PubMed Central

    Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.

    2013-01-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319

  12. Prevalence of Leptospira interrogans antibodies in free-ranging Tayassu pecari of the Southern Pantanal, Brazil, an ecosystem where wildlife and cattle interact.

    PubMed

    de Freitas, Tatiana P Tavares; Keuroghlian, Alexine; Eaton, Donald P; de Freitas, Emanuel Barbosa; Figueiredo, Aline; Nakazato, Luciano; de Oliveira, Jacqueline M; Miranda, Flávia; Paes, Rita Cassia S; Monteiro, Leticia A R Carneiro; Lima, José Vergílio B; da C Neto, Aparecida A; Dutra, Valéria; de Freitas, Julio Cesar

    2010-12-01

    We surveyed a wild population of white-lipped peccaries (Tayassu pecari) in the Brazilian Pantanal for evidence of Leptospira interrogans. Serum samples from 71 free-ranging T. pecari were obtained between 2003 and 2005 in the southern Pantanal of Mato Grosso do Sul state. We used microscopic microagglutination to test for antibodies against 14 L. interrogans serovars (antibody titers ≥ 1:100 were considered seropositive). Seventy percent of captured animals tested positive for leptospirosis antibodies. Antibodies against icterohaemorrhagiae and autumnalis serovars were the most prevalent. We used log-linear analyses to test for associations among seropositivity, age class, and sex of captured animals. Seropositivity was strongly associated with animal age class, but independent of sex. Forty-six percent of animals less than 2 years old, 63% of adults during peak reproductive years, and 100% of the oldest age class were seropositive. A nonparametric multivariate procedure (MRPP) showed that the composition of serovar antibody types changed with age, and ANOVA models demonstrated that antibody titers increased with age, suggesting long-term exposure to a greater number and variety (i.e., serovar types) of L. interrogans infections. This study presents the first quantitative survey of antibodies against L. interrogans serovars in a T. pecari population of the Pantanal. The high prevalence of leptospirosis antibodies in free-ranging white-lipped peccaries and the potential impacts on reproduction and population dynamics emphasize the need for further studies investigating the roles of Pantanal wildlife and livestock in the transmission and maintenance of L. interrogans in the environment. PMID:20596776

  13. Identification of Seroreactive Proteins of Leptospira interrogans Serovar Copenhageni Using a High-Density Protein Microarray Approach

    PubMed Central

    Lessa-Aquino, Carolina; Borges Rodrigues, Camila; Pablo, Jozelyn; Sasaki, Rie; Jasinskas, Algis; Liang, Li; Wunder, Elsio A.; Ribeiro, Guilherme S.; Vigil, Adam; Galler, Ricardo; Molina, Douglas; Liang, Xiaowu; Reis, Mitermayer G.; Ko, Albert I.; Medeiros, Marco Alberto; Felgner, Philip L.

    2013-01-01

    Background Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease. Methodology In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results. Principal findings We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients. Conclusions Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for

  14. SEROPREVALENCE OF NINE LEPTOSPIRA INTERROGANS SEROVARS IN WILD CARNIVORES, UNGULATES, AND PRIMATES FROM A ZOO POPULATION IN A METROPOLITAN REGION OF CHILE.

    PubMed

    Moreno-Beas, Eduardo; Abalos, Pedro; Hidalgo-Hermoso, Ezequiel

    2015-12-01

    Serum samples from 130 individuals representing 42 species of carnivores, ungulates, and primates from a population of captive mammals in Metropolitan Region in Chile were tested for antibodies against nine serovars of Leptospira interrogans using the microscopic agglutination test. Ten percent of the animals were seropositive to one or more serovars. Seroprevalence was significantly higher in ungulates (20.4%) compared to carnivores (3.8%) and primates (3.4%). There were no significant differences in seroprevalence among sex and age ranges. The most frequent serovar detected was Autumnalis, present in 53.4% of antibody-positive animals. Most positive animals had titers of ≤1 : 200, except for a maned wolf ( Chrysocyon brachyurus ) with titers of 1 : 400 against serovar Hardjo. To the authors' knowledge, this is the first report of Leptospira exposure detected in native endangered pudu ( Pudu puda ) and the first confirmation of exposure to L. interrogans in captive wild mammals in Chile. Leptospirosis should be considered as a differential diagnosis in future disease presentation for hepatitis or abortions in captive mammals in Chile. PMID:26667533

  15. Multiple-locus variable-number tandem repeat analysis (MLVA) of Leptospira interrogans serovar Pomona from Argentina reveals four new genotypes.

    PubMed

    Pavan, María Elisa; Cairó, Fabián; Brihuega, Bibiana; Samartino, Luis

    2008-01-01

    Outbreaks of leptospirosis occur regularly in Argentina, but little is known about their epidemiological relationships. We have analyzed the genetic diversity of a collection of 16 strains of Leptospira interrogans serovar Pomona isolated from animals and humans in Argentina during the past 45 years. Genotyping was performed by multiple-locus variable-number tandem repeat analysis (MLVA) using the loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31, as described by Majed et al. [Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005;43:539-45]. Clustering analysis revealed four new distinct MLVA genotypes, with a dominant one. Strains with this genotype were consistently isolated since 1960 to the present, mainly from cows and pigs, but also from humans, representing 75% of the total strains studied. These strains coexisted temporally and geographically with isolates presenting the other new genotypes. VNTR4 locus, with four different alleles, presented the highest diversity between the VNTR loci analyzed. MLVA patterns obtained will be useful for future diagnostic and epidemiological tracing analysis. PMID:17531318

  16. Safety and efficacy of a new octavalent combined Erysipelas, Parvo and Leptospira vaccine in gilts against Leptospira interrogans serovar Pomona associated disease and foetal death.

    PubMed

    Jacobs, A A C; Harks, F; Hoeijmakers, M; Collell, M; Segers, R P A M

    2015-07-31

    The safety and protective efficacy of a new octavalent combination vaccine containing inactivated Erysipelothrix rhusiopathiae, Parvovirus, and Leptospira interrogans (sensu lato) serogroups Canicola, Icterohaemorrhagiae, Australis (Bratislava), Grippotyphosa, Pomona and Tarassovi - Porcilis(®) Ery+Parvo+Lepto - was evaluated in laboratory studies and under field conditions. The safety (2× overdose and repeated dose) was tested in 26 gilts. In this study, neither vaccine related temperature increase nor other systemic reactions were observed after intramuscular vaccination. No local reactions were observed except for one animal that had a small local reaction (2cm diameter) that lasted for 5 days after the third vaccination. Efficacy was tested in 40 gilts. A group of 20 gilts was vaccinated at 20 and 24 weeks of age with Porcilis(®) Ery+Parvo+Lepto and a group of 20 age- and source-matched animals served as the control group. The gilts were inseminated at 41 weeks or 66 weeks of age and were challenged with serovar Pomona 10 weeks after insemination, corresponding to 6 months (n=2×10) and 12 months (n=2×10) after the last vaccination. After both the 6- and 12-month challenges the control animals developed clinical signs (fever, lethargy and anorexia) and leptospiraemia as determined by positive blood culture. In addition, both the 6- and 12-month challenges resulted in death of 21% and 27% of the total number of foetuses in the control groups, respectively. Clinical signs and leptospiraemia were statistically significantly lower in vaccinated gilts after both the 6- and 12-month challenges. In addition, foetal death was statistically significantly lower (3% and 2%, respectively) in vaccinated gilts after both the 6- and 12 month challenges. The vaccine was tested further under field conditions on a Portuguese farm with a history of an increasing abortion rate associated with a Leptospira serovar Pomona infection (confirmed by PCR and serology). This study was

  17. Live Imaging of Bioluminescent Leptospira interrogans in Mice Reveals Renal Colonization as a Stealth Escape from the Blood Defenses and Antibiotics

    PubMed Central

    Ratet, Gwenn; Veyrier, Frédéric J.; Fanton d'Andon, Martine; Kammerscheit, Xavier; Nicola, Marie-Anne; Picardeau, Mathieu; Boneca, Ivo G.; Werts, Catherine

    2014-01-01

    Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Some animals asymptomatically carry L. interrogans in their kidneys and excrete bacteria in their urine, which contaminates the environment. Humans are infected through skin contact with leptospires and develop mild to severe leptospirosis. Previous attempts to construct fluorescent or bioluminescent leptospires, which would permit in vivo visualization and investigation of host defense mechanisms during infection, have been unsuccessful. Using a firefly luciferase cassette and random transposition tools, we constructed bioluminescent chromosomal transformants in saprophytic and pathogenic leptospires. The kinetics of leptospiral dissemination in mice, after intraperitoneal inoculation with a pathogenic transformant, was tracked by bioluminescence using live imaging. For infective doses of 106 to 107 bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2×108 bacteria, the septicemia led to the death of mice within 3 days post-infection. In surviving mice, one week after infection, pathogenic leptospires reemerged only in the kidneys, where they multiplied and reached a steady state, leading to a sustained chronic renal infection. These experiments reveal that a fraction of the leptospiral population escapes the potent blood defense, and colonizes a defined number of niches in the kidneys, proportional to the infective dose. Antibiotic treatments failed to eradicate leptospires that colonized the kidneys, although they were effective against L. interrogans if administered before or early after infection. To conclude, mice infected with bioluminescent L. interrogans proved to be a novel model to study both acute and chronic leptospirosis, and revealed that, in the kidneys, leptospires are protected from antibiotics. These bioluminescent leptospires represent a powerful new tool to

  18. "Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions"

    PubMed Central

    2012-01-01

    Background Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro. PMID:22463075

  19. A Model System for Studying the Transcriptomic and Physiological Changes Associated with Mammalian Host-Adaptation by Leptospira interrogans Serovar Copenhageni

    PubMed Central

    Caimano, Melissa J.; Sivasankaran, Sathesh K.; Allard, Anna; Hurley, Daniel; Hokamp, Karsten; Grassmann, André A.; Hinton, Jay C. D.; Nally, Jarlath E.

    2014-01-01

    Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of “core” housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for

  20. Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence.

    PubMed

    Pappas, Christopher J; Picardeau, Mathieu

    2015-11-01

    Leptospirosis is a zoonotic disease that affects ∼1 million people annually, with a mortality rate of >10%. Currently, there is an absence of effective genetic manipulation tools for targeted mutagenesis in pathogenic leptospires. Transcription activator-like effectors (TALEs) are a recently described group of repressors that modify transcriptional activity in prokaryotic and eukaryotic cells by directly binding to a targeted sequence within the host genome. To determine the applicability of TALEs within Leptospira spp., two TALE constructs were designed. First, a constitutively expressed TALE gene specific for the lacO-like region upstream of bgaL was trans inserted in the saprophyte Leptospira biflexa (the TALEβgal strain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALEβgal strain. Second, to study the role of LigA and LigB in pathogenesis, a constitutively expressed TALE gene with specificity for the homologous promoter regions of ligA and ligB was cis inserted into the pathogen Leptospira interrogans (TALElig). LigA and LigB expression was studied by using three independent clones: TALElig1, TALElig2, and TALElig3. Immunoblot analysis of osmotically induced TALElig clones demonstrated 2- to 9-fold reductions in the expression levels of LigA and LigB, with the highest reductions being noted for TALElig1 and TALElig2, which were avirulent in vivo and nonrecoverable from animal tissues. This study reconfirms galactosidase activity in the saprophyte and suggests a role for LigA and LigB in pathogenesis. Collectively, this study demonstrates that TALEs are effective at reducing the expression of targeted genes within saprophytic and pathogenic strains of Leptospira spp., providing an additional genetic manipulation tool for this genus. PMID:26341206

  1. Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

    PubMed Central

    Pappas, Christopher J.

    2015-01-01

    Leptospirosis is a zoonotic disease that affects ∼1 million people annually, with a mortality rate of >10%. Currently, there is an absence of effective genetic manipulation tools for targeted mutagenesis in pathogenic leptospires. Transcription activator-like effectors (TALEs) are a recently described group of repressors that modify transcriptional activity in prokaryotic and eukaryotic cells by directly binding to a targeted sequence within the host genome. To determine the applicability of TALEs within Leptospira spp., two TALE constructs were designed. First, a constitutively expressed TALE gene specific for the lacO-like region upstream of bgaL was trans inserted in the saprophyte Leptospira biflexa (the TALEβgal strain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALEβgal strain. Second, to study the role of LigA and LigB in pathogenesis, a constitutively expressed TALE gene with specificity for the homologous promoter regions of ligA and ligB was cis inserted into the pathogen Leptospira interrogans (TALElig). LigA and LigB expression was studied by using three independent clones: TALElig1, TALElig2, and TALElig3. Immunoblot analysis of osmotically induced TALElig clones demonstrated 2- to 9-fold reductions in the expression levels of LigA and LigB, with the highest reductions being noted for TALElig1 and TALElig2, which were avirulent in vivo and nonrecoverable from animal tissues. This study reconfirms galactosidase activity in the saprophyte and suggests a role for LigA and LigB in pathogenesis. Collectively, this study demonstrates that TALEs are effective at reducing the expression of targeted genes within saprophytic and pathogenic strains of Leptospira spp., providing an additional genetic manipulation tool for this genus. PMID:26341206

  2. Stray dogs as reservoirs of the zoonotic agents Leptospira interrogans, Trypanosoma cruzi, and Aspergillus spp. in an urban area of Chiapas in southern Mexico.

    PubMed

    Jimenez-Coello, Matilde; Ortega-Pacheco, Antonio; Guzman-Marin, Eugenia; Guiris-Andrade, Dario M; Martinez-Figueroa, Laura; Acosta-Viana, Karla Y

    2010-03-01

    This investigation determined the presence and prevalence of the zoonotic agents Leptospira interrogans, Trypanosoma cruzi, and Aspergillus spp. in the stray dog population (a total of 224 stray dogs) in an urban area of Southern Mexico. Blood serum samples were taken from all dogs, and root hair samples were taken from dogs with skin lesions and partial alopecia. IgG antibodies for L. interrogans from 10 serovars were detected using the microscopic agglutination test. Immunofluorescence antibody test and Western blot assay were used for serologic diagnosis of T. cruzi. The Sabouraud medium was used to isolate Aspergillus spp. Prevalence of L. interrogans was 4.9%, which was determined by identifying only serovars Pyrogenes, which accounted for 3.6%, and Tarassovi, which constituted 1.3%, with titers from 1:100 to 1:800. Additionally, T. cruzi antibodies were detected in 4.5% of the dogs. Skin lesions were found in 43% of the dogs (98/224), and 35 cultures were positive for Aspergillus spp. (35.7%, p < 0.05, 95% confidence interval 2.45-3.67), identified as A. niger (82.8%), A. flavus (14.3%), and A. terreus (2.9%). This study demonstrates the presence of certain zoonotic agents (bacteria, protozoa, and fungi) in stray dogs living within the studied area. Dogs play an important role in the transmission of diseases that are potentially harmful to humans. Although the prevalence of canine leptospirosis and trypanosomiasis is not high in Southern Mexico compared with other tropical regions of Mexico, the presence of these zoonotic agents in the stray dog population demonstrates that the stray dog population in this region is a significant reservoir and potential source of infection in humans. Special care should be taken when handling stray dogs that exhibit skin lesions with partial alopecia, since a pathological Aspergillus sp. fungus may be present. PMID:19514808

  3. Effect of exposure to Neospora caninum, Salmonella, and Leptospira interrogans serovar Hardjo on the economic performance of Irish dairy herds.

    PubMed

    O' Doherty, E; Sayers, R; O' Grady, L; Shalloo, L

    2015-04-01

    The objective of the current study was to quantify the effects of exposure to Salmonella, Neospora caninum, and Leptospira interrogans serovar Hardjo (L. hardjo) on dairy farm profitability and to simulate the effect of vaccination for Salmonella and L. hardjo on dairy farm profitability. The production effects associated with exposure to each of these pathogens in study herds were defined under 3 categories: (1) milk production effects, (2) reproduction effects (including culling), and (3) mortality effects. The production effects associated with exposure to Salmonella, N. caninum, and L. hardjo were incorporated into the Moorepark Dairy Systems Model. In the analysis, herds negative for exposure to Salmonella, N. caninum, and L. hardjo were assumed baseline herds, with all results presented relative to this base. In simulations examining the effect of vaccination for Salmonella and L. hardjo on farm profitability, vaccinated herds (vaccination costs included) were considered as baseline herds and results were presented relative to this base. Total annual profits in unvaccinated herds were reduced by €77.31, €94.71, and €112.11 per cow at milk prices of €0.24, €0.29, and €0.34/L, respectively, as a result of exposure to Salmonella. In the current study, herds positive for exposure to Salmonella recorded a 316-kg reduction in milk yield, whereas no association was detected between exposure to N. caninum or L. hardjo and milk production. Exposure to both N. caninum and L. hardjo was associated with compromised reproductive performance. Herds positive for exposure to N. caninum and Salmonella had greater rates of adult cow mortality and calf mortality, respectively. Vaccination for both Salmonella and L. hardjo was associated with improved performance in study herds. Exposure to N. caninum resulted in a reduction in annual farm profits of €11.55, €12, and €12.44 per cow at each milk price, whereas exposure to L. hardjo resulted in a reduction in

  4. Identification of Leptospira interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death

    PubMed Central

    Ojcius, David M.; Zhao, Xin; Sun, Dexter; Ge, Yu-Mei; Zheng, Lin-Li; Lin, Xu’ai; Li, Lan-Juan; Yan, Jie

    2013-01-01

    Background Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca2+ concentration ([Ca2+]i) elevation induced by infection can cause cell death, but [Ca2+]i changes and high [Ca2+]i-induced death of macrophages due to infection of Leptospira have not been previously reported. Methodology/Principal Findings We first used a Ca2+-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca2+]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca2+]i elevation was caused by both extracellular Ca2+ influx through the purinergic receptor, P2X7, and Ca2+ release from the endoplasmic reticulum, as seen by suppression of [Ca2+]i elevation when receptor-gated calcium channels were blocked or P2X7 was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3), which in turn induces intracellular Ca2+ release from endoplasmic reticulum, with the Km of 199 µM and Kcat of 8.566E-5 S-1. Secretion of L-PI-PLC from the spirochete into supernatants of leptospire-macrophage co-cultures and cytosol of infected macrophages was also observed by Western Blot assay. Lower [Ca2+]i elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca2+]i. Moreover, PI-PLCs (PI-PLC-β3 and PI-PLC-γ1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca2+]i increases induced apoptosis and necrosis of macrophages, while mild [Ca2+]i elevation only caused apoptosis. Conclusions/Significance This study demonstrated that L. interrogans infection induced [Ca2

  5. Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans

    PubMed Central

    Fontana, Célia; Lambert, Ambroise; Benaroudj, Nadia; Gasparini, David; Gorgette, Olivier; Cachet, Nathalie; Bomchil, Natalia; Picardeau, Mathieu

    2016-01-01

    Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence. PMID:27044038

  6. Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans.

    PubMed

    Fontana, Célia; Lambert, Ambroise; Benaroudj, Nadia; Gasparini, David; Gorgette, Olivier; Cachet, Nathalie; Bomchil, Natalia; Picardeau, Mathieu

    2016-01-01

    Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence. PMID:27044038

  7. Leptospira interrogans Lsa23 protein recruits plasminogen, factor H and C4BP from normal human serum and mediates C3b and C4b degradation.

    PubMed

    Siqueira, Gabriela H; Atzingen, Marina V; de Souza, Gisele O; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2016-02-01

    It has been reported that pathogenic Leptospira are resistant to normal human serum (NHS) due to their ability to evade the complement immune system by interacting with factor H (FH) and C4b-binding protein (C4BP) regulators. Moreover, plasmin generation on the leptospiral surface diminishes C3b and IgG deposition, decreasing opsonophagocytosis by immune competent cells. We have previously reported that Lsa23 (LIC11360) is a multipurpose protein capable of binding purified extracellular matrix molecules, FH, C4BP and plasminogen (PLG)/plasmin in the presence of PLG activators. In this work, we provide further evidence that Lsa23 is located at the bacterial surface by using immunofluorescence microscopy. We show that Lsa23 has the ability to acquire FH, C4BP and PLG from NHS, and use these interactions to evade innate immunity. The binding with the complement regulators FH and C4BP preserves factor I (FI) activity, leading to C3b and C4b degradation products, respectively. C3b and C4b alpha-chain cleavage was also observed when Lsa23 bound to PLG generating plasmin, an effect blocked by the protease inhibitor aprotinin. Lsa23 also inhibited lytic activity by NHS mediated by both classical and alternative complement pathways. Thus, Lsa23 has the ability to block both pathways of the complement system, and may help pathogenic Leptospira to escape complement-mediated clearance in human hosts. Indeed, NHS treated with Lsa23 confers a partial serum resistance phenotype to Leptospira biflexa, whereas blocking this protein with anti-Lsa23 renders pathogenic L. interrogans more susceptible to complement-mediated killing. Thus, Lsa23 is a multifunctional protein involved in many pathways, featuring C4b cleavage by plasmin, knowledge that may help in the development of preventive approaches to intervene with human complement escape by this versatile pathogen. PMID:26614523

  8. Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.

    PubMed

    Matsunaga, James; Schlax, Paula J; Haake, David A

    2013-11-01

    The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double

  9. In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis.

    PubMed

    Raja, Veerapandian; Shanmughapriya, Santhanam; Kanagavel, Murugesan; Artiushin, Sergey C; Velineni, Sridhar; Timoney, John F; Natarajaseenivasan, Kalimuthusamy

    2016-01-01

    Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection. PMID:26607308

  10. In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis

    PubMed Central

    Raja, Veerapandian; Shanmughapriya, Santhanam; Kanagavel, Murugesan; Artiushin, Sergey C.; Velineni, Sridhar; Timoney, John F.

    2015-01-01

    Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection. PMID:26607308

  11. Comparison of Bacterial Burden and Cytokine Gene Expression in Golden Hamsters in Early Phase of Infection with Two Different Strains of Leptospira interrogans

    PubMed Central

    Fujita, Rie; Koizumi, Nobuo; Sugiyama, Hiromu; Tomizawa, Rina; Sato, Ryoichi; Ohnishi, Makoto

    2015-01-01

    Leptospirosis, a zoonotic infection with worldwide prevalence, is caused by pathogenic spirochaetes of Leptospira spp., and exhibits an extremely broad clinical spectrum in human patients. Although previous studies indicated that specific serovars or genotypes of Leptospira spp. were associated with severe leptospirosis or its outbreak, the mechanism underlying the difference in virulence of the various Leptospira serotypes or genotypes remains unclear. The present study addresses this question by measuring and comparing bacterial burden and cytokine gene expression in hamsters infected with strains of two L. interrogans serovars Manilae (highly virulent) and Hebdomadis (less virulent). The histopathology of kidney, liver, and lung tissues was also investigated in infected hamsters. A significantly higher bacterial burden was observed in liver tissues of hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.01). The average copy number of the leptospiral genome was 1,302 and 20,559 in blood and liver, respectively, of hamsters infected with serovar Manilae and 1,340 and 4,896, respectively, in hamsters infected with serovar Hebdomadis. The expression levels of mip1alpha in blood; tgfbeta, il1beta, mip1alpha, il10, tnfalpha and cox2 in liver; and tgfbeta, il6, tnfalpha and cox2 in lung tissue were significantly higher in hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.05). In addition, infection with serovar Manilae resulted in a significantly larger number of hamsters with tnfalpha upregulation (p = 0.04). Severe distortion of tubular cell arrangement and disruption of renal tubules in kidney tissues and hemorrhage in lung tissues were observed in Manilae-infected hamsters. These results demonstrate that serovar Manilae multiplied more efficiently in liver tissues and induced significantly higher expression of genes encoding pro- and anti-inflammatory cytokines than serovar Hebdomadis

  12. Fine Mapping of the Interaction between C4b-Binding Protein and Outer Membrane Proteins LigA and LigB of Pathogenic Leptospira interrogans

    PubMed Central

    Breda, Leandro C. D.; Hsieh, Ching-Lin; Castiblanco Valencia, Mónica M.; da Silva, Ludmila B.; Barbosa, Angela S.; Blom, Anna M.; Yung-Fu, Chang; Isaac, Lourdes

    2015-01-01

    The complement system consists of more than 40 proteins that participate in the inflammatory response and in pathogen killing. Complement inhibitors are necessary to avoid the excessive consumption and activation of this system on host cells. Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Pathogenic leptospires are able to escape from complement activation by binding to host complement inhibitors Factor H [FH] and C4b-binding protein (C4BP) while non-pathogenic leptospires are rapidly killed in the presence of fresh serum. In this study, we demonstrate that complement control protein domains (CCP) 7 and 8 of C4BP α-chain interact with the outer membrane proteins LcpA, LigA and LigB from the pathogenic leptospire L. interrogans. The interaction between C4BP and LcpA, LigA and LigB is sensitive to ionic strength and inhibited by heparin. We fine mapped the LigA and LigB domains involved in its binding to C4BP and heparin and found that both interactions are mediated through the bacterial immunoglobulin-like (Big) domains 7 and 8 (LigA7-8 and LigB7-8) of both LigA and LigB and also through LigB9-10. Therefore, C4BP and heparin may share the same binding sites on Lig proteins. PMID:26517116

  13. Identification of a 35-kilodalton serovar-cross-reactive flagellar protein, FlaB, from Leptospira interrogans by N-terminal sequencing, gene cloning, and sequence analysis.

    PubMed Central

    Lin, M; Surujballi, O; Nielsen, K; Nadin-Davis, S; Randall, G

    1997-01-01

    During the screening of antibodies to pathogenic leptospires, a murine monoclonal antibody (designated M138) was found to react with various serovars. An antigen of approximately 35 kDa from Leptospira interrogans serovar pomona, which reacted strongly with M138, was characterized by N-terminal amino acid sequencing and identified as a flagellin, a class B polypeptide subunit (FlaB) of the periplasmic flagella. The gene encoding the FlaB protein, flaB, was amplified from the genomic DNA of several pathogenic serovars by PCR with a single pair of oligonucleotide primers, suggesting that FlaB is highly conserved among these serovars. Cloning and sequence analysis of flaB from serovar pomona revealed that it contains an 849-bp open reading frame with a G + C content of 46.88% which encodes a 283-amino-acid protein with a calculated molecular mass of 31.297 kDa and a predicted pI of 9.065. A sequence comparison of flagellin proteins revealed that the amino acid sequence is most variable in the central portion of the serovar pomona FlaB, which is believed to contain specific sequence information and which may thus be useful in the design of DNA or synthetic peptide probes suitable for the detection of infection with pathogenic leptospires. PMID:9317049

  14. [The serovars of Leptospira interrogans isolated from cases of human leptospirosis in São Paulo, Brazil].

    PubMed

    Sakata, E E; Yasuda, P H; Romero, E C; Silva, M V; Lomar, A V

    1992-01-01

    Eighteen strains of L. interrogans isolated from human cases were serotyped by the agglutinin-absorption test at Instituto Adolfo Lutz in São Paulo, Brazil. Fourteen were identified as serovar copenhageni (icterohaemorrhagiae serogroup), 2 as canicola (canicola serogroup), 1 as castellonis (Ballum serogroup) and 1 as pomona serogroup (serovar not yet defined). The frequency of serovar copenhageni in 100% of the isolates in icterohaemorrhagiae serogroup is emphasized and more studies to verify the real serovars prevalence as subsidy to the epidemiology of this infection are suggested by the authors. PMID:1342073

  15. Structural characterization of a novel subfamily of leucine-rich repeat proteins from the human pathogen Leptospira interrogans.

    PubMed

    Miras, Isabelle; Saul, Frederick; Nowakowski, Mireille; Weber, Patrick; Haouz, Ahmed; Shepard, William; Picardeau, Mathieu

    2015-06-01

    Pathogenic Leptospira spp. are the agents of leptospirosis, an emerging zoonotic disease. Analyses of Leptospira genomes have shown that the pathogenic leptospires (but not the saprophytes) possess a large number of genes encoding proteins containing leucine-rich repeat (LRR) domains. In other pathogenic bacteria, proteins with LRR domains have been shown to be involved in mediating host-cell attachment and invasion, but their functions remain unknown in Leptospira. To gain insight into the potential function of leptospiral LRR proteins, the crystal structures of four LRR proteins that represent a novel subfamily with consecutive stretches of a 23-amino-acid LRR repeat motif have been solved. The four proteins analyzed adopt the characteristic α/β-solenoid horseshoe fold. The exposed residues of the inner concave surfaces of the solenoid, which constitute a putative functional binding site, are not conserved. The various leptospiral LRR proteins could therefore recognize distinct structural motifs of different host proteins and thus serve separate and complementary functions in the physiology of these bacteria. PMID:26057675

  16. Leptospira Protein Expression During Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  17. Complete Genome Sequences of Low-Passage Virulent and High-Passage Avirulent Variants of Pathogenic Leptospira interrogans Serovar Manilae Strain UP-MMC-NIID, Originally Isolated from a Patient with Severe Leptospirosis, Determined Using PacBio Single-Molecule Real-Time Technology.

    PubMed

    Satou, Kazuhito; Shimoji, Makiko; Tamotsu, Hinako; Juan, Ayaka; Ashimine, Noriko; Shinzato, Misuzu; Toma, Claudia; Nohara, Toshitsugu; Shiroma, Akino; Nakano, Kazuma; Teruya, Kuniko; Terabayashi, Yasunobu; Ohki, Shun; Koizumi, Nobuo; Okano, Shou; Suzuki, Toshihiko; Hirano, Takashi

    2015-01-01

    Here, we report the complete genome sequences of low-passage virulent and high-passage avirulent variants of pathogenic Leptospira interrogans serovar Manilae strain UP-MMC-NIID, a major causative agent of leptospirosis. While there were no major differences between the genome sequences, the levels of base modifications were higher in the avirulent variant. PMID:26272567

  18. Complete Genome Sequences of Low-Passage Virulent and High-Passage Avirulent Variants of Pathogenic Leptospira interrogans Serovar Manilae Strain UP-MMC-NIID, Originally Isolated from a Patient with Severe Leptospirosis, Determined Using PacBio Single-Molecule Real-Time Technology

    PubMed Central

    Shimoji, Makiko; Tamotsu, Hinako; Juan, Ayaka; Ashimine, Noriko; Shinzato, Misuzu; Toma, Claudia; Nohara, Toshitsugu; Shiroma, Akino; Nakano, Kazuma; Teruya, Kuniko; Terabayashi, Yasunobu; Ohki, Shun; Koizumi, Nobuo; Okano, Shou; Suzuki, Toshihiko; Hirano, Takashi

    2015-01-01

    Here, we report the complete genome sequences of low-passage virulent and high-passage avirulent variants of pathogenic Leptospira interrogans serovar Manilae strain UP-MMC-NIID, a major causative agent of leptospirosis. While there were no major differences between the genome sequences, the levels of base modifications were higher in the avirulent variant. PMID:26272567

  19. Physiological Osmotic Induction of Leptospira interrogans Adhesion: LigA and LigB Bind Extracellular Matrix Proteins and Fibrinogen▿

    PubMed Central

    Choy, Henry A.; Kelley, Melissa M.; Chen, Tammy L.; Møller, Annette K.; Matsunaga, James; Haake, David A.

    2007-01-01

    Transmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenic Leptospira spp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of the Staphylococcus aureus FnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests. PMID:17296754

  20. The recombinant LIC10508 is a plasma fibronectin, plasminogen, fibrinogen and C4BP-binding protein of Leptospira interrogans.

    PubMed

    Siqueira, Gabriela H; Teixeira, Aline F; Fernandes, Luis G; de Souza, Gisele O; Kirchgatter, Karin; Romero, Eliete C; Vasconcellos, Silvio A; Vieira, Monica L; Nascimento, Ana Lucia T O

    2016-03-01

    Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. In this study, we report that the recombinant proteins LIC10507, LIC10508 and LIC10509 are recognized by confirmed leptospirosis serum samples at both phases of the disease. The recombinant rLIC10508 and rLIC10507 are plasminogen (PLG)-binding proteins, capable of generating plasmin in the presence of a PLG activator. The proteins bind to PLG in a dose-dependent and saturable manner, fulfilling host-ligand interaction. Furthermore, rLIC10508 interacts with fibrinogen (Fg), plasma fibronectin and C4b binding protein (C4BP). The binding of rLIC10508 to Fg decreases the fibrin clotting in a thrombin-catalyzed reaction. The incubation with 4 μM of protein promoted 40% inhibition upon clotting formation. C4BP bound to rLIC10508 retained its cofactor activity for factor I promoting the cleavage of C4b protein, which may reduce the membrane attack complex formation. Although these proteins have high amino acid sequence similarity, rLIC10508 is the most talented of the three, a behavior that might be explained by its unique putative 3D structure, whereas structures of rLIC10507 and rLIC10509 are very similar. Plasmin generation (rLIC10507 and rLIC10508), together with decreasing fibrin clot formation (rLIC10508) and impairment of the complement system (rLIC10508) may help the bacteria to overcome host defense, facilitating the infection process. PMID:26657108

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

    PubMed Central

    2013-01-01

    Background Leptospirosis 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. Methods Acute 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. Results The 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. Conclusions The 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

  2. Plasmin cleaves fibrinogen and the human complement proteins C3b and C5 in the presence of Leptospira interrogans proteins: A new role of LigA and LigB in invasion and complement immune evasion.

    PubMed

    Castiblanco-Valencia, Mónica Marcela; Fraga, Tatiana Rodrigues; Pagotto, Ana Helena; Serrano, Solange Maria de Toledo; Abreu, Patricia Antonia Estima; Barbosa, Angela Silva; Isaac, Lourdes

    2016-05-01

    Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host. PMID:26822552

  3. Physical and genetic maps of the Leptospira interrogans serovar icterohaemorrhagiae strain Ictero no.1 chromosome and sequencing of a 19-kb region of the genome containing the 5S rRNA gene.

    PubMed

    Takahashi, Y; Akase, K; Hirano, H; Fukunaga, M

    1998-07-17

    We report the construction of physical and genetic maps of the chromosome of Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No.1 using pulsed-field gel electrophoresis of DNA fragments generated by digestion with enzymes SrfI, AscI, FseI, and NotI and using reciprocal hybridization. We also sequenced the 19-kilobase (kb) DNA segment including the one gene for 5S rRNA (rrf) of pathogenic Leptospira. The size of the chromosome of the strain Ictero No.1 was estimated to be 4673kb and was found to be similar to those of the chromosomes of the leptospira strains Verdun (serovar icterohaemorrhagiae) and RZ11 (serovar pomona). The strains Verdun and RZ11 carry a small 350-kb replicon (minichromosome), and the strain Ictero No.1 also contained the same kind of molecule together with the chromosome. The physical maps of the strains Ictero No.1 and Verdun were almost identical, as were the locations of the selected genes, except for the location of one of the 16S rRNA genes. Overall, the genetic organization appeared to be conserved within the serovar icterohaemorrhagiae strains. In the sequenced region, we identified 10 putative ORFs and one rrf sequence, and the transcription orientations were all the same. A homology search for the products deduced from the sequenced data revealed that the orf H exhibited high similarity to malic acid enzyme of Haemophilus influenzae and fumarate hydratase of Escherichia coli (orf J). The rest of the putative products encoded by ORFs in the sequenced region showed little similarity with the proteins contained in the databases and were considered to be unknown proteins. PMID:9666070

  4. An ortholog of the Leptospira interrogans lipoprotein LipL32 aids in the colonization of Pseudoalteromonas tunicata to host surfaces

    PubMed Central

    Gardiner, Melissa; Hoke, David E.; Egan, Suhelen

    2014-01-01

    The bacterium Pseudoalteromonas tunicata is a common surface colonizer of marine eukaryotes, including the macroalga Ulva australis.Genomic analysis of P. tunicata identified genes potentially involved in surface colonization, including genes with homology to bacterial virulence factors that mediate attachment. Of particular interest is the presence of a gene, designated ptlL32, encoding an ortholog to the Leptospira lipoprotein LipL32, which has been shown to facilitate the interaction of Leptospira sp. with host extracellular matrix (ECM) structures and is thought to be an important virulence trait for pathogenic Leptospira. To investigate the role of PtlL32 in the colonization by P. tunicata we constructed and characterized a ΔptlL32 mutant strain. Whilst P. tunicata ΔptlL32 bound to an abiotic surface with the same capacity as the wild type strain, it had a marked effect on the ability of P. tunicata to bind to ECM, suggesting a specific role in attachment to biological surfaces. Loss of PtlL32 also significantly reduced the capacity for P. tunciata to colonize the host algal surface demonstrating a clear role for this protein as a host-colonization factor. PtlL32 appears to have a patchy distribution across specific groups of environmental bacteria and phylogenetic analysis of PtlL32 orthologous proteins from non-Leptospira species suggests it may have been acquired via horizontal gene transfer between distantly related lineages. This study provides the first evidence for an attachment function for a LipL32-like protein outside the Leptospira and thereby contributes to the understanding of host colonization in ecologically distinct bacterial species. PMID:25071736

  5. Leptospirosis in Kuala Lumpur and the comparative evaluation of two rapid commercial diagnostic kits against the MAT test for the detection of antibodies to leptospira interrogans.

    PubMed

    Sekhar, W Y; Soo, E H; Gopalakrishnan, V; Devi, S

    2000-08-01

    The aim of the study was to look into the epidemiology of serodiagnosed cases of leptospirosis at the University Hospital and compare two commercial ELISA Assays to the Microscopic Agglutination Test (MAT). Demographic data for all serodiagnosed cases for the years 1991-1997 were collected. From this data, 104 sera (n = 104) were selected as samples for comparative evaluation of the commercial ELISAs (INDX Dip-S-Ticks and PanBio ELISA) to the MAT test. Thirty two (n = 32) negative control sera were selected from serodiagnosed cases of other differential diagnosis of leptospira infection. The MAT test is a standard test that detects agglutination antibodies to leptospira biflexa, while the INDX Dip-S-Ticks is an ELISA dot test assaying for total anti-leptospira antibodies. The PanBio ELISA is a colorometric assay in test well strips to detect anti-leptospira IgM. The sensitivity, specificity, and efficiency of tests were calculated at a MAT cut-off value of 1:320. Demographic data showed that leptospirosis peaks during March-May and Aug-Nov coinciding with the inter-monsoon period with more men being infected than women and more adults than children. The sensitivity, specificity, and efficiency of test for the INDX Dip-S-Ticks were 83.3%, 93.8% and 87.5% while the values for the PanBio ELISA were 54.2%, 96.9% and 71.3%. The suboptimal PanBio result could be related to the blocking effect of high IgG titres or could be related to the diagnostic MAT cut-off values used in this study. The data hence reflects a pattern of transmission that is related to "wet" occupational risk factors. The commercial assays evaluated, are easier to perform but interpretation of results should be based on level of endemicity. The INDX Dip-S-Ticks allows this flexibility and is a practical alternative to the MAT test. PMID:11256343

  6. Detection of reactive canines to Leptospira in Campeche City, Mexico.

    PubMed

    Blum Domínguez, Selene Del C; Chi Dzib, María Y; Maldonado Velázquez, María G; Nuñez Oreza, Luis A; Gómez Solano, Mónica I; Caballero Poot, Rebeca I; Tamay Segovia, Paulino

    2013-01-01

    Leptospira reactivity in stray and household dogs in Campeche as well as associated risk factors to the seropositivity in household dogs have been herein determined. The survey included 323 dogs, 142 of which were stray dogs and 181 household dogs. Nine Leptospira interrogans serovars were tested by the microagglutination test. Reactivity was 21.3 % (69/323), 17.2 % corresponded to household dogs and 26.7 % to stray dogs. Leptospira Canicola (29 %), Leptospira Hardjo (22.58 %), and Leptospira Icterohaemorrhagiae (16.12 %) were the most common serovars reacting against the serum of household animals, while Leptospira Canicola (15.78 %), Leptospira icterohaemorrhagiae (13.15 %), and Leptospira Pomona (7.89 %) were those reacting in stray dogs. Results showed that all dogs have been in contact with different Leptospira serovars and outdoor exposure is the main infection risk factor. PMID:23560786

  7. Radiometric method for the rapid detection of Leptospira organisms

    SciTech Connect

    Manca, N.; Verardi, R.; Colombrita, D.; Ravizzola, G.; Savoldi, E.; Turano, A.

    1986-02-01

    A rapid and sensitive radiometric method for detection of Leptospira interrogans serovar pomona and Leptospira interrogans serovar copenhageni is described. Stuart's medium and Middlebrook TB (12A) medium supplemented with bovine serum albumin, catalase, and casein hydrolysate and labeled with /sup 14/C-fatty acids were used. The radioactivity was measured in a BACTEC 460. With this system, Leptospira organisms were detected in human blood in 2 to 5 days, a notably shorter time period than that required for the majority of detection techniques.

  8. Three-Dimensional Structures of Pathogenic and Saprophytic Leptospira Species Revealed by Cryo-Electron Tomography

    PubMed Central

    Raddi, Gianmarco; Morado, Dustin R.; Yan, Jie; Haake, David A.

    2012-01-01

    Leptospira interrogans is the primary causative agent of the most widespread zoonotic disease, leptospirosis. An in-depth structural characterization of L. interrogans is needed to understand its biology and pathogenesis. In this study, cryo-electron tomography (cryo-ET) was used to compare pathogenic and saprophytic species and examine the unique morphological features of this group of bacteria. Specifically, our study revealed a structural difference between the cell envelopes of L. interrogans and Leptospira biflexa involving variations in the lipopolysaccharide (LPS) layer. Through cryo-ET and subvolume averaging, we determined the first three-dimensional (3-D) structure of the flagellar motor of leptospira, with novel features in the flagellar C ring, export apparatus, and stator. Together with direct visualization of chemoreceptor arrays, DNA packing, periplasmic filaments, spherical cytoplasmic bodies, and a unique “cap” at the cell end, this report provides structural insights into these fascinating Leptospira species. PMID:22228733

  9. Genome-wide transposon mutagenesis in pathogenic Leptospira species.

    PubMed

    Murray, Gerald L; Morel, Viviane; Cerqueira, Gustavo M; Croda, Julio; Srikram, Amporn; Henry, Rebekah; Ko, Albert I; Dellagostin, Odir A; Bulach, Dieter M; Sermswan, Rasana W; Adler, Ben; Picardeau, Mathieu

    2009-02-01

    Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans. PMID:19047402

  10. Expansion of the in vitro assay for Leptospira potency testing to other Serovars: Case study with Leptospira hardjo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Code for Federal Regulations (9 CFR 113:101-104) specifies how vaccine potency is evaluated in a hamster model for evaluation of leptospiral vaccines against pomona, icterohaemorrhagiae, canicola, and grippotyphosa serotypes of Leptospira interrogans. There are several issues which complicate th...

  11. First Isolates of Leptospira spp., from Rodents Captured in Angola.

    PubMed

    Fortes-Gabriel, Elsa; Carreira, Teresa; Vieira, Maria Luísa

    2016-05-01

    Rodents play an important role in the transmission of pathogenic Leptospira spp. However, in Angola, neither the natural reservoirs of these spirochetes nor leptospirosis diagnosis has been considered. Regarding this gap, we captured rodents in Luanda and Huambo provinces to identify circulating Leptospira spp. Rodent kidney tissue was cultured and DNA amplified and sequenced. Culture isolates were evaluated for pathogenic status and typing with rabbit antisera; polymerase chain reaction (PCR) and sequencing were also performed. A total of 37 rodents were captured: Rattus rattus (15, 40.5%), Rattus norvegicus (9, 24.3%), and Mus musculus (13, 35.2%). Leptospiral DNA was amplified in eight (21.6%) kidney samples. From the cultures, we obtained four (10.8%) Leptospira isolates belonging to the Icterohaemorrhagiae and Ballum serogroups of Leptospira interrogans and Leptospira borgpetersenii genospecies, respectively. This study provides information about circulating leptospires spread by rats and mice in Angola. PMID:26928840

  12. First Isolates of Leptospira spp., from Rodents Captured in Angola

    PubMed Central

    Fortes-Gabriel, Elsa; Carreira, Teresa; Vieira, Maria Luísa

    2016-01-01

    Rodents play an important role in the transmission of pathogenic Leptospira spp. However, in Angola, neither the natural reservoirs of these spirochetes nor leptospirosis diagnosis has been considered. Regarding this gap, we captured rodents in Luanda and Huambo provinces to identify circulating Leptospira spp. Rodent kidney tissue was cultured and DNA amplified and sequenced. Culture isolates were evaluated for pathogenic status and typing with rabbit antisera; polymerase chain reaction (PCR) and sequencing were also performed. A total of 37 rodents were captured: Rattus rattus (15, 40.5%), Rattus norvegicus (9, 24.3%), and Mus musculus (13, 35.2%). Leptospiral DNA was amplified in eight (21.6%) kidney samples. From the cultures, we obtained four (10.8%) Leptospira isolates belonging to the Icterohaemorrhagiae and Ballum serogroups of Leptospira interrogans and Leptospira borgpetersenii genospecies, respectively. This study provides information about circulating leptospires spread by rats and mice in Angola. PMID:26928840

  13. [Detection of leptospira by culture of vitreous humor and detection of antibodies against leptospira in vitreous humor and serum of 225 horses with equine recurrent uveitis].

    PubMed

    Dorrego-Keiter, Elisa; Tóth, József; Dikker, Lieke; Sielhorst, Jutta; Schusser, Gerald Fritz

    2016-01-01

    In the ongoing discussion regarding the aetiopathogenesis of equine recurrent uveitis (ERU) it was the aim of the present study to elucidate the relationship of leptospira infection and ERU. In a population of 225 horses leptospira were examined in vitreous humor by culture and leptospira antibody were detected in vitreous humor and serum samples. Preoperative serum samples were collected from 221/225 ERU patients of different age, gender and breed. Undiluted vitreous humor was aseptically taken from 198/225 patients that underwent pars plana vitrectomy at the beginning of surgery and from 27/225 patients' eyeball after enucleation: Serum and vitreous humor were tested for specific leptospiral antibodies by microscopic agglutination test (MAT). Furthermore, vitreous humor was examined by culture. 20 patients which were euthanized due to a live-threatening disease other than ERU served as a control group. A total of 127/221 (57.5%) horses had serum antibodies (≥ 1:100). Most frequently antibodies against L. interrogans serovar Grippotyphosa were detected (79/127), followed by L. interrogans serovar lcterohaemorrhagiae (34/127) and L. interrogans serovar Bratislava (29/127). Only 79/225 horses (35.1%) had leptospiral antibodies in vitreous humor, in which L. interrogans serovar Grippotyphosa (67/79) was identified most frequently followed by L. interrogans serovar Pomona (18/79) and L. interrogans serovar lcterohaemorrhagiae (8/79) which was identified as single or multiple reaction. Isolation of leptospira from vitreous humor was positive in 34/212 horses (16%). 10/20 control horses had a positive antibody titer against leptospira in serum and 2/20 horses in vitreous humor, whereas there was no leptospira detected in culture. The result of 84% negative cultures from vitreous humor of 212 ERU patients is decisive for the diagnosis and therapy of ERU. PMID:27344913

  14. Conservation of the S10-spc-alpha Locus within Otherwise Highly Pastic Genomes Provides Phylogenetic Insight into the Genus Leptospira

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comparative analysis of the Leptospira interrogans S10-spc-alpha operon was performed by PCR using primer sets covering the whole operon. Correctly sized fragments were obtained by PCR from all of L. interrogans strains for each primer set indicating that the S10-spc-alpha locus is well conserved ...

  15. Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C

    SciTech Connect

    Stamm, L.V.; Charon, N.W.

    1988-03-01

    The habitats for the two major Leptospira spp. differ. The main habitat of L. biflexa is soil and water, whereas L. interrogans primarily resides in the renal tubules of animals. We investigated whether these two species, along with L. illini (species incertae sedis), differ with respect to their sensitivity to UV radiation. The doses of UV resulting in 37, 10 and 1% survival were determined for representive serovars from each species. L. interrogans serovar pomona was 3.0 to 4.8 times more sensitive to UV than the other Leptospira species under the 37, 10, and 1% survival parameters. In comparison to other bacteria, L. interrogans serovar pomona is among the most sensitive to UV. In a qualitative UV sensitivity assay., L. interrogans serovars were found to be in general more sensitive than L. biflexa serovars. All three species were found to have a photoreactivation DNA repair mechanism. Since organisms that are resistant to UV are often resistant to the DNA cross-linking agent mitomycin C, we tested the relative sensitivity of several Leptospira serovars to this compound. With few exceptions, L. biflexa and L. illini serovars were considerably more resistant to mitomycin C than the L. interrogans serovars. The mitomycin C sensitivity assay could be a useful addition to current characterization tests used to differentiate the Leptospira species.

  16. Draft Genome Sequence of the First Pathogenic Leptospira Isolates from Ecuador

    PubMed Central

    Barragan, Veronica; Sahl, Jason W.; Wiggins, Kristin; Chiriboga, Jorge; Salinas, Ana; Cantos, Nancy E.; Loor, Mariana N.; Intriago, Bertha I.; Morales, Melba; Trueba, Gabriel

    2016-01-01

    Pathogenic Leptospira spp. cause leptospirosis upon contact with mucosa through wounds or ingestion, leading to headaches, fever, jaundice, kidney or liver failure, or death in about 1.3 million people each year. Here, we present the draft genomes of one L. santarosai isolate and two L. interrogans isolates from Ecuador. PMID:27151788

  17. Draft Genome Sequence of the First Pathogenic Leptospira Isolates from Ecuador.

    PubMed

    Barragan, Veronica; Sahl, Jason W; Wiggins, Kristin; Chiriboga, Jorge; Salinas, Ana; Cantos, Nancy E; Loor, Mariana N; Intriago, Bertha I; Morales, Melba; Trueba, Gabriel; Pearson, Talima

    2016-01-01

    Pathogenic Leptospira spp. cause leptospirosis upon contact with mucosa through wounds or ingestion, leading to headaches, fever, jaundice, kidney or liver failure, or death in about 1.3 million people each year. Here, we present the draft genomes of one L. santarosai isolate and two L. interrogans isolates from Ecuador. PMID:27151788

  18. Molecular characterization of Leptospira sp by multilocus variable number tandem repeat analysis (MLVA) from clinical samples: a case report.

    PubMed

    Pailhoriès, Hélène; Buzelé, Rodolphe; Picardeau, Mathieu; Robert, Sylvie; Mercier, Emmanuelle; Mereghetti, Laurent; Lanotte, Philippe

    2015-08-01

    Leptospirosis is a zoonotic infection for which diagnosis is difficult. It has appeared as a global emerging infectious disease over recent years. Genotype determination often requires a Leptospira strain obtained by culture, which is a long and fastidious technique. A method based on multilocus variable number tandem repeat analysis (MLVA) to determine the genotype of Leptospira interrogans, performed directly on blood or urine samples, is proposed. This method was applied to a fatal case of leptospirosis for which the geographical origin of infection was unknown. This technique will allow a genotype to be obtained for L. interrogans, even when cultures remain negative. PMID:26159846

  19. Distribution of Plasmids in Distinct Leptospira Pathogenic Species

    PubMed Central

    Wang, Yanzhuo; Zhuang, Xuran; Zhong, Yi; Zhang, Cuicai; Zhang, Yan; Zeng, Lingbing; Zhu, Yongzhang; He, Ping; Dong, Ke; Pal, Utpal; Guo, Xiaokui; Qin, Jinhong

    2015-01-01

    Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups—pathogens, non-pathogens, and intermediates—based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological

  20. Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

    PubMed

    Wang, Yanzhuo; Zhuang, Xuran; Zhong, Yi; Zhang, Cuicai; Zhang, Yan; Zeng, Lingbing; Zhu, Yongzhang; He, Ping; Dong, Ke; Pal, Utpal; Guo, Xiaokui; Qin, Jinhong

    2015-11-01

    Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups--pathogens, non-pathogens, and intermediates--based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological

  1. Comparative analyses of transport proteins encoded within the genomes of Leptospira species.

    PubMed

    Buyuktimkin, Bora; Saier, Milton H

    2016-09-01

    Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they all have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity arose in Leptospira correlating to progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles. PMID:27296707

  2. Is it too cold for Leptospira interrrogans transmission on the Faroese Islands?

    PubMed

    Jensen, Per M; Magnussen, Eydfinn

    2016-02-01

    Leptospira interrogans is a bacterium that can infect most mammal species. Brown rats are considered to be one of the most important reservoirs of Leptospira because they frequently are infected and live in close proximity to humans. Past studies of prevalence of Leptospira in brown rats indicate that temperature--both high and low--may negatively affect the prevalence rate in rats, so that Leptospira is rare or even absent at temperatures below 7-8 °C. Here we investigated the prevalence of infection in brown rats on the Faroese Islands (mean temperature of 6.5 °C) and did not find any infected animals in a sample of 95 animals. We propose that prevalence rates of Leptospira are very low in rural brown rats in the cooler Scandinavian regions, even though urban/sewer rats might be highly infected in the same regions. PMID:26442766

  3. Epidemiology of Leptospira Transmitted by Rodents in Southeast Asia

    PubMed Central

    Mielcarek, Mathilde; Tatard, Caroline; Chaval, Yannick; Suputtamongkol, Yupin; Buchy, Philippe; Jittapalapong, Sathaporn; Herbreteau, Vincent; Morand, Serge

    2014-01-01

    Background Leptospirosis is the most common bacterial zoonoses and has been identified as an important emerging global public health problem in Southeast Asia. Rodents are important reservoirs for human leptospirosis, but epidemiological data is lacking. Methodology/Principal Findings We sampled rodents living in different habitats from seven localities distributed across Southeast Asia (Thailand, Lao PDR and Cambodia), between 2009 to 2010. Human isolates were also obtained from localities close to where rodents were sampled. The prevalence of Leptospira infection was assessed by real-time PCR using DNA extracted from rodent kidneys, targeting the lipL32 gene. Sequencing rrs and secY genes, and Multi Locus Variable-number Tandem Repeat (VNTR) analyses were performed on DNA extracted from rat kidneys for Leptospira isolates molecular typing. Four species were detected in rodents, L. borgpetersenii (56% of positive samples), L. interrogans (36%), L. kirschneri (3%) and L. weilli (2%), which were identical to human isolates. Mean prevalence in rodents was approximately 7%, and largely varied across localities and habitats, but not between rodent species. The two most abundant Leptospira species displayed different habitat requirements: L. interrogans was linked to humid habitats (rice fields and forests) while L. borgpetersenii was abundant in both humid and dry habitats (non-floodable lands). Conclusion/Significance L. interrogans and L. borgpetersenii species are widely distributed amongst rodent populations, and strain typing confirmed rodents as reservoirs for human leptospirosis. Differences in habitat requirements for L. interrogans and L. borgpetersenii supported differential transmission modes. In Southeast Asia, human infection risk is not only restricted to activities taking place in wetlands and rice fields as is commonly accepted, but should also include tasks such as forestry work, as well as the hunting and preparation of rodents for consumption, which

  4. Comparative genomic analyses of transport proteins encoded within the genomes of Leptospira species.

    PubMed

    Buyuktimkin, Bora; Saier, Milton H

    2015-11-01

    Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity was accompanied by progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles. PMID:26247102

  5. Molecular and serological characterization of the first Leptospira santarosai strain isolated from a dog.

    PubMed

    Miotto, Bruno Alonso; Moreno, Luisa Zanolli; Guilloux, Aline Gil Alves; Sousa, Gisele Oliveira de; Loureiro, Ana Paula; Moreno, Andrea Micke; Lilenbaum, Walter; Vasconcellos, Silvio Arruda; Heinemann, Marcos Bryan; Hagiwara, Mitika Kuribayashi

    2016-10-01

    Leptospirosis is a zoonotic disease of global importance caused by pathogenic Leptospira species. Dogs can become asymptomatically infected, acting like reservoir hosts for pathogenic Leptospira, notably Leptospira interrogans serovar Canicola. Identification of such individuals and characterization of leptospires involved in chronic infections may unravel the role of dogs in the epidemiology of particular leptospiral strains. The aim of the present work was to describe the first Leptospira santarosai strain isolated from a dog. The dog was kept in a public shelter in São Paulo city, Brazil, and presented asymptomatic urinary shedding detected by PCR. Prospective evaluation was performed to fully characterize its chronic carrier state. The dog did not present anti-Leptospira titles or clinical/laboratorial abnormalities during the evaluations; nevertheless long-term urinary shedding was confirmed by PCR and leptospires were recovered from two occasions. The isolated strain was molecularly characterized by partial 16S rRNA and secY gene sequencing and MLST analysis. Serogroup identification was performed using polyclonal antibodies. The strain was identified as Leptospira santarosai, serogroup Sejroe. This is the first evidence in the literature of the isolation of L. santarosai in dogs. Our findings show that dogs can persistently harbor leptospires other than L. interrogans. PMID:27282095

  6. The association between rainfall and seropositivity to Leptospira in outdoor reared pigs.

    PubMed

    Boqvist, Sofia; Eliasson-Selling, Lena; Bergström, Karin; Magnusson, Ulf

    2012-07-01

    Outdoor reared pigs were used as indicators for investigating the effect of weather conditions in the seroprevalence of Leptospira. Over the period February to March 2008, sera from 386 sows on 11 farms in southern Sweden were tested for antibodies to the following Leptospira serovars: L. interrogans serovar (sv) Bratislava, L. kirschneri sv Grippotyphosa, L. interrogans sv Icterohaemorrhagiae, L. interrogans sv Pomona, L. borgpetersenii sv Tarassovi and one domestic strain (mouse 2A) related to L. borgpetersenii sv Sejroe and L. borgpetersenii sv Istrica. The highest seroprevalence was to this strain (8.0%) followed by sv Bratislava (3.9%). Six of the 11 farms had sows which were seropositive to at least one of the Leptospira serovars. Data on rainfall and temperature were retrieved for the respective farms. For each millimetre of extra rainfall, there was an increase in the odds ratio (OR) for seropositivity to sv Bratislava of 4.3 (95% CI 1.9-10), and to strain mouse 2A of 2.5 (95% CI 1.0-6.4). There was no association between seropositivity and temperature. This study indicates that different climate conditions within the northern temperate climate zone may be of importance for the presence of Leptospira-seropositivity in mammals. PMID:22227225

  7. Transcriptional Response of Leptospira interrogans to Different Iron Sources

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospirosis is a globally important zoonotic disease. Humans can become infected via exposure to infected animals or contaminated water or soil. Iron is an essential element for many cellular processes and its sequestration in the host environment constitutes an immune defence mechanism. Pathoge...

  8. Transcriptional Response of Leptospira interrogans to Different Iron Sources

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800, Australia (1), Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Victoria 3800, Australia (2), Davi...

  9. Use of a New High Resolution Melting Method for Genotyping Pathogenic Leptospira spp.

    PubMed Central

    Naze, Florence; Desvars, Amélie; Picardeau, Mathieu; Bourhy, Pascale; Michault, Alain

    2015-01-01

    Background Leptospirosis is a worldwide zoonosis that is endemic in tropical areas, such as Reunion Island. The species Leptospira interrogans is the primary agent in human infections, but other pathogenic species, such as L. kirschner and L. borgpetersenii, are also associated with human leptospirosis. Methods and Findings In this study, a melting curve analysis of the products that were amplified with the primer pairs lfb1 F/R and G1/G2 facilitated an accurate species classification of Leptospira reference strains. Next, we combined an unsupervised high resolution melting (HRM) method with a new statistical approach using primers to amplify a two variable-number tandem-repeat (VNTR) for typing at the subspecies level. The HRM analysis, which was performed with ScreenClust Software, enabled the identification of genotypes at the serovar level with high resolution power (Hunter-Gaston index 0.984). This method was also applied to Leptospira DNA from blood samples that were obtained from Reunion Island after 1998. We were able to identify a unique genotype that is identical to that of the L. interrogans serovars Copenhageni and Icterohaemorrhagiae, suggesting that this genotype is the major cause of leptospirosis on Reunion Island. Conclusions Our simple, rapid, and robust genotyping method enables the identification of Leptospira strains at the species and subspecies levels and supports the direct genotyping of Leptospira in biological samples without requiring cultures. PMID:26154161

  10. The Characteristics of Ubiquitous and Unique Leptospira Strains from the Collection of Russian Centre for Leptospirosis

    PubMed Central

    Voronina, Olga L.; Kunda, Marina S.; Aksenova, Ekaterina I.; Ryzhova, Natalia N.; Semenov, Andrey N.; Petrov, Evgeny M.; Didenko, Lubov V.; Lunin, Vladimir G.; Ananyina, Yuliya V.; Gintsburg, Alexandr L.

    2014-01-01

    Background and Aim. Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity of Leptospira in natural and anthropurgic foci was not clearly defined. Methods. The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS). Results. The most wide spread and prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmental Leptospira strain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species. Conclusions. Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions. PMID:25276806

  11. Improvement of trivalent leptospira vaccine by removal of anaphylactic agents.

    PubMed

    Moazenijula, Gholamreza; Jabbari, A R; Geravand, M Moradi; Banihashemi, R; Hajizadeh, A

    2011-12-01

    The anaphylactic reactions in cattle following leptospira vaccination mostly booster dose in different parts of Iran have been reported. The serum proteins as allergic substances are components of liquid phase of the vaccine. Therefore, the vaccine was modified by washing the whole cultures by centrifugations. The modified vaccine was safe in laboratory animals and cattle as well as under field conditions. Microagglutination test revealed a similar pattern of antibody response to the three Leptospira interrogans serovars (Canicola, Grippotyphosa, and Sejro hardjo) in all vaccinated cattle groups while was higher than the response of control animals. The results of the present investigation revealed that we can minimize postvaccination shock in vaccinated cattle populations with removing the shock proteins. PMID:21698521

  12. Genotypes of Leptospira spp. strains isolated from dogs in Buenos Aires, Argentina.

    PubMed

    Grune Loffler, Sylvia; Passaro, Diego; Samartino, Luis; Soncini, Analía; Romero, Graciela; Brihuega, Bibiana

    2014-01-01

    Leptospirosis is an infectious disease of wide global distribution, which is endemic in Argentina. The objective of this study was to obtain the genetic profiles of Leptospira spp. strains isolated from clinical cases of dogs in the province of Buenos Aires by the multiple-locus variable-number tandem repeat analysis (MLVA). Eight isolated canine strains were genotyped by MLVA, obtaining the identical profile of Leptospira interrogans serovar Canicola Hond Utrecht IV in the strains named Dogy and Mayo. The strains named Bel, Sarmiento, La Plata 4581 and La Plata 5478 were identical to the profile of the genotype of L. interrogans serovar Portlandvere MY 1039.The strain named Avellaneda was identical to the genotype profile of L. interrogans serovar Icterohaemorrhagiae RGA and the strain named SB had the same profile as the L. interrogans serovar Pomona Baires genotype and was similar to the profile of serovar Pomona Pomona genotype. It would be useful to include a larger number of isolates from different dog populations in various provinces of Argentina and to characterize the genetic profiles of the strains circulating in the country. The information obtained will be useful for the control of leptospirosis in the dog population. PMID:25444128

  13. Functional thioredoxin reductase from pathogenic and free-living Leptospira spp.

    PubMed

    Sasoni, Natalia; Iglesias, Alberto A; Guerrero, Sergio A; Arias, Diego G

    2016-08-01

    Low molecular mass thiols and antioxidant enzymes have essential functions to detoxify reactive oxygen and nitrogen species maintaining cellular redox balance. The metabolic pathways for redox homeostasis in pathogenic (Leptospira interrogans) and free-living (Leptospira biflexa) leptospires species were not functionally characterized. We performed biochemical studies on recombinantly produced proteins to in depth analyze kinetic and structural properties of thioredoxin reductase (LinTrxR) and thioredoxin (LinTrx) from L. interrogans, and two TrxRs (LbiTrxR1 and LbiTrxR2) from L. biflexa. All the TrxRs were characterized as homodimeric flavoproteins, with LinTrxR and LbiTrxR1 catalyzing the NADPH dependent reduction of LinTrx and DTNB. The thioredoxin system from L. interrogans was able to use glutathione disulfide, lipoamide disulfide, cystine and bis-γ-glutamyl cysteine and homologous peroxiredoxin as substrates. Classic TrxR activity of LinTrxR2 had not been evidenced in vitro, but recombinant Escherichia coli cells overexpressing LbiTrxR2 showed high tolerance to oxidative stress. The enzymatic systems herein characterized could play a key role for the maintenance of redox homeostasis and the function of defense mechanisms against reactive oxidant species in Leptospira spp. Our results contribute to the general knowledge about redox biochemistry in these bacteria, positioning TrxR as a critical molecular target for the development of new anti-leptospiral drugs. PMID:27178006

  14. Though not Reservoirs, Dogs might Transmit Leptospira in New Caledonia

    PubMed Central

    Gay, Noellie; Soupé-Gilbert, Marie-Estelle; Goarant, Cyrille

    2014-01-01

    Leptospira has been a major public health concern in New Caledonia for decades. However, few multidisciplinary studies addressing the zoonotic pattern of this disease were conducted so far. Here, pig, deer and dog samples were collected. Analyses were performed using molecular detection and genotyping. Serological analyses were also performed for dogs. Our results suggest that deer are a reservoir of L. borgpetersenii Hardjobovis and pigs a reservoir of L. interrogans Pomona. Interestingly, 4.4% of dogs were renal carriers of Leptospira. In dog populations, MAT results confirmed the circulation of the same Leptospira serogroups involved in human cases. Even if not reservoirs, dogs might be of significance in human contamination by making an epidemiological link between wild or feral reservoirs and humans. Dogs could bring pathogens back home, shedding Leptospira via their urine and in turn increasing the risk of human contamination. We propose to consider dog as a vector, particularly in rural areas where seroprevalence is significantly higher than urban areas. Our results highlight the importance of animal health in improving leptospirosis prevention in a One Health approach. PMID:24747539

  15. Genotypes of pathogenic Leptospira spp isolated from rodents in Argentina

    PubMed Central

    Loffler, Sylvia Grune; Pavan, Maria Elisa; Vanasco, Bibiana; Samartino, Luis; Suarez, Olga; Auteri, Carmelo; Romero, Graciela; Brihuega, Bibiana

    2014-01-01

    Leptospirosis is the most widespread zoonosis in the world and significant efforts have been made to determine and classify pathogenic Leptospira strains. This zoonosis is maintained in nature through chronic renal infections of carrier animals, with rodents and other small mammals serving as the most important reservoirs. Additionally, domestic animals, such as livestock and dogs, are significant sources of human infection. In this study, a multiple-locus variable-number tandem repeat analysis (MLVA) was applied to genotype 22 pathogenic Leptospira strains isolated from urban and periurban rodent populations from different regions of Argentina. Three MLVA profiles were identified in strains belonging to the species Leptospira interrogans (serovars Icterohaemorrhagiae and Canicola); one profile was observed in serovar Icterohaemorrhagiae and two MLVA profiles were observed in isolates of serovars Canicola and Portlandvere. All strains belonging to Leptospira borgpetersenii serovar Castellonis exhibited the same MLVA profile. Four different genotypes were isolated from urban populations of rodents, including both mice and rats and two different genotypes were isolated from periurban populations. PMID:24676656

  16. Genotypes of pathogenic Leptospira spp isolated from rodents in Argentina.

    PubMed

    Loffler, Sylvia Grune; Pavan, Maria Elisa; Vanasco, Bibiana; Samartino, Luis; Suarez, Olga; Auteri, Carmelo; Romero, Graciela; Brihuega, Bibiana

    2014-04-01

    Leptospirosis is the most widespread zoonosis in the world and significant efforts have been made to determine and classify pathogenic Leptospira strains. This zoonosis is maintained in nature through chronic renal infections of carrier animals, with rodents and other small mammals serving as the most important reservoirs. Additionally, domestic animals, such as livestock and dogs, are significant sources of human infection. In this study, a multiple-locus variable-number tandem repeat analysis (MLVA) was applied to genotype 22 pathogenic Leptospira strains isolated from urban and periurban rodent populations from different regions of Argentina. Three MLVA profiles were identified in strains belonging to the species Leptospira interrogans (serovars Icterohaemorrhagiae and Canicola); one profile was observed in serovar Icterohaemorrhagiae and two MLVA profiles were observed in isolates of serovars Canicola and Portlandvere. All strains belonging to Leptospira borgpetersenii serovar Castellonis exhibited the same MLVA profile. Four different genotypes were isolated from urban populations of rodents, including both mice and rats and two different genotypes were isolated from periurban populations. PMID:24676656

  17. Though not reservoirs, dogs might transmit Leptospira in New Caledonia.

    PubMed

    Gay, Noellie; Soupé-Gilbert, Marie-Estelle; Goarant, Cyrille

    2014-04-01

    Leptospira has been a major public health concern in New Caledonia for decades. However, few multidisciplinary studies addressing the zoonotic pattern of this disease were conducted so far. Here, pig, deer and dog samples were collected. Analyses were performed using molecular detection and genotyping. Serological analyses were also performed for dogs. Our results suggest that deer are a reservoir of L. borgpetersenii Hardjobovis and pigs a reservoir of L. interrogans Pomona. Interestingly, 4.4% of dogs were renal carriers of Leptospira. In dog populations, MAT results confirmed the circulation of the same Leptospira serogroups involved in human cases. Even if not reservoirs, dogs might be of significance in human contamination by making an epidemiological link between wild or feral reservoirs and humans. Dogs could bring pathogens back home, shedding Leptospira via their urine and in turn increasing the risk of human contamination. We propose to consider dog as a vector, particularly in rural areas where seroprevalence is significantly higher than urban areas. Our results highlight the importance of animal health in improving leptospirosis prevention in a One Health approach. PMID:24747539

  18. Genotyping of Leptospira directly in urine samples of cattle demonstrates a diversity of species and strains in Brazil.

    PubMed

    Hamond, C; Pestana, C P; Medeiros, M A; Lilenbaum, W

    2016-01-01

    The aim of this study was to identify Leptospira in urine samples of cattle by direct sequencing of the secY gene. The validity of this approach was assessed using ten Leptospira strains obtained from cattle in Brazil and 77 DNA samples previously extracted from cattle urine, that were positive by PCR for the genus-specific lipL32 gene of Leptospira. Direct sequencing identified 24 (31·1%) interpretable secY sequences and these were identical to those obtained from direct DNA sequencing of the urine samples from which they were recovered. Phylogenetic analyses identified four species: L. interrogans, L. borgpetersenii, L. noguchii, and L. santarosai with the most prevalent genotypes being associated with L. borgpetersenii. While direct sequencing cannot, as yet, replace culturing of leptospires, it is a valid additional tool for epidemiological studies. An unexpected finding from this study was the genetic diversity of Leptospira infecting Brazilian cattle. PMID:26076668

  19. Prevention of renal infection and urinary shedding in dogs by a Leptospira vaccination.

    PubMed

    Schreiber, Paul; Martin, Virginie; Najbar, Wojciech; Sanquer, Annaelle; Gueguen, Sylvie; Lebreux, Bernard

    2005-06-15

    Prevention of urinary shedding of Leptospira interrogans spp. by chronically infected dogs remains a key objective of the vaccination in dogs against leptospirosis which is a zoonotic disease. An inactivated bivalent vaccine composed of Leptospira interrogans serovars icterohaemorrhagiae [L. icterohaemorrhagiae] and canicola [L. canicola] bacterins was tested for its ability to protect puppies against a challenge exposure with L. icterohaemorrhagiae. The vaccine was administered twice at a 3-week interval to six puppies aged from 8 to 9 weeks. Six other puppies were used as unvaccinated controls. All puppies were challenged 2 weeks after the second vaccine injection by intraperitoneal (IP) administration of L. icterohaemorrhagiae (day 0). Clinical signs, haematological and biochemical changes and evidence of Leptospira in blood, urine and kidney were monitored for 4 weeks after the challenge exposure (days 0-28). Puppies were euthanised on day 28 for post-mortem and histological examinations of liver and kidney. Control group presented clinical pictures of severe or subclinical infection. One dog developed severe clinical signs (hypothermia, depression, anorexia, abdominal pain, dehydration, icterus, weight loss) and died on post-infection day (PID) 7 due to an acute renal failure. Gross and microscopic lesions were in accordance with this clinical pattern. In the five remaining control dogs, the challenge exposure induced mainly a systemic infection including leptospiraemia, leptospiruria and renal carriage. The vaccinated group remained healthy throughout the study period. In conclusion, immunisation with a Leptospira vaccine was shown to protect dogs against symptomatology and leptospiraemia, urine shedding and renal infection. PMID:15917139

  20. Investigation of reservoir animals of Leptospira in the northern part of Miyazaki Prefecture.

    PubMed

    Koizumi, Nobuo; Muto, Maki; Yamamoto, Seigo; Baba, Yoshitaka; Kudo, Momotoshi; Tamae, Yoshinobu; Shimomura, Koji; Takatori, Ichiro; Iwakiri, Akira; Ishikawa, Koji; Soma, Hirotoshi; Watanabe, Haruo

    2008-11-01

    We surveyed reservoir animals of leptospires in the northern part of Miyazaki Prefecture, where a cluster of human leptospirosis had occurred during the summer of 2006. Leptospira was isolated from 6 of 57 large Japanese field mice (Apodemus speciosus). The serogroups of the isolates were Autumnalis (5 strains) and Hebdomadis (1 strain) and the partial nucleotide sequences of their flaB genes suggested that the isolates belonged to L. interrogans. The human patient sera reacted specifically with the Leptospira strain isolated from the mice captured around the area where each patient occurred, suggesting that mice are the source of human infection. We also detected leptospiral DNAs by flaB-polymerase chain reaction in the kidneys of large feral animals; wild boars (positive ratio 10.3%; 4 of 39) and deer (19.2%; 10 of 52). The Leptospira spp. harbored by these animals were deduced to be L. interrogans (in 5 animals) and L. borgpetersenii (in 9 animals) by the nucleotide sequences of the amplicons. Anti-Leptospira antibodies were also detected among symptomatic hound dogs. These results suggest that these feral animals may cause leptospirosis and pose a potential risk to hunters and workers in the meat processing industry. PMID:19050356

  1. Prevalence of antibody to six Leptospira servovars in Swedish wild boars.

    PubMed

    Boqvist, Sofia; Bergström, Karin; Magnusson, Ulf

    2012-04-01

    Zoonotic Leptospira bacteria are pathogens that may increase in importance with climate change. We investigated the prevalence of antibody to six Leptospira serovars (sv) in the Swedish wild boar (Sus scrofa) population, which is increasing in number and geographic distribution. The serovars we selected cause disease in pigs or may be of use as sentinel serovars to measure the potential spread in Swedish fauna. In total, 386 serum samples from wild boars collected between 2005 and 2007 were investigated using a microscopic agglutination test for Leptospira interrogans sv Bratislava strain Jez Bratislava, sv Icterohaemorrhagiae strain Kantorowicz, sv Pomona strain Pomona, Leptospira kirschneri sv Grippotyphosa strain Duyster, and Leptospira borgpetersenii sv Tarassovi strain Perepelitsin, and a domestic strain closely related to sv Sejroe. Twelve (3.1%) of the analyzed samples were antibody-positive. Of those, nine (2.3%) were positive for sv Bratislava and 0.8% for sv Icterohaemorrhagiae. All antibody-positive samples originated from areas where wild boars are reported to be common. We conclude that Leptospira infection is less common in Swedish wild boar than in continental Europe. However, we recommend continuous surveillance to follow the effects of climate change and an increasing wild boar population. PMID:22493129

  2. Cloning and Sequence Analysis of LipL32, a Surface–Exposed Lipoprotein of Pathogenic Leptospira Spp

    PubMed Central

    Khodaverdi Darian, Ebrahim; Forghanifard, Mohammad Mahdi; Moradi Bidhendi, Soheila; Chang, Yung-Fu; Yahaghi, Emad; Esmaelizad, Majid; Khaleghizadeh, Maryam; Khaki, Pejvak

    2013-01-01

    Background Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira species. A major challenge of this disease is the application of basic research to improve diagnostic methods and related vaccine development. Outer membrane proteins of Leptospira are potential candidates that may be useful as diagnostic or immunogenic factors in treatment and analysis of the disease. Objectives To develop an effective subunit vaccine against prevalent pathogenic Leptospira species, we sequenced and analyzed the LipL32 gene from three different Leptospira interrogans (L.interrogans) vaccinal serovars in Iran. Materials and Methods Following DNA extraction from these three serovars, the related LipL32 genes were amplified and cloned in the pTZ57R/T vector. Recombinant clones were confirmed by colony- PCR and DNA sequencing. The related sequences were subjected to homology analysis by comparing them to sequences in the Genbank database. Results The LipL32 sequences were >94% homologous among the vaccinal and other pathogenic Leptospira serovars in GenBank. This result indicates the conservation of this gene within the pathogenic Leptospires. Conclusions The cloned gene in this study may provide a potentially suitable platform for development of a variety of applications such as serological diagnostic tests or recombinant vaccines against leptospirosis. PMID:24719688

  3. Similarities in Leptospira Serogroup and Species Distribution in Animals and Humans in the Indian Ocean Island of Mayotte

    PubMed Central

    Desvars, Amélie; Naze, Florence; Vourc'h, Gwenaël; Cardinale, Eric; Picardeau, Mathieu; Michault, Alain; Bourhy, Pascale

    2012-01-01

    Our objective was to identify local animal reservoirs of leptospirosis to explain the unusual features of Leptospira strains recently described among patients on the island of Mayotte. By means of a microscopic agglutination test using local clinical isolates, we found that 11.2% of black rats were seropositive to Leptospira, whereas 10.2% of flying foxes, 2% of lemurs, 93.1% of domestic dogs, and 87.5% of stray dogs were seropositive. As observed in humans, Mini was the main serogroup circulating in animals, whereas serogroup Icterohaemorrhagiae was absent. Using quantitative polymerase chain reaction, we also showed that 29.8% of rats carried leptospires in their kidneys. The sequencing of 16S rRNA gene sequences of Leptospira found in black rat kidneys identified four genomospecies (Leptospira borgpetersenii, Leptospira interrogans, Leptospira kirschneri, and L. borgpetersenii group B), which established black rats as the major source of leptospirosis transmission to humans. The origins of such a genetic diversity in Leptospira strains are discussed. PMID:22764304

  4. CATALASE ACTIVITY IN LEPTOSPIRA

    PubMed Central

    Rao, P. J.; Larson, A. D.; Cox, C. D.

    1964-01-01

    Rao, P. J. (University of Illinois, Urbana), A. D. Larson, and C. D. Cox. Catalase activity in Leptospira. J. Bacteriol. 88:1045–1048. 1964.—A number of serotypes of Leptospira were found to possess catalase activity, although considerable variation in activity existed among various serotypes. Catalase activity of L. pomona was reduced by inhibitors commonly employed for arresting catalase activity in other biological systems. Catalase activity was increased three to five times by growing cultures under conditions of oxygen availability; however, aeration had no beneficial effect on total viable cell crop. The relationship of oxygen to metabolism and future studies on virulence of the leptospirae is discussed. PMID:14219017

  5. Interaction of Bovine Peripheral Blood Polymorphonuclear Cells and Leptospira Species; Innate Responses in the Natural Bovine Reservoir Host

    PubMed Central

    Wilson-Welder, Jennifer H.; Frank, Ami T.; Hornsby, Richard L.; Olsen, Steven C.; Alt, David P.

    2016-01-01

    Cattle are the reservoir hosts of Leptospira borgpetersenii serovar Hardjo, and can also be reservoir hosts of other Leptospira species such as L. kirschneri, and Leptospira interrogans. As a reservoir host, cattle shed Leptospira, infecting other animals, including humans. Previous studies with human and murine neutrophils have shown activation of neutrophil extracellular trap or NET formation, and upregulation of inflammatory mediators by neutrophils in the presence of Leptospira. Humans, companion animals and most widely studied models of Leptospirosis are of acute infection, hallmarked by systemic inflammatory response, neutrophilia, and septicemia. In contrast, cattle exhibit chronic infection with few outward clinical signs aside from reproductive failure. Taking into consideration that there is host species variation in innate immunity, especially in pathogen recognition and response, the interaction of bovine peripheral blood polymorphonuclear cells (PMNs) and several Leptospira strains was evaluated. Studies including bovine-adapted strains, human pathogen strains, a saprophyte and inactivated organisms. Incubation of PMNs with Leptospira did induce slight activation of neutrophil NETs, greater than unstimulated cells but less than the quantity from E. coli P4 stimulated PMNs. Very low but significant from non-stimulated, levels of reactive oxygen peroxides were produced in the presence of all Leptospira strains and E. coli P4. Similarly, significant levels of reactive nitrogen intermediaries (NO2) was produced from PMNs when incubated with the Leptospira strains and greater quantities in the presence of E. coli P4. PMNs incubated with Leptospira induced RNA transcripts of IL-1β, MIP-1α, and TNF-α, with greater amounts induced by live organisms when compared to heat-inactivated leptospires. Transcript for inflammatory cytokine IL-8 was also induced, at similar levels regardless of Leptospira strain or viability. However, incubation of Leptospira strains

  6. Interaction of Bovine Peripheral Blood Polymorphonuclear Cells and Leptospira Species; Innate Responses in the Natural Bovine Reservoir Host.

    PubMed

    Wilson-Welder, Jennifer H; Frank, Ami T; Hornsby, Richard L; Olsen, Steven C; Alt, David P

    2016-01-01

    Cattle are the reservoir hosts of Leptospira borgpetersenii serovar Hardjo, and can also be reservoir hosts of other Leptospira species such as L. kirschneri, and Leptospira interrogans. As a reservoir host, cattle shed Leptospira, infecting other animals, including humans. Previous studies with human and murine neutrophils have shown activation of neutrophil extracellular trap or NET formation, and upregulation of inflammatory mediators by neutrophils in the presence of Leptospira. Humans, companion animals and most widely studied models of Leptospirosis are of acute infection, hallmarked by systemic inflammatory response, neutrophilia, and septicemia. In contrast, cattle exhibit chronic infection with few outward clinical signs aside from reproductive failure. Taking into consideration that there is host species variation in innate immunity, especially in pathogen recognition and response, the interaction of bovine peripheral blood polymorphonuclear cells (PMNs) and several Leptospira strains was evaluated. Studies including bovine-adapted strains, human pathogen strains, a saprophyte and inactivated organisms. Incubation of PMNs with Leptospira did induce slight activation of neutrophil NETs, greater than unstimulated cells but less than the quantity from E. coli P4 stimulated PMNs. Very low but significant from non-stimulated, levels of reactive oxygen peroxides were produced in the presence of all Leptospira strains and E. coli P4. Similarly, significant levels of reactive nitrogen intermediaries (NO2) was produced from PMNs when incubated with the Leptospira strains and greater quantities in the presence of E. coli P4. PMNs incubated with Leptospira induced RNA transcripts of IL-1β, MIP-1α, and TNF-α, with greater amounts induced by live organisms when compared to heat-inactivated leptospires. Transcript for inflammatory cytokine IL-8 was also induced, at similar levels regardless of Leptospira strain or viability. However, incubation of Leptospira strains

  7. Evaluation of Cell Binding Activities of Leptospira ECM Adhesins

    PubMed Central

    Robbins, Gregory T.; Hahn, Beth L.; Evangelista, Karen V.; Padmore, Lavinia; Aranda, Patrick S.; Coburn, Jenifer

    2015-01-01

    Pathogenic spirochetes of the genus Leptospira are the causative agents of leptospirosis, a zoonotic infection that occurs globally. The bacteria colonize the renal proximal tubules of many animals and are shed in the urine. Contact with the urine, or with water contaminated with the urine of infected animals can cause infection of new host animals, including humans. Mechanisms of colonization of the proximal tubule and other tissues are not known, but specific interactions between bacterial adhesins and host substrates are likely to be critical in this process. Several extracellular matrix (ECM) adhesins have been previously identified, but more recently, it has been shown that Leptospira bind more efficiently to cells than ECM. In this work, recombinant forms of five putative Leptospira ECM adhesins, namely LipL32, Loa22, OmpL1, p31/LipL45, and LenA were evaluated for binding to cells as well as an expanded variety of ECM components. Reproducible and significant adhesin activity was demonstrated only for OmpL1, which bound to both mammalian cell lines tested and to glycosaminoglycans (GAGs). While determination of biologically significant bacterial adhesion activity will require generation of site-directed mutant strains, our results suggest that OmpL1 is a strong candidate for future evaluation regarding the roles of the adhesin activity of the protein during L. interrogans infection. PMID:25875373

  8. Rodents and Leptospira transmission risk in Terceira island (Azores).

    PubMed

    Collares-Pereira, M; Mathias, M L; Santos-Reis, M; Ramalhinho, M G; Duarte-Rodrigues, P

    2000-01-01

    The role of rodents as Leptospira renal carriers in Terceira island was evaluated (1993-1995) through kidney culture and serology [microscopic aglutination test (MAT)] of 94 mice and rats. Fifty-nine animals were positive (n = 41 by serology + culturing; n = 11 serology; n = 7 culturing), presenting a wide distribution in man-made and natural areas. House mice had the highest bacteriological (82.9%) and serological (90.9%) rates, being strictly related to serovar arborea. Black rats were involved in the dispersion of all isolated L. interrogans sensu lato serovars (arborea, copenhageni and icterohaemorrhagiae). Logistic regression analysis and non-metric multi-dimensional scaling, relating Leptospira infection with biological and environmental variables, expressed that adult males Mus domesticus, sexually active and living in humid biotopes, mainly above 500 m, are the most likely reservoirs. This study emphasizes the role of house-mice in the epidemiology of leptospirosis in Terceira and the need of reducing the risk of Leptospira transmission through integrated control programmes, primarily focusing on adult house-mice in peri-domestic environments, before the breeding season. PMID:11484805

  9. Leptospira spp. in rodents and shrews in Germany.

    PubMed

    Mayer-Scholl, Anne; Hammerl, Jens Andre; Schmidt, Sabrina; Ulrich, Rainer G; Pfeffer, Martin; Woll, Dietlinde; Scholz, Holger C; Thomas, Astrid; Nöckler, Karsten

    2014-08-01

    Leptospirosis is an acute, febrile disease occurring in humans and animals worldwide. Leptospira spp. are usually transmitted through direct or indirect contact with the urine of infected reservoir animals. Among wildlife species, rodents act as the most important reservoir for both human and animal infection. To gain a better understanding of the occurrence and distribution of pathogenic leptospires in rodent and shrew populations in Germany, kidney specimens of 2973 animals from 11 of the 16 federal states were examined by PCR. Rodent species captured included five murine species (family Muridae), six vole species (family Cricetidae) and six shrew species (family Soricidae). The most abundantly trapped animals were representatives of the rodent species Apodemus flavicollis, Clethrionomys glareolus and Microtus agrestis. Leptospiral DNA was amplified in 10% of all animals originating from eight of the 11 federal states. The highest carrier rate was found in Microtus spp. (13%), followed by Apodemus spp. (11%) and Clethrionomys spp. (6%). The most common Leptospira genomospecies determined by duplex PCR was L. kirschneri, followed by L. interrogans and L. borgpetersenii; all identified by single locus sequence typing (SLST). Representatives of the shrew species were also carriers of Leptospira spp. In 20% of Crocidura spp. and 6% of the Sorex spp. leptospiral DNA was detected. Here, only the pathogenic genomospecies L. kirschneri was identified. PMID:25062275

  10. Development of Leptospira in vitro potency assays: EU/industry experience and perspectives.

    PubMed

    Klaasen, H L B M; van der Veen, M; Molkenboer, M J C H; Bruderer, U

    2013-09-01

    Nobivac® Lepto (MSD Animal Health) is a non-adjuvanted canine leptospirosis vaccine containing inactivated whole cells of Leptospira interrogans serogroup Canicola serovar Portlandvere and L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. The current standard in vivo potency test is a hamster challenge test associated with major drawbacks such as animal suffering and poor reproducibility. Here, the quantification of antigenic mass by ELISA as a new in vitro potency test is described, supporting the 3Rs concept (replacement, reduction, and refinement of animal tests) and in accordance with European Pharmacopoeia Monograph 0447 (Canine Leptospirosis Vaccine [Inactivated]). The two corresponding sandwich ELISAs are based on monoclonal antibodies specific for immunodominant leptospiral lipopolysaccharide epitopes. Protection in passive immunization experiments demonstrate that these monoclonal antibodies recognize key protective antigens in currently licensed human and veterinary whole cell Leptospira vaccines. The high precision and robustness renders the two ELISAs much more reliable correlates of potency in dogs than the hamster potency test. The recent approval of these assays for a new canine leptospirosis vaccine is an important contribution to the 3Rs in quality control testing of Leptospira vaccines. PMID:23867758

  11. Human Leptospirosis Caused by a New, Antigenically Unique Leptospira Associated with a Rattus Species Reservoir in the Peruvian Amazon

    PubMed Central

    Cespedes, Manuel; Diaz, M. Monica; Galloway, Renee L.; Saito, Mayuko; Steigerwalt, Arnold G.; Patra, Kailash P.; Ore, Carlos Vidal; Gotuzzo, Eduardo; Gilman, Robert H.; Levett, Paul N.; Vinetz, Joseph M.

    2008-01-01

    As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species “Leptospira licerasiae” serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010T, which has been deposited into internationally accessible culture collections. By microscopic agglutination test, “Leptospira licerasiae” serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti–L. fainei serovar Hurstbridge at a titer of 1∶100. LipL32, although not detectable by PCR, was detectable in “Leptospira licerasiae” serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against “Leptospira licerasiae” serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon. PMID:18382606

  12. Use of PCR to identify Leptospira in kidneys of big brown bats (Eptesicus fuscus) in Kansas and Nebraska, USA.

    PubMed

    Harkin, Kenneth R; Hays, Michael; Davis, Rolan; Moore, Michael

    2014-07-01

    Bats have been implicated as potential carriers of Leptospira as a result of surveys, mostly in Australia and South America. We measured the prevalence of pathogenic leptospires in kidneys of bats from Kansas and Nebraska. From 7 August 2012 to 21 August 2012, we extracted DNA from kidneys of 98 big brown bats (Eptesicus fuscus) submitted and found negative for rabies. The DNA was processed in a two-step, seminested PCR assay with a dual-labeled Taqman probe specific for pathogenic leptospires. As a negative control, we used a saprophytic leptospire (Leptospira biflexa Patoc) and, as a pathogenic control, Leptospira interrogans Canicola. All bat kidneys were negative for pathogenic leptospires, suggesting that it is unlikely that the big brown bat, one of the most prevalent bat species in North America, is a reservoir for transmission of leptospires to dogs or humans. PMID:24807182

  13. Prevalence and Genotype Allocation of Pathogenic Leptospira Species in Small Mammals from Various Habitat Types in Germany.

    PubMed

    Obiegala, Anna; Woll, Dietlinde; Karnath, Carolin; Silaghi, Cornelia; Schex, Susanne; Eßbauer, Sandra; Pfeffer, Martin

    2016-03-01

    Small mammals serve as most important reservoirs for Leptospira spp., the causative agents of Leptospirosis, which is one of the most neglected and widespread zoonotic diseases worldwide. The knowledge about Leptospira spp. occurring in small mammals from Germany is scarce. Thus, this study's objectives were to investigate the occurrence of Leptospira spp. and the inherent sequence types in small mammals from three different study sites: a forest in southern Germany (site B1); a National Park in south-eastern Germany (site B2) and a renaturalised area, in eastern Germany (site S) where small mammals were captured. DNA was extracted from kidneys of small mammals and tested for Leptospira spp. by real-time PCR. Positive samples were further analysed by duplex and conventional PCRs. For 14 positive samples, multi locus sequence typing (MLST) was performed. Altogether, 1213 small mammals were captured: 216 at site B1, 456 at site B2 and 541 at site S belonging to following species: Sorex (S.) araneus, S. coronatus, Apodemus (A.) flavicollis, Myodes glareolus, Microtus (Mi.) arvalis, Crocidura russula, Arvicola terrestris, A. agrarius, Mustela nivalis, Talpa europaea, and Mi. agrestis. DNA of Leptospira spp. was detected in 6% of all small mammals. At site B1, 25 small mammals (11.6%), at site B2, 15 small mammals (3.3%) and at site S, 33 small mammals (6.1%) were positive for Leptospira spp. Overall, 54 of the positive samples were further determined as L. kirschneri, nine as L. interrogans and four as L. borgpetersenii while five real-time PCR-positive samples could not be further determined by conventional PCR. MLST results revealed focal occurrence of L. interrogans and L. kirschneri sequence type (ST) 117 while L. kirschneri ST 110 was present in small mammals at all three sites. Further, this study provides evidence for a particular host association of L. borgpetersenii to mice of the genus Apodemus. PMID:27015596

  14. Prevalence and Genotype Allocation of Pathogenic Leptospira Species in Small Mammals from Various Habitat Types in Germany

    PubMed Central

    Karnath, Carolin; Silaghi, Cornelia; Schex, Susanne; Eßbauer, Sandra; Pfeffer, Martin

    2016-01-01

    Small mammals serve as most important reservoirs for Leptospira spp., the causative agents of Leptospirosis, which is one of the most neglected and widespread zoonotic diseases worldwide. The knowledge about Leptospira spp. occurring in small mammals from Germany is scarce. Thus, this study’s objectives were to investigate the occurrence of Leptospira spp. and the inherent sequence types in small mammals from three different study sites: a forest in southern Germany (site B1); a National Park in south-eastern Germany (site B2) and a renaturalised area, in eastern Germany (site S) where small mammals were captured. DNA was extracted from kidneys of small mammals and tested for Leptospira spp. by real-time PCR. Positive samples were further analysed by duplex and conventional PCRs. For 14 positive samples, multi locus sequence typing (MLST) was performed. Altogether, 1213 small mammals were captured: 216 at site B1, 456 at site B2 and 541 at site S belonging to following species: Sorex (S.) araneus, S. coronatus, Apodemus (A.) flavicollis, Myodes glareolus, Microtus (Mi.) arvalis, Crocidura russula, Arvicola terrestris, A. agrarius, Mustela nivalis, Talpa europaea, and Mi. agrestis. DNA of Leptospira spp. was detected in 6% of all small mammals. At site B1, 25 small mammals (11.6%), at site B2, 15 small mammals (3.3%) and at site S, 33 small mammals (6.1%) were positive for Leptospira spp. Overall, 54 of the positive samples were further determined as L. kirschneri, nine as L. interrogans and four as L. borgpetersenii while five real-time PCR-positive samples could not be further determined by conventional PCR. MLST results revealed focal occurrence of L. interrogans and L. kirschneri sequence type (ST) 117 while L. kirschneri ST 110 was present in small mammals at all three sites. Further, this study provides evidence for a particular host association of L. borgpetersenii to mice of the genus Apodemus. PMID:27015596

  15. Prevalence of antibodies against Leptospira sp. in snakes, lizards and turtles in Slovenia

    PubMed Central

    2013-01-01

    Background Leptospiral infections in poikilothermic (cold blooded) animals have received very little attention and the literature concerning natural infections of these animals is limited. The aim of this study was to determine the prevalence of leptospiral antibodies in reptiles, imported into Slovenia and intended to be pets in close contact with humans. A total of 297 reptiles (22 snakes, 210 lizards and 65 turtles) were tested for specific antibodies against serovars of Leptospira interrogans sensu stricto using the microscopic agglutination test (MAT). Live cultures of different serovars were used as antigens. MAT was performed according to standard procedures and the degree of reaction was interpreted by estimating the percentage of agglutinated leptospires. Samples showing titres of ≥ 50 against one or more serovars were considered as positive. Results Antibodies against seven pathogenic serovars of L. interrogans sensu stricto were detected in 46 of 297 reptiles. Among 22 snakes, specific antibodies against pathogenic serovars of three Leptospira species (L. interrogans, L. kirschneri and L. borgpetersenii) at titre levels from 1:50 to 1:400 were detected in 6 snakes. In 31 of 210 lizards, specific antibodies were found in titres from 1:50 to 1:1000 and, finally, among 65 turtles (terrapins and tortoises), 9 had specific antibodies at titre levels between 1:50 and 1:1600. Animals imported from non-EU countries showed significantly higher prevalence (25.0%; 95 confidence interval: 16.7–33.3%) than animals from EU member states (10.4%; confidence interval: 6.1–14.7%). Conclusions Reptiles may be considered as potential reservoirs of L. interrogans sensu stricto. Origin of the animals is a risk factor for presence of leptospiral antibodies, especially in lizards. Special attention should be focused on animals from non-EU member states. PMID:24020619

  16. Highly Pathogenic Leptospira Found in Urban Brown Rats (Rattus norvegicus) in the Largest Cities of Sweden.

    PubMed

    Strand, Tanja M; Löhmus, Mare; Persson Vinnersten, Thomas; Råsbäck, Therese; Sundström, Karin; Bergström, Tomas; Lundkvist, Åke

    2015-12-01

    Leptospirosis is an emerging zoonosis of global concern; however, its contemporary occurrence in Sweden, a European country partly located north of the Arctic Circle, is poorly known. Four out of 30 brown rats, captured within urban districts in Sweden, were found to be positive for antibodies to Leptospira interrogans serovar Icterohaemorrhagiae. This serovar causes Weil's disease in humans, a severe infection with jaundice, renal failure, and hemorrhage. Our study is the first finding of this highly pathogenic serovar in Swedish rats since the 1930s. PMID:26579782

  17. [Significance of human leptospirosis in Mexico. Detection of Leptospira antibodies in a blood donor population].

    PubMed

    Gavaldón, D G; Cisneros, M A; Rojas, N; Moles-Cervantes, L P

    1995-01-01

    The presence of specific serum antibodies has been used as a diagnostic test for human leptospirosis. The presence of these antibodies in humans is indicative of an active natural infection. Its detection after exposure denotes the presence of immunity. Serum samples from 206 adult blood donors were analyzed with a microscopic agglutination assay against 7 serovars of Leptospira interrogans. A total of 7% were positive with the following serovar distribution; shermani 53%, canicola 33%, pyrogens 20%, pomona 13% and icterohaemorrhagiae 6%. The highest frequency of seropositivity was found in the 20 year to 39 age group. These results in asymptomatic individuals show that leptospirosis is a frequent zoonosis in Mexico. PMID:8582567

  18. Prevalence of Leptospira spp. in the kidneys of wild boars and deer in Japan.

    PubMed

    Koizumi, Nobuo; Muto, Maki; Yamada, Akio; Watanabe, Haruo

    2009-06-01

    We surveyed the prevalence of Leptospira spp. from 2005 to 2008 in wild boars and deer in Japan using polymerase chain reaction. Leptospiral flaB was detected in the kidneys of wild boars (positive ratio, 15.2%; 22 of 145) from 9 prefectures and a deer (1.1%; 1 of 94) from 1 prefecture in Japan. There was no annual change in the prevalence of positive animals during the investigation period (chi-squared test, p=0.94) or in the prevalence in male and female wild boars in the 2007 to 2008 season (Fisher's exact test, P=0.45). The Leptospira species harbored by these animals were deduced to be L. interrogans (from 22 animals) and L. borgpetersenii (from 1 animal). PMID:19578291

  19. Serologic and Molecular Studies of Leptospira and Leptospirosis among Rats in the Philippines

    PubMed Central

    Villanueva, Sharon Y. A. M.; Ezoe, Hirokazu; Baterna, Rubelia A.; Yanagihara, Yasutake; Muto, Maki; Koizumi, Nobuo; Fukui, Takashi; Okamoto, Yoshihiro; Masuzawa, Toshiyuki; Cavinta, Lolita L.; Gloriani, Nina G.; Yoshida, Shin-ichi

    2010-01-01

    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. PMID:20439972

  20. Distribution and Diversity of Pathogenic Leptospira Species in Peri-domestic Surface Waters from South Central Chile

    PubMed Central

    Mason, Meghan R.; Encina, Carolina; Sreevatsan, Srinand; Muñoz-Zanzi, Claudia

    2016-01-01

    Background Leptospirosis is a neglected zoonosis affecting animals and humans caused by infection with Leptospira. The bacteria can survive outside of hosts for long periods of time in soil and water. While identification of Leptospira species from human cases and animal reservoirs are increasingly reported, little is known about the diversity of pathogenic Leptospira species in the environment and how surveillance of the environment might be used for monitoring and controlling disease. Methods and Findings Water samples (n = 104) were collected from the peri-domestic environment of 422 households from farms, rural villages, and urban slums participating in a broader study on the eco-epidemiology of leptospirosis in the Los Rios Region, Chile, between October 2010 and April 2012. The secY region of samples, previously detected as pathogenic Leptospira by PCR, was amplified and sequenced. Sequences were aligned using ClustalW in MEGA, and a minimum spanning tree was created in PHYLOViZ using the goeBURST algorithm to assess sequence similarity. Sequences from four clinical isolates, 17 rodents, and 20 reference strains were also included in the analysis. Overall, water samples contained L. interrogans, L. kirschneri, and L. weilii, with descending frequency. All species were found in each community type. The distribution of the species differed by the season in which the water samples were obtained. There was no evidence that community-level prevalence of Leptospira in dogs, rodents, or livestock influenced pathogen diversity in the water samples. Conclusions This study reports the presence of pathogenic Leptospira in the peri-domestic environment of households in three community types and the differences in Leptospira diversity at the community level. Systematic environmental surveillance of Leptospira can be used for detecting changes in pathogen diversity and to identify and monitor contaminated areas where an increased risk of human infection exists. PMID

  1. Genus I. Leptospira

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospira comprise a diverse group of bacteria. Some species cause serious infections in animals and humans. These bacteria are aerobes that consume long-chain fatty acids and alcohols as carbon and energy sources. This genus is distinguished from Leptonema or Turneriella by lack of similarity u...

  2. Rapid Leptospira identification by direct sequencing of the diagnostic PCR products in New Caledonia

    PubMed Central

    2010-01-01

    Background Most of the current knowledge of leptospirosis epidemiology originates from serological results obtained with the reference Microscopic Agglutination Test (MAT). However, inconsistencies and weaknesses of this diagnostic technique are evident. A growing use of PCR has improved the early diagnosis of leptospirosis but a drawback is that it cannot provide information on the infecting Leptospira strain which provides important epidemiologic data. Our work is aimed at evaluating if the sequence polymorphism of diagnostic PCR products could be used to identify the infecting Leptospira strains in the New Caledonian environment. Results Both the lfb1 and secY diagnostic PCR products displayed a sequence polymorphism that could prove useful in presumptively identifying the infecting leptospire. Using both this polymorphism and MLST results with New Caledonian isolates and clinical samples, we confirmed the epidemiological relevance of the sequence-based identification of Leptospira strains. Additionally, we identified one cluster of L. interrogans that contained no reference strain and one cluster of L. borgpetersenii found only in the introduced Rusa deer Cervus timorensis russa that is its probable reservoir. Conclusions The sequence polymorphism of diagnostic PCR products proved useful in presumptively identifying the infecting Leptospira strains. This could contribute to a better understanding of leptospirosis epidemiology by providing epidemiological information that cannot be directly attained from the use of PCR as an early diagnostic test for leptospirosis. PMID:21176235

  3. Targeted mutagenesis in pathogenic Leptospira species: disruption of the LigB gene does not affect virulence in animal models of leptospirosis.

    PubMed

    Croda, Julio; Figueira, Claudio Pereira; Wunder, Elsio A; Santos, Cleiton S; Reis, Mitermayer G; Ko, Albert I; Picardeau, Mathieu

    2008-12-01

    The pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetically manipulating pathogenic Leptospira species. Thus, homologous recombination between introduced DNA and the corresponding chromosomal locus has never been demonstrated for this pathogen. Leptospiral immunoglobulin-like repeat (Lig) proteins were previously identified as putative Leptospira virulence factors. In this study, a ligB mutant was constructed by allelic exchange in L. interrogans; in this mutant a spectinomycin resistance (Spc(r)) gene replaced a portion of the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluorescence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster model of leptospirosis. In addition, inoculation of rats with the ligB mutant induced persistent colonization of the kidneys. Finally, LigB was not required to mediate bacterial adherence to cultured cells. Taken together, our data provide the first evidence of site-directed homologous recombination in pathogenic Leptospira species. Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the host and in the development of acute disease manifestations or persistent renal colonization. PMID:18809657

  4. Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins during in vitro Culture Attenuation

    PubMed Central

    Lehmann, Jason S.; Corey, Victoria C.; Ricaldi, Jessica N.; Vinetz, Joseph M.; Winzeler, Elizabeth A.; Matthias, Michael A.

    2016-01-01

    Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12–25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis. PMID:26711524

  5. Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins During in vitro Culture Attenuation.

    PubMed

    Lehmann, Jason S; Corey, Victoria C; Ricaldi, Jessica N; Vinetz, Joseph M; Winzeler, Elizabeth A; Matthias, Michael A

    2016-02-01

    Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12-25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis. PMID:26711524

  6. Detection of Pathogenic Leptospira Bacteria in Pinniped Populations via PCR and Identification of a Source of Transmission for Zoonotic Leptospirosis in the Marine Environment▿

    PubMed Central

    Cameron, Caroline E.; Zuerner, Richard L.; Raverty, Stephen; Colegrove, Kathleen M.; Norman, Stephanie A.; Lambourn, Dyanna M.; Jeffries, Steven J.; Gulland, Frances M.

    2008-01-01

    Leptospirosis, caused by the spirochete Leptospira, is a geographically widespread disease that affects a broad range of mammals, including marine mammals. Among pinniped populations, periodic epizootics of leptospirosis are responsible for significant die-offs. Along the west coast of North America, the most recent leptospirosis epizootic occurred in 2004, during which samples were collected from cases ranging from California to British Columbia. The primary objective of this study was to use this well-defined sample set to determine the feasibility of using PCR techniques to diagnose Leptospira infection among pinniped populations in comparison with diagnostic methodologies commonly used for marine mammals. Successful amplification was achieved from a variety of samples, including freshly collected urine, urine stored at −80°C for less than 6 months, and kidney (freshly collected, frozen, and decomposed), as well as feces- and urine-contaminated sand collected in the vicinity of a live-stranded animal. Pathological examination of tissue collected from Leptospira-infected animals revealed the presence of leptospiral antigen in the kidneys. The use of species-specific primer pairs revealed a pattern of host specificity for Leptospira interrogans in sea lions and Leptospira kirschneri in elephant seals. These studies indicate PCR is a sensitive and specific diagnostic tool for the detection of Leptospira infection in pinnipeds and reveal a potential source for epizootic, enzootic, and zoonotic spread of leptospirosis in a marine environment. PMID:18367568

  7. Detection of pathogenic Leptospira bacteria in pinniped populations via PCR and identification of a source of transmission for zoonotic leptospirosis in the marine environment.

    PubMed

    Cameron, Caroline E; Zuerner, Richard L; Raverty, Stephen; Colegrove, Kathleen M; Norman, Stephanie A; Lambourn, Dyanna M; Jeffries, Steven J; Gulland, Frances M

    2008-05-01

    Leptospirosis, caused by the spirochete Leptospira, is a geographically widespread disease that affects a broad range of mammals, including marine mammals. Among pinniped populations, periodic epizootics of leptospirosis are responsible for significant die-offs. Along the west coast of North America, the most recent leptospirosis epizootic occurred in 2004, during which samples were collected from cases ranging from California to British Columbia. The primary objective of this study was to use this well-defined sample set to determine the feasibility of using PCR techniques to diagnose Leptospira infection among pinniped populations in comparison with diagnostic methodologies commonly used for marine mammals. Successful amplification was achieved from a variety of samples, including freshly collected urine, urine stored at -80 degrees C for less than 6 months, and kidney (freshly collected, frozen, and decomposed), as well as feces- and urine-contaminated sand collected in the vicinity of a live-stranded animal. Pathological examination of tissue collected from Leptospira-infected animals revealed the presence of leptospiral antigen in the kidneys. The use of species-specific primer pairs revealed a pattern of host specificity for Leptospira interrogans in sea lions and Leptospira kirschneri in elephant seals. These studies indicate PCR is a sensitive and specific diagnostic tool for the detection of Leptospira infection in pinnipeds and reveal a potential source for epizootic, enzootic, and zoonotic spread of leptospirosis in a marine environment. PMID:18367568

  8. Cross-Species Surveillance of Leptospira in Domestic and Peri-Domestic Animals in Mahalla City, Gharbeya Governorate, Egypt

    PubMed Central

    Felt, Stephen A.; Wasfy, Momtaz O.; El-Tras, Wael F.; Samir, Ahmed; Rahaman, Bassem Abdel; Boshra, Marie; Parker, Tina M.; Hatem, Mahmoud Essam; El-Bassiouny, Ahmed Ahmed; Murray, Clinton K.; Pimentel, Guillermo

    2011-01-01

    A survey of 179 animals (black rats, dogs, sheep, buffaloes, cattle, donkeys, weasels, and cats) for Leptospira infection was conducted in Mahalla City (Lower Egypt). Blood, urine, and kidney were collected and tested by culture, microscopic agglutination test (MAT), and/or polymerase chain reaction (PCR). Among rats, 26% were positive by PCR, including 7% that were also positive by culture for L. interrogans serovars Grippotyphosa, Pyrogenes, and Icterohaemorrhagiae. L. borpetersenii serovar Polonica was isolated for the first time in Egypt in three rats. MAT titers ≥ 1:800 were observed in 11% of rats and 12% of dogs. L. interrogans serovar Grippotyphosa was detected in one cat. Sheep and donkeys were negative for leptospirosis by all methods. Buffaloes and cattle were seropositive in 20% and 44% of animals, respectively. Data indicate that several pathogenic serovars are circulating in the animals, which may pose exposure risks and account for high rates of acute febrile illness. PMID:21363980

  9. A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species

    PubMed Central

    Amornchai, Premjit; Wuthiekanun, Vanaporn; Bailey, Mark S.; Holden, Matthew T. G.; Zhang, Cuicai; Jiang, Xiugao; Koizumi, Nobuo; Taylor, Kyle; Galloway, Renee; Hoffmaster, Alex R.; Craig, Scott; Smythe, Lee D.; Hartskeerl, Rudy A.; Day, Nicholas P.; Chantratita, Narisara; Feil, Edward J.; Aanensen, David M.; Spratt, Brian G.; Peacock, Sharon J.

    2013-01-01

    Background The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. Methodology and Findings We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. Conclusion The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis. PMID:23359622

  10. A replicative plasmid vector allows efficient complementation of pathogenic Leptospira strains.

    PubMed

    Pappas, Christopher J; Benaroudj, Nadia; Picardeau, Mathieu

    2015-05-01

    Leptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp. PMID:25724960

  11. Leptospira species and serovars identified by MALDI-TOF mass spectrometry after database implementation

    PubMed Central

    2014-01-01

    Background Leptospirosis, a spirochaetal zoonotic disease of worldwide distribution, endemic in Europe, has been recognized as an important emerging infectious disease, though yet it is mostly a neglected disease which imparts its greatest burden on impoverished populations from developing countries. Leptospirosis is caused by the infection with any of the more than 230 serovars of pathogenic Leptospira sp. In this study we aimed to implement the MALDI-TOF mass spectrometry (MS) database currently available in our laboratory with Leptospira reference pathogenic (L. interrogans, L. borgpetersenii, L. kirschneri, L. noguchii), intermediate (L. fainei) and saprophytic (L. biflexa) strains of our collection in order to evaluate its possible application to the diagnosis of leptospirosis and to the typing of strains. This was done with the goal of understanding whether this methodology could be used as a tool for the identification of Leptospira strains, not only at species level for diagnostic purposes, but also at serovar level for epidemiological purposes, overcoming the limits of serological and molecular conventional methods. Twenty Leptospira reference strains were analysed by MALDI-TOF MS. Statistical analysis of the protein spectra was performed by ClinProTools software. Results The spectra obtained by the analysis of the reference strains tested were grouped into 6 main classes corresponding to the species analysed, highlighting species-specific protein profiles. Moreover, the statistical analysis of the spectra identified discriminatory peaks to recognize Leptospira strains also at serovar level extending previously published data. Conclusions In conclusion, we confirmed that MALDI-TOF MS could be a powerful tool for research and diagnostic in the field of leptospirosis with broad applications ranging from the detection and identification of pathogenic leptospires for diagnostic purposes to the typing of pathogenic and non-pathogenic leptospires for

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

    PubMed Central

    Ferreira, Ana Sofia; Costa, Pedro; Rocha, Teresa; Amaro, Ana; Vieira, Maria Luísa; Ahmed, Ahmed; Thompson, Gertrude; Hartskeerl, Rudy A.; Inácio, João

    2014-01-01

    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. PMID:25398140

  13. Direct detection and differentiation of pathogenic Leptospira species using a multi-gene targeted real time PCR approach.

    PubMed

    Ferreira, Ana Sofia; Costa, Pedro; Rocha, Teresa; Amaro, Ana; Vieira, Maria Luísa; Ahmed, Ahmed; Thompson, Gertrude; Hartskeerl, Rudy A; Inácio, João

    2014-01-01

    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. PMID:25398140

  14. Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

    PubMed

    Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

    2015-01-01

    The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. PMID:26655756

  15. Molecular detection of Leptospira spp. in the urine of cattle in northern Iran

    PubMed Central

    Shafighi, T; Zahraei Salehi, T; Abdollahpour, G; Asadpour, L; Akbarein, H; Salehzadeh, A

    2014-01-01

    Leptospirosis is a zoonosis of worldwide distribution, caused by Leptospira interrogans and is considered as an emerging global public health problem. Transmission usually results from direct or indirect exposure to the urine or other body fluids of leptospiruric animals which may become a source of infection for human or other animals. Having a humid climate with plenty of annual rainfall, Guilan province is a suitable environment for maintaining Leptospira spp. Hence, early detection of Leptospira spp. in the host prompts control and protection, and the polymerase chain reaction (PCR) is a suitable method. The present report aimed to demonstrate the PCR analysis of bovine urine for detection of leptospiral DNA. A total of 98 urine samples were randomly collected from cattle bladder in Rasht abattoir of Iran and the presence of leptospiral DNA was assayed by PCR amplification of rrs (16S rRNA) gene and the results confirmed by nested PCR. Out of 98 urine samples in 42 samples leptospires DNA was identified with the frequency of 43%. The high presence of the organism in the urine of carriers is a serious threat to the dairy farms and to the public health which requires an effective control measure in the north provinces of Iran. PMID:27175139

  16. Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

    PubMed Central

    Carroll, James A.; Olano, L. Rennee; Sturdevant, Daniel E.; Rosa, Patricia A.

    2015-01-01

    The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. PMID:26655756

  17. Comparison of Immunoprotection of Leptospira Recombinant Proteins with conventional vaccine in experimental animals.

    PubMed

    Parthiban, M; Kumar, S Senthil; Balachandran, C; Kumanan, K; Aarthi, K S; Nireesha, G

    2015-12-01

    Leptospirosis is a bacterial disease caused by bacteria of the genus Leptospira affecting humans and animals. Untreated leptospirosis may result in severe kidney damage, meningitis, liver failure, respiratory distress, and even death. Virulent leptospirosis can rapidly enter kidney fibroblasts and induce a programmed cell death. Thus, it is a challenge for immunologists to develop an effective and safe leptospirosis vaccine. Here, we compared the commercial canine leptospira vaccine and recombinant proteins (OmpL1 and LipL41) with and without adjuvant in terms of immune response and challenge studies in hamsters and immune response studies alone in experimental dogs. The outer membrane proteins viz., lipL41 and OmpL1 of leptospira interrogans serovars icterohaemorrhagiae were amplified. The primers were designed in such a way that amplified products of OmpL1 and lipL41 were ligated and cloned simultaneously into a single vector. The cloned products were expressed in E. coli BL21 cells. The immunoprotection studies were conducted for both recombinant proteins and commercial vaccine. The challenge experiment studies revealed that combination of both rLip41 and rOmpL1 and commercial vaccine gave 83% and 87% protection, respectively. Histopathological investigation revealed mild sub lethal changes were noticed in liver and kidney in commercially vaccinated group alone. The immune responses against recombinant leptospiral proteins were also demonstrated in dogs. PMID:26742322

  18. DIVERSITY OF BAT-ASSOCIATED LEPTOSPIRA IN THE PERUVIAN AMAZON INFERRED BY BAYESIAN PHYLOGENETIC ANALYSIS OF 16S RIBOSOMAL DNA SEQUENCES

    PubMed Central

    MATTHIAS, MICHAEL A.; DÍAZ, M. MÓNICA; CAMPOS, KALINA J.; CALDERON, MARITZA; WILLIG, MICHAEL R.; PACHECO, VICTOR; GOTUZZO, EDUARDO; GILMAN, ROBERT H.; VINETZ, JOSEPH M.

    2008-01-01

    The role of bats as potential sources of transmission to humans or as maintenance hosts of leptospires is poorly understood. We quantified the prevalence of leptospiral colonization in bats in the Peruvian Amazon in the vicinity of Iquitos, an area of high biologic diversity. Of 589 analyzed bats, culture (3 of 589) and molecular evidence (20 of 589) of leptospiral colonization was found in the kidneys, yielding an overall colonization rate of 3.4%. Infection rates differed with habitat and location, and among different bat species. Bayesian analysis was used to infer phylogenic relationships of leptospiral 16S ribosomal DNA sequences. Tree topologies were consistent with groupings based on DNA-DNA hybridization studies. A diverse group of leptospires was found in peri-Iquitos bat populations including Leptospira interrogans (5 clones), L. kirschneri (1), L. borgpetersenii (4), L. fainei (1), and two previously undescribed leptospiral species (8). Although L. kirschenri and L. interrogans have been previously isolated from bats, this report is the first to describe L. borgpetersenii and L. fainei infection of bats. A wild animal reservoir of L. fainei has not been previously described. The detection in bats of the L. interrogans serovar Icterohemorrhagiae, a leptospire typically maintained by peridomestic rats, suggests a rodent-bat infection cycle. Bats in Iquitos maintain a genetically diverse group of leptospires. These results provide a solid basis for pursuing molecular epidemiologic studies of bat-associated Leptospira, a potentially new epidemiologic reservoir of transmission of leptospirosis to humans. PMID:16282313

  19. Isolation of a Seawater Tolerant Leptospira spp. from a Southern Right Whale (Eubalaena australis)

    PubMed Central

    Rago, Virginia; Uhart, Marcela

    2015-01-01

    Leptospirosis is the most widespread zoonotic disease in the world. It is caused by pathogenic spirochetes of the genus Leptospira spp. and is maintained in nature through chronic renal infection of carrier animals. Rodents and other small mammals are the main reservoirs. Information on leptospirosis in marine mammals is scarce; however, cases of leptospirosis have been documented in pinniped populations from the Pacific coast of North America from southern California to British Columbia. We report the isolation of a Leptospira spp. strain, here named Manara, from a kidney sample obtained from a Southern Right Whale (Eubalaena australis) calf, which stranded dead in Playa Manara, Península Valdés, Argentina. This strain showed motility and morphology typical of the genus Leptospira spp. under dark-field microscopy; and grew in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and Fletcher medium after 90 days of incubation at 28°C. Considering the source of this bacterium, we tested its ability to grow in Fletcher medium diluted with seawater at different percentages (1%, 3%, 5%, 7% and 10% v/v). Bacterial growth was detected 48 h after inoculation of Fletcher medium supplemented with 5% sea water, demonstrating the halophilic nature of the strain Manara. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the pathogenic species of the genus Leptospira spp., with sequence similarities within the range 97–100%, and closely related to L. interrogans. Two different PCR protocols targeting genus-specific pathogenic genes (G1-G2, B64I-B64II and LigB) gave positive results, which indicates that the strain Manara is likely pathogenic. Further studies are needed to confirm this possibility as well as determine its serogroup. These results could modify our understanding of the epidemiology of this zoonosis. Until now, the resistance and ability to grow in seawater for long periods of time had been proven for the strain

  20. Isolation of a Seawater Tolerant Leptospira spp. from a Southern Right Whale (Eubalaena australis).

    PubMed

    Grune Loffler, Sylvia; Rago, Virginia; Martínez, Mara; Uhart, Marcela; Florin-Christensen, Monica; Romero, Graciela; Brihuega, Bibiana

    2015-01-01

    Leptospirosis is the most widespread zoonotic disease in the world. It is caused by pathogenic spirochetes of the genus Leptospira spp. and is maintained in nature through chronic renal infection of carrier animals. Rodents and other small mammals are the main reservoirs. Information on leptospirosis in marine mammals is scarce; however, cases of leptospirosis have been documented in pinniped populations from the Pacific coast of North America from southern California to British Columbia. We report the isolation of a Leptospira spp. strain, here named Manara, from a kidney sample obtained from a Southern Right Whale (Eubalaena australis) calf, which stranded dead in Playa Manara, Península Valdés, Argentina. This strain showed motility and morphology typical of the genus Leptospira spp. under dark-field microscopy; and grew in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and Fletcher medium after 90 days of incubation at 28°C. Considering the source of this bacterium, we tested its ability to grow in Fletcher medium diluted with seawater at different percentages (1%, 3%, 5%, 7% and 10% v/v). Bacterial growth was detected 48 h after inoculation of Fletcher medium supplemented with 5% sea water, demonstrating the halophilic nature of the strain Manara. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the pathogenic species of the genus Leptospira spp., with sequence similarities within the range 97-100%, and closely related to L. interrogans. Two different PCR protocols targeting genus-specific pathogenic genes (G1-G2, B64I-B64II and LigB) gave positive results, which indicates that the strain Manara is likely pathogenic. Further studies are needed to confirm this possibility as well as determine its serogroup. These results could modify our understanding of the epidemiology of this zoonosis. Until now, the resistance and ability to grow in seawater for long periods of time had been proven for the strain

  1. Cross-Sectional Study of Leptospira Seroprevalence in Humans, Rats, Mice, and Dogs in a Main Tropical Sea-Port City

    PubMed Central

    Romero-Vivas, Claudia M. E.; Cuello-Pérez, Margarett; Agudelo-Flórez, Piedad; Thiry, Dorothy; Levett, Paul N.; Falconar, Andrew K. I.

    2013-01-01

    Samples were collected from 128 symptomatic humans, 83 dogs, 49 mice, and 20 rats (Rattus rattus: 16; Rattus norvegicus: 4) in neighborhoods where human leptospirosis have been reported within the principal sea-port city of Colombia. Seroprevalences were assessed against 19 pathogenic, 1 intermediate pathogenic, and 1 saprophytic Leptospira serogroups. Pathogenic Leptospira were confirmed using conventional Leptospira-specific polymerase chain-reaction and pulsed-field gel electrophoresis analysis was used for serovar identification. Seroprevalences of 20.4%, 12.5%, 25.0%, 22.9%, and 12.4% were obtained against one to seven different serogroups in mice, R. rattus, R. norvegicus, dogs, and humans, respectively. The DNA was confirmed to be from pathogenic Leptospira by detecting the lipL32 gene in 12.5%, 3.7%, and 0.03% of the R. rattus, dog, and human samples, respectively. The first genetically typed Colombian isolate was obtained from a rat and identified as Leptospira interrogans serovar Icterohaemorrhagiae/Copenhageni. PMID:23149584

  2. Characterization of Leptospira isolates from serovar hardjo by ribotyping, arbitrarily primed PCR, and mapped restriction site polymorphisms.

    PubMed Central

    Perolat, P; Merien, F; Ellis, W A; Baranton, G

    1994-01-01

    Leptospira serovar hardjo isolates of the hardjoprajitno and hardjobovis genotypes were characterized by ribotyping, arbitrarily primed PCR (AP-PCR) fingerprinting, and the study of mapped restriction site polymorphisms (MRSPs) in rrs and rrl genes. After restriction of chromosomal DNA with BglII, EcoRI, or HindIII, each genotype was individualized with a distinct ribotype. The fingerprints produced by AP-PCR with seven primers clearly separated the two groups; primers KF and RSP produced species-specific products which assigned hardjoprajitno and hardjobovis isolates to the species L. interrogans sensu stricto and L. borgpetersenii, respectively. Furthermore, AP-PCR fingerprints gave evidence of a considerable genomic heterogeneity at the strain level among the hardjobovis group. Conversely, the hardjoprajitno group was homogeneous. MRSP profiles in ribosomal genes indicated that hardjoprajitno and hardjobovis isolates belonged to L. interrogans MRSP group B and L. borgpetersenii group C, respectively. AP-PCR and determination of MRSPs in ribosomal genes proved to be quick and reliable methods for typing Leptospira strains and for studying intraspecific population structures. Images PMID:7989548

  3. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    PubMed

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen. PMID:26527082

  4. Severe Leptospira interrogans serovar Icterohaemorrhagiae infection with hepato-renal-pulmonary involvement treated with corticosteroids

    PubMed Central

    Schulze, Marco H; Raschel, Heribert; Langen, Heinz-Jakob; Stich, August; Tappe, Dennis

    2014-01-01

    Key Clinical Message The traditional concept of immediate antibiotic treatment in suspected leptospirosis seems to be especially important for patients up to day 4 of clinical illness. As immune mechanisms probably play a crucial role in advanced leptospirosis with presumed pulmonary hemorrhages, patients might benefit from corticosteroids or other immunosuppressive agents beside antibiotics. PMID:25614810

  5. [Evaluation of the vaccine potentiality of 2 strains from Leptospira interrogans serogroup Ballum].

    PubMed

    Rodríguez, Andrés González; Jiménez, Yoandra Rodríguez; Santiesteban, Niurka Batista; Abreu, Yolanda Valdés; Osenes, Juan Francisco Núñez; González, Marta González

    2005-01-01

    Two vacine candidate strains from Ballum serogroup were characterized and their immunogenicity and protective power were studied in hamster. The monovalent vaccine formulations obtained showed a high immunogenicity and capacity of homologous protection and, in a lesser degree, of cross protection. PMID:17966485

  6. Geographical Dissemination of Leptospira interrogans serovar Pomona During Seasonal Migration of California Sea Lions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospirosis is one of the most widespread bacterial zoonoses in the world, and affects both terrestrial and marine mammals. Little information is available describing the geographical spread of leptospirosis, but it is logical to assume that animal migrations contribute to this process. There ha...

  7. Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate

    PubMed Central

    Oliveira, Thaís Larré; Grassmann, André Alex; Schuch, Rodrigo Andrade; Seixas Neto, Amilton Clair Pinto; Mendonça, Marcelo; Hartwig, Daiane Drawanz; McBride, Alan John Alexander; Dellagostin, Odir Antônio

    2015-01-01

    The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37’s ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis. PMID:26588685

  8. Leptospira spp.: Novel insights into host-pathogen interactions.

    PubMed

    Fernandes, Luis G; Siqueira, Gabriela H; Teixeira, Aline R F; Silva, Lucas P; Figueredo, Jupciana M; Cosate, Maria R; Vieira, Monica L; Nascimento, Ana L T O

    2016-08-01

    Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is an important infectious disease that affects humans and animals. The disease causes economic losses as it affects livestock, with decreased milk production and death. Our group is investigating the genome sequences of L. interrogans targeting surface-exposed proteins because, due to their location, these proteins are capable to interact with several host components that could allow establishment of the infection. These interactions may involve adhesion of the bacteria to extracellular matrix (ECM) components and, hence, help bacterial colonization. The bacteria could also react with the host fibrinolytic system and/or with the coagulation cascade components, such as, plasminogen (PLG) and fibrinogen (Fg), respectively. The binding with the first system generates plasmin (PLA), increasing the proteolytic power of the bacteria, while the second interferes with clotting in a thrombin-catalyzed reaction, which may promote hemorrhage foci and increase bacterial dissemination. Interaction with the complement system negative regulators may help bacteria to evade the host immune system, facilitating the invasion. This work compiles the main described leptospiral proteins that could act as adhesins, as PLG and fibrinogen receptors and as complement regulator binding proteins. We present models in which we suggest possible mechanisms of how leptospires might colonize and invade host tissues, causing the disease. Understanding leptospiral pathogenesis will help to identify antigen candidates that would contribute to the development of more effective vaccines and diagnostic tests. PMID:26727033

  9. Amino acid biosynthesis in the spirochete Leptospira: evidence for a novel pathway of isoleucine biosynthesis.

    PubMed

    Charon, N W; Johnson, R C; Peterson, D

    1974-01-01

    Radioactive carbon dioxide was incubated with growing cells of Leptospira interrogans serotypes semaranga and tarassovi, and the specific activities and distribution of the label within the cellular amino acids were determined. The origins of the carbon skeletons of all the acid-stable amino acids except isoleucine were found to be consistent with known biosynthetic pathways for these amino acids. Experiments using radioactive carbon dioxide and other tracers indicated that most of the isoleucine was synthesized by a pathway not involving threonine. The origin of the carbon skeleton of isoleucine consisted of two residues of pyruvate (carbons 2 and 3) and acetate of acetyl-coenzyme A by this pathway. Isotope competition studies indicated that the pathway was regulated by isoleucine. The results are discussed in relation to two proposed pathways of isoleucine biosynthesis involving citramalate as an intermediate. PMID:4808901

  10. Urea Utilization by Leptospira

    PubMed Central

    Kadis, Solomon; Pugh, William L.

    1974-01-01

    One representative of each of five different pathogenic serotypes of Leptospira as well as one saprophytic strain were capable of growing on medium containing urea in place of an ammonium salt as a nitrogen source. Growth of all of the organisms tested on 1% urea was substantial, but only those that exhibited strong urease activity could grow to any appreciable extent on urea at a concentration as high as 2%. Intact urea-grown cells of the pathogenic serotypes tested (grippotyphosa and icterohaemorrhagiae) exhibited urease activity, with the level of activity of the former being considerably greater. No urease could be detected in cells of the saprophytic strain. When the pathogenic leptospires were sonicated or treated with toluene, the urease activity was greatly enhanced. When cultivated on NH4Cl, neither intact nor disrupted cells of any of the strains tested exhibited any urease activity. Cells of the grippotyphosa and icterohaemorrhagiae strains exhibited diauxic growth when cultivated in the presence of both NH4Cl and urea, whereas only monophasic growth could be detected for the saprophytic test strain. The experimental data on urea utilization and urease activity, when considered in the light of previously reported findings on leptospiral pathology, renal physiology, and the role of urease in other bacterial infections, suggests a significant role for leptospiral urease (in addition to other factors) in determining localization of the organism in the kidney and contributing to the resultant kidney pathology. PMID:4426709

  11. Passive immunization with Leptospira LPS-specific agglutinating but not non-agglutinating monoclonal antibodies protect guinea pigs from fatal pulmonary hemorrhages induced by serovar Copenhageni challenge.

    PubMed

    Challa, Sreerupa; Nally, Jarlath E; Jones, Carroll; Sheoran, Abhineet S

    2011-06-15

    Leptospira interrogans serovar Copenhageni causes pulmonary hemorrhages with respiratory failure, a major cause of death in leptospirosis patients. Protective immunity to Leptospira is known to correlate with the production of leptospiral lipopolysaccharide (L-LPS)-specific agglutinating antibodies. We generated L-LPS-specific mouse monoclonal antibodies (MAbs) and investigated if these MAbs can protect guinea pigs against fatal pulmonary hemorrhages caused by serovar Copenhageni. The MAbs L8H4 and L9B11 against 22kDa L-LPS agglutinated leptospires and completely protected guinea pigs from the development of fatal pulmonary hemorrhages by serovar Copenhageni, whereas the MAb L4C1 against 8kDa L-LPS neither agglutinated the bacteria nor protected the animals against the fatal pulmonary hemorrhages. PMID:21549788

  12. Comparison of Two Multilocus Sequence Based Genotyping Schemes for Leptospira Species

    PubMed Central

    Boonsilp, Siriphan; Wuthiekanun, Vanaporn; Nalam, Kishore; Spratt, Brian G.; Aanensen, David M.; Smythe, Lee D.; Ahmed, Niyaz; Feil, Edward J.; Hartskeerl, Rudy A.; Peacock, Sharon J.

    2011-01-01

    Background Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results. Methods and Findings A total of 48 isolates consisting of L. interrogans (n = 40) and L. kirschneri (n = 8) were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively). Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs) by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence) was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability) of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5–96.5) vs. 93.5 (95% CI 88.6–98.4). The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L. Conclusions This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages. PMID:22087342

  13. Reactivity of heat-stable Leptospira antigenic preparation used in enzyme-linked immunosorbent assay for detection of antibodies in swine serum.

    PubMed

    Wasiński, B; Pejsak, Z

    2012-01-01

    Serology plays an important role in laboratory diagnosis of leptospirosis. Apart from the most often used microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA) seems to be useful especially in screenings of animal herds. The ELISA used for detection of antibodies against selected Leptospira serogroups in swine serum samples was investigated during the study. An essential element of this test is heat-stable antigenic preparation from cultures of Leptospira interrogans serovars Icterohaemorrhagiae, Pomona and L. borgpetersenii serovar Sejroe. The aim of the present study was to identify and analyze ELISA heat-stable antigen fractions playing a role in the reaction with leptospiral antibodies indicated in swine serum. Reactivity of the three-component antigenic preparation was compared in immunoblotting with reactivity of six heat-stable antigenic preparations made from the following single serovars: L. interrogans serovars Icterohaemorrhagiae, Pomona, Canicola, L. borgpetersenii serovars Sejroe, Tarassovi and L. kirshneri serovar Grippotyphosa. All antigenic preparations were submitted to SDS-PAGE and transferred to a nitrocellulose membrane using a semidry system. After the transfer, the membrane was incubated with diluted swine serum containing antibodies specific for one of the six above mentioned Leptospira serovars. For the three-component antigenic preparation and antigens prepared from single serovars the immunoblot revealed reaction of sera with fractions of the 20-26 kDa region and around the 14.5 kDa region. The investigated heat-stable Leptospira antigenic preparation contains fractions demonstrating serogroup- and species-specificity. Fraction 20-26 kDa showed serogroup-specific activity, whereas the fraction around 14.5 kDa showed species-specific activity. PMID:22708354

  14. Risk of infection and associated influenza-like disease among abattoir workers due to two Leptospira species.

    PubMed

    Dreyfus, A; Heuer, C; Wilson, P; Collins-Emerson, J; Baker, M G; Benschop, J

    2015-07-01

    The aims of this study were to determine the annual incidence of infection with Leptospira interrogans serovar Pomona and/or Leptospira borgpetersenii serovar Hardjo and its association with influenza-like illness (ILI) in meat workers in New Zealand. Sera were collected twice, 50-61 weeks apart, from 592 workers at eight abattoirs slaughtering sheep (n = 4), cattle (n = 2) and deer (n = 2), and tested by the microscopic agglutination test for Hardjo and Pomona. Forty-nine (8·3%) participants either seroconverted or had at least a twofold increased serological titre against either serovar. The worker infection risk was higher in sheep abattoirs (11·9%) than in abattoirs processing deer (0%) or cattle (1·2%) (P < 0·01). The annualized risk of mild (ILI) or severe clinical disease attributable to the two Leptospira serovars was 2·7%. This study has demonstrated that meat workers are at substantial risk of infection and clinical disease, suggesting further investigation of infection sources and preventive measures are warranted. PMID:25266854

  15. A novel flagellar sheath protein, FcpA, determines filament coiling, translational motility and virulence for the Leptospira spirochete.

    PubMed

    Wunder, Elsio A; Figueira, Cláudio P; Benaroudj, Nadia; Hu, Bo; Tong, Brian A; Trajtenberg, Felipe; Liu, Jun; Reis, Mitermayer G; Charon, Nyles W; Buschiazzo, Alejandro; Picardeau, Mathieu; Ko, Albert I

    2016-08-01

    Leptospira are unique among bacteria based on their helical cell morphology with hook-shaped ends and the presence of periplasmic flagella (PF) with pronounced spontaneous supercoiling. The factors that provoke such supercoiling, as well as the role that PF coiling plays in generating the characteristic hook-end cell morphology and motility, have not been elucidated. We have now identified an abundant protein from the pathogen L. interrogans, exposed on the PF surface, and named it Flagellar-coiling protein A (FcpA). The gene encoding FcpA is highly conserved among Leptospira and was not found in other bacteria. fcpA(-) mutants, obtained from clinical isolates or by allelic exchange, had relatively straight, smaller-diameter PF, and were not able to produce translational motility. These mutants lost their ability to cause disease in the standard hamster model of leptospirosis. Complementation of fcpA restored the wild-type morphology, motility and virulence phenotypes. In summary, we identified a novel Leptospira 36-kDa protein, the main component of the spirochete's PF sheath, and a key determinant of the flagella's coiled structure. FcpA is essential for bacterial translational motility and to enable the spirochete to penetrate the host, traverse tissue barriers, disseminate to cause systemic infection and reach target organs. PMID:27113476

  16. Interaction of Human Complement Factor H Variants Tyr402 and His402 with Leptospira spp.

    PubMed Central

    Silva, Aldacilene Souza; Valencia, Mónica Marcela Castiblanco; Cianciarullo, Aurora Marques; Vasconcellos, Sílvio Arruda; Barbosa, Angela Silva; Isaac, Lourdes

    2011-01-01

    Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. The disease represents a serious public health problem in underdeveloped tropical countries. Leptospires infect hosts through small abrasions in the skin or mucous membranes and they rapidly disseminate to target organs. The capacity of some pathogenic leptospiral strains to acquire the negative complement regulators factor H (FH) and C4b binding protein correlates with their ability to survive in human serum. In this study we assessed the functional consequences of the age macular degeneration-associated polymorphism FH His402 or FH Tyr402 on FH–Leptospira interactions. In binding assays using sub-saturating amounts of FH, the FH Tyr402 variant interacted with all the strains tested more strongly than the FH His402 variant. At higher concentrations, differences tended to disappear. We then compared cofactor activities displayed by FH His402 and FH Tyr402 bound to the surface of L. interrogans. Both variants exhibit similar activity as cofactors for Factor I-mediated cleavage of C3b, thus indicating that they do not differ in their capacity to regulate the complement cascade. PMID:22566834

  17. Isolation and Characterization of New Leptospira Genotypes from Patients in Mayotte (Indian Ocean)

    PubMed Central

    Bourhy, Pascale; Collet, Louis; Clément, Sabine; Huerre, Michel; Ave, Patrick; Giry, Claude; Pettinelli, François; Picardeau, Mathieu

    2010-01-01

    Background Leptospirosis has been implicated as a severe and fatal form of disease in Mayotte, a French-administrated territory located in the Comoros archipelago (southwestern Indian Ocean). To date, Leptospira isolates have never been isolated in this endemic region. Methods and Findings Leptospires were isolated from blood samples from 22 patients with febrile illness during a 17-month period after a PCR-based screening test was positive. Strains were typed using hyper-immune antisera raised against the major Leptospira serogroups: 20 of 22 clinical isolates were assigned to serogroup Mini; the other two strains belonged to serogroups Grippotyphosa and Pyrogenes, respectively. These isolates were further characterized using partial sequencing of 16S rRNA and ligB gene, Multi Locus VNTR Analysis (MLVA), and pulsed field gel electrophoresis (PFGE). Of the 22 isolates, 14 were L. borgpetersenii strains, 7 L. kirschneri strains, and 1, belonging to serogoup Pyrogenes, was L. interrogans. Results of the genotyping methods were consistent. MLVA defined five genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes. PFGE fingerprint patterns of clinical strains did not match any of the patterns in the reference strains belonging to the same serogroup, suggesting that the strains were novel serovars. Conclusions Preliminary PCR screening of blood specimen allowed a high isolation frequency of leptospires among patients with febrile illness. Typing of leptospiral isolates showed that causative agents of leptospirosis in Mayotte have unique molecular features. PMID:20582311

  18. Urinary shedding of leptospires and presence of Leptospira antibodies in healthy dogs from Upper Bavaria.

    PubMed

    Llewellyn, Julia-Rebecca; Krupka-Dyachenko, Inke; Rettinger, Anna Lena; Dyachenko, Viktor; Stamm, Ivonne; Kopp, Peter Andreas; Straubinger, Reinhard Konrad; Hartmann, Katrin

    2016-01-01

    Leptospirosis is classified as a re-emerging zoonotic disease with global impor- tance. The aim of this study was to determine urinary shedding of leptospires in healthy dogs and to identify the shedded leptospire species. Furthermore, antibody presence against leptospires was evaluated. In a prospective study urine samples of 200 healthy dogs from Upper Bavaria were randomly collected and evaluated by real-time polymerase chain reaction (PCR) specific for the lipL32 gene of pathogenic Leptospira (L) spp. Positive samples were further character- ized via multilocus sequence typing (MLST) to identify the Leptospira species. Microagglutination test (MAT) was performed to determine serum antibody titers. Three of 200 urine samples were found to be PCR-positive resulting in a urinary shedding prevalence of 1.5% (95% confidence interval 0.3-4.5%). All three dogs had been vaccinated before with a bivalent vaccine, covering the serogroups Canicola and lcterohaemorrhagiae. One dog shed leptospires of the species L. borgpetersenii, and two of the species L. interrogans. Of all dogs, 17.0% had antibody titers ≥ 1:100, and 3.5% titers ≥ 1:400 to serovars of non-vaccinal sero- groups. Healthy dogs that shed leptospires represent a possible risk for humans and other animals. The study emphasizes the importance of general hygiene measures in veterinary practice while handling urine of all dogs, and the use of vaccines that protect against a broader range of serogroups and that prevent urinary shedding. PMID:27344919

  19. 9 CFR 113.105 - Leptospira Hardjo Bacterin.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Leptospira Hardjo Bacterin. 113.105... Inactivated Bacterial Products § 113.105 Leptospira Hardjo Bacterin. Leptospira Hardjo Bacterin shall be produced from a culture of Leptospira hardjo which has been inactivated and is nontoxic. Each serial...

  20. 9 CFR 113.105 - Leptospira Hardjo Bacterin.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Hardjo Bacterin. 113.105... Inactivated Bacterial Products § 113.105 Leptospira Hardjo Bacterin. Leptospira Hardjo Bacterin shall be produced from a culture of Leptospira hardjo which has been inactivated and is nontoxic. Each serial...

  1. 9 CFR 113.105 - Leptospira Hardjo Bacterin.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Leptospira Hardjo Bacterin. 113.105... Inactivated Bacterial Products § 113.105 Leptospira Hardjo Bacterin. Leptospira Hardjo Bacterin shall be produced from a culture of Leptospira hardjo which has been inactivated and is nontoxic. Each serial...

  2. 9 CFR 113.105 - Leptospira Hardjo Bacterin.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Leptospira Hardjo Bacterin. 113.105... Inactivated Bacterial Products § 113.105 Leptospira Hardjo Bacterin. Leptospira Hardjo Bacterin shall be produced from a culture of Leptospira hardjo which has been inactivated and is nontoxic. Each serial...

  3. 9 CFR 113.105 - Leptospira Hardjo Bacterin.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Leptospira Hardjo Bacterin. 113.105... Inactivated Bacterial Products § 113.105 Leptospira Hardjo Bacterin. Leptospira Hardjo Bacterin shall be produced from a culture of Leptospira hardjo which has been inactivated and is nontoxic. Each serial...

  4. Neutrophil Extracellular Traps are Involved in the Innate Immune Response to Infection with Leptospira

    PubMed Central

    Scharrig, Emilia; Carestia, Agostina; Ferrer, María F.; Cédola, Maia; Pretre, Gabriela; Drut, Ricardo; Picardeau, Mathieu; Schattner, Mirta; Gómez, Ricardo M.

    2015-01-01

    NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden. PMID:26161745

  5. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

    PubMed

    Matsunaga, James; Barocchi, Michele A; Croda, Julio; Young, Tracy A; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A; Reis, Mitermayer G; Riley, Lee W; Haake, David A; Ko, Albert I

    2003-08-01

    Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

  6. Molecular and serological investigation of Leptospira and leptospirosis in dogs in Japan.

    PubMed

    Koizumi, Nobuo; Muto, Maki Mizutani; Akachi, Shigehiro; Okano, Shou; Yamamoto, Seigo; Horikawa, Kazumi; Harada, Seiya; Funatsumaru, Sadayuki; Ohnishi, Makoto

    2013-04-01

    Canine leptospirosis, which is caused by infection with pathogenic Leptospira species, occurs worldwide, but information regarding the causative Leptospira serotypes and genotypes and their effects on virulence in dogs remains limited. Monitoring acute leptospirosis in dogs as sentinels can also aid in estimating the risk of human leptospirosis, particularly when the disease is rare, as it currently is in Japan. Among 283 clinically suspected cases of leptospirosis diagnosed from August 2007 to March 2011 in Japan, 83 cases were laboratory diagnosed as leptospirosis by blood culture, a rise in antibody titres in paired sera using a microscopic agglutination test (MAT) and/or DNA detection using flaB-nested PCR. The infected dogs comprised hunting dogs (31 dogs) and companion animals (50 dogs) and two unknown; 63.4 % of the infected dogs were males. The mortality rate was 53.2 %. A rise of at least fourfold in MAT titre was detected in 30 dogs whose paired serum samples were obtained, and the predominant reactive serogroup was Hebdomadis (53.3 %), followed by Australis (16.7 %) and Autumnalis (16.7 %). Leptospira interrogans was isolated from 45 dogs of the following serogroups: Australis (16), Autumnalis (six), Canicola (one), Hebdomadis (21) and Icterohaemorrhagiae (one). All of these serogroups caused lethal infections (57.1-100 %). Genetic heterogeneity was demonstrated in serogroups Australis, Autumnalis and Hebdomadis by multilocus sequence typing (MLST) and/or RFLP analysis based on PFGE. In serogroup Hebdomadis, each genotype determined by MLST had a unique mortality rate in the infected dogs. Although classic canine leptospirosis is associated with serovars Canicola and Icterohaemorrhagiae, serogroup Hebdomadis has become the predominant serogroup causing high mortality in Japan. This study suggests that the virulence of members of serogroup Hebdomadis in dogs may be associated with the genotypes in this serogroup. PMID:23264455

  7. Protein typing of major outer membrane lipoproteins from Chinese pathogenic Leptospira spp. and characterization of their immunogenicity.

    PubMed

    Luo, Dongjiao; Xue, Feng; Ojcius, David M; Zhao, Jinfang; Mao, Yafei; Li, Liwei; Lin, Xuai; Yan, Jie

    2009-12-10

    Leptospirosis, caused by different Leptospira species, is one of the most widespread zoonotic infections worldwide. Here we expressed three major leptospiral lipoproteins and examined their immunogenicity. All the pathogenic Leptospira strains tested possess the lipL21, lipL32 and lipL41 genes, but the latter two can be further divided into different gene types (lipL32-1, lipL32-2, lipL41-1, lipL41-2). Microscopic agglutination test revealed that rLipLs antisera had extensive cross-immunoagglutination among the 178 leptospiral strains in which rLipL32-1 contributed the highest agglutination titer. The rLipLs-based ELISAs established in this study demonstrated that in the sera of 385 leptospirosis patients infected with different serovars of Leptospira interrogans, rLipL32-1 had the highest positive rates for IgG and IgM (89.4-98.7%), followed by the IgG/IgM positive rates of rLipL21 (87.0-96.1%) and rLipL32-2 (86.5-96.9%), while the two rLipL41s presented the lowest IgG/IgM positive rates (69.9-83.9%). The immunoprotective levels in guinea pigs of rLipL32-1 (58.3% and 66.7%) were the highest, compared to those of the other rLipLs (25.0-58.3%). Multiple different rLipLs would increase immunoprotective levels (from 58.3% and 66.7% to 83.3% and 91.7%). The data suggest that all the rLipLs are the genus-specific superficial antigens of pathogenic Leptospira species and should be considered in designing universal vaccines against leptospirosis. PMID:19796723

  8. The usefulness of semi-solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka, Japan.

    PubMed

    Saito, Mitsumasa; Villanueva, Sharon Y A M; Masuzawa, Toshiyuki; Haraguchi, Yusuke; Ita, Shuhei; Miyahara, Satoshi; Ozuru, Ryo; Yamaguchi, Takayoshi; Yoshimura, Michinobu; Ikejiri, Mami; Aramaki, Natsumi; Amran, Muhammad Yunus; Muslich, Lisa Tenriesa; Iida, Ken-ichiro; Yanagihara, Yasutake; Gloriani, Nina G; Yoshida, Shin-ichi

    2015-06-01

    Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi-solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira-positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi-solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi-solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats. PMID:25890990

  9. Isolation and molecular characterization of Leptospira borgpetersenii serovar Hardjo strain Hardjobovis in the urine of naturally infected cattle in Brazil.

    PubMed

    Chideroli, R T; Pereira, U P; Gonçalves, D D; Nakamura, A Y; Alfieri, A A; Alfieri, A F; Freitas, J C

    2016-01-01

    Most epidemiologic studies on bovine leptospirosis are based on serological tests that use antibodies against several serotypes, including the serovar Hardjo, which is widespread and considered to be the most adapted to bovine hosts. However, using only serological studies is not sufficient to identify and distinguish species of leptospires. The aim of this study was report the first isolation in Brazil of two strains serovar Hardjo obtained in urine samples from naturally infected cows in a small Brazilian dairy herd and find the genetic species and consequently the type strain Hardjobovis by molecular characterization. Fifteen dairy cows with a history of reproductive failure, such as abortion and infertility, were selected. Urine samples obtained from each animal were immediately seeded in tubes containing Ellinghausen-McCullough-Johnson-Harris culture medium. The identification of the isolates was performed by Multilocus variable-number tandem-repeat analysis (MLVA) technique and phylogenetic analysis of partial sequence of gene sec Y. From the 15 urine samples evaluated, two Leptospira were found and identified as the Londrina 49 and Londrina 54 strains. The MLVA profiles and sequencing of gene sec Y characterized the isolates as L. borgpetersenii serovar Hardjo strain Hadjobovis because it has different genetic pattern of Leptospira interrogans serovar Hardjo strain Hardjoprajitno. Therefore, more studies are needed including isolation and molecular characterization from regional strains to obtain a better knowledge about epidemiology of serovar Hardjo in bovine which may assist in future strategies of prevention and control of bovine leptospirosis. PMID:26909976

  10. Antimicrobial Disk Susceptibility Testing of Leptospira spp. Using Leptospira Vanaporn Wuthiekanun (LVW) Agar.

    PubMed

    Wuthiekanun, Vanaporn; Amornchai, Premjit; Langla, Sayan; White, Nicholas J; Day, Nicholas P J; Limmathurotsakul, Direk; Peacock, Sharon J

    2015-08-01

    Leptospira Vanaporn Wuthiekanun (LVW) agar was used to develop a disk diffusion assay for Leptospira spp. Ten pathogenic Leptospira isolates were tested, all of which were susceptible to 17 antimicrobial agents (amoxicillin/clavulanic acid, amoxicillin, azithromycin, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, doripenem, doxycycline, gentamicin, linezolid, nitrofurantoin, penicillin, piperacillin/tazobactam, and tetracycline). All 10 isolates had no zone of growth inhibition for four antimicrobials (fosfomycin, nalidixic acid, rifampicin, and trimethoprim/sulfamethoxazole). Of the ten Leptospira, seven had a growth inhibition zone of ≤ 21 mm for aztreonam, the zone diameter susceptibility break point for Enterobacteriaceae. This assay could find utility as a simple screening method during the epidemiological surveillance of antimicrobial resistance in Leptospira spp. PMID:26055750

  11. Leptospira immunoglobulin-like proteins as a serodiagnostic marker for acute leptospirosis.

    PubMed

    Croda, Julio; Ramos, João G R; Matsunaga, James; Queiroz, Adriano; Homma, Akira; Riley, Lee W; Haake, David A; Reis, Mitermayer G; Ko, Albert I

    2007-05-01

    There is an urgent need for improved diagnosis of leptospirosis, an emerging infectious disease which imparts a large disease burden in developing countries. We evaluated the use of Leptospira immunoglobulin (Ig)-like (Lig) proteins as a serodiagnostic marker for leptospirosis. Lig proteins have bacterial immunoglobulin-like (Big) tandem repeat domains, a moiety found in virulence factors in other pathogens. Sera from patients identified during urban outbreaks in Brazil reacted strongly with immunoblots of a recombinant fragment comprised of the second to sixth Big domains of LigB from L. interrogans serovar Copenhageni, the principal agent for transmission in this setting. Furthermore, the sera recognized an analogous LigB fragment derived from L. kirschneri serovar Grippotyphosa, a pathogenic serovar which is not endemic to the study area. The immunoblot assay detected anti-LigB IgM antibodies in sera from 92% (95% confidence interval, 85 to 96%) of patients during acute-phase leptospirosis. The assay had a sensitivity of 81% for sera from patients with less than 7 days of illness. Anti-LigB antibodies were found in sera from 57% of the patients who did not have detectable anti-whole-Leptospira responses as detected by IgM enzyme-linked immunosorbent assay and microagglutination test. The specificities of the assay were 93 to 100% and 90 to 97% among sera from healthy individuals and patients with diseases that have clinical presentations that overlap with those of leptospirosis, respectively. These findings indicate that the antibody response to this putative virulence determinant is a sensitive and specific marker for acute infection. The use of this marker may aid the prompt and timely diagnosis required to reduce the high mortality associated with severe forms of the disease. PMID:17360842

  12. The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue.

    PubMed

    Fearnley, C; Wakeley, P R; Gallego-Beltran, J; Dalley, C; Williamson, S; Gaudie, C; Woodward, M J

    2008-08-01

    A LightCycler real-time PCR hybridization probe-based assay that detects a conserved region of the16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n=180) and aborted pig foetuses (n=24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n=7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n=30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. PMID:17961617

  13. A novel tetravalent Leptospira bacterin protects against infection and shedding following challenge in dogs

    PubMed Central

    Klaasen, H. L. B. M.; van der Veen, M.; Molkenboer, M. J. C. H.; Sutton, D.

    2013-01-01

    Recent evidence based on the current epidemiological situation suggests that vaccines against canine leptospirosis in Europe should be directed against infection with Leptospira interrogans (sensu lato) serogroups Canicola, Icterohaemorrhagiae, Grippotyphosa and Australis. In the eight studies presented here, dogs were vaccinated with Nobivac L4 (MSD Animal Health), a new tetravalent inactivated vaccine containing antigen from four strains representing these four serogroups. The dogs were then challenged, together with unvaccinated control dogs, using heterologous strains from the same four serogroups. In four of the studies, pups without agglutinating antibodies against the four serogroups were vaccinated with Nobivac L4 vaccine. In a further four studies, Nobivac L4 vaccine was given 48 hours after administration of antiserum from vaccinated dogs designed to mimic the serological status of pups with maternally derived antibodies against these serogroups. In all eight studies, vaccine efficacy was assessed in terms of antibody response, clinical signs, fever, thrombocyte count, frequency of positive isolation of challenge organisms from blood, urine and kidney and frequency of interstitial nephritis. The results demonstrate that Nobivac L4 vaccine induces sterile immunity against leptospiraemia and renal infection with strains of serogroups Canicola, Icterohaemorrhagiae and Grippotyphosa, and induces sterile immunity against leptospiraemia with a strain of serogroup Australis. Since sterile immunity was achieved in pups pretreated with antiserum as well, it can be concluded that this vaccine is also likely to be efficacious in the face of maternally derived antibodies in pups from the age of six weeks. PMID:23180149

  14. Intermediate Energy Metabolism of Leptospira

    PubMed Central

    Baseman, J. B.; Cox, C. D.

    1969-01-01

    Metabolic studies were performed on three representative serotypes of Leptospira: a water isolate designated B16 and two pathogenic serotypes, pomona and schueffneri. Examination of whole cells of B16 for their ability to oxidize various substrates revealed that oleate significantly stimulated oxygen uptake. The respiratory quotient of 0.7 implied that oleate was degraded to carbon dioxide and water. Other substrates, such as carbohydrates, alcohols, intermediates of the citric acid cycle, and short-chain acids, including selected amino acids, did not stimulate endogenous respiration of whole cells. No oxygen uptake could be measured when cell-free extracts were tested with the substrates used with whole cells. Enzymatic analyses of cell-free extracts of the three strains demonstrated enzymes of the citric acid cycle, enzymes of the glycolytic and pentose pathways, and the general acyl coenzyme A dehydrogenase required for β-oxidation of fatty acids. Strain B16 and the two pathogenic serotypes appeared to possess similar metabolic capabilities. Enzymatic data might also explain the apparent inability of B16 to oxidize other substrates; kinases necessary for activation of common nonphosphorylated compounds were not detected in leptospiral extracts. These findings emphasized the dependence of leptospiral growth upon long-chain fatty acids. PMID:5776541

  15. Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer membrane protein OmpL32

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer membrane proteins has been shown to modulate the effectiveness of the host immu...

  16. Isolation of Leptospira from a phocid: acute renal failure and mortality from Leptospirosis in rehabilitated northern elephant seals (Mirounga angustirostris), California, USA.

    PubMed

    Delaney, Martha A; Colegrove, Kathleen M; Spraker, Terry R; Zuerner, Richard L; Galloway, Renee L; Gulland, Frances M D

    2014-07-01

    During rehabilitation, acute renal failure due to leptospirosis occurred in eight male northern elephant seals (Mirounga angustirostris) that stranded along the central California coast in 2011. Characteristic histologic lesions including renal tubular degeneration, necrosis, and mineralization, and mild lymphoplasmacytic interstitial nephritis were noted in the six animals examined. Immunohistochemistry, bacterial culture, and PCR were positive in 2/3, 2/3, and 3/4 seals, respectively, and 6/8 had high serum antibody titers to Leptospira interrogans serovar pomona. Pulsed-field gel electrophoresis confirmed one isolate as serovar pomona. Variable number tandem repeat (VNTR) analysis showed both elephant seal isolates were identical to each other but distinct from those isolated from California sea lions (Zalophus californianus). The time from stranding to onset of azotemia was 1 to 38 (median=24) days, suggesting some seals were infected at the rehabilitation facility. Based on temporal and spatial incidence of infection, transmission among elephant seals likely occurred during rehabilitation. Molecular (VNTR) analysis of the two isolates indicates there is a unique L. interrogans serovar pomona genotype in elephant seals, and sea lions were not the source of infection prior to or during rehabilitation. This study confirms the susceptibility of northern elephant seals to leptospirosis, indicates intraspecies transmission during rehabilitation, and reports the first isolation and preliminary characterization of leptospires from elephant seals. PMID:24807176

  17. First Isolation and Direct Evidence for the Existence of Large Small-Mammal Reservoirs of Leptospira sp. in Madagascar

    PubMed Central

    Rahelinirina, Soanandrasana; Léon, Albertine; Harstskeerl, Rudy A.; Sertour, Natacha; Ahmed, Ahmed; Raharimanana, Claudine; Ferquel, Elisabeth; Garnier, Martine; Chartier, Loïc; Duplantier, Jean-Marc; Rahalison, Lila; Cornet, Muriel

    2010-01-01

    Background Leptospirosis has long been a major public health concern in the southwestern Indian Ocean. However, in Madagascar, only a few, old studies have provided indirect serological evidence of the disease in humans or animals. Methodology/Principal Findings We conducted a large animal study focusing on small-mammal populations. Five field trapping surveys were carried out at five sites, from April 2008 to August 2009. Captures consisted of Rattus norvegicus (35.8%), R. rattus (35.1%), Mus musculus (20.5%) and Suncus murinus (8.6%). We used microbiological culture, serodiagnosis tests (MAT) and real-time PCR to assess Leptospira infection. Leptospira carriage was detected by PCR in 91 (33.9%) of the 268 small mammals, by MAT in 17 of the 151 (11.3%) animals for which serum samples were available and by culture in 9 of the 268 animals (3.3%). Rates of infection based on positive PCR results were significantly higher in Moramanga (54%), Toliara (48%) and Mahajanga (47.4%) than in Antsiranana (8.5%) and Toamasina (14%) (p = 0.001). The prevalence of Leptospira carriage was significantly higher in R. norvegicus (48.9%), S. murinus (43.5%) and R. rattus (30.8%) than in M. musculus (9.1%) (p<0.001). The MAT detected antibodies against the serogroups Canicola and Icterohaemorrhagiae. Isolates were characterized by serology, secY sequence-based phylogeny, partial sequencing of rrs, multi-locus VNTR analysis and pulsed field gel electrophoresis. The 10 isolates obtained from nine rats were all identified as species L. interrogans serogroup Canicola serovar Kuwait and all had identical partial rrs and secY sequences. Conclusions/Significance We present here the first direct evidence of widespread leptospiral carriage in small mammals in Madagascar. Our results strongly suggest a high level of environmental contamination, consistent with probable transmission of the infection to humans. This first isolation of pathogenic Leptospira strains in this country may

  18. [Immunogenecity of expressed protein p68 from recombinant plasmid rpDJt in L. interrogans serovar lai].

    PubMed

    Jiang, N; Dai, B; Li, S; Zhao, H; Fang, Z; Wu, W; Ye, D; Liu, J; Song, S; Yang, Y; Zhang, Y; Liu, F; Tu, Y; Yang, H; Huang, Z; Liang, L; Hu, L; Zhao, M

    1997-06-01

    There are two types of infection caused by pathogenic microorganisms, intracellular infection and intercellular infection. Infection of pathogenic leptospira is an intercellular infection. The immunological reaction of host to intercellular infection is unique. The potential immunogen of an expressed protein should meet three criteria: it can be degraded (by antigen-present cells in the host); it should have antigenic epitope which can be recognized by specific antibodies and have at least one epitope that can be recognized by an MHC II protein and T cell receptor. In this study we report the cloning of an L. interrogans protein in plasmid rpDJt and the immunogencity of the expressed protein derivative. A genomic library of L. interrogans serovar lai strain 017 was constructed with the plasmid vector pUC18. Recombinant plasmids, designated pDJH2 and pDJ8 were screened from the bank. EcoRI-inserted fragment of 1. 9 kb recombinant DNA of pDJH2 was ligated into T7 RNA polymerase/promoter vectors (pT7-7). Then they were transformed into E. coli JM109 (De3), one of subclones, designated rpDJt was achieved. SDS-PAGE showed that the molecular weights of expression proteins were 68 kd and 23 kd respectively, designated p68 and p23. Purifying and isolating p68 and p23, we separated them from SDS-Polyacrylamide gels by using Side-Strip method. After fragmenting and electroeluting, p68 and p23 were injected into guinea pigs and rabbits. An extremely strong immune response to p68 was obtained since an anti-p68 antibody response could be detected to a dilution 1:524,288 (guinea pigs) and 1:262,144 (rabbits) by ELISA while anti-P23 antibody being 1:1024 (the same to guinea pigs and rabbits). The results of improved MTT and conA 3HTdR transformation methods showed the activities and proliferation of Th-cells were increased in guinea pigs after p68 immunization (IL-6, 83.25 IU/ml, IL-2, 28.75 IU/ml; RPI, 2.04, SI, 65.62%) Thlymphocyte existed in two subclasses, the Th1- and Th2

  19. Leptospira Exposure and Gardeners: A Case-Control Seroprevalence Study

    PubMed Central

    Alvarado-Esquivel, Cosme; Hernandez-Tinoco, Jesus; Sanchez-Anguiano, Luis Francisco; Ramos-Nevarez, Agar; Cerrillo-Soto, Sandra Margarita; Guido-Arreola, Carlos Alberto

    2016-01-01

    Background Leptospira can be found in soil. However, it is unclear whether occupational exposure to soil may represent a risk for Leptospira infection in humans. Therefore, we sought to determine the association of Leptospira IgG seroprevalence with the occupation of gardener, and to determine the epidemiological characteristics of gardeners associated with Leptospira exposure. Methods We performed a case-control study in 168 gardeners and 168 age- and gender-matched control subjects without gardening occupation in Durango City, Mexico. The seroprevalence of anti-Leptospira IgG antibodies in cases and controls was determined using an enzyme immunoassay. Bivariate and multivariate analyses were used to assess the association of Leptospira exposure and the characteristics of the gardeners. Results Anti-Leptospira IgG antibodies were found in 10 (6%) of 168 gardeners and in 15 (8.9%) of 168 control subjects (odds ratio (OR): 0.64; 95% confidence interval (CI): 0.28 - 1.48; P = 0.40). Multivariate analysis showed that Leptospira seropositivity was positively associated with female gender (OR: 5.82; 95% CI: 1.11 - 30.46; P = 0.03), and negatively associated with eating while working (OR: 0.21; 95% CI: 0.05 - 0.87; P = 0.03). In addition, multivariate analysis showed that high anti-Leptospira levels were associated with consumption of boar meat (OR: 28.00; 95% CI: 1.20 - 648.80; P = 0.03). Conclusions This is the first case-control study of Leptospira exposure in gardeners. Results do not support an association of Leptospira exposure with the occupation of gardener. However, further studies to confirm the lack of this association are needed. The potential role of consumption of boar meat in Leptospira infection deserves further investigation. PMID:26668679

  20. Studies of the binding of ficolin-2 and ficolin-3 from the complement lectin pathway to Leptospira biflexa, Pasteurella pneumotropica and Diarrheagenic Escherichia coli.

    PubMed

    Sahagún-Ruiz, Alfredo; Breda, Leandro Carvalho Dantas; Valencia, Mónica Marcela Castiblanco; Elias, Waldir P; Munthe-Fog, Lea; Garred, Peter; Barbosa, Angela Silva; Isaac, Lourdes

    2015-10-01

    Ficolins recognize pathogen associated molecular patterns and activate the lectin pathway of complement system. However, our knowledge regarding pathogen recognition of human ficolins is still limited. We therefore set out to explore and investigate the possible interactions of the two main serum ficolins, ficolin-2 and ficolin-3 with different Gram-negative bacteria. We used recombinant ficolin molecules and normal human serum, which were detected with anti-ficolin monoclonal antibodies. In addition we investigated the capacity of these pathogens to activate the lectin pathway of complement system. We show for the first time that human ficolin-2 recognizes the nonpathogenic spirochete Leptospira biflexa serovar Patoc, but not the pathogenic Leptospira interrogans serovar Kennewicki strain Fromm. Additionally, human ficolin-2 and ficolin-3 recognize pathogenic Pasteurella pneumotropica, enteropathogenic Escherichia coli (EPEC) serotype O111ab:H2 and enteroaggregative E. coli (EAEC) serogroup O71 but not four enterohemorrhagic E. coli, three EPEC, three EAEC and two nonpathogenic E. coli strains (DH5α and HB101). The lectin pathway was activated by Pasteurella pneumotropica, EPEC O111ab:H2 and EAEC O71 after incubation with C1q depleted human serum. In conclusion, this study provide novel insight in the binding and complement activating capacity of the lectin pathway initiation molecules ficolin-2 and ficolin-3 towards relevant Gram-negative pathogens of pathophysiological relevance. PMID:26074063

  1. Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32.

    PubMed

    Kumar, Vikram; Damodharan, S; Pandaranayaka, Eswari P J; Madathiparambil, Madanan G; Tennyson, Jebasingh

    2016-01-01

    Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6. PMID:25692534

  2. [Leptospira, does it simulate or cause leukemia?].

    PubMed

    Velasco-Castrejón, Oscar; Rivas-Sánchez, Beatriz; Gutiérrez, Esther; Chávez, Laura; Duarte, Paulo; Chavarria, Salvador; Rivera-Reyes, Héctor Hugo

    2005-01-01

    Two cases of chronic leptospirosis in bicitopenic and pancitopenic patients, respectively, with mucocutaneous and visceral bleedings were presented. They were diagnosed myeloblastic leukemia M3 and acute lymphoblastic leukemia L2 by bone marrow aspiration and they were treated as such at the hematology department of a general hospital. Both patients died after one of them had considerably improved on being treated with sodium crystalline penicillin at high doses. The histopathological studies could not demonstrate the presence of neoplastic cells in bone marrow but leptospiras were found by means of silver stained preparations (Warthin - Starry) and immunofluorescence, both in this organ and in other tissues studied: kidney., spleen, liver and lungs. The histopathological diagnoses were generalized leptospirosis with medullary aplasia and generalized leptospirosis with myelodisplastic syndrome, respectively. It was reviewed the possibility that leptospira could cause leukemoid syndromes and/or leukemia. Based on these results, it is recommended to hematologists, infectologists, pathologists, and others, to use again the technques of argentic impregnation, immunohystochemistry and immunofluorescence to study the bone marrow and other tissues in order to detect the possible presence of leptospira that would allow to treat the patients more effectively, particularly, patients like these that had serological titres considered negatives so as to avoid the false security existing in the official health institutions about the minimum or null impact that leptospirosis cause in public health, which imply the absence of control systems for this spirochetosis. PMID:17966470

  3. Reservoir hosts of Leptospira inadai in India.

    PubMed

    Gangadhar, N L; Rajasekhar, M; Smythe, L D; Norris, M A; Symonds, M L; Dohnt, M F

    2000-12-01

    Isolation of Leptospira from the kidneys of Rattus rattus wroughtoni hinton, Rattus rattus rufescens, Bandicota bengalensis and Bandicota indica was attempted in Bangalore in southern India. In total, 296 spirochaetes were isolated from 1,348 kidney cultures (an isolation rate of 22%). A batch of fifty-six isolates from India was identified, based on serological and polymerase chain reaction analysis, of which twenty-three isolates were identified as L. inadai by the World Health Organization/Food and Agriculture Organization Collaborating Centre for Reference and Research on Leptospirosis, in Brisbane. This is the first record of isolation of L. inadai from rodents. The preponderance of L. inadai in four different species of rodents suggests that these animals could be the natural reservoir hosts of L. inadai, and raises a critical question as to the likely impact of this species of Leptospira on the renal carrier status of other Leptospira pathogenic to humans and animals in this part of India. Virulence studies conducted at the University of Trieste in Italy, revealed that isolates of L. inadai from India were moderately or totally serum resistant when subjected to a serum killing test. To establish the possible seroprevalence of this species in the population, the inclusion of L. inadai in the battery of leptospiral antigens used for sero-epidemiological studies is recommended. PMID:11107622

  4. Molecular Typing of Pathogenic Leptospira Serogroup Icterohaemorrhagiae Strains Circulating in China during the Past 50 Years

    PubMed Central

    Zhang, Cuicai; Yang, Huimian; Li, Xiuwen; Cao, Zhiqiang; Zhou, Haijian; Zeng, Linzi; Xu, Jianmin; Xu, Yinghua; Chang, Yung-Fu; Guo, Xiaokui; Zhu, Yongzhang; Jiang, Xiugao

    2015-01-01

    Background Leptospirosis is one of the most important neglected tropical infectious diseases worldwide. Icterohaemorrhagiae has been throughout recent history, and still is, the predominant serogroup of this pathogen in China. However, very little in detail is known about the serovars or genotypes of this serogroup. Methodology/Principal Findings In this study, 120 epidemic strains from five geographically diverse regions in China collected over a 50 year period (1958~2008), and 8 international reference strains characterized by 16S rRNA sequencing and MLST analysis. 115, 11 and 2 strains were identified as L. interrogans, L. borgpetersenii, and L. kirschneri, respectively. 17 different STs were identified including 69 ST1 strains, 18 ST17, 18 ST128, 9 ST143 and 2 ST209. The remaining 12 strains belonged to 12 different STs. eBURST analysis demonstrated that, among the clonal complexes isolated (CCs), CC1 accounted for 73.3% (88/120) strains representing three STs: ST1, ST128 and ST98. ST1 was the most likely ancestral strain of this CC, followed by singleton CC17 (17/120) and CC143 (11/120). Further analysis of adding 116 serogroup Icterohaemorrhagiae strains in the MLST database and studies previously described using global eBURST analysis and MST dendrogram revealed relatively similar ST clustering patterns with five main CCs and 8 singletons among these 244 strains. CC17 was found to be the most prevalent clone of pathogenic Leptospira circulating worldwide. This is the first time, to our knowledge, that ST1 and ST17 strains were distributed among 4 distinct serovars, indicating a highly complicated relationship between serovars and STs. Conclusions/Significance Our studies demonstrated a high level of genetic diversity in the serogroup Icterohaemorrhagiae strains. Distinct from ST17 or ST37 circulating elsewhere, ST1 included in CC1, has over the past 50 years or so, proven to be the most prevalent ST of pathogenic leptospires isolated in China. Moreover, the

  5. Shedding and seroprevalence of pathogenic Leptospira spp. in sheep and cattle at a New Zealand Abattoir.

    PubMed

    Fang, F; Collins-Emerson, J M; Cullum, A; Heuer, C; Wilson, P R; Benschop, J

    2015-06-01

    A cross-sectional study was carried out on sheep and cattle slaughtered at a New Zealand abattoir from September to November 2010 to investigate the supplier-specific shedding rate, renal carriage rate and seroprevalence of leptospires. In the 2008/2009 season, this abattoir experienced three human leptospirosis cases from 20 staff, of which two were hospitalized. Urine, kidney and blood samples were collected from carcasses of 399 sheep (six suppliers, 17 slaughter lines) and 146 cattle (three suppliers, 22 slaughter lines). The urine and kidney samples were tested by quantitative real-time PCR (qPCR), while serum samples (from coagulated blood samples) were tested by microscopic agglutination test (MAT). In total, 27% (73/274; 95% CI: 18-37) of urine samples tested positive by qPCR. Species-specific shedding rates (prevalence of positive urine qPCR) were 31% (95% CI: 17-48) for sheep and 21% (95% CI: 14-30) for cattle. For 545 kidney samples tested, 145 were qPCR positive (27%; 95% CI: 17-39). The average prevalence of kidney qPCR positivity was 29% (95% CI: 17-45) for sheep and 21% (95% CI: 15-28) for cattle. Three hundred and thirty of 542 sampled sheep and cattle had antibodies against Leptospira borgpetersenii serovar Hardjobovis (Hardjobovis) and/or Leptospira interrogans serovar Pomona (Pomona), based on reciprocal MAT titre ≥1 : 48 (overall seroprevalence of 61%; 95% CI: 48-73). Seroprevalence was 57% (95% CI: 40-72) for sheep and 73% (95% CI: 59-83) for cattle. Among the seropositive animals, 41% (70/170; 95% CI: 30-54) were shedding (tested positive by urine qPCR) and 42% (137/330; 95% CI: 30-54) had renal carriage (tested positive by kidney qPCR). Some risk management options for abattoirs or farms to prevent human leptospirosis infections include vaccination of maintenance hosts, the use of personal protective equipment, and the application of urine qPCR to detect shedding status of stock as surveillance and as an alert. PMID:25043226

  6. Genome Sequence of the Saprophyte Leptospira Biflexa Provides Insights into the Evolution of Leptospira and the Pathogenesis of Leptospirosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospira biflexa is a free-living spirochete found broadly distributed in aquatic environments, and is the primary model for studying gene function in Leptospira spp. The L. biflexa genome has 3,590 protein-coding genes (excluding transposases) distributed across three circular replicons: two of w...

  7. Screening of a Leptospira biflexa Mutant Library To Identify Genes Involved in Ethidium Bromide Tolerance

    PubMed Central

    Pětrošová, Helena

    2014-01-01

    Leptospira spp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyte L. biflexa is a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants in L. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduce L. biflexa susceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n = 11), transport (n = 6), membrane structure (n = 5), stress response (n = 2), DNA damage repair (n = 1), and other processes (n = 3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth of L. biflexa in the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenic L. interrogans. PMID:25063661

  8. Adenosine deaminase activity in serum, erythrocytes and lymphocytes of rats infected with Leptospira icterohaemorrhagiae.

    PubMed

    Tonin, Alexandre A; Pimentel, Victor C; da Silva, Aleksandro S; de Azevedo, Maria Isabel; Souza, Viviane C G; Wolkmer, Patrícia; Rezer, João F P; Badke, Manoel R T; Leal, Daniela B R; Schetinger, Maria Rosa C; Monteiro, Silvia G; Lopes, Sonia T A

    2012-04-01

    Leptospirosis is a systemic disease of humans and domestic animals, mainly dogs, cattle and swine. The course of human leptospirosis varies from mild to severe fatal forms and the most severe form of human leptospirosis is principally caused by Leptospira interrogans serovar icterohaemorrhagiae (L. icterohaemorrhagiae). The enzyme adenosine deaminase (ADA) plays an important role in the production and differentiation of blood cells. The aim of this study was to evaluate the activity of ADA in serum, erythrocytes and lymphocytes of rats infected with L. icterohaemorrhagiae, as compared with non-infected rats. Twenty-four adult rats, divided into two uniform groups (A and B) were used for the enzymatic assays. The animals in Group B were inoculated intraperitoneally with 2×10(8) leptospires/rat, and the rodents in Group A (control) were not-inoculated. Blood collection was performed on days 5 and 15 post-infection (PI) and the blood used to assess the ADA activity. The infection by L.icterohaemorrhagiae altered erythrocyte count, hemoglobin concentration and hematocrit, causing a decrease in all these parameters on day 15 PI. Lymphocytes decreased significantly on day 15 PI, and ADA activity in serum was inhibited in infected rats on days 5 and 15 PI and its activity in erythrocytes were increased on day 5 PI. On day 5 PI, we found an increase in ADA activity in erythrocytes of infected rats. No correlation was observed between hematocrit and erythrocyte ADA activity on days 5 and 15 PI. The ADA activity was inhibited in rats infected on day 15 PI. A positive correlation (r(2)=60) was also observed between the number of lymphocytes and ADA activity in lymphocytes on day 15 PI (P<0.05). In conclusion, our results showed that the ADA activity is altered in serum, lymphocytes and erythrocytes in experimental infection by L.icterohaemorrhagiae in rats, concomitantly with hematological parameters. PMID:21320715

  9. Synanthropic Cockroaches (Blattidae: Periplaneta spp.) Harbor Pathogenic Leptospira in Colombia.

    PubMed

    Gonzalez-Astudillo, Viviana; Bustamante-Rengifo, Javier A; Bonilla, Álvaro; Lehmicke, Anna Joy J; Castillo, Andrés; Astudillo-Hernández, Miryam

    2016-01-01

    Leptospirosis cases in Colombia are typically linked to peridomestic rodents; however, empirical data suggest that Leptospira-infected patients with no apparent exposure to these reservoirs are common. Cockroaches (Periplaneta spp.) have equal or greater interaction with humans than rodents, yet their potential role as carriers of Leptospira has not been assessed. We determined if pathogenic Leptospira is harbored by Periplaneta spp. in Cali (Colombia) and the variables influencing this relationship. Fifty-nine cockroaches were captured from seven sites and DNA was extracted from the body surface and digestive tract for a multiplex polymerase chain reaction, targeting genes secY and flaB. Logistic regression models and proportion tests showed a higher likelihood for Leptospira to be isolated from body surfaces (P > 0.001) and from individuals inside houses (six times more likely). These findings are the first to demonstrate an association between Periplaneta spp. and Leptospira, suggesting the need to investigate the potential for cockroaches to serve as reservoirs or transport hosts for Leptospira. PMID:26553295

  10. Microcinematographic analysis of tethered Leptospira illini.

    PubMed Central

    Charon, N W; Daughtry, G R; McCuskey, R S; Franz, G N

    1984-01-01

    A model of Leptospira motility was recently proposed. One element of the model states that in translating cells the anterior spiral-shaped end gyrates counterclockwise and the posterior hook-shaped end gyrates clockwise. We tested these predictions by analyzing cells tethered to a glass surface. Leptospira illini was incubated with antibody-coated latex beads (Ab-beads). These beads adhered to the cells, and subsequently some cells became attached to either the slide or the cover glass via the Ab-beads. As previously reported, these cells rapidly moved back and forth across the surface of the beads. In addition, a general trend was observed: cells tethered to the cover glass rotated clockwise around the Ab-bead; cells tethered to the slide rotated counterclockwise around the Ab-bead. A computer-aided microcinematographic analysis of tethered cells indicated that the direction of rotation of cells around the Ab-bead was a function of both the surface of attachment and the shape of the cell ends. The results can best be explained by assuming that the gyrating ends interact with the glass surface to cause rotation around the Ab-beads. The analysis obtained indicates that the hook- and spiral-shaped ends rotate in the directions predicted by the model. In addition, the tethered cell assay permitted detection of rapid, coordinated reversals of the cell ends, e.g., cells rapidly switched from a hook-spiral configuration to a spiral-hook configuration. These results suggest the existance of a mechanism which coordinates the shape of the cell ends of L. illini. Images PMID:6501226

  11. Leptospira spp. infection in sheep herds in southeast Brazil

    PubMed Central

    2014-01-01

    Background With the aim of studying Leptospira spp. infection in sheep herds, blood samples and respective kidney and liver fragments were collected from 100 animals from twenty different properties during slaughter at a meat company in the Sorocaba region, São Paulo state, southeast Brazil. The microscopic agglutination test (MAT) was performed with 29 strains of Leptospira spp. To identify the agent in the liver and kidney, 100 samples of each tissue were submitted to culture in Fletcher medium and analyzed by the polymerase chain reaction (PCR) for Leptospira spp. Results MAT detected 23 samples serologically positive for one or more Leptospira spp. serovars and significantly more for Autumnalis. Eight (4%) samples were positive in culture (four kidneys and four livers), corresponding to five animals with positive serology (one animal simultaneously positive for both kidney and liver) and two negatives. PCR detected Leptospira spp. in 14 samples (seven kidneys and seven livers) corresponding to 12 positive animals (two animals simultaneously positive for kidney and liver), of which ten were serologically positive and two negative. Conclusions PCR was faster, more practical and more sensitive than culture for detecting leptospires. The results reinforce the importance of sheep in the epidemiological context of leptospirosis. PMID:24822059

  12. Leptospira seroprevalence and associations between seropositivity, clinical disease and host factors in horses

    PubMed Central

    Båverud, V; Gunnarsson, A; Engvall, E Olsson; Franzén, P; Egenvall, A

    2009-01-01

    Background A cross-sectional study was carried out to determine the seroprevalence of different serovars of Leptospira spp. and their association with clinical disease and host factors in Swedish horses. Methods Sera from 2017 horses brought to equine clinics during 1997–98 were investigated. The sera were examined by microscopic agglutination test for the presence of antibodies against the following L. interrogans serovars: Bratislava strain Jez, Icterohaemorrhagiae strain Kantorowicz and Pomona strain Pomona and also L. kirschneri sv Grippotyphosa strain Duyster and L. borgpetersenii sv Sejroe strain M 84. Host factors, disease factors, season, pasture access and outdoor confinement variables were analysed with respect to seropositivity to sv Bratislava and Icterohaemorrhagiae. Multivariable logistic regression was used to model seropositivity to sv Bratislava and Icterohaemorrhagiae (seroprevalence > 8%). Results The seroprevalence, at a cut-off 1:100, were for sv Bratislava (16.6%), Icterohaemorrhagiae (8.3%), Sejroe (1.2%), Pomona (0.5%) and Grippotyphosa (0.4%). In the multivariable analysis, it was demonstrated that seroprevalence increased with age for sv Bratislava and Icterohaemorrhagiae. For sv Bratislava the seasons April – June and October – December and for sv Icterohaemorrhagiae October – December had higher seroprevalences than other seasons. Horses not used for racing had higher levels of seropositivity to sv Bratislava. Furthermore, horses with respiratory problems as well as horses with fatigue had higher levels of seropositivity to sv Bratislava. Ponies and coldbloods, and horses with access to pasture, had lower seroprevalence for sv Icterohaemorrhagiae. Healthy horses had lower seroprevalence for sv Icterohaemorrhagiae, than non-healthy horses. Conclusion There was no significant association between clinical signs and disease and positive titres to sv Bratislava (except for the association between respiratory problems and fatigue and

  13. Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum.

    PubMed

    Castiblanco-Valencia, Mónica M; Fraga, Tatiana R; Breda, Leandro C D; Vasconcellos, Sílvio A; Figueira, Cláudio P; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I; Barbosa, Angela S; Isaac, Lourdes

    2016-05-01

    Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. PMID:26976804

  14. Seroepidemiology of Leptospira Exposure in General Population in Rural Durango, Mexico.

    PubMed

    Alvarado-Esquivel, Cosme; Sánchez-Anguiano, Luis Francisco; Hernández-Tinoco, Jesús

    2015-01-01

    The magnitude of Leptospira exposure in rural Mexico is largely unknown. We sought to determine the seroprevalence of Leptospira IgG antibodies in adults in rural Durango, Mexico, and to determine the sociodemographic, behavioral, and housing characteristics of the subjects associated with Leptospira seropositivity. We performed a cross-sectional study in 282 adults living in rural Durango, Mexico. Sera from participants were analyzed for Leptospira IgG antibodies using a commercially available enzyme immunoassay. Seroprevalence association with the characteristics of the subjects was analyzed by bivariate and multivariate analyses. Of the 282 rural subjects (42.91 ± 17.53 years old) studied, 44 (15.6%) had anti-Leptospira IgG antibodies. Seropositivity to Leptospira was not associated with gender, educational level, employment, socioeconomic status, contact with animals or soil, or type of floors at home. In contrast, multivariate analysis showed that Leptospira exposure was associated with national trips (OR = 2.09; 95% CI: 1.05-4.16; P = 0.03) and poor education of the head of the family (OR = 2.96; 95% CI: 1.51-5.78; P = 0.001). We demonstrated serological evidence of Leptospira exposure in adults in rural northern Mexico. The contributing factors associated with Leptospira exposure found in the present study may be useful for optimal planning of preventive measures against Leptospira infection. PMID:26240822

  15. Seroepidemiology of Leptospira Exposure in General Population in Rural Durango, Mexico

    PubMed Central

    Alvarado-Esquivel, Cosme; Sánchez-Anguiano, Luis Francisco; Hernández-Tinoco, Jesús

    2015-01-01

    The magnitude of Leptospira exposure in rural Mexico is largely unknown. We sought to determine the seroprevalence of Leptospira IgG antibodies in adults in rural Durango, Mexico, and to determine the sociodemographic, behavioral, and housing characteristics of the subjects associated with Leptospira seropositivity. We performed a cross-sectional study in 282 adults living in rural Durango, Mexico. Sera from participants were analyzed for Leptospira IgG antibodies using a commercially available enzyme immunoassay. Seroprevalence association with the characteristics of the subjects was analyzed by bivariate and multivariate analyses. Of the 282 rural subjects (42.91 ± 17.53 years old) studied, 44 (15.6%) had anti-Leptospira IgG antibodies. Seropositivity to Leptospira was not associated with gender, educational level, employment, socioeconomic status, contact with animals or soil, or type of floors at home. In contrast, multivariate analysis showed that Leptospira exposure was associated with national trips (OR = 2.09; 95% CI: 1.05–4.16; P = 0.03) and poor education of the head of the family (OR = 2.96; 95% CI: 1.51–5.78; P = 0.001). We demonstrated serological evidence of Leptospira exposure in adults in rural northern Mexico. The contributing factors associated with Leptospira exposure found in the present study may be useful for optimal planning of preventive measures against Leptospira infection. PMID:26240822

  16. What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira

    PubMed Central

    Fouts, Derrick E.; Matthias, Michael A.; Adhikarla, Haritha; Adler, Ben; Amorim-Santos, Luciane; Berg, Douglas E.; Bulach, Dieter; Buschiazzo, Alejandro; Chang, Yung-Fu; Galloway, Renee L.; Haake, David A.; Haft, Daniel H.; Hartskeerl, Rudy; Ko, Albert I.; Levett, Paul N.; Matsunaga, James; Mechaly, Ariel E.; Monk, Jonathan M.; Nascimento, Ana L. T.; Nelson, Karen E.; Palsson, Bernhard; Peacock, Sharon J.; Picardeau, Mathieu; Ricaldi, Jessica N.; Thaipandungpanit, Janjira; Wunder, Elsio A.; Yang, X. Frank; Zhang, Jun-Jie; Vinetz, Joseph M.

    2016-01-01

    Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade’s refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic

  17. What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira.

    PubMed

    Fouts, Derrick E; Matthias, Michael A; Adhikarla, Haritha; Adler, Ben; Amorim-Santos, Luciane; Berg, Douglas E; Bulach, Dieter; Buschiazzo, Alejandro; Chang, Yung-Fu; Galloway, Renee L; Haake, David A; Haft, Daniel H; Hartskeerl, Rudy; Ko, Albert I; Levett, Paul N; Matsunaga, James; Mechaly, Ariel E; Monk, Jonathan M; Nascimento, Ana L T; Nelson, Karen E; Palsson, Bernhard; Peacock, Sharon J; Picardeau, Mathieu; Ricaldi, Jessica N; Thaipandungpanit, Janjira; Wunder, Elsio A; Yang, X Frank; Zhang, Jun-Jie; Vinetz, Joseph M

    2016-02-01

    Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic

  18. 9 CFR 113.101 - Leptospira Pomona Bacterin.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira pomona organisms, using a dose of 10-10,000 hamster...

  19. 9 CFR 113.101 - Leptospira Pomona Bacterin.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira pomona organisms, using a dose of 10-10,000 hamster...

  20. 9 CFR 113.103 - Leptospira Canicola Bacterin.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira canicola organisms, using a dose of 10-10,000...

  1. 9 CFR 113.101 - Leptospira Pomona Bacterin.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira pomona organisms, using a dose of 10-10,000 hamster...

  2. 9 CFR 113.101 - Leptospira Pomona Bacterin.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira pomona organisms, using a dose of 10-10,000 hamster...

  3. 9 CFR 113.103 - Leptospira Canicola Bacterin.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira canicola organisms, using a dose of 10-10,000...

  4. 9 CFR 113.103 - Leptospira Canicola Bacterin.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira canicola organisms, using a dose of 10-10,000...

  5. 9 CFR 113.103 - Leptospira Canicola Bacterin.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira canicola organisms, using a dose of 10-10,000...

  6. 9 CFR 113.103 - Leptospira Canicola Bacterin.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira canicola organisms, using a dose of 10-10,000...

  7. 9 CFR 113.101 - Leptospira Pomona Bacterin.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either... least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls. (3... intraperitoneally with a suspension of virulent Leptospira pomona organisms, using a dose of 10-10,000 hamster...

  8. 77 FR 43827 - International Workshop on Alternative Methods for Leptospira

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-26

    ...The NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) announces an ``International Workshop on Alternative Methods for Leptospira Vaccine Potency Testing: State of the Science and the Way Forward.'' This workshop, the second in a series of specialized vaccine workshops, will review recent advances and innovations in science and technology that can be......

  9. THE ORGANIZATION OF LEPTOSPIRA AT A GENOMIC LEVEL

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete nucleotide sequences of three, and in the not too distant future, six strains from the genus Leptospira will be available. Managing and maintaining these data will be a perpetual problem unless a system is devised to address this issue. We propose a central role for the IUMS Subcommitte...

  10. GENOMIC SEQUENCE ANALYSIS OF LEPTOSPIRA BORGPETERSENII SEROVAR HARDJO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A genomic library from Leptospira borgpetersenii serovar hardjo strain JB197 was prepared by mechanically shearing the DNA and inserting it into a positive selection vector. DNA was prepared from approximately 22,000 random clones and used as templates for automated sequencing. Sequence data was c...

  11. [Occurrance of antibodies against Leptospira in horses in Middle Germany].

    PubMed

    Pikalo, Jutta; Sattler, Tatjana; Eichinger, Michaela; Loitsch, Angelika; Sun, Hao; Schmoll, Friedrich; Schusser, Gerald Fritz

    2016-01-01

    Aim of the study was to detect antibodies and potential risk factors for an infec- tion with Leptospira in horses in Middle Germany. Serum samples of 314 horses were examined retrospectively by microscopic agglutination test for the presence of antibodies against eight Leptospira serovars. In total, 17.2% (n = 54) of the horses were positive for one or more of the serovars analyzed. The most prevalent serovar was lcterohaemorrhagiae (11.1%), followed by serovar Bratislava (9.6 %) and Grippotyphosa (1.9%). Mares showed a significantly higher occurrence of antibodies (p < 0.05) than geldings or stallions. Horses used for breeding have a significantly lower risk than horses used in sport or horses used for leisure activity. There was also a significantly higher prevalence (p < 0.05) in summer than in the other seasons. No significant influence of breed, husbandry conditions and age on the antibody occurrence was observed (p > 0.05). The clinical chemical parameters did not differ significantly between horses with positive or negative Leptospira antibody result (p > 0.05). It became apparent that horses can be infected with Leptospira without developing of clinical symptoms. PMID:27344912

  12. GLOBAL PATTERNS OF LEPTOSPIRA PREVALENCE IN VERTEBRATE RESERVOIR HOSTS.

    PubMed

    Andersen-Ranberg, Emilie U; Pipper, Christian; Jensen, Per M

    2016-07-01

    Leptospirosis is a widespread emerging bacterial zoonosis. As the transmission is believed to be predominantly waterborne, human incidence is expected to increase in conjunction with global climate change and associated extreme weather events. Providing more accurate predictions of human leptospirosis requires more detailed information on animal reservoirs that are the source of human infection. We evaluated the prevalence of Leptospira in vertebrates worldwide and its association with taxonomy, geographic region, host biology, ambient temperature, and precipitation patterns. A multivariate regression analysis with a meta-analysis-like approach was used to analyze compiled data extracted from 300 Leptospira-related peer reviewed papers. A fairly uniform Leptospira infection prevalence of about 15% was found in the majority of mammalian families. Higher prevalence was frequently associated with species occupying urban habitats, and this may explain why climatic factors were not significantly correlated with prevalence as consistently as expected. Across different approaches of the multiple regression analyses, the variables most frequently correlated with Leptospira infection prevalence were the host's ability to swim, minimum ambient temperature, and methodologic quality of the study. Prevalence in carnivores was not associated with any climatic variable, and the importance of environmental risk factors were indicated to be of lesser consequence in nonhuman mammals. The dataset is made available for further analysis. PMID:27187029

  13. 21 CFR 866.3350 - Leptospira spp. serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Leptospira spp. serological reagents. 866.3350 Section 866.3350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3350...

  14. 21 CFR 866.3350 - Leptospira spp. serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leptospira spp. serological reagents. 866.3350 Section 866.3350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3350...

  15. 21 CFR 866.3350 - Leptospira spp. serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Leptospira spp. serological reagents. 866.3350 Section 866.3350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3350...

  16. 21 CFR 866.3350 - Leptospira spp. serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Leptospira spp. serological reagents. 866.3350 Section 866.3350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3350...

  17. 21 CFR 866.3350 - Leptospira spp. serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Leptospira spp. serological reagents. 866.3350 Section 866.3350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3350...

  18. Direct Measurement of Helical Cell Motion of the Spirochete Leptospira

    PubMed Central

    Nakamura, Shuichi; Leshansky, Alexander; Magariyama, Yukio; Namba, Keiichi; Kudo, Seishi

    2014-01-01

    Leptospira are spirochete bacteria distinguished by a short-pitch coiled body and intracellular flagella. Leptospira cells swim in liquid with an asymmetric morphology of the cell body; the anterior end has a long-pitch spiral shape (S-end) and the posterior end is hook-shaped (H-end). Although the S-end and the coiled cell body called the protoplasmic cylinder are thought to be responsible for propulsion together, most observations on the motion mechanism have remained qualitative. In this study, we analyzed the swimming speed and rotation rate of the S-end, protoplasmic cylinder, and H-end of individual Leptospira cells by one-sided dark-field microscopy. At various viscosities of media containing different concentrations of Ficoll, the rotation rate of the S-end and protoplasmic cylinder showed a clear correlation with the swimming speed, suggesting that these two helical parts play a central role in the motion of Leptospira. In contrast, the H-end rotation rate was unstable and showed much less correlation with the swimming speed. Forces produced by the rotation of the S-end and protoplasmic cylinder showed that these two helical parts contribute to propulsion at nearly equal magnitude. Torque generated by each part, also obtained from experimental motion parameters, indicated that the flagellar motor can generate torque >4000 pN nm, twice as large as that of Escherichia coli. Furthermore, the S-end torque was found to show a markedly larger fluctuation than the protoplasmic cylinder torque, suggesting that the unstable H-end rotation might be mechanically related to changes in the S-end rotation rate for torque balance of the entire cell. Variations in torque at the anterior and posterior ends of the Leptospira cell body could be transmitted from one end to the other through the cell body to coordinate the morphological transformations of the two ends for a rapid change in the swimming direction. PMID:24411236

  19. USDA regulatory guidelines and practices for veterinary Leptospira vaccine potency testing.

    PubMed

    Srinivas, G B; Walker, A; Rippke, B

    2013-09-01

    Batch-release potency testing of leptospiral vaccines licensed by the United States Department of Agriculture (USDA) historically was conducted through animal vaccination-challenge models. The hamster vaccination-challenge assay was Codified in 1974 for bacterins containing Leptospira pomona, Leptospira icterohaemorrhagiae, and Leptospira canicola, and in 1975 for bacterins containing Leptospira grippotyphosa. In brief, 10 hamsters are vaccinated with a specified dilution of bacterin. After a holding period, the vaccinated hamsters, as well as nonvaccinated controls, are challenged with virulent Leptospira and observed for mortality. Eighty percent of vaccinated hamsters must survive in the face of a valid challenge. The high cost of the Codified tests, in terms of monetary expense and animal welfare, prompted the Center for Veterinary Biologics (CVB) to develop ELISA alternatives for them. Potency tests for other serogroups, such as Leptospira hardjo-bovis, that do not have Codified requirements for potency testing continue to be examined on a case-by-case basis. PMID:23838570

  20. Distribution of Leptospira Serogroups in Cattle Herds and Dogs in France

    PubMed Central

    Ayral, Florence C.; Bicout, Dominique J.; Pereira, Helena; Artois, Marc; Kodjo, Angeli

    2014-01-01

    A retrospective study was conducted to identify and describe the distribution pattern of Leptospira serogroups in domestic animals in France. The population consisted of cattle herds and dogs with clinically suspected leptospirosis that were tested at the “Laboratoire des Leptospires” between 2008 and 2011. The laboratory database was queried for records of cattle and dogs in which seroreactivity in Leptospira microagglutination tests was consistent with a recent or current infection, excluding vaccine serogroups in dogs. A total of 394 cattle herds and 232 dogs were diagnosed with clinical leptospirosis, and the results suggested infection by the Leptospira serogroup Australis in 43% and 63%, respectively; by the Leptospira serogroup Grippotyphosa in 17% and 9%, respectively; and by the Leptospira serogroup Sejroe in 33% and 6%, respectively. This inventory of infecting Leptospira serogroups revealed that current vaccines in France are not fully capable of preventing the clinical form of the disease. PMID:25092816

  1. Distribution of Leptospira serogroups in cattle herds and dogs in France.

    PubMed

    Ayral, Florence C; Bicout, Dominique J; Pereira, Helena; Artois, Marc; Kodjo, Angeli

    2014-10-01

    A retrospective study was conducted to identify and describe the distribution pattern of Leptospira serogroups in domestic animals in France. The population consisted of cattle herds and dogs with clinically suspected leptospirosis that were tested at the "Laboratoire des Leptospires" between 2008 and 2011. The laboratory database was queried for records of cattle and dogs in which seroreactivity in Leptospira microagglutination tests was consistent with a recent or current infection, excluding vaccine serogroups in dogs. A total of 394 cattle herds and 232 dogs were diagnosed with clinical leptospirosis, and the results suggested infection by the Leptospira serogroup Australis in 43% and 63%, respectively; by the Leptospira serogroup Grippotyphosa in 17% and 9%, respectively; and by the Leptospira serogroup Sejroe in 33% and 6%, respectively. This inventory of infecting Leptospira serogroups revealed that current vaccines in France are not fully capable of preventing the clinical form of the disease. PMID:25092816

  2. Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts.

    PubMed

    Mgode, Georgies F; Machang'u, Robert S; Mhamphi, Ginethon G; Katakweba, Abdul; Mulungu, Loth S; Durnez, Lies; Leirs, Herwig; Hartskeerl, Rudy A; Belmain, Steven R

    2015-12-01

    The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries. PMID:26624890

  3. Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts

    PubMed Central

    Mgode, Georgies F.; Machang’u, Robert S.; Mhamphi, Ginethon G.; Katakweba, Abdul; Mulungu, Loth S.; Durnez, Lies; Leirs, Herwig; Hartskeerl, Rudy A.; Belmain, Steven R.

    2015-01-01

    The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries. PMID:26624890

  4. Leptospira Exposure and Waste Pickers: A Case-Control Seroprevalence Study in Durango, Mexico

    PubMed Central

    Alvarado-Esquivel, Cosme; Hernandez-Tinoco, Jesus; Sanchez-Anguiano, Luis Francisco; Ramos-Nevarez, Agar; Cerrillo-Soto, Sandra Margarita; Guido-Arreola, Carlos Alberto

    2015-01-01

    Background Infection with Leptospira may occur by contact with Leptospira-infected animals. Waste pickers are in contact with rodents and dogs while picking in the garbage. Whether waste pickers are at risk for Leptospira infection is largely unknown. This study was aimed to determine the association of Leptospira IgG seroprevalence with the occupation of waste picking, and to determine the epidemiological characteristics of the waste pickers with Leptospira exposure. Methods Through a case-control study, we determined the seroprevalence of anti-Leptospira IgG antibodies in 90 waste pickers and 90 age- and gender-matched control subjects in Durango City, Mexico using an enzyme immunoassay. Data were analyzed by bivariate and multivariate analyses. Results The prevalence of anti-Leptospira IgG antibodies was similar in waste pickers (4/90: 4.4%) to that in control subjects (5/90: 5.6%) (P = 1.00). Bivariate analysis showed that Leptospira exposure in waste pickers was associated with increasing age (P = 0.009), no education (P = 0.008), and consumption of rat meat (P = 0.04). However, these associations were no longer found by multivariate analysis. Leptospira exposure in waste pickers was not associated with health status, duration in the activity, wearing hand gloves and facemasks, history of injuries with sharp material of the garbage, or contact with animals or soil. Conclusions This is the first study about Leptospira exposure in waste pickers. Results suggest that waste pickers are not at increasing risk for Leptospira exposure in Durango City, Mexico. Further research with a larger sample size to elucidate the association of Leptospira exposure with waste picking activity is needed. PMID:26124911

  5. High Seroprevalence of Leptospira Exposure in Meat Workers in Northern Mexico: A Case-Control Study

    PubMed Central

    Alvarado-Esquivel, Cosme; Hernandez-Tinoco, Jesus; Sanchez-Anguiano, Luis Francisco; Ramos-Nevarez, Agar; Cerrillo-Soto, Sandra Margarita; Saenz-Soto, Leandro; Martinez-Ramirez, Lucio

    2016-01-01

    Background The seroepidemiology of Leptospira infection in workers occupationally exposed to raw meat has been poorly studied. This work aimed to determine the association between Leptospira exposure and the occupation of meat worker, and to determine the seroprevalence association with socio-demographic, work, clinical and behavioral characteristics of the meat workers studied. Methods We performed a case-control study in 124 meat workers and 124 age- and gender-matched control subjects in Durango City, Mexico. Sera of cases and controls were analyzed for anti-Leptospira IgG antibodies using a commercially available enzyme immunoassay. Data of meat workers were obtained with the aid of a questionnaire. The association of Leptospira exposure with the characteristics of meat workers was analyzed by bivariate and multivariate analyses. Results Anti-Leptospira IgG antibodies were found in 22 (17.7%) of 124 meat workers and in eight (6.5%) of 124 controls (OR = 3.12; 95% CI: 1.33 - 7.33; P = 0.006). Seroprevalence of Leptospira infection was similar between male butchers (17.6%) and female butchers (18.2%) (P = 1.00). Multivariate analysis of socio-demographic, work and behavioral variables showed that Leptospira exposure was associated with duration in the activity, rural residence, and consumption of snake meat and unwashed raw fruits. Conclusions This is the first case-control study of the association of Leptospira exposure with the occupation of meat worker. Results indicate that meat workers represent a risk group for Leptospira exposure. Risk factors for Leptospira exposure found in this study may help in the design of optimal preventive measures against Leptospira infection. PMID:26858797

  6. Leptospira and Rodents in Cambodia: Environmental Determinants of Infection

    PubMed Central

    Ivanova, Svilena; Herbreteau, Vincent; Blasdell, Kim; Chaval, Yannick; Buchy, Philippe; Guillard, Bertrand; Morand, Serge

    2012-01-01

    We investigated infection of rodents and shrews by Leptospira spp. in two localities of Cambodia (Veal Renh, Kaev Seima) and in four types of habitat (forests, non-flooded lands, lowland rain-fed paddy fields, houses) during the wet and the dry seasons. Habitat preference was common, and rodent and shrew species were found only in houses or in rain-fed paddy fields or in forests. Among 649 small mammals trapped belonging to 12 rodent species and 1 shrew species, 71 of 642 animals tested were carriers of Leptospira according to the 16S ribosomal RNA marker used. Rodent infection was higher in low-slope locations, corresponding to rain-fed paddy fields, especially in the rainy season and in Kaev Seima. Rodents (Rattus exulans) and shrews (Suncus murinus) inhabiting households showed significantly low levels of infections, whereas rodents living in and near to forests (shrubby wasteland, orchards) showed high levels of infection. PMID:22665613

  7. Seroprevalence of Leptospira spp. in clinically healthy horses in Switzerland.

    PubMed

    Blatti, S; Overesch, G; Gerber, V; Frey, J; Hüssy, D

    2011-10-01

    A retrospective, cross-sectional study was conducted to determine the leptospiral seroprevalence in clinically healthy horses in Switzerland. A representative sample of 615 horse sera was examined by microscopic agglutination test for the presence of antibodies against 15 Leptospira spp. serovars. In total, 58.5 % (n = 360) of the horses were positive for one or more of the antigens analysed, with 20.3 % of them showing titres >= 400. The most prevalent serovar was Pyrogenes (22.6 %), followed by serovars Canicola (22.1 %) and Australis (19.2 %). Older horses, mares, ponies and animals spending increased time on pasture exhibited significantly higher prevalence rates (p < 0.05). Moreover, the prevalence was higher in summer and autumn (p = 0.003). The high seroprevalence in healthy horses indicates that they are often exposed to or infected with Leptospira spp. without developing signs of disease. Therefore, other laboratory and clinical data should always be taken into consideration when interpreting serological test results for Leptospira spp. PMID:21971672

  8. Modulation of Hemostatic and Inflammatory Responses by Leptospira Spp.

    PubMed Central

    Vieira, Mônica L.; Naudin, Clément; Mörgelin, Matthias; Romero, Eliete C.; Nascimento, Ana Lucia T. O.; Herwald, Heiko

    2016-01-01

    Leptospirosis is a worldwide spread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. In severe infections, hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. These complications often occur when the host response is controlled and/or modulated by the bacterial pathogen. In the present investigation, we aimed to analyze the modulation of the hemostatic and inflammatory host responses by the bacterial pathogen Leptospira. The effects of leptospires and their secreted products on stimulation of human intrinsic and extrinsic pathways of coagulation were investigated by means of altered clotting times, assembly and activation of contact system and induction of tissue factor. We show that both extrinsic and intrinsic coagulation cascades are modulated in response to Leptospira or leptospiral secreted proteins. We further find that the pro-inflammatory mediator bradykinin is released following contact activation at the bacterial surface and that pro-coagulant microvesicles are shed from monocytes in response to infection. Also, we show that human leptospirosis patients present higher levels of circulating pro-coagulant microvesicles than healthy individuals. Here we show that both pathways of the coagulation system are modulated by leptospires, possibly leading to altered hemostatic and inflammatory responses during the disease. Our results contribute to the understanding of the leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments for the severe manifestations of the disease. PMID:27167223

  9. Modulation of Hemostatic and Inflammatory Responses by Leptospira Spp.

    PubMed

    Vieira, Mônica L; Naudin, Clément; Mörgelin, Matthias; Romero, Eliete C; Nascimento, Ana Lucia T O; Herwald, Heiko

    2016-05-01

    Leptospirosis is a worldwide spread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. In severe infections, hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. These complications often occur when the host response is controlled and/or modulated by the bacterial pathogen. In the present investigation, we aimed to analyze the modulation of the hemostatic and inflammatory host responses by the bacterial pathogen Leptospira. The effects of leptospires and their secreted products on stimulation of human intrinsic and extrinsic pathways of coagulation were investigated by means of altered clotting times, assembly and activation of contact system and induction of tissue factor. We show that both extrinsic and intrinsic coagulation cascades are modulated in response to Leptospira or leptospiral secreted proteins. We further find that the pro-inflammatory mediator bradykinin is released following contact activation at the bacterial surface and that pro-coagulant microvesicles are shed from monocytes in response to infection. Also, we show that human leptospirosis patients present higher levels of circulating pro-coagulant microvesicles than healthy individuals. Here we show that both pathways of the coagulation system are modulated by leptospires, possibly leading to altered hemostatic and inflammatory responses during the disease. Our results contribute to the understanding of the leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments for the severe manifestations of the disease. PMID:27167223

  10. Leptospira infections in freshwater fish in Morogoro Tanzania: a hidden public health threat.

    PubMed

    Mgode, Georgis F; Mhamphi, Ginethon G; Katkweba, Abdul S; Thomas, Michael

    2014-04-01

    Leptospirosis caused by spirochete bacterium of genus Leptospira affects humans and animals worldwide. Rodents are major reservoirs of leptospires whereas wetland and aquatic migratory birds also carry and transmit leptospires. Leptospirosis studies in fish are lacking in African countries despite favourable environment and abundant reservoirs, which can spread leptospires into aquatic habitats and infect fish. The objectives of this study were to determine presence of Leptospira in fish; the prevalent Leptospira serovars and whether are related to serovars reported in animals; and to ascertain potential public health risk. Live tilapia, catfish and eel fish (n = 48) were caught in Mindu Dam in Morogoro Municipality in eastern Tanzania. Blood sample was collected using syringes and needles to obtain serum for serological detection of leptospirosis using gold standard microagglutination test utilizing local and reference Leptospira serovars Sokoine, Kenya, Pomona and Hebdomadis. Twenty-six fish (54.2%) were positive for serovar Kenya (29.2%) and Sokoine (25%). Leptospira prevalence was high in both catfish (58.3%) and tilapia fish (47.8%). Thus, different fish types are infected with Leptospira found in animals. Fish could be source of Leptospira infection to humans since tilapia and catfish are the common fish type widely consumed in Tanzania. Further study covering lakes, rivers and dams is required to better understand the prevalence of Leptospira in fish and actual public health threats. PMID:26875305

  11. Leptospira Exposure and Patients with Liver Diseases: A Case-Control Seroprevalence Study.

    PubMed

    Alvarado-Esquivel, Cosme; Sánchez-Anguiano, Luis Francisco; Hernández-Tinoco, Jesús; Ramos-Nevárez, Agar; Margarita Cerrillo-Soto, Sandra; Alberto Guido-Arreola, Carlos

    2016-06-01

    The seroepidemiology of Leptospira infection in patients suffering from liver disease has been poorly studied. Information about risk factors associated with infection in liver disease patients may help in the optimal planning of preventive measures. We sought to determine the association of Leptospira IgG seroprevalence and patients with liver diseases, and to determine the characteristics of the patients with Leptospira exposure. We performed a case-control study of 75 patients suffering from liver diseases and 150 age- and gender-matched control subjects. Diagnoses of liver disease included liver cirrhosis, steatosis, chronic hepatitis, acute hepatitis, and amoebic liver abscess. Sera of participants were analyzed for the presence of anti- Leptospira IgG antibodies using a commercially available enzyme immunoassay. Anti-Leptospira IgG antibodies were found in 17 (22.7%) of 75 patients and in 15 (10.0%) of 150 control subjects (OR = 2.32; 95% CI: 1.09-4.94; P=0.03). This is the first age- and gender-matched case control study about Leptospira seroprevalence in patients with liver diseases. Results indicate that Leptospira infection is associated with chronic and acute liver diseases. Results warrants for additional studies on the role of Leptospira exposure in chronic liver disease. PMID:27493589

  12. Leptospira Exposure and Patients with Liver Diseases: A Case-Control Seroprevalence Study

    PubMed Central

    Alvarado-Esquivel, Cosme; Sánchez-Anguiano, Luis Francisco; Hernández-Tinoco, Jesús; Ramos-Nevárez, Agar; Margarita Cerrillo-Soto, Sandra; Alberto Guido-Arreola, Carlos

    2016-01-01

    The seroepidemiology of Leptospira infection in patients suffering from liver disease has been poorly studied. Information about risk factors associated with infection in liver disease patients may help in the optimal planning of preventive measures. We sought to determine the association of Leptospira IgG seroprevalence and patients with liver diseases, and to determine the characteristics of the patients with Leptospira exposure. We performed a case-control study of 75 patients suffering from liver diseases and 150 age- and gender-matched control subjects. Diagnoses of liver disease included liver cirrhosis, steatosis, chronic hepatitis, acute hepatitis, and amoebic liver abscess. Sera of participants were analyzed for the presence of anti- Leptospira IgG antibodies using a commercially available enzyme immunoassay. Anti-Leptospira IgG antibodies were found in 17 (22.7%) of 75 patients and in 15 (10.0%) of 150 control subjects (OR = 2.32; 95% CI: 1.09-4.94; P=0.03). This is the first age- and gender-matched case control study about Leptospira seroprevalence in patients with liver diseases. Results indicate that Leptospira infection is associated with chronic and acute liver diseases. Results warrants for additional studies on the role of Leptospira exposure in chronic liver disease. PMID:27493589

  13. Development of Chronic and Acute Golden Syrian Hamster Infection Models with Leptospira borgpetersenii serovar Hardjo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The golden Syrian hamster (Mesocricetus auratus) is frequently used as a model to study virulence for several species of Leptospira. Onset of an acute, lethal infection following infection with several pathogenic Leptospira species has been widely adopted for vaccine testing. An important exceptio...

  14. Isolation of Leptospira hardjo from kidneys of Ontario cattle at slaughter.

    PubMed Central

    Prescott, J F; Miller, R B; Nicholson, V M

    1987-01-01

    Kidneys from 117 cattle from 110 Ontario farms were examined at slaughter for leptospires. Leptospira hardjo (hardjo-bovis A) was isolated from 11 kidneys and L. kennewicki from one. The isolations were all made (12/89, 13.5%) from beef cattle from feedlots, no isolates being obtained from dairy or beef cattle from extensive farms (0/28). Isolations were only made from cattle with antibody titers (greater than or equal to 20) against the serovars recovered. Isolation was more sensitive than immunofluorescence in identifying leptospira, particularly in animals with low antibody titers against L. hardjo. Leptospira were isolated from two kidneys with multiple gross lesions of focal nephritis, but there was no correlation between the presence of scanty kidney lesions and isolations of leptospira. Leptospira hardjo infection appears to be common in Ontario feedlot cattle. PMID:3300922

  15. Molecular Characterization of Leptospira spp. in Environmental Samples from North-Eastern Malaysia Revealed a Pathogenic Strain, Leptospira alstonii.

    PubMed

    Azali, Muhammad Azharuddin; Yean Yean, Chan; Harun, Azian; Aminuddin Baki, Nurul Najian; Ismail, Nabilah

    2016-01-01

    The presence of pathogenic Leptospira spp. in the environment poses threats to human health. The aim of this study was to detect and characterize Leptospira spp. from environmental samples. A total of 144 samples comprised of 72 soil and 72 water samples were collected from markets and recreational areas in a north-eastern state in Malaysia. Samples were cultured on Ellinghausen and McCullough modified by Johnson and Harris media. Leptospires were positive in 22.9% (n = 33) of the isolates. Based on partial sequences of 16S rRNA, a pathogenic leptospire, Leptospira alstonii (n = 1/33), was identified in 3% of the isolates followed by intermediate leptospire (L. wolffii, n = 1/33, and L. licerasiae, n = 7/33) and nonpathogenic leptospire, L. meyeri (n = 22/33) in 24.2% and 66.7%, respectively. This study demonstrates the presence of a clinically significant pathogenic L. alstonii in the environments which could pose health risks to the occupants and visitors. PMID:27127522

  16. Molecular Characterization of Leptospira spp. in Environmental Samples from North-Eastern Malaysia Revealed a Pathogenic Strain, Leptospira alstonii

    PubMed Central

    Azali, Muhammad Azharuddin; Yean Yean, Chan; Aminuddin Baki, Nurul Najian

    2016-01-01

    The presence of pathogenic Leptospira spp. in the environment poses threats to human health. The aim of this study was to detect and characterize Leptospira spp. from environmental samples. A total of 144 samples comprised of 72 soil and 72 water samples were collected from markets and recreational areas in a north-eastern state in Malaysia. Samples were cultured on Ellinghausen and McCullough modified by Johnson and Harris media. Leptospires were positive in 22.9% (n = 33) of the isolates. Based on partial sequences of 16S rRNA, a pathogenic leptospire, Leptospira alstonii (n = 1/33), was identified in 3% of the isolates followed by intermediate leptospire (L. wolffii, n = 1/33, and L. licerasiae, n = 7/33) and nonpathogenic leptospire, L. meyeri (n = 22/33) in 24.2% and 66.7%, respectively. This study demonstrates the presence of a clinically significant pathogenic L. alstonii in the environments which could pose health risks to the occupants and visitors. PMID:27127522

  17. Comparative Analysis of Leptospira Strains Isolated from Environmental Soil and Water in the Philippines and Japan

    PubMed Central

    Villanueva, Sharon Y. A. M.; Chakraborty, Antara; Miyahara, Satoshi; Segawa, Takaya; Asoh, Tatsuma; Ozuru, Ryo; Gloriani, Nina G.; Yanagihara, Yasutake; Yoshida, Shin-ichi

    2013-01-01

    There have been few reports on the epidemiological analysis of environmental Leptospira isolates. This is probably because the isolation of leptospires from the environment was usually unsuccessful due to the overgrowth of contaminants and the slow growth of Leptospira. In this study, we collected a total of 88 samples of soil and water from three sites: Metro Manila and Nueva Ecija, Philippines (an area where Leptospira is now endemic), and Fukuoka, Japan (an area where Leptospira was once endemic). We succeeded in isolating Leptospira from 37 samples by using the novel combination of five antimicrobial agents reported in 2011. The frequencies of positive isolation of Leptospira in the Philippines and Japan were 40 and 46%, respectively. For Leptospira-positive samples, five colonies from each sample were isolated and analyzed by pulsed-field gel electrophoresis (PFGE). The isolates from each area showed their respective characteristics in phylogenetic trees based on the PFGE patterns. Some isolates were closely related to each other across borders. Based on 16S rRNA gene-based phylogenetic analysis, four isolates in Fukuoka were identified as a pathogenic species, L. alstonii; however, its virulence had been lost. One isolate from Nueva Ecija was identified as the intermediate pathogenic species Leptospira licerasiae. Most of the isolates from the environment belonged to nonpathogenic Leptospira species. We also investigated the strain variation among the isolates in a puddle over 5 months. We demonstrated, using PFGE analysis, that Leptospira survived in the wet soil on dry days and appeared in the surface water on rainy days. These results showed that the soil could be a reservoir of leptospires in the environment. PMID:23144130

  18. Opportunities and strategies to further reduce animal use for Leptospira vaccine potency testing.

    PubMed

    Walker, A; Srinivas, G B

    2013-09-01

    Hamsters are routinely infected with virulent Leptospira for two purposes in the regulation of biologics: the performance of Codified potency tests and maintenance of challenge culture for the Codified potency tests. Options for reducing animal use in these processes were explored in a plenary lecture at the "International Workshop on Alternative Methods for Leptospira Vaccine Potency Testing: State of the Science and the Way Forward" held at the Center for Veterinary Biologics in September 2012. The use of validated in vitro potency assays such as those developed by the U.S. Department of Agriculture for Leptospira (L.) canicola, Leptospira grippotyphosa, Leptospira pomona, and Leptospira icterohaemorrhagiae rather than the Codified hamster vaccination-challenge assay was encouraged. Alternatives such as reduced animal numbers in the hamster vaccination-challenge testing were considered for problematic situations. Specifically, the merits of sharing challenge controls, reducing group sizes, and eliminating animals for concurrent challenge dose titration were assessed. Options for maintaining virulent, stable cultures without serial passage through hamsters or with decreased hamster use were also discussed. The maintenance of virulent Leptospira without the use of live animals is especially difficult since a reliable means to maintain virulence after multiple in vitro passages has not yet been identified. PMID:23891496

  19. Seroprevalence of Brucella abortus and Leptospira hardjo in cattle

    PubMed Central

    Pandian, S. Jegaveera; Ray, Pradeep Kumar; Chandran, P. C.; Kumar, Manoj

    2015-01-01

    Aim: The aim was to assess the seroprevalence of B. abortus and Leptospira hardjo in the cattle population of Bihar, this work was carried out. Materials and Methods: Randomly selected 450 cattle from nine districts of Bihar were serologically screened for antibodies against L. hardjo and B. abortus. DAS-ELISA for leptospira and AB-ELISA for brucella were carried out. Based on the results prevalence in each district and the state are reported herewith. Results and Discussion: In this study, it was found that the seroprevalence of L. hardjo was 9.11% and that of B. abortus was 12.2% in Bihar. Indigenous cattle were found to be less susceptible to leptospirosis and brucellosis even though they accounted for 83.11% of the study population. Conclusion: Although there was no acute disease, antibodies detected against L. hardjo and B. abortus in the cattle population indicated the presence of chronic and subclinical infection, which could challenge the fertility of the animals. PMID:27047076

  20. Draft Genome Sequence of Brazilian Leptospira noguchii Serogroup Panama Strain U73, Isolated from Cattle

    PubMed Central

    Moreno, Luisa Z.; Loureiro, Ana P.; Miraglia, Fabiana; Matajira, Carlos E. C.; Kremer, Frederico S.; Eslabao, Marcos R.; Dellagostin, Odir A.; Lilenbaum, Walter

    2015-01-01

    Leptospira noguchii is a current zoonotic pathogen in Brazil. Here, we report the draft genome sequence of the Brazilian L. noguchii serogroup Panama strain U73, isolated from asymptomatic cattle urine. PMID:26472831

  1. Prevalence of antibodies to Leptospira in wild mammals trapped on livestock farms in Ontario, Canada.

    PubMed

    Allen, Samantha E; Ojkic, Davor; Jardine, Claire M

    2014-07-01

    To determine the prevalence and diversity of Leptospira serogroups circulating in wildlife on farms in Ontario, we tested samples from 51 raccoons (Procyon lotor), seven skunks (Mephitis mephitis), four rats (Rattus norvegicus), and three opossums (Didelphis virginiana) that were trapped on 27 livestock (swine [Sus scrofa], cattle [Bos taurus]) farms in 2010. Seventeen of 51 raccoons (33%; 95% confidence interval [CI], 21-48%) sampled were positive for at least one Leptospira serogroup using the microscopic agglutination test. None of the other 14 animals had detectable Leptospira antibodies. On swine farms, 13 of 30 raccoons (43%; 95% CI, 27-61%) were antibody positive, and on cattle farms, four of 21 raccoons (19%; 95% CI, 8-40%) were positive. Leptospira antibody prevalence in raccoons did not differ between swine and cattle farms. Raccoons were positive to serovars representative of serogroups Grippotyphosa, Australis, Icterohaemorrhagiae, and Pomona and were negative to serovars of serogroups Autumnalis, Canicola, and Sejroe. The prevalence of Leptospira antibodies in raccoons in this study is similar to what has been reported previously; however, the diversity of serogroups was higher in this study than what has been reported in raccoons from an urban area of Ontario, Canada. Understanding the prevalence and distribution of Leptospira serogroups in wildlife in Ontario, Canada, is important for the development and maintenance of appropriate disease management strategies in humans, livestock, and companion animals. PMID:24807356

  2. Malagasy bats shelter a considerable genetic diversity of pathogenic Leptospira suggesting notable host-specificity patterns.

    PubMed

    Gomard, Yann; Dietrich, Muriel; Wieseke, Nicolas; Ramasindrazana, Beza; Lagadec, Erwan; Goodman, Steven M; Dellagi, Koussay; Tortosa, Pablo

    2016-04-01

    Pathogenic Leptospira are the causative agents of leptospirosis, a disease of global concern with major impact in tropical regions. Despite the importance of this zoonosis for human health, the evolutionary and ecological drivers shaping bacterial communities in host reservoirs remain poorly investigated. Here, we describe Leptospira communities hosted by Malagasy bats, composed of mostly endemic species, in order to characterize host-pathogen associations and investigate their evolutionary histories. We screened 947 individual bats (representing 31 species, 18 genera and seven families) for Leptospira infection and subsequently genotyped positive samples using three different bacterial loci. Molecular identification showed that these Leptospira are notably diverse and include several distinct lineages mostly belonging to Leptospira borgpetersenii and L. kirschneri. The exploration of the most probable host-pathogen evolutionary scenarios suggests that bacterial genetic diversity results from a combination of events related to the ecology and the evolutionary history of their hosts. Importantly, based on the data set presented herein, the notable host-specificity we have uncovered, together with a lack of geographical structuration of bacterial genetic diversity, indicates that the Leptospira community at a given site depends on the co-occurring bat species assemblage. The implications of such tight host-specificity on the epidemiology of leptospirosis are discussed. PMID:26902801

  3. Pathogenic and saprophytic Leptospira species in water and soils from selected urban sites in peninsular Malaysia.

    PubMed

    Benacer, Douadi; Woh, Pei Yee; Mohd Zain, Siti Nursheena; Amran, Fairuz; Thong, Kwai Lin

    2013-01-01

    Leptospira species were studied in water and soils from selected urban sites in Malaysia. A total of 151 water (n=121) and soil (n=30) samples were collected from 12 recreational lakes and wet markets. All samples were filtered and inoculated into semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media supplemented with additional 5-fluorouracil. The cultures were then incubated at 30°C and observed under a dark field microscope with intervals of 10 days. A PCR assay targeting the rrs gene was used to confirm the genus Leptospira among the isolates. Subsequently, the pathogenic status of the isolates was determined using primer sets G1/G2 and Sapro1/Sapro2, which target the secY and rrs genes, respectively. The isolates were identified at serogroup level using the microscopic agglutination test (MAT) while their genetic diversity was assessed by pulsed field gel electrophoresis (PFGE). Based on dark field microscopy, 23.1% (28/121) water and 23.3% (7/30) soil cultures were positive for Leptospira spp. Of the 35 positive cultures, only 8 were pure and confirmed as Leptospira genus by PCR assay. Two out of 8 isolates were confirmed as pathogenic, 5 were saprophytic and one was intermediate. These 8 isolates were negative for the 25 reference hyperimmune rabbit sera tested in the MAT. PFGE showed that all 8 of these environmental Leptospira spp. were genetically diverse. In conclusion, the presence of pathogenic Leptospira spp. in the urban Malaysian environment may indicate and highlight the importance of water screening, especially in recreational lakes, in order to minimize any chance of Leptospira infection. PMID:23363618

  4. Whole-genome Sequencing of the Saprophyte L. biflexa and Comparative Genomics of Pathogoenic and Saprophytic Leptospira Species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present the complete genome sequences of Leptospira biflexa, a saprophyte that is used as a model for gene functional analysis in Leptospira spp. The L. biflexa genome has 3,730 protein-coding genes distributed across three circular replicons: two of which are chromosomes (3,600- and 278-kb in si...

  5. Impact of Environment and Social Gradient on Leptospira Infection in Urban Slums

    PubMed Central

    Felzemburgh, Ridalva D. M.; Santana, Francisco S.; Mohr, Sharif; Melendez, Astrid X. T. O.; Queiroz, Adriano; Santos, Andréia C.; Ravines, Romy R.; Tassinari, Wagner S.; Carvalho, Marília S.; Reis, Mitermayer G.; Ko, Albert I.

    2008-01-01

    Background Leptospirosis has become an urban health problem as slum settlements have expanded worldwide. Efforts to identify interventions for urban leptospirosis have been hampered by the lack of population-based information on Leptospira transmission determinants. The aim of the study was to estimate the prevalence of Leptospira infection and identify risk factors for infection in the urban slum setting. Methods and Findings We performed a community-based survey of 3,171 slum residents from Salvador, Brazil. Leptospira agglutinating antibodies were measured as a marker for prior infection. Poisson regression models evaluated the association between the presence of Leptospira antibodies and environmental attributes obtained from Geographical Information System surveys and indicators of socioeconomic status and exposures for individuals. Overall prevalence of Leptospira antibodies was 15.4% (95% confidence interval [CI], 14.0–16.8). Households of subjects with Leptospira antibodies clustered in squatter areas at the bottom of valleys. The risk of acquiring Leptospira antibodies was associated with household environmental factors such as residence in flood-risk regions with open sewers (prevalence ratio [PR] 1.42, 95% CI 1.14–1.75) and proximity to accumulated refuse (1.43, 1.04–1.88), sighting rats (1.32, 1.10–1.58), and the presence of chickens (1.26, 1.05–1.51). Furthermore, low income and black race (1.25, 1.03–1.50) were independent risk factors. An increase of US$1 per day in per capita household income was associated with an 11% (95% CI 5%–18%) decrease in infection risk. Conclusions Deficiencies in the sanitation infrastructure where slum inhabitants reside were found to be environmental sources of Leptospira transmission. Even after controlling for environmental factors, differences in socioeconomic status contributed to the risk of Leptospira infection, indicating that effective prevention of leptospirosis may need to address the social

  6. Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR.

    PubMed Central

    Woo, T H; Patel, B K; Smythe, L D; Symonds, M L; Norris, M A; Dohnt, M F

    1997-01-01

    Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected. PMID:9399509

  7. Leptospira Contamination in Household and Environmental Water in Rural Communities in Southern Chile

    PubMed Central

    Muñoz-Zanzi, Claudia; Mason, Meghan R.; Encina, Carolina; Astroza, Angel; Romero, Alex

    2014-01-01

    Leptospirosis is a zoonosis of global distribution that affects tropical and temperate areas. Under suitable conditions, Leptospira can survive in water and soil and contribute to human and animal infections. The objective of this study was to describe the presence of pathogenic Leptospira in peri-domestic water samples from rural households in southern Chile. Water samples, including puddles, containers, animal troughs, rivers, canals, and drinking water were collected from 236 households and tested for Leptospira using a PCR assay targeting the lipL32 gene. Evidence of Leptospira presence was detected in all sample types; overall, 13.5% (77/570) samples tested positive. A total of 10/22 (45.5%) open containers, 12/83 (14.5%) animal drinking sources, 9/47 (19.1%) human drinking sources, and 36/306 (19.3%) puddles tested positive. Lower income (OR = 4.35, p = 0.003), increased temperature (OR = 1.23, p < 0.001), and presence of dogs (OR = 15.9, p = 0.022) were positively associated with positive puddles. Increased number of rodent signs was associated with positive puddles in the household (OR = 3.22); however, only in the lower income households. There was no association between PCR positive rodents and puddles at the household level. Results revealed the ubiquity of Leptospira in the household environment and highlight the need to develop formal approaches for systematic monitoring. PMID:24972030

  8. Characterization of Leptospira santarosai Serogroup Grippotyphosa Serovar Bananal Isolated from Capybara ( Hydrochaeris hydrochaeris ) in Brazil.

    PubMed

    Moreno, Luisa Z; Miraglia, Fabiana; Marvulo, Maria F V; Silva, Jean C R; Paula, Catia D; Costa, Barbara L P; Morais, Zenaide M; Ferreira, Fernando; Neto, José S Ferreira; Dellagostin, Odir A; Hartskeerl, Rudy A; Vasconcellos, Silvio A; Moreno, Andrea M

    2016-07-01

    Leptospirosis is a widespread zoonosis caused by bacteria of the genus Leptospira. Rodents appear to be the most important reservoirs of infection. They contaminate the environment and food and can transmit the pathogen when they are consumed by carnivores. Capybara ( Hydrochaeris hydrochaeris ) are efficient reservoirs of Leptospira, and because they are in close contact with farm animals and are found in semiurban areas, they represent a risk to public health. We isolated five Leptospira strains from capybara kidneys in Sao Paulo State, Brazil, in 2001 and typed them using serologic and molecular techniques. These strains include the Leptospira santarosai serogroup Grippotyphosa serovar Bananal. Pulsed field gel electrophoresis resulted in a unique pattern distinct from the reference strains, and the isolates clustered with greater than 85% similarity. The isolates also presented higher growth rates than other Leptospira serovars, with high minimal inhibitory concentration values for most of the tested antibiotics, with the exception of penicillin and ampicillin. This isolation and characterization of the L. santarosai serogroup Grippotyphosa serovar Bananal from capybara, highlights the importance of wild and sinantropic rodents as carriers of pathogenic leptospires. PMID:27258408

  9. Leptospira contamination in household and environmental water in rural communities in southern Chile.

    PubMed

    Muñoz-Zanzi, Claudia; Mason, Meghan R; Encina, Carolina; Astroza, Angel; Romero, Alex

    2014-07-01

    Leptospirosis is a zoonosis of global distribution that affects tropical and temperate areas. Under suitable conditions, Leptospira can survive in water and soil and contribute to human and animal infections. The objective of this study was to describe the presence of pathogenic Leptospira in peri-domestic water samples from rural households in southern Chile. Water samples, including puddles, containers, animal troughs, rivers, canals, and drinking water were collected from 236 households and tested for Leptospira using a PCR assay targeting the lipL32 gene. Evidence of Leptospira presence was detected in all sample types; overall, 13.5% (77/570) samples tested positive. A total of 10/22 (45.5%) open containers, 12/83 (14.5%) animal drinking sources, 9/47 (19.1%) human drinking sources, and 36/306 (19.3%) puddles tested positive. Lower income (OR = 4.35, p = 0.003), increased temperature (OR = 1.23, p < 0.001), and presence of dogs (OR = 15.9, p = 0.022) were positively associated with positive puddles. Increased number of rodent signs was associated with positive puddles in the household (OR = 3.22); however, only in the lower income households. There was no association between PCR positive rodents and puddles at the household level. Results revealed the ubiquity of Leptospira in the household environment and highlight the need to develop formal approaches for systematic monitoring. PMID:24972030

  10. Whole genome sequencing revealed host adaptation-focused genomic plasticity of pathogenic Leptospira

    PubMed Central

    Xu, Yinghua; Zhu, Yongzhang; Wang, Yuezhu; Chang, Yung-Fu; Zhang, Ying; Jiang, Xiugao; Zhuang, Xuran; Zhu, Yongqiang; Zhang, Jinlong; Zeng, Lingbing; Yang, Minjun; Li, Shijun; Wang, Shengyue; Ye, Qiang; Xin, Xiaofang; Zhao, Guoping; Zheng, Huajun; Guo, Xiaokui; Wang, Junzhi

    2016-01-01

    Leptospirosis, caused by pathogenic Leptospira spp., has recently been recognized as an emerging infectious disease worldwide. Despite its severity and global importance, knowledge about the molecular pathogenesis and virulence evolution of Leptospira spp. remains limited. Here we sequenced and analyzed 102 isolates representing global sources. A high genomic variability were observed among different Leptospira species, which was attributed to massive gene gain and loss events allowing for adaptation to specific niche conditions and changing host environments. Horizontal gene transfer and gene duplication allowed the stepwise acquisition of virulence factors in pathogenic Leptospira evolved from a recent common ancestor. More importantly, the abundant expansion of specific virulence-related protein families, such as metalloproteases-associated paralogs, were exclusively identified in pathogenic species, reflecting the importance of these protein families in the pathogenesis of leptospirosis. Our observations also indicated that positive selection played a crucial role on this bacteria adaptation to hosts. These novel findings may lead to greater understanding of the global diversity and virulence evolution of Leptospira spp. PMID:26833181

  11. Whole genome sequencing revealed host adaptation-focused genomic plasticity of pathogenic Leptospira.

    PubMed

    Xu, Yinghua; Zhu, Yongzhang; Wang, Yuezhu; Chang, Yung-Fu; Zhang, Ying; Jiang, Xiugao; Zhuang, Xuran; Zhu, Yongqiang; Zhang, Jinlong; Zeng, Lingbing; Yang, Minjun; Li, Shijun; Wang, Shengyue; Ye, Qiang; Xin, Xiaofang; Zhao, Guoping; Zheng, Huajun; Guo, Xiaokui; Wang, Junzhi

    2016-01-01

    Leptospirosis, caused by pathogenic Leptospira spp., has recently been recognized as an emerging infectious disease worldwide. Despite its severity and global importance, knowledge about the molecular pathogenesis and virulence evolution of Leptospira spp. remains limited. Here we sequenced and analyzed 102 isolates representing global sources. A high genomic variability were observed among different Leptospira species, which was attributed to massive gene gain and loss events allowing for adaptation to specific niche conditions and changing host environments. Horizontal gene transfer and gene duplication allowed the stepwise acquisition of virulence factors in pathogenic Leptospira evolved from a recent common ancestor. More importantly, the abundant expansion of specific virulence-related protein families, such as metalloproteases-associated paralogs, were exclusively identified in pathogenic species, reflecting the importance of these protein families in the pathogenesis of leptospirosis. Our observations also indicated that positive selection played a crucial role on this bacteria adaptation to hosts. These novel findings may lead to greater understanding of the global diversity and virulence evolution of Leptospira spp. PMID:26833181

  12. Evaluation of colostral immunity in swine with commercial anti-leptospira polyvalent whole-bacteria vaccine.

    PubMed

    Martins Soto, Francisco Rafael; Regina Pinheiro, Sônia; Honma Ito, Fumio; Maria Moraes, Zenaide; Paldes Gonçales, Amane; Santos de Azevedo, Sérgio; Bernardi, Fernanda; Rodrigues Camargo, Sebastião; Arruda Vasconcellos, Silvio

    2008-07-01

    The intensity and duration of passive immunity against swine leptospirosis were investigated by the microscopic agglutination test and in vitro leptospira growth inhibition. Twenty-one females at first parturition were divided into three groups: Group A (n=08): received two doses with 30 days interval of the commercial anti-leptospira bacterin A. Group B (n=06) received two doses with 30 days interval of the commercial anti-leptospira bacterin B and Group C (n=07) was the control. In all groups the colostrums were collected. Blood collection of piglets was performed in four different ages. Agglutinin antibodies were equally detected in sera and colostrums for serovars Canicola, Grippotyphosa, Copenhageni, Icterohaemorrhagiae and Pomona (Group A) and Canicola, Copenhageni, Icterohaemorrhagiae, Pomona and Hardjo (Group B). Mean neutralizing antibodies titers were low. Passive immunity was low duration. PMID:17521726

  13. Preliminary Investigations on the Distribution of Leptospira Serovars in Domestic Animals in North-west Morocco.

    PubMed

    Benkirane, A; Noury, S; Hartskeerl, R A; Goris, M G A; Ahmed, A; Nally, J E

    2016-04-01

    Leptospirosis is a neglected zoonosis of global importance with a complex epidemiology that affects humans, domestic and wild mammals. However, due to the diversity of clinical signs and difficulties of establishing a confirmatory laboratory diagnosis, the disease remains poorly investigated, particularly in the developing world. In Morocco, a descriptive study of the seroprevalence of Leptospira infection in animals has never been undertaken. To fill this gap, the current study was conducted on a subset of animals in north-west Morocco as a preliminary step towards understanding the epidemiological patterns of animal leptospirosis in the country. The study was conducted on 289 serum samples collected between January and April 2012 from dogs, cattle, sheep, goats and donkeys in the areas of Rabat-Temara, Sidi Kacem and Oulmes. All serum samples were tested by the MAT with 14 reference strains of the most prevalent pathogenic serovars of Leptospira and two serovars of non-pathogenic Leptospira. The overall seroprevalence of Leptospira in cattle, sheep, goats, dogs and donkeys was 15%, 18%, 20%, 21% and 20%, respectively. The most prevalent serogroups found in each species were Ballum, Sejroe, and Australis in cattle, Ballum, Australis and Sejroe in sheep, Australis and Ballum in goats, Javanica and Australis in donkey and Australis, Ballum and Canicola in dogs. Of all the serogroups tested in this study, Icterohaemorrhagiae, the only serogroup which has been previously reported in humans in Morocco, was rarely reactive. The majority of reactive sera were collected from low land areas. A large number of sera samples classified as seronegative when tested against pathogenic leptospires were positive when tested against non-pathogenic leptospires; this is suggestive of possible novel, as yet unclassified, Leptospira serovars in Morocco. Eleven of thirteen sheep urine samples were positive by real-time PCR confirming their role as Leptospira carriers in Morocco. PMID

  14. Rapid identification of some Leptospira isolates from cattle by random amplified polymorphic DNA fingerprinting.

    PubMed

    Corney, B G; Colley, J; Djordjevic, S P; Whittington, R; Graham, G C

    1993-11-01

    We compared random amplified polymorphic DNA (RAPD) fingerprinting with cross-absorption agglutination and restriction enzyme analysis for typing bovine leptospires. Using RAPD fingerprinting, we examined a number of Leptospira serovars, namely, hardjo genotypes bovis and prajitno, pomona, balcanica, tarassovi, swajizak, kremastos, australis, and zanoni, which are likely to be isolated from Australian cattle. Each serovar and genotype had a unique RAPD profile. Of 26 field isolates of Leptospira, 23 were identified as hardjo genotype bovis subtype A, 2 were identified as zanoni, and 1 was identified as pomona by RAPD fingerprinting, and their types were confirmed by cross-absorption agglutination and restriction enzyme analysis. PMID:8263177

  15. Leptospira: The Dawn of the Molecular Genetics Era for an Emerging Zoonotic Pathogen

    PubMed Central

    Ko, Albert I.; Goarant, Cyrille; Picardeau, Mathieu

    2011-01-01

    Leptospirosis is a zoonotic disease which has emerged as a major cause of morbidity and mortality among impoverished populations. One centenary after the discovery of the causative spirochaetal agent, little is understood of Leptospira pathogenesis, which in turn has hampered the identification of new intervention strategies to address this neglected disease. However the recent availability of complete genome sequences for Leptospira and discovery of genetic tools to transform the pathogen has led to major insights into the biology and pathogenesis of this pathogen. We discuss the life cycle of the bacterium and the new advances that have been made and their implications for the future prevention of this disease. PMID:19756012

  16. Rapid identification of some Leptospira isolates from cattle by random amplified polymorphic DNA fingerprinting.

    PubMed Central

    Corney, B G; Colley, J; Djordjevic, S P; Whittington, R; Graham, G C

    1993-01-01

    We compared random amplified polymorphic DNA (RAPD) fingerprinting with cross-absorption agglutination and restriction enzyme analysis for typing bovine leptospires. Using RAPD fingerprinting, we examined a number of Leptospira serovars, namely, hardjo genotypes bovis and prajitno, pomona, balcanica, tarassovi, swajizak, kremastos, australis, and zanoni, which are likely to be isolated from Australian cattle. Each serovar and genotype had a unique RAPD profile. Of 26 field isolates of Leptospira, 23 were identified as hardjo genotype bovis subtype A, 2 were identified as zanoni, and 1 was identified as pomona by RAPD fingerprinting, and their types were confirmed by cross-absorption agglutination and restriction enzyme analysis. Images PMID:8263177

  17. Low-stringency PCR with diagnostically useful primers for identification of Leptospira serovars.

    PubMed Central

    de Caballero, O L; Dias Neto, E; Koury, M C; Romanha, A J; Simpson, A J

    1994-01-01

    Primers proposed for the diagnosis of the pathogenic spirochete Leptospira spp. (C. Gravekamp, H. V. D. Kemp, M. Franzen, D. Carrington, G.J. Schoone, G.J.J.M. Van Eys, C. O. R. Everard, R.A. Hartskeel, and W.J. Terpstra, J. Gen. Microbiol. 139:1691-1700, 1993) have been found to produce complex serovar-specific patterns under low-stringency PCR conditions. Such patterns obtained by low-stringency PCR, which maintain the specific band as an internal control, offer, an approach to the standardized identification of Leptospira serovars in clinical laboratories. Images PMID:8051272

  18. Protein-free and low-protein media for the cultivation of Leptospira.

    PubMed Central

    Bey, R F; Johnson, R C

    1978-01-01

    A protein-free medium composed of charcoal-detoxified Tweens (polysorbates), vitamins B12 and B1, inorganic salts, and organic buffer is described that supports the growth and subculture of pathogenic and saprophytic Leptospira. Growth was initiated from small inocula, and cell densities of 10(9) organisms per ml were attained. Antigenicity and immunogenicity of Leptospira cultivated in this medium were similar to those of cells cultivated in serum-containing media. The protein-free medium was converted to a low-protein medium by the addition of 0.1% bovine serum albumin. PMID:631889

  19. Epidemiology of Leptospira weilii serovar Topaz infections in Australia.

    PubMed

    Slack, Andrew T; Symonds, Meegan L; Dohnt, Michael F; Corney, Bruce G; Smythe, Lee D

    2007-06-01

    Leptospirosis is a zoonotic disease with a worldwide distribution. Leptospira weilii serovar (sv.) Topaz is a newly described serovar first isolated in the far north of Queensland, Australia. The epidemiology of L. weilii sv. Topaz infections in Australia was characterised through the use of surveillance questionnaires and molecular studies. There have been 24 human and 2 animal (bovine and bandicoot) L. weilii sv. Topaz infections diagnosed since 1991. The majority of these infections have occurred in Far North Queensland, with the remaining infections occurring in South East Queensland and in Western Australia. The majority of patients with L. weilii sv. Topaz infections presented with classical leptospirosis symptoms including; fever, headaches, sweats, chills and myalgia. The occupations of human cases of L. weilii sv. Topaz infection included banana farming, dairy and beef cattle production and tourist related activities. Fluorescent amplified fragment length polymorphism (FAFLP) was performed on 15 L. weilii sv. Topaz isolates including 2 animal isolates. Clustering analysis grouped the 15 isolates into 5 main clades with 13 unique FAFLP profiles. A high level of relatedness was demonstrated between 2 animal and 2 human isolates. PMID:17724998

  20. Seroprevalence of anti-Leptospira spp. antibodies in dogs in Bahia, Brazil.

    PubMed

    Oliveira Lavinsky, Manuella; Said, Roueda Abou; Strenzel, Gil Marcelo Reuss; Langoni, Helio

    2012-09-01

    The aim of this study was to determine the prevalence of anti-Leptospira spp. antibodies in dogs living in the urban area of the city of Ilhéus, Bahia, Brazil using the microscopic agglutination test (MAT) to investigate 24 serovars. A semi-structured questionnaire was administered to dog owners to collect data about demography, husbandry and environmental factors. The prevalence of anti-Leptospira spp. antibodies in the population of 282 dogs was 7.1% (95% confidence interval: 4.4-10.7%). Serovar Copenhageni was the most prevalent, followed by serovars Bratislava, Canicola and Gryppotyphosa. No risk factor was detected with regard to demography (age, gender and breed), husbandry (Leptospira vaccinations, food and water exposure through their environment, hunting habits, contact with other animals and contact with rats) and environmental factors (sewage network, garbage collection, history of flooding, river proximity and wastelands). Despite the low prevalence found in this study, the seroprevalence of Leptospira spp. in healthy dogs in Ilhéus indicates the presence of this agent in the environment, which may be a source of human infection. Knowledge of the serovars present in this environment is important for understanding the epidemiology of leptospirosis and establishing public health policies aimed at its control. PMID:22510215

  1. In vitro susceptibilities of five Leptospira strains to 16 antimicrobial agents.

    PubMed Central

    Oie, S; Hironaga, K; Koshiro, A; Konishi, H; Yoshii, Z

    1983-01-01

    The in vitro activities of 16 antibiotics against five serovar strains of the genus Leptospira were determined. Five of the antibiotics (ampicillin, cefmetazole, moxalactam, ceftizoxime, and cefotaxime) exhibited a lower minimal inhibitory concentration than did penicillin G. In tests for minimal bactericidal concentration, ceftizoxime and cefotaxime were found to be more effective than penicillin G, streptomycin, tetracycline, ampicillin, and cefmetazole. PMID:6660857

  2. Use of polymerase chain reaction for the identification of Leptospira sp. in urine of carriers.

    PubMed

    Ruiz, Victor Manuel Banda; Vega, Laura Elena Orozco; Velázquez, Rosa Maria Urrutia

    2005-01-01

    The objectives of the present work were to detect Leptospira seropositive animals. The ELISA results report only IgG antibodies, which could be attributable to chronic infections or else, that they are healthy carriers. All polymerase chain reaction positive animals should be considered potential sources of infection. PMID:17966477

  3. Prevalence and risk factors for Leptospira exposure in New Zealand veterinarians.

    PubMed

    Sanhueza, J M; Heuer, C; Wilson, P R; Benschop, J; Collins-Emerson, J M

    2015-07-01

    This study assessed seroprevalence and risk factors for Leptospira (serovars Hardjo, Pomona, Ballum, Copenhageni, Tarassovi) exposure in New Zealand veterinarians. Veterinarians (n = 277) at one of two conferences were voluntarily enrolled and blood samples taken. Microscopic agglutination test (MAT) titres ⩾48 were considered seropositive. Fourteen veterinarians (5·1%, 95% confidence interval 2·8-8·3) were seropositive to Leptospira. Home slaughter of cattle or pigs were significant risk factors for Leptospira exposure. There were no clear relationships between the animal species handled at work and serostatus. However, veterinarians spending a 'mid to high' proportion of their time (>50% to ⩽75%) with pets had higher odds of being seropositive than those not working with pets. A borderline positive association (P = 0·09) was observed between seropositivity and clinical influenza-like illness (⩾3 days off work) in the 18 months before the study. Assuming causality, this suggests that 8·3% of these cases may be attributed to Leptospira exposure. PMID:25804406

  4. Analysis of Multilocus Sequence Typing for Identification of Leptospira Isolates in Brazil ▿

    PubMed Central

    Romero, E. C.; Blanco, R. M.; Galloway, R. L.

    2011-01-01

    A collection of 101 Leptospira isolates was tested by multilocus sequence typing (MLST) and by traditional serotyping. MLST divided the isolates into 4 sequence types (STs), while serotyping classified them into 6 serogroups. Two isolates failed to generate products for some genes by MLST. MLST was less discriminatory than serotyping for uncommonly occurring isolates from humans in Brazil. PMID:21880969

  5. Presence of antibodies against Leptospira serovars in Chaetophractus villosus (Mammalia, Dasypodidae), La Pampa province, Argentina.

    PubMed

    Kin, Marta S; Brihuega, Bibiana; Fort, Marcelo; Delgado, Fernando; Bedotti, Daniel; Casanave, Emma B

    2015-01-01

    Leptospirosis is a zoonosis of worldwide distribution. The aim of this study was to examine the presence of antibodies against 21 Leptospira reactive serovars in Chaetophractus villosus in La Pampa province, Argentina, using the microscopic agglutination test (MAT). Pathologic changes compatible with leptospirosis and in situ detection of the agent by immunohistochemistry were studied in 24 and 3 individuals respectively. Only 35/150 (23.3%) serum samples had antibodies against Leptospira sp. Six percent of the samples reacted with serovar Canicola, 4.7% with serovar Castellonis, 1.3% with serovar Icterohemorrhagieae and 0.7% with serovar Hardjo. Sixteen (10.6%) serum samples agglutinated with Castellonis-Icterohemorrhagiae and Canicola-Castellonis serovars, both with 4.7%, and Canicola-Hardjo and Castellonis-Canicola-Icterohemorrhagiae both with 0.6%. Fourteen animals had variable degrees of lesions, which were more severe in animals with higher serological titers (3200), and Leptospira sp. was detected in 3 animals by immunohistochemistry. These results represent the first record of the presence of Leptospira in C. villosus in La Pampa. PMID:25754485

  6. Comparison of Mucosal, Subcutaneous and Intraperitoneal Routes of Rat Leptospira Infection.

    PubMed

    Zilber, Anne-Laure; Belli, Patrick; Grezel, Delphine; Artois, Marc; Kodjo, Angeli; Djelouadji, Zoheira

    2016-03-01

    Leptospirosis is a zoonosis found worldwide that is caused by a spirochete. The main reservoirs of Leptospira, which presents an asymptomatic infection, are wild rodents, including the brown rat (Rattus norvegicus). Experimental studies of the mechanisms of its renal colonization in rats have previously used an intraperitoneal inoculation route. However, knowledge of rat-rat transmission requires the use of a natural route of inoculation, such as a mucosal or subcutaneous route. We investigated for the first time the effects of subcutaneous and mucosal inoculation routes compared to the reference intraperitoneal route during Leptospira infection in adult rats. Infection characteristics were studied using Leptospira renal isolation, serology, and molecular and histological analyses. Leptospira infection was asymptomatic using each inoculation route, and caused similar antibody production regardless of renal colonization. The observed renal colonization rates were 8 out of 8 rats, 5 out of 8 rats and 1 out of 8 rats for the intraperitoneal, mucosal and subcutaneous inoculation routes, respectively. Thus, among the natural infection routes studied, mucosal inoculation was more efficient for renal colonization associated with urinary excretion than the subcutaneous route and induced a slower-progressing infection than the intraperitoneal route. These results can facilitate understanding of the infection modalities in rats, unlike the epidemiological studies conducted in wild rats. Future studies of other natural inoculation routes in rat models will increase our knowledge of rat-rat disease transmission and allow the investigation of infection kinetics. PMID:27031867

  7. Development of Hamster Models for Acute and Chronic Infections with Leptospira borgpetersenii serovar Hardjo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Golden Syrian hamster is frequently used as a small animal model to study acute leptospirosis. However, use of this small animal model to study Leptospira borgpetersenii serovar Hardjo infections has not been well documented. Cattle are the normal maintenance hosts of L. borgpetersenii serovar...

  8. Comparison of Mucosal, Subcutaneous and Intraperitoneal Routes of Rat Leptospira Infection

    PubMed Central

    Zilber, Anne-Laure; Belli, Patrick; Grezel, Delphine; Artois, Marc; Kodjo, Angeli; Djelouadji, Zoheira

    2016-01-01

    Leptospirosis is a zoonosis found worldwide that is caused by a spirochete. The main reservoirs of Leptospira, which presents an asymptomatic infection, are wild rodents, including the brown rat (Rattus norvegicus). Experimental studies of the mechanisms of its renal colonization in rats have previously used an intraperitoneal inoculation route. However, knowledge of rat-rat transmission requires the use of a natural route of inoculation, such as a mucosal or subcutaneous route. We investigated for the first time the effects of subcutaneous and mucosal inoculation routes compared to the reference intraperitoneal route during Leptospira infection in adult rats. Infection characteristics were studied using Leptospira renal isolation, serology, and molecular and histological analyses. Leptospira infection was asymptomatic using each inoculation route, and caused similar antibody production regardless of renal colonization. The observed renal colonization rates were 8 out of 8 rats, 5 out of 8 rats and 1 out of 8 rats for the intraperitoneal, mucosal and subcutaneous inoculation routes, respectively. Thus, among the natural infection routes studied, mucosal inoculation was more efficient for renal colonization associated with urinary excretion than the subcutaneous route and induced a slower-progressing infection than the intraperitoneal route. These results can facilitate understanding of the infection modalities in rats, unlike the epidemiological studies conducted in wild rats. Future studies of other natural inoculation routes in rat models will increase our knowledge of rat-rat disease transmission and allow the investigation of infection kinetics. PMID:27031867

  9. Seroprevalence of Leptospira Hardjo in the Irish suckler cattle population

    PubMed Central

    2012-01-01

    Background Prior to the present study, the seroprevalence of leptospirosis in Irish suckler herds was unknown. In this study, we describe the herd and animal-level prevalence of Leptospira Hardjo infection in the Irish suckler cattle population. For the purposes of the study, the 26 counties of the Republic of Ireland were divided into 6 regions from which a representative number of herds were selected. A herd was considered eligible for sampling if it was not vaccinating against leptospirosis and if it contained ≥ 9 breeding animals of beef breed ≥ 12 months of age. In total, 288 randomly selected herds were eligible for inclusion in the seroprevalence dataset analysis. Serological testing was carried out using a commercially available monoclonal antibody-capture ELISA, (sensitivity 100%; specificity 86.67%). Results Herds were categorised as either “Free from Infection” or “Infected” using the epidemiological software tool, FreeCalc 2.0. Using this classification, 237 herds were “Infected” (82.29%). The South West and South East regions had the highest herd prevalence. The regional effect on herd prevalence was largely mirrored by breeding herd size. A true animal-level prevalence of 41.75% was calculated using the epidemiological software tool, TruePrev. There was a statistically significant regional trend, with true prevalence being highest in the South East (P < 0.05). The median Breeding Herd Size (BHS), when categorised into quartiles, had a statistically significant influence on individual animal true seroprevalence (P < 0.001); true seroprevalence increased with increasing BHS. Conclusions Leptospirosis is a widespread endemic disease in the Republic of Ireland. It is possible that economic losses due to leptospirosis in unvaccinated Irish suckler herds may be underestimated. PMID:22546216

  10. Leptospira Seroprevalence and Risk Factors in Health Centre Patients in Hoima District, Western Uganda

    PubMed Central

    Pearson, Raewynne; Kankya, Clovice; Kajura, Charles; Alinaitwe, Lordrick; Kakooza, Steven; Pelican, Katharine M.; Travis, Dominic A.; Mahero, Michael; Boulware, David R.; Mugisha, Lawrence

    2016-01-01

    Background The burden of human leptospirosis in Uganda is unknown. We estimated the seroprevalence of Leptospira antibodies, probable acute/recent leptospirosis, and risk factors for seropositivity in humans in rural Western Uganda. Methodology and Principal Findings 359 non-pregnant adults visiting the Kikuube and Kigorobya Health Centers were sequentially recruited during March and April 2014. A health history survey and serum were collected from consented participants. Overall, 69% reported having fever in the past year, with 49% reporting malaria, 14% malaria relapse, 6% typhoid fever, 3% brucellosis, and 0% leptospirosis. We tested sera by microscopic agglutination test (MAT) against eight Leptospira serovars representing seven serogroups. Leptospira seroprevalence was 35% (126/359; 95%CI 30.2–40.3%) defined as MAT titer ≥ 1:100 for any serovar. The highest prevalence was against L. borgpetersenii Nigeria (serogroup Pyrogenes) at 19.8% (71/359; 95%CI 15.9–24.4%). The prevalence of probable recent leptospirosis (MAT titer ≥1:800) was 1.9% (95%CI 0.9–4.2%) and uniquely related to serovar Nigeria (serogroup Pyrogenes). Probable recent leptospirosis was associated with having self-reported malaria within the past year (p = 0.048). Higher risk activities included skinning cattle (n = 6) with 12.3 higher odds (95%CI 1.4–108.6; p = 0.024) of Leptospira seropositivity compared with those who had not. Participants living in close proximity to monkeys (n = 229) had 1.92 higher odds (95%CI 1.2–3.1; p = 0.009) of seropositivity compared with participants without monkeys nearby. Conclusions/Significance The 35% prevalence of Leptospira antibodies suggests that exposure to leptospirosis is common in rural Uganda, in particular the Nigeria serovar (Pyrogenes serogroup). Leptospirosis should be a diagnostic consideration in febrile illness and “smear-negative malaria” in rural East Africa. PMID:27487398

  11. Molecular Detection of Leptospira in Two Returned Travelers: Higher Bacterial Load in Cerebrospinal Fluid versus Serum or Plasma

    PubMed Central

    Waggoner, Jesse J.; Soda, Elizabeth A.; Seibert, Ryan; Grant, Philip; Pinsky, Benjamin A.

    2015-01-01

    Leptospirosis is a potentially severe illness in returned travelers. Patients often present with fever, headache, and neck pain, which may lead to a workup for meningitis including the acquisition of cerebrospinal fluid (CSF). Although Leptospira DNA has been detected in CSF by polymerase chain reaction (PCR), little data exist regarding the utility of testing CSF in addition to serum or plasma obtained on presentation. In this report, we present two cases of leptospirosis in returned travelers presenting with fever and headache. Our first patient had neutrophilic meningitis, and Leptospira was detectable only in CSF obtained on admission. The second patient had a normal CSF profile, but Leptospira was detected in CSF at a bacterial load 5- to 10-fold higher than that in plasma. CSF is an important specimen for the diagnosis of Leptospira by molecular methods and may yield an actionable diagnosis in the absence of leptospiremia. PMID:26033024

  12. Molecular Detection of Leptospira in Two Returned Travelers: Higher Bacterial Load in Cerebrospinal Fluid Versus Serum or Plasma.

    PubMed

    Waggoner, Jesse J; Soda, Elizabeth A; Seibert, Ryan; Grant, Philip; Pinsky, Benjamin A

    2015-08-01

    Leptospirosis is a potentially severe illness in returned travelers. Patients often present with fever, headache, and neck pain, which may lead to a workup for meningitis including the acquisition of cerebrospinal fluid (CSF). Although Leptospira DNA has been detected in CSF by polymerase chain reaction (PCR), little data exist regarding the utility of testing CSF in addition to serum or plasma obtained on presentation. In this report, we present two cases of leptospirosis in returned travelers presenting with fever and headache. Our first patient had neutrophilic meningitis, and Leptospira was detectable only in CSF obtained on admission. The second patient had a normal CSF profile, but Leptospira was detected in CSF at a bacterial load 5- to 10-fold higher than that in plasma. CSF is an important specimen for the diagnosis of Leptospira by molecular methods and may yield an actionable diagnosis in the absence of leptospiremia. PMID:26033024

  13. Comparative genomic analysis of Brazilian Leptospira kirschneri serogroup Pomona serovar Mozdok.

    PubMed

    Moreno, Luisa Z; Kremer, Frederico S; Miraglia, Fabiana; Loureiro, Ana P; Eslabao, Marcus R; Dellagostin, Odir A; Lilenbaum, Walter; Moreno, Andrea M

    2016-08-01

    Leptospira kirschneri is one of the pathogenic species of the Leptospira genus. Human and animal infection from L. kirschneri gained further attention over the last few decades. Here we present the isolation and characterisation of Brazilian L. kirschneri serogroup Pomona serovar Mozdok strain M36/05 and the comparative genomic analysis with Brazilian human strain 61H. The M36/05 strain caused pulmonary hemorrhagic lesions in the hamster model, showing high virulence. The studied genomes presented high symmetrical identity and the in silico multilocus sequence typing analysis resulted in a new allelic profile (ST101) that so far has only been associated with the Brazilian L. kirschneri serogroup Pomona serovar Mozdok strains. Considering the environmental conditions and high genomic similarity observed between strains, we suggest the existence of a Brazilian L. kirschneri serogroup Pomona serovar Mozdok lineage that could represent a high public health risk; further studies are necessary to confirm the lineage significance and distribution. PMID:27581124

  14. Successful development and validation of an in vitro replacement assay for Leptospira vaccine potency tests.

    PubMed

    Kulpa-Eddy, J

    2012-01-01

    The standard requirement for serial release potency testing of Leptospira bacterins in the United States is the hamster vaccination challenge test. It is a test that uses a large number of animals experiencing pain or distress, takes weeks to conduct, can be expensive and requires that laboratory personnel handle a viable zoonotic pathogen. In an effort to address these concerns, the United States Department of Agriculture (USDA) developed an in vitro method for potency testing of four Leptospira serovars. This enzyme-linked immunosorbent assay (ELISA) was subsequently validated in the target species. USDA informed their biologics licensees, permittees and applicants of the availability of reference bacterins and the regulatory acceptance regarding this alternative test method in notices issued in 2007 and 2009. This presentation describes how the initial research and subsequent development and validation work were accomplished. PMID:22888601

  15. Widespread detection of antibodies to Leptospira in feral swine in the United States.

    PubMed

    Pedersen, K; Pabilonia, K L; Anderson, T D; Bevins, S N; Hicks, C R; Kloft, J M; Deliberto, T J

    2015-07-01

    As feral swine continue to expand their geographical range and distribution across the United States, their involvement in crop damage, livestock predation, and pathogen transmission is likely to increase. Despite the relatively recent discovery of feral swine involvement in the aetiology of a variety of pathogens, their propensity to transmit and carry a wide variety of pathogens is disconcerting. We examined sera from 2055 feral swine for antibody presence to six serovars of Leptospira that can also infect humans, livestock or domestic animals. About 13% of all samples tested positive for at least one serovar, suggesting that Leptospira infection is common in feral swine. Further studies to identify the proportion of actively infected animals are needed to more fully understand the risk they pose. PMID:25518910

  16. Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses.

    PubMed

    Evangelista, Karen V; Coburn, Jenifer

    2010-09-01

    Leptospirosis, the most widespread zoonosis in the world, is an emerging public health problem, particularly in large urban centers of developing countries. Several pathogenic species of the genus Leptospira can cause a wide range of clinical manifestations, from a mild, flu-like illness to a severe disease form characterized by multiorgan system complications leading to death. However, the mechanisms of pathogenesis of Leptospira are largely unknown. This article will address the animal models of acute and chronic leptospire infections, and the recent developments in the genetic manipulation of the bacteria, which facilitate the identification of virulence factors involved in pathogenesis and the assessment of their potential values in the control and prevention of leptospirosis. PMID:20860485

  17. Comparative genomic analysis of Brazilian Leptospira kirschneri serogroup Pomona serovar Mozdok.

    PubMed

    Moreno, Luisa Z; Kremer, Frederico S; Miraglia, Fabiana; Loureiro, Ana P; Eslabao, Marcus R; Dellagostin, Odir A; Lilenbaum, Walter; Moreno, Andrea M

    2016-07-11

    Leptospira kirschneri is one of the pathogenic species of the Leptospira genus. Human and animal infection from L. kirschneri gained further attention over the last few decades. Here we present the isolation and characterisation of Brazilian L. kirschneri serogroup Pomona serovar Mozdok strain M36/05 and the comparative genomic analysis with Brazilian human strain 61H. The M36/05 strain caused pulmonary hemorrhagic lesions in the hamster model, showing high virulence. The studied genomes presented high symmetrical identity and the in silico multilocus sequence typing analysis resulted in a new allelic profile (ST101) that so far has only been associated with the Brazilian L. kirschneri serogroup Pomona serovar Mozdok strains. Considering the environmental conditions and high genomic similarity observed between strains, we suggest the existence of a Brazilian L. kirschneri serogroup Pomona serovar Mozdok lineage that could represent a high public health risk; further studies are necessary to confirm the lineage significance and distribution. PMID:27409843

  18. Comparative genomic analysis of Brazilian Leptospira kirschneri serogroup Pomona serovar Mozdok

    PubMed Central

    Moreno, Luisa Z; Kremer, Frederico S; Miraglia, Fabiana; Loureiro, Ana P; Eslabao, Marcus R; Dellagostin, Odir A; Lilenbaum, Walter; Moreno, Andrea M

    2016-01-01

    Leptospira kirschneri is one of the pathogenic species of the Leptospira genus. Human and animal infection from L. kirschneri gained further attention over the last few decades. Here we present the isolation and characterisation of Brazilian L. kirschneri serogroup Pomona serovar Mozdok strain M36/05 and the comparative genomic analysis with Brazilian human strain 61H. The M36/05 strain caused pulmonary hemorrhagic lesions in the hamster model, showing high virulence. The studied genomes presented high symmetrical identity and the in silico multilocus sequence typing analysis resulted in a new allelic profile (ST101) that so far has only been associated with the Brazilian L. kirschneri serogroup Pomona serovar Mozdok strains. Considering the environmental conditions and high genomic similarity observed between strains, we suggest the existence of a Brazilian L. kirschneri serogroup Pomona serovar Mozdok lineage that could represent a high public health risk; further studies are necessary to confirm the lineage significance and distribution. PMID:27581124

  19. High Prevalence of Intermediate Leptospira spp. DNA in Febrile Humans from Urban and Rural Ecuador

    PubMed Central

    Chiriboga, Jorge; Barragan, Verónica; Arroyo, Gabriela; Sosa, Andrea; Birdsell, Dawn N.; España, Karool; Mora, Ana; Espín, Emilia; Mejía, María Eugenia; Morales, Melba; Pinargote, Carmina; Gonzalez, Manuel; Hartskeerl, Rudy; Keim, Paul; Bretas, Gustavo; Eisenberg, Joseph N.S.

    2015-01-01

    Leptospira spp., which comprise 3 clusters (pathogenic, saprophytic, and intermediate) that vary in pathogenicity, infect >1 million persons worldwide each year. The disease burden of the intermediate leptospires is unclear. To increase knowledge of this cluster, we used new molecular approaches to characterize Leptospira spp. in 464 samples from febrile patients in rural, semiurban, and urban communities in Ecuador; in 20 samples from nonfebrile persons in the rural community; and in 206 samples from animals in the semiurban community. We observed a higher percentage of leptospiral DNA–positive samples from febrile persons in rural (64%) versus urban (21%) and semiurban (25%) communities; no leptospires were detected in nonfebrile persons. The percentage of intermediate cluster strains in humans (96%) was higher than that of pathogenic cluster strains (4%); strains in animal samples belonged to intermediate (49%) and pathogenic (51%) clusters. Intermediate cluster strains may be causing a substantial amount of fever in coastal Ecuador. PMID:26583534

  20. Infection with Leptospira kirschneri Serovar Mozdok: First Report from the Southern Hemisphere.

    PubMed

    da Cunha, Carlos Eduardo Pouey; Felix, Samuel Rodrigues; Neto, Amilton Clair Pinto Seixas; Campello-Felix, Anelize; Kremer, Frederico Schmitt; Monte, Leonardo Garcia; Amaral, Marta Gonçalves; de Oliveira Nobre, Márcia; da Silva, Éverton Fagonde; Hartleben, Cláudia Pinho; McBride, Alan John Alexander; Dellagostin, Odir Antonio

    2016-03-01

    Leptospirosis is a global zoonosis caused by pathogenic Leptospira spp. In this study, we characterized two Leptospira kirschneri serogroup Pomona serovar Mozdok isolates, one obtained from a dog and the other from a patient with severe leptospirosis, 4 years later. Histopathological analysis showed that both isolates caused severe tissue damage when used to infect hamsters. While L. kirschneri serogroup Pomona serovar Mozdok is endemic in animals in Europe, there is only one report of human leptospirosis in the literature. Although strains belonging to L. kirschneri serogroup Pomona have been identified in cases of human leptospirosis in Europe, serovar Mozdok has not yet been implicated. The 4-year interval between isolations and the fact that this is the first report of serovar Mozdok as the causative agent of human leptospirosis in the southern hemisphere, demonstrates its epidemiological importance to public health. Moreover, the presence of serovar Mozdok in Brazil has the potential to affect vaccine and diagnostic test development. PMID:26755566

  1. Deciphering Morphological Determinants of the Helix-Shaped Leptospira ▿ †

    PubMed Central

    Slamti, Leyla; de Pedro, Miguel A.; Guichet, Emilande; Picardeau, Mathieu

    2011-01-01

    Leptospira spp. are thin, highly motile, slow-growing spirochetes that can be distinguished from other bacteria on the basis of their unique helical shape. Defining the mechanisms by which these bacteria generate and maintain this atypical morphology should greatly enhance our understanding of the fundamental physiology of these pathogens. In this study, we showed that peptidoglycan sacculi from Leptospira spp. retain the helical shape of intact cells. Interestingly, the distribution of muropeptides was different from that in the Escherichia coli model, indicating that specific enzymes might be active on the peptidoglycan macromolecule. We could alter the shape of Leptospira biflexa with the broad-spectrum β-lactam antibiotic penicillin G and with amdinocillin and aztreonam, which are β-lactams that preferentially target penicillin-binding protein 2 (PBP2) and PBP3, respectively, in some species. Although genetic manipulations of Leptospira spp. are scarce, we were able to obtain mutants with alterations in genes encoding PBPs, including PBP3. Loss of this protein resulted in cell elongation. We also generated an L. biflexa strain that conditionally expresses MreB. Loss of the MreB function was correlated with morphological abnormalities such as a localized increased diameter and heterogeneous length. A prolonged depletion of MreB resulted in cell lysis, suggesting that this protein is essential. These findings indicate that important aspects of leptospiral cell morphology are determined by the cytoskeleton and the murein layer, thus providing a starting point for a better understanding of the morphogenesis in these atypical bacteria. PMID:21926230

  2. Serologic evidence of Leptospira spp. serovars in brown bears (Ursus arctos) from Croatia.

    PubMed

    Slavica, Alen; Konjevic, Dean; Huber, Duro; Milas, Zoran; Turk, Nenad; Sindicic, Magda; Severin, Kresimir; Dezdek, Danko; Masek, Tomislav

    2010-01-01

    Serum samples from 52 free-ranging brown bears (Ursus arctos) collected in Croatia over a period of 10 yr (1998-2007) were tested by microscopic agglutination test for specific antibodies (Ab) to 12 Leptospira spp. pathogenic serovars. At titers ranging from 1:100 to 1:2,000, 19 samples (36.5%) were Abpositive to at least one serovar. Antibodies for 10 Leptospira spp. serovars were detected: Icterohaemorrhagiae, Australis, Sejroe, Canicola, Poi, Hardjo, Ballum, Saxkoebing, Pomona, and Grippotyphosa. In comparison to previous reports, the prevalence of Ab to serovar Icterohaemorrhagiae (52.6%) was significantly higher. Other common serovars were Australis (47.4%) and Sejroe (42.1%). High Ab titers for serovars Canicola (1:500) and Grippotyphosa (1:1,000) were detected for the first time in free-ranging bears from Croatia. A significant correlation between the age of the bears and detection of Ab to Leptospira spp. serovars suggested the presence of pathogenic agents in the natural habitats, whereas increasing trends of Ab prevalence for specific serovars (Icter-ohaemorrhagiae, Australis, and Sejroe) confirmed cohabitation of bears with rats and other small terrestrial mammals on garbage dumps and at bear feeding stations. To prevent cohabitation of bears and rodents, improvements in Croatian waste treatment, big game management, and rodent control programs are strongly recommended, especially in Lika and Gorski Kotar, regions that have high-quality natural habitats for brown bears in Croatia. PMID:20090039

  3. Altodouro, a new Leptospira serovar of the Pomona serogroup isolated from rodents in northern Portugal.

    PubMed

    Paiva-Cardoso, Maria das Neves; Arent, Zbigniew; Gilmore, Colm; Hartskeerl, Rudy; Ellis, William A

    2013-01-01

    A Leptospira strain (designated RIM 139) was isolated from the kidney of a house-mouse, Mus musculus, in Trás-os-Montes e Alto Douro region of northern Portugal. The isolate showed typical leptospiral motility and morphology under dark-field microscopy and was pathogenic for hamsters. Species determination was carried out on basis of PCR products generated by species-specific primers and by sequencing of the secY gene. Putative serogroup typing was performed using the microscopic agglutination test (MAT) with a panel of rabbit anti-Leptospira sera representative of the major pathogenic serogroups. Serovar identification was carried out by a combination of monoclonal antibodies and cross-agglutinin absorption test (CAAT). The novel nature of the strain was confirmed by restriction endonuclease analysis (REA). Results showed that RIM 139 represents a new serovar. The name Altodouro is proposed for this new serovar. Speciation findings - PCR analysis of the ompL1 gene and sequencing of the secY gene - indicated that it belonged to Leptospira kirschneri as did amplification with G1/G2 and B64-I/B64-II the primer sets. PMID:23070280

  4. Seroprevalence of Leptospira spp. and Borrelia burgdorferi sensu lato in Italian horses.

    PubMed

    Ebani, Valentina V; Bertelloni, Fabrizio; Pinzauti, Paolo; Cerri, Domenico

    2012-01-01

    The aim of the study was to determine the seroprevalence of Leptospira spp. and Borrelia burgdorferi sensu lato in healthy horses living in 7 provinces of central Italy. In the period 2007-2009, sera from 386 horses were tested by microagglutination test (MAT) to detect antibodies to Leptospira spp., employing the following serovars as antigens: Bratislava, Ballum, Canicola, Icterohaemorrhagiae, Grippotyphosa, Hardjo, Pomona, Tarassovi. 3 animals were positive for the serovars Icterohaemorrhagiae, 2 to Bratislava, and 1 to Pomona, for a total 1.5% seroprevalence. All sera were examined by immunofluorence antibody test (IFAT) to reveal anti-B. burgdorferi s.l. antibodies. 94 (24.3%) horses were positive with antibody titres ranging from 1:64 to 1:1,024. The seroprevalence was significantly higher in >10 year-old horses compared to younger subjects. No significant differences in the mean seroprevalence were observed in the respective years. The total mean seroprevalence were strictly related to the environmental conditions of the areas in which the horses lived. No cross-reactions between Leptospira and Borrelia were observed. This is the first serological survey on antibodies to B. burgdorferi s.l. in Italian horses. PMID:22742794

  5. Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

    PubMed Central

    Jung, Lenice Roteia Cardoso; Bomfim, Maria Rosa Quaresma; Kroon, Erna Geessien; Nunes, Álvaro Cantini

    2015-01-01

    Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars. PMID:26273261

  6. International regulatory requirements for Leptospira vaccine potency testing. Roundtable: current requirements and opportunity for harmonization.

    PubMed

    Draayer, Hans A; Bruckner, Lukas; Peña-Moctezuma, Alejandro de la; Srinivas, Geetha

    2013-09-01

    Progress continues to be made in the ongoing efforts to replace, reduce, or refine the use of laboratory animals for Leptospira vaccine potency testing in certain markets/regions. Leptospira-containing vaccines, as with many veterinary vaccines, are manufactured and distributed both on a regional basis by local manufacturers and internationally by large multinational firms. Three general scenarios exist for the international testing and distribution of veterinary vaccines including: 1) the importing country recognizes the country of origin's testing and batch release data with no additional testing; 2) the importing country requires the manufacturer to conduct a specific potency assay based on the current importing market's regulations for the importing country or 3) the importing country requires retesting of the product in country prior to distribution. Scenarios 2 and 3 both have the potential to significantly increase the usage of laboratory animals for what may be considered redundant testing. Specific requirements for the importation of Leptospira vaccines in the United States, Europe, and Mexico were presented as well as efforts to reduce the use of laboratory animal testing through the availability of internationally recognized tests. PMID:23890730

  7. LipL41, a Hemin Binding Protein from Leptospira santarosai serovar Shermani

    PubMed Central

    Lin, Ming-Hsing; Chang, Yuan-Chih; Hsiao, Chwan-Deng; Huang, Shih-Hsun; Wang, Min-Shi; Ko, Yi-Ching; Yang, Chih-Wei; Sun, Yuh-Ju

    2013-01-01

    Leptospirosis is one of the most widespread zoonotic diseases in the world. It is caused by the pathogen Leptospira that results in multiple-organ failure, in particular of the kidney. Outer membrane lipoprotein is the suspected virulence factor of Leptospira. In Leptospira spp LipL41 is one major lipoprotein and is highly conserved. Previous study suggests that LipL41 bears hemin-binding ability and might play a possible role in iron regulation and storage. However, the characterization of hemin-binding ability of LipL41 is still unclear. Here the hemin-binding ability of LipL41 was examined, yielding a Kd = 0.59 ± 0.14 μM. Two possible heme regulatory motifs (HRMs), C[P/S], were found in LipL41 at 140Cys-Ser and 220Cys-Pro. The mutation study indicates that Cys140 and Cys220 might be cooperatively involved in hemin binding. A supramolecular assembly of LipL41 was determined by transmission electron microscopy. The LipL41 oligomer consists of 36 molecules and folds as a double-layered particle. At the C-terminus of LipL41, there are two tetratricopeptide repeats (TPRs), which might be involved in the protein-protein interaction of the supramolecular assembly. PMID:24349474

  8. The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira: A Prospective Evaluation

    PubMed Central

    Dittrich, Sabine; Rudgard, William E.; Woods, Kate L.; Silisouk, Joy; Phuklia, Weerawat; Davong, Viengmon; Vongsouvath, Manivanh; Phommasone, Koukeo; Rattanavong, Sayaphet; Knappik, Michael; Craig, Scott B.; Weier, Steven L.; Tulsiani, Suhella M.; Dance, David A. B.; Newton, Paul N.

    2016-01-01

    Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N = 109) and negative (N = 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55–76) and 59% (95% CI: 49–68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N = 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used. PMID:26880775

  9. The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira: A Prospective Evaluation.

    PubMed

    Dittrich, Sabine; Rudgard, William E; Woods, Kate L; Silisouk, Joy; Phuklia, Weerawat; Davong, Viengmon; Vongsouvath, Manivanh; Phommasone, Koukeo; Rattanavong, Sayaphet; Knappik, Michael; Craig, Scott B; Weier, Steven L; Tulsiani, Suhella M; Dance, David A B; Newton, Paul N

    2016-04-01

    Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N= 109) and negative (N= 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55-76) and 59% (95% CI: 49-68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N= 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used. PMID:26880775

  10. Leptospira in breast tissue and milk of urban Norway rats (Rattus norvegicus).

    PubMed

    DE Oliveira, D; Figueira, C P; Zhan, L; Pertile, A C; Pedra, G G; Gusmão, I M; Wunder, E A; Rodrigues, G; Ramos, E A G; Ko, A I; Childs, J E; Reis, M G; Costa, F

    2016-08-01

    Leptospirosis is a zoonosis caused by bacteria of the genus Leptospira. The disease is globally distributed and a major public health concern. The Norway rat (Rattus norvegicus) is the main reservoir of the pathogen in urban slums of developing and developed countries. The potential routes of intra-specific leptospire transmission in rats are largely unknown. Herein, we identified pathogenic Leptospira spp. in breast tissue and milk of naturally infected rats. We examined kidney, breast tissue and milk from 24 lactating rats for the presence of leptospires using immunofluorescence, immunohistochemistry, polymerase chain reaction (PCR) and scanning electronic microscopy. All 24 rats had evidence for Leptospira in the kidneys, indicating chronic carriage. The majority of kidney-positive rats had detectable leptospires in milk (18, 75%) and breast tissue (16, 67%), as evidenced by immunofluorescence assay and immunohistochemistry. Four (17%) milk samples and two (8%) breast tissue samples were positive by quantitative real-time PCR. Scanning electron microscopy confirmed the presence of leptospires in breast tissue. No major pathological changes in breast tissue were found. This study, for the first time, identified leptospires in the milk and breast tissue of wild Norway rats, suggesting the possibility of milk-borne transmission of leptospirosis to neonates. PMID:27019024

  11. Development of Lyophilized Loop-Mediated Isothermal Amplification Reagents for the Detection of Leptospira.

    PubMed

    Chen, Hua-Wei; Weissenberger, Giulia; Ching, Wei-Mei

    2016-05-01

    Leptospirosis is considered to be the most widespread zoonosis. This worldwide emerging infectious disease is caused by the pathogenic species belonging to the genus Leptospira. Polymerase chain reaction (PCR)-based diagnostic assays have been developed for detecting Leptospira DNA in cell cultures and clinical samples. Because PCR requires specialized equipment and extensive end-user training, it is not suitable for routine work in resource-limited areas. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of Leptospira in patient, animal and environmental samples using lyophilized reagents at a single temperature of around 63°C with a heating block. The sensitivity of this LAMP assay is very similar to the PCR method. The amplified DNA products can be visualized with the naked eyes using hydroxy naphthol blue or detected by the fluorescence signal of SYBR green dye in the reaction when an ultraviolet lamp or compact fluorescence tube scanner is used. This LAMP assay is simple, rapid, and can be performed with a water bath or heating block. The lyophilized LAMP reagents are stable for 3 months when stored at 4°C and 1 month when stored at 25°C, respectively. It is ideal for resource-limited settings where leptospirosis is endemic. PMID:27168577

  12. In vitro susceptibilities of Leptospira spp. and Borrelia burgdorferi isolates to amoxicillin, tilmicosin, and enrofloxacin.

    PubMed

    Kim, Doo; Kordick, Dorsey; Divers, Thomas; Chang, Yung Fu

    2006-12-01

    Antimicrobial susceptibility testing was conducted with 6 different spirochetal strains (4 strains of Leptospira spp. and 2 strains of Borrelia burgdorferi) against 3 antimicrobial agents, commonly used in equine and bovine practice. The ranges of MIC and MBC of amoxicillin against Leptospira spp. were 0.05 - 6.25 microgram/ml and 6.25 - 25.0 microgram/ml, respectively. And the ranges of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of amoxicillin against B. burgdorferi were 0.05 - 0.39 microgram/ml and 0.20 - 0.78 microgram/ml, respectively. The ranges of MIC and MBC of enrofloxacin against Leptospira spp. were 0.05 - 0.39 microgram/ml and 0.05 - 0.39 microgram/ml, respectively. Two strains of B. burgdorferi were resistant to enrofloxacin at the highest concentration tested for MBC (>or=100 microgram/ml). Therefore, the potential role of tilmicosin in the treatment of leptospirosis and borreliosis should be further evaluated in animal models to understand whether the in vivo studies will confirm in vitro results. All spirochetal isolates were inhibited (MIC) and were killed (MBC) by tilmicosin at concentrations below the limit of testing (

  13. Prevalence and risk factors associated with Leptospira spp. infection in technified swine farms in the state of Alagoas, Brazil: risk factors associated with Leptospira spp. in swine farms.

    PubMed

    Valença, R M B; Mota, R A; Castro, V; Anderlini, G A; Pinheiro Júnior, J W; Brandespim, D F; Valença, S R F A; Guerra, M M P

    2013-02-01

    The aim of this study was to determine the prevalence and to identify the risk factors associated with Leptospira spp. infection in technified pig farms in the state of Alagoas, Brazil. To compose sample for the prevalence study, 342 pigs were used (312 sows and 30 boars) proceeding from seven swine farms distributed in five districts of the state of Alagoas, Brazil. The infection's serological diagnosis was performed by microscopic agglutination test. The risk factors analysis was performed using research questionnaires consisting of objective questions related to the breeder, the general characteristics of the property, and the productive, reproductive and sanitary management. Prevalence of 16.1% (55/342) of pigs seropositive was obtained. The associated risk factors were not performing quarantine (P = 0.003, OR = 5.43, CI = 1.79-16.41) and the use of artificial insemination (P = 0.023, OR = 3.38, CI = 1.18-9.66). A significant association of sow infection with the increased number of stillborn and mummified foetuses was found, as well as with the increased frequency of oestrus recurrence and the increased weaning-to-oestrus interval of seropositive sows. One might state that Leptospira spp. infection is disseminated in technified pig farms in the State of Alagoas, favouring reproductive failures and the impairment of zootechnical performance in these properties. The risk factors identified in this study are facilitators in the infecting agent dissemination and should be adjusted to control the disease in the herds studied. PMID:22469011

  14. Serovar Diversity of Pathogenic Leptospira Circulating in the French West Indies

    PubMed Central

    Bourhy, Pascale; Herrmann Storck, Cécile; Theodose, Rafaelle; Olive, Claude; Nicolas, Muriel; Hochedez, Patrick; Lamaury, Isabelle; Zinini, Farida; Brémont, Sylvie; Landier, Annie; Cassadou, Sylvie; Rosine, Jacques; Picardeau, Mathieu

    2013-01-01

    Background Leptospirosis is one of the most important neglected tropical bacterial diseases in Latin America and the Caribbean. However, very little is known about the circulating etiological agents of leptospirosis in this region. In this study, we describe the serological and molecular features of leptospires isolated from 104 leptospirosis patients in Guadeloupe (n = 85) and Martinique (n = 19) and six rats captured in Guadeloupe, between 2004 and 2012. Methods and Findings Strains were studied by serogrouping, PFGE, MLVA, and sequencing 16SrRNA and secY. DNA extracts from blood samples collected from 36 patients in Martinique were also used for molecular typing of leptospires via PCR. Phylogenetic analyses revealed thirteen different genotypes clustered into five main clades that corresponded to the species: L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchi, and L. santarosai. We also identified L. kmetyi in at least two patients with acute leptospirosis. This is the first time, to our knowledge, that this species has been identified in humans. The most prevalent genotypes were associated with L. interrogans serovars Icterohaemorrhagiae and Copenhageni, L. kirschneri serovar Bogvere, and L. borgpetersenii serovar Arborea. We were unable to identify nine strains at the serovar level and comparison of genotyping results to the MLST database revealed new secY alleles. Conclusions The overall serovar distribution in the French West Indies was unique compared to the neighboring islands. Typing of leptospiral isolates also suggested the existence of previously undescribed serovars. PMID:23516654

  15. Influence of Household Rat Infestation on Leptospira Transmission in the Urban Slum Environment

    PubMed Central

    Costa, Federico; Ribeiro, Guilherme S.; Felzemburgh, Ridalva D. M.; Santos, Norlan; Reis, Renato Barbosa; Santos, Andreia C.; Fraga, Deborah Bittencourt Mothe; Araujo, Wildo N.; Santana, Carlos; Childs, James E.; Reis, Mitermayer G.; Ko, Albert I.

    2014-01-01

    Background The Norway rat (Rattus norvegicus) is the principal reservoir for leptospirosis in many urban settings. Few studies have identified markers for rat infestation in slum environments while none have evaluated the association between household rat infestation and Leptospira infection in humans or the use of infestation markers as a predictive model to stratify risk for leptospirosis. Methodology/Principal Findings We enrolled a cohort of 2,003 urban slum residents from Salvador, Brazil in 2004, and followed the cohort during four annual serosurveys to identify serologic evidence for Leptospira infection. In 2007, we performed rodent infestation and environmental surveys of 80 case households, in which resided at least one individual with Leptospira infection, and 109 control households. In the case-control study, signs of rodent infestation were identified in 78% and 42% of the households, respectively. Regression modeling identified the presence of R. norvegicus feces (OR, 4.95; 95% CI, 2.13–11.47), rodent burrows (2.80; 1.06–7.36), access to water (2.79; 1.28–6.09), and un-plastered walls (2.71; 1.21–6.04) as independent risk factors associated with Leptospira infection in a household. We developed a predictive model for infection, based on assigning scores to each of the rodent infestation risk factors. Receiver operating characteristic curve analysis found that the prediction score produced a good/excellent fit based on an area under the curve of 0.78 (0.71–0.84). Conclusions/Significance Our study found that a high proportion of slum households were infested with R. norvegicus and that rat infestation was significantly associated with the risk of Leptospira infection, indicating that high level transmission occurs among slum households. We developed an easily applicable prediction score based on rat infestation markers, which identified households with highest infection risk. The use of the prediction score in community-based screening may

  16. Frequency and type of renal lesions in dogs naturally infected with leptospira species.

    PubMed

    Ortega-Pacheco, A; Colin-Flores, R F; Gutiérrez-Blanco, E; Jiménez-Coello, M

    2008-12-01

    The aim of this study was to determine the frequency and type of renal lesions associated with positive titers against Leptospira sp. in a stray dog population. Three hundred fifty pairs of kidneys and an equal number of serum samples were collected from dogs captured by the dog pound of Merida, Yucatan, Mexico. Euthanasia of dogs was performed following the regulations of the Official Mexican Health Ministry (NOM-033-ZOO-1995). Serum samples were evaluated with the microscopic agglutination test, and tissue samples were processed and fixed in paraffin. After staining with hematoxylin and eosin, the frequency of renal lesions was determined and classified. As an additional evaluation, samples with interstitial nephritis were stained by the Warthin-Starry method in order to observe the presence of spirochete forms that could be morphologically compatible with Leptospira spp. We found that 98% of cases presented at least one type of lesion. The main histological lesions found were mesangial proliferative glomerulonephritis (MPGN) in 63.7% (n= 223), mesangial proliferative glomerulonephritis and interstitial nephritis (MPGN+IN) in 34% (n= 119), nephrosclerosis in 0.57% (n= 2), mesangial glomerulonephritis in 0.28% (n= 1), and interstitial nephritis (IN) in 0.28% (n= 1). Thirty-four percent (n= 122) of the dogs were seropositive to Leptospira sp., mainly against serovar canicola. Among dogs with IN (alone or associated with MPGN) (n= 120), 49.1% were seropositive to Leptospira sp., but only 17% of them showed spirochete forms compatible with the bacteria. A statistical association between seropositive dogs and the presence of MPGN+IN was determined (P < 0.0001; odds ratio 2.7, confidence interval 1.7-4.5). We concluded that the frequency of renal lesions found in this study is high and L. canicola is probably the most common circulating serovar in dogs from this area. Dogs that have been in contact with Leptospira spp. have a higher risk of developing renal lesions of

  17. Contrasting Patterns in Mammal–Bacteria Coevolution: Bartonella and Leptospira in Bats and Rodents

    PubMed Central

    Lei, Bonnie R.; Olival, Kevin J.

    2014-01-01

    Background Emerging bacterial zoonoses in bats and rodents remain relatively understudied. We conduct the first comparative host–pathogen coevolutionary analyses of bacterial pathogens in these hosts, using Bartonella spp. and Leptospira spp. as a model. Methodology/Principal Findings We used published genetic data for 51 Bartonella genotypes from 24 bat species, 129 Bartonella from 38 rodents, and 26 Leptospira from 20 bats. We generated maximum likelihood and Bayesian phylogenies for hosts and bacteria, and tested for coevoutionary congruence using programs ParaFit, PACO, and Jane. Bartonella spp. and their bat hosts had a significant coevolutionary fit (ParaFitGlobal = 1.9703, P≤0.001; m2 global value = 7.3320, P≤0.0001). Bartonella spp. and rodent hosts also indicated strong overall patterns of cospeciation (ParaFitGlobal = 102.4409, P≤0.001; m2 global value = 86.532, P≤0.0001). In contrast, we were unable to reject independence of speciation events in Leptospira and bats (ParaFitGlobal = 0.0042, P = 0.84; m2 global value = 4.6310, P = 0.5629). Separate analyses of New World and Old World data subsets yielded results congruent with analysis from entire datasets. We also conducted event-based cophylogeny analyses to reconstruct likely evolutionary histories for each group of pathogens and hosts. Leptospira and bats had the greatest number of host switches per parasite (0.731), while Bartonella and rodents had the fewest (0.264). Conclusions/Significance In both bat and rodent hosts, Bartonella exhibits significant coevolution with minimal host switching, while Leptospira in bats lacks evolutionary congruence with its host and has high number of host switches. Reasons underlying these variable coevolutionary patterns in host range are likely due to differences in disease-specific transmission and host ecology. Understanding the coevolutionary patterns and frequency of host-switching events between bacterial pathogens and

  18. Is Leptospira able to survive in raw milk? Study on the inactivation at different storage times and temperatures.

    PubMed

    Fratini, Filippo; Turchi, Barbara; Ferrone, Martina; Galiero, Alessia; Nuvoloni, Roberta; Torracca, Beatrice; Cerri, Domenico

    2016-09-01

    The consumption of raw milk is currently increasing due to several beneficial aspects, such as nutritional qualities, taste, and health benefits. However, some authors highlight the potential risk associated with raw milk consumption. In Italy, while the absence of some pathogen microorganisms is set by the regional regulation DGR 381/2007, for other microorganisms, such as Leptospira, no recommendations are provided. Leptospira is not ascribed among classical milk pathogens; however, it can potentially be present in raw milk. The aim of this study was to evaluate the survival in raw milk of six serovars of Leptospira after storage at different temperatures (4 °C ± 2 °C, 20 °C ± 2 °C, and 30 °C ± 2 °C) for different incubation times (20 min, 45 min, 1 h, and 1 h and 30 min), in order to determine the potential risk for consumers. Moreover, the immediate effect of bovine, goat, and donkey raw milk on tested Leptospira serovars was visually evaluated. After incubation, all samples were subcultured in EMJH and incubated aerobically at 30 °C for 3 months. All inoculated media were weekly examined by dark-field microscope in order to assess Leptospira survival. Extemporary observation of strains' behavior in milk allowed to detect an almost immediate motility loss, and no leptospires were detected by microscopic observations carried out weekly during the trial period. According to our results, it could be possible to exclude raw milk as a source of Leptospira infection for consumers. PMID:26888018

  19. A cross sectional observational study to estimate herd level risk factors for Leptospira spp. serovars in small holder dairy cattle farms in southern Chile

    PubMed Central

    2014-01-01

    Background The south of Chile constitutes the main cattle milk producing area of the country. Regarding leptospirosis control in Chile, there is neither an official program nor an epidemiological characterization of smallholder dairy farms. This study was carried out to determine Leptospira seroprevalence and to evaluate risk factors associated with seropositivity at herd level in smallholder bovine dairy herds in southern Chile. A cross-sectional study was conducted, and a convenient sample of 1,537 apparently healthy dairy cows was included in the study. Individual blood samples were taken and examined for six selected reference Leptospira serovars by the Microscopic Agglutination Test (MAT). Results Of the included herds 75% (52/69) showed serological titers against one or more Leptospira serovar. Leptospira borgpetersenii serovar Hardjo was the serovar most frequently (81%) reported from animals with positive results. The variables considered risk factors for Leptospira seropositivity were calve natural breeding system, using a specific calving area and vaccination against Leptospira. Adult cows in contact with calves weaned, proved to be a protective factor against infection. Conclusions Herds neglecting the management practices mentioned in this study could represent an important source of Leptospira infection for other herds in the same geographic area, as well as for other animal species. PMID:24906684

  20. OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

    PubMed Central

    Fernandes, Luis G. V.; Vieira, Monica L.; Kirchgatter, Karin; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Romero, Eliete C.

    2012-01-01

    Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical β-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 ± 871.03 nM and 1,239.23 ± 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 ± 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection. PMID:22802342

  1. Potent Innate Immune Response to Pathogenic Leptospira in Human Whole Blood

    PubMed Central

    Hartskeerl, Rudy A.; van Gorp, Eric C. M.; Schuller, Simone; Monahan, Avril M.; Nally, Jarlath E.; van der Poll, Tom; van 't Veer, Cornelis

    2011-01-01

    Background Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires. Methodology/Principal Findings We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires. Conclusions/Significance Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response. PMID:21483834

  2. An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples.

    PubMed

    Riediger, Irina N; Hoffmaster, Alex R; Casanovas-Massana, Arnau; Biondo, Alexander W; Ko, Albert I; Stoddard, Robyn A

    2016-01-01

    Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from <1 cell /ml for river water to 36 cells/mL for ultrapure water with E. coli as a carrier. In conclusion, we optimized a method to quantify pathogenic Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden. PMID:27487084

  3. An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples

    PubMed Central

    Riediger, Irina N.; Hoffmaster, Alex R.; Biondo, Alexander W.; Ko, Albert I.; Stoddard, Robyn A.

    2016-01-01

    Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from <1 cell /ml for river water to 36 cells/mL for ultrapure water with E. coli as a carrier. In conclusion, we optimized a method to quantify pathogenic Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden. PMID:27487084

  4. Orientia, rickettsia, and leptospira pathogens as causes of CNS infections in Laos: a prospective study

    PubMed Central

    Dittrich, Sabine; Rattanavong, Sayaphet; Lee, Sue J; Panyanivong, Phonepasith; Craig, Scott B; Tulsiani, Suhella M; Blacksell, Stuart D; Dance, David A B; Dubot-Pérès, Audrey; Sengduangphachanh, Amphone; Phoumin, Phonelavanh; Paris, Daniel H; Newton, Paul N

    2015-01-01

    Summary Background Scrub typhus (caused by Orientia tsutsugamushi), murine typhus (caused by Rickettsia typhi), and leptospirosis are common causes of febrile illness in Asia; meningitis and meningoencephalitis are severe complications. However, scarce data exist for the burden of these pathogens in patients with CNS disease in endemic countries. Laos is representative of vast economically poor rural areas in Asia with little medical information to guide public health policy. We assessed whether these pathogens are important causes of CNS infections in Laos. Methods Between Jan 10, 2003, and Nov 25, 2011, we enrolled 1112 consecutive patients of all ages admitted with CNS symptoms or signs requiring a lumbar puncture at Mahosot Hospital, Vientiane, Laos. Microbiological examinations (culture, PCR, and serology) targeted so-called conventional bacterial infections (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, S suis) and O tsutsugamushi, Rickettsia typhi/Rickettsia spp, and Leptospira spp infections in blood or cerebrospinal fluid (CSF). We analysed and compared causes and clinical and CSF characteristics between patient groups. Findings 1051 (95%) of 1112 patients who presented had CSF available for analysis, of whom 254 (24%) had a CNS infection attributable to a bacterial or fungal pathogen. 90 (35%) of these 254 infections were caused by O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp. These pathogens were significantly more frequent than conventional bacterial infections (90/1051 [9%] vs 42/1051 [4%]; p<0·0001) by use of conservative diagnostic definitions. CNS infections had a high mortality (236/876 [27%]), with 18% (13/71) for R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp combined, and 33% (13/39) for conventional bacterial infections (p=0·076). Interpretation Our data suggest that R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp infections are important causes of CNS infections in Laos

  5. Immune responses to Leptospira infection: roles as biomarkers for disease severity.

    PubMed

    Chirathaworn, Chintana; Kongpan, Sutthikarn

    2014-01-01

    Various leptospiral components have been identified and shown to be involved in tissue destruction. In addition, immune responses to leptospires have been implicated in target organ damages in severe leptospirosis cases. Several inflammatory mediators were shown to be higher in susceptible animals than in resistant hosts. Moreover, cytokines/chemokines and serum proteins induced following Leptospira infection were suggested to be biomarkers for disease severity in human leptospirosis. This review focuses on the role of immune responses in the severity of leptospirosis. Studies in both animal models and humans are discussed. PMID:24275371

  6. Transcriptome Sequencing Reveals Wide Expression Reprogramming of Basal and Unknown Genes in Leptospira biflexa Biofilms

    PubMed Central

    Spangenberg, Lucía; Lopes Bastos, Bruno; Graña, Martín; Vasconcelos, Larissa; Almeida, Áurea; Greif, Gonzalo; Robello, Carlos; Ristow, Paula

    2016-01-01

    ABSTRACT The genus Leptospira is composed of pathogenic and saprophytic spirochetes. Pathogenic Leptospira is the etiological agent of leptospirosis, a globally spread neglected disease. A key ecological feature of some pathogenic species is their ability to survive both within and outside the host. For most leptospires, the ability to persist outside the host is associated with biofilm formation, a most important bacterial strategy to face and overcome hostile environmental conditions. The architecture and biochemistry of leptospiral biofilms are rather well understood; however, the genetic program underpinning biofilm formation remains mostly unknown. In this work, we used the saprophyte Leptospira biflexa as a model organism to assess over- and underrepresented transcripts during the biofilm state, using transcriptome sequencing (RNA-seq) technology. Our results showed that some basal biological processes like DNA replication and cell division are downregulated in the mature biofilm. Additionally, we identified significant expression reprogramming for genes involved in motility, sugar/lipid metabolism, and iron scavenging, as well as for outer membrane-encoding genes. A careful manual annotation process allowed us to assign molecular functions to many previously uncharacterized genes that are probably involved in biofilm metabolism. We also provided evidence for the presence of small regulatory RNAs in this species. Finally, coexpression networks were reconstructed to pinpoint functionally related gene clusters that may explain how biofilm maintenance is regulated. Beyond elucidating some genetic aspects of biofilm formation, this work reveals a number of pathways whose functional dissection may impact our understanding of leptospiral biology, in particular how these organisms adapt to environmental changes. IMPORTANCE In this work, we describe the first transcriptome based on RNA-seq technology focused on studying transcriptional changes associated with biofilm

  7. Transcriptome Sequencing Reveals Wide Expression Reprogramming of Basal and Unknown Genes in Leptospira biflexa Biofilms.

    PubMed

    Iraola, Gregorio; Spangenberg, Lucía; Lopes Bastos, Bruno; Graña, Martín; Vasconcelos, Larissa; Almeida, Áurea; Greif, Gonzalo; Robello, Carlos; Ristow, Paula; Naya, Hugo

    2016-01-01

    The genus Leptospira is composed of pathogenic and saprophytic spirochetes. Pathogenic Leptospira is the etiological agent of leptospirosis, a globally spread neglected disease. A key ecological feature of some pathogenic species is their ability to survive both within and outside the host. For most leptospires, the ability to persist outside the host is associated with biofilm formation, a most important bacterial strategy to face and overcome hostile environmental conditions. The architecture and biochemistry of leptospiral biofilms are rather well understood; however, the genetic program underpinning biofilm formation remains mostly unknown. In this work, we used the saprophyte Leptospira biflexa as a model organism to assess over- and underrepresented transcripts during the biofilm state, using transcriptome sequencing (RNA-seq) technology. Our results showed that some basal biological processes like DNA replication and cell division are downregulated in the mature biofilm. Additionally, we identified significant expression reprogramming for genes involved in motility, sugar/lipid metabolism, and iron scavenging, as well as for outer membrane-encoding genes. A careful manual annotation process allowed us to assign molecular functions to many previously uncharacterized genes that are probably involved in biofilm metabolism. We also provided evidence for the presence of small regulatory RNAs in this species. Finally, coexpression networks were reconstructed to pinpoint functionally related gene clusters that may explain how biofilm maintenance is regulated. Beyond elucidating some genetic aspects of biofilm formation, this work reveals a number of pathways whose functional dissection may impact our understanding of leptospiral biology, in particular how these organisms adapt to environmental changes. IMPORTANCE In this work, we describe the first transcriptome based on RNA-seq technology focused on studying transcriptional changes associated with biofilm growth

  8. Household Characteristics Associated with Rodent Presence and Leptospira Infection in Rural and Urban Communities from Southern Chile

    PubMed Central

    Muñoz-Zanzi, Claudia; Mason, Meghan; Encina, Carolina; Gonzalez, Marcelo; Berg, Sergey

    2014-01-01

    Rodents are well-recognized reservoirs of Leptospira, contributing to its maintenance in endemic areas and playing a role in the public health risk associated with the infection. This study sought to provide some insights into rodent populations from Chile and their Leptospira carriage. In total, 393 rodents were trapped in 177 households. Higher rodent counts were associated with year 2 of the study, rainfall, and number of rodent signs. There was an inverse correlation with the number of cats. The number of rodents was higher in villages compared with slums (rate ratio = 3.23) but modified by average household age. Eighty rodents (20.4%) tested positive for Leptospira: 19.7% on the farms, 25.9% in villages, and 12.3% in the slums. Prevalence was 22.5% in Mus musculus, 20.7% in Rattus rattus, 21.1% in wild rodents, and 10.3% in R. norvegicus. Seasonal and temporal effects were the major determinants of Leptospira infection in rodent populations. PMID:24445209

  9. Plasminogen Binding Proteins and Plasmin Generation on the Surface of Leptospira spp.: The Contribution to the Bacteria-Host Interactions

    PubMed Central

    Vieira, Monica L.; Atzingen, Marina V.; Oliveira, Rosane; Mendes, Renata S.; Domingos, Renan F.; Vasconcellos, Silvio A.; Nascimento, Ana L. T. O.

    2012-01-01

    Leptospirosis is considered a neglected infectious disease of human and veterinary concern. Although extensive investigations on host-pathogen interactions have been pursued by several research groups, mechanisms of infection, invasion and persistence of pathogenic Leptospira spp. remain to be elucidated. We have reported the ability of leptospires to bind human plasminogen (PLG) and to generate enzimatically active plasmin (PLA) on the bacteria surface. PLA-coated Leptospira can degrade immobilized ECM molecules, an activity with implications in host tissue penetration. Moreover, we have identified and characterized several proteins that may act as PLG-binding receptors, each of them competent to generate active plasmin. The PLA activity associated to the outer surface of Leptospira could hamper the host immune attack by conferring the bacteria some benefit during infection. The PLA-coated leptospires obstruct complement C3b and IgG depositions on the bacterial surface, most probably through degradation. The decrease of leptospiral opsonization might be an important aspect of the immune evasion strategy. We believe that the presence of PLA on the leptospiral surface may (i) facilitate host tissue penetration, (ii) help the bacteria to evade the immune system and, as a consequence, (iii) permit Leptospira to reach secondary sites of infection. PMID:23118516

  10. Preliminary Characterization of Mus musculus–Derived Pathogenic Strains of Leptospira borgpetersenii Serogroup Ballum in a Hamster Model

    PubMed Central

    da Silva, Éverton F.; Félix, Samuel R.; Cerqueira, Gustavo M.; Fagundes, Michel Q.; Neto, Amilton C. P. S.; Grassmann, André A.; Amaral, Marta G.; Gallina, Tiago; Dellagostin, Odir A.

    2010-01-01

    Human and animal leptospirosis caused by Leptospira spp. belonging to serogroup Ballum has increased worldwide in the past decade. We report the isolation and serologic and molecular characterization of four L. borgpetersenii serogroup Ballum isolates obtained from Mus musculus, and preliminary virulence studies. These isolates are useful for diagnosis of leptospirosis and for epidemiologic studies of its virulence and pathogenic mechanisms. PMID:20682877

  11. A Leptospira borgpetersenii Serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic infection of cattle with Leptospira borgpetersenii serovar Hardjo reduces animal production through reproductive failure and presents a persistent health threat to workers in the animal industry. Cattle are maintenance hosts for serovar Hardjo and development of a protective vaccine has bee...

  12. Household characteristics associated with rodent presence and Leptospira infection in rural and urban communities from Southern Chile.

    PubMed

    Muñoz-Zanzi, Claudia; Mason, Meghan; Encina, Carolina; Gonzalez, Marcelo; Berg, Sergey

    2014-03-01

    Rodents are well-recognized reservoirs of Leptospira, contributing to its maintenance in endemic areas and playing a role in the public health risk associated with the infection. This study sought to provide some insights into rodent populations from Chile and their Leptospira carriage. In total, 393 rodents were trapped in 177 households. Higher rodent counts were associated with year 2 of the study, rainfall, and number of rodent signs. There was an inverse correlation with the number of cats. The number of rodents was higher in villages compared with slums (rate ratio = 3.23) but modified by average household age. Eighty rodents (20.4%) tested positive for Leptospira: 19.7% on the farms, 25.9% in villages, and 12.3% in the slums. Prevalence was 22.5% in Mus musculus, 20.7% in Rattus rattus, 21.1% in wild rodents, and 10.3% in R. norvegicus. Seasonal and temporal effects were the major determinants of Leptospira infection in rodent populations. PMID:24445209

  13. Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection of pathogenic Leptospira spp. in marine mammals is challenging: microbiological culture can take 3-6 months and has low sensitivity, immunohistochemical staining of kidney to detect leptospires is invasive and time consuming, and serological methods, such as the microscopic agglutina...

  14. Waterborne Leptospirosis: Survival and Preservation of the Virulence of Pathogenic Leptospira spp. in Fresh Water.

    PubMed

    Andre-Fontaine, Genevieve; Aviat, Florence; Thorin, Chantal

    2015-07-01

    Many studies have implicated fresh water as a source of leptospirosis outbreaks. To estimate the survival and the preservation of the virulence of pathogenic Leptospira spp. maintained in water, we selected five still waters with various pH and mineral profiles. The water samples were artificially inoculated with a culture of a pathogenic strain belonging to serovar Icterohaemorrhagiae. Samples were stored for 20 months at 4, 20 or 30 °C. The survival and preservation of virulence of this pathogenic strain was estimated by subculturing these stored samples. After 14 and 20 months of storage, the strain Icterohaemorrhagiae was re-isolated, and its virulence was determined using an animal model. In these waters, the mean survival was 130 days for storage at 4 °C, 263 days at 20 °C, and 316 days at 30 °C. Unexpectedly, the mean survival was 344 days for a final pH < 7 and 129 days for pH ≥ 7. Moreover, the pathogenic strain remained fully virulent and was able to induce a lethal disease in gerbils even when the pH of the contaminated waters decreased to <6. These data showed that despite unfavourable storage conditions such as cold, nutrient-poor acidic waters, the survival and virulence of pathogenic Leptospira spp. was fully preserved over at least 20 months. PMID:26003629

  15. Development of a Recombinase Polymerase Amplification Assay for the Detection of Pathogenic Leptospira

    PubMed Central

    Ahmed, Ahmed; van der Linden, Hans; Hartskeerl, Rudy A.

    2014-01-01

    Detection of leptospires based on DNA amplification techniques is essential for the early diagnosis of leptospirosis when anti-Leptospira antibodies are below the detection limit of most serological tests. In middle and low income countries where leptospirosis is endemic, routine implementation of real-time PCR is financially and technically challenging due to the requirement of expensive thermocycler equipment. In this study we report the development and evaluation of a novel isothermal recombinase polymerase amplification assay (RPA) for detection of pathogenic Leptospira based on TwistAmp chemistry. RPA enabled the detection of less than two genome copies per reaction. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity and specificity of 94.7% and 97.7%, respectively) compared to culturing as the reference standard. RPA presents a powerful tool for the early diagnosis of leptospirosis in humans and in animals. Furthermore, it enables the detection of the causative agent in reservoirs and environment, and as such is a valuable adjunct to current tools for surveillance and early outbreak warning. PMID:24814943

  16. Prevalence of Leptospira antibodies in wild boars (Sus scrofa) from Northern Portugal: risk factor analysis.

    PubMed

    Vale-Gonçalves, H M; Cabral, J A; Faria, M C; Nunes-Pereira, M; Faria, A S; Veloso, O; Vieira, M L; Paiva-Cardoso, Md N

    2015-07-01

    Leptospirosis is a zoonosis of worldwide distribution, caused by infection with pathogenic spirochaetes of the genus Leptospira. The wild boar (Sus scrofa), an important hunting species in Europe, seems to play a significant role in the epidemiological cycle of leptospirosis. A total of 101 serum samples from wild boar hunted in Northern Portugal were analysed for leptospiral antibodies detection by microscopic agglutination test. Sera were collected during hunting seasons (2011-2013) and tested with 17 different pathogenic serovars of Leptospira. Antibodies against nine serovars were detected in 66 (65·4%) of these sera. Serovars Tarassovi and Altodouro exhibited the highest seroreactivity rates (23·8% and 16·8%, respectively), followed by Autumnalis (7·9%) and Bratislava (6·9%). Age and district of origin were found to be risk factors for the presence of leptospiral antibodies in contrast to gender. From a One Health perspective, this study revealed that wild boar should be considered as a potential source of leptospirosis dissemination for humans and animal species (domestic and wild) in shared environments, particularly in the Trás-os-Montes region. PMID:25519057

  17. Antimicrobial susceptibility of Leptospira isolates from dogs and rats to 12 antimicrobial agents.

    PubMed

    Suepaul, S M; Carrington, C; Campbell, M; Borde, G; Adesiyun, A A

    2015-03-01

    This study determined the antimicrobial susceptibilities of 67 isolates of Leptospira from dogs (suspect canine cases: n=7 and stray dogs: n=6) and rodents (n=54) in Trinidad to 12 antimicrobial agents using broth microdilution and macrodilution techniques. Commonly used antimicrobial agents such as the penicillin G and ceftriaxone had relatively low minimal inhibitory concentrations (MICs) while doxycycline displayed a relatively higher value but was still considered to be effective. While imipenem was the most effective with low MIC values in vitro, sulphamethoxazole-trimethoprim had the highest i.e. least effective. Based on these results, the drugs commonly used in the treatment of leptospirosis (penicillin G, penicillin-streptomycin, doxycycline and ceftriaxone) in both humans and animals in Trinidad appear to have similar MICs and MBCs in vitro when compared with published reports. The serovar of Leptospira spp. and in most cases the origin of the isolates did not significantly (P>0.05) influence their susceptibilities to the antimicrobial agents tested. PMID:25801249

  18. Detection of wild animals as carriers of Leptospira by PCR in the Pantanal biome, Brazil.

    PubMed

    Vieira, Anahi S; Narduche, Lorena; Martins, Gabriel; Schabib Péres, Igor A H F; Zimmermann, Namor P; Juliano, Raquel S; Pellegrin, Aiesca O; Lilenbaum, Walter

    2016-11-01

    Leptospiral infection is widespread in wildlife. In this context, wild ecosystems in tropical countries hold a vast biodiversity, including several species that may act as potential reservoirs of leptospires. The Pantanal biome presents highly favorable environmental conditions for the occurrence of leptospirosis, such as high temperatures, constant flooding, and high biodiversity. The purpose of this study was to detect wild animals as carriers of Leptospira sp. using direct methods (PCR and culture) in the Pantanal biome, Brazil. A total of 35 animals were studied, namely Cerdocyon thous, Nasua nasua, Ozotoceros bezoarticus, and Sus scrofa species. Blood for serology (MAT) and urine for bacteriological culturing and PCR was sampled. The most prevalent serogroups were Javanica and Djasiman. Additionally, 40.6% of these animals presented PCR positive reactions. Seroreactivity associated with the high frequency of leptospiral carriers among the different studied species suggests a high level of exposure of the studied animals to pathogenic Leptospira strains. Our results are still limited and the actual role of the studied animals in the epidemiology of leptospirosis in the Pantanal region remains to be elucidated. PMID:27496621

  19. Development of a recombinase polymerase amplification assay for the detection of pathogenic Leptospira.

    PubMed

    Ahmed, Ahmed; van der Linden, Hans; Hartskeerl, Rudy A

    2014-05-01

    Detection of leptospires based on DNA amplification techniques is essential for the early diagnosis of leptospirosis when anti-Leptospira antibodies are below the detection limit of most serological tests. In middle and low income countries where leptospirosis is endemic, routine implementation of real-time PCR is financially and technically challenging due to the requirement of expensive thermocycler equipment. In this study we report the development and evaluation of a novel isothermal recombinase polymerase amplification assay (RPA) for detection of pathogenic Leptospira based on TwistAmp chemistry. RPA enabled the detection of less than two genome copies per reaction. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity and specificity of 94.7% and 97.7%, respectively) compared to culturing as the reference standard. RPA presents a powerful tool for the early diagnosis of leptospirosis in humans and in animals. Furthermore, it enables the detection of the causative agent in reservoirs and environment, and as such is a valuable adjunct to current tools for surveillance and early outbreak warning. PMID:24814943

  20. Serological investigation of Leptospira infection and its circulation in one intensive-type water buffalo farm in the Philippines.

    PubMed

    Villanueva, Marvin A; Mingala, Claro N; Gloriani, Nina G; Yanagihara, Yasutake; Isoda, Norikazu; Nakajima, Chie; Suzuki, Yasuhiko; Koizumi, Nobuo

    2016-02-01

    Water buffalo is an indispensable livestock in the Philippines. Leptospirosis is a serious zoonosis that can be fatal to humans and cause reproductive problems in livestock. Leptospirosis has been reported in some countries where water buffaloes are commercially raised, highlighting the Leptospira prevalence in this farming system, but information on leptospirosis in water buffalo farms in the Philippines is limited. In this study, we collected blood samples from rats (n = 21), and water buffaloes (n = 170) from different groups and locations in one intensive-type buffalo farm in the Philippines. Serum was analyzed by microscopic agglutination test (MAT). Anti-Leptospira antibodies reacting with serogroups Canicola, Icterohaemorrhagiae and Pomona were found in sera of 30% tested rats, and 48% of water buffalo sera tested positive for at least one Leptospira strain, in which serogroups Mini, Hebdomadis, Tarassovi and Pyrogenes were predominantly agglutinated. The number of seropositive young water buffaloes (< 1 year-old) was lower than that of older seropositive ones. Furthermore, sera from younger water buffaloes were reactive with single serotypes with low MAT titers, but older animals were reactive with multiple Leptospira strains with variable MAT titers. In addition, antibodies against serogroups Icterohaemorrhagiae and Pomona were detected in both animals. Finally, Leptospira infection was found associated with age and animal grouping, highlighting the impact of management in the persistence of leptospirosis at intensive-type buffalo farm settings in the Philippines. Further investigation and appropriate control strategies are required to prevent leptospirosis from causing risks to public health and economic losses to the water buffalo farming industry. PMID:27348885

  1. Letter to the Editor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We would like to express several concerns regarding the article “Efficacy of vaccination of cattle with the Leptospira interrogans serovar Hardjo type hardjoprajitno component of a pentavalent Leptospira bacterin against experimental challenge with Leptospira borgpetersenii serovar Hardjo type hardj...

  2. Identification of Tenrec ecaudatus, a Wild Mammal Introduced to Mayotte Island, as a Reservoir of the Newly Identified Human Pathogenic Leptospira mayottensis

    PubMed Central

    Lagadec, Erwan; Gomard, Yann; Le Minter, Gildas; Cordonin, Colette; Cardinale, Eric; Ramasindrazana, Beza; Dietrich, Muriel; Goodman, Steven M; Tortosa, Pablo; Dellagi, Koussay

    2016-01-01

    Leptospirosis is a bacterial zoonosis of major concern on tropical islands. Human populations on western Indian Ocean islands are strongly affected by the disease although each archipelago shows contrasting epidemiology. For instance, Mayotte, part of the Comoros Archipelago, differs from the other neighbouring islands by a high diversity of Leptospira species infecting humans that includes Leptospira mayottensis, a species thought to be unique to this island. Using bacterial culture, molecular detection and typing, the present study explored the wild and domestic local mammalian fauna for renal carriage of leptospires and addressed the genetic relationships of the infecting strains with local isolates obtained from acute human cases and with Leptospira strains hosted by mammal species endemic to nearby Madagascar. Tenrec (Tenrec ecaudatus, Family Tenrecidae), a terrestrial mammal introduced from Madagascar, is identified as a reservoir of L. mayottensis. All isolated L. mayottensis sequence types form a monophyletic clade that includes Leptospira strains infecting humans and tenrecs on Mayotte, as well as two other Malagasy endemic tenrecid species of the genus Microgale. The lower diversity of L. mayottensis in tenrecs from Mayotte, compared to that occurring in Madagascar, suggests that L. mayottensis has indeed a Malagasy origin. This study also showed that introduced rats (Rattus rattus) and dogs are probably the main reservoirs of Leptospira borgpetersenii and Leptospira kirschneri, both bacteria being prevalent in local clinical cases. Data emphasize the epidemiological link between the two neighbouring islands and the role of introduced small mammals in shaping the local epidemiology of leptospirosis. PMID:27574792

  3. Identification of Tenrec ecaudatus, a Wild Mammal Introduced to Mayotte Island, as a Reservoir of the Newly Identified Human Pathogenic Leptospira mayottensis.

    PubMed

    Lagadec, Erwan; Gomard, Yann; Le Minter, Gildas; Cordonin, Colette; Cardinale, Eric; Ramasindrazana, Beza; Dietrich, Muriel; Goodman, Steven M; Tortosa, Pablo; Dellagi, Koussay

    2016-08-01

    Leptospirosis is a bacterial zoonosis of major concern on tropical islands. Human populations on western Indian Ocean islands are strongly affected by the disease although each archipelago shows contrasting epidemiology. For instance, Mayotte, part of the Comoros Archipelago, differs from the other neighbouring islands by a high diversity of Leptospira species infecting humans that includes Leptospira mayottensis, a species thought to be unique to this island. Using bacterial culture, molecular detection and typing, the present study explored the wild and domestic local mammalian fauna for renal carriage of leptospires and addressed the genetic relationships of the infecting strains with local isolates obtained from acute human cases and with Leptospira strains hosted by mammal species endemic to nearby Madagascar. Tenrec (Tenrec ecaudatus, Family Tenrecidae), a terrestrial mammal introduced from Madagascar, is identified as a reservoir of L. mayottensis. All isolated L. mayottensis sequence types form a monophyletic clade that includes Leptospira strains infecting humans and tenrecs on Mayotte, as well as two other Malagasy endemic tenrecid species of the genus Microgale. The lower diversity of L. mayottensis in tenrecs from Mayotte, compared to that occurring in Madagascar, suggests that L. mayottensis has indeed a Malagasy origin. This study also showed that introduced rats (Rattus rattus) and dogs are probably the main reservoirs of Leptospira borgpetersenii and Leptospira kirschneri, both bacteria being prevalent in local clinical cases. Data emphasize the epidemiological link between the two neighbouring islands and the role of introduced small mammals in shaping the local epidemiology of leptospirosis. PMID:27574792

  4. Circulating serovars of Leptospira in cart horses of central and southern Ethiopia and associated risk factors.

    PubMed

    Tsegay, K; Potts, A D; Aklilu, N; Lötter, C; Gummow, B

    2016-03-01

    Little work has been done on diseases of horses in Ethiopia or tropical regions of the world. Yet, Ethiopia has the largest horse population in Africa and their horses play a pivotal role in their economy as traction animals. A serological and questionnaire survey was therefore conducted to determine the circulating serovars of Leptospira and their association with potential risk factors in the cart horse population of Central and Southern Ethiopia. A total of 184 out of 418 cart horses from 13 districts had antibody titres of 1:100 or greater to at least one of 16 serovars of Leptospira species in Central and Southern Ethiopian horses. A significantly higher seropositivity (62.1%) was noted in horses from the highland agroecology followed by midland (44.4%) and lowland (39.8%). Serovar Bratislava (34.5%) was the predominant serovar followed by serovars Djasiman (9.8%), Topaz (5.98%) and Pomona (5.3%). Age and location proved to be associated with seropositive horses with older horses being more commonly affected and the districts of Ziway (Batu) (Apparent Prevalence (AP)=65.5%), Shashemene (AP=48.3%) and Sebeta (AP=41.4%) having the highest prevalence. Multivariable logistic regression found risk factors significantly associated with Leptospira seropositive horses were drinking river water (OR=2.8) and horses 7-12 years old (OR=5) and risk factors specifically associated with serovar Bratislava seropositive horses were drinking river water (OR=2.5), horses ≥13 years (OR=3.5) and the presence of dogs in adjacent neighbouring properties (OR=0.3). Dogs had a protective effect against seropositivity to serovars Bratislava and Djasiman, which may be due to their ability to control rodents. The high seroprevalence confirm that leptospirosis is endemic among horses of Central and Southern Ethiopia. The predominance of serovar Bratislava supports the idea that serovar Bratislava may be adapted to and maintained by the horse population of Central and Southern Ethiopia

  5. Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing

    PubMed Central

    Waggoner, Jesse J.; Balassiano, Ilana; Mohamed-Hadley, Alisha; Vital-Brazil, Juliana Magalhães; Sahoo, Malaya K.; Pinsky, Benjamin A.

    2015-01-01

    Background Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens. Methods/Principal Findings 478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%). Conclusions/Significance This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize

  6. Leptospira spp. in Domestic Cats from Different Environments: Prevalence of Antibodies and Risk Factors Associated with the Seropositivity

    PubMed Central

    Azócar-Aedo, Lucía; Monti, Gustavo; Jara, Ronald

    2014-01-01

    Simple Summary Although Leptospira infection occurs in domestic cat populations, studies on leptospirosis are very limited in felines and the role of cats in the epidemiology of this zoonosis has not received much attention. The present work is an epidemiologic study intended to determine the prevalence of anti-Leptospira antibodies and risk factors related with the seropositivity in cats from urban and rural environments. A higher prevalence in rural cats was detected (25.2%) compared with urban animals (1.8%). Characteristics of the habitat of the animals and some agricultural activities performed by cat’s owners were found to be risk factors associated with the seropositivity. Abstract Leptospirosis is an emerging zoonotic disease of worldwide distribution. A cross-sectional study was conducted in urban and rural environments in southern Chile (1) to detect domestic cats with serologic evidence of exposure to Leptospira spp.; (2) to determine the prevalence of anti-Leptospira antibodies; (3) to describe seroprevalences according to different characteristics of the animals, and (4) to identify risk factors associated with the seropositivity in the Microscopic Agglutination Test (MAT). Blood samples were taken from 124 owned cats. A frequentist and Bayesian approach were applied for prevalence estimation. The overall apparent prevalence of anti-Leptospira antibodies was 8.1% (95% Confident Interval = 3.9–4.3). With the Bayesian approach, the overall True Prevalence (TP) was 5.2% (95% Credibility Interval (CrI) = 0.6–12.4). The TP for urban cats was 1.8% (95% CrI = 0.1–7.2) and the TP for rural felines was 25.2% (95% CrI = 9.3–46.6). Cats that live in a place where agricultural activities are performed with water that flows in streams or backwater and cats that live in places near flooded areas had a higher risk of seropositivity in MAT. The exposure to Leptospira spp. in domestic cats of urban and rural origin in Southern Chile is a public health concern

  7. Interlaboratory Agreement of Pulsed-Field Gel Electrophoresis Identification of Leptospira Serovars

    PubMed Central

    Mende, Katrin; Galloway, Renee L.; Becker, Sara J.; Beckius, Miriam L.; Murray, Clinton K.; Hospenthal, Duane R.

    2013-01-01

    Leptospirosis may be caused by > 250 Leptospira serovars. Serovar classification is a complex task that most laboratories cannot perform. We assessed the interlaboratory reproducibility of a pulsed-field gel electrophoresis (PFGE) identification technique developed by the Centers for Disease Control and Prevention (CDC). Blinded exchange of 93 Leptospiraceae strains occurred between San Antonio Military Medical Center (SAMMC) and the CDC. PFGE was performed and gel images were analyzed and compared with patterns present in each laboratory's database (CDC database: > 800 strain patterns; SAMMC database: > 300 strain patterns). Overall, 93.7% (74 of 79) of strains present in each receiving laboratory's database were correctly identified. Five isolates were misidentified, and two isolates did not match serovar PFGE patterns in the receiving laboratory's database. Patterns for these seven isolates were identical between laboratories; four serovars represented misidentified reference strains. The PFGE methodology studied showed excellent interlaboratory reproducibility, enabling standardization and data sharing between laboratories. PMID:23817329

  8. A multilocus variable number tandem repeat analysis assay provides high discrimination for genotyping Leptospira santarosai strains

    PubMed Central

    Hamond, Camila; Pinna, Melissa; Medeiros, Marco Alberto; Bourhy, Pascale; Lilenbaum, Walter

    2015-01-01

    Considering the prevalence of Leptospira santarosai infections in the Americas and the scarce information about the species, we aimed to apply a multilocus variable number tandem repeat (VNTR) analysis (MLVA) for the molecular typing of L. santarosai isolates from various sources. Amplification of three VNTR loci selected from L. santarosai genome sequences resulted in a wide range of sizes for the amplified products amongst the 21 L. santarosai strains analysed. This suggested a variation in tandem repeat copy numbers in the VNTR loci. secY sequencing also showed a high nucleotide diversity, confirming the MLVA data. In conclusion, this novel MLVA provided a high level of discrimination between L. santarosai isolates, and this new typing tool could be used to investigate leptospirosis in regions where L. santarosai predominates. PMID:25721051

  9. HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

    PubMed Central

    Noda, Angel Alberto; Rodríguez, Islay; Rodríguez, Yaindrys; Govín, Anamays; Fernández, Carmen; Obregón, Ana Margarita

    2014-01-01

    This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries. PMID:25229221

  10. Leptospira borgpetersenii serovar hardjo type hardjobovis in bovine embryos fertilized in vitro.

    PubMed Central

    Bielanski, A B; Surujballi, O

    1998-01-01

    The association of Leptospira borgpetersenii serovar hardjo type hardjobovis with bovine embryos produced by in vitro fertilization was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Morula stage embryos with an intact zona pellucida (ZP) were exposed to this spirochete for 24 h in culture medium, washed by the standard washing procedure as recommended by the International Embryo Transfer Society, and then examined. SEM showed typical helicoid leptospires on the surface and in the pores of the ZP. TEM showed cross and longitudinal sections of leptospires in the matrix and channels of the ZP, in the perivitelline and intercellular spaces, on the vitellus and in the embryonic cells. Some of the embryos that were penetrated showed damage to the membranes and the cytoplasm. The ineffectiveness of the washing procedure, for the removal of hardjobovis from exposed embryos may be of importance to the industry. Images Figure 1. Figure 2. Figure 3. Figure 4. PMID:9684055

  11. Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement.

    PubMed

    Vital-Brazil, Juliana Magalhães; Balassiano, Ilana Teruszkin; Oliveira, Fabiano Sutter de; Costa, Alberto Dias de Souza; Hillen, Leandro; Pereira, Martha Maria

    2010-05-01

    The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies. PMID:20512254

  12. Anti-Leptospira spp. antibodies in Crotalus durissus collilineatus kept in captivity and its zoonotic relevance.

    PubMed

    Rodrigues, T C S; Santos, A L Q; Lima, A M C; Gomes, D O; Brites, V L C

    2016-06-01

    Leptospirosis is a worldwide spread zoonosis that can affect all groups of vertebrates, including reptiles. Because it has been little studied in snakes, this study focused on determining the occurrence of anti-Leptospira spp. antibodies in 64 Crotalus durissus collilineatus kept in captivity and on identifying the most common serovars in these animals, using the microscopic agglutination test. Of these, almost 90% were positive and there were reactions to the 22 serovars used in the study. The most common serovar in these snakes was Javanica, Andamana and Patoc. Most frequent titers were 25 and 50, although high titers (such as 1600) were also recorded, despite the absence of clinical symptoms. The possibility should be considered of captive snakes serving as a serious source of leptospiral infection in humans, which is why it is essential to study, prevent and control the disease in breeding centers and serpentariums. PMID:26875762

  13. Aseptic meningitis caused by Leptospira spp diagnosed by polymerase chain reaction.

    PubMed

    Romero, Eliete Caló; Blanco, Roberta Morozetti; Yasuda, Paulo Hideki

    2010-12-01

    Leptospirosis is a zoonotic disease caused by the pathogenic Leptospira spp. The clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. The neurological leptospirosis forms are usually neglected. The aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (PCR). Thirty-nine cerebrospinal fluid (CSF) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and PCR. Leptospira spp DNA was detected in 23 (58.97%) of the CSF samples. Anti-leptospiral antibodies were found in 13 (33.33%) CSF samples. Twelve CSF samples were positive by PCR assay and negative by microscopic agglutination test (MAT) assay. Two CSF samples were positive by MAT and negative by PCR. The positive and negative agreement between both tests was 11 and 14, respectively. CSF samples from six cases of unknown diagnosis were positive by PCR assay. Eight cases showed positive results using PCR and MAT. Leptospirosis could be detected by PCR assay from the 3rd-26th day after illness onset. The sensitivity of the PCR was assessed with confirmed cases of leptospirosis (by MAT) and found to be 89.5%. All CSFs were negative by culture. PCR was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. We recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive. PMID:21225195

  14. Detection of Leptospira spp. in wildlife reservoir hosts in Ontario through comparison of immunohistochemical and polymerase chain reaction genotyping methods.

    PubMed

    Shearer, Karen E; Harte, Michael J; Ojkic, Davor; Delay, Josepha; Campbell, Douglas

    2014-03-01

    A total of 460 kidney samples from wildlife (beavers, coyotes, deer, foxes, opossums, otters, raccoons, skunks) were obtained from road-kill and hunter/trapper donations in Ontario between January 2010 and November 2012. The objectives of the study were to detect Leptospira spp. by immunohistochemistry and polymerase chain reaction (PCR), to map presence of leptospires in wildlife relative to livestock and human populations, and to characterize positive samples by sequencing and comparison to leptospires known to affect domestic animals and humans. The proportion of samples that tested positive ranged from 0% to 42%, with the highest rates in skunks and raccoons. Leptospira spp. were present in kidneys of wildlife across Ontario, particularly in areas of high human density, and areas in which livestock populations are abundant. The PCR was too weak in most samples to permit genotyping and examination of the relationship between the leptospires found in this study and those affecting domestic animals and humans. PMID:24587507

  15. Product-specific validation of a serological potency test for release of Leptospira vaccines in the European Union.

    PubMed

    Stirling, Catrina; Novokova, Viera

    2013-09-01

    Historically in the European Union, all Leptospira vaccines were released using the European Pharmacopoeia (Ph. Eur.) hamster potency assay. Recently, there has been a shift toward alternatives that offer either refinement of testing or replacement of animals for product release. This is being driven by animal welfare concerns but also by a drive to have more consistent, cheaper, and faster batch release tests. This publication discusses one such example of a multicomponent canine vaccine that includes three Leptospira serovars and has recently been registered in the European Union. The potency release test is a refinement because it uses rabbit serology rather than hamster challenge. This publication covers the principles of the test method, challenges faced during its development and registration, and discussion about benefits and limitations of this method. It concludes with a view of how the use of serology testing could fit into an overall strategy to move to fully in vitro testing by adopting a consistency approach. PMID:23849308

  16. PATHOGENIC LEPTOSPIRA SEROVARS IN FREE-LIVING SEA LIONS IN THE GULF OF CALIFORNIA AND ALONG THE BAJA CALIFORNIA COAST OF MEXICO.

    PubMed

    Avalos-Téllez, Rosalía; Carrillo-Casas, Erika M; Atilano-López, Daniel; Godínez-Reyes, Carlos R; Díaz-Aparicio, Efrén; Ramírez-Delgado, David; Ramírez-Echenique, María F; Leyva-Leyva, Margarita; Suzán, Gerardo; Suárez-Güemes, Francisco

    2016-04-28

    The California sea lion ( Zalophus californianus ), a permanent inhabitant of the Gulf of California in Mexico, is susceptible to pathogenic Leptospira spp. infection, which can result in hepatic and renal damage and may lead to renal failure and death. During summer 2013, we used the microscopic agglutination test (MAT) to investigate the prevalence of anti-Leptospira antibodies in blood of clinically healthy sea lion pups from seven rookery islands on the Pacific Coast of Baja California (Pacific Ocean) and in the Gulf of California. We also used PCR to examine blood for Leptospira DNA. Isolation of Leptospira in liquid media was unsuccessful. We found higher antibody prevalence in sea lions from the rookery islands in the gulf than in those from the Pacific Coast. Antibodies against 11 serovars were identified in the Gulf of California population; the most frequent reactions were against serovars Bataviae (90%), Pyrogenes (86%), Wolffi (86%), Celledoni (71%), and Pomona (65%). In the Pacific Ocean population, MAT was positive against eight serovars, where Wolffi (88%), Pomona (75%), and Bataviae (70%) were the most frequent. Serum samples agglutinated with more than one Leptospira serovar. The maximum titer was 3,200. Each island had a different serology profile, and islands combined showed a distinct profile for each region. We detected pathogenic Leptospira DNA in 63% of blood samples, but we found no saprophytic Leptospira. Positive PCR results were obtained in blood samples with high and low MAT titers. Together, these two methods enhance the diagnosis and interpretation of sea lion leptospirosis. Our results may be related to human activities or the presence of other reservoirs with which sea lions interact, and they may also be related to sea lion stranding. PMID:26967136

  17. Molecular Characterization and Serology of Leptospira kirschneri (Serogroup Grippotyphosa) Isolated from Urine of a Mare Post-Abortion in Brazil.

    PubMed

    Hamond, C; Martins, G; Bremont, S; Medeiros, M A; Bourhy, P; Lilenbaum, W

    2016-05-01

    A strain of Leptospira kirschneri (serogroup Grippotyphosa) was cultured from urine of a mare post-abortion in Brazil and characterized by serogrouping, multiple-locus variable-number tandem repeat analysis, PGFE, and sequencing of genes rrs and secY. Strains of L. kirschneri have apparently never been recovered from horses in tropical area, only in Europe and USA. Knowledge of local epidemiology is important to interpret genetic profiles of leptospires circulating in an area. PMID:26355500

  18. Detection of antibodies against Brucella abortus, Leptospira spp., and Apicomplexa protozoa in water buffaloes in the Northeast of Argentina.

    PubMed

    Konrad, José L; Campero, Lucía M; Caspe, Gastón S; Brihuega, Bibiana; Draghi, Graciela; Moore, Dadin P; Crudeli, Gustavo A; Venturini, María C; Campero, Carlos M

    2013-11-01

    Water buffalo industry has become a profitable activity worldwide, including the Northeast of Argentina (NEA). However, research on diseases affecting this species is scarce. The aim of the present study was to detect antibodies against Brucella abortus, Leptospira spp., Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. in 500 water buffalo cows from five ranches (100 animals each) in the NEA. Serum samples were tested for B. abortus by fluorescence polarization assay, Leptospira spp. by microagglutination test, and N. caninum, T. gondii, and Sarcocystis spp. by indirect fluorescent antibody tests. Overall, the proportion of seropositive animals was 6.4, 22.2, 42.2, 25.4, and 50.8 % for brucellosis, leptospirosis, neosporosis, toxoplasmosis, and sarcocystosis, respectively. The proportion of seropositive animals for all diseases was statistically different among herds (p < 0.05). Statistical differences were also detected among age groups for brucellosis and neosporosis (p < 0.05). The detection of specific antibodies to B. abortus, Leptospira spp., and several Apicomplexa protozoans in water buffaloes in the NEA is reported in this study. PMID:23765549

  19. Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

    PubMed Central

    Waggoner, Jesse J.; Balassiano, Ilana; Abeynayake, Janaki; Sahoo, Malaya K.; Mohamed-Hadley, Alisha; Liu, Yuanyuan; Vital-Brazil, Juliana Magalhães; Pinsky, Benjamin A.

    2014-01-01

    Background Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species. Methodology/Principal Findings For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. Conclusions/Significance The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests. PMID:25379890

  20. Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials

    PubMed Central

    Ahmed, Ahmed; Engelberts, Mirjam F. M.; Boer, Kimberly R.; Ahmed, Niyaz; Hartskeerl, Rudy A.

    2009-01-01

    Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the

  1. DETECTION OF Leptospira spp. AND Brucella abortus ANTIBODIES IN FREE-LIVING JAGUARS (Panthera onca) IN TWO PROTECTED AREAS OF NORTHERN PANTANAL, BRAZIL

    PubMed Central

    ONUMA, Selma Samiko Miyazaki; KANTEK, Daniel Luis Zanella; CRAWSHAW, Peter Gransden; MORATO, Ronaldo Gonçalves; MAY-JÚNIOR, Joares Adenilson; de MORAIS, Zenaide Maria; FERREIRA, José Soares; de AGUIAR, Daniel Moura

    2015-01-01

     This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection. PMID:25923900

  2. Seroprevalence of Leptospira hardjo in cattle and African buffalos in southwestern Uganda.

    PubMed

    Atherstone, Christine; Picozzi, Kim; Kalema-Zikusoka, Gladys

    2014-02-01

    Leptospirosis, caused by the spirochete bacterium Leptospira spp. is a zoonosis, distributed worldwide and classified as an emerging infectious disease. Fatal outcomes to leptospiral infection do occur and the disease can cause abortion and other reproductive problems in cattle, goats, and pigs. In humans the symptoms range from subclinical infection to acute febrile illness, pulmonary hemorrhage and renal failure. Leptospirosis has never been officially reported to the World Health Organization (WHO) or the World Animal Health Organization in animals or humans in Uganda. However, favorable ecological conditions and suitable animal hosts can be found within the country. A commercially available enzyme-linked immunosorbent (ELISA) kit was used to screen sera samples from domesticated cattle and African buffalo (Syncerus caffer) at two locations in southwestern Uganda, collected over a 4-year period. Positive samples were found in both cattle and African buffalo samples, from both locations and across the sampling period. Overall seroprevalence was 42.39% in African buffalo and 29.35% in cattle. PMID:24323512

  3. Pathogenic Leptospira Species Acquire Factor H and Vitronectin via the Surface Protein LcpA

    PubMed Central

    da Silva, Ludmila Bezerra; Miragaia, Lidia dos Santos; Breda, Leandro Carvalho Dantas; Abe, Cecilia Mari; Schmidt, Mariana Costa Braga; Moro, Ana Maria; Monaris, Denize; Conde, Jonas Nascimento; Józsi, Mihály; Isaac, Lourdes; Abreu, Patrícia Antônia Estima

    2014-01-01

    Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn2+-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion. PMID:25534939

  4. Variables Associated with Infections of Cattle by Brucella abortus., Leptospira spp. and Neospora spp. in Amazon Region in Brazil.

    PubMed

    Chiebao, D P; Valadas, S Y O B; Minervino, A H H; Castro, V; Romaldini, A H C N; Calhau, A S; De Souza, R A B; Gennari, S M; Keid, L B; Soares, R M

    2015-10-01

    The frequency of Neospora spp., Leptospira spp. and Brucella abortus infections in adult cattle was determined in herds of the State of Pará, Brazil, which is an important region for cattle production located in the Amazon region. A total of 3466 adult female cattle from 176 herds were tested, leading to a frequency of seropositive animals of 14.7%, 3.7% and 65.5% and a herd positivity of 87.4%, 41.3% and 98.8% for infections caused by Neospora spp., B. abortus and Leptospira spp., respectively. The five most frequently diagnosed serologic responses to Leptospira spp. were those against serovars hardjo, wolfii, grippotyphosa, hebdomadis and shermani. The following associations were found: practice of artificial insemination, large farm size, large herd size, large number of dogs and high number of total abortions per year with the presence of antibodies against serovar hardjo; positive results to serovar grippotyphosa with the presence of dogs; inappropriate disposal of aborted foetuses with positivity to serovar hebdomadis. Serovar grippotyphosa was also associated with number of episodes of abortions. Neospora spp. positive herds were associated with episodes of abortion and B. abortus infection with the disposal of dead animals and aborted foetuses on pastures and with the use of artificial insemination. In conclusion, the high frequency of brucellosis, leptospirosis and neosporosis in the region may be a consequence of social, natural and raising conditions as: (i) climate conditions that favour the survival and spread of pathogens in the environment; (ii) farms located in regions bordering forest areas; (iii) farms in areas of difficult access to the veterinary service; (iv) extensive beef herds raised at pastures with different age and productive groups inter-mingled; and (v) minimal concerns regarding hygiene practices and disease prevention measures. PMID:26302373

  5. Report on the international workshop on alternative methods for Leptospira vaccine potency testing: state of the science and the way forward.

    PubMed

    Stokes, William; Srinivas, Geetha; McFarland, Richard; Kulpa-Eddy, Jodie; Casey, Warren; Walker, Angela; Draayer, Hans; Sebring, Randy; Brown, Karen; Balks, Elisabeth; Stirling, Catrina; Klaasen, Eric; Hill, Richard; Rippke, Byron; Ruby, Kevin; Alt, David; Mukhopadhyay, Suman; Kojima, Hajime; Johnson, Nelson; Rinckel, Lori; Doelling, Vivian; Jones, Brett

    2013-09-01

    Routine potency testing of Leptospira vaccines is mostly conducted using a vaccination-challenge test that involves large numbers of hamsters and unrelieved pain and distress. NICEATM, ICCVAM, and their international partners organized a workshop to review the state of the science of alternative methods that might replace, reduce, and refine the use of animals for veterinary Leptospira vaccine potency testing and to identify ways to advance improved alternative methods. Vaccine manufacturers were encouraged to initiate or continue product-specific validation using in vitro enzyme-linked immunosorbent assays as replacements for potency testing of four common Leptospira serogroups. Participants discussed the potential for eliminating the back-titration procedure in the hamster challenge assay, which could reduce animal use by 50% for each individual potency test. Further animal reduction may also be possible by using cryopreserved Leptospira stock to replace continual passaging through hamsters. Serology assays were identified as a way to further reduce and refine animal use but should be considered only after attempting in vitro assays. Workshop participants encouraged consideration of analgesics and use of earlier humane endpoints when the hamster vaccination-challenge potency assay is used. International harmonization of alternative potency methods was recommended to avoid duplicative potency testing to meet regionally different requirements. PMID:23890729

  6. Detection of Pathogenic Leptospira Bacteria in Pinniped Populations via PCR and Identification of a Source of Transmission for Zoonotic Leptospirosis in the Marine Environment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospirosis, caused by the spirochete bacterium Leptospira, is a geographically widespread disease that affects a broad range of mammals, including marine mammals. During 2004 an outbreak of leptospirosis occurred among select pinniped populations along the West Coast of North America, with cases...

  7. Serosurvey of Smooth Brucella, Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil.

    PubMed

    Furtado, Mariana Malzoni; Gennari, Solange Maria; Ikuta, Cassia Yumi; Jácomo, Anah Tereza de Almeida; de Morais, Zenaide Maria; Pena, Hilda Fátima de Jesus; Porfírio, Grasiela Edith de Oliveira; Silveira, Leandro; Sollmann, Rahel; de Souza, Gisele Oliveira; Tôrres, Natália Mundim; Ferreira Neto, José Soares

    2015-01-01

    This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature. PMID:26605787

  8. Serosurvey of Smooth Brucella, Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil

    PubMed Central

    Furtado, Mariana Malzoni; Gennari, Solange Maria; Ikuta, Cassia Yumi; Jácomo, Anah Tereza de Almeida; de Morais, Zenaide Maria; Pena, Hilda Fátima de Jesus; Porfírio, Grasiela Edith de Oliveira; Silveira, Leandro; Sollmann, Rahel; de Souza, Gisele Oliveira; Tôrres, Natália Mundim; Ferreira Neto, José Soares

    2015-01-01

    This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature. PMID:26605787

  9. Detection of leptospiral antigen (L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae) by immunoelectron microscopy in the liver and kidney of experimentally infected guinea-pigs.

    PubMed Central

    De Brito, T.; Prado, M. J.; Negreiros, V. A.; Nicastri, A. L.; Sakata, E. E.; Yasuda, P. H.; Santos, R. T.; Alves, V. A.

    1992-01-01

    Guinea-pigs were experimentally infected with L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae and their liver and kidney were studied by immunoelectron microscopy using the post embedding indirect immunogold labelling technique. Primary antibody was a purified rabbit anti-serum produced against the same leptospiral strain used in the inoculum. Gold-labelled leptospiral antigen (LAg) was found close to cell membranes of hepatocytes, kidney tubular cells and endothelial cells of the interstitial capillaries of the kidney. Afterwards it was internalized by hepatic and tubular cells, and eventually found in lysosomes. Phagolysosomes of Kupffer cells were also found to contain remnants of degraded leptospires and gold-labelled LAg. Gold-labelled intact leptospires were detected at the enlarged intercellular spaces between hepatocytes at the areas of hepatic cell plate disarray, showing the potential for leptospiral migration during the septicaemic phase of the disease potentially contributing to the pathogenesis of the lesions. The affinity of leptospiral antigenic material for cell membranes suggests an initial interaction with cell surface proteins followed by its internalization and cell damage. The nature of antigenic material detected, however, remains undefined; it may be a toxin, an enzyme or any other factor/s involved in leptospiral virulence. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:1419779

  10. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira.

    PubMed

    Nurul Najian, A B; Engku Nur Syafirah, E A R; Ismail, Nabilah; Mohamed, Maizan; Yean, Chan Yean

    2016-01-15

    In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device. PMID:26709307

  11. Application of monoclonal antibodies in a rapid sandwich dot-enzyme linked immunosorbent assay for identification and antigen detection of Leptospira serovars.

    PubMed

    Sharma, Rashmi; Tuteja, Urmil; Khushiramani, Rekha; Shukla, Jyoti; Batra, H V

    2008-04-01

    Monoclonal antibodies (MAbs) were produced by fusing SP2/0 myeloma cells with spleen cells of BALB/c mice that were immunized with live whole cells of the four most prevalent Leptospira serovars--namely, autumnalis, australis, grippotyphosa, and icterohaemorrhagiae. A total of 26 MAbs (10 autumnalis, 5 australis, 4 grippotyphosa, and 7 icterohaemorrhagiae) were produced that showed specific, restricted, or broad cross-reactivity when tested with 19 standard pathogenic and 3 standard saprophytic serovars by MAT and dot-ELISA. Monoclonal antibodies like AT4 and AT5 against serovar autumnalis; AS1 and AS2 against serovar australis; GR1, GR3, and GR4 raised against serovar grippotyphosa; and also the MAbs IC3 to IC7 against serovar icterohaemorrhagiae were all usable as typing reagents in a rapid dot-ELISA. Selected MAbs were subsequently utilized in a rapid sandwich dot-ELISA for identification of Leptospira serovars as well as for antigen detection in experimentally infected mice and guinea pigs. Results of rapid sandwich dot-ELISA were compared with dark field microscopy, culture, and PCR in experimentally infected animals and sandwich dot-ELISA detected the presence of Leptospira antigen during the bacteremia stage in all experimental animals. Besides detecting antigens in animals infected with homologous serovars, the sandwich dot-ELISA employing pooled capture and revealing antibodies also detected Leptospira in the group of animals infected separately with the serovars australis and icterohaemorrhagiae. Results showed PCR to be a reliable and rapid test for demonstration of Leptospira in the plasma samples. The rapid sandwich dot-ELISA appeared more advantageous over PCR in being simple, rapid, field based, and economical. This method shows better promise of being used as a bedside test for routine diagnostic purposes. PMID:18642676

  12. PCR and Culture Identification of Pathogenic Leptospira spp. from Coastal Soil in Leyte, Philippines, after a Storm Surge during Super Typhoon Haiyan (Yolanda)

    PubMed Central

    Miyahara, Satoshi; Villanueva, Sharon Y. A. M.; Aramaki, Natsumi; Ikejiri, Mami; Kobayashi, Yoshie; Guevarra, Jonathan P.; Masuzawa, Toshiyuki; Gloriani, Nina G.; Yanagihara, Yasutake; Yoshida, Shin-ichi

    2014-01-01

    Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where Leptospira spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthof's medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic Leptospira species was detected in 11 samples (47.8%) by analysis of flaB by nested PCR. Eventually, two pathogenic Leptospira strains were isolated and showed the highest 16S rRNA gene sequence similarity to Leptospira kmetyi. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area. PMID:25172869

  13. Evaluation of three 5' exonuclease-based real-time polymerase chain reaction assays for detection of pathogenic Leptospira species in canine urine.

    PubMed

    Fink, Jamie M; Moore, George E; Landau, Ruth; Vemulapalli, Ramesh

    2015-03-01

    Leptospirosis is caused by several pathogenic Leptospira species, and is an important infectious disease of dogs. Early detection of infection is crucial for an effective antibiotic treatment of the disease. Though different polymerase chain reaction (PCR) assays have been developed for detection of pathogenic Leptospira spp., thorough evaluation of the performance of these assays using dog urine samples has not been carried out. In the current study, the performance of 3 real-time PCR (qPCR) assays was assessed, 1 targeting the 16S ribosomal RNA (rRNA) gene and the other 2 targeting the lipL32 gene, a gene for the LipL32 outer membrane protein. With DNA extracted from laboratory-cultured pathogenic Leptospira spp., all 3 qPCR assays showed 100% specificity and had identical lower limits of detection. Compared to a conventional, gel-based PCR assay, all 3 qPCR assays were 100-fold more sensitive. There was a 100% agreement in the results of the 3 assays when tested on urine samples collected aseptically from 30 dogs suspected for leptospirosis. However, when tested on 30 urine samples that were collected by the free-catch method, the 16S rRNA-based assay falsely detected 13.3% of the samples as positive for pathogenic Leptospira spp. Nucleotide sequence analysis of the amplified DNA fragments showed that the assay resulted in false positives because of unrelated bacteria. All urine samples collected from 100 apparently healthy dogs at a local animal shelter tested negative for pathogenic Leptospira spp. These results highlight the importance of sample-specific validation of PCR-based diagnostic assays and the application of appropriately validated assays for more reliable pathogen detection. PMID:25776541

  14. The adaptor molecule Trif contributes to murine host defense during Leptospiral infection.

    PubMed

    Jayaraman, Priya A; Devlin, Amy A; Miller, Jennifer C; Scholle, Frank

    2016-09-01

    Leptospirosis is a zoonotic disease and is caused by pathogenic species of the Leptospira genus, including Leptospira interrogans (L. interrogans). Humans, domestic and wild animals are susceptible to acute or chronic infection. The innate immune response is a critical defense mechanism against Leptospira interrogans, and has been investigated in mouse models. Murine Toll-like receptors (TLRs) have been shown to be key factors in sensing and responding to L. interrogans infection. Specifically, TLR2, TLR4 and the TLR adaptor molecule MyD88 are essential for host defense against L. interrogans; however, the role of the TLR adaptor molecule TIR-domain-containing adaptor-inducing interferon β (TRIF) in the response to L. interrogans has not been previously determined. In the present study, TRIF was found to play an important role during leptospiral infection. Following challenge with L. interrogans, Trif(-/-) mice exhibited delayed weight gain compared to wild-type mice. Moreover, Trif(-/-) mice exhibited an increase in L. interrogans burden in the kidneys, lungs, and blood at early time points (less than 7days post infection). Multiple components of the innate immune responses were dampened in response to leptospiral infection including transcription and production of cytokines, and the humoral response, which suggested that TRIF contributes to expression and production of cytokines important for the host defense against L. interrogans. PMID:27259371

  15. Isolation and clinical sample typing of human leptospirosis cases in Argentina.

    PubMed

    Chiani, Yosena; Jacob, Paulina; Varni, Vanina; Landolt, Noelia; Schmeling, María Fernanda; Pujato, Nazarena; Caimi, Karina; Vanasco, Bibiana

    2016-01-01

    Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6 Leptospira interrogans, one Leptospira wolffii and one Leptospira broomii) and 58 out of 85 (68.2%) clinical samples (55 L. interrogans, 2 Leptospira meyeri, and one Leptospira kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients. PMID:26658064

  16. Case Series of Fatal Leptospira spp./Dengue Virus Co-Infections—Puerto Rico, 2010–2012

    PubMed Central

    Pérez Rodríguez, Nicole M.; Galloway, Renee; Blau, Dianna M.; Traxler, Rita; Bhatnagar, Julu; Zaki, Sherif R.; Rivera, Aidsa; Torres, Jose V.; Noyd, David; Santiago-Albizu, Xavier E.; García, Brenda Rivera; Tomashek, Kay M.; Bower, William A.; Sharp, Tyler M.

    2014-01-01

    Co-infection with pathogens that cause acute febrile illness creates a diagnostic challenge as a result of overlapping clinical manifestations. Here, we describe four fatal cases of Leptospira species/dengue virus co-infection in Puerto Rico. Although all patients sought care early, antibiotic administration was delayed for most. Steroids were administered to all patients, in most cases before antibiotics. These cases show the need for clinicians evaluating patients in or recently returned from the tropics with acute febrile illness to consider both dengue and leptospirosis. Furthermore, they illustrate the need for nucleic acid- or antigen-based rapid diagnostic tests to enable timely patient diagnosis and management. In particular, antibiotic therapy should be initiated early for patients with suspected leptospirosis, and steroids should not be administered to patients with suspected dengue. PMID:25092820

  17. Urban Market Gardening and Rodent-Borne Pathogenic Leptospira in Arid Zones: A Case Study in Niamey, Niger

    PubMed Central

    Dobigny, Gauthier; Garba, Madougou; Tatard, Caroline; Loiseau, Anne; Galan, Max; Kadaouré, Ibrahima; Rossi, Jean-Pierre; Picardeau, Mathieu; Bertherat, Eric

    2015-01-01

    Leptospirosis essentially affects human following contact with rodent urine-contaminated water. As such, it was mainly found associated with rice culture, recreational activities and flooding. This is also the reason why it has mainly been investigated in temperate as well as warm and humid regions, while arid zones have been only very occasionally monitored for this disease. In particular, data for West African countries are extremely scarce. Here, we took advantage of an extensive survey of urban rodents in Niamey, Niger, in order to look for rodent-borne pathogenic Leptospira species presence and distribution across the city. To do so, we used high throughput bacterial 16S-based metabarcoding, lipL32 gene-targeting RT-PCR, rrs gene sequencing and VNTR typing as well as GIS-based multivariate spatial analysis. Our results show that leptospires seem absent from the core city where usual Leptospira reservoir rodent species (namely R. rattus and M. natalensis) are yet abundant. On the contrary, L. kirschneri was detected in Arvicanthis niloticus and Cricetomys gambianus, two rodent species that are restricted to irrigated cultures within the city. Moreover, the VNTR profiles showed that rodent-borne leptospires in Niamey belong to previously undescribed serovars. Altogether, our study points towards the importance of market gardening in maintain and circulation of leptospirosis within Sahelian cities. In Africa, irrigated urban agriculture constitutes a pivotal source of food supply, especially in the context of the ongoing extensive urbanization of the continent. With this in mind, we speculate that leptospirosis may represent a zoonotic disease of concern also in arid regions that would deserve to be more rigorously surveyed, especially in urban agricultural settings. PMID:26437456

  18. Serologic survey for Leptospira spp. in captive neotropical felids in Foz do Iguaçu, Paraná, Brazil.

    PubMed

    Ullmann, Leila Sabrina; Hoffmann, Juliano L; de Moraes, Wanderlei; Cubas, Zalmir S; dos Santos, Leonilda Correia; da Silva, Rodrigo Costa; Moreira, Nei; Guimaraes, Ana Marcia Sa; Camossi, Lucilene Granuzzio; Langoni, Helio; Biondo, Alexander W

    2012-06-01

    Leptospirosis is a bacterial zoonosis of worldwide distribution and is endemic in tropical countries, where rodents and other wild mammals are abundant and may act as reservoirs. Leptospirosis has become a concern in captive wild animals, due mostly to their exposure to contaminated urine or environment. Although domestic cats (Felis catus) have been reported refractory to leptospirosis, serology and disease in captive wild felids is still unclear. In this study 57 adult, clinically healthy felids, including 1 Geoffroy's cat (Leopardus geoffroyi), 3 jaguarundis (Puma yagouaroundi), 17 margays (Leopardus wiedii), 22 little spotted cats (Leopardus tigrinus), and 14 ocelots (Leopardus pardalis) kept in captivity at the Sanctuary at the Itaipu Binacional hydroelectric power plant (Bela Vista Biological Sanctuary), Foz do Iguacu City, Paraná State, Brazil, were serologically surveyed for the presence of antibodies against 28 serovars of Leptospira spp. by microagglutination test (MAT). Two animals (3.5%) were seropositive: one male ocelot to the serovar Cynopteri (titer 100) and one female margay to Autumnalis (100) and Butembo (200). The captive-born, 5-yr-old ocelot had been solitary housed in an individual cage. The approximately 21-yr-old wild-caught margay was also kept individually. None of the tested animals showed signs ofleptospirosis. During a study conducted 4 yr previously in the same facility, this particular margay also tested positive for the same two serovars, among others. The present study indicates that the felids tested for Leptospira spp. by MAT were exposed to serovars, but did not demonstrate clinical signs of disease. Comparison with a previous study suggests that serovar titers may vary over time and that leptospirosis dynamics remains unclear in wild felids. PMID:22779223

  19. Urban Market Gardening and Rodent-Borne Pathogenic Leptospira in Arid Zones: A Case Study in Niamey, Niger.

    PubMed

    Dobigny, Gauthier; Garba, Madougou; Tatard, Caroline; Loiseau, Anne; Galan, Max; Kadaouré, Ibrahima; Rossi, Jean-Pierre; Picardeau, Mathieu; Bertherat, Eric

    2015-01-01

    Leptospirosis essentially affects human following contact with rodent urine-contaminated water. As such, it was mainly found associated with rice culture, recreational activities and flooding. This is also the reason why it has mainly been investigated in temperate as well as warm and humid regions, while arid zones have been only very occasionally monitored for this disease. In particular, data for West African countries are extremely scarce. Here, we took advantage of an extensive survey of urban rodents in Niamey, Niger, in order to look for rodent-borne pathogenic Leptospira species presence and distribution across the city. To do so, we used high throughput bacterial 16S-based metabarcoding, lipL32 gene-targeting RT-PCR, rrs gene sequencing and VNTR typing as well as GIS-based multivariate spatial analysis. Our results show that leptospires seem absent from the core city where usual Leptospira reservoir rodent species (namely R. rattus and M. natalensis) are yet abundant. On the contrary, L. kirschneri was detected in Arvicanthis niloticus and Cricetomys gambianus, two rodent species that are restricted to irrigated cultures within the city. Moreover, the VNTR profiles showed that rodent-borne leptospires in Niamey belong to previously undescribed serovars. Altogether, our study points towards the importance of market gardening in maintain and circulation of leptospirosis within Sahelian cities. In Africa, irrigated urban agriculture constitutes a pivotal source of food supply, especially in the context of the ongoing extensive urbanization of the continent. With this in mind, we speculate that leptospirosis may represent a zoonotic disease of concern also in arid regions that would deserve to be more rigorously surveyed, especially in urban agricultural settings. PMID:26437456

  20. Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the Spirochete Leptospira biflexa by Random Insertional Mutagenesis

    PubMed Central

    Louvel, Hélène; Saint Girons, Isabelle; Picardeau, Mathieu

    2005-01-01

    The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources. PMID:15838052

  1. Comparison of agglutinating and neutralizing antibodies to serovar hardjo in sows immunized with two commercial whole culture polivalent anti-leptospira bacterins.

    PubMed

    Soto, Francisco Rafael Martins; Pinheiro, Sônia Regina; Morais, Zenaide Maria; Gonçales, Amane Paldês; de Azevedo, Sérgio Santos; Bernardi, Fernanda; Camargo, Sebastião Rodrigues; Vasconcellos, Silvio Arruda

    2008-07-01

    It was performed the comparison of the intensity and duration of agglutinating and neutralizing antibodies to serovar Hardjo in swines vaccinated with two commercial anti-leptospira bacterins. Sows no reactive to 24 Leptospira sp serovars in the microscopic agglutination test (MAT) were divided in three groups: Group A (n=08): received two vaccine A doses with 30 days interval, Group B (n=08) two vaccine B doses with 30 days interval and Group C (n=08): control no vaccinated against leptospirosis.Blood samples were collected each 30 days during six months following the first vaccination. The sera were tested by MAT and growth inhibition test (GIT) to serovar Hardjo in order to evaluate respectively agglutinating and neutralizing antibodies. It was found that neutralizing antibodies persisted for a longer time than the agglutinating ones and that the absence of agglutinating antibodies does not means in the absence of the neutralizing. The peaks of agglutinating antibodies was obtained at least 30 days earlier than that produced by neutralizing. The duration of both kinds of antibodies measured differed between the two bacterines tested. The period for inducing neutralizing antibodies against serovar Hardjo indicated that gilts must be immunized with two doses of whole culture anti-leptospira bacterines applied 30 days each other at least 90 days before the first mating. For the maintenance of hight levels of neutralizing antibodies the revaccinations must be performed every six months after the first vaccination. PMID:24031250

  2. Seroprevalence and risk factors of antibodies against Leptospira spp. in ovines from Uberlândia municipality, Minas Gerais state, Brazil

    PubMed Central

    Salaberry, Sandra R.S.; Castro, Vanessa; Nassar, Alessandra F.C.; Castro, Jacqueline R.; Guimarães, Ednaldo C.; Lima-Ribeiro, Anna M.C.

    2011-01-01

    The objectives of the present study were to verify the seroprevalence of anti-Leptospira spp. antibodies, identify the most frequent serovars and the risk factors associated with the infection in Uberlândia, Minas Gerais, Brazil. A total of 334 ovines blood samples were collected in 12 farms from Uberlândia municipality to be evaluated by means the Microscopic Agglutination Test (MAT) against 22 serovars of Leptospira spp. and an epidemiologic questionnaire was applied for each farm in order to correlate with risk factors of leptospirosis: sex, age and breed as well as contact with cattle, contact with dogs and presence of rodents. The prevalence of seropositive to MAT was found in seventy four ovines (22.2%; CI 95% 17.6–26.4%), with titers ranging from 100 to 3200. The most frequent serovars identified were: Hardjo, Autumnalis, Hardjo and Wolffi association and Grippotyphosa. Statistically significant differences were found in males, pure breeds and presence of rodents (p<0.05). The prevalence of anti-Leptospira spp. antibodies found in the present study demonstrated that this bacterium occurs in ovines of Uberlândia municipality, MG, Brazil. The need for the adoption of efficient management for the control of rodents and infection in ovines in order to avoid leptospirosis in the local flocks and future transmission to humans. PMID:24031773

  3. Comparison of agglutinating and neutralizing antibodies to serovar hardjo in sows immunized with two commercial whole culture polivalent anti-leptospira bacterins

    PubMed Central

    Soto, Francisco Rafael Martins; Pinheiro, Sônia Regina; Morais, Zenaide Maria; Gonçales, Amane Paldês; de Azevedo, Sérgio Santos; Bernardi, Fernanda; Camargo, Sebastião Rodrigues; Vasconcellos, Silvio Arruda

    2008-01-01

    It was performed the comparison of the intensity and duration of agglutinating and neutralizing antibodies to serovar Hardjo in swines vaccinated with two commercial anti-leptospira bacterins. Sows no reactive to 24 Leptospira sp serovars in the microscopic agglutination test (MAT) were divided in three groups: Group A (n=08): received two vaccine A doses with 30 days interval, Group B (n=08) two vaccine B doses with 30 days interval and Group C (n=08): control no vaccinated against leptospirosis.Blood samples were collected each 30 days during six months following the first vaccination. The sera were tested by MAT and growth inhibition test (GIT) to serovar Hardjo in order to evaluate respectively agglutinating and neutralizing antibodies. It was found that neutralizing antibodies persisted for a longer time than the agglutinating ones and that the absence of agglutinating antibodies does not means in the absence of the neutralizing. The peaks of agglutinating antibodies was obtained at least 30 days earlier than that produced by neutralizing. The duration of both kinds of antibodies measured differed between the two bacterines tested. The period for inducing neutralizing antibodies against serovar Hardjo indicated that gilts must be immunized with two doses of whole culture anti-leptospira bacterines applied 30 days each other at least 90 days before the first mating. For the maintenance of hight levels of neutralizing antibodies the revaccinations must be performed every six months after the first vaccination. PMID:24031250

  4. Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease

    PubMed Central

    Miyahara, Satoshi; Saito, Mitsumasa; Kanemaru, Takaaki; Villanueva, Sharon Y A M; Gloriani, Nina G; Yoshida, Shin-ichi

    2014-01-01

    Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection. PMID:24945433

  5. An in vitro growth inhibition test for measuring the potency of Leptospira spp. Sejroe group vaccine in buffaloes.

    PubMed

    de Nardi, Geraldo; Genovez, Margareth Elide; Ribeiro, Marcio Garcia; Castro, Vanessa; Jorge, André Mendes

    2010-07-01

    Leptospira spp. serovars Hardjo and Wollfi from Sejroe serogroup have been detected in livestock in Brazil, where the main control procedures rely on vaccination. The potency of two commercial vaccines available in this country was monitored by microagglutination test-MAT and in vitro growth inhibition test-GIT in serum samples from 33 female buffaloes divided into: G1-unvaccinated control; G2-vaccinated with Leptobac-6 containing serovars Hardjo and Wolffi and G3-vaccinated with Triangle-9 containing serovar Hardjo. G2 and G3 animals were vaccinated on day zero, and received a booster and two revaccinations on days 30, 210 and 390 and G1 animals received phosphate buffered saline. Serum samples were collected at 15-day intervals between days 0 and 60; and at 30-day intervals between days 60 and 540 and were tested by MAT and GIT with serovars Hardjo and Wolffi. G1 remained negative throughout the experiment. Both vaccines were able to induce agglutinating and growth inhibition antibodies. Six months after the last revaccination, all animals tested negative by MAT, but still were positive by GIT until the end of experimental period. GIT could be a good tool to evaluate the potency and to monitor antibodies responses of vaccines of Sejroe group serovars. PMID:20332068

  6. Restriction fragment length polymorphisms distinguish Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates from different geographical locations.

    PubMed Central

    Zuerner, R L; Ellis, W A; Bolin, C A; Montgomery, J M

    1993-01-01

    Genetic variability among Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates representing several geographical regions was determined by restriction endonuclease analysis. Five previously unidentified EcoRI digestion patterns and one previously unidentified HhaI digestion pattern were seen with the various isolates. The copy number and genomic distribution of an L. borgpetersenii insertion sequence (IS1533) was determined. Hardjo-bovis isolate 033 (the type strain for hardjo-bovis) contained 40 well dispersed copies of IS1533. IS1533 probes were used to compare hardjo-bovis isolates by DNA blot hybridization analysis. Use of these probes showed the presence of additional genetic heterogeneity among hardjo-bovis isolates, which restriction endonuclease analysis did not show. Pulsed-field gel electrophoretic analysis of DNAs from several isolates suggested that some polymorphisms arose by genomic rearrangements. All hardjo-bovis isolates were categorized into 14 distinct groups on the basis of common hybridization and endonuclease digestion patterns. Most of these groups were isolated from distinct geographical regions, suggesting that several different clonal populations of hardjo-bovis exist. Images PMID:7681437

  7. Antibodies to Leptospira among blood donors in higher-risk areas of Australia: possible implications for transfusion safety

    PubMed Central

    Faddy, Helen; Seed, Clive; Lau, Colleen; Racloz, Vanessa; Flower, Robert; Smythe, Lee; Burns, Mary-Anne; Dohnt, Michael; Craig, Scott; Harley, Robert; Weinstein, Philip

    2015-01-01

    Background Leptospirosis is one of the most common bacterial zoonoses worldwide, and clinical manifestations range from asymptomatic infection to acute febrile illness, multi-organ failure and death. Asymptomatic, acute bacteraemia in a blood donor provides a potential for transfusion-transmission, although only a single such case from India has been recorded. Human leptospirosis is uncommon in developed countries; however, the state of Queensland in Australia has one of the highest rates among developed countries, especially after increased rainfall. This study examined the prevalence of antibodies to Leptospira spp. in blood donors residing in higher-risk areas of Australia, to evaluate the appropriateness of current blood safety guidelines. Materials and methods Plasma samples collected from blood donors residing in higher-risk areas of Australia during 2009 and 2011 were included in the study. All samples were tested for the presence of antibodies to 22 leptospiral serovars using the microscopic agglutination test. Result No sample had antibody titres suggestive of a current or recent infection, however, seven samples (1.44%, 95% CI: 0.38–2.50%) had titres suggestive of a past infection. Discussion This study provides data that may support the appropriateness of current relevant donor selection policies in Australia. Given that the risk profile for leptospirosis is expanding and that the infection is likely to become more prevalent with climate change, this disease may become more of a concern for transfusion safety in the future. PMID:24960651

  8. Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease.

    PubMed

    Miyahara, Satoshi; Saito, Mitsumasa; Kanemaru, Takaaki; Villanueva, Sharon Y A M; Gloriani, Nina G; Yoshida, Shin-ichi

    2014-08-01

    Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection. PMID:24945433

  9. Experimental Hamster Infection with a Strain of Leptospira borgpetersenii Ballum Isolated from a Reservoir Mouse in New Caledonia

    PubMed Central

    Matsui, Mariko; Roche, Louise; Soupé-Gilbert, Marie-Estelle; Roudier, Martine; Moniquet, Vincent; Goarant, Cyrille

    2015-01-01

    Leptospirosis is a neglected zoonosis caused by pathogenic Leptospira. In this study, we characterized the virulence of isolate B3-13S obtained from a wild mouse (Mus musculus) captured in New Caledonia, subsequently identified as a bacterium belonging to the L. borgpetersenii serogroup Ballum. Hamsters were infected with an intraperitoneal injection of 2 × 108 bacteria, resulting in severe histopathological organ damages consistent with tissue lesions previously observed with other strains. Hamsters were also injected with 1 × 108 or 5 × 107 bacteria and animals that recovered showed renal carriage of leptospires in concentrations similar to the bacterial load quantified in mouse kidneys, with urinary shedding of bacteria up to 4 weeks postinfection. The serogroup Ballum is increasingly reported in human leptospirosis, and these results highlight the use of the B3-13S isolate for the development of models resulting in either severe acute or chronic forms of the infection, allowing for better characterization of its pathogenesis. PMID:25758655

  10. Seroprevalence of bovine leptospiral antibodies by microscopic agglutination test in Southeast of Iran

    PubMed Central

    Khalili, Mohammad; Sakhaee, Ehsanollah; Aflatoonian, Mohammad Reza; Abdollahpour, Gholamreza; Tabrizi, Saeed Sattari; Damaneh, Elham Mohammadi; Hossini-nasab, Sajad

    2014-01-01

    Objective To evaluate serological findings of bovine leptospirosis which is a zoonotic disease with worldwide distribution caused by Leptospira interrogans. Methods One hundred and sixty seven sera were collected from 9 commercial dairy herds in jiroft suburbs, from July to October 2011. Microscopic agglutination test (MAT) was used to evaluates serological findings of bovine leptospirosis in Jiroft suburb dairy farms, Kerman province, Iran. Results Antibodies were found by MAT at least against one serovar of Leptospira interrogans in 29 samples (17.36%) among 167 sera at a dilution 1:100 or higher, and Leptospira pomona was the most prevalent serovar. Positive titers against more than one serovar were detected in 6 sera of the positive samples. Conclusion This study is the first report of leptospirosis in Southeast Iran and showed that Leptospira pomona was the most and Leptospira icterohaemorrhagiae the least prevalent serovars in Southeast Iran. PMID:25182718

  11. Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

    PubMed Central

    Haake, D A; Champion, C I; Martinich, C; Shang, E S; Blanco, D R; Miller, J N; Lovett, M A

    1993-01-01

    Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane

  12. Separating Putative Pathogens from Background Contamination with Principal Orthogonal Decomposition: Evidence for Leptospira in the Ugandan Neonatal Septisome

    PubMed Central

    Schiff, Steven J.; Kiwanuka, Julius; Riggio, Gina; Nguyen, Lan; Mu, Kevin; Sproul, Emily; Bazira, Joel; Mwanga-Amumpaire, Juliet; Tumusiime, Dickson; Nyesigire, Eunice; Lwanga, Nkangi; Bogale, Kaleb T.; Kapur, Vivek; Broach, James R.; Morton, Sarah U.; Warf, Benjamin C.; Poss, Mary

    2016-01-01

    Neonatal sepsis (NS) is responsible for over 1 million yearly deaths worldwide. In the developing world, NS is often treated without an identified microbial pathogen. Amplicon sequencing of the bacterial 16S rRNA gene can be used to identify organisms that are difficult to detect by routine microbiological methods. However, contaminating bacteria are ubiquitous in both hospital settings and research reagents and must be accounted for to make effective use of these data. In this study, we sequenced the bacterial 16S rRNA gene obtained from blood and cerebrospinal fluid (CSF) of 80 neonates presenting with NS to the Mbarara Regional Hospital in Uganda. Assuming that patterns of background contamination would be independent of pathogenic microorganism DNA, we applied a novel quantitative approach using principal orthogonal decomposition to separate background contamination from potential pathogens in sequencing data. We designed our quantitative approach contrasting blood, CSF, and control specimens and employed a variety of statistical random matrix bootstrap hypotheses to estimate statistical significance. These analyses demonstrate that Leptospira appears present in some infants presenting within 48 h of birth, indicative of infection in utero, and up to 28 days of age, suggesting environmental exposure. This organism cannot be cultured in routine bacteriological settings and is enzootic in the cattle that often live in close proximity to the rural peoples of western Uganda. Our findings demonstrate that statistical approaches to remove background organisms common in 16S sequence data can reveal putative pathogens in small volume biological samples from newborns. This computational analysis thus reveals an important medical finding that has the potential to alter therapy and prevention efforts in a critically ill population. PMID:27379237

  13. Separating Putative Pathogens from Background Contamination with Principal Orthogonal Decomposition: Evidence for Leptospira in the Ugandan Neonatal Septisome.

    PubMed

    Schiff, Steven J; Kiwanuka, Julius; Riggio, Gina; Nguyen, Lan; Mu, Kevin; Sproul, Emily; Bazira, Joel; Mwanga-Amumpaire, Juliet; Tumusiime, Dickson; Nyesigire, Eunice; Lwanga, Nkangi; Bogale, Kaleb T; Kapur, Vivek; Broach, James R; Morton, Sarah U; Warf, Benjamin C; Poss, Mary

    2016-01-01

    Neonatal sepsis (NS) is responsible for over 1 million yearly deaths worldwide. In the developing world, NS is often treated without an identified microbial pathogen. Amplicon sequencing of the bacterial 16S rRNA gene can be used to identify organisms that are difficult to detect by routine microbiological methods. However, contaminating bacteria are ubiquitous in both hospital settings and research reagents and must be accounted for to make effective use of these data. In this study, we sequenced the bacterial 16S rRNA gene obtained from blood and cerebrospinal fluid (CSF) of 80 neonates presenting with NS to the Mbarara Regional Hospital in Uganda. Assuming that patterns of background contamination would be independent of pathogenic microorganism DNA, we applied a novel quantitative approach using principal orthogonal decomposition to separate background contamination from potential pathogens in sequencing data. We designed our quantitative approach contrasting blood, CSF, and control specimens and employed a variety of statistical random matrix bootstrap hypotheses to estimate statistical significance. These analyses demonstrate that Leptospira appears present in some infants presenting within 48 h of birth, indicative of infection in utero, and up to 28 days of age, suggesting environmental exposure. This organism cannot be cultured in routine bacteriological settings and is enzootic in the cattle that often live in close proximity to the rural peoples of western Uganda. Our findings demonstrate that statistical approaches to remove background organisms common in 16S sequence data can reveal putative pathogens in small volume biological samples from newborns. This computational analysis thus reveals an important medical finding that has the potential to alter therapy and prevention efforts in a critically ill population. PMID:27379237

  14. Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool.

    PubMed

    Romero, Eliete C; Yasuda, Paulo H

    2006-06-01

    A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in São Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5%) and one to serogroup Sejroe (2.5%). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey. PMID:16951806

  15. Risk factor analysis for antibodies to Brucella, Leptospira and C. burnetii among cattle in the Adamawa Region of Cameroon: a cross-sectional study.

    PubMed

    Mazeri, Stella; Scolamacchia, Francesca; Handel, Ian G; Morgan, Kenton L; Tanya, Vincent N; Bronsvoort, Barend M deC

    2013-02-01

    Brucellosis, leptospirosis and Q fever are important livestock diseases, commonly responsible for significant production losses, yet their epidemiology in sub-Saharan Africa is largely unknown. Animal reservoirs pose the main risk of transmission to humans, where serious disease can occur. In the developing world setting, the flu-like symptoms of the acute stages of these diseases can be misdiagnosed as malaria, which can result in the administration of the wrong treatment, prolonged disease and increase in antibiotic resistance. Multivariable mixed-effects logistic regression models in this study revealed potential risk factors associated with the aforementioned pathogens in cattle in the Adamawa Region of Cameroon, with wildlife, namely, buffaloes, playing a major role in both Brucella and Coxiella burnetii seropositivity. Cattle mixing with other herds at night and cattle grazing in an area on a route taken by herds on transhumance appear to be positively associated with Leptospira seropositivity, while female cows and whether buffaloes are seen during grazing or transhumance are positively associated with C. burnetii seropositivity. On the other hand, animals that have been on transhumance in the past year and animals belonging to herdsmen of the Fulbe ethnic group appear to be protected against Leptospira and C. burnetii, respectively. Cattle of more than 2 years old appear to have increased odds of being seropositive to either pathogen. Further research is needed to confirm these findings and improve the knowledge of the epidemiology of these three pathogens in Africa, taking particular consideration of the wildlife involvement in the disease transmission. PMID:23117621

  16. Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR.

    PubMed

    Suwancharoen, Duangjai; Sittiwicheanwong, Busara; Wiratsudakul, Anuwat

    2016-09-01

    Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle. PMID:27150208

  17. Human Leptospirosis on Reunion Island, Indian Ocean: Are Rodents the (Only) Ones to Blame?

    PubMed Central

    Cordonin, Colette; Le Minter, Gildas; Gomard, Yann; Pagès, Frédéric; Jaffar-Bandjee, Marie-Christine; Tortosa, Pablo; Dellagi, Koussay

    2016-01-01

    Background Although leptospirosis is a zoonosis of major concern on tropical islands, the molecular epidemiology of the disease aiming at linking human cases to specific animal reservoirs has been rarely explored within these peculiar ecosystems. Methodology/Principal Findings Five species of wild small mammals (n = 995) as well as domestic animals (n = 101) were screened for Leptospira infection on Reunion Island; positive samples were subsequently genotyped and compared to Leptospira from clinical cases diagnosed in 2012–2013 (n = 66), using MLST analysis. We identified two pathogenic species in human cases, namely Leptospira interrogans and Leptospira borgpetersenii. Leptospira interrogans was by far dominant both in clinical samples (96.6%) and in infected animal samples (95.8%), with Rattus spp and dogs being its exclusive carriers. The genetic diversity within L. interrogans was apparently limited to two sequence types (STs): ST02, identified among most clinical samples and in all rats with complete MLST, and ST34, identified in six humans, but not in rats. Noteworthy, L. interrogans detected in two stray dogs partially matched with ST02 and ST34. Leptospira borgpetersenii was identified in two clinical samples only (3.4%), as well as in cows and mice; four haplotypes were identified, of which two seemingly identical in clinical and animal samples. Leptospira borgpetersenii haplotypes detected in human cases were clearly distinct from the lineage detected so far in the endemic bat species Mormopterus francoismoutoui, thus excluding a role for this volant mammal in the local human epidemiology of the disease. Conclusions/Significance Our data confirm rats as a major reservoir of Leptospira on Reunion Island, but also pinpoint a possible role of dogs, cows and mice in the local epidemiology of human leptospirosis. This study shows that a comprehensive molecular characterization of pathogenic Leptospira in both clinical and animal samples helps to gaining

  18. SEROLOGICAL AND MOLECULAR SURVEY OF Leptospira spp. AMONG CART HORSES FROM AN ENDEMIC AREA OF HUMAN LEPTOSPIROSIS IN CURITIBA, SOUTHERN BRAZIL

    PubMed Central

    Finger, Mariane Angélica; de Barros, Ivan Roque; Leutenegger, Christian; Estrada, Marko; Ullmann, Leila Sabrina; Langoni, Hélio; Kikuti, Mariana; Dornbush, Peterson Triches; Deconto, Ivan; Biondo, Alexander Welker

    2014-01-01

    Introduction: Cart horses are a re-emerging population employed to carry recyclable material in cities. Methods: Sixty-two horses were sampled in an endemic area of human leptospirosis. The microscopic agglutination test (MAT) and real-time polymerase chain reaction (qPCR) were performed. Results: A seropositivity of 75.8% with serovar Icterohaemorrhagiae in 80.8% of the horses was observed. Blood and urine were qPCR negative. MAT showed positive correlations with rainfall (p = 0.02) and flooding (p = 0.03). Conclusions: Although horses may be constantly exposed to Leptospira spp. in the environment mostly because of rainfall and flooding, no leptospiremia or leptospiruria were observed in this study. PMID:25351539

  19. Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents

    PubMed Central

    SHIOKAWA, Kanae; GAMAGE, Chandika D.; KOIZUMI, Nobuo; SAKODA, Yoshihiro; SHIMIZU, Kenta; TSUDA, Yoshimi; YOSHIMATSU, Kumiko; ARIKAWA, Jiro

    2015-01-01

    The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87–188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83% . A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P. pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction. PMID:26412049

  20. Monoclonal antibodies against LipL32, the major outer membrane protein of pathogenic Leptospira: production, characterization, and testing in diagnostic applications.

    PubMed

    Fernandes, Cláudia P H; Seixas, Fabiana K; Coutinho, Mariana L; Vasconcellos, Flávia A; Seyffert, Núbia; Croda, Julio; McBride, Alan J; Ko, Albert I; Dellagostin, Odir A; Aleixo, José A G

    2007-02-01

    Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysaccharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 in its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (MAbs). Three MAbs against rLipL32 were produced, isotyped, and evaluated for further use in diagnostic tests of leptospirosis using different approaches. MAbs were conjugated to peroxidase and evaluated in a native protein enzyme-linked immunosorbent assay (ELISA) with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for indirect immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 MAbs conjugated to peroxidase or used as primary antibody bound to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. In immunofluorescence assay, MAbs labeled bacterial cells either intact or methanol fixed. Two MAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by polymerase chain reaction (PCR) amplification. Results of this study suggest that the MAbs produced can be useful for the development of diagnostic tests based on detection of LipL32 leptospiral antigen in biological fluids. PMID:17316084

  1. Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents.

    PubMed

    Shiokawa, Kanae; Gamage, Chandika D; Koizumi, Nobuo; Sakoda, Yoshihiro; Shimizu, Kenta; Tsuda, Yoshimi; Yoshimatsu, Kumiko; Arikawa, Jiro

    2016-03-01

    The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87-188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83% . A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P. pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction. PMID:26412049

  2. EMJH medium with 5-fluorouracil and nalidixic acid associated with serial dilution technique used to recover Leptospira spp from experimentally contaminated bovine semen

    PubMed Central

    Miraglia, Fabiana; de Moraes, Zenaide Maria; Melville, Priscilla Anne; Dias, Ricardo Augusto; Vasconcellos, Silvio Arruda

    2009-01-01

    Bovine semen experimentally contaminated with Leptospira santarosai serovar Guaricura was submitted to the modified EMJH medium with 5-fluorouracil (300mg/L) and nalidixic acid (20mg/L), named as “selective medium” and using the serial dilution technique, in order to evaluate the percentage of recovery of the added microorganism. The selective EMJH medium was found with higher percentage of recovery of leptospiras and minor losses of samples due to contamination with opportunistic microorganisms than the non-selective EMJH medium: 151/376 (40.0%) of positive growth; and 38/376 (10.0%) contamination and 58/376 (15%) and 129/376 (34.0%), respectively. These results were statistically significant (p<0. 0001; Fisher). Differences were found when the frequencies of positive leptospires recovery have been compared in the serial dilution technique (10-1 to 10-4) between the selective and non-selective media at different dilution factors. At 1/10th dilution the percentages found were (0%, 0/80) and (38%, 30/80), at 1/100th dilution, (3%, 2/80) and (49%, 39/ 80) and at 1/1,000th dilution, (25%, 20/80) and (50%, 40/80), respectively. The percentage of recovery of leptospires was found to be directly proportional to the dilution used. The methodology of the serial dilution technique (setting at least three dilutions) and the use of selective EMJH medium have been found to be efficient for the isolation of leptospires from the bovine semen samples. PMID:24031342

  3. Production of reactive oxygen (H2O2) and nitrogen (NO) intermediates and tnf-α in mice genetically selected for high (H) and low (L) antibody response and experimentally infected with Leptospira serovar pomona

    PubMed Central

    Haanwinckel, Maria Cristina Santos; de Oliveira, Silvio Luis

    2011-01-01

    The aim of the present study was to evaluate the activity of macrophages, and the production of TNF-α and antibodies against experimental infection by Leptospira serovar Pomona in mice genetically selected for High (H) or Low (L) humoral immune response. To evaluate macrophagic activity, peritoneal and splenic lavages were performed for determination of oxygen (H2O2) and nitrogen (NO) intermediates. The production of the tumor necrosis factor (TNF-α) was investigated through bioassays in serum and homogenates of splenic and hepatic cells of control and infected animals, as was as specific antibodies production. The immune response against serovar Pomona in those lines, was characterized by high antibody production, especially in later periods of the infectious process, whereas values of bacterial recovery in culture medium were lower. The production of reactives oxygen and nitrogen intermediate, also helped to eliminate Leptospira Pomona in both lines; H2O2 production an important factor in HIV-A, as well as NO production in LIV-A, especially in later post-inoculation periods. The same was detected for TNF-α. Results suggest that such lines could be an important model to investigate the pathogenesis and the immune response of animals against the several Leptospira serovars. PMID:24031688

  4. Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species.

    PubMed Central

    Shang, E S; Summers, T A; Haake, D A

    1996-01-01

    We report the cloning of the gene encoding a surface-exposed leptospiral lipoprotein, designated LipL41. In a previous study, a 41-kDa protein antigen was identified on the surface of Leptospira kirschneri (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe.A Lambda ZAP II library containing EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL41 gene was identified. The deduced amino acid sequence of LipL41 would encode a 355-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinant His6-LipL41 fusion protein was expressed in Escherichia coli in order to generate specific rabbit antiserum. LipL41 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL41 exclusively into the detergent phase. At least eight proteins, including LipL41 and the other major Triton X-114 detergent phase proteins, are intrinsically labeled during incubation of L. kirschneri in media containing [3H] palmitate. Processing of LipL41 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. Triton X-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin), LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36, only LipL41 and OmpL1 were exposed on the surface of intact organisms. Immunoblot analysis of a panel of Leptospira species reveals that LipL41 expression is highly conserved among leptospiral pathogens. PMID:8675344

  5. Leptospirosis in raccoons in Quebec: 2 case reports and seroprevalence in a recreational area.

    PubMed Central

    Mikaelian, I; Higgins, R; Lequient, M; Major, M; Lefebvre, F; Martineau, D

    1997-01-01

    Raccoons may represent a source of leptospires for humans and domestic animals. We describe a case of severe interstitial nephritis associated with the serovar bratislava of Leptospira interrogans (1st report in wildlife), and the seroprevalence to 4 leptospire serovars in a recreational area in Quebec. Images Figure 1. p442-a PMID:9220134

  6. Three cases of canine leptospirosis in Quebec.

    PubMed Central

    Kalin, M; Devaux, C; DiFruscia, R; Lemay, S; Higgins, R

    1999-01-01

    Three dogs from different locations with acute renal failure were hospitalized in autumn 1996 and 1997. Leptospira interrogans serovar pomona was detected by the microscopic agglutination test. All dogs recovered after antibiotic treatment. The importance of the development of vaccines adapted to emerging serovars in dogs should be addressed. PMID:10086220

  7. Opsonization of Treponema pallidum is mediated by immunoglobulin G antibodies induced only by pathogenic treponemes.

    PubMed Central

    Shaffer, J M; Baker-Zander, S A; Lukehart, S A

    1993-01-01

    Rabbit antisera to Leptospira interrogans, Borrelia hermsii, and Treponema phagedenis biotype Reiter, reactive to shared spirochetal antigens, failed to enhance phagocytosis of Treponema pallidum by macrophages, while immunoglobulin G to Treponema pallidum subsp. pertenue and Treponema paraluiscuniculi promoted phagocytosis. Opsonic antibodies are directed to pathogen-restricted, not shared spirochetal, antigens. PMID:8423106

  8. Introduction of a rapid dipstick assay for the detection of Leptospira-specific immunoglobulin m antibodies in the laboratory diagnosis of leptospirosis in a hospital in Makassar, Indonesia.

    PubMed

    Hatta, M; Smits, H L; Gussenhoven, G C; Gooskens, J

    2000-09-01

    An easy, rapid and robust dipstick assay for detection of leptospira-specific immunoglobulin M (IgM) antibodies was evaluated on 403 patients admitted for hospitalization because of fever. The clinical symptoms and signs of 35 patients were consistent with leptospirosis. The final diagnosis for the remaining patients was as follows: 136 with typhoid fever, 82 with hepatitis, 74 with malaria, 48 with infections of the respiratory tract, and 20 with fever of unknown origin. The clinical diagnosis of leptospirosis was confirmed for 24 (68.6%) patients by the combined results of the microscopic agglutination test (MAT), the reference test for leptospirosis, and of IgM ELISA, a standard laboratory test for the serodiagnosis of leptospirosis. In addition, serum specimens from 8 (2.2%) patients with a final clinical diagnosis other than leptospirosis were found to be positive in MAT and/or IgM ELISA. Compared with the results of MAT and IgM ELISA a sensitivity of 91.6% and specificity of 93.6% was calculated for the dipstick assay. Most of the serum samples from the laboratory confirmed patients gave a moderate to strong staining intensity of the antigen band of the dipstick and were easy to read. The results demonstrate that the dipstick assay is convenient to use and allows the rapid and accurate confirmation of patients with clinical suspicion of leptospirosis in areas where the disease is endemic. PMID:11289012

  9. Protective Killed Leptospira borgpetersenii Vaccine Induces Potent Th1 Immunity Comprising Responses by CD4 and γδ T Lymphocytes

    PubMed Central

    Naiman, Brian M.; Alt, David; Bolin, Carole A.; Zuerner, Richard; Baldwin, Cynthia L.

    2001-01-01

    Leptospira borgpetersenii serovar hardjo is the most common cause of bovine leptospirosis and also causes zoonotic infections of humans. A protective killed vaccine against serovar hardjo was shown to induce strong antigen-specific proliferative responses by peripheral blood mononuclear cells (PBMC) from vaccinated cattle by 2 months after the first dose of vaccine. This response was absent from nonvaccinated control cattle. The mean response peaked by 2 months after completion of the two-dose vaccination regimen, and substantial proliferation was measured in in vitro cultures throughout the 7 months of the study period. Variations in magnitude of the response occurred among the vaccinated animals, but by 7 months postvaccination there was a substantial antigen-specific response with PBMC from all vaccinated animals. Up to one-third of the PBMC from vaccinated animals produced gamma interferon (IFN-γ) after 7 days in culture with antigen, as ascertained by flow cytometric analysis, and significant levels of IFN-γ were measured in culture supernatants by enzyme-linked immunosorbent assay. Two-color immunofluorescence revealed that one-third of the IFN-γ-producing cells were γδ T cells, with the remaining cells being CD4+ T cells. The significance of this study is the very potent Th1-type immune response induced and sustained following vaccination with a killed bacterial vaccine adjuvanted with aluminum hydroxide and the involvement of γδ T cells in the response. Moreover, induction of this Th1-type cellular immune response is associated with the protection afforded by the bovine leptospiral vaccine against L. borgpetersenii serovar hardjo. PMID:11705932

  10. Estudio de la estructura logica utilizada en la ensenanza y el aprendizaje de los conceptos sobre el comportamiento de gases en el curso introductorio de quimica a nivel universitario

    NASA Astrophysics Data System (ADS)

    Costa Diaz, Agnes

    El estudio que se presenta es de caracter cualitativo, un estudio multicasos donde se estudia la estructura logica utilizada por cuatro (4) profesores universitarios que ensenan el curso introductorio de quimica, en la planificacion, presentacion y evaluacion del tema sobre el comportamiento de los gases. Se utilizaron varias fuentes de informacion como: cuestionarios de profesores y estudiantes, entrevistas, grabaciones videomagnetofonicas, materiales didacticos y una prueba conceptual, entre otros. La informacion recopilada fue analizada de acuerdo al orden logico del contenido presentado, el estilo de ensenanza del profesor, las tecnicas y estrategias utilizadas para el desarrollo de destrezas de pensamiento, el ambiente fisico en el salon de clase y los instrumentos de evaluacion y avaluo. El estudio demuestra que lo que los profesores piensan y planifican para hacer sus presentaciones no necesariamente es lo que ocurre en el salon de clases. El desarrollo de destrezas de pensamiento, que constituye una prioridad de los profesores, no se elaboran efectivamente. El uso de las estrategias de resolucion de problemas numericos predomino. La participacion del estudiante en el salon de clases fue limitada y no se logro demostrar el desarrollo de las destrezas de pensamiento deseadas. Aunque los profesores tienen su propio estilo de ensenanza, el orden logico del contenido presentado en clase fue el mismo o siguio muy de cerca el orden establecido por el libro de texto. Los profesores utilizaron preferentemente la tiza y la pizarra para sus presentaciones y la dinamica en el salon de clases fue esencialmente tradicional. Los profesores hicieron su presentacion y los estudiantes copiaron pasivamente la informacion. Las evaluaciones de los estudiantes fueron esencialmente, pruebas escritas de seleccion multiple de acuerdo con el estilo en que se les enseno. El avaluo fue casi inexistente. La prueba conceptual administrada revela un aprendizaje pobre en los conceptos mas

  11. A cross sectional study of leptospirosis and fetal death in Yucatan, Mexico

    PubMed Central

    Vado-Solis, Ignacio; Pérez-Osorio, Carlos; Peniche-Lara, Gaspar; Segura-Correa, José

    2016-01-01

    Introduction: Leptospirosis is a zoonotic disease affecting mainly to low income human population. Acute leptospiral infection during pregnancy has been associated with spontaneous abortion and fetal death during the first trimester and the abortion may occur as consequence of systemic failure. Objective: To estimate the frequency of Leptospira interrogans infection in women with spontaneous abortion in the state of Yucatan, Mexico. Methods: A cross sectional study on women with spontaneous abortion was conducted. Serum samples were tested for Leptospirosis by the microaglutination test, to estimate the frequency of the infecting serovar. The indirect ELISA IgM was used to detect recent infection by L. interrogans. DNA was extracted from paraffin-embedded tissue of placenta for PCR detection of L. interrogans. Results: Overall frequency of infection with L. interrogans in the 81 women with abortion was 13.6%. Five of the 12 serovars evaluated were found and included. Two of the 11 women with abortion and positive to microaglutination test were also positive to the ELISA IgM test. None samples were positive for PCR Leptospira diagnosis. Conclusion: two women could be associated with spontaneous abortion due to leptospirosis, because they showed antibodies against L. interrogans in the microaglutination test and ELISA IgM assays. Differences between regions were found with respect to the prevalences of lesptospirosis. PMID:27226658

  12. An outbreak of severe leptospirosis in capuchin (Cebus) monkeys

    PubMed Central

    Szonyi, Barbara; Agudelo-Flórez, Piedad; Ramírez, Marcela; Moreno, Natali; Ko, Albert I.

    2013-01-01

    Naturally acquired acute leptospirosis in monkeys is uncommon. This study reports an outbreak of severe leptospirosis among 52 capuchin (Cebus) monkeys that had been rescued from homes and housed in a wildlife rehabilitation center in Colombia in 2007. Case confirmation consisted of Leptospira isolation followed by a polymerase chain reaction targeting the LipL32 gene. The attack and mortality rates were 71% and 27%, respectively. Sixteen cases were confirmed. Necropsy revealed diffuse jaundice and pulmonary hemorrhage. Multi-locus sequence typing identified the agent to be Leptospira interrogans sequence type 17, indicating rats as the source of infection. An environmental survey confirmed rodent infestation as the cause of the outbreak. The extent of Leptospira transmission between humans and monkeys is unknown. Improper husbandry of non-human primates could create new reservoirs and transmission routes for Leptospira threatening conservation efforts and public health. PMID:20554228

  13. Serological survey of Bartonella spp., Borrelia burgdorferi, Brucella spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Echinococcus, Hanta-, TBE- and XMR-virus infection in employees of two forestry enterprises in North Rhine-Westphalia, Germany, 2011-2013.

    PubMed

    Jurke, Annette; Bannert, N; Brehm, K; Fingerle, V; Kempf, V A J; Kömpf, D; Lunemann, M; Mayer-Scholl, A; Niedrig, M; Nöckler, K; Scholz, H; Splettstoesser, W; Tappe, D; Fischer, Silke F

    2015-10-01

    We initiated a survey to collect basic data on the frequency and regional distribution of various zoonoses in 722 employees of forestry enterprises in the German state of North Rhine-Westphalia (NRW) from 2011 to 2013. Exposures associated with seropositivity were identified to give insight into the possible risk factors for infection with each pathogen. 41.2% of participants were found to be seropositive for anti-Bartonella IgG, 30.6% for anti-Borrelia burgdorferi IgG, 14.2% for anti-Leptospira IgG, 6.5% for anti-Coxiella burnetii IgG, 6.0% for anti-Hantavirus IgG, 4.0% for anti-Francisella tularensis IgG, 3.4% for anti-TBE-virus IgG, 1.7% for anti-Echinococcus IgG, 0.0% for anti-Brucella IgG and anti-XMRV IgG. Participants seropositive for B. burgdorferi were 3.96 times more likely to be professional forestry workers (univariable analysis: OR 3.96; 95% CI 2.60-6.04; p<0.001); and participants seropositive for Hantavirus 3.72 times more likely (univariable analysis: OR 3.72; 95% CI 1.44-9.57; p=0.007). This study found a surprisingly high percentage of participants seropositive for anti-B. henselae IgG and for anti-F. tularensis IgG. The relatively high seroprevalence for anti-Leptospira IgG seen in this study could be related to living conditions rather than to exposure at work. No specific risk for exposure to C. burnetii and Echinococcus was identified, indicating that neither forestry workers nor office workers represent a risk population and that NRW is not a typical endemic area. Forestry workers appear to have higher risk for contact with B. burgdorferi-infected ticks and a regionally diverse risk for acquiring Hantavirus-infection. The regional epidemiology of zoonoses is without question of great importance for public health. Knowledge of the regional risk factors facilitates the development of efficient prevention strategies and the implementation of such prevention measures in a sustainable manner. PMID:26422407

  14. [Leptospirosis outbreak in calves from Corrientes Province, Argentina].

    PubMed

    Draghi, María G; Brihuega, Bibiana; Benítez, Daniel; Sala, Juan M; Biotti, Graciela M; Pereyra, Matilde; Homse, Alberto; Guariniello, Luciano

    2011-01-01

    Leptospirosis is an infectious disease resulting in significant economic losses in livestock production. This disease causes abortion, embryo death, death of calves within the first few days of life and mastitis. We report a leptospirosis outbreak in calf growing and fattening. Histopathological and hemoparasite studies, immunofluorescence, and bacterial cultures were performed. A strain of Leptospira interrogans serovar Pomona was isolated from samples collected from dead calves. PMID:21491066

  15. Serologic survey for cross-species pathogens in urban coyotes (Canis latrans), Colorado, USA.

    PubMed

    Malmlov, Ashley; Breck, Stewart; Fry, Tricia; Duncan, Colleen

    2014-10-01

    Abstract As coyotes (Canis latrans) adapt to living in urban environments, the opportunity for cross-species transmission of pathogens may increase. We investigated the prevalence of antibodies to pathogens that are either zoonotic or affect multiple animal species in urban coyotes in the Denver metropolitan area, Colorado, USA, in 2012. We assayed for antibodies to canine parvovirus-2, canine distemper virus, rabies virus, Toxoplasma gondii, Yersinia pestis, and serotypes of Leptospira interrogans. Overall, 84% of the animals had antibodies to canine parvovirus-2, 44% for canine distemper virus, 20% for T. gondii (IgG), 28% for Y. pestis, and 4% for L. interrogans serotype Grippotyphosa. No neutralizing antibodies were detected to rabies virus, T. gondii (IgM), or L. interrogans serotypes other than Grippotyphosa. With 88% of animals exposed to at least one pathogen, our results suggest that coyotes may serve as important reservoirs and sentinels for etiologic agents. PMID:25121408

  16. Serologic evidence for selected infectious diseases in Marsican brown bears (Ursus arctos marsicanus) in Italy (2004-09).

    PubMed

    Di Francesco, Cristina Esmeralda; Gentile, Leonardo; Di Pirro, Vincenza; Ladiana, Lara; Tagliabue, Silvia; Marsilio, Fulvio

    2015-01-01

    We tested 30 serum samples collected during 2004-09 from 22 free-ranging Marsican brown bears (Ursus arctos marsicanus) in the National Park of Abruzzo, Lazio, and Molise, Italy, for antibodies against canine distemper virus (CDV), canine adenovirus type 2 (CAV-2), canine parvovirus type 2 (CPV-2), Brucella spp., and eight Leptospira interrogans sensu lato serovars. Antibody to CDV was detected in 11 samples (37%); only two bears (10%) had detectable CAV-2 and Brucella spp. antibodies; three bears were positive for L. interrogans serovar Bratislava; and one sample had antibody against L. interrogans serovar Copenhageni. All samples were positive for CPV-2 antibody. The CPV-2 antibody titers varied from 1∶640 to 1∶10,240, suggesting that transmission was still active. Fifty percent of bears were positive for antibody to two or more pathogens. Our results highlight the need to consider infectious diseases as a potential risk for Marsican brown bear conservation. PMID:25375945

  17. Seroepidemiology of leptospirosis in Minnesota wolves

    USGS Publications Warehouse

    Khan, M.A.; Goyal, S.M.; Diesch, S.L.; Mech, L.D.; Fritts, S.H.

    1991-01-01

    Serum samples (n = 457) from wolves (Canis lupus) in northern Minnesota were collected from 1972 through 1986 and were tested for antibodies against Leptospira interrogans using a microtiter agglutination test. Twelve serovars included in the study were: australis, autumnalis, ballum, bataviae, bratislava, canicola, copenhageni, grippotyphosa, hardjo, pomona, pyrogenes, and tarassovi. Fifty-two (11%) sera had antibody titers of greater than or equal to 1:50 against one or more serovars of L. interrogans. The seroprevalence of different serovars in decreasing order was: grippotyphosa, bratislava, autumnalis, canicola, pomona, ballum, pyrogenes, hardjo, and copenhageni. No antibodies were found against australis, bataviae, and tarassovi. These results indicate that L. interrogans infection may occur in wolves of Minnesota.

  18. Molecular characterisation and disease severity of leptospirosis in Sri Lanka.

    PubMed

    Bandara, Kanchana Kumari; Weerasekera, Manjula; Gunasekara, Chinthika P; Ranasinghe, Nilantha; Marasinghe, Chamil; Fernando, Neluka

    2015-06-01

    Leptospirosis is a re-emerging zoonotic disease all over the world, important in tropical and subtropical areas. A majority of leptospirosis infected patients present as subclinical or mild disease while 5-10% may develop severe infection requiring hospitalisation and critical care. It is possible that several factors, such as the infecting serovar, level of leptospiraemia, host genetic factors and host immune response, may be important in predisposition towards severe disease. Different Leptospira strains circulate in different geographical regions contributing to variable disease severity. Therefore, it is important to investigate the circulating strains at geographical locations during each outbreak for epidemiological studies and to support the clinical management of the patients. In this study immunochromatography, microscopic agglutination test and polymerase chain reaction were used to diagnose leptospirosis. Further restriction fragment length polymorphism and DNA sequencing methods were used to identify the circulating strains in two selected geographical regions of Sri Lanka. Leptospira interrogans, Leptospira borgpetersenii and Leptospira kirschneri strains were identified to be circulating in western and southern provinces. L. interrogans was the predominant species circulating in western and southern provinces in 2013 and its presence was mainly associated with renal failure. PMID:26061234

  19. Molecular characterisation and disease severity of leptospirosis in Sri Lanka

    PubMed Central

    Bandara, Kanchana Kumari; Weerasekera, Manjula; Gunasekara, Chinthika P; Ranasinghe, Nilantha; Marasinghe, Chamil; Fernando, Neluka

    2015-01-01

    Leptospirosis is a re-emerging zoonotic disease all over the world, important in tropical and subtropical areas. A majority of leptospirosis infected patients present as subclinical or mild disease while 5-10% may develop severe infection requiring hospitalisation and critical care. It is possible that several factors, such as the infecting serovar, level of leptospiraemia, host genetic factors and host immune response, may be important in predisposition towards severe disease. Different Leptospira strains circulate in different geographical regions contributing to variable disease severity. Therefore, it is important to investigate the circulating strains at geographical locations during each outbreak for epidemiological studies and to support the clinical management of the patients. In this study immunochromatography, microscopic agglutination test and polymerase chain reaction were used to diagnose leptospirosis. Further restriction fragment length polymorphism and DNA sequencing methods were used to identify the circulating strains in two selected geographical regions of Sri Lanka. Leptospira interrogans, Leptospira borgpetersenii and Leptospira kirschneri strains were identified to be circulating in western and southern provinces. L. interrogans was the predominant species circulating in western and southern provinces in 2013 and its presence was mainly associated with renal failure. PMID:26061234

  20. SEROLOGIC SURVEY AND RESULTS OF URINARY PCR TESTING FOR LEPTOSPIROSIS IN CAPTIVE BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS).

    PubMed

    Olds, June E; Sun, Yaxuan; Baum, David H; Gauger, Phillip

    2015-12-01

    Leptospirosis is an important zoonotic disease occurring clinically and subclinically in humans and a wide variety of mammal species worldwide. Often, rodents and wild animals are identified as important reservoirs for the disease. Twenty-two captive black-tailed prairie dogs (Cynomys ludovicianus) housed within a zoo were examined as part of a routine census and preventive medicine program. During examinations, blood and urine were collected to screen for exposure to, or infection with, leptospirosis. All animals were apparently healthy at the time of examination. Leptospira microscopic agglutination test identified 12 of 22 (54.5%) prairie dogs with antibody titers ≥1 : 100 against Leptospira interrogans serovar bratislava on initial serologic examination. All prairie dogs within this collection were serologically negative for L. interrogans serovars canicola, hardjo, icterohaemorrhagiae, and pomona and Leptospira kirschneri serovar grippotyphosa. Leptospira polymerase chain reaction (PCR) testing of urine was negative in all animals tested. This report describes evidence that captive prairie dogs may be exposed to leptospirosis, most likely from wild rodent reservoirs; however, serum titers are low, and lack of leptospiral DNA detected by PCR indicates that these captive animals are unlikely to be important reservoirs for the disease. PMID:26667541

  1. Evaluation of Type 1 Immune Response in Naïve and Vaccinated Animals following Challenge with Leptospira borgpetersenii Serovar Hardjo: Involvement of WC1+ γδ and CD4 T Cells

    PubMed Central

    Naiman, Brian M.; Blumerman, Seth; Alt, David; Bolin, Carole A.; Brown, Rachel; Zuerner, Richard; Baldwin, Cynthia L.

    2002-01-01

    Organisms within the Hardjo serovar of Leptospira species are harbored in cattle throughout the world, causing abortion in pregnant animals as well as being shed in the urine, thereby providing sources of zoonotic infection for humans. We recently showed that sterile immunity in vaccinated cattle is associated with induction of a type 1 (Th1) cell-mediated immune response. Here naïve and previously vaccinated pregnant cattle were challenged with a virulent strain of serovar Hardjo and subsequently evaluated for expression of a type 1 immune response. Lymphocytes that responded in a recall response to antigen by undergoing blast transformation were evident in cultures of peripheral blood mononuclear cells (PBMC) from vaccinated cattle throughout the postchallenge test period while those from naïve cattle were evident at one time point only. Nevertheless, beginning at 2 weeks after challenge, gamma interferon (IFN-γ) was measured in supernatants of antigen-stimulated PBMC cultures from nonvaccinated animals although the amount produced was always less than that in cultures of PBMC from vaccinated animals. IFN-γ+ cells were also evident in antigen-stimulated cultures of PBMC from vaccinated but not from nonvaccinated animals throughout the postchallenge period. The IFN-γ+ cells included CD4+ and WC1+ γδ T cells, and a similar proportion of these two subpopulations were found among the dividing cells in antigen-stimulated cultures as ascertained by carboxyfluorescein succinimidyl ester loading. Finally, while naïve and vaccinated animals had similar levels of antigen-specific immunoglobulin G1 (IgG1) following challenge, vaccinated animals had twofold-more IgG2. In conclusion, while infection may induce a type 1 response we suggest that it is too weak to prevent establishment of chronic infection. PMID:12379692

  2. Leptospira Immunoglobulin-Like Protein B (LigB) Binds to Both the C-Terminal 23 Amino Acids of Fibrinogen αC Domain and Factor XIII: Insight into the Mechanism of LigB-Mediated Blockage of Fibrinogen α Chain Cross-Linking.

    PubMed

    Hsieh, Ching-Lin; Chang, Eric; Tseng, Andrew; Ptak, Christopher; Wu, Li-Chen; Su, Chun-Li; McDonough, Sean P; Lin, Yi-Pin; Chang, Yung-Fu

    2016-09-01

    The coagulation system provides a primitive but effective defense against hemorrhage. Soluble fibrinogen (Fg) monomers, composed of α, β and γ chains, are recruited to provide structural support for the formation of a hemostatic plug. Fg binds to platelets and is processed into a cross-linked fibrin polymer by the enzymatic clotting factors, thrombin and Factor XIII (FXIII). The newly formed fibrin-platelet clot can act as barrier to protect against pathogens from entering the bloodstream. Further, injuries caused by bacterial infections can be confined to the initial wound site. Many pathogenic bacteria have Fg-binding adhesins that can circumvent the coagulation pathway and allow the bacteria to sidestep containment. Fg expression is upregulated during lung infection providing an attachment surface for bacteria with the ability to produce Fg-binding adhesins. Fg binding by leptospira might play a crucial factor in Leptospira-associated pulmonary hemorrhage, the main factor contributing to lethality in severe cases of leptospirosis. The 12th domain of Leptospira immunoglobulin-like protein B (LigB12), a leptospiral adhesin, interacts with the C-terminus of FgαC (FgαCC). In this study, the binding site for LigB12 was mapped to the final 23 amino acids at the C-terminal end of FgαCC (FgαCC8). The association of FgαCC8 with LigB12 (ELISA, KD = 0.76 μM; SPR, KD = 0.96 μM) was reduced by mutations of both charged residues (R608, R611 and H614 from FgαCC8; D1061 from LigB12) and hydrophobic residues (I613 from FgαCC8; F1054 and A1065 from LigB12). Additionally, LigB12 bound strongly to FXIII and also inhibited fibrin formation, suggesting that LigB can disrupt coagulation by suppressing FXIII activity. Here, the detailed binding mechanism of a leptospiral adhesin to a host hemostatic factor is characterized for the first time and should provide better insight into the pathogenesis of leptospirosis. PMID:27622634

  3. Leptospirosis and tularaemia in raccoons (Procyon lotor) of Larimer County, [corrected] Colorado.

    PubMed

    Duncan, C; Krafsur, G; Podell, B; Baeten, L A; LeVan, I; Charles, B; Ehrhart, E J

    2012-02-01<